SEMINAR CONTRIBUTIONS TO THE
UNDERSTANDING OF IMMUNE CELL
SIGNALING AND PROGRAMMED CELL
DEATH, NOT GOING INTO MANY
DISCOVERIES BUT JUST HIGHLIGHT
TWO EXAMPLES, HE DISCOVERED
CASPASE 3 AND ITS INVOLVEMENT IN
THE APOPTOSIS DOWN STREAM OF
RECEPTORS AND MORE RECENTLY
DISCOVERED THE PATHWAY OF
NON-CANONICAL PYROPTOSIS.
THANK YOU FOR COMING, WE'RE
LOOKING FORWARD TO YOUR TALK.
>> THANKS FOR ACCOMMODATING ME.
IT'S AN UNUSUAL HOUR.
WHAT I THOUGHT I WOULD DO TODAY
IS DISCUSS SOME RELATIVELY
RECENT WORK ON FORMATION AND
MEMBRANES, HOW THAT RELATES TO
INNATE IMMUNE MECHANISMS.
I'LL GIVE YOU A BIT OF A
HISTORICAL CONTEXT AND THEN I'LL
FINISH UP WITH SOME MORE RECENT
DATA.
SO, THE FIRST QUESTION I'D LIKE
TO ADDRESS IS THIS QUESTION THAT
HAS LOOMED LARGE IN CELL BIOLOGY
AND IMMUNOLOGY FOR DECADES
REALLY, WHICH IS HOW A
LEADERLESS CYTOKINE, CYTOKINES
THAT LACK A SIGNAL PEPTIDE,
PROTOTYPICAL EXAMPLES BEING IL-1
BETA AND IL-18, HOW ARE THEY
RELEASED?
SO, BEFORE I GET TO THIS
QUESTION, I NEED TO GIVE YOU A
BACK GROUND ON IL-1 BETA AND
IL-18, JUST TO REMIND YOU THESE
CYTOKINES DISCOVERED IN THE
'80s WERE INITIALLY IDENTIFIED
AS EXISTING IN A SENSE IN A PRO
FORM WHERE THEY WERE
BIOLOGICALLY INERT, AND THEY HAD
TO BE PROTEOLYTICALLY PROCESSED
TO GIVE YOU MATURE CYTOKINE IL-1
AND IL-18.
IT WAS REASONED IF ONE WAS ABLE
TO IDENTIFY PROTEASE RESPONSIBLE
FOR THIS PROCESSING AND INHIBIT
THAT PROTEASE, THEN YOU WOULD
FREEZE THE CYTOKINES IN THIS
INACTIVE PRO FORM, THAT WOULD BE
BE A EFFECTIVE
ANTI-INFLAMMATORY.
AND SO THE RACE WAS ON TO
IDENTIFY THIS PROTEASE, AND IT
TURNED OUT TO BE A MOST UNUSUAL
PROTEASE, INTERLEUKIN 1
CONVERTING ENZYME CLONED BY
BLACK AND THORNBERRY.
IT CLEAVED ACID RESIDUE, VERY
UNUSUAL SUBSTRATE SPECIFICITY.
CYSTEINE PROTEASE, BUT UNUSUAL
SUBSTRATE SPECIFICITY.
UNBEKNOWNST TO THEM THERE WAS A
RACE ON TO IDENTIFY GENES
RESPONSIBLE FOR CELL DEATH, IN
THE DEVELOPMENTAL ORGANISM C.
ELEGANS.
THEY HAD IDENTIFY THIS PROTEASE
THAT ALSO EXISTS IN THE XYMOGEN
FORM.
AS I MENTIONED, THEY WERE
UNAWARE THERE WAS AN INTEREST IN
CELL DEATH IN THE WORM, YOU
COULDN'T BE FURTHER AFIELD FROM
WHAT THEY WERE THINKING ABOUT.
AND YET THIS DISCOVERY OF
INTERLEUKIN 1 CONVERTING ENZYME
WAS FUNDAMENTAL IMPORTANCE
BECAUSE WITH IT CAME REALIZATION
THAT THE CENTRAL EXECUTOR OF
CELL DEATH THAT PREVIOUSLY HAD
NO KNOWN FUNCTION, WAS A
MYSTERIOUS GENE IN THE WORM, WAS
ACTUALLY -- HAD HOMOLOGY TO
INTERLEUKIN 1 BETA CONVERTING
ENZYME.
CED-3 WAS A PROTEASE, A DEATH
PROTEASE, THAT CLEAVED ACID
RESIDUE.
THIS IS A GREAT EXAMPLE HOW
SEEMINGLY UNRELATED AREAS OF
RESEARCH CAN COME TOGETHER AND
ILLUMINATE A WHOLE NEW HIGHWAY
OF DISCOVERY.
SINCE THEN LOT OF CASPASES HAVE
BEEN IDENTIFIED.
THEY ARE A FAMILY NOW, MAMMALIAN
CASPASES.
AND YOU'RE PROBABLY FAMILIAR
WITH THE ONES THAT PLAY A
PROMINENT ROLE IN APOPTOSIS SUCH
AS CASPASE 3 AND CASPASE 7.
BUT THE GRANDDADDY OF THEM ALL,
OF COURSE, IS INTERLEUKIN 1
CONVERTING ENZYME, CASPASE 1,
THAT'S HOW IT ALL STARTED.
AND CASPASE 1 PLAYS A PROMINENT
ROLE IN INFLAMMATION, AND THERE
ARE OTHER CASPASES THAT DO SO AS
WELL.
AND THESE ARE CASPASE-4 AND 5 IN
THE HUMAN REPRESENTED AS A
SINGLE CASPASE, CASPASE-11 IN
THE MOUSE, AND THIS -- THESE ARE
CASPASES THAT FUNDAMENTALLY ARE
IMPORTANT IN NON-CANONICAL
INFLAMMASOME PATHWAY.
SO, AGAIN, I'LL EMPHASIZE THESE
ARE CYSTEINE PROTEASES, AND THEY
ALL CLEAVE ACID RESIDUE.
CASPASE 1 IS THE CASPASE THAT IS
RESPONSIBLE FOR THE PROTEOLYTIC
MATURATION OF IL-1 BETA AND
IL-18.
I NEED TO INTRODUCE YOU TO THE
INFLAMMASOME, SO THE
INFLAMMASOME CAN BE THOUGHT OF
AS A CYTOSOLIC APPARATUS THAT IS
RESIDENT IN THE CYTOPLASMIC
COMPARTMENT OF INNATE IMMUNE
CELLS, INCLUDES END THEY'LLAL
CHYLAL -- EPITHELIAL CELLS.
IT RESPONDS TO VIOLATIONS BY
ACTIVATING INNATE IMMUNE SYSTEM,
IN THIS CASE HERALDED BY THE
GENERATION OF MATURE IL-1 BETA
AND IL-18.
