May 28, 2021 - Free Activators

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May 28, 2021 - Free Activators

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May 28, 2021 - Free Activators -

Tavern Brawl is one of Hearthstone's lighter game modes. A weekly event, a new Tavern Brawl begins each Wednesday (Thursday in Asian regions). See the schedule for this week's times.

Each Tavern Brawl presents a limited time opportunity to face other players in special matches with unique rules. These might be requiring players to use special pre-made decks, adding random cards or minions, or changing the very rules of ability activation or mana use. Some Tavern Brawls allow the player to construct special decks especially for the challenge from within the game mode itself, while others provide powerful premade decks, or entirely random decks, and some Brawls even see players take control of special heroes with their own Hero Powers. Some Brawls feature Tavern Brawl cards, special cards not playable normally, as well as boss cards from adventures such as One Night in Karazhan. Each week sees a fresh Tavern Brawl, with its own unique rules.

As well as being matched against other random opponents, players are able to challenge players on their friends list to a Tavern Brawl, as a special type of Friendly Challenge.[1]

An intentionally "fun" game mode, Tavern Brawls offer unique limited time experiences, with far less of a competitive focus than other game modes. This makes them good for quick matches, friendly games and cumulative wins. With many Brawls incorporating random card generation or minion summoning, winning a Tavern Brawl often involves a fair amount of luck, as well as the ability to adapt to and deal with the situation at hand. Tavern Brawls help to break up the focused rigours of Hearthstone's other game modes by providing a fresh twist on the game's rules each week.

Tavern Brawls are intended to be highly accessible, even for players without extensive card collections.[2] However, Tavern Brawls are not unlocked until the player reaches level 20 with at least one class (including Demon Hunter class which is level 20 after completing the prologue).

For a compact list of unique Tavern Brawls, see Brawls list.

Schedule[]

Each week, the Tavern Brawl is open for nearly the entire week, with only one hour of downtime, according to the following schedule by region:[3]

RegionOpenClose
AmericasWed 9:00 AM PTWed 8:00 AM PT
EuropeWed 8:00 PM CETWed 7:00 PM CET
ChinaThu 5:00 AM CSTThu 4:00 AM CST
AsiaThu 6:00 AM KSTThu 5:00 AM KST

Converted to Coordinated Universal Time (UTC) the schedule is as follows:

RegionOpenClose
AmericasWed 4:00 PM UTC (3 PM UTC during daylight savings)Wed 3:00 PM UTC (2 PM during daylight savings)
EuropeWed 7:00 PM UTCWed 6:00 PM UTC
ChinaWed 9:00 PM UTCWed 8:00 PM UTC
AsiaWed 9:00 PM UTCWed 8:00 PM UTC
  • Daylight Savings Time in the U.S. starts on the 2nd Sunday of March and ends on the 1st Sunday of November each year.
  • Daylight Savings Time in Europe (CEST) generally starts on the last Sunday of March and ends on the last Sunday of October each year.

History[]

#OpenCloseTavern BrawlType
336November 17, 2021November 24, 2021Reprise: Battle of Tol BaradConstructed
335November 10, 2021November 17, 2021Reprise: Just a HallucinationRandomized
334November 3, 2021November 10, 2021Fairytale BrawlPremade
333October 27, 2021November 3, 2021Reprise: The Haunted CarouselRandomized
332October 20, 2021October 27, 2021Reprise: Rise of the ZombeastsRandomized
331October 13, 2021October 20, 2021Reprise: Valeera's Bag of Burgled SpellsRandomized
330October 6, 2021October 13, 2021Reprise: Battle of the BansConstructed
329September 29, 2021October 6, 2021Reprise: Half & HalfConstructed
328September 22, 2021September 29, 2021Reprise: Three WishesRandomized
327September 15, 2021September 22, 2021Reprise: Party Portals!Randomized
326September 8, 2021September 15, 2021Reprise: All-Star SquadConstructed
325September 1, 2021September 8, 2021A Recipe United!Premade
324August 25, 2021September 1, 2021Reprise: Cloneball!Randomized
323August 18, 2021August 25, 2021Reprise: Brawl Block: Gods, Gadgetzan, GurubashiConstructed
322August 11, 2021August 18, 2021Reprise: Shake, Deathrattle and Roll!Randomized
321August 4, 2021August 11, 2021Reprise: RandomoniumRandomized
320July 28, 2021August 4, 2021Reprise: When Clones Attack!Randomized
319July 21, 2021July 28, 2021Reprise: Clockwork Card DealerConstructed
318July 14, 2021July 21, 2021The Duelist Burndown!Premade
317July 7, 2021July 14, 2021Reprise: Ragnaros' Fire FestivalConstructed
316June 30, 2021July 7, 2021Reprise: BY THE POWER OF RAGNAROSConstructed
315June 23, 2021June 30, 2021Reprise: Send In The Murlocs!Randomized
314June 16, 2021June 23, 2021Reprise: Battle of the BansConstructed
313June 9, 2021June 16, 2021Reprise: Blood MagicConstructed
312June 2, 2021June 9, 2021Reprise: Just MacawsRandomized
311May 26, 2021June 2, 2021Reprise: Showdown at Blackrock MountainPremade
310May 19, 2021May 26, 2021Reprise: Captain Blackheart's TreasureRandomized
309May 12, 2021May 19, 2021Reprise: UnidentifiaBrawlRandomized
308May 5, 2021May 12, 2021Reprise: A Chance EncounterRandomized
307April 28, 2021May 5, 2021Reprise: Battle of Tol BaradConstructed
306April 21, 2021April 28, 2021Reprise: Valeera's Bag of Burgled SpellsRandomzied
305April 14, 2021April 21, 2021Reprise: Top 3Constructed
304April 7, 2021April 14, 2021A Recipe of Tenacity!Premade
303March 31, 2021April 7, 2021Reprise: Everybunny Get in Here!Randomized
302March 24, 2021March 31, 2021Reprise: Three WishesRandomized
301March 17, 2021March 24, 2021Reprise: A Temporus ShiftConstructed
300March 10, 2021March 17, 2021Reprise: Just a HallucinationRandomized
299March 3, 2021March 10, 2021Reprise: Brawl Block: Tinyfin Mode!Constructed
298February 24, 2021March 3, 2021Reprise: Cart Crash at the CrossroadsRandomized
297February 17, 2021February 24, 2021Reprise:: The Great AmalgamationConstructed
296February 10, 2021February 17, 2021Reprise: Lunar Blessings!Randomized
295February 3, 2021February 10, 2021Reprise: All-Star SquadConstructed
294January 27, 2021February 3, 2021Reprise: Cloneball!Randomized
293January 20, 2021January 27, 2021Reprise: When Clones Attack!Randomized
292January 13, 2021January 20, 2021Reprise: A Less Friendly Game of ChessConstructed
291January 6, 2021January 13, 2021Visions of SaygePremade
290December 30, 2020January 6, 2021Reprise: A New Year BashCooperative
289December 23, 2020December 30, 2020Reprise: Wacky Waxy Winter's VeilConstructed
288December 16, 2020December 23, 2020Reprise: Gift ExchangeConstructed
287December 9, 2020December 16, 2020Reprise: Battle of the BansConstructed
286December 2, 2020December 9, 2020Reprise: Shake, Deathrattle and Roll!Randomized
285November 25, 2020December 2, 2020Half & HalfConstructed

Older Tavern Brawls[]

#OpenCloseTavern BrawlType
284November 18, 2020November 25, 2020Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
283November 11, 2020November 18, 2020Reprise: Gone Fishin'Constructed
282November 4, 2020November 11, 2020Reprise: Brawl of GaudinessRandomized
281October 28, 2020November 4, 2020Reprise: Send In The Murlocs!Randomized
280October 21, 2020October 28, 2020Reprise: The Haunted CarouselRandomized
279October 14, 2020October 21, 2020Boss Battle Royale 3Premade
278October 7, 2020October 14, 2020Hallow's End Masquerade BallConstructed
277September 30, 2020October 7, 2020Reprise: Rise of the ZombeastsRandomized
276September 23, 2020September 30, 2020Heroic BrawliseumBrawliseum
275September 16, 2020September 23, 2020Reprise: RandomoniumRandomized
274September 9, 2020September 16Reprise: Blood MagicConstructed
273September 2, 2020September 9, 2020Reprise: UnidentifiaBrawlRandomized
272August 26, 2020September 2, 2020Reprise: Top 3Constructed
271August 19, 2020August 26, 2020Reprise: Party Portals!Randomized
270August 12, 2020August 19, 2020Reprise: Who's the Boss Now?Constructed
269August 5, 2020August 12, 2020Reprise: Valeera's Bag of Burgled SpellsRandomized
268July 29, 2020August 5, 2020Reprise: Just MacawsRandomized
267July 22, 2020July 29, 2020Reprise: Just a HallucinationRandomized
266July 15, 2020July 22, 2020Reprise: A Peek to the PastConstructed
265July 7, 2020July 15, 2020Reprise: Decks Assemble!Randomized
264July 1, 2020July 8, 2020Rumbledome!Premade
263June 24, 2020July 1, 2020The Outlandish BurndownPremade
262June 17, 2020June 24, 2020An Outlandish RecipePremade
261June 10, 2020June 17, 2020Reprise: Captain Blackheart's TreasureRandomized
260June 3, 2020June 10, 2020Reprise: Send In The Murlocs!Randomzied
259May 27, 2020June 3, 2020Reprise: A Cavalcade of Brawls!Randomized
258May 20, 2020May 27, 2020Reprise: Cloneball!Randomized
257May 13, 2020May 20, 2020Reprise: Battle of the BansConstructed
256May 6, 2020May 13, 2020Reprise: A Chance EncounterRandomized
255April 29, 2020May 6, 2020Brawl Block: Feeling Adventurous?Constructed
254April 22, 2020April 29, 2020Reprise: Battle of Tol BaradConstructed
253April 15, 2020April 22, 2020Reprise: Dr. Boom's Ignoblegarden!Randomized
252April 8, 2020April 15, 2020The Fury of Kael'thasConstructed
251April 1, 2020April 8, 2020Reprise: Brawl of Champions Grand FinalsPremade
250March 25, 2020April 1, 2020Reprise: Return of Mechazod!Cooperative
249March 18, 2020March 25, 2020Reprise: Three WishesRandomized
248March 11, 2020March 18, 2020Reprise: Hall of ChampionsPremade
247March 4, 2020March 11, 2020The Burndown - RekindledPremade
246February 26, 2020March 4, 2020Reprise: Shiftcon WestRandomized
245February 19, 2020February 26, 2020Reprise: When Clones Attack!Randomized
244February 12, 2020February 19, 2020Brawl of GaudinessRandomized
243February 5, 2020February 12, 2020Reprise: All-Star SquadConstructed
242January 29, 2020February 5, 2020Lunar Blessings!Randomized
241January 22, 2020January 29, 2020Road to NorthrendSingle-player
240January 15, 2020January 22, 2020Reprise: Pick a Hand, Any HandConstructed
239January 8, 2020January 15, 2020Just MacawsRandomized
238January 1, 2020January 8, 2020Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
237December 25, 2019January 1, 2020Reprise: A New Year BashCooperative
236December 18, 2019December 25, 2019Reprise: Gift ExchangeConstructed
235December 11, 2019December 18, 2019Decorating Dalaran!Single-player
234December 4, 2019December 11, 2019Reprise: Heart of the SunwellConstructed
233November 27, 2019December 4, 2019Reprise: Encounter at the CrossroadsRandomized
232November 20, 2019November 27, 2019Shake, Deathrattle and Roll!Randomized
231November 13, 2019November 20, 2019Reprise: Top 3Constructed
230November 6, 2019November 13, 2019Reprise: Send In The Murlocs!Randomized
229October 30, 2019November 6, 2019Reprise: Captain Blackheart's TreasureRandomized
228October 23, 2019October 30, 2019The Haunted CarouselRandomized
227October 16, 2019October 23, 2019Doom in the Tomb (week 2)Single-player
226October 9, 2019October 16, 2019Doom in the TombSingle-player
225October 2, 2019October 9, 2019Reprise: Six-ShooterConstructed
224September 25, 2019October 2, 2019Reprise: Rise of the ZombeastsRandomized
223September 18, 2019September 25, 2019Brawl Block: Tinyfin Mode!Constructed
222September 11 , 2019September 18, 2019Three WishesRandomized
221September 4, 2019September 11, 2019The League of Explorers: Revived!Single-player
220August 28, 2019September 4, 2019Reprise: Valeera's Bag of Burgled SpellsRandomized
219August 21, 2019August 28, 2019The Great AmalgamationConstructed
218August 14, 2019August 21, 2019Reprise: Cart Crash at the CrossroadsRandomized
217August 7, 2019August 14, 2019Reprise: Your Standard BrawliseumBrawliseum
216July 31, 2019August 7, 2019Reprise: A Less Friendly Game of ChessConstructed
215July 24, 2019July 31, 2019The Burndown!Premade
214July 17, 2019July 24, 2019Blackrock Crash (week 2)Single-player
213July 10, 2019July 17, 2019Blackrock CrashSingle-player
212July 3, 2019July 10, 2019Reprise: Decks Assemble!Randomized
211June 26, 2019July 1, 2019Reprise: A Temporus ShiftConstructed
210June 19, 2019June 24, 2019Brawl Block: Gods, Gadgetzan, GurubashiConstructed
209June 12, 2019June 17, 2019Rise of the MechPremade
208June 5, 2019June 10, 2019Reprise: Nefarian Rises!Cooperative
207May 29, 2019June 3, 2019The 207th Brawl Spectacular!Randomized
206May 22, 2019May 27, 2019Reprise: Clockwork Card DealerConstructed
205May 15, 2019May 20, 2019Reprise: Cloneball!Randomized
204May 8, 2019May 13, 2019Henchmania!Premade
203May 1, 2019May 6, 2019Reprise: Showdown at Blackrock MountainPremade
202April 24, 2019April 29, 2019Reprise: Hall of ChampionsPremade
201April 17, 2019April 22, 2019Dr. Boom's Ignoblegarden!Randomized
200April 10, 2019April 15, 2019Reprise: Your Standard BrawliseumBrawliseum
199April 3, 2019April 8, 2019Reprise: RandomoniumRandomized
198March 27, 2019April 1, 2019Brawl Block: Year of the MammothConstructed
197March 20, 2019March 25, 2019Battle of the BansConstructed
196March 13, 2019March 18, 2019Reprise: BY THE POWER OF RAGNAROSConstructed
195March 6, 2019March 11, 2019Brawliseum - Miniature WarfareBrawliseum
194February 27, 2019March 4, 2019Brawl of Champions Grand FinalsPremade
193February 20, 2019February 25, 2019Brawl of Champions Round TwoPremade
192February 13, 2019February 18, 2019Brawl of Champions Round OnePremade
191February 6, 2019February 11, 2019Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
190January 30, 2019February 4, 2019Reprise: The Void SingularityConstructed
189January 23, 2019January 28, 2019Shiftcon WestRandomized
188January 16, 2019January 21, 2019Reprise: A Chance EncounterRandomized
187January 9, 2019January 14, 2019Brawl Block: Old and NewConstructed
186January 2, 2019January 7, 2019Reprise: Just a HallucinationRandomized
185December 26, 2018December 31, 2018Reprise: Gift ExchangeConstructed
184December 19, 2018December 24, 2018Returns & ExchangesConstructed
183December 12, 2018December 17, 2018A Royal RecipePremade
182December 5, 2018December 10, 2018Reprise: Your Standard BrawliseumBrawliseum
181November 28, 2018December 3, 2018Reprise: Return of Mechazod!Cooperative
180November 21, 2018November 26, 2018When Clones Attack!Randomized
179November 14, 2018November 19, 2018A Temporus ShiftConstructed
178November 7, 2018November 12, 2018Send In The Murlocs!Randomized
177October 31, 2018November 5, 2018Reprise: Decks Assemble!Randomized
176October 24, 2018October 29, 2018Reprise: The Headless Horseman Rides!Single-player
175October 17, 2018October 22, 2018Monster SmashPremade
174October 10, 2018October 15, 2018Reprise: Boss Battle Royale 2Premade
173October 3, 2018October 8, 2018Reprise: Spiders, Spiders, EVERYWHERE!Randomized
172September 26, 2018October 1, 2018Six-ShooterConstructed
171September 19, 2018September 24, 2018Duel of the Death KnightsRandomized
170September 12, 2018September 17, 2018Reprise: Pick a Hand, Any HandConstructed
169September 5, 2018September 10, 2018Reprise: Too Many Portals!Constructed
168August 29, 2018September 3, 2018Reprise: Double Deathrattler BattlerConstructed
167August 22, 2018August 27, 2018A Scientific RecipePremade
166August 15, 2018August 20, 2018Reprise: Your Standard BrawliseumBrawliseum
165August 8, 2018August 13, 2018Reprise: A New Year BashCooperative
164August 1, 2018August 6, 2018Reprise: Valeera's Bag of Burgled SpellsRandomized
163July 25, 2018July 30, 2018A Brawl of Fire and IceSingle-player
162July 18, 2018July 23, 2018Reprise: Ragnaros' Fire FestivalConstructed
161July 11, 2018July 16, 2018Reprise: ShiftConRandomized
160July 4, 2018July 9, 2018Reprise: Blood Magic - StandardConstructed
159June 27, 2018July 2, 2018A Less Friendly Game of ChessConstructed
158June 20, 2018June 24, 2018Reprise: Shadow ReflectionConstructed
157June 13, 2018June 17, 2018Rise of the ZombeastsRandomized
156June 6, 2018June 10, 2018Reprise: RandomoniumRandomized
155May 30, 2018June 4, 2018Gone Fishin'Constructed
154May 22, 2018May 28, 2018Reprise: Cloneball!Randomized
153May 16, 2018May 21, 2018Top 2 - StandardConstructed
152May 9, 2018May 14, 2018Everybunny Get in Here!Randomized
151May 2, 2018May 6, 2018Your Standard BrawliseumBrawliseum
150April 25, 2018April 29, 2018A Witchy RecipePremade
149April 18, 2018April 22, 2018Reprise: Idols of AzerothPremade
148April 11, 2018April 15, 2018A Chance EncounterRandomized
147April 4, 2018April 9, 2018Blessing of the RavenRandomized
146March 28, 2018April 2, 2018Reprise: Miniature Warfare - StandardConstructed
145March 21, 2018March 26, 2018Portals to Another DimensionRandomized
144March 14, 2018March 19, 2018Reprise: Cart Crash at the CrossroadsRandomized
143March 7, 2018March 12, 2018Reprise: Showdown at Blackrock MountainPremade
142February 28, 2018March 5, 2018The Wild BrawliseumBrawliseum
141February 21, 2018February 25, 2018Venture into the WildPremade
140February 15, 2018February 19, 2018Boss Battle Royale 2Premade
139February 7, 2018February 12, 2018Reprise: Just a HallucinationRandomized
138January 31, 2018February 5, 2018UnidentifiaBrawlRandomized
137January 24, 2018January 29, 2018Reprise: Nefarian Rises!Cooperative
136January 17, 2018January 21, 2018Hall of ChampionsPremade
135January 10, 2018January 15, 2018Shadow ReflectionConstructed
134January 3, 2018January 8, 2018Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
133December 27, 2017January 1, 2018Reprise: Idols of AzerothPremade
132December 20, 2017December 25, 2017Wacky Waxy Winter's VeilConstructed
131December 13, 2017December 18, 2017Reprise: Valeera's Bag of Burgled SpellsRandomized
130December 6, 2017December 11, 2017Reprise: Yellow-Brick BrawlConstructed
129November 29, 2017December 4, 2017The Void SingularityConstructed
128November 22, 2017November 27, 2017Reprise: Decks Assemble!Randomized
127November 15, 2017November 20, 2017Treasure in the Catacombs!Randomized
126November 8, 2017November 13, 2017Reprise: The Great Summoner CompetitionConstructed
125November 1, 2017November 6, 2017Reprise: Miniature WarfareConstructed
124October 25, 2017October 30, 2017The Headless Horseman Rides!Single-player
123October 18, 2017October 23, 2017Reprise: ShiftConRandomized
122October 11, 2017October 16, 2017Reprise: Spellbook DuelConstructed
121October 4, 2017October 9, 2017Reprise: A Peek to the PastConstructed
120September 27, 2017October 1, 2017Reprise: Encounter at the CrossroadsRandomized
119September 20, 2017September 25, 2017Reprise: The Great Stone of ChallengeConstructed
118September 13, 2017September 18, 2017A Frozen RecipePremade
117September 6, 2017September 11, 2017Reprise: An Evil ExchangePremade
116August 30, 2017September 4, 2017Reprise: Blood Magic - StandardConstructed
115August 23, 2017August 28, 2017BY THE POWER OF RAGNAROSConstructed
114August 16, 2017August 21, 2017Encounter at the CrossbonesRandomized
113August 9, 2017August 14, 2017Reprise: ShiftConRandomized
112August 2, 2017August 7, 2017Battle for the CraterRandomized
111July 26, 2017July 31, 2017Reprise: Showdown at Blackrock MountainPremade
110July 19, 2017July 24, 2017Ahune's Superior BrawlPremade
109July 12, 2017July 17, 2017Reprise: Clash of the MinionsConstructed
108July 5, 2017July 10, 2017Ragnaros' Fire FestivalConstructed
107June 28, 2017July 3, 2017Reprise: All-Star SquadConstructed
106June 21, 2017June 26, 2017Reprise: Spiders, Spiders, EVERYWHERE!Randomized
105June 14, 2017June 18, 2017Reprise: Cart Crash at the CrossroadsRandomized
104June 6, 2017June 12, 2017Just a HallucinationRandomized
103May 31, 2017June 5, 2017Reprise: RandomoniumRandomized
102May 24, 2017May 29, 2017A Peek to the PastConstructed
101May 17, 2017May 22, 2017Reprise: An Evil ExchangePremade
100May 10, 2017May 15, 2017A Cavalcade of Brawls!Randomized
99May 3, 2017May 8, 2017Heroic Tavern Brawl - Wild formatBrawliseum
98April 26, 2017May 1, 2017Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
97April 19, 2017April 24, 2017An Ancient RecipePremade
96April 12, 2017April 17, 2017Reprise: Valeera's Bag of Burgled SpellsRandomized
95April 5, 2017April 10, 2017Reprise: The Masked BallConstructed
94March 29, 2017April 3, 2017A Mammoth of a New YearCooperative
93March 22, 2017March 27, 2017Decorating StormwindSingle-player
92March 15, 2017March 20, 2017Reprise: Cart Crash at the CrossroadsRandomized
91March 8, 2017March 13, 2017Reprise: Heroic Tavern BrawlBrawliseum
90March 1, 2017March 6, 2017Gadgetzan Throw-DownPremade
89February 22, 2017February 27, 2017Reprise: Cloneball!Randomized
88February 15, 2017February 20, 2017Blood MagicConstructed
87February 8, 2017February 13, 2017Reprise: Battle of Tol BaradConstructed
86February 1, 2017February 6, 2017Reprise: Gift ExchangeConstructed
85January 25, 2017January 30, 2017Boss Battle RoyalePremade
84January 18, 2017January 23, 2017Spellbook DuelConstructed
83January 11, 2017January 16, 2017Reprise: Cart Crash at the CrossroadsRandomized
82January 4, 2017January 9, 2017The Dark WandererSingle-player
81December 28, 2016January 2, 2017Reprise: Party Portals!Randomized
80December 21, 2016December 26, 2016Reprise: Clockwork Card Dealer - StandardConstructed
79December 14, 2016December 19, 2016The Battle for GadgetzanPremade
78December 7, 2016December 12, 2016Reprise: Double Deathrattler BattlerConstructed
77November 30, 2016December 5, 2016Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
76November 23, 2016November 28, 2016Valeera's Bag of Burgled SpellsRandomized
75November 16, 2016November 21, 2016Heroic Tavern BrawlBrawliseum
74November 9, 2016November 14, 2016Reprise: Miniature WarfareConstructed
73November 2, 2016November 7, 2016Yellow-Brick BrawlConstructed
72October 26, 2016October 31, 2016Reprise: Captain Blackheart's TreasureRandomized
71October 19, 2016October 24, 2016Reprise: ShiftConRandomized
70October 12, 2016October 17, 2016All-Star SquadConstructed
69October 5, 2016October 10, 2016A Friendly Game of ChessPremade
68September 28, 2016October 3, 2016Reprise: Boom Bot vs. Annoy-o-Tron!Premade
67September 21, 2016September 26, 2016Reprise: RandomoniumRandomized
66September 14, 2016September 19, 2016Top 3Constructed
65September 7, 2016September 12, 2016Reprise: Cart Crash at the CrossroadsRandomized
64August 31, 2016September 5, 2016Party Portals!Randomized
63August 24, 2016August 29, 2016Reprise: Captain Blackheart's TreasureRandomized
62August 17, 2016August 22, 2016Nefarian Rises!Cooperative
61August 10, 2016August 15, 2016A Dark RecipePremade
60August 3, 2016August 8, 2016Storming StormwindSingle-player
59July 27, 2016August 1, 2016Reprise: RandomoniumRandomized
58July 20, 2016July 25, 2016Reprise: Idols of AzerothPremade
57July 13, 2016July 18, 2016'Servant of Yogg-Saron' TryoutsRandomized
56July 6, 2016July 11, 2016Reprise: Clockwork Card DealerConstructed
55June 29, 2016July 4, 2016ShiftConRandomized
54June 22, 2016June 27, 2016Pick a Hand, Any HandConstructed
53June 15, 2016June 20, 2016Reprise: Heart of the SunwellConstructed
52June 8, 2016June 13, 2016Cloneball!Randomized
51June 1, 2016June 6, 2016Cart Crash at the CrossroadsRandomized
50May 25, 2016May 30, 2016Shadow TowersConstructed
49May 18, 2016May 23, 2016Reprise: Unite Against Mechazod!Cooperative
48May 11, 2016May 16, 2016Top 2Constructed
47May 4, 2016May 9, 2016Reprise: Spiders, Spiders, EVERYWHERE!Randomized
46April 27, 2016May 2, 2016Reprise: Encounter at the CrossroadsRandomized
45April 20, 2016April 25, 2016Idols of AzerothPremade
44April 13, 2016April 18, 2016The Great Stone of ChallengeConstructed
43April 6, 2016April 11, 2016Reprise: Too Many Portals!Randomized
42March 30, 2016April 4, 2016You Must be This Tall to BrawlConstructed
41March 23, 2016March 28, 2016An Evil ExchangePremade
40March 16, 2016March 21, 2016Reprise: Battle of Tol BaradConstructed
39March 9, 2016March 14, 2016Reprise: RandomoniumRandomized
38March 2, 2016March 7, 2016Deal Your FateConstructed
37February 24, 2016February 29, 2016Clash of the MinionsConstructed
36February 17, 2016February 22, 2016Lead from the FrontConstructed
35February 10, 2016February 15, 2016Reprise: Decks Assemble!Randomized
34February 3, 2016February 8, 2016Blingtron's Beauteous BrawlConstructed
33January 27, 2016February 1, 2016Miniature WarfareConstructed
32January 20, 2016January 25, 2016Reprise: Captain Blackheart's TreasureRandomized
31January 13, 2016January 18, 2016Return of Mechazod!Cooperative
30January 6, 2016January 11, 2016Battle of the BuildsConstructed
29December 30, 2015January 4, 2016Reprise: Who's the Boss Now?Constructed
28December 23, 2015December 28, 2015Reprise: RandomoniumRandomized
27December 16, 2015December 21, 2015Gift ExchangeConstructed
26December 9, 2015December 14, 2015Reprise: Encounter at the CrossroadsRandomized
25December 2, 2015December 7, 2015Decks Assemble!Randomized
24November 25, 2015November 30, 2015Battle of Tol BaradConstructed
23November 18, 2015November 23, 2015Captain Blackheart's TreasureRandomized
22November 11, 2015November 16, 2015Reprise: Showdown at Blackrock MountainPremade
21November 4, 2015November 9, 2015Unite Against Mechazod!Cooperative
20October 28, 2015November 2, 2015Reprise: Spiders, Spiders, EVERYWHERE!Randomized
19October 21, 2015October 26, 2015Who's the Boss Now?Constructed
18October 14, 2015October 19, 2015RandomoniumRandomized
17October 7, 2015October 12, 2015Clockwork Card DealerConstructed
16September 30, 2015October 5, 2015Boom Bot vs. Annoy-o-Tron!Premade
15September 23, 2015September 28, 2015Underdog RulesConstructed
14September 16, 2015September 21, 2015Reprise: Too Many Portals!Randomized
13September 9, 2015September 14, 2015Double Deathrattler BattlerConstructed
12September 2, 2015September 7, 2015It's Raining Mana!Constructed
11August 26, 2015August 31, 2015Reprise: Encounter at the CrossroadsRandomized
10August 19, 2015August 24, 2015Grand Tournament MatchPremade
9August 12, 2015August 17, 2015Reprise: The Great Summoner CompetitionConstructed
8August 5, 2015August 10, 2015The Masked BallConstructed
7July 29, 2015August 3, 2015Too Many Portals!Randomized
6July 22, 2015July 27, 2015Heart of the SunwellConstructed
5July 15, 2015July 20, 2015Encounter at the CrossroadsRandomized
4July 8, 2015July 13, 2015The Great Summoner CompetitionConstructed
3July 1, 2015July 6, 2015Spiders, Spiders, EVERYWHERE!Randomized
2June 24, 2015June 29, 2015Banana Brawl!Constructed
1June 17, 2015June 22, 2015Showdown at Blackrock MountainPremade