SO, IT'S A COMPLEX TOPIC, AND I
WILL AT THE RISK OF
OVERSIMPLIFICATION, BECAUSE IT
ISN'T THAT BUT THE REST OF MY
TALK I'LL JUST REMIND PEOPLE
THAT THERE'S A NON-CANONICAL
PATHWAY THAT RESPONDS TO
INTRACELLULAR LPS AND ENGAGES
CASPASE-11 AND CANONICAL PATHWAY
THAT RESPONDS TO BEWILDERING
ARRAY OF PAMPS AND DAMPS,
TOXINS, ENGAGES CASPASE 1.
AND CASPASE-11 IN FACT
COMMUNICATES WITH CASPASE 1,
THIS CONVERTING ENZYME IS
RESPONSIBLE FOR THE PROTEOLYTIC
MATURATION OF THESE CYTOKINES.
I WANTS TO MAKE AN IMPORTANT
POINT EVEN AFTER PROTEOLYTIC
MATURATION THESE CYTOKINES LACK
A SIGNAL PEPTIDE.
SO, THERE'S NO ER GOLGI PATH FOR
THEIR RELEASE.
HOW ARE THEY GETTING OUT?
AND WHAT ONE REALIZED IN ABOUT
2015 WAS THAT BOTH CASPASE-11
AND CASPASE-1, ONCE ACTIVATED,
CAN ENGAGE A FORM OF NECROTIC
DEATH TERMED PYROPTOSIS THAT IS
CHARACTERIZED BY INTENSE
MEMBRANE PERFORATION.
AND IT WAS PRESUMED THAT THIS
MEMBRANE PERFORATION PROVIDES
THE CHANNELS FOR THE EXIT OF
THESE MATURE CYTOKINES.
SO THE RELEASE OF LEADERLESS
CYTOKINES WAS THROUGH A
MECHANISM THAT INVOLVED MEMBRANE
PERFORATION, AND THEIR ACCESS TO
THE INTRACELLULAR MILIEU.
ANOTHER QUESTION WAS, WELL, HOW
ARE THE MEMBRANES GETTING
PERFORATED?
WELL, IN ORDER TO ADDRESS THAT,
WE CARRIED OUT A FORWARD GENETIC
SCREEN.
THIS IS PUBLISHED SO I'LL GO
OVER IT QUICKLY.
THIS INDUCES POINTS MUTATIONS,
IT'S A VERY LARGE PROGRAM.
TENS OF THOUSANDS OF PEDIGREES
WERE GENERATED.
AND THE QUESTION WAS SIMPLE.
WE ISOLATED MACROPHAGES FROM
THESE MUTAGENIZED MICE AND ASKED
IF THEIR ABILITY TO RELEASE IL-1
BETA WAS COMPROMISED.
THIS IS ONE EXAMPLE.
A FEW HUNDRED MICE HERE.
YOU STIMULATE THEM WITH
CYTOSOLIC LPS, THEY MAKE COPIOUS
BUCKET LOADS OF IL-1 BETA THAT'S
RELEASED.
NOW, IN RESPONSE TO LPS
CASPASE-11 MUTANT DOESN'T MAKE
IL-BETA.
CASPASE-11 WAS DOWNSTREAM OF
LPS.
BUT WHAT CAUGHT OUR ATTENTION
WAS ANOTHER MUTANT STRAIN B1 1,
AND AT THIS JUNCTURE WE WERE
QUITE SURPRISED, WE NEVER HEARD
OF GASDERMIN-B, NO KNOWN
FUNCTION.
AND SO THE QUESTION WE WERE
FACED WITHS THAT GASDERMIN-D
RESPOND FOR RELEASE OF
LEADERLESS CYTOKINES, ADDRESSED
WITH SIMPLE EXPERIMENT.
REMEMBER, WE CAN MONITOR
PROTEOLYTIC MATURATION OF IL-1
BETA, SO IT GOES FROM THE PRO
FORM, ABOUT 31KD, TO MATURE
FORM, 17 KD.
MATURE FORM DOESN'T HAVE A
SIGNAL PEP TIDE BUT WE CAN
MONITOR FROM PRO TO MATURE BY
WESTERN BLOT AND MONITOR RELEASE
BY ELISA.
LET'S COMPARE.
WILDTYPE CELLS, YOU'LL NOTICE
THAT THERE'S A TRANSITION FROM
THE 31K TO THE MATURE 17K FORM
ON INFLAMMASOME ACTIVATION THAT
IS SHOWN HERE.
YOU'RE GETTING THE MATURE 17K
FORM THAT.
IS RELEASED INTO THE
SUPERNATANT.
SO IT'S BEHAVING AS EXPECTED.
WE'RE GOING TO DO THE SAME
EXPERIMENT IN GASDERMIN-D NULL
MACROPHAGE.
YOU GENERATE MATURE IL-1 BETA, B
17, AS MUCH AS IN THE WILDTYPE.
BUT WHEN YOU MEASURE BY ELISA,
RELEASE IS GREATLY COMPROMISED.
FROM THIS WE SURMISED THAT
GASDERMIN-D, WHATEVER IT DOES,
SOMEHOW MEDIATES RELEASE OF
LEADERLESS CYTOKINES LIKE IL-1.
THE OTHER UNEXPECTED FINDING WAS
GASDERMIN-D UNDERWENT CLEAVAGE
ON INFLAMMASOME ACTIVATION.
I'LL SHOW YOU DATA FOR
CASPASE-11, WESTERN BLOT, NULL
MACROPHAGES.
WESTERN BLOT FOR GASDERMIN-D,
THE NULL STRAINS LACK THE BAND.
IF YOU LOOK AT THE RIGHT BAND,
IN THE WILDTYPE SITUATION,
NOTICE WHAT HAPPENS TO WILDTYPE
GASDERMIN-D WHEN WE STIMULATE
THE INFLAMMASOME WITH LPS.
THERE'S A DIMINUTION OF THE
FILAMENT BAND AND CONCOMITANT
APPEARANCE OF A P30 FRAGMENT.
ANTIBODY SPECIFIC FOR P30,
THAT'S WHAT WE SEE.
THAT DOESN'T HAPPEN WHEN
CASPASE-11 NULL MACROPHAGES ARE
STIMULATED WITH LPS.
SO, FROM THIS, WE SURMISE THAT
CASPASE-11 ON ACTIVATION LIKELY
CLEAVES GASDERMIN-D TO GIVE YOU
THIS P30 FRAGMENT.
THE ANTIBODY WAS SPECIFIC FOR
P30 SO WE DIDN'T SEE THE OTHER
P20 FRAGMENT.
BUT IF YOU TAKE RECOMBINANT
GASDERMIN-D AND CASPASE-11, YOU
GET EMERGENCE OF FRAGMENT.
IF YOU MAP THE CLEAVAGE SITE,
CLEAVAGE IS RESIDUE, 276.
SO WHAT THIS DATA SAYS IS THAT
ON INFLAMMASOME ACTIVATION,
GASDERMIN-D HAS BEEN CLEAVED TO
P30 AND P20.
IT DOESN'T SAY WHETHER THAT
CLEAVAGE IS NECESSARY FOR
RELEASE OF IL-1 BETA.
FOR THAT WE HAVE TO UNDERTAKE
THE NEXT EXPERIMENT IN WHICH WE
GENERATE A NON-CLEAVABLE FORM OF
GASDERMIN-D, WHERE WE MAKE A
KNOCK-IN MOUSE, WHERE IT IS
CONVERTED TO ALLENASE, IS
GASDERMIN-D NECESSARY AND
CLEAVAGE NECESSARY FOR RELEASE
OF LEADERLESS CYTOKINES?
SO WE ISOLATE MACROPHAGES FROM
WILDTYPE CELLS AND STIMULATE
WITH LPS AND THEN MAKE --
RELEASE A TON OF IL-1 BETA.
THE GASDERMIN-D, THE KNOCKOUT,
AS I SHOWED BEFORE, THEY DON'T
RELEASE IL-1 BETA.
BUT LOOK AT THIS.
THE MICE EXPRESSING THE
PROCESSING MUTANT, SO EXPRESSING
GASDERMIN-D, BUT WHICH CANNOT BE
CLEAVED, ALSO CANNOT RELEASE
IL-1 BETA.
SO, WHAT THIS SAYS IS THAT NOT
ONLY IS GASDERMIN-D REQUIRED FOR
THE RELEASE OF LEADERLESS
CYTOKINES LIKE IL-1 BETA AND
IL-1, 18.
IT HAS COULD BE CLEAVED, OR YOU
DON'T GET CLEAVED.
IT DAWNED ON US THEN MAYBE WE'RE
DEALING WITH CHANNEL FORMING
PROTEIN HERE.
AND WE TESTED THAT AGAINST QUITE
SIMPLY THAT WE SAID MAYBE ONE OF
THE FRAGMENTS, P30 OR P20, IS
ASSEMBLING INTO A PERFORATOR
THAT ALLOWS FOR THE RELEASE OF
IL-1 BETA.
AND IN ORDER TO ADDRESS THAT
QUESTION, WE JUST OVEREXPRESSED
THE FULL LENGTH GASDERMIN-D P30
OR P20 AND EXAMINED THE UPTAKE
OF NUCLEAR DYE.
WHEN YOU OVEREXPRESS P30, THE
NUCLEAR DYE IS UPTAKEN,
SUGGESTING THAT IT IS P30 THAT
IS RESPONSIBLE FOR MEMBRANE
DISRUPTION.
SO TO SUMMARIZE, CASPASE-11
CLEAVES GASDERMIN-D, YOU
GENERATE THE P30 FRAGMENT, AND
THIS IS THE P30 FRAGMENT, THE
PERFORATOR.
OKAY.
SO, WE WERE QUITE INTERESTED IN
THE MECHANISM THAT ASSEMBLED THE
CHANNEL, P30, THE EVIDENCE WAS
POSITIVE.
IF WE TOOK PURIFIED LIPOSOMES,
INCUBATED THEM WITH THE P30
FRAGMENT OF GASDERMIN-D, AND
EXAMINED THE PREPARATION BY
NEGATIVE STAIN ELECTRON
MICROSCOPY, THE IMAGE WAS
STUNNING.
WE COULD SEE THE LIPOSOME
MEMBRANES, WITH WITHIN IT COULD
SEE ASSEMBLED PORE-LIKE
STRUCTURED, CALCULATED IT WAS 24
P30s COMING TOGETHER AND
GIVING YOU A PORE OF ABOUT 15 TO
20 NANOMETERS INTERNAL DIAMETER,
SO FAIRLY LARGE PORE.
NOW, BY THIS TIME THE CAT WAS
OUT OF THE BAG, AND THERE WAS A
LOT OF COMP -- COMPETITION ON
THE HORIZON.
THE ATOMIC STRUCTURE WAS
GENERATED BY WU AND COLLEAGUES
AT CAR HARD, BUT ESSENTIALLY
IT -- HARVARD.
THE FULL LENGTH MOLECULE IS P30
AND P20, AND P30 IS HELD IN
CONSTRAINED MANNER BY P20.
ONCE YOU HAVE THIS PROTEOLYTIC
CLEAVAGE, P20 FLIES OFF, AND P30
UNDERGOES ENORMOUS
CONFIRMATIONAL CHANGE.
THERE'S SORT OF AN UNVEILING,
UNFURLING OF THIS N-TERMINUS
INTO BETA SHEETS THAT'S INSERTED
INTO THE MEMBRANE.
THIS IS HOW WU'S STRUCTURE, 33
P30S COMING TOGETHER.
I THINK THE PROBLEM WAS SOLVED
IN A SENSE THAT THE PORE IS
GENERATED, THE CHANNEL IS
GENERATED BY THIS WHAT WAS AN
UNKNOWN PROTEIN IN 2015,
GASDERMIN-D, GENERATED BY P30
FRAGMENT COMING TOGETHER,
OLIGOMERRIZING AND GIVING YOU A
PORE IN THE MEMBRANE.
AND SO THE ANSWER TO THE
QUESTION OF HOW A LEADERLESS
CYTOKINE LIKE IL-1 BETA AND
IL-18 RELEASE I THINK IS NOW
ANSWERED, WHETHER YOU ACTIVATE
OR CANONICAL OR NON-CANONICAL
PATHWAY, AS LONG AS YOU HAVE
CASPASES, THIS WILL LEAD TO
CLEAVAGE OF GASDERMIN-D, FORMING
A PORE, LARGE ENOUGH THAT IT'S
GOING TO ALLOW FOR PASSAGE OF
4.5-NANOMETER IL-1 BETA AND
IL-18.
OBVIOUSLY IF THE PORE IS
PERSISTENT, YOU'LL COLLAPSE THE
ELECTRO CHEMICAL GRADIENT,
LEADING TO LYSIS OF THE CELLS
WHICH ARE GENERALLY MEASURED BY
LDH RELEASE.
TWO PAPERS ANNOUNCING DISCOVERY
FROM MY OWN GROUP AND A GROUP
USING DIFFERENT METHODS, FORWARD
GENETICS, HE USE CRISPR, SAME
CONCLUSIONS, PUBLISHED IN 2015,
SINCE THEN STUDY OF GASDERMINS
HAVE BEEN A COTTAGE INDUSTRY
WITH A FLURRY, EXPLOSION OF
PUBLICATION.
I WANTED TO BRING YOUR ATTENTION
TO ONE PUBLICATION FROM A COUPLE
YEARS AGO, WHICH WAS THE
DISCOVERY THAT BACTERIA HAVE
GASDERMIN.
AND QUITE REMARKABLY, THE
BACTERIAL GASDERMIN EXISTS ON AN
OPERON IN WHICH IS ALSO ENCODED
A CASPASE-LIKE PROTEASE, SO ON
PHAGE INFECTION THE CASPASE-LIKE
PROTEASES IS ACTIVATED,
BACTERIAL GASDERMIN IS CLEAVED,
N-TERMINAL DOMAIN OF BACTERIAL
GASDERMIN MAKES A PORE IN THE
MEMBRANE OF THE BACTERIA,
LEADING TO ITS DEMISE, AND THE
REPLICATIVE NICHE IS DENIED.