Repeated Tavern Brawls[]

While most weeks see a brand new Tavern Brawl, just under half are reprises of previous Brawls. Since each Brawl is only available for 7 days (originally 5), this allows players to enjoy old favourites again, and avoids Brawls becoming overly similar due to a desire to explore past mechanics. Most reprises are simple repeat outings, identical to the original Brawl, but some involve changes, such as fixing bugs or balance issues, or even adding in extra elements.

The following table lists Tavern Brawls that have appeared more than once, including brief notes of any known changes.

Tavern BrawlNumber of appearancesKnown changes
Randomonium10The 8th appearance awarded a Rise of Shadows card pack.
Encounter at the Crossroads8The 5th appearance featured Party Crashers, and awarded a Journey to Un'Goro card pack.

The 6th appearance was renamed to "Encounter at the Crossbones".

Valeera's Bag of Burgled Spells8
Captain Blackheart's Treasure7The 5th appearance restricted cards to the standard format.
Cart Crash at the Crossroads7
Cloneball!7
'Servant of Yogg-Saron' Tryouts7
Battle of Tol Barad6
Decks Assemble!6The 3rd appearance awarded a Kobolds & Catacombs card pack.
Just a Hallucination6The 3rd appearance awarded a Rastakhan's Rumble card pack.
Showdown at Blackrock Mountain6Starting Mana Crystals for Nefarian reduced in the 2nd appearance.
All-Star Squad5
Battle of the Bans5
Blood Magic52nd, 3rd, and 4th appearances used standard format decks.
Clockwork Card Dealer5
Gift Exchange5
Send In The Murlocs!5
ShiftCon5
Spiders, Spiders, EVERYWHERE!5
Unite Against Mechazod! / Return of Mechazod!5Assassinate replaced by Prioritize in Mechazod's deck.
Your Standard Brawliseum5
A Chance Encounter4
A New Year Bash (A Mammoth of a New Year)4The 2nd appearance renamed the Brawl. The 4th appearance changed the decks for each player.
Idols of Azeroth4
Heroic Brawliseum (Heroic Tavern Brawl)4The 3rd appearance used wild format decks. The 4th appearance renamed the Brawl.
Miniature Warfare4
Party Portals!4In the second appearance, changes were made to Romulo, Party Elemental, Party Elemental and Mime
Rise of the Zombeasts42nd appearance lowered the starting health to 20.
Three Wishes4
Top 34
When Clones Attack!4
A Less Friendly Game of Chess3
A Peek to the Past3
A Temporus Shift3In the 2nd appearance of the Brawl, the first player no longer has an extra turn on turn 1.
An Evil Exchange3The 2nd appearance changed Kel'Thuzad's Hero Power from "Resurrect a friendly minion that died this game" to "Add a random Naxxramas minion to your hand", Rafaam's Hero Power at one and two pieces were changed, Noth the Plaguebringer's cost was increased, and three of Kel'Thuzad's cards in his deck were changed.

The 3rd appearance buffed Kel'Thuzad's Hero Power to decrease the generated card's cost by 3.

BY THE POWER OF RAGNAROS3
Double Deathrattler Battler33rd appearance used standard format decks.
Hall of Champions3
Heart of the Sunwell3
Just Macaws3
Nefarian Rises!3
Pick a Hand, Any Hand3
Ragnaros' Fire Festival3
Shake, Deathrattle and Roll!3
The Great Summoner Competition3The 3rd appearance used standard format decks
The Haunted Carousel3
Too Many Portals!3
UnidentifiaBrawl3
Who's the Boss Now?3
A Cavalcade of Brawls!2The 2nd appearance changed the first line of the description to "The cavalcade returns!"
Boom Bot vs. Annoy-o-Tron!2
Boss Battle Royale 22The 2nd appearance changed the druid hero and the warlock hero received changes.
Brawl Block: Gods, Gadgetzan, Gurubashi2
Brawl Block: Tinyfin Mode!2
Brawl of Champions Grand Finals2
Clash of the Minions2
Brawl of Gaudiness2
Dr. Boom's Ignoblegarden!2
Everybunny Get in Here!2
Gone Fishin'2
Half & Half2
Lunar Blessings!2
Shadow Reflection2
Shiftcon West2
Six-Shooter2
Spellbook Duel2
The Burndown!22nd appearance renamed the brawl The Burndown - Rekindled and used a different selection of cards
The Great Amalgamation2
The Great Stone of Challenge2
The Headless Horseman Rides!2
The Masked Ball2
The Void Singularity2
Top 2 / Top 2 - Standard2
Wacky Waxy Winter's Veil2
Yellow-Brick Brawl22nd appearance used standard format decks

Notes[]

Access[]

  • Tavern Brawls have free admission.[1][4][5] Very rarely, a Brawl such as Heroic Tavern Brawl may charge an admission fee.
  • Tavern Brawls are only available to players with at least one level 20 hero.[1]
  • Players can play each Tavern Brawl as many times as they like while it is active.[6][7]
  • Only the current Tavern Brawl is accessible to players. Players are not able to access past Brawls, or challenge other players to take part in them.[8]
  • While each Brawl has a limited run, some Tavern Brawls are repeated later on. Reprise selection appears to be largely based on popularity.

Decks[]

  • Tavern Brawls use unique decks, which do not take up any of the player's regular deck slots.[9][10] Players create Tavern Brawls within the Tavern Brawl interface, and the deck is lost when the Brawl is retired.
  • The composition of Tavern Brawl decks depends on the Brawl. Many are constructed partially or wholly from the player's own collection, with some seeing random cards added to the deck made. Other Brawls feature premade decks, either with specific (and often unique) cards, or entirely randomised.
    • Brawl decks will not require or allow the player to use more than 2 copies of a card from their own collection, or more than 1 in the case of legendaries.[11]
  • Most Brawls require the player to choose a class, either during deck construction or in place of deck construction, but some instead randomly assign players to take control of special heroes, with their own decks.
  • Tavern Brawl cards are similar to Boss cards, special cards specific to Tavern Brawls are not available in other types of play. See below for more information.

Matchmaking[]

  • Unless specifically challenging a friend, players will be randomly matched against other participating players "of similar skill level".[12]
  • Initial player seeding is apparently based on rankings in other types of play.[13] This is most likely based on Casual or Ranked mode play, or a combination of the two.
  • After the initial placement, player matchmaking is controlled by the player's success in Tavern Brawls, using a hidden MMR.[14] Tavern Brawl MMR is ongoing, and carries over from week to week.[15]
  • Cooperative Tavern Brawls are an exception, and do not use any MMR.[16]

Rewards and quests[]

  • Players earn a free Classiccard pack for their first Tavern Brawl win each week except some Tavern Brawls before new expansion launch - those Tavern Brawls rewards a pack of the upcoming expansion.
    • No additional rewards are offered after the initial win. This is intended to prevent players from feeling they have to play,[17] and may also be in order to allow design space for unusual and potentially uneven arrangements, which could otherwise prove frustrating for those seeking to earn repeated rewards through wins.
    • Very rarely a Brawl will offer other rewards, such as a unique card back, in place of the usual card pack.
    • The card pack reward was originally announced to end when the "launch event" ended.[1][18] However, having begun in June 2015, it seems likely the reward is now intended to be ongoing.
  • Tavern Brawls are eligible to count toward daily quests.[19][20] Wins also count toward the "10 gold per 3 wins" reward.[21]
  • There is one Brawl-specific quest, "Everybody! Get in here!", which awards 60 gold for playing 3 Tavern Brawls.
  • Challenging a friend to a Tavern Brawl will not be eligible for most quests, as these matches are considered Friendly Challenges for these purposes.[22]
  • Players can earn experience in Tavern Brawl.[23]

General[]

  • Tavern Brawls are released in the Americas region first, followed by the Europe, Taiwan/Korea and China regions, simultaneously, around 4 hours later. As a result, the Americas region sometimes experiences technical issues following the release of a new Brawl, serving as a testbed for the latest modifications. Despite not directly modifying other game modes, the release of a Tavern Brawl nevertheless involves changes which can cause disconnections and extended downtime for the entire game, making this testing period necessary to limit disruption to a single region. The four hour delay following the Americas release is intended to allow time for the developers to attend to any stability issues or severe bugs, before launching the Brawl in other regions.
    • As of December 2015, additional testing measures are planned, to reduce related stability issues.[24] However, the changes will "take some time to implement".[24]

Tavern Brawl cards[]

Tavern Brawl cards are special cards, heroes and Hero Powers only available to players when participating in a specific Tavern Brawl. These are usually provided through pre-made decks, although they may be granted to players directly during matches. Tavern Brawl cards usually match the theme of the Brawl, and often feature fun or outlandish powers not seen in regular cards.

For a full list of known cards, see Tavern Brawl card. Featured Tavern Brawl cards for each Brawl can be found on individual Brawl pages, or in the Previous Brawls section.

[]

Main article: Fireside Brawl

Fireside Brawls are special Tavern Brawls that can only be played at Fireside Gatherings.

Each month there will be a different Fireside Brawl to try—check the Fireside Gatherings website to find out about the brawl that’s running this month. These special brawls run the whole month with no breaks, and they co-exist with the weekly Tavern Brawls.

The first Fireside Brawl that got a large amount of attention was Tavern vs. Lich King.

Design[]

"I'm not exaggerating when I say that each week, people tell me that we've created the greatest brawl ever, at the same time that others are saying it's by far the worst." - Ben Brode[25]

As of October 2016, Tavern Brawls are primarily designed by Pat Nagle (the namesake of Nat Pagle) and Dan Emmons.[26] The two designers "sit in a room and brainstorm", with additional inspiration from the internet and other team members. The ideas are then playtested and iterated upon with the rest of Team 5.[27][28]Tim Erskine serves as producer for Tavern Brawl content.[29]

According to designers Emmons and Nagle, the most important goal of Tavern Brawls is to provide variety and freshness, "mixing up" the standard game experience of ladder or Arena play, and providing players a reason and reward for logging in regularly.[26] The "Surprise" of the new Brawl each week is also "really big" for the developers.[26]

The designers originally considered having a ranked ladder for Tavern Brawl mode, including a Legend rank.[30] However, they decided not to in order to avoid players feeling pressure to play, instead adopting a strong focus on "fun".[30] For example, the card pack reward for a single win provides a reward for trying each Brawl, without creating any sense of compulsion for players who didn't that week's Brawl.

The Tavern Brawl mode is also designed to help with the game's accessibility. This is partly achieved through Brawls that don't require players to own a lot of cards, and also in the 'free' card pack reward each week, helping players to build their collections.[30]

Tavern Brawls allow for experimentation, and are sometime used to test or develop new mechanics, allowing the developers to observe player reactions. This time spent developing the required technology makes it relatively simple to then implement the mechanic elsewhere in the game.[31] Examples of this process include Discover. However, the developers balance the ease of experimentation with a desire to keep new mechanics fresh for players to discover in new sets. Ben Brode explains:

"There are pros and cons. The pros are obviously that we would get more confidence on balance. One of the cons is that part of the fun of card games is exploration. If we are putting it out there and a lot of it gets solved or figured out, then when we release a set it isn’t as crazy and exciting as it could be."[32]

The brevity of Brawls also frees the developers to experiment with a variety of designs, since "even if [the developers] make a really bad one" it will only last a few days.[26] This allows the designers to create Brawls that target specific groups of players, and take risks with their designs.[26]

The designers learned from early Brawls like Spiders, Spiders, EVERYWHERE! with decks composed solely of minions, incorporating a small number of random spells into later minion-focused Brawls such as ShiftCon.[30]

History[]

Tavern Brawls were being discussed by the developers prior to the launch of Hearthstone.[33] However, it was not until June 10, 2015, more than a year after the game's release, that the feature would be announced to players.

The Tavern Brawl system was added with Patch 2.7.0.9166 on June 15, but was not activated until June 17, when the first Tavern Brawl, Showdown at Blackrock Mountain, went live. In the interim period before the first Brawl, the as yet blank fourth button on the game box would begin to sparkle each time the player entered the main menu.

Captain Blackheart's Treasure was planned for release in October 2015, in order to debut the upcoming Discover mechanic.[27] However, technical problems prevented this, and the keyword was instead announced at BlizzCon 2015 with its accompanying adventure, The League of Explorers.

On November 4, 2015, the first cooperative Tavern Brawl was released, Unite Against Mechazod!, marking the first cooperative two-player experience within the game. At the time of its creation, this was the most difficult Brawl to create yet.[27]

In December 2015, with disconnections and downtime in the Europe region coinciding with the release of the latest Tavern Brawl becoming a regular occurrence, Blizzard announced that they would be adding additional testing measures to help prevent this in the future.[24] Shortly afterwards it was announced that the release times for Tavern Brawls in each region would be changing: instead of debuting in the Europe region at 4pm UTC, followed by Americas at 6pm and other regions at 7pm, new Brawls would now debut in the Americas region at 5pm UTC, then in all other regions 9pm.[34][35]

In October 2016 Heroic Tavern Brawl was announced, marking a new hybridisation of the Tavern Brawl and Arena game modes. This was also the first known Brawl to use Standard format, rather than the usual Wild format. The first explicitly Standard format Brawl followed in December 2016 with Clockwork Card Dealer - Standard, a simple variant of the original Clockwork Card Dealer.

On July 1, 2019, a new schedule was implemented for Tavern Brawls, reducing the weekly downtime from roughly 2.25 days to a short time span of 1 hour.[3]

Trivia[]

  • As of October 2016 Randomonium is "by far the most popular Brawl", both in terms of number of games played and hours in total spent playing.[36][37] Designer Dean Ayala explains, "[Randomonium] continues to be one of the mostly highly played brawls by unique user in addition to one of the most highly played brawls by individual user. Basically this means a ton of people play it, and the ones that do play it play a ton of games."[38]
  • The developers considered placing the Tavern Brawl button in the third slot (currently occupied by the Arena button), which would have provided a more symmetrical appearance given the smaller amount of space available for lettering on the fourth button. However, they decided against it, possibly due to length issues in languages other than English.[39]
  • On July 29, 2015, Showdown at Blackrock Mountain was mistakenly reprised as the seventh Tavern Brawl, sparking widespread complaint from players, who assumed this to be the intended Brawl. The correct Brawl, Too Many Portals!, replaced it around an hour later, but not until many players had already played and won games in the former Brawl, earning their weekly card pack. Zeriyah remarked, "Sometimes when you play with dimensional space, the wrong Tavern Brawl gets sucked through the portal. Whoops."[40] A similar error has taken place with subsequent Brawls. On July 20, 2016, Battle of the Builds was reprised as the fifty-eighth Tavern Brawl, but bugs quickly became evident. Blizzard announced a few hours later that the Brawl "wasn't behaving" and replaced it with another reprise, Idols of Azeroth.[41]
  • Following the Gift Exchange Tavern Brawl, the 2015 winter break saw a repeat of Randomonium rather than another new Tavern Brawl. Ben Brode explained that this was partly due to there being "nobody in the office over the break to fix things if they go wrong, which new brawls are more likely to do."[42]
  • The 5th reprise of Encounter at the Crossroads was the first Brawl that didn't offer a Classic card pack, but instead a card pack from an upcoming expansion.
  • The popularity of the Tavern Brawl game mode in Hearthstone has led to its adoption in other Blizzard games. Overwatch and Heroes of the Storm saw the introduction of the "Weekly Brawl" and "Heroes Brawl" game modes on March 22 and April 10, 2016, respectively, less than a year after the Hearthstone format's launch;[43][44] while "PvP Brawls" were added to World of Warcraft on April 5, 2017.[45] While the Tavern label is specific to Hearthstone, the term Brawl has thus become shorthand for an "alternative way to play", featuring special and regularly changing twists on the game's normal ruleset, with an emphasis on fun, "crazy" and "unique" matches.[43]
  • Before the patch update on July 1, 2019, Tavern Brawls originally would end the following Monday and be unavailable from Monday to Wednesday.

Gallery[]

Patch changes[]

References[]

External links[]

Источник: https://hearthstone.fandom.com/wiki/Tavern_Brawl

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Источник: https://pubmed.ncbi.nlm.nih.gov/34085031/

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TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation

Abstract

The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.

Introduction

NOD-, LRR-, and pyrin domain-containing protein 3 (NLRP3) is a crucial pattern recognition receptor, which can sense pathogen-associated molecular patterns (PAMPs) and endogenous danger signals (danger-associated molecular patterns, DAMPs) to initiate the formation of a multi-protein complex named the NLRP3 inflammasome1,2,3,4,5. Canonical activation of the NLRP3 inflammasome requires two sequential steps. PAMPs or the activation of cytokines provides the first signal (priming signal), which activates nuclear factor (NF)-κB and induces the expression of the NLRP3, an integral part of the NLRP3 inflammasome, pro-interleukin-1β (pro-IL-1β), and pro-IL-18. The second signal (activation signal) is provided by a wide range of stimuli, such as particulates, crystals, and adenosine 5’-triphosphate disodium (ATP). Upon activation, NLRP3 inflammasome assembly activates caspase-1, which in turn cleaves pro-IL-1β and pro-IL-18. NLRP3 inflammasome has been implicated in multiple diseases and its activity should be tightly controlled1.

Various posttranslational modifications (PTMs) of NLRP3, including phosphorylation, ubiquitination, and SUMOylation, play crucial roles in controlling inflammasome activation1. The level of intracellular NLRP3 protein is considered as a key rate-limiting step for canonical activation of NLRP3 inflammasome6,7. In resting macrophages, NLRP3 is ubiquitylated and its protein level is relatively low, which limits NLRP3 inflammasome activation. Upon priming and activation, NLRP3 is deubiquitylated, and that enhances NLRP3 expression and facilitates inflammasome activation8,9. The E3 ubiquitin ligases tripartite motif containing protein 31 (TRIM31) and membrane-associated RING finger protein 7 (MARCH7) induce Lys(K)48-linked ubiquitylation of NLRP3 and in turn promotes its degradation via proteasome and autophagy, respectively10,11. The ubiquitin-specific peptidase 1 (USP1)-associated factor 1 (UAF1) /USP1 deubiquitinase complex selectively inhibits K48-linked ubiquitination and protein degradation of NLRP3 enhances cellular NLRP3 levels12, which are indispensable for subsequent NLRP3 inflammasome assembly and activation. Recently, small ubiquitin-like modifier (SUMO) protein-conjugated modifications, which are termed as SUMOylation, were identified in NLRP3 during inflammasome activation13,14. SUMOylation, a type of PTM that is similar to ubiquitination, is mediated by E1-activating enzymes, a sole E2 ligase UBC9, and E3 SUMO ligases15,16,17. Three SUMO proteins, including SUMO1, SUMO2, and SUMO3, can be covalently conjugated to proteins as a single moiety (SUMO1) or as polymeric SUMO chains (SUMO2 and SUMO3). SUMOylation contributes significantly to mediating protein interaction, regulating protein localization in cells, regulating the activity of transcription factors, maintaining genomic stability, and transcriptional regulation15. The E3 SUMO protein ligase MUL1 (also known as MAPL) catalyzes SUMO2/3 modification of NLRP3 and restrains NLRP3 activation, whereas SUMO-1-catalyzed SUMOylation of NLRP3 facilitates inflammasome activation. Although SUMO1 modification of NLRP3 is required for inflammasome activation, both the mechanism details and the E3 SUMO ligase that mediates SUMO1-catalyzed SUMOylation of NLRP3 are unclear.

Here we identify the E3 SUMO ligase TRIM28 (also known as Kruppel-associated box-associated protein 1 (KAP1) or transcriptional intermediary factor 1β (TIF1β)) as an enhancer of inflammasome activity by promoting NLRP3 SUMOylation. TRIM28 interacts with NLRP3, facilitates SUMO1 and SUMO2/3 catalyzed SUMOylation of NLRP3, and thereby inhibits its ubiquitination and proteasomal degradation. TRIM28 stabilizes NLRP3 protein expression and enhances subsequent inflammasome activation. These data provide a mechanism by which SUMOylation enhances NLRP3 inflammasome activation and indicate that TRIM28 might be a target for the treatment of diseases caused by aberrant inflammasome activation.

Results

TRIM28 interacts with NLRP3

To identify potential interactors of NLRP3, we incubated a specific anti-NLRP3 antibody with total cell lysates of mouse primary peritoneal macrophages (PMs) stimulated with lipopolysaccharide (LPS). The co-immunoprecipitated complex was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The E3 SUMO ligase TRIM28 was identified as a potential NLRP3 interactor by liquid chromatography coupled with tandem mass spectrometry (Fig. 1a). LPS markedly induced TRIM28 expression (Fig. 1b), suggesting that TRIM28 may be involved in the process of NLRP3 inflammasome activation. However, Trim28 mRNA decreased at first and increased subsequently upon LPS stimulation (Supplementary Fig. 1a), indicating the existence of other regulatory mechanisms for TRIM28 protein levels in response to LPS stimulation. We next examined the interaction between TRIM28 and NLRP3 in vivo. Confocal analysis demonstrated the colocalization between TRIM28 and NLRP3 (Fig. 1c). Next, the interaction between TRIM28 and NLRP3 was investigated under physiological conditions by immunoprecipitation (IP). An association between TRIM28 and NLRP3 was detected in both resting LPS-primed and ATP-activated mouse PMs (Fig. 1d). TRIM28 along with NLRP3, ASC, or caspase-1 were cotransfected in HEK293T cells. TRIM28 could immunoprecipitate with NLRP3 but not ASC and caspase-1 (Fig. 1e). Taken together, these data suggest that TRIM28 could interact with NLRP3.

a Identification of TRIM28 as a potential NLRP3 interactor in LPS-stimulated mouse peritoneal macrophages (PMs) by mass spectrum primed with LPS. b Immunoblot analysis of TRIM28 expression in LPS-stimulated mouse PMs (n = 3 independent experiments). c Confocal microscopic analysis of the colocalization of NLRP3 (Red) and TRIM28 (Green) in unstimulated mouse embryonic fibroblasts (MEFs). Scale bar, 10 μm. d IP analysis of endogenous association between TRIM28 and NLRP3 in LPS-stimulated or LPS-primed and ATP-activated mouse PMs (n = 3 independent experiments). e Immunoprecipitation (IP) analysis of the association between TRIM28 and NLRP3, ASC or Caspase-1 in HEK293T cells transfected with the indicated plasmids. Similar results were obtained from three independent experiments.

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TRIM28 has no effects on NLRP3 inflammasome priming

Next, we first investigated the potential functions of TRIM28 in the priming of NLRP3 inflammasome. Trim28fl/fl mice were crossed with Lyz2cre mice to specifically knockout Trim28 in myeloid cells (called “Trim28CKO” here). Trim28 deficiency had no effects on LPS-induced phosphorylation of IκBα and total IκBα (Fig. 2a) and transcription of Il1b, Nlrp3, Tnf, and Il6 in PMs (Fig. 2b and Supplementary Fig. 1b). To further investigate the function of TRIM28, small interfering RNA (siRNA)-knockdown experiments were performed. Synthesized siRNA targeting mouse Trim28 was used to suppress endogenous TRIM28 expression in PMs (Supplementary Fig. 1c). Trim28 knockdown also did not affect phosphorylation of IκBα and total IκBα expression and transcription of Il1b, Nlrp3, Tnf, and Il6 (Fig. 2c, d and Supplementary Fig. 1d). Collectively, these data indicate that TRIM28 could not affect LPS-induced NF-κB activation and the priming process of NLRP3 inflammasome activation.

a, c Immunoblot analysis of p-IκBα and IκBα in LPS-stimulated mouse PMs from WT or Trim28CKO mice (a) or PMs transfected with Ctrl siRNA or Trim28 siRNA (c). b, d RT-PCR analysis of Ilb and Nlrp3 mRNA in LPS-stimulated mouse PMs from WT or Trim28CKO mice (b) or PMs transfected with Ctrl siRNA or Trim28 siRNA (d). All data are represented as mean ± SD in b, d. Similar results were obtained from five independent experiments (a, c) and three independent experiments (b, d).

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TRIM28 promotes NLRP3 inflammasome activation

Next, we tested the effect of TRIM28 on NLRP3 inflammasome activation. Mouse PMs from both wild-type (WT) and Trim28CKO mice were primed by LPS and then stimulated by NLRP3 inflammasome activators (ATP or nigericin (Nig)) or poly(dA:dT) (an AIM2 inflammasome activator). Trim28 deficiency markedly inhibited NLRP3 inflammasome-induced IL-1β secretion, while had no effects on AIM2 inflammasome activation (Fig. 3a). However, tumor necrosis factor (TNF) and IL-6 secretion were not influenced by Trim28 deficiency (Fig. 3b). Activated caspase-1 cleaves gasdermin D (GSDMD) and releases its N-terminal regions, which then punch holes in the membrane, resulting in the release of IL-1β and pyroptosis18. Trim28 deficiency suppressed both caspase-1 and GSDMD cleavage following NLRP3 inflammasome activation (Fig. 3c). Meanwhile, we observed that Trim28 deficiency did not influence IL-1β but greatly attenuated LPS-induced NLRP3 expression (Fig. 3c), suggesting that TRIM28 may selectively enhance NLRP3 inflammasome activation by promoting NLRP3 expression. Trim28 knockdown also reduced IL-1β secretion following LPS priming and ATP activation in mouse PMs (Fig. 3d), with no effects on both TNF and IL-6 secretion (Supplementary Fig. 1e). Taken together, these data indicate that TRIM28 facilitates NLRP3 inflammasome activation.

a, b ELISA analysis of IL-1β, TNF, and IL-6 in supernatants of mouse PMs from WT or Trim28CKO mice following priming with LPS for 7 h and subsequent stimulation with ATP, Nig, or poly(dA:dT) for 1 h (a two-tailed t test WT vs. Trim28CKO, *p = 0.011241, **p = 0.005688). c Immunoblot analysis of supernatants (SN) and cell lysates (CL) of mouse PMs from WT or Trim28CKO mice, following priming with LPS and subsequent stimulation with ATP (n = 3 independent experiments). d ELISA analysis of IL-1β in supernatants from mouse PMs transfected with control (Ctrl) siRNA or Trim28 siRNA for 48 h, followed by priming with LPS for 7 h and subsequent stimulation with ATP for 1 h (two-tailed t test Ctrl siRNA vs. Trim28 siRNA, **p = 0.005134). e ELISA analysis of serum levels of IL-1β, TNF, and IL-6 of WT or Trim28CKO mice after i.p. LPS injection (PBS, n = 3; LPS, n = 8 per group, two-tailed t test WT vs. Trim28CKO, ***p = 6 × 10−6). All data are represented as mean ± SD in a, b, d, e (*p < 0.05; **p < 0.01; ***p < 0.001). Similar results were obtained from three independent experiments.

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We next investigated the function of TRIM28 on NLRP3 inflammasome activation in vivo. The secretion of IL-1β by intraperitoneal (i.p.) injection of LPS is NLRP3 dependent. The secretion of IL-1β in the serum of Trim28CKO mice decreased upon LPS challenge, while TNF and IL-6 remained unchanged (Fig. 3e). These results demonstrate that Trim28 deficiency attenuates NLRP3 inflammasome activation in vivo.

TRIM28 inhibits proteasomal degradation of NLRP3

Next, we determined whether TRIM28 regulates NLRP3 protein expression. TRIM28 overexpression increased NLRP3 protein expression in HEK293T cell (Fig. 4a). Consistently, both Trim28 deficiency and knockdown inhibited LPS-induced NLRP3 protein expression in mouse PMs (Fig. 4b, c). Interestingly, NLRP3 protein expression in unstimulated macrophages was also considerably attenuated by Trim28 deficiency or knockdown (Supplementary Fig. 2a, b). Although TRIM28 has transcriptional regulatory functions19, it did not enhance NLRP3 transcription (Fig. 2b, d). We then investigated the effects of TRIM28 on NLRP3 protein degradation by cycloheximide (CHX) chase assay. Trim28 deficiency markedly promoted NLRP3 protein degradation (Fig. 4d, e). Ubiquitin–proteasome and autophagy–lysosome are major pathways for NLRP3 degradation. We then further clarified which pathway was affected by TRIM28. MG132, a selective inhibitor of proteasome, but not the lysosome inhibitor chloroquine and autophagy inhibitor 3-methyladenine (3-MA), restored the NLRP3 protein expression in LPS-stimulated Trim28 deficiency macrophages (Fig. 4f, g and Supplementary Fig. 2c). Collectively, these data indicate that TRIM28 selectively inhibits NLRP3 protein degradation in proteasome.

a Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3 and increasing amount of Flag-TRIM28 plasmid. b Immunoblot analysis of lysates of mouse PMs from WT or Trim28CKO mice, following stimulation with LPS. c Immunoblot analysis of lysates of mouse PMs transfected with Ctrl siRNA or Trim28 siRNA for 48 h, following stimulation with LPS. d, e Immunoblot analysis of NLRP3 expression in mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h and then treated cycloheximide (CHX) for the indicated time periods. NLRP3 expression was quantitated by measuring band intensities using the “ImageJ” software. The values were normalized to Actin. Data are represented as mean ± SD (two-tailed t test WT vs. Trim28CKO, ***p = 1 × 10−12, 3 × 10−17). f Immunoblot analysis of NLRP3 expression in mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h, together with DMSO or MG132 (10 μM) treatment for 4 h. g Immunoblot analysis of NLRP3 expression in mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h, together with DMSO or chloroquine (50 μM) treatment for 4 h. Similar results were obtained from three independent experiments.

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TRIM28 promotes SUMOylation of NLRP3

Ubiquitination is required for the protein degradation via the ubiquitin–proteasome pathway. We therefore examined the potential roles of TRIM28 on NLRP3 ubiquitination. Trim28 deficiency facilitated NLRP3 ubiquitination in unstimulated, LPS-primed, and ATP-activated macrophages (Fig. 5a). K48-linked ubiquitination is required for proteasomal degradation of target proteins. Next, to clarify whether TRIM28 could inhibit K48-linked ubiquitination, the ubiquitin mutant vectors K48 and K63, which contain arginine substitutions of all of its lysine residues except the one at positions 48 and 63 respectively, were used in the transfection assays. TRIM28 inhibited both total and K48-linked ubiquitination of NLRP3, whereas it had no effects on K63-linked ubiquitination of NLRP3 (Fig. 5b). TRIM28 is an E3 SUMOylation ligase20 and the cysteine (C) at position 651 (C651) is required for its enzymatic activity. TRIM28 point mutant (C651) with substitution of the cysteine residue with alanine (A) at position 651 could not bind to the key E2 SUMO binding enzyme UBC9 (Supplementary Fig. 3), and thus its E3 SUMO ligase activity was inactivated21. TRIM28 C651 lost the ability to inhibit NLRP3 ubiquitination and degradation (Fig. 5c, d), suggesting that TRIM28 selectively attenuates K48-linked ubiquitination of NLRP3 and its proteasomal degradation, via its E3 SUMO ligase activity.

a Immunoblot analysis of lysates of mouse PMs from WT or Trim28CKO mice, primed with LPS for 4 h, together with MG132 (10 μM) treatment for 3 h, and then stimulation with ATP for 1 h, and followed by IP with NLRP3 antibody. b Immunoblot analysis of lysates from HEK293T cells transfected with HA-Ub (WT), HA-tagged K48-linked ubiquitin (K48-Ub), or HA-tagged K63-linked ubiquitin (K63-Ub), Myc-NLRP3, and Flag-TRIM28, together with MG132 (10 μM) treatment for 4 h and followed by IP with Myc antibody. c Immunoblot analysis of lysates from HEK293T cells transfected with HA-tagged ubiquitin (HA-Ub), Myc-NLRP3, and Flag-TRIM28 WT or C651A, together with MG132 (10 μM) treatment for 4 h, and followed by IP with Myc antibody. d Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3, together with Flag-TRIM28 WT or C651A. Similar results were obtained from three independent experiments.