SO, THIS I FIND IS QUITE
REMARKABLE BECAUSE WHEN WE THINK
OF BACTERIAL INNATE IMMUNITY,
YOU KNOW, IT'S BEEN VERY
POWERFUL IN TERMS OF TOOLS IT
HAS GENERATED.
RESTRICTION ENZYMES, CRISPR.
BUT YOU AND I, THEY ARE NOT
CONSERVED ENOUGH, WE DON'T HAVE
RESTRICTION ENZYMES OR CRISPR,
BUT HERE IS A MECHANISM OF
INNATE IMMUNITY IN BACTERIA
THAT'S PRESENT IN YOU AND I.
QUITE REMARKABLE.
ALL RIGHT.
SO, NOW, OBVIOUSLY FROM WHAT
I'VE INTONATED THROUGH THIS
GASDERMIN-D PORE COMES OUT A
PLETHORA OF CYTOKINES.
ONCE YOU FORM THIS NOT ONLY DO
YOU GET IL-1 BETA AND IL-18,
IL-1 ALPHA, OTHER
PRO-INFLAMMATORY SUBSTANCES THAT
POUR OUT, AND THIS IS REALLY
CONTRIBUTES TO CYTOKINE STORM
RESPONSIBLE FOR SO MANY CRITICAL
ILLNESSES LIKE SEPSIS AND ARDS.
AND I THINK THE NOTION IN OUR
MIND IS THAT IF ONE COULD
DEVELOP AN INHIBITOR TO
GASDERMIN-D, IN ONE FELL SWOOP
YOU WOULD ATTENUATE THE RELEASE
OF THESE CYTOKINES, IT MAY BE A
BENEFIT IN THESE ACUTE
SYNDROMES.
THERE'S A LOT OF THE ACTIVITY
TRYING TO DEVELOP
PHARMACOLOGICALLY,
THERAPEUTICALLY EFFICACIOUS,
INHIBITORS THAT HAVE
SPECIFICITY.
NO I'M GOING TO SOMETHING
UNEXPECTED.
I'LL STILL NOT SURE OF THE
RAMIFICATIONS OF THIS DISCOVERY
SO I THOUGHT I WOULD SHARE WITH
YOU.
WHEN WE DO SCREENS OF TENS OF
THOUSANDS OF MICE, REMEMBER WHAT
WE'RE DOING IS WE'RE GIVING THEM
INDUSTRIAL LOADS OF CYTOSOLIC
LPS, SO WE CAN MEASURE THE
RELEASE OF IL-1 BETA, THEY MAKE
BUCKET LOADS OF IL-1 BETA, AND
WE'RE LOOKING FOR SITUATIONS
WHERE THE INFLAMMASOME IS
BROKEN.
THAT'S THE ASSAY.
BUT, THESE CELLS IN WHICH WE'VE
GIVEN -- EXPOSED TO INDUSTRIAL
LEVELS OF CYTOSOLIC LPS ARE
DESTINED TO DIE.
HAVE YOU PRECIPITOUS ACTIVATION
OF INFLAMMATORY CASPASES.
GENERATION OF GASDERMIN-D PORES.
AND THEN THE CELLS LYSE.
A SANITY CHECK, WE MEASURE LDH
RELEASE.
EVERY TIME YOU GET INDUSTRIAL
AMOUNTS OF IL-1 BETA RELEASE YOU
GET HDA RELEASE.
OKAY, WELL, YEAH, THAT'S A
SANITY CHECK.
IT CAME TO ME ONE THEY, THEY
SAID WE'VE GOT A STRAIN THAT
RELEASES IL-1 BETA.
BUT DOESN'T RELEASE LDH.
IT'S A STRAIN, MUTATION, THIS
NINJ1 PROTEIN.
HOW CAN THAT BE?
ARE THE CELLS DEAD?
YEAH, THEY ARE DEAD.
I SAID, YOU'RE TELLING ME THAT
YOU'RE MEASURING CELLS FROM LDH,
YOU DON'T SEE A SIGNAL?
LDH IS USED IN THE SIGNAL, THE
CLINIC, IT'S HOW WE MEASURE CELL
LYSIS.
DEAD CELLS LYSE, RELEASE LDH.
YOU'RE NOT TO TELL ME YOU HAVE
DEAD CELLS THAT ARE NOT
RELEASING LDH.
JUST CAN'T BE.
RIGHT?
DOESN'T MAKE ANY SENSE.
SO, ONE THING I SHOULD SAY IL-1
BETA IS RELATIVELY SMALL, 17KD,
LDH IS BIG, 140KD.
NOW, WE'VE GOTTEN NINJ1 FROM A
FORWARD GENETIC SCREENING, IT'S
NOT GETTING INTO THE GENETICS,
OFTEN THE STRAINS CARRY MULTIPLE
MUTATIONS.
I'M NOT GOING TO BELIEVE THIS
TILL YOU MAKE A KNOCKOUT.
MAYBE TWO OR THREE INDEPENDENT
KNOCKOUT STRAINS TO BE SURE
BECAUSE THIS IS WHACK-O.
HERE IS THE DATA.
SO, WE'RE GOING TO LOOK AT IL-1
BETA RELEASE, OPEN BARS ARE
WILDTYPE MACROPHAGES, RED BARS
ARE NINJ1 NULL MACROPHAGES,
STIMULATE WITH LPS, IN WILD TYPE
AND NINJ1 KNOCKOUT YOU'RE IL-1
BETA RELEASE.
WE SEE THAT THE GENERATION OF
THE P30 FRAGMENT IN WILD TYPE
AND NINJ1 NULL MACROPHAGES OF
GASDERMIN-D IS EQUIVALENT.
IN THE NINJ1 NULL MACROPHAGES
THAT ARE DYING YOU'RE GETTING
GASDERMIN-D PORES.
LET'S LOOK AT LDH RELEASE.
WILD TYPE MACROPHAGE RELEASED,
NULL MACROPHAGES DO NOT.
I'M LIKE, MAN, THIS IS JUST
SOMETHING WEIRD ABOUT LDH.
I JUST CANNOT BUY THIS RESULT.
BUT THEN WE LOOKED AT THE
SECRETOME.
WE LOOKED AT THE RELEASE OF
UNBIASED, REALLY OF ALL PROTEINS
RELEASED INTO THE EXTRACELLULAR
MEDIA BY LPS.
INDEED, IN THE ABSENCE OF NINJ1,
THERE'S A LARGE SUBSTANTIAL
NUMBER OF PROTEINS WHOSE RELEASE
IS COMPROMISED.
SO IT'S NOT JUST LDH.
I'M LIKE, YOU KNOW, THIS IS
REALLY WEIRD.
WE'VE GOT TO LOOK AT THE CELLS.