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NLRP3 could be modified by SUMO1 and SUMO2/3 in macrophages (Fig. 6a, b), which is consistent with previous reports13,14. Interestingly, we found that LPS stimulation enhanced both SUMO1 and SUMO2/3 modification of NLRP3, while SUMO1 modification further increased and SUMO2/3 modification decreased following ATP stimulation (Fig. 6a, b and Supplementary Fig. 4). These data suggest a dynamic change of NLRP3 SUMOylation status during the priming and activation step of NLRP inflammasome. 2′,3′,4′-Trihydroxy flavone (2-D08), an inhibitor of protein SUMOylation, dose-dependently decreased NLRP3 expression (Fig. 6c), suggesting the potential role of SUMOylation on NLRP3 expression. We have identified E3 SUMO ligase TRIM28 as a NLRP3-associated protein and that promoted us to investigate whether TRIM28 could mediate the SUMOylation of NLRP3. Both Trim28 deficiency and knockdown attenuated SUMO1 and SUMO2/3 modification of NLRP3 in macrophages (Fig. 6a, b and Supplementary Fig. 4). Furthermore, TRIM28 promoted SUMO1, SUMO2, and SUMO3 modification in HEK293T cells, while TRIM28 C651 mutant lost the ability to mediate NLRP3 SUMOylation (Fig. 6d–f). Therefore, TRIM28 induced NLRP3 SUMOylation, inhibited NLRP3 ubiquitination and degradation, and thus facilitated inflammasome activation (see a model in Fig. 7).

a, b Immunoblot analysis of lysates of mouse peritoneal macrophages from WT or Trim28CKO mice, primed with LPS for 4 h, together with MG132 (10 μM) treatment for 3 h, and then stimulation with ATP for 1 h, and followed by SUMOylation assays with NLRP3 antibody. c Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3 and stimulated with an increasing amount of 2-D08 for 24 h. df Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3, His-UBC9 and Flag-TRIM28 WT or C651A, and HA-SUMO1, HA-SUMO2, or HA-SUMO3, followed by MG132 (10 μM) treatment for 4 h, and then performed SUMOylation assays with Myc antibody. Similar results were obtained from three independent experiments.

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TRIM28 binds to NLRP3, promotes SUMOylation of NLRP3, and thus inhibits NLRP3 ubiquitination and degradation in proteasome, resulting in the enhancement of NLRP3 inflammasome activation.

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Discussion

Various types of PTMs are important for the regulation of protein folding, stability, localization, and functional activities. NLRP3 is subjected to multiple PTMs, including phosphorylation, ubiquitination, alkylation, S-nitrosylation, and SUMOylation, to tightly control optimal activation of NLRP3 inflammasome. But the correlations and interactions among various types of PTMs of NLRP3 remain less clarified. SUMOylation interacts with other PTMs, such as ubiquitination, phosphorylation, and acetylation, to regulate protein functions15. For example, ubiquitinated p53 recruits the E3 SUMO ligase PLASy to promote SUMOylation of p5322. E3 SUMO ligase PLAS4-mediated SUMOylation of MDC1 recruits E3 ubiquitin-ligase RNF4 to promote MDC ubiquitination in DNA damage16. However, SUMO1 modification usually blocks ubiquitination of its substrate. For example, SUMO1-modified IκBα cannot be ubiquitinated and is resistant to proteasome-mediated degradation23. SUMO1 modification of cGAS and STING prevent their polyubiquitination and degradation during viral infection24. SUMO1 and Ub both bind to the lysine (K) residues of their substrates and that leads to competitive inhibition. In this study, we reveal the correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation. The E3 SUMO ligase TRIM28 facilitates SUMO1 and SUMO2/3-catalyzed SUMOylation of NLRP3, whereby attenuates K48-linked ubiquitination of NLRP3, resulting in enhancement of NLRP3 stability.

During NLRP3 inflammasome priming (such as LPS stimulation), SUMO1/2/3 modification and ubiquitination are all increased. Following activation (ATP stimulation), SUMO1 modification is further enhanced, while SUMO2/3 modification and ubiquitination were decreased, suggesting that dynamic changes of SUMOylation and ubiquitination during NLRP3 inflammasome activation. MUL1-mediated SUMO2/3-catalyzed NLRP3 SUMOylation was reported to inhibit NLRP3 inflammasome activation13. But SUMO1-catalyzed NLRP3 SUMOylation promotes inflammasome activation, which is consistent with a previous report14. SUMO1 modified Lys (K) residues cannot be SUMOylated or ubiquitinated, which is different to the characteristics of SUMO2/3 modification and ubiquitination25,26. Therefore, SUMO1 modification of NLRP3 may block the conjugation of ubiquitin by competing with the same K sites of NLRP3 and inhibits ubiquitous degradation of NLRP3 and therefore facilitates inflammasome activation. Although MUL1-mediated SUMO2/3-catalyzed SUMOylation of NLRP3 inhibited inflammasome activation13, we found that TRIM28-mediated SUMO2/3-catalyzed SUMOylation facilitated inflammasome activation. In addition, TRIM28, but not MUL1, inhibited the ubiquitination degradation of NLRP3. Therefore, TRIM28 and MUL1 regulate NLRP3 activity by different mechanisms. Multiple potential SUMOylation sites exist in NLRP3 protein (such as K204 and K689, etc.), and TRIM28 and MUL1 may mediate NLRP3 SUMOylation at different lysine sites. SUMOylation mediated by TRIM28 may block the ubiquitination site of NLRP3 and thus inhibits NLRP3 proteasomal degradation. Further analysis of the SUMOylation and ubiquitination sites of NLRP3 will help to elucidate the underlying mechanisms. In addition, TRIM28 promotes SUMO1/2/3-catalyzed SUMOylation of NLRP3, and SUMO1 blocks other PTMs (including ubiquitination) of NLRP3, resulting in the stability of NLRP3 and enhancement of inflammasome activation. Based on the experimental data, we propose a working model to illustrate how TRIM28-mediated SUMOylation enhances NLRP3 inflammasome activation (Fig. 7). TRIM28 binds to NLRP3, promotes its SUMOylation, and thereby inhibits NLRP3 ubiquitination and subsequent degradation in proteasome. However, in Trim28-deficiency macrophages, NLRP3 is still synthesized normally, and NLRP3 degradation is enhanced. Therefore, the absence of TRIM28 does not completely eliminate NLRP3 expression. Thus, enhanced cellular level of NLRP3 facilitates inflammasome assembly and enhances inflammasome activation.

In addition to its E3 SUMO ligase activity, TRIM28 is a well-known epigenetic suppressor. TRIM28 can bind to KRAB zinc-finger proteins (KZNFs) and recruit the histone methyltransferase SETDB1 to recognize and bind to H3k9me3, resulting in transcriptional repression27,28,29. TRIM28 works as a master epigenetic regulator of endogenous retroviral elements (ERVs) to maintain ERVs in a silent state and thereby protects cells from detrimental genome instability and aberrant interferon responses. Furthermore, TRIM28 possesses important epigenetic roles in the differentiation and functions of regulatory T cells and Th17 cells, and genetic deletion of KAP1 in T cells results in autoimmune phenotype30,31,32,33. However, in our study, we found that TRIM28 had no effect on the transcription of key components of inflammasome, including Il1b and Nlrp3.

In summary, we identify the E3 SUMO ligase TRIM28 as a stabilizer of NLRP3 by promoting its SUMOylation. Concordantly, TRIM28 enhances inflammasome activation via enhancing cellular level of NLRP3. Given the pivotal roles of inflammasome in the occurrence and development of a variety of diseases, modulation of NLRP3 activation by targeting SUMOylation or TRIM28 will be a priming target for the treatment of diseases caused by aberrant NLRP3 activity.

Methods

Mice

Trim28fl/fl mice34 (Stock Number:018552) and Lyz2cre mice (Stock Number: 004781) were from Jackson Laboratory. Trim28fl/fl mice were crossed with Lyz2cre transgenic mice to obtain Trim28CKO mice with Trim28 deficiency in myeloid cells. Mice were kept in specific pathogen-free-level Laboratory Animal Room with 12-/12-h light and dark alternating time, temperature 18–22 °C and humidity 45–65%. The background strain of all mice used is C57BL/6J. All mice were used at 6–11 weeks of age, and both male and female mice were used. All animal experiments were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the School of Basic Medical Science, Shandong University, Jinan, Shandong Province, China.

Reagents and antibodies

ATP (A1852), Nig (N7143), Z-Leu-Leu-Leu-al (MG132, C2211), LPS (Escherichia coli, O111:B4, L4130), NAnti-Flag and anti-Myc are the same isotypes.

Cell culture

To obtain mouse primary PMs, C57BL/6J mice (male, 6–8 weeks old) were injected i.p. with 3% Brewer’s thiogly-collate broth. Three days later, peritoneal exudate cells were harvested and incubated. Two hours later, nonadherent cells were removed and the adherent monolayer cells were used as PMs. Human embryonic kidney (HEK293T) cells were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum (Invitrogen-Gibco). The concentration of agonists or stimuli were used as below: LPS 200 ng/ml for mouse primary PMs, LPS 1 μg/ml for mouse embryonic fibroblasts (MEFs), ATP 2.5 mM, Nig 50 mM, MG132 10 μM, chloroquine 50 μM, 3-MA 10 mM. Poly(dA:dT) were transfected into macrophages with the final concentration as 200 ng/ml.

Plasmid construction and transfection

TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added. TRIM28 C651A was generated using the KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan). Expression plasmids for NLRP3, ASC, Caspase-1, HA-Ub WT, mutant K48, and mutant K63 were described previously10,12. HA-SUMO1 (HG13095-NY), HA-SUMO2 (HG13443-NY), HA-SUMO3 (HG12782-NY), and His-UBC9 (HG143205-NH) were from Sino Biological. All constructs were confirmed by DNA sequencing. Plasmids were transiently transfected into HEK293T cell with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.

RNA interference assay

siRNAs were synthesized as following: murine Trim28: 5′-GACAUCGUGGAGAAUUAUU-3′, negative control: 5′-UUCUCCGAACGUGUCACGU-3′. These siRNA duplexes were transfected into mouse PMs using INTERFERin reagents (Polyplus-transfection) according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of mouse IL-1β (cat. number: 1210123), TNF (cat. number: 1217202), and IL-6 (cat. number: 1210603) were measured using ELISA kits (Dakewe Biotech Company Ltd., Shenzhen, China) according to the manufacturer’s instructions.

RNA quantitation

RNA from macrophages was extracted by using the RNAfast200 RNA Extraction Kit according to the manufacturer’s instructions. Quantitative real-time PCR analysis was carried out using the Applied Biosystems StepOnePlus Real-Time PCR System and SYBR RT-PCR kits (Roche). Specific primers used for real-time PCR assays are listed in Supplementary Table 1. Data are normalized to β-Actin expression in each sample.

Immunofluorescent and confocal microscopy

MEFs, which were plated on glass coverslips in 24-well plates, were fixed with Immunol Staining Fix Solution (Beyotime). Cells were permeabilized with 0.5% Triton-X 100 in phosphate-buffered saline (PBS) and blocked with Immunol Staining Blocking Buffer (Beyotime) for 1 h. Then the cells were incubated with anti-TRIM28 (Mouse) and anti-NLRP3 (Rabbit) diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) at 4 °C overnight. Next, the cells were stained with secondary Abs (anti-Mouse conjugated to Alexa Fluor 488 and anti-Rabbit conjugated to Alexa Fluor 633) and nuclei were stained with DAPI (Beyotime)35. Then the cells were subjected to microscopic analysis with the Zeiss LSM780 confocal laser microscope, which was provided by the Micro Characterization Facility of Shandong University.

IP and immunoblot analysis

For IP, whole-cell lysates were lysed in IP buffer containing 1.0% (vol/vol) Nonidet P 40, 50 mM Tris–HCl, pH 7.4, 50 mM EDTA, 150 mM NaCl, and a protease inhibitor “cocktail” (Sigma). After 15 min centrifugation at 12,000 × g at 4 °C, protein concentrations in the lysates were measured with a bicinchoninic acid assay (Pierce, Rockford, IL), and then supernatants were collected and incubated with protein G Plus-Agarose IP reagent together with specific antibody overnight. Beads were washed five times with IP buffer. Immunoprecipitates were eluted by boiling with 1% (wt/vol) SDS sample buffer. For immunoblot analysis, cells were lysed with RIPA Reagent (Pierce, Rockford, IL) supplemented with a protease inhibitor “cocktail.” After 15 min centrifugation at 12,000 × g at 4 °C, protein concentrations in the lysates were measured with a bicinchoninic acid assay. Equal amounts of lysates were separated by SDS–PAGE and then were transferred onto nitrocellulose membranes for immunoblot analysis. Cell culture supernatants were harvested and concentrated for immunoblot with Amicon Ultra 10K from Millipore. NLRP3 expression in Chase assay was quantitated by measuring band intensities using the “ImageJ” software. The values were normalized to Actin.

SUMOylation assays

Cells cultured in 6-cm plates were transfected with the indicated plasmids (HEK293T cells) or primed with LPS and ATP (mouse PMs). Whole-cell lysates were lysed in 40 μl SDS lysis buffer containing 5.0% (vol/vol) Glycerol, 50 mM Tris–HCl, pH 6.8, 40 mM DTT, 1.0% (wt/vol) SDS, 20 μM NEM, and a protease inhibitor “cocktail.” After 15 min centrifugation at 12,000 × g at 4 °C, lysates were put at 100 °C for 10 min. Protein concentrations in the lysates were measured with a bicinchoninic acid assay and then diluted 1:10 with IP buffer and supernatants were collected and incubated with protein G Plus-Agarose IP reagent together with specific antibody overnight. Beads were washed five times with IP buffer. Immunoprecipitates were eluted by boiling with 1% (wt/vol) SDS sample buffer.

In vivo LPS challenge

WT or Trim28CKO mice (females, 6–8 weeks old) were i.p. injected with 25 mg/kg LPS or PBS. After 4–6 h, mice were sacrificed, and blood was collected for measurement of serum cytokines IL-1β, TNF, and IL-6 by ELISA.

Statistical analysis

Statistical significance between groups was determined by two-tailed Student’s t test by using GraphPad Prism 6.0. Values of p < 0.05 were considered to be statistically significant.

ImageJ software

The protocol of ImageJ for western blotting: (1) Open an image. (2) Select the strips by rectangle. (3) Click Analyze-Gels-Select First Lane. (4) Click Analyze-Gels-Plot Lanes. (5) Seal off every area by straight line. (6) Select every area wand tool. (7) Get the results from a new window.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files or from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (81622030, 31570867, 31870866, and 81861130369). W.Z. is a Newton Advanced Fellow awarded by the Academy of Medical Sciences (NAF007\1003).

Author information

Affiliations

  1. Department of Pathogenic Biology and Key Laboratory of Infection and Immunity of Shandong Province, School of Basic Medical Science, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China

    Ying Qin, Qi Li, Wenbo Liang, Rongzhen Yan, Li Tong, Mutian Jia, Chunyuan Zhao & Wei Zhao

  2. Department of Cell Biology, School of Basic Medical Science, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China

    Chunyuan Zhao

Contributions

W.Z. designed and supervised the research; Y.Q., Q.L., W.L., R.Y., L.T., M.J., and C.Z. performed the experiments; Y.Q. and W.Z analyzed the data and wrote the paper.

Corresponding author

Correspondence to Wei Zhao.

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Peer review informationNature Communications thanks Domenico Delfino and Gloria Lopez-Castejón for their contribution to the peer review of this work. Peer reviewer reports are available.

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Qin, Y., Li, Q., Liang, W. et al. TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation. Nat Commun12, 4794 (2021). https://doi.org/10.1038/s41467-021-25033-4

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New technology exposes liars through telltale activation of facial muscles

Researchers at Tel Aviv University detected 73% of the lies told by trial participants based on the contraction of their facial muscles—achieving a higher rate of detection than any known method. The study identified two different groups of 'liars': those who activate their cheek muscles when they lie, and those who activate their eyebrows. According to the researchers, the technology has great potential for detecting deception in real-life contexts, such as security and crime.

The study was conducted by a team of experts from Tel Aviv University headed by Prof. Yael Hanein of the Center of Nanoscience and Nanotechnology and School of Electrical Engineering, Iby and Aladar Fleischman Faculty of Engineering, and Prof. Dino Levy from the Coller School of Management. The team included Dr. Anastasia Shuster, Dr. Lilach Inzelberg, Dr. Uri Ossmy and Ph.D. candidate Liz Izakon. The paper was published in Brain and Behavior.

The new study was founded upon a groundbreaking innovation from Prof. Hanein's laboratory: stickers printed on soft surfaces containing electrodes that monitor and measure the activity of muscles and nerves. The technology, already commercialized by X-trodes Ltd., has many applications, such as monitoring sleep at home and early diagnosis of neurological diseases. This time, the researchers chose to explore its effectiveness in a different arena—lie detection.

Prof. Levy says, "Many studies have shown that it's almost impossible for us to tell when someone is lying to us. Even experts, such as police interrogators, do only a little better than the rest of us. Existing lie detectors are so unreliable that their results are not admissible as evidence in courts of law—because just about anyone can learn how to control their pulse and deceive the machine. Consequently, there is a great need for a more accurate deception-identifying technology. Our study is based on the assumption that facial muscles contort when we lie, and that so far, no electrodes have been sensitive enough to measure these contortions."

The researchers attached the novel stickers with their special electrodes to two groups of facial muscles: the cheek muscles close to the lips, and the muscles over the eyebrows. Participants were asked to sit in pairs facing one another, with one wearing headphones through which the words 'line' or 'tree' were transmitted. When the wearer heard 'line' but said 'tree' or vice versa he was obviously lying, and his partner's task was to try and detect the lie. Then the two subjects switched roles.

As expected, participants were unable to detect their partners' lies with any statistical significance. However, the electrical signals delivered by the electrodes attached to their face identified the lies at an unprecedented success rate of 73%.

Prof. Levy says, "Since this was an initial study, the lie itself was very simple. Usually when we lie in real life, we tell a longer tale which includes both deceptive and truthful components. In our study, we had the advantage of knowing what the participants heard through the headsets, and therefore also knowing when they were lying. Thus, using advanced machine learning techniques, we trained our program to identify lies based on EMG (electromyography) signals coming from the electrodes. Applying this method, we achieved an accuracy of 73%—not perfect, but much better than any existing technology. Another interesting discovery was that people lie through different facial muscles: Some lie with their cheek muscles and others with their eyebrows."

The researchers believe that their results can have dramatic implications in many spheres of our lives. In the future, the electrodes may become redundant, with video software trained to identify lies based on the actual movements of facial muscles. Prof. Levy says, "In the bank, in police interrogations, at the airport, or in online job interviews, high-resolution cameras trained to identify movements of facial muscles will be able to tell truthful statements from lies. Right now, our team's task is to complete the experimental stage, train our algorithms and do away with the electrodes. Once the technology has been perfected, we expect it to have numerous, highly diverse applications."



More information: Anastasia Shuster et al, Lie to my face: An electromyography approach to the study of deceptive behavior, Brain and Behavior (2021). DOI: 10.1002/brb3.2386

Provided by Tel Aviv University

Citation: New technology exposes liars through telltale activation of facial muscles (2021, November 19) retrieved 19 November 2021 from https://medicalxpress.com/news/2021-11-technology-exposes-liars-telltale-facial.html

This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no part may be reproduced without the written permission. The content is provided for information purposes only.

Источник: https://medicalxpress.com/news/2021-11-technology-exposes-liars-telltale-facial.html

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Pat Bowden

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New technology exposes liars through telltale activation of facial muscles

Researchers at Tel Aviv University detected 73% of the lies told by trial participants based on the contraction of their facial muscles—achieving a higher rate of detection than any known method. The study identified two different groups of 'liars': those who activate their cheek muscles when they lie, and those who activate their eyebrows. According to the researchers, the technology has great potential for detecting deception in real-life contexts, such as security and crime.