SO, I'LL JUST REMIND YOU THAT
CELLS WERE GIVEN NECROTIC
STIMULUS, WE'RE LOOKING AT BONE
MARROW DERIVED MACROPHAGES,
INITIALLY BALLOON AND COLLAPSE.
LET'S ORIENT OURSELVES WITH THAT
IMAGE.
THEY BALLOON UP.
AND THEN THEY COLLAPSE.
THEY UNDERGO LYSIS.
NOW WE'RE GOING TO LOOK AT A LOW
POWER VIEW OF BONE MARROW
DERIVED MACROPHAGES, NIGERICIN.
THEY BALLOON BUT COLLAPSE INTO
THE SCRAMBLED EGG APPEARANCE.
IF YOU DO THE EXPERIMENT WITH
THE NINJ1 NULL CELLS THEY
BALLOON, BALLOON UP, AND BALLOON
UP.
THIS IS OVERNIGHT.
IT'S NOT LIKE WE SAID, YOU KNOW,
WE'VE GOT A WINDOW OF 10 MINUTES
TO MAKE THIS FIGURE.
NO, YOU CAN GO HOME, SPEND THE
NIGHT, COME BACK, THEY ARE STILL
BALLOONED.
EVENTUALLY OF COURSE THEY WILL
LYSE.
BUT THE WINDOW OF BALLOONING IS
ENORMOUS.
I REMEMBER WHEN WE FIRST
PUBLISHED THIS PAPER, BECAUSE OF
TIME, CRISPR, PEOPLE DIDN'T
BELIEVE IT.
IT'S BULLSHIT.
EVERYBODY DID A CRISPR
EXPERIMENT, INDEED ABLE TO
DUPLICATE THE WORK.
NOW, THE BALLOONED CELLS ARE
DEAD.
THEY ARE DEADER THAN DOORNAILS.
BUT THEY ARE BALLOONED.
THIS IS NOT LYSING.
VERY, VERY ODD.
I'M LIKE, WHAT THE HELL IS
NINJ1?
AGAIN, UNEXPECTED DISCOVERY.
LIBRARY TELLS YOU NOTHING.
IT'S 157 AMINO ACIDS, SMALL
PROTEIN.
I'LL GET TO THIS LATER, ONE OF
THE TOPOLOGY PROGRAMS PREDICTS
IT TO HAVE THE N-TERMINUS AND
C-TERMINUS OUT, AND WITH AMPHI
PATTIC DOMAIN THAT'S EXPERIMENT.
IT'S CONSERVED.
DROSOPHILAS THAT THREE, NINJ A,
B, C.
WHAT IS IT DOING?
SO, THIS GOT ME THINKING, US
THINKING.
MAYBE NINJ1 IS NOT ONLY
DOWNSTREAM OF GASDERMIN-D FOR
CELL LYSIS BUT IT'S THE FINAL
COMMON COMPONENT THAT YOU NEED
THIS FOR CELLS TO LYSE NO MATTER
HOW THEY DIE.
NO MATTER HOW THEY DIE YOU NEED
NINJ1 FOR CELL LYSIS.
PRETTY WILD.
LET'S GO THROUGH PERCENTAGE LDH
RELEASE, OPEN BARS ARE WILDTYPE
CELLS, RED NINJ1 KNOCKOUTS.
IN ABSENCE OF TREATMENT, THE
RELEASE IS 18%.
NIGERICIN, YOU SAW RELEASE OF
NINJ1, NULL CELLS COMPROMISED,
USED STREPTOLYSIN OR LISTERIA,
BUT LOOK AT THIS, CYST CISPLATIN
INDUCED DEATH HAS NOTHING TO DO
WITH GASDERMIN-D, YET THOSE
CELLS, THE LYSIS OF THOSE CELLS,
THE LYSIS OF DEAD CELLS, FROM
CISPLATIN EXPOSURE HAS A NINJ1
DEPENDENCE.
OKAY.
SO, WHY IS THIS SO SURPRISING?
BECAUSE YOU REMEMBER MISS JONES,
OUR HIGH SCHOOL BIOLOGY TEACHER.
SHE SAID WHEN CELLS DIE, THEY
LYSE, AND IT'S A MECHANISM, A
PASSIVE MECHANISM, AND WRITE IT
DOWN IN YOUR NOTEBOOK, BOYS AND
GIRLS, WE'LL CALL IT OSMOTIC
LYSIS.
THAT'S WHAT WE LEARNED.
RIGHT?
[LAUGHTER]
THAT'S WHAT HAPPENS.
YEAH.
OKAY.
WHAT I I'M SAYING IS SOMETHING
RADICALLY DIFFERENT.
LYSIS, EVEN FOLLOWING DEATH, IS
AN ACTIVE EVENT AND IT'S A
PROGRAMMED EVENT.
NOW, HOW ARE WE GOING TO STUDY
THIS?
WELL, A PREDICTION WOULD BE IF
NINJ1 REALLY IS RESPONSIBLE FOR
CELL LYSIS, IF YOU OVEREXPRESS
IT, IT SHOULD SHATTER CELLS.
INDEED THAT'S THE CASE.
TAKE 293T CELLS, A HUMAN NINJ1,
FOR THAT MATTER FLY NINJ A OR B,
YOU SHATTER THE CELLS.
THIS BECOMES A SURROGATE
BIOCHEMICAL ASSAY, BY WHICH WE
CAN INTERROGATE IT THROUGH
ALANINE SCANNING MUTAGENESIS
I'LL SHOW YOU ON THE NEXT SLIDE.
BUT ALSO I WANT TO EMPHASIZE
THAT NINJ1 IN ITS VARIOUS GUISES
IS PRESENT ALL THE WAY DOWN TO
THE FLY.
SO, AGAIN, NINJ1 GOING FROM THE
N-TERMINUS TO C-TERMINUS HAS
THIS EXTERNAL AMPIPATHIC HELIX,
A TRANSMEMBRANE DOMAIN 1 AND
TRANSMEMBRANE DOMAIN 2, IF YOU
LOOK AT CONSERVATION SCORE,
LOWER THE SCORE, HIGHER
CONSERVATION, THE AMPIPATHIC
HELIX IS HIGHLY CONSERVED AMONG
SPECIES.
NOW WE'RE GOING TO DO AN
ALLOSCAN, CHANGE FIVE AMINO
ACIDS AT A TIME TO ALANINE,
STARTING FROM THE N-TERMINUS,
GOING TO THE C-TERMINUS, AND
WE'RE GOING TO ASSAY THE
PROTEINS BY THEIR ABILITY TO
INDUCE CELL DEATH.
SO WHEN YOU OVEREXPRESS WILDTYPE
NINJ1, YOU INDUCE CELL DEATH.
IF YOU MUTATE THE N-TERMINUS,
IT'S QUITE RESISTANT, QUITE
ACCOMMODATING, TO MUTATION.
BUT AS SOON AS YOU MESS WITH THE
AMPIPATHIC HELIX, NINJ1 IS NO
LONGER ABLE TO LYSE CELLS.
SO THE AMPIPATHIC HEEL IS IS
IMPORTANT.