The study was conducted by a team of experts from Tel Aviv University headed by Prof. Yael Hanein of the Center of Nanoscience and Nanotechnology and School of Electrical Engineering, Iby and Aladar Fleischman Faculty of Engineering, and Prof. Dino Levy from the Coller School of Management. The team included Dr. Anastasia Shuster, Dr. Lilach Inzelberg, Dr. Uri Ossmy and Ph.D. candidate Liz Izakon. The paper was published in Brain and Behavior.

The new study was founded upon a groundbreaking innovation from Prof. Hanein's laboratory: stickers printed on soft surfaces containing electrodes that monitor and measure the activity of muscles and nerves. The technology, already commercialized by X-trodes Ltd., has many applications, such as monitoring sleep at home and early diagnosis of neurological diseases. This time, the researchers chose to explore its effectiveness in a different arena—lie detection.

Prof. Levy says, "Many studies have shown that it's almost impossible for us to tell when someone is lying to us. Even experts, such as police interrogators, do only a little better than the rest of us. Existing lie detectors are so unreliable that their results are not admissible as evidence in courts of law—because just about anyone can learn how to control their pulse and deceive the machine. Consequently, there is a great need for a more accurate deception-identifying technology. Our study is based on the assumption that facial muscles contort when we lie, and that so far, no electrodes have been sensitive enough to measure these contortions."

The researchers attached the novel stickers with their special electrodes to two groups of facial muscles: the cheek muscles close to the lips, and the muscles over the eyebrows. Participants were asked to sit in pairs facing one another, with one wearing headphones through which the words 'line' or 'tree' were transmitted. When the wearer heard 'line' but said 'tree' or vice versa he was obviously lying, and his partner's task was to try and detect the lie. Then the two subjects switched roles.

As expected, participants were unable to detect their partners' lies with any statistical significance. However, the electrical signals delivered by the electrodes attached to their face identified the lies at an unprecedented success rate of 73%.

Prof. Levy says, "Since this was an initial study, the lie itself was very simple. Usually when we lie in real life, we tell a longer tale which includes both deceptive and truthful components. In our study, we had the advantage of knowing what the participants heard through the headsets, and therefore also knowing when they were lying. Thus, using advanced machine learning techniques, we trained our program to identify lies based on EMG (electromyography) signals coming from the electrodes. Applying this method, we achieved an accuracy of 73%—not perfect, but much better than any existing technology. Another interesting discovery was that people lie through different facial muscles: Some lie with their cheek muscles and others with their eyebrows."

The researchers believe that their results can have dramatic implications in many spheres of our lives. In the future, the electrodes may become redundant, with video software trained to identify lies based on the actual movements of facial muscles. Prof. Levy says, "In the bank, in police interrogations, at the airport, or in online job interviews, high-resolution cameras trained to identify movements of facial muscles will be able to tell truthful statements from lies. Right now, our team's task is to complete the experimental stage, train our algorithms and do away with the electrodes. Once the technology has been perfected, we expect it to have numerous, highly diverse applications." May 28



More information: Anastasia Shuster et al, Lie to my face: An electromyography approach to the study of deceptive behavior, Brain and Behavior (2021). DOI: 10.1002/brb3.2386

Provided by Tel Aviv University

Citation: New technology exposes liars through telltale activation of facial muscles (2021, November 19) Akeytsu 20.3.5 Free Download with Crack retrieved 19 November 2021 parted magic 2019 - Activators Patch from https://medicalxpress.com/news/2021-11-technology-exposes-liars-telltale-facial.html

This document is subject to copyright. Apart from any fair dealing for the purpose of private study or research, no part may be reproduced without the written permission. The content is provided for information purposes only.

Источник: https://medicalxpress.com/news/2021-11-technology-exposes-liars-telltale-facial.html

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Sensing Enzyme Activation Heat Capacity at the Single-Molecule Level Using Gold-Nanorod-Based Optical Whispering Gallery Modes

ActivePresenter Free Download Here, we report a label-free gold nanoparticle-based single-molecule optical platform to study the immobilization, activity, and thermodynamics of single enzymes. The sensor uses plasmonic gold nanoparticles coupled to optical whispering gallery modes (WGMs) to probe enzyme conformational dynamics during turnover at a microsecond time resolution. Using a glucosidase enzyme as the model system, we explore the temperature dependence of the enzyme turnover at the single-molecule (SM) level. A recent physical model for understanding enzyme temperature dependencies (macromolecular rate theory; MMRT) has emerged as a powerful tool to study the relationship between enzyme turnover and thermodynamics. Using WGMs, SM enzyme measurements enable us to accurately track turnover as a function of conformational changes and therefore to quantitatively probe the key feature of the MMRT model, the activation heat capacity, at the ultimate level of SM. Our data shows that WGMs are extraordinarily sensitive to protein conformational change and can discern both multiple steps with turnover as well as microscopic conformational substates within those steps. The temperature dependence studies show that the MMRT model can be applied to a range of steps within turnover at the SM scale that is associated with conformational change. Our study validates the notion that MMRT captures differences in dynamics between states. The WGM sensors provide a platform for the quantitative analysis of SM activation heat capacity, applying MMRT to the label-free sensing of microsecond substates of active enzymes.

Источник: https://pubmed.ncbi.nlm.nih.gov/34085031/

TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation

Abstract

The cellular NLRP3 protein level is crucial for assembly and activation of the NLRP3 inflammasome. Various posttranslational modifications (PTMs), including phosphorylation and ubiquitination, control NLRP3 protein degradation and inflammasome activation; however, the function of small ubiquitin-like modifier (SUMO) modification (called SUMOylation) in controlling NLRP3 stability and subsequent inflammasome activation is unclear. Here, we show that the E3 SUMO ligase tripartite motif-containing protein 28 (TRIM28) is an enhancer of NLRP3 inflammasome activation by facilitating NLRP3 expression. TRIM28 binds NLRP3, promotes SUMO1, SUMO2 and SUMO3 modification of NLRP3, and thereby inhibits NLRP3 ubiquitination and proteasomal degradation. Concordantly, Trim28 deficiency attenuates NLRP3 inflammasome activation both in vitro and in vivo. These data identify a mechanism by which SUMOylation controls the cellular NLRP3 level and inflammasome activation, and reveal correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation.

Introduction

NOD- LRR- and pyrin domain-containing protein 3 (NLRP3) is a crucial pattern recognition receptor, which can sense pathogen-associated molecular patterns (PAMPs) and endogenous danger signals (danger-associated molecular patterns, DAMPs) to initiate the formation of a multi-protein complex named Privacy Drive Free Download NLRP3 inflammasome1,2,3,4,5. Canonical activation of the NLRP3 inflammasome requires two sequential steps. PAMPs or the activation of cytokines provides the first signal (priming signal), which activates nuclear factor (NF)-κB and induces the expression of the NLRP3, an integral part of the NLRP3 inflammasome, pro-interleukin-1β (pro-IL-1β), and pro-IL-18. The second signal (activation signal) is provided by a wide range of stimuli, such as particulates, crystals, and adenosine 5’-triphosphate disodium (ATP). Upon activation, NLRP3 inflammasome assembly activates caspase-1, which in turn cleaves pro-IL-1β and pro-IL-18. NLRP3 inflammasome has been implicated in multiple diseases and its activity should be tightly controlled1.

Various posttranslational modifications (PTMs) of NLRP3, including phosphorylation, ubiquitination, and SUMOylation, play crucial roles in controlling inflammasome activation1. The level of intracellular NLRP3 protein is considered as a key rate-limiting step for canonical activation of NLRP3 inflammasome6,7. In resting macrophages, NLRP3 is ubiquitylated and its protein level is relatively low, which limits NLRP3 inflammasome activation. Upon priming and activation, NLRP3 is deubiquitylated, and that enhances NLRP3 expression and facilitates inflammasome activation8,9. The E3 ubiquitin ligases tripartite motif containing protein 31 (TRIM31) and membrane-associated RING finger protein 7 (MARCH7) induce Lys(K)48-linked ubiquitylation of NLRP3 and in turn promotes its degradation via proteasome and autophagy, respectively10,11. The ubiquitin-specific peptidase 1 (USP1)-associated factor 1 (UAF1) /USP1 deubiquitinase complex selectively inhibits K48-linked ubiquitination and protein degradation of NLRP3 enhances cellular NLRP3 levels12, which are indispensable for subsequent NLRP3 inflammasome assembly and activation. Recently, small ubiquitin-like modifier (SUMO) protein-conjugated modifications, which are termed as SUMOylation, were identified in NLRP3 during inflammasome activation13,14. SUMOylation, a type of PTM that is similar to ubiquitination, is mediated by E1-activating enzymes, a sole E2 ligase UBC9, and E3 SUMO ligases15,16,17. Three Vidmore Video Converter Crack proteins, including SUMO1, SUMO2, and SUMO3, can be covalently conjugated to proteins as a single moiety (SUMO1) or as polymeric SUMO chains (SUMO2 and SUMO3). SUMOylation contributes significantly to mediating protein interaction, regulating protein localization in cells, regulating the activity of transcription factors, maintaining genomic stability, and transcriptional regulation15. The E3 SUMO protein ligase MUL1 (also known as MAPL) catalyzes SUMO2/3 modification of NLRP3 and restrains NLRP3 activation, whereas SUMO-1-catalyzed SUMOylation of NLRP3 facilitates inflammasome activation. Although SUMO1 modification of NLRP3 is required for inflammasome activation, both the mechanism details and the E3 SUMO ligase that mediates SUMO1-catalyzed SUMOylation of NLRP3 are unclear.

Here we identify the E3 SUMO ligase TRIM28 (also known as Kruppel-associated box-associated protein 1 (KAP1) or transcriptional intermediary factor 1β (TIF1β)) as an enhancer of inflammasome activity by promoting NLRP3 SUMOylation. TRIM28 interacts with NLRP3, facilitates SUMO1 and SUMO2/3 catalyzed SUMOylation of NLRP3, and thereby inhibits its ubiquitination and proteasomal degradation. TRIM28 stabilizes NLRP3 protein expression and enhances subsequent inflammasome activation. These data provide a mechanism by which SUMOylation enhances NLRP3 inflammasome activation and indicate that TRIM28 might be a target for the treatment of diseases caused by aberrant inflammasome activation.

Results

TRIM28 interacts with NLRP3

To identify potential interactors of NLRP3, we incubated a specific anti-NLRP3 antibody with total cell lysates of mouse primary peritoneal macrophages (PMs) stimulated with lipopolysaccharide (LPS). The co-immunoprecipitated complex was separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The E3 SUMO ligase TRIM28 was identified as a potential NLRP3 interactor by liquid chromatography coupled with tandem mass spectrometry (Fig. 1a). LPS markedly induced TRIM28 expression (Fig. 1b), suggesting that TRIM28 may be involved in the process of NLRP3 inflammasome activation. However, Trim28 mRNA decreased at first and increased subsequently upon LPS stimulation (Supplementary Fig. 1a), indicating the existence of other regulatory mechanisms for TRIM28 protein levels in response to LPS stimulation. We next examined the interaction between TRIM28 and NLRP3 in vivo. Confocal analysis demonstrated the colocalization between TRIM28 and NLRP3 (Fig. 1c). Next, the interaction between TRIM28 and NLRP3 was investigated under physiological conditions by immunoprecipitation (IP). An association between TRIM28 and NLRP3 was detected in both resting LPS-primed and ATP-activated mouse PMs (Fig. 1d). TRIM28 along with NLRP3, ASC, or caspase-1 were cotransfected in HEK293T cells. TRIM28 could immunoprecipitate with NLRP3 but not ASC and caspase-1 (Fig. 1e). Taken together, these data suggest that TRIM28 could interact with NLRP3.

a Identification of TRIM28 as a potential NLRP3 interactor in LPS-stimulated mouse peritoneal macrophages (PMs) by mass spectrum primed with LPS. b Immunoblot analysis of TRIM28 expression in LPS-stimulated mouse PMs (n = 3 independent experiments). c Confocal microscopic analysis of the colocalization of NLRP3 (Red) and TRIM28 (Green) in unstimulated mouse embryonic fibroblasts (MEFs). Scale bar, 10 μm. d IP analysis of endogenous association 2021 - Free Activators TRIM28 and NLRP3 in LPS-stimulated or LPS-primed and ATP-activated mouse PMs (n = 3 independent experiments). e Immunoprecipitation (IP) analysis of the association between TRIM28 and NLRP3, ASC or Caspase-1 in HEK293T cells transfected with the indicated plasmids. Similar results were obtained from three independent experiments.

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TRIM28 has no effects on NLRP3 inflammasome priming

Next, we first investigated the potential functions of TRIM28 in the priming of NLRP3 inflammasome. Trim28fl/fl mice were crossed with Lyz2cre mice to specifically knockout Trim28 in myeloid cells (called “Trim28CKO” here). Trim28 deficiency had no effects on LPS-induced phosphorylation of IκBα and total IκBα (Fig. 2a) and transcription of Il1b, Nlrp3, Tnf, and Il6 in PMs (Fig. 2b and Supplementary Fig. 1b). To further investigate the function of TRIM28, small interfering RNA (siRNA)-knockdown experiments were performed. Synthesized siRNA targeting mouse Trim28 was used to suppress endogenous TRIM28 expression in PMs (Supplementary Fig. 1c). Trim28 knockdown also did not affect phosphorylation of IκBα and total IκBα expression and transcription of Il1b, Nlrp3, Tnf, and Il6 (Fig. 2c, d and Supplementary Fig. 1d). Collectively, these data indicate that TRIM28 could not affect LPS-induced NF-κB activation and the priming process of NLRP3 inflammasome activation.

a, c Immunoblot analysis of p-IκBα manycam pro crack mac IκBα in LPS-stimulated mouse PMs from WT or Trim28CKO mice (a) or PMs transfected with Ctrl siRNA or Trim28 siRNA (c). b, d RT-PCR analysis of Ilb and Nlrp3 mRNA in LPS-stimulated mouse PMs from WT or Trim28CKO mice (b) or PMs transfected with Ctrl siRNA or Trim28 siRNA (d). All data are represented as mean ± SD in b, d. Similar results were obtained from five independent experiments (a, c) and three independent experiments (b, d).

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TRIM28 promotes NLRP3 inflammasome activation

Next, we tested the effect of TRIM28 on NLRP3 inflammasome activation. Mouse PMs from both wild-type (WT) and Trim28CKO mice were primed by LPS and then stimulated by NLRP3 inflammasome activators (ATP or nigericin (Nig)) or poly(dA:dT) (an AIM2 inflammasome activator). Trim28 deficiency markedly inhibited NLRP3 inflammasome-induced IL-1β secretion, while had no effects on AIM2 inflammasome activation (Fig. 3a). However, tumor necrosis factor (TNF) and IL-6 secretion were not influenced by Trim28 deficiency (Fig. 3b). Activated caspase-1 cleaves gasdermin D (GSDMD) and releases its N-terminal regions, which then punch holes in the membrane, resulting in the release of IL-1β and pyroptosis18. Trim28 deficiency suppressed both caspase-1 and GSDMD cleavage following NLRP3 inflammasome activation (Fig. 3c). Meanwhile, we observed that Trim28 deficiency did not influence IL-1β but greatly attenuated LPS-induced NLRP3 expression (Fig. 3c), suggesting that TRIM28 may selectively enhance NLRP3 inflammasome activation by promoting NLRP3 expression. Trim28 knockdown also reduced IL-1β secretion following LPS priming and ATP activation in mouse PMs (Fig. 3d), with no effects on both TNF and IL-6 secretion (Supplementary Fig. 1e). Taken together, these data indicate that TRIM28 facilitates NLRP3 inflammasome activation.

a, b ELISA analysis of IL-1β, TNF, and IL-6 in supernatants of mouse PMs from WT or Trim28CKO mice following priming with LPS for 7 h and subsequent stimulation with ATP, Nig, or poly(dA:dT) for 1 h (a two-tailed t test WT vs. Trim28CKO, *p = 0.011241, **p = 0.005688). c Immunoblot analysis of supernatants (SN) and cell lysates (CL) of mouse PMs from WT or Trim28CKO mice, following priming with LPS and subsequent stimulation with ATP (n = 3 independent experiments). d ELISA analysis of IL-1β in supernatants from mouse PMs transfected with control (Ctrl) siRNA or Trim28 siRNA for 48 h, followed by priming with LPS for 7 h and subsequent stimulation with ATP for 1 h (two-tailed t test Ctrl siRNA vs. Trim28 siRNA, **p = 0.005134). e ELISA analysis of serum levels of IL-1β, TNF, and IL-6 of WT or Trim28CKO mice after i.p. LPS injection (PBS, n = 3; LPS, n = 8 per group, two-tailed t test WT vs. Trim28CKO, ***p = 6 × 10−6). All data are represented as mean ± SD in a, b, d, e (*p < 0.05; **p < 0.01; ***p < 0.001). Similar results were obtained from three independent experiments.

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We next investigated the function of TRIM28 on NLRP3 inflammasome activation in vivo. The secretion of IL-1β by intraperitoneal (i.p.) injection of LPS is NLRP3 dependent. The secretion of IL-1β in the serum of Trim28CKO mice decreased upon LPS challenge, while TNF and IL-6 remained unchanged (Fig. 3e). These results demonstrate that Trim28 deficiency attenuates NLRP3 inflammasome activation in vivo.

TRIM28 inhibits proteasomal degradation of NLRP3

Next, we determined whether TRIM28 regulates NLRP3 protein expression. TRIM28 overexpression increased NLRP3 protein expression in HEK293T cell (Fig. 4a). Consistently, both Trim28 deficiency and knockdown inhibited LPS-induced NLRP3 protein expression in mouse PMs (Fig. 4b, c). Interestingly, NLRP3 protein expression in unstimulated macrophages was also considerably attenuated by Trim28 deficiency or knockdown (Supplementary Fig. 2a, b). Although TRIM28 has transcriptional regulatory functions19, it did not enhance NLRP3 transcription (Fig. 2b, d). We then investigated the effects of TRIM28 on NLRP3 protein degradation by cycloheximide (CHX) chase assay. Trim28 deficiency markedly promoted NLRP3 protein degradation (Fig. 4d, e). Ubiquitin–proteasome and autophagy–lysosome are major pathways for NLRP3 degradation. We then further clarified which pathway was affected by TRIM28. MG132, a selective inhibitor of proteasome, but not the lysosome inhibitor chloroquine and autophagy inhibitor 3-methyladenine (3-MA), restored the NLRP3 protein expression in LPS-stimulated Trim28 deficiency macrophages (Fig. 4f, g and Supplementary Fig. 2c). Collectively, these data indicate that TRIM28 selectively inhibits NLRP3 protein degradation in proteasome.

a Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3 and increasing amount of Flag-TRIM28 plasmid. b Immunoblot analysis of lysates of mouse PMs from WT or Trim28CKO mice, following stimulation with LPS. c Immunoblot analysis of lysates of mouse PMs transfected with Ctrl siRNA or Trim28 siRNA for 48 h, following stimulation with LPS. d, e Immunoblot analysis of NLRP3 expression VMware Workstation Crack 6.1.2 Build 17801498 + Key 2021 mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h and then treated cycloheximide (CHX) for the indicated time periods. NLRP3 expression was quantitated by measuring band intensities using the “ImageJ” software. The values were normalized to Actin. Data are represented as mean ± SD (two-tailed t test WT vs. Trim28CKO, ***p = 1 × 10−12, 3 × 10−17). f Immunoblot analysis of NLRP3 expression in mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h, together with DMSO or MG132 (10 μM) treatment for 4 h. g Immunoblot analysis of NLRP3 expression in mouse PMs from WT or Trim28CKO mice stimulated with LPS for 4 h, together with DMSO or chloroquine (50 μM) treatment for 4 h. Similar results were obtained from three independent experiments.

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TRIM28 promotes SUMOylation of NLRP3

Ubiquitination is required for the protein degradation via the ubiquitin–proteasome pathway. We therefore examined the potential roles of TRIM28 on NLRP3 ubiquitination. Trim28 deficiency facilitated NLRP3 ubiquitination in unstimulated, LPS-primed, and ATP-activated macrophages (Fig. 5a). K48-linked ubiquitination is required for proteasomal degradation of target proteins. Next, to clarify whether TRIM28 could inhibit K48-linked ubiquitination, the ubiquitin mutant vectors K48 and K63, which contain arginine substitutions of all of its lysine residues except the one at positions 48 and 63 respectively, were used in the transfection assays. TRIM28 inhibited both total and K48-linked ubiquitination of NLRP3, whereas it had no effects on K63-linked ubiquitination of NLRP3 (Fig. 5b). TRIM28 is an E3 SUMOylation ligase20 and the cysteine (C) at position 651 (C651) is required for its enzymatic activity. TRIM28 point mutant (C651) with substitution of the cysteine residue with alanine (A) at position 651 could not bind to the key E2 SUMO binding enzyme UBC9 (Supplementary Fig. 3), and thus its E3 SUMO ligase activity was inactivated21. TRIM28 C651 lost the ability to inhibit NLRP3 ubiquitination and degradation (Fig. 5c, d), suggesting that TRIM28 selectively attenuates K48-linked ubiquitination of NLRP3 and its proteasomal degradation, via its E3 SUMO ligase activity.

a Immunoblot analysis of lysates of mouse PMs from WT or Trim28CKO mice, primed with LPS for 4 h, together with MG132 (10 μM) treatment for 3 h, and then stimulation with ATP for 1 h, and followed by IP with NLRP3 antibody. b Immunoblot analysis of lysates from HEK293T cells transfected with HA-Ub (WT), HA-tagged K48-linked ubiquitin (K48-Ub), or HA-tagged K63-linked ubiquitin (K63-Ub), Myc-NLRP3, and Flag-TRIM28, together with MG132 (10 μM) treatment for 4 h and followed by IP with Myc antibody. c Immunoblot analysis of lysates from HEK293T cells transfected with HA-tagged ubiquitin (HA-Ub), Myc-NLRP3, and Flag-TRIM28 WT or C651A, together with MG132 (10 μM) treatment for 4 h, and followed by IP with Myc antibody. d Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3, together with Flag-TRIM28 WT or C651A. Similar results were obtained from three independent experiments.

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NLRP3 could be modified by SUMO1 and SUMO2/3 in macrophages (Fig. 6a, b), which is consistent with previous reports13,14. Interestingly, we found that LPS stimulation enhanced both SUMO1 and SUMO2/3 modification of NLRP3, while SUMO1 modification further increased and SUMO2/3 modification decreased following ATP stimulation (Fig. 6a, b and Supplementary Fig. 4). These data suggest a dynamic change of NLRP3 SUMOylation status during the priming and activation step of NLRP inflammasome. 2′,3′,4′-Trihydroxy flavone (2-D08), an inhibitor of protein SUMOylation, dose-dependently decreased NLRP3 expression (Fig. 6c), suggesting the potential role of SUMOylation on NLRP3 expression. We have identified E3 SUMO ligase TRIM28 as a NLRP3-associated protein and that promoted us to investigate whether TRIM28 could mediate the SUMOylation of NLRP3. Both Trim28 deficiency and knockdown attenuated SUMO1 and SUMO2/3 modification of NLRP3 in macrophages (Fig. 6a, b and Supplementary Fig. 4). Furthermore, TRIM28 promoted SUMO1, SUMO2, and SUMO3 modification in HEK293T cells, while TRIM28 C651 mutant lost the ability to mediate NLRP3 SUMOylation (Fig. 6d–f). Therefore, TRIM28 induced NLRP3 SUMOylation, inhibited NLRP3 ubiquitination and degradation, and Actual Window Menu Licenses key facilitated inflammasome activation (see a model in Fig. 7).

a, b 2021 - Free Activators analysis of lysates of mouse peritoneal macrophages from WT or Trim28CKO mice, primed with LPS for 4 h, together with MG132 (10 μM) treatment for 3 h, and then stimulation with ATP for 1 h, and followed by SUMOylation assays with NLRP3 antibody. c Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3 and stimulated with an increasing amount of 2-D08 for 24 h. df Immunoblot analysis of lysates from HEK293T cells transfected with Myc-NLRP3, His-UBC9 and Flag-TRIM28 WT or C651A, and HA-SUMO1, HA-SUMO2, or HA-SUMO3, followed by MG132 (10 μM) treatment for 4 h, and then performed SUMOylation assays with Myc antibody. Similar results were obtained from three independent experiments.

Full size image

TRIM28 binds to NLRP3, promotes SUMOylation of NLRP3, and thus inhibits NLRP3 ubiquitination and degradation in proteasome, resulting in the enhancement of NLRP3 inflammasome activation.

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Discussion

Various types of PTMs are important for the regulation of protein folding, stability, localization, and functional activities. NLRP3 is subjected to multiple PTMs, including phosphorylation, ubiquitination, alkylation, S-nitrosylation, and SUMOylation, to tightly control optimal activation of NLRP3 inflammasome. But the correlations and interactions among various types of PTMs of NLRP3 remain less clarified. SUMOylation interacts with other PTMs, such as ubiquitination, phosphorylation, and acetylation, to regulate protein functions15. Barcode software free download full version example, ubiquitinated p53 recruits the E3 SUMO ligase PLASy to promote SUMOylation of p5322. E3 SUMO ligase PLAS4-mediated SUMOylation of MDC1 recruits E3 ubiquitin-ligase RNF4 to promote MDC ubiquitination in DNA damage16. However, SUMO1 modification usually blocks ubiquitination of its substrate. For example, SUMO1-modified IκBα cannot be ubiquitinated and is resistant to proteasome-mediated degradation23. SUMO1 modification of cGAS and STING prevent their polyubiquitination and degradation during viral infection24. SUMO1 and Ub both bind to the lysine (K) residues of their substrates and that leads to competitive inhibition. In this study, we reveal the correlations and interactions of NLRP3 SUMOylation and ubiquitination during inflammasome activation. The E3 SUMO ligase TRIM28 facilitates SUMO1 and SUMO2/3-catalyzed SUMOylation of NLRP3, whereby attenuates K48-linked ubiquitination of NLRP3, resulting in enhancement of NLRP3 stability.

During NLRP3 inflammasome priming (such as LPS stimulation), SUMO1/2/3 modification and ubiquitination are all increased. Following activation (ATP stimulation), SUMO1 modification is further enhanced, while SUMO2/3 modification and ubiquitination were decreased, suggesting that dynamic changes of SUMOylation and ubiquitination during NLRP3 inflammasome activation. MUL1-mediated SUMO2/3-catalyzed NLRP3 SUMOylation was reported to inhibit NLRP3 inflammasome activation13. But SUMO1-catalyzed NLRP3 SUMOylation promotes inflammasome activation, which is consistent with a previous report14. SUMO1 modified Lys (K) residues cannot be SUMOylated or ubiquitinated, which is different to the characteristics of SUMO2/3 modification and ubiquitination25,26. Therefore, SUMO1 modification of NLRP3 may block the conjugation of ubiquitin by competing with the same K sites of NLRP3 and inhibits ubiquitous degradation of NLRP3 and therefore facilitates inflammasome activation. Although MUL1-mediated SUMO2/3-catalyzed SUMOylation of NLRP3 inhibited inflammasome activation13, we found that TRIM28-mediated SUMO2/3-catalyzed SUMOylation facilitated inflammasome activation. In addition, TRIM28, but not MUL1, inhibited the ubiquitination degradation of NLRP3. Therefore, TRIM28 and MUL1 regulate NLRP3 activity by different mechanisms. Multiple potential SUMOylation sites exist in NLRP3 protein (such as K204 and K689, etc.), and TRIM28 and MUL1 may mediate NLRP3 SUMOylation at different Remo Recover 6.1 Crack License Free Download 2021 sites. SUMOylation mediated by TRIM28 may block the ubiquitination site of NLRP3 and thus inhibits NLRP3 proteasomal degradation. Further analysis of the SUMOylation and ubiquitination sites of NLRP3 will help to elucidate the underlying mechanisms. In addition, TRIM28 promotes SUMO1/2/3-catalyzed SUMOylation of NLRP3, and SUMO1 blocks other PTMs (including ubiquitination) of NLRP3, resulting in the stability of NLRP3 and enhancement of inflammasome activation. Based on the experimental data, we propose a working model to illustrate how TRIM28-mediated SUMOylation enhances NLRP3 inflammasome activation (Fig. 7). TRIM28 binds to NLRP3, promotes its SUMOylation, and thereby inhibits NLRP3 ubiquitination and subsequent degradation in proteasome. However, in Trim28-deficiency macrophages, NLRP3 is still synthesized normally, and NLRP3 degradation is enhanced. Therefore, the absence of TRIM28 does not completely eliminate NLRP3 expression. Thus, enhanced cellular level of NLRP3 facilitates inflammasome assembly and enhances inflammasome activation.

In addition to its E3 SUMO ligase activity, TRIM28 is a well-known epigenetic suppressor. TRIM28 can bind to KRAB zinc-finger proteins (KZNFs) and recruit the histone methyltransferase SETDB1 to recognize and bind to H3k9me3, resulting in transcriptional repression27,28,29. TRIM28 works as a master epigenetic regulator of endogenous retroviral elements (ERVs) to maintain ERVs in a silent state and thereby protects cells from detrimental genome instability and aberrant interferon responses. Furthermore, TRIM28 possesses important epigenetic roles in the differentiation and functions of regulatory T cells and Th17 cells, and genetic deletion of KAP1 in T cells results in autoimmune phenotype30,31,32,33. However, in our study, we found that TRIM28 had no effect on the transcription of key components of inflammasome, including Il1b and Nlrp3.

In summary, we identify the E3 SUMO ligase TRIM28 as a stabilizer of NLRP3 by promoting its SUMOylation. Concordantly, TRIM28 enhances inflammasome activation via enhancing cellular level of NLRP3. Given the pivotal roles of inflammasome in the occurrence and development of a variety of diseases, modulation of NLRP3 activation by targeting SUMOylation or TRIM28 will be a priming target for the treatment of diseases caused by aberrant NLRP3 activity.

Methods

Mice

Trim28fl/fl mice34 (Stock Number:018552) and Lyz2cre mice (Stock Number: 004781) were from Jackson Laboratory. Trim28fl/fl mice were crossed with Lyz2cre transgenic mice to obtain Trim28CKO mice with Trim28 deficiency in myeloid cells. Mice were kept in specific pathogen-free-level Laboratory Animal Room with 12-/12-h light and dark alternating time, temperature 18–22 °C and humidity 45–65%. The background strain of all mice used is C57BL/6J. All mice were used at 6–11 weeks of age, and both male and female mice were used. All animal experiments were undertaken in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, with the approval of the Scientific Investigation Board of the School of Basic Medical Science, Shandong University, Jinan, Shandong Province, China.

Reagents and antibodies

ATP (A1852), Nig (N7143), Z-Leu-Leu-Leu-al (MG132, C2211), LPS (Escherichia coli, O111:B4, L4130), NAnti-Flag and anti-Myc are the same isotypes.

Cell culture

To obtain mouse primary PMs, C57BL/6J mice (male, 6–8 weeks old) were injected i.p. with 3% Brewer’s thiogly-collate broth. Three days later, peritoneal exudate cells were harvested and incubated. Two hours later, nonadherent cells were removed and the adherent monolayer cells were used as PMs. Human embryonic kidney (HEK293T) cells were obtained from American Type Culture Collection (Manassas, VA). The cells were cultured at 37 °C under 5% CO2 in Dulbecco’s Modified Eagle Medium supplemented with 10% fetal calf serum (Invitrogen-Gibco). The concentration of agonists or stimuli were used as below: LPS 200 ng/ml for mouse primary PMs, LPS 1 μg/ml for mouse embryonic fibroblasts (MEFs), ATP 2.5 mM, Nig 50 mM, MG132 10 μM, chloroquine 50 μM, 3-MA 10 mM. Poly(dA:dT) were transfected into macrophages with the final concentration as 200 ng/ml.

Plasmid construction and transfection

TRIM28 expression plasmid (SC321588) was purchased from Origene and then Flag-tag was added. TRIM28 C651A was generated using the KOD-Plus-Mutagenesis Kit (Toyobo, Osaka, Japan). Expression plasmids for NLRP3, ASC, Caspase-1, HA-Ub WT, mutant K48, and mutant K63 were described previously10,12. HA-SUMO1 (HG13095-NY), HA-SUMO2 (HG13443-NY), HA-SUMO3 (HG12782-NY), and His-UBC9 (HG143205-NH) were from Sino Biological. All constructs were confirmed by DNA sequencing. Plasmids were transiently transfected into HEK293T cell with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions.

RNA interference assay

siRNAs were synthesized as following: murine Trim28: 5′-GACAUCGUGGAGAAUUAUU-3′, negative control: 5′-UUCUCCGAACGUGUCACGU-3′. These siRNA duplexes were transfected into mouse PMs using INTERFERin reagents (Polyplus-transfection) according to the manufacturer’s instructions.

Enzyme-linked immunosorbent assay (ELISA)

The concentrations of mouse IL-1β (cat. number: 1210123), TNF (cat. number: 1217202), and IL-6 (cat. number: 1210603) were measured using ELISA kits (Dakewe Biotech Company Ltd., Shenzhen, China) according to the manufacturer’s instructions.

RNA quantitation

RNA from macrophages was extracted by using the RNAfast200 RNA Extraction Kit according to the manufacturer’s instructions. Quantitative real-time PCR analysis was carried out using the Applied Biosystems StepOnePlus Real-Time PCR System and SYBR RT-PCR kits (Roche). Specific primers used for real-time PCR assays are listed in Supplementary Table 1. Data are normalized to β-Actin expression in each sample.

Immunofluorescent and confocal microscopy

MEFs, which were plated on glass coverslips in 24-well plates, were fixed with Immunol Staining Fix Solution (Beyotime). Cells were permeabilized with 0.5% Triton-X 100 in phosphate-buffered saline (PBS) and blocked with Immunol Staining Blocking Buffer (Beyotime) for 1 h. Then the cells were incubated with anti-TRIM28 (Mouse) and anti-NLRP3 (Rabbit) diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) at 4 °C overnight. Next, the cells were stained with secondary Abs (anti-Mouse conjugated to Alexa Fluor 488 and anti-Rabbit conjugated to Alexa Fluor 633) and nuclei were stained with DAPI (Beyotime)35. Then the cells were subjected to microscopic analysis with the Zeiss LSM780 confocal laser microscope, which was provided by the Micro Characterization Facility of Shandong University.

IP and immunoblot analysis

For IP, whole-cell lysates were lysed in IP buffer containing 1.0% (vol/vol) Nonidet P 40, 50 mM Tris–HCl, pH 7.4, 50 mM EDTA, 150 mM NaCl, and a protease inhibitor “cocktail” (Sigma). After 15 min centrifugation at 12,000 × g at 4 °C, protein concentrations in the lysates were measured with a bicinchoninic acid assay (Pierce, Rockford, IL), and then supernatants were collected and incubated with protein G Plus-Agarose IP reagent together with specific antibody overnight. Beads were washed five times with IP buffer. Immunoprecipitates were eluted by boiling with 1% (wt/vol) SDS sample buffer. For immunoblot analysis, cells were lysed with RIPA Reagent (Pierce, Rockford, IL) supplemented with a protease inhibitor “cocktail.” After 15 min centrifugation at 12,000 × g at 4 °C, protein concentrations in the lysates were measured with a bicinchoninic acid assay. Equal amounts of lysates were separated by SDS–PAGE and artlantis 2019 crack only - Free Activators were transferred onto nitrocellulose membranes for immunoblot analysis. Cell culture supernatants were harvested and concentrated for immunoblot with Amicon Ultra 10K from Millipore. NLRP3 expression in Chase assay was quantitated by measuring band intensities using the “ImageJ” software. The values were normalized to Actin.

SUMOylation assays

Cells cultured in 6-cm plates were transfected with the indicated plasmids (HEK293T cells) or primed with LPS and ATP (mouse PMs). Whole-cell lysates were lysed in 40 μl SDS lysis buffer containing 5.0% (vol/vol) Glycerol, 50 mM Tris–HCl, pH 6.8, 40 mM DTT, 1.0% (wt/vol) SDS, 20 μM NEM, and a protease inhibitor “cocktail.” After 15 min centrifugation at 12,000 × g at 4 °C, lysates were put at 100 °C for 10 min. Protein concentrations in the lysates were measured with a bicinchoninic acid assay and then diluted 1:10 with IP buffer and supernatants were collected and incubated with protein G Plus-Agarose IP reagent together with specific antibody overnight. Beads were washed five times with IP buffer. Immunoprecipitates were eluted by boiling with 1% (wt/vol) SDS sample buffer.

In vivo LPS challenge

WT or Trim28CKO mice (females, 6–8 weeks old) were i.p. injected with 25 mg/kg LPS or PBS. After 4–6 h, mice were sacrificed, and blood was collected for measurement of serum cytokines IL-1β, TNF, and IL-6 by ELISA.

Statistical analysis

Statistical significance between groups was determined by two-tailed Student’s t test by using GraphPad Prism 6.0. Values of p < 0.05 were considered to be statistically significant.

ImageJ software

The protocol of ImageJ for western blotting: (1) Open an image. (2) Select the strips by rectangle. (3) Click Analyze-Gels-Select First Lane. (4) Click Analyze-Gels-Plot Lanes. (5) Seal off every area by straight line. (6) Select every area wand tool. (7) Get the results from a new window.

Data availability

The authors declare that the data supporting the findings of this study are available within the paper and its supplementary information files or from the corresponding author upon reasonable request. Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (81622030, 31570867, 31870866, and 81861130369). W.Z. is a Newton Advanced Fellow awarded by the Academy of Medical Sciences (NAF007\1003).

Author information

Affiliations

  1. Department of Pathogenic Biology and Key Laboratory of Infection and Immunity of Shandong Province, School of Basic Medical Science, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China

    Ying Qin, Qi Li, Wenbo Liang, Rongzhen Yan, Li Tong, Mutian Jia, Chunyuan Zhao & Wei Zhao

  2. Department of Cell Biology, School of Basic Medical Science, Cheeloo College of Medicine, Shandong University, Jinan, Shandong, China

    Chunyuan Zhao

Contributions

W.Z. designed and supervised the research; Y.Q., Q.L., W.L., R.Y., L.T., M.J., and C.Z. performed the experiments; Y.Q. and W.Z analyzed the data and wrote the paper.

Corresponding author

Correspondence to Wei Zhao.

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The authors declare no competing interests.

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Qin, Y., Li, Q., Liang, W. et al. TRIM28 SUMOylates and stabilizes NLRP3 to facilitate inflammasome activation. Nat Commun12, 4794 (2021). https://doi.org/10.1038/s41467-021-25033-4

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Tavern Brawl is one of Hearthstone's lighter game modes. A weekly event, a new Tavern Brawl begins each Wednesday (Thursday in Asian regions). See the schedule for this week's times.