WHAT'S THE ENDOGENOUS MOLECULE
LOOKING LIKE?
HOW DOES THAT BEHAVE?
SO, LET'S LOOK AT BONE
MARROW-DERIVED MACROPHAGES.
WE TREAT THEM WITH POISON
NIGERICIN, ONE HOUR, TWO HOUR,
BLOTTING FOR ENDOGENOUS NINJ1
AND WE'RE SEPARATING OUT THE
PRECIPITATE ON INNATIVE BLUE
PAGE GEL.
HEALTH CELLS NINJ1 IS A MONOMER
DIMER.
WITHIN ONE HOUR FORMS POLYMERIC
STRUCTURE, RIGHT BANDS, NOT DEAD
IN THE KNOCKOUT.
IF WE LOOK BY IMMUNOSTAINING WE
SEE IN CONTROL CELLS IF I HAVE
USED ON PLASMA MEMBRANE BUT DEAD
CELLS FOLLOWING NIGERICIN
EXPOSURE PUNCTATE FOCI ON CELL
MEMBRANE.
IT'S NOT THERE IN THE NINJ1
KNOCKOUTS, THE STAINING IS
SPECIFIC.
SO, WE WERE SIDETRACKED FOR SOME
TIME BECAUSE HOW DOES THIS
HAPPEN?
HOW DO YOU GO FROM MONOMER TO
POLYMER SO QUICKLY?
THERE MUST BE A SECOND
MESSENGER.
THERE MUST BE A PHOSPHORYLATION,
UBIQUITINATION, SOMETHING MUST
HAVE CHANGED.
AND WE HAVEN'T BEEN ABLE TO
DETECT IT.
I'LL COME BACK TO WHAT OUR
THINKING IS RIGHT NOW.
THERE ISN'T A TRIGGER OF
PROTEOLYTIC CLEAVAGE,
PHOSPHORYLATION, YOUR --
UBIQUITINATION IS IS
RESPONSIBLE, MONOMER GOING TO
POLYMER.
YOU HAVE TO ASK YOURSELF A VERY
IMPORTANT QUESTION.
WHY WOULD THIS BE COULD BE
SERVED IN EVOLUTIONARY TIME?
BECAUSE WHAT I'M SAYING IS WHAT
WE'VE CONSERVED IS A MECHANISM
THAT MAKES A DEAD CELL DEADER.
THAT DOESN'T MAKE ANY SENSE.
YOU'RE DEAD.
WHY WOULD EVOLUTION CARE?
WELL, I THINK THE REASON FOR
THAT IS THAT AS YOU KNOW, WE
HAVE INJURED AND DEFECTIVE CELLS
ALL THE TIME BEING GENERATED.
EITHER INNOCUOUSLY OR THROUGH
TRAUMA, INEFFECT, ET CETERA. --
INFECTION, ET CETERA, TAKEN UP
BY ENDOTHELIAL SYSTEM OR
NEIGHBORING CELLS, A BUFFER
TAKES UP INJURED CELLS AND
DIGESTING THEM AND THERE IS NO
INFLAMMATORY RESPONSE.
THINK OF APOPTOTIC CELLS.
BUT IF YOU EXCEED A CERTAIN
THRESHOLD AND THAT THRESHOLD IS
THE ABILITY OF THESE CELLS TO
ENGULF INJURED CELLS,
AFROCYTOSIS, THAT'S A SIGNAL FOR
THE BODY.
IT SAYS WE HAVE INJURY HERE,
BUFFER SYSTEM IS SATURATED,
CELLS RAPIDLY LYSE, RELEASE
MEDIATOR OF INFLAMMATION, THE
DAMPs.
YOU'RE CALLING IN THE IMMUNE
RESPONSE.
SO, THAT'S THE THINKING.
AN EXTENSION OF THAT THINKING IF
YOU'RE ABLE TO INHIBIT LYSIS OF
DEAD CELLS, THAT WOULD
POTENTIALLY BE
ANTI-INFLAMMATORY.
SO I THINK NINJ1 IS CONSERVED IN
EVOLUTION, THERE IN THE FLY,
BECAUSE IT INDUCES RAPID CELL
LYSIS AND THIS SIGNALS, IT'S A
PRIMITIVE WAY TO SIGNAL
INFLAMMATION.
SO A LOT OF THIS IS PUBLISHED, I
WON'T GO INTO IT.
CONSISTENT WITH THIS MODEL IS
THE FACT THAT NINJ1 NULL MICE
ARE SUSCEPTIBLE TO INFECTIONS,
ESPECIALLY INTRACELLULAR
PATHOGENS, RESISTANT TO STERILE
INFLAMMATION.
DATA ON STERILE INFLAMMATION, TO
ADDRESS THIS IN A DEFINITIVE
MANNER WE USED BOTH KNOCKOUT
STUDIES AND WE GENERATED A VERY
POWERFUL TOOL.
WE GENERATED AN ANTIBODY THAT IS
CAPABLE OF NEUTRALIZING NINJ1.
IN ORDER TO DO THAT, WE
EXPRESSED NINJ1 PROTEIN IN
EXTRACELLULAR VESICLES,
GENERATED A LARGE NUMBER OF
RECOMBINANT MONOCLONAL
ANTIBODIES, ASSAYED FOR THEIR
ABILITY TO INHIBIT LDH RELEASE.
THIS IS JUST THE RELEASE OF LDH
ON THE Y-AXIS.
THIS IS THE NINJ1 KNOCKOUT, SO
THERE'S A COME PRO MIGHT HAVE
HAD -- COMPROMISED RELEASE, AND
JUST AS COMPROMISED IS CLONE D1,
IN THE PRESENCE OF D1 YOU
NEUTRALIZE THE ABILITY OF NINJ1
TO LYSE MEMBRANES TO THE SAME
EXTENT AS THE KNOCKOUT.
SO, D1 INHABITS PLASMA MEMBRANE
RUPTURE.
I'LL SHOW YOU DATA, A FUNCTION
OF LPS, UNTREATED CELLS YOU GET
TREMENDOUS RELEASE OF LDH ON
EXPOSURE TO CYTOSOLIC LPS, THE
NINJ1 KNOCKOUT IS THE SOLID
BLACK BAR, PROTECTED, BUT ALMOST
AS WELL PROTECTED IS CELLS
EXPOSED TO THE ANTIBODY ONE
MICROGRAM OR TEN MICROGRAMS PER
MILL.
OKAY.
NOW, HOW THE ANTIBODY WORKS IS
WE BELIEVE IT SUPPRESSES
OLIGOMERIZATION.
IF YOU PURIFY, IT COMES OUT AS A
VERY LARGE MOLECULAR WEIGHT
OBJECT, UNDER SCANNING, UNDER
TRANSMISSION ELECTRON MICROSCOPY
YOU CAN SEE THESE ARE
CHOCK-A-BLOCK FULL OF
POLYMERIZED PROTEINS OFTEN IN
CIRCULAR CONFIGURATIONS, IF YOU
DO THE SAME EXPERIMENT BUT NOW
IN THE PRESENCE OF ANTIBODY,
THEN THERE'S A DEPOLYMERIZATION,
NO EVIDENCE FOR NINJ1 OLIGOMER
FORMATION BY GEL STAINING OR
ELECTROMICROSCOPY.