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RegionOpenClose
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EuropeWed 8:00 PM CETWed 7:00 PM CET
ChinaThu 5:00 AM CSTThu 4:00 AM CST
AsiaThu 6:00 AM KSTThu 5:00 AM KST

Converted to Coordinated Universal Time (UTC) the schedule is as follows:

RegionOpenClose
AmericasWed 4:00 PM UTC (3 PM UTC during daylight savings)Wed 3:00 PM UTC (2 PM during daylight savings)
EuropeWed 7:00 PM UTCWed 6:00 PM UTC
ChinaWed 9:00 PM UTCWed 8:00 PM UTC
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  • Daylight Savings Time in the U.S. starts on the 2nd Sunday of March and ends on the 1st Sunday of November each year.
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325September 1, 2021September 8, 2021A Recipe United!Premade
324August 25, 2021September 1, 2021Reprise: Cloneball!Randomized
323August 18, 2021August 25, 2021Reprise: Brawl Block: Gods, Gadgetzan, GurubashiConstructed
322August 11, 2021August 18, 2021Reprise: Shake, Deathrattle and Roll!Randomized
321August 4, 2021August 11, 2021Reprise: RandomoniumRandomized
320July 28, 2021August 4, 2021Reprise: When Clones Attack!Randomized
319July 21, 2021July 28, 2021Reprise: Clockwork Card DealerConstructed
318July 14, 2021July 21, 2021The Duelist Burndown!Premade
317July 7, 2021July 14, 2021Reprise: Ragnaros' Fire FestivalConstructed
316June 30, 2021July 7, 2021Reprise: BY THE POWER OF RAGNAROSConstructed
315June 23, 2021June 30, 2021Reprise: Send In The Murlocs!Randomized
314June 16, 2021June 23, 2021Reprise: Battle of the BansConstructed
313June 9, 2021June 16, 2021Reprise: Blood MagicConstructed
312June 2, 2021June 9, 2021Reprise: Just MacawsRandomized
311May 26, 2021June 2, 2021Reprise: Showdown at Blackrock MountainPremade
310May 19, 2021May 26, 2021Reprise: Captain Blackheart's TreasureRandomized
309May 12, 2021May 19, 2021Reprise: UnidentifiaBrawlRandomized
308May 5, 2021May 12, 2021Reprise: A Chance EncounterRandomized
307April 28, 2021May 5, 2021Reprise: Battle of Tol BaradConstructed
306April 21, 2021April 28, 2021Reprise: Valeera's Bag of Burgled SpellsRandomzied
305April 14, 2021April 21, 2021Reprise: Top 3Constructed
304April 7, 2021April 14, 2021A Recipe of Tenacity!Premade
303March 31, 2021April 7, 2021Reprise: Everybunny Get in Here!Randomized
302March 24, 2021March 31, 2021Reprise: Three WishesRandomized
301March 17, 2021March 24, 2021Reprise: A Temporus ShiftConstructed
300March 10, 2021March 17, 2021Reprise: Just a HallucinationRandomized
299March 3, 2021March 10, 2021Reprise: Brawl Block: Tinyfin Mode!Constructed
298February 24, 2021March 3, 2021Reprise: Cart Crash at the CrossroadsRandomized
297February 17, 2021February 24, 2021Reprise:: The Great AmalgamationConstructed
296February 10, 2021February 17, 2021Reprise: Lunar Blessings!Randomized
295February 3, 2021February 10, 2021Reprise: All-Star SquadConstructed
294January 27, 2021February 3, 2021Reprise: Cloneball!Randomized
293January 20, 2021January 27, 2021Reprise: When Clones Attack!Randomized
292January 13, 2021January 20, 2021Reprise: A Less Friendly Game of ChessConstructed
291January 6, 2021January 13, 2021Visions of SaygePremade
290December 30, 2020January 6, 2021Reprise: A New Year 2021 - Free Activators
289December 23, 2020December 30, 2020Reprise: Wacky Waxy Winter's VeilConstructed
288December 16, 2020December 23, 2020Reprise: Gift ExchangeConstructed
287December 9, 2020December 16, 2020Reprise: Battle of the BansConstructed
286December 2, 2020December 9, 2020Reprise: Shake, Deathrattle and Roll!Randomized
285November 25, 2020December 2, 2020Half & HalfConstructed

Older Tavern Brawls[]

#OpenCloseTavern BrawlType
284November 18, 2020November 25, 2020Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
283November 11, 2020November 18, 2020Reprise: Gone Fishin'Constructed
282November 4, 2020November 11, 2020Reprise: Brawl of GaudinessRandomized
281October 28, 2020November 4, 2020Reprise: Send In The Murlocs!Randomized
280October 21, 2020October 28, 2020Reprise: The Haunted CarouselRandomized
279October 14, 2020October 21, 2020Boss Battle Royale 3Premade
278October 7, 2020October 14, 2020Hallow's End Masquerade BallConstructed May 28 30, 2020October 7, 2020Reprise: Rise of the ZombeastsRandomized
276September 23, 2020September 30, 2020Heroic BrawliseumBrawliseum
275September 16, 2020September 23, 2020Reprise: RandomoniumRandomized
274September 9, 2020September 16Reprise: Blood MagicConstructed
273September 2, 2020September 9, 2020Reprise: UnidentifiaBrawlRandomized
272August 26, 2020September 2, 2020Reprise: Top 3Constructed
271August 19, 2020August 26, 2020Reprise: Party Portals!Randomized
270August 12, 2020August 19, 2020Reprise: Who's the Boss Now?Constructed
269August 5, 2020August 12, 2020Reprise: Valeera's Bag of Burgled SpellsRandomized
268July 29, 2020August 5, 2020Reprise: Just MacawsRandomized
267July 22, 2020July 29, 2020Reprise: Just a HallucinationRandomized
266July 15, 2020July 22, 2020Reprise: A Peek to the PastConstructed
265July 7, 2020July 15, 2020Reprise: Decks Assemble!Randomized
264July 1, 2020July 8, 2020Rumbledome!Premade
263June 24, 2020July 1, 2020The Outlandish BurndownPremade
262June 17, 2020June 24, 2020An Outlandish RecipePremade
261June 10, 2020June 17, 2020Reprise: Captain Blackheart's TreasureRandomized
260June 3, 2020June 10, 2020Reprise: Send In The Murlocs!Randomzied
259May 27, 2020June 3, 2020Reprise: A Cavalcade of Brawls!Randomized
258May 20, 2020May 27, 2020Reprise: Cloneball!Randomized
257May 13, 2020May 20, 2020Reprise: Battle of the BansConstructed
256May 6, 2020May 13, 2020Reprise: A Chance EncounterRandomized
255April 29, 2020May 6, 2020Brawl Block: Feeling Adventurous?Constructed
254April 22, 2020April 29, 2020Reprise: Battle of Tol BaradConstructed
253April 15, 2020April 22, 2020Reprise: Dr. Boom's Ignoblegarden!Randomized
252April 8, 2020April 15, 2020The Fury of Kael'thasConstructed
251April 1, 2020April 8, 2020Reprise: Brawl of Champions Grand FinalsPremade
250March 25, 2020April 1, 2020Reprise: Return of Mechazod!Cooperative
249March 18, 2020March 25, 2020Reprise: Three WishesRandomized
248March 11, 2020March 18, 2020Reprise: Hall of ChampionsPremade
247March 4, 2020March 11, 2020The Burndown - RekindledPremade
246February 26, 2020March 4, 2020Reprise: Shiftcon WestRandomized
245February 19, 2020February 26, 2020Reprise: When Clones Attack!Randomized
244February 12, 2020February 19, 2020Brawl of GaudinessRandomized
243February 5, 2020February 12, 2020Reprise: All-Star SquadConstructed
242January 29, 2020February 5, 2020Lunar Blessings!Randomized
241January 22, 2020January 29, 2020Road to NorthrendSingle-player
240January 15, 2020January 22, 2020Reprise: Pick a Hand, Any HandConstructed
239January 8, 2020January 15, 2020Just MacawsRandomized
238January 1, 2020January 8, 2020Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
237December 25, 2019January 1, 2020Reprise: A New Year BashCooperative
236December 18, 2019December 25, 2019Reprise: Gift ExchangeConstructed
235December 11, 2019December 18, 2019Decorating Dalaran!Single-player
234December 4, 2019December 11, 2019Reprise: Heart of the SunwellConstructed
233November 27, 2019December 4, 2019Reprise: Encounter at the CrossroadsRandomized
232November 20, 2019November 27, 2019Shake, Deathrattle and Roll!Randomized
231November 13, 2019November 20, 2019Reprise: Top 3Constructed
230November 6, 2019November 13, 2019Reprise: Send In The Murlocs!Randomized
229October 30, 2019November 6, 2019Reprise: Captain Blackheart's TreasureRandomized
228October 23, 2019October 30, 2019The Haunted CarouselRandomized
227October 16, 2019October 23, 2019Doom in the Tomb (week 2)Single-player
226October 9, 2019October 16, 2019Doom in the TombSingle-player
225October 2, 2019October 9, 2019Reprise: Six-ShooterConstructed
224September 25, 2019October 2, 2019Reprise: Rise of the ZombeastsRandomized
223September 18, 2019September 25, 2019Brawl Block: Tinyfin Mode!Constructed
222September 112019September 18, 2019Three WishesRandomized
221September 4, 2019September 11, 2019The League of Explorers: Revived!Single-player
220August 28, 2019September 4, 2019Reprise: Valeera's Bag of Burgled SpellsRandomized
219August 21, 2019August 28, 2019The Great AmalgamationConstructed
218August 14, 2019August 21, 2019Reprise: Cart Crash at the CrossroadsRandomized
217August 7, 2019August 14, 2019Reprise: Your Standard BrawliseumBrawliseum
216July 31, 2019August 7, 2019Reprise: A Less Friendly Game of ChessConstructed
215July 24, 2019July 31, 2019The Burndown!Premade
214July 17, 2019July 24, 2019Blackrock Crash (week 2)Single-player
213July 10, 2019July 17, 2019Blackrock CrashSingle-player
212July 3, 2019July 10, 2019Reprise: Decks Assemble!Randomized
211June 26, 2019July 1, 2019Reprise: A Temporus ShiftConstructed
210June 19, 2019June 24, 2019Brawl Block: Gods, Gadgetzan, GurubashiConstructed
209June 12, 2019June 17, 2019Rise of the MechPremade
208June 5, 2019June 10, 2019Reprise: Nefarian Rises!Cooperative
207May 29, 2019June 3, 2019The 207th Brawl Spectacular!Randomized
206May 22, 2019May 27, 2019Reprise: Clockwork Card DealerConstructed
205May 15, 2019May 20, 2019Reprise: Cloneball!Randomized
204May 8, 2019May 13, 2019Henchmania!Premade
203May 1, 2019May 6, 2019Reprise: Showdown at Blackrock MountainPremade
202April 24, 2019April 29, 2019Reprise: Hall of ChampionsPremade
201April 17, 2019April 22, 2019Dr. Boom's Ignoblegarden!Randomized
200April 10, 2019April 15, 2019Reprise: Your Standard BrawliseumBrawliseum
199April 3, 2019April 8, 2019Reprise: RandomoniumRandomized
198March 27, 2019April 1, 2019Brawl Block: Year of the MammothConstructed
197March 20, 2019March 25, 2019Battle of the BansConstructed
196March 13, 2019March 18, 2019Reprise: BY THE POWER OF RAGNAROSConstructed
195March 6, 2019March 11, 2019Brawliseum - Miniature WarfareBrawliseum
194February 27, 2019March 4, 2019Brawl of Champions Grand FinalsPremade
193February 20, 2019February 25, May 28 of Champions Round TwoPremade
192February 13, 2019February 18, 2019Brawl of Champions Round OnePremade
191February 6, 2019February 11, 2019Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
190January 30, 2019February 4, 2019Reprise: The Void SingularityConstructed
189January 23, 2019January 28, 2019Shiftcon WestRandomized
188January 16, 2019January 21, 2019Reprise: A Chance EncounterRandomized
187January 9, 2019January 14, 2019Brawl Block: Old and NewConstructed
186January 2, 2019January 7, 2019Reprise: Just a HallucinationRandomized
185December 26, 2018December 31, 2018Reprise: Gift ExchangeConstructed
184December 19, 2018December 24, 2018Returns & ExchangesConstructed
183December 12, 2018December 17, 2018A Royal RecipePremade
182December 5, 2018December 10, 2018Reprise: Your Standard BrawliseumBrawliseum
181November 28, 2018December 3, 2018Reprise: Return of Mechazod!Cooperative
180November 21, 2018November 26, 2018When Clones Attack!Randomized
179November 14, 2018November 19, 2018A Temporus ShiftConstructed
178November 7, 2018November 12, 2018Send In The Murlocs!Randomized
177October 31, 2018November 5, 2018Reprise: Decks Assemble!Randomized
176October 24, 2018October 29, 2018Reprise: The Headless Horseman Rides!Single-player
175October 17, 2018October 22, 2018Monster SmashPremade
174October 10, 2018October 15, 2018Reprise: Boss Battle Royale 2Premade
173October 3, 2018October 8, 2018Reprise: 2021 - Free Activators, Spiders, EVERYWHERE!Randomized
172September 26, 2018October 1, 2018Six-ShooterConstructed
171September 19, 2018September 24, 2018Duel of the Death KnightsRandomized
170September 12, 2018September 17, 2018Reprise: Pick a Hand, Any HandConstructed
169September 5, 2018September 10, 2018Reprise: Too Many Portals!Constructed
168August 29, 2018September 3, 2018Reprise: Double Deathrattler BattlerConstructed
167August 22, 2018August 27, 2018A Scientific RecipePremade
166August 15, 2018August 20, 2018Reprise: Your Standard BrawliseumBrawliseum
165August 8, 2018August 13, 2018Reprise: A New Year BashCooperative
164August 1, 2018August 6, 2018Reprise: Valeera's Bag of Burgled SpellsRandomized
163July 25, 2018July 30, 2018A Brawl of Fire and IceSingle-player
162July 18, 2018July 23, 2018Reprise: Ragnaros' Fire FestivalConstructed
161July 11, 2018July 16, 2018Reprise: ShiftConRandomized
160July 4, 2018July 9, 2018Reprise: Blood Magic - StandardConstructed
159June 27, 2018July 2, 2018A Less Friendly Game of ChessConstructed
158June 20, 2018June 24, 2018Reprise: Shadow ReflectionConstructed
157June 13, 2018June 17, 2018Rise of the ZombeastsRandomized
156June 6, 2018June 10, 2018Reprise: RandomoniumRandomized
155May 30, 2018June 4, 2018Gone Fishin'Constructed
154May 22, 2018May 28, 2018Reprise: Cloneball!Randomized
153May 16, 2018May 21, 2018Top 2 - StandardConstructed
152May 9, 2018May 14, 2018Everybunny Get in Here!Randomized
151May 2, 2018May 6, 2018Your Standard BrawliseumBrawliseum
150April 25, 2018April 29, 2018A Witchy RecipePremade
149April 18, 2018April 22, 2018Reprise: Idols of AzerothPremade
148April 11, 2018April 15, 2018A Chance EncounterRandomized
147April 4, 2018April 9, 2018Blessing of the RavenRandomized
146March 28, 2018April 2, 2018Reprise: Miniature Warfare - StandardConstructed
145March 21, 2018March 26, 2018Portals to Another DimensionRandomized
144March 14, 2018March 19, 2018Reprise: May 28 Crash at the CrossroadsRandomized
143March 7, 2018March 12, 2018Reprise: Showdown at Blackrock MountainPremade
142February 28, 2018March 5, 2018The Wild BrawliseumBrawliseum
141February 21, 2018February 25, 2018Venture into the WildPremade
140February 15, 2018February 19, 2018Boss Battle Royale 2Premade
139February 7, 2018February 12, 2018Reprise: Just a HallucinationRandomized
138January 31, 2018February 5, 2018UnidentifiaBrawlRandomized
137January 24, 2018January 29, 2018Reprise: Nefarian Rises!Cooperative
136January 17, 2018January 21, 2018Hall of ChampionsPremade
135January 10, 2018January 15, 2018Shadow ReflectionConstructed
134January 3, 2018January 8, 2018Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
133December 27, 2017January 1, 2018Reprise: Idols of AzerothPremade
132December 20, 2017December 25, 2017Wacky Waxy Winter's VeilConstructed
131December 13, 2017December 18, 2017Reprise: Valeera's Bag of Burgled SpellsRandomized
130December 6, 2017December 11, 2017Reprise: Yellow-Brick BrawlConstructed
129November 29, 2017December 4, 2017The Void SingularityConstructed
128November 22, 2017November 27, 2017Reprise: Decks Assemble!Randomized
127November 15, 2017November 20, 2017Treasure in the Catacombs!Randomized
126November 8, 2017November 13, 2017Reprise: The Great Summoner CompetitionConstructed
125November 1, 2017November 6, 2017Reprise: Miniature WarfareConstructed
124October 25, 2017October 30, 2017The Headless Horseman Rides!Single-player
123October 18, 2017October 23, 2017Reprise: ShiftConRandomized
122October 11, 2017October 16, 2017Reprise: Spellbook DuelConstructed
121October 4, 2017October 9, 2017Reprise: A Peek to the PastConstructed
120September 27, 2017October 1, 2017Reprise: Encounter at the CrossroadsRandomized
119September 20, 2017September 25, 2017Reprise: The Great Stone of ChallengeConstructed
118September 13, 2017September 18, 2017A Frozen RecipePremade
117September 6, 2017September 11, 2017Reprise: An Evil ExchangePremade
116August 30, 2017September Avira Prime Free Download, 2017Reprise: Blood Magic - StandardConstructed
115August 23, 2017August 28, 2017BY THE POWER OF RAGNAROSConstructed
114August 16, 2017August 21, 2017Encounter at the CrossbonesRandomized
113August 9, 2017August 14, 2017Reprise: ShiftConRandomized
112August 2, 2017August 7, 2017Battle for the CraterRandomized
111July 26, 2017July 31, 2017Reprise: Showdown at Blackrock MountainPremade
110July 19, 2017July 24, 2017Ahune's Superior BrawlPremade
109July 12, 2017July 17, 2017Reprise: Clash of the MinionsConstructed
108July 5, 2017July 10, 2017Ragnaros' Fire FestivalConstructed
107June 28, 2017July 3, 2017Reprise: All-Star SquadConstructed
106June 21, 2017June 26, 2017Reprise: Spiders, Spiders, EVERYWHERE!Randomized
105June 14, 2017June 18, 2017Reprise: Cart Crash at the CrossroadsRandomized
104June 6, 2017June 12, 2017Just a HallucinationRandomized
103May 31, 2017June 5, 2017Reprise: RandomoniumRandomized
102May 24, 2017May 29, 2017A Peek to the PastConstructed
101May 17, 2017May 22, 2017Reprise: An Evil ExchangePremade
100May 10, 2017May 15, 2017A Cavalcade of Brawls!Randomized
99May 3, 2017May 8, 2017Heroic Tavern Brawl - Wild formatBrawliseum
98April 26, 2017May 1, 2017Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
97April 19, 2017April 24, 2017An Ancient RecipePremade
96April 12, 2017April 17, 2017Reprise: Valeera's Bag of Burgled SpellsRandomized
95April 5, 2017April 10, 2017Reprise: The Masked BallConstructed
94March 29, 2017April 3, 2017A Mammoth of a New YearCooperative
93March 22, 2017March 27, 2017Decorating StormwindSingle-player
92March 15, 2017March 20, 2017Reprise: Cart Crash at the CrossroadsRandomized
91March 8, 2017March 13, 2017Reprise: Heroic Tavern BrawlBrawliseum
90March 1, 2017March 6, 2017Gadgetzan Throw-DownPremade
89February 22, 2017February 27, 2017Reprise: Cloneball!Randomized
88February 15, 2017February 20, 2017Blood MagicConstructed
87February 8, 2017February 13, 2017Reprise: Battle of Tol BaradConstructed
86February 1, 2017February 6, 2017Reprise: Gift ExchangeConstructed
85January 25, 2017January 30, 2017Boss Battle RoyalePremade
84January 18, 2017January 23, 2017Spellbook DuelConstructed
83January 11, 2017January 16, 2017Reprise: Cart Crash at the CrossroadsRandomized
82January 4, 2017January 9, 2017The Dark WandererSingle-player
81December 28, 2016January 2, 2017Reprise: Party Portals!Randomized
80December 21, 2016December 26, 2016Reprise: Clockwork 2021 - Free Activators Dealer - StandardConstructed
79December 14, 2016December 19, 2016The Battle for GadgetzanPremade
78December 7, 2016December 12, 2016Reprise: Double Deathrattler BattlerConstructed
77November 30, 2016December 5, 2016Reprise: 'Servant of Yogg-Saron' TryoutsRandomized
76November 23, 2016November 28, 2016Valeera's Bag of Burgled SpellsRandomized
75November 16, 2016November 21, 2016Heroic Tavern BrawlBrawliseum
74November 9, 2016November 14, 2016Reprise: Miniature WarfareConstructed
73November 2, 2016November 7, 2016Yellow-Brick BrawlConstructed
72October 26, 2016October 31, 2016Reprise: Captain Blackheart's TreasureRandomized
71October 19, 2016October 24, 2016Reprise: ShiftConRandomized
70October 12, 2016October 17, 2016All-Star SquadConstructed
69October 5, 2016October 10, 2016A Friendly Game of ChessPremade
68September 28, 2016October 3, 2016Reprise: Boom Bot vs. Annoy-o-Tron!Premade
67September 21, 2016September 26, 2016Reprise: RandomoniumRandomized
66September 14, 2016September 19, 2016Top 3Constructed
65September 7, 2016September 12, 2016Reprise: Cart Crash at the CrossroadsRandomized
64August 31, 2016September 5, 2016Party Portals!Randomized
63August 24, 2016August 29, 2016Reprise: Captain Blackheart's TreasureRandomized
62August 17, 2016August 22, 2016Nefarian Rises!Cooperative
61August 10, 2016August 15, 2016A Dark RecipePremade
60August 3, 2016August 8, 2016Storming StormwindSingle-player
59July 27, 2016August 1, 2016Reprise: RandomoniumRandomized
58July 20, 2016July 25, 2016Reprise: Idols of AzerothPremade
57July 13, 2016July 18, 2016'Servant of Yogg-Saron' TryoutsRandomized
56July 6, 2016July 11, 2016Reprise: Clockwork Card DealerConstructed
55June 29, 2016July 4, 2016ShiftConRandomized
54June 22, 2016June 27, 2016Pick a Hand, Any HandConstructed
53June 15, 2016June 20, 2016Reprise: Heart of the SunwellConstructed
52June 8, 2016June 13, 2016Cloneball!Randomized
51June 1, 2016June 6, 2016Cart Crash at the CrossroadsRandomized
50May 25, 2016May 30, 2016Shadow TowersConstructed
49May 18, 2016May 23, 2016Reprise: Unite Against Mechazod!Cooperative
48May 11, 2016May 16, 2016Top 2Constructed
47May 4, 2016May 9, 2016Reprise: Spiders, Spiders, EVERYWHERE!Randomized
46April 27, 2016May 2, 2016Reprise: Encounter at the CrossroadsRandomized
45April 20, 2016April 25, 2016Idols of AzerothPremade
44April 13, 2016April 18, 2016The Great Stone of ChallengeConstructed
43April 6, 2016April 11, 2016Reprise: Too Many Portals!Randomized
42March 30, 2016April 4, 2016You Must be This Tall to BrawlConstructed
41March 23, 2016March 28, 2016An Evil ExchangePremade
40March 16, 2016March 21, 2016Reprise: Battle of Tol BaradConstructed
39March 9, 2016March 14, 2016Reprise: RandomoniumRandomized
38March 2, 2016March 7, 2016Deal Your FateConstructed
37February 24, 2016February 29, 2016Clash of the MinionsConstructed
36February 17, 2016February 22, 2016Lead from the FrontConstructed
35February 10, 2016February 15, 2016Reprise: Decks Assemble!Randomized
34February 3, 2016February 8, 2016Blingtron's Beauteous BrawlConstructed
33January 27, 2016February 1, 2016Miniature WarfareConstructed
32January 20, 2016January 25, 2016Reprise: Captain Blackheart's TreasureRandomized
31January 13, 2016January 18, 2016Return of Mechazod!Cooperative
30January 6, 2016January 11, 2016Battle of the BuildsConstructed
29December 30, 2015January 4, 2016Reprise: Who's the Boss Now?Constructed
28December 23, 2015December 28, 2015Reprise: RandomoniumRandomized
27December 16, 2015December 21, 2015Gift ExchangeConstructed
26December 9, 2015December 14, 2015Reprise: Encounter at the CrossroadsRandomized
25December 2, 2015December 7, 2015Decks Assemble!Randomized
24November 25, 2015November 30, 2015Battle of Tol BaradConstructed
23November 18, 2015November 23, 2015Captain Blackheart's TreasureRandomized
22November 11, 2015November 16, 2015Reprise: Showdown at Blackrock MountainPremade
21November 4, 2015November 9, 2015Unite Against Mechazod!Cooperative
20October 28, 2015November 2, 2015Reprise: Spiders, Spiders, EVERYWHERE!Randomized
19October 21, 2015October 26, 2015Who's the Boss Now?Constructed
18October 14, 2015October 19, 2015RandomoniumRandomized
17October 7, 2015October 12, 2015Clockwork Card DealerConstructed
16September 30, 2015October 5, 2015Boom Bot vs. Annoy-o-Tron!Premade
15September 23, 2015September 28, 2015Underdog RulesConstructed
14September 16, 2015September 21, 2015Reprise: Too Many Portals!Randomized
13September 9, 2015September 14, 2015Double Deathrattler BattlerConstructed
12September 2, 2015September 7, 2015It's Raining Mana!Constructed
11August 26, 2015August 31, 2015Reprise: Encounter at the CrossroadsRandomized
10August 19, 2015August 24, 2015Grand Tournament MatchPremade
9August 12, 2015August 17, 2015Reprise: The Great Summoner CompetitionConstructed
8August 5, 2015August 10, 2015The Masked BallConstructed
7July 29, 2015August 3, 2015Too Many Portals!Randomized
6July 22, 2015July 27, 2015Heart of the SunwellConstructed
5July 15, 2015July 20, 2015Encounter at the CrossroadsRandomized
4July 8, 2015July 13, 2015The Great Summoner CompetitionConstructed
3July 1, 2015July 6, 2015Spiders, Spiders, EVERYWHERE!Randomized
2June 24, 2015June 29, 2015Banana Brawl!Constructed
1June 17, 2015June 22, 2015Showdown at Blackrock MountainPremade