IN THE U.K. BIOBANK
POLYMORPHISMS IN NINJ1 WERE
ASSOCIATED WITH PEOPLE THAT HAD
LOW LEVELS OF CIRCULATING LIVER
ENZYMES, THAT YOU NORMALLY
MEASURE FOR LIVER DAMAGE.
WE THOUGHT MAYBE LIVER INJURY
WOULD BE A PLACE WE SHOULD LOOK
AT NEUTRALIZING A NINJ1.
SO THE FIRST EXPERIMENT WAS A
GENETIC EXPERIMENT WHERE WE TOOK
OUR NINJ1 KNOCKOUT MICE, AND WE
TREATED THE MICE WITH TNF D
GALACTOSAMINE.
STAINING IS NOT THERE IN THE
KNOCKOUT, IT IS SPECIFIC.
LOOK AT THIS DATA.
IF YOU EXPOSE THE MICE TO TNF
AND GALACTOSAMINE, THEY ARE
GREATLY SUBDUED ALMOST TO
CONTROL LEVELS IN THE NINJ1
KNOCKOUT.
SO, WHAT ABOUT THE ANTIBODY?
NOW, AGAIN, WE USE ANOTHER MODEL
FOR LIVER INJURY.
THIS IS -- WE'RE INJURING THE
LIVER THROUGH CON-A.
THIS RESEMBLES CTL INDUCED
KILLING OF HEPATOCYTES.
AND AGAIN IN THE GENETIC MODEL
YOU SEE SUBSTANTIAL PROTECTION
IN THIS MODEL.
NOW, HERE'S THE EXPERIMENT WITH
THE ANTIBODY, ALBEIT IT IS A
PRE-TREATMENT REGIMEN, SO IT'S A
RELATIVELY LOW BAR, SO TAKE IT
FOR WHAT IT'S WORTH.
WE GIVE THE ANTIBODY FIRST AND
THEN GIVE THE CON-A.
I HOPE YOU'LL REALIZE WHEN YOU
LOOK AT LDH OR LIVER ENZYMES,
THAT IN THE PRESENCE OF THE
ANTIBODY SHOWN HERE IN BLUE
THERE'S A DRAMATIC DIFFERENCE,
DRAMATIC SUPPRESSION OF LIVER
INJURY.
THIS IS CORROBORATED BY RELEASE
OF CYTOKINES AND SIMPLY JUST
STAINING THE LIVER FOR
NEUTROPHILS.
SO YOU SUPPRESS IMMUNE INJURY.
SO, THIS SUGGESTS THAT NINJ1 IS
POTENTIALLY A DRUGGABLE TARGET.
AND IT BRINGS TO THE FORE THE
CONCEPT THAT WHEN YOU HAVE
OVERWHELMING DEATH OF CELLS,
THAT THE PHAGOCYTIC SYSTEM CAN
NO LONGER DEAL WITH EXCESS DEAD
CELLS, RAPID LYSIS MEDIATED BY
NINJ1, LEADS TO THE RELEASE OF
DAMPs, AND INITIATION OF
TISSUE INFLAMMATION.
AND THAT THIS INJURY IN CASES OF
STERILE INFLAMMATION COULD BE
BENEFICIALLY BLOCKED BY
NEUTRALIZING NINJ1 WITH AN
ANTIBODY TO NINJ1.
BUT WHERE FROM HERE?
HOW IS NINJ1 GETTING ACTIVATED,
$64,000 QUESTION.
NOW, I DON'T THINK IT'S A
POST-TRANSLATIONAL MODIFICATION.
THERE'S SO MANY ROADS TO NINJ1
ACTIVATION, WHETHER YOU KILL THE
CELLS WITH CISPLATIN OR
LISTERIA, VERY LITTLE IN COMMON
BETWEEN WHAT'S DOING THE
KILLING.
I THINK NINJ1 IS MEASURING,
RESPONDING TO THE HEALTH OF THE
MEMBRANE.
NOW, THE QUESTION IS, IS IT
CHANGES IN MEMBRANE TENSION OR
MEMBRANE COMPOSITION, OR THE
BINDING, OR ABSENCE OF PARTNER,
WHAT IS IT THAT IS PRECISELY
RESPONSIBLE?
AND I THINK THIS IS THE QUESTION
I WOULD BRING YOUR ATTENTION TO
BECAUSE IT'S BEGINNING TO BE
ANSWERED.
NOW, THE FIRST ATTEMPT AT THAT
WAS TO -- WHICH WAS VERY
GRATIFYING FOR US BECAUSE WE
WERE ON A LIMB SAYING NINJ1
FORMS MEGA PORES, INVOLVED WITH
CELL LYSIS, A PAPER THAT DID
STRUCTURE OF ACTIVATED
POLYMERIC NINJ1, THERE ARE
PAPERS IN PRESS FROM WU AND ZING
HONG DAI WITH SIMILAR
CONCLUSIONS.
GENERALLY THEY SAY THIS.
IN THE ACTIVE POLYMERIC STATE,
THE ARCHITECTURE OF NINJ1 IS SO.
YOU HAVE TWO TRANSMEMBRANE
HELICES I TALKED ABOUT, BUT THE
AMPIPATHIC IS EMBEDDED IN THE
MEMBRANE, ACTUALLY TWO HELICES.
THERE'S A KINK.
THIS KINK IS IMPORTANT BECAUSE
THE KINKED HELIX ACTS AS A
CROSS-BRACE BECAUSE IT INTERACTS
CAN A JOINT TRANSMEMBRANE
DOMAIN, YOU CAN SEE HOW IT COULD
TRIGGER POLYMERIZATION.
SO IT HAS THE APPROPRIATE
AMPIPATHIC CHARACTER, AND COULD
POTENTIALLY GENERATE VERY LARGE
PORES, THEY ESTIMATE
500-NANOMETER PORES ESSENTIALLY
FOR MEMBRANE LYSIS.
SO I THINK THIS IS AN IMPORTANT
STEP, CERTAINLY DOESN'T ANSWER
THE QUESTION.
AND WHAT WE'RE SORT OF PRESENTLY
CONCERNED WITH IS THAT HOW SURE
ARE WE THAT THE N-TERMINUS IS
OUTSIDE?
BECAUSE THIS IS ONE MODEL, THE
N-TERMINUS IS OUTSIDE.
SO WE WOULD SAY IN THE INACTIVE
MONOMERIC STATE, THE N-TERMINUS
IS OUTSIDE.
THAT'S THIS.
BUT IN THE ACTIVE STATE IT'S
INSIDE, AND IT'S KINKED.
THAT'S A HUGE CLAIM TO MAKE.
ALMOST THERMODYNAMICALLY
IMPOSSIBLE FOR THIS TO HAPPEN.