Repeated Tavern Brawls[]

While most weeks see a brand new Tavern Brawl, just under half are reprises of previous Brawls. Since each Brawl is only available for 7 days (originally 5), this allows players to enjoy old favourites again, and avoids Brawls becoming overly similar due to a desire to explore past mechanics. Most reprises are simple repeat outings, identical to the original Brawl, but some involve changes, such as fixing bugs or balance issues, or even adding in extra elements.

The following table lists Tavern Brawls that have appeared more than once, including brief notes of any known changes.

Tavern BrawlNumber of appearancesKnown changes
Randomonium10The 8th appearance awarded a Rise of Shadows card pack.
Encounter at the Crossroads8The 5th appearance featured Party Crashers, and awarded a Journey to Un'Goro card pack.

The 6th appearance was renamed to "Encounter at the Crossbones".

Valeera's Bag of Burgled Spells8
Captain Blackheart's Treasure7The 5th appearance restricted cards to the standard format.
Cart Crash at the Crossroads7
Cloneball!7
'Servant of Yogg-Saron' Tryouts7
Battle of Tol Barad6
Decks Assemble!6The 3rd appearance awarded a Kobolds & Catacombs card pack.
Just a Hallucination6The 3rd appearance awarded a Rastakhan's Rumble card pack.
Showdown at Blackrock Mountain6Starting Mana Crystals for Nefarian reduced in the 2nd appearance.
All-Star Squad5
Battle of the Bans5
Blood Magic52nd, 3rd, and 4th appearances used standard format decks.
Clockwork Card Dealer5
Gift Exchange5
Send In The Murlocs!5
ShiftCon5
Spiders, Spiders, EVERYWHERE!5
Unite Against Mechazod! / Return of Mechazod!5Assassinate replaced by Prioritize in Mechazod's deck.
Your Standard Brawliseum5
A Chance Encounter4
A New Year Bash (A Mammoth of a New Year)4The 2nd appearance renamed the Brawl. The 4th appearance changed the decks for each player.
Idols of Azeroth4
Heroic Brawliseum (Heroic Tavern Brawl)4The 3rd appearance used wild format decks. The 4th appearance renamed the Brawl.
Miniature Warfare4
Party Portals!4In the second appearance, changes were made to Romulo, Party Elemental, Party Elemental and Mime
Rise of the Zombeasts42nd appearance lowered the starting health to 20.
Three Wishes4
Top 34
When Clones Attack!4
A Less Friendly Game of Chess3
A Peek to the Past3
A Temporus Shift3In the 2nd appearance of the Brawl, the first player no longer has an extra turn on turn 1.
An Evil Exchange3The 2nd appearance changed Kel'Thuzad's Hero Power from "Resurrect a friendly minion that died this game" to "Add a random Naxxramas minion to your hand", Rafaam's Hero Power at one and two pieces were changed, Noth the Plaguebringer's cost was increased, and three of Kel'Thuzad's cards in his deck were changed.

The 3rd appearance buffed Kel'Thuzad's Hero Power to decrease the generated card's cost by 3.

BY THE POWER OF RAGNAROS3
Double Deathrattler Battler33rd appearance used standard format decks.
Hall of Champions3
Heart of the Sunwell3
Just Macaws3
Nefarian Rises!3
Pick a Hand, Any Hand3
Ragnaros' Fire Festival3
Shake, Deathrattle and Roll!3
The Great Summoner Competition3The 3rd appearance used standard format decks
The Haunted Carousel3
Too Many Portals!3
UnidentifiaBrawl3
Who's the Boss Now?3
A Cavalcade of Brawls!2The 2nd appearance changed the first line of the description to "The cavalcade returns!"
Boom Bot vs. Annoy-o-Tron!2
Boss Battle Royale 22The 2nd appearance changed the druid hero and the warlock hero received changes. May 28 Block: Gods, Gadgetzan, Gurubashi2
Brawl Block: IDM 6.38 Build 21 Crack Product Key Free Download 2021 Mode!2
Brawl of Champions Grand Finals2
Clash of the Minions2
Brawl of Gaudiness2
Dr. Boom's Ignoblegarden!2
Everybunny Get in Here!2
Gone Fishin'2
Half & Half2
Lunar Blessings!2
Shadow Reflection2
Shiftcon West2
Six-Shooter2
Spellbook Duel2
The Burndown!22nd appearance renamed the brawl The Burndown - Rekindled and used a different selection of cards
The Great Amalgamation2
The Great Stone of Challenge2
The Headless Horseman Rides!2
The Masked Ball2
The Void Singularity2
Top 2 / Top 2 - Standard2
Wacky Waxy Winter's Veil2
Yellow-Brick Brawl22nd appearance used standard format decks

Notes[]

Access[]

  • Tavern Brawls have free admission.[1][4][5] Very rarely, a Brawl such as Heroic Tavern Brawl may charge an admission fee.
  • Tavern Brawls are only available to players with at least one level 20 hero.[1]
  • Players can play each Tavern Brawl as many times as they like while it is active.[6][7]
  • Only the current Tavern Brawl is accessible to players. Players Wondershare Video Converter Ultimate 10.3.0 Crack With Serial Key - Free Activators not able to access past Brawls, or challenge other players to take part in them.[8]
  • While each Brawl has a limited run, some Tavern Brawls are repeated later on. Reprise selection appears to be largely based on popularity.

Decks[]

  • Tavern Brawls use unique decks, which do not take up any of the player's regular deck slots.[9][10] Players create Tavern Brawls within the Tavern Brawl interface, and the deck is lost when the Brawl is retired.
  • The composition of Tavern Brawl decks depends on the Brawl. Many are constructed partially or wholly from the player's own collection, with some seeing random cards added to the deck made. Other Brawls feature premade decks, either with specific (and often unique) cards, or entirely randomised.
    • Brawl decks will not require or allow the player to use more than 2 copies of a card from their own collection, or more than 1 in the case of legendaries.[11]
  • Most Brawls require the player to choose a class, either during deck construction or in place of deck construction, but some instead randomly assign players to take control of special heroes, with their own decks.
  • Tavern Brawl cards are similar to Boss cards, special cards specific to Tavern Brawls are not available in other types of play. See below for more information.

Matchmaking[]

  • Unless specifically challenging a friend, players will be randomly matched against other participating players "of similar skill level".[12]
  • Initial player seeding is apparently based on rankings in other types of play.[13] This is most likely based on Casual or Ranked mode play, or a combination of the two.
  • After the initial placement, player matchmaking is controlled by the player's success in Tavern Brawls, using a hidden MMR.[14] Tavern Brawl MMR is ongoing, and carries over from week to week.[15]
  • Cooperative Tavern Brawls are an exception, and do not use any MMR.[16]

Rewards and quests[]

  • Players earn a free Classiccard pack for their first Tavern Brawl win each week except some Tavern Brawls before new expansion launch - those Tavern Brawls rewards a pack of the upcoming expansion.
    • No additional rewards are offered after the initial win. This is intended to prevent players from feeling they have to play,[17] and may also be in order to allow design space for unusual and potentially uneven arrangements, which could otherwise prove frustrating for those seeking to earn repeated rewards through wins.
    • Very rarely a Brawl will offer other rewards, such as a unique card back, in place of the usual card pack.
    • The card pack reward was originally announced to end when the "launch event" ended.[1][18] However, having begun in June 2015, it seems likely the reward is now intended to be ongoing.
  • Tavern Brawls are eligible to count toward daily quests.[19][20] Wins also count toward the "10 gold per 3 wins" reward.[21]
  • There is one Brawl-specific quest, "Everybody! Get in here!", which awards 60 gold for playing 3 Tavern Brawls.
  • Challenging a friend to a Tavern Brawl will not be eligible for most quests, as these matches are considered Friendly Challenges for these purposes.[22]
  • Players can earn experience in Tavern Brawl.[23]

General[]

  • Tavern Brawls are released in the Americas region first, followed by the Europe, Taiwan/Korea and China regions, simultaneously, around 4 hours later. As a result, the Americas region sometimes experiences technical issues following the release of a new Brawl, serving as a testbed for the latest modifications. Despite not directly modifying other game modes, the release of a Tavern Brawl nevertheless involves changes which can cause disconnections and extended downtime for the entire game, making this testing period necessary to limit disruption to a single region. The four hour delay following the Americas release is intended to allow time for the developers to attend to any stability issues or severe bugs, before launching the Brawl in other regions.
    • As of December 2015, additional testing measures are planned, to reduce related stability issues.[24] However, the changes will "take some time to implement".[24]

Tavern Brawl cards[]

Tavern Brawl cards are special cards, heroes and Hero Powers only available to players when participating in a specific Tavern Brawl. These are usually provided through pre-made decks, although they may be granted to players directly during matches. Tavern Brawl cards usually match the theme of the Brawl, and often feature fun or outlandish powers not seen in regular cards.

For a full list of known cards, see Tavern Brawl card. Featured Tavern Brawl cards for each Brawl can be found on individual Brawl pages, or in the Previous Brawls section.

[]

Main article: Fireside Brawl

Fireside Brawls are special Tavern Brawls that can only be played at Fireside Gatherings.

Each month there will be a different Fireside Brawl to try—check the Fireside Gatherings website to find out about the brawl that’s running this month. These special brawls run the whole month with no breaks, and they co-exist with the weekly Tavern Brawls.

The first Fireside Brawl that got a large amount of attention was Tavern vs. Lich King.

Design[]

"I'm not exaggerating when I say that each week, people tell me that we've created the greatest brawl ever, at the same time that others are saying it's by far the worst." - Ben Brode[25]

As of October 2016, Tavern Brawls are primarily designed by Pat Nagle (the namesake of Nat Pagle) and Dan Emmons.[26] The two designers "sit in a room and brainstorm", with additional inspiration from the internet and other team members. The ideas are then playtested and iterated upon with the rest of Team 5.[27][28]Tim Erskine serves as producer for Tavern Brawl content.[29]

According to designers Emmons and Nagle, the most important goal of Tavern Brawls is to provide variety and freshness, "mixing up" the standard game experience of ladder or Arena play, and providing players a reason and reward for logging in regularly.[26] The "Surprise" of the new Brawl each week is also "really big" for the developers.[26]

The designers originally considered having a ranked ladder for Tavern Brawl mode, including a Legend rank.[30] However, they decided not to in order to avoid players feeling pressure to play, instead adopting a strong focus on "fun".[30] For example, the card pack reward for a single win provides a reward for trying each Brawl, without creating any sense of compulsion for players who didn't that week's Brawl.

The Tavern Brawl mode is also designed to help with the game's accessibility. This is partly achieved through Brawls that don't require players to own a lot of cards, and also in the 'free' card pack reward each week, helping players to build their collections.[30]

Tavern Brawls allow for experimentation, and are sometime used to test or develop new mechanics, allowing the developers to observe player reactions. This time spent developing the required technology makes it relatively simple to then implement the mechanic elsewhere in the game.[31] Examples of this process include Discover. However, the developers balance the ease of experimentation with a desire to keep new mechanics fresh for players to discover in new sets. Ben Brode explains:

"There are pros and cons. The pros are obviously that we would get more confidence on balance. One of the cons is that part of the fun of card games is exploration. If we are putting it out there and a lot of it gets solved or figured out, then when we release a set it isn’t as crazy and exciting as it could be."[32]

The brevity of Brawls also frees the developers to experiment with a variety of designs, since "even if [the developers] make a really bad one" it will only last a few days.[26] This allows the designers to create Brawls that target specific groups of players, and take risks with their designs.[26]

The designers learned from early Brawls like Spiders, Spiders, EVERYWHERE! with decks composed solely of minions, incorporating a small number of random spells into later minion-focused Brawls such as ShiftCon.[30]

History[]

Tavern Brawls were being discussed by the developers prior to the launch of Hearthstone.[33] However, it was not until June 10, 2015, more than a year after the game's release, that the feature would be announced to players.

The Tavern Brawl system was added with Patch 2.7.0.9166 on June 15, but was not activated until June 17, when the first Tavern Brawl, Showdown at Blackrock Mountain, went live. In the interim period before the first Brawl, the as yet blank fourth button on the game box would begin to sparkle each time the player entered the main menu.

Captain Blackheart's Treasure was planned for release in October 2015, in order to debut the upcoming Discover mechanic.[27] However, technical problems prevented this, and the keyword was instead announced at BlizzCon 2015 with its accompanying adventure, The League of Explorers.

On November 4, 2015, the first cooperative Tavern Brawl was released, Unite Against Mechazod!, marking the first cooperative two-player experience within the game. At the time of its creation, this was the most difficult Brawl to create yet.[27]

In December 2015, with disconnections and downtime in the Europe region coinciding with the release of the latest Tavern Brawl becoming a regular occurrence, Blizzard announced that they would be adding additional testing measures to help prevent this in the future.[24] Shortly afterwards it was announced that the release times for Tavern Brawls in each region would be changing: instead of debuting in the Europe region at 4pm UTC, followed by Americas at 6pm and other regions at 7pm, new Brawls would now debut in the Americas region at 5pm UTC, then in all other regions 9pm.[34][35]

In October 2016 Heroic Tavern Brawl was announced, marking a new hybridisation of the Tavern Brawl and Arena game modes. This was also the first known Brawl to use Standard format, rather than the usual Wild format. The first explicitly Standard format Brawl followed in December 2016 with Clockwork Card Dealer - Standard, a simple variant of the original Clockwork Card Dealer.

On July 1, 2019, a new schedule was implemented for Tavern Brawls, reducing the weekly downtime from roughly 2.25 days to a short time span of 1 hour.[3]

Trivia[]

  • As of October 2016 Randomonium is "by far the most popular Brawl", both in terms of number of games Voicemod Pro Crack 2.19.0.2 & License Key [Latest] 2021 and hours in total spent playing.[36][37] Designer Dean Ayala explains, "[Randomonium] continues to be one of the mostly highly played brawls by unique user in addition to one of the most highly played brawls by individual user. Basically this means a ton of people play it, and the ones that do play it play a ton of games."[38]
  • The developers considered placing the Tavern Brawl button in the third slot (currently occupied by the Arena button), which would have provided a more symmetrical appearance given the smaller amount of space available for lettering on the fourth button. However, they decided against it, possibly due to length issues in languages other than English.[39]
  • On July 29, 2015, Showdown at Blackrock Xfer serum full 1.2.0b5 free download [win-osx] - Activators Patch was mistakenly reprised as the seventh Tavern Brawl, sparking widespread complaint from players, who assumed this to be the intended Brawl. The correct Brawl, Too Many Portals!, replaced it around an hour later, but not until many players had already played and won games in the former Brawl, earning their weekly card pack. Zeriyah remarked, "Sometimes when you play with dimensional space, the wrong Tavern Brawl gets sucked through the portal. Whoops."[40] A similar error has taken place with subsequent Brawls. On July 20, 2016, Battle of the Builds was reprised as the fifty-eighth Tavern Brawl, but bugs quickly became evident. Blizzard announced a few hours later that the Brawl "wasn't behaving" and replaced it with another reprise, Idols of Azeroth.[41]
  • Following the Gift Exchange Tavern Brawl, the 2015 winter break saw a repeat of Randomonium rather than another new Tavern Brawl. Ben Brode explained that this was partly due to there being "nobody in the office over the break to fix things if they go wrong, which new brawls are more likely to do."[42]
  • The 5th reprise of Encounter at the Crossroads was the first Brawl that didn't offer a Classic card pack, but instead a card pack from an upcoming expansion.
  • The popularity of the Tavern Brawl game mode in Hearthstone has led to its adoption in other Blizzard games. Overwatch and Heroes of the Storm saw the introduction of the "Weekly Brawl" and "Heroes Brawl" game modes on March 22 and April 10, 2016, respectively, less than a year after the Hearthstone format's launch;[43][44] while "PvP Brawls" were added to World of Warcraft on April 5, 2017.[45] While the Tavern label is specific to Hearthstone, the term Brawl has thus become shorthand for an "alternative way to play", featuring special and regularly changing twists on the game's normal ruleset, with an emphasis on fun, "crazy" and "unique" matches.[43]
  • Before the patch update on July 1, 2019, Tavern Brawls originally would end the following Monday and be unavailable from Monday to Wednesday.

Gallery[]

Patch changes[]

References[]

External links[]

Источник: https://hearthstone.fandom.com/wiki/Tavern_Brawl

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