I DON'T HAVE THE ANSWER BUT I'LL
SHOW YOU THE EVIDENCE.
WE BELIEVE THAT AT LEAST A
FRACTION OF NINJ1 HAS THIS
ORIENTATION WITH THE N-TERMINUS
AMPIPATHIC ON THE OUTSIDE, AND
MAY BE THAT IT'S JUST HUGGING
YOU THE PLASMA MEMBRANE,
PARTIALLY EMBEDDED IN THE PLASMA
MEMBRANE.
WHEN WE DO FAX WITH CLONE 25 WE
SEE A SIGNAL.
IF WE C-TERMINUS, RETAINED,
N-TERMINUS IT'S GONE.
THERE ARE OTHER INTERPRETATIONS,
OPEN WHERE WE'RE AT RIGHT NOW.
SO WE'RE AT THE VERY CUSP OF
HAVING SOLVED THE MONOMERIC
STRUCTURE, AND I'M HOPING THAT
IN THE NEXT WEEK OR TWO WE'LL BE
ABLE TO GET BETTER INSIGHT INTO
MOLECULAR GYMNASTICS BUT IT
WON'T ADDRESS THE QUESTION OF
WHAT TRIGGERS IT.
IS IT TENSION, IS IT
COMPOSITION, WHAT IS IT?
AND I THINK THAT WILL BE THE
SUBJECT OF A LOT OF PAPERS BY
OTHERS AND HOPEFULLY BY
OURSELVES.
SO, TWO CONCLUSIONS FROM THE
TALK.
LEADERLESS CYTOKINES ARE
RELEASED BY GASDERMIN-D PORES.
AND THE SECOND CONCLUSION, VERY
SURPRISING, THAT CELL LYSIS
FOLLOWING CELL DEATH IS NOT A
PASSIVE EVENT, LIKE MISS JONES
TAUGHT US IN HIGH SCHOOL, THAT'S
ONE GREATLY ACCELERATED BY
NINJ1.
OKAY.
I'LL END THERE.
HOW AM I DOING FOR TIME?
ALL RIGHT.
SO THE WORK HAS BEEN ONGOING,
PLENTY OF COLLABORATION WITH MY
COLLEAGUE KAYAGAKI AND HIS
GROUP, STOWE AND LEE, OPHER WAS
INSTRUMENTAL IN DISCOVERY OF
NINJ1.
AND WE'VE HAD THE FORWARD
GENETIC SCREEN, MASSIVE EFFORT
DONE IN COLLABORATION WITH ANU,
CANBERRA.
STRUCTURAL WORK DONE BY ISHAN
AND KAMELA, RYAN AND LEE AND
PATHOLOGY BY JOSH WEBSTER.
CHRIS GOODNOW IS PART OF THE
CANBERRA GROUP BUT HAS SINCE
MOVED TO SYDNEY.
THE WORK WOULDN'T HAVE BEEN
POSSIBLE WITHOUT THEM.
I HOPE I'VE TAKEN YOU ON A
PERSONAL JOURNEY OF DISCOVERY.
I DIDN'T SHOW SIGNAL CELL
RNAseq, DID I?
WELL, I'LL OLD WORLD, WHAT CAN I
SAY?
THANK YOU.
[APPLAUSE]
>> GREAT TALK.
I APPRECIATED ALL THE WESTERN
BLOTS OF COURSE.
I WAS WONDERING, TWO QUESTIONS.
ONE, SO CELL DEATH, APOPTOSIS IS
ALSO IMPORTANT DURING
DEVELOPMENT.
FROM YOUR MOUSE MODELS AND SO ON
DO YOU HAVE ANY INDICATION THAT
NINJ1 COULD ALSO PLAY A ROLE
THERE?
DO YOU SEE RATIOS?
TO THE MECHANISM I HOPE I DIDN'T
MISS IT, BUT YOU HAVE ANOTHER
MONOCLONAL ANTIBODY, CLONE 25,
DID YOU ALSO TEST THAT?
DOES THAT ALSO INHIBIT THE
OLIGOMERIZATION?
>> CLONE 25 DOESN'T WORK AT AS
WELL, PARTIAL INHIBITOR, THAT'S
WHY WE WENT TO D1.
AND YOUR FIRST QUESTION WAS --
>> IS THERE ANY WAY THAT NINJ1
COULD ALSO PLAY A ROLE IN --
>> OH, YEAH, IN DEVELOPMENT.
YOU KNOW, I THINK NINJ1 COMES
INTO EFFECT WHEN DEATH IS
OVERWHELMING, SO NORMALLY IN
DEVELOPMENTAL DEATH, THE
SURROUNDING CELLS WILL ENGULF
IT.
YOU KNOW, A SLAUGHTERHOUSE IS
99.9% OF CELLS ARE GOING TO
UNDERGO APOPTOTIC DEMISE.
IF YOU STAINED FOR CLEAVED
CASPASE 3, YOU SEE VERY FEW,
IT'S SO RAPID.
I TELL PEOPLE THERE ARE A
MILLION CELLS THAT UNDERGO APOPS
APOPTOSIS EVERY SECOND, THAT
DOESN'T RESONATE.
THIS IS THE NUMBER OF CELLS THAT
UNDERGOES APOPTOSIS, IT'S TWO
EGGS WORTH OF APOPTOSISU HAPPENS
IN YOU EVERY DAY.
YOU'RE NOT INFLAMED.
THAT IS BECAUSE YOUR SYSTEM OF
CLEARING IS THAT EFFICIENT.
BUT WHEN YOU HAVE OVERWHELMING
INJURY, SYSTEM SATURATED, YOU
GET LYSIS AND SIGNALING.
>> BEAUTIFUL, WONDERFUL WORK.
I HAVE A QUESTION.
MAYBE I MISSED SOMETHING BECAUSE
YOU MENTIONED THAT THIS NINJ1
MAYBE THE LAST STEP TO CLEAR
WHEN CELLS ARE INJURED OR
APOPTOTIC.
WE KNOW IF THE CELLS BETWEEN
INJURED OR NECROTIC CELLS GET
QUITE DIFFERENT OUTCOME.
MY QUESTION IS DO YOU HAVE
ANY -- MAYBE I MISSED SOMETHING.
>> NO, YOU DIDN'T MISS
SOMETHING.
YOU'RE RIGHT.
I WANT TO MAKE ANOTHER IMPORTANT
POINT, EMPHASIZED IN THE REVIEW
CELL DEATH PUBLISHED TWO MONTHS
AGO.
SO HAVE A LOOK AT THAT.
IF YOU HAVE OVERWHELMING
APOPTOSIS YOU WILL HAVE
SECONDARY NECROSIS IN AN
ORGANISM.
SO WHILE WE TRY TO TEND TO THINK
OF APOPTOTIC DEATH AS
IMMUNOLOGICALLY SILENT, OVER A
CERTAIN THRESHOLD, IT WILL
INDUCE INFLAMMATION BECAUSE
THOSE APOPTOTIC CELLS WILL
UNDERGO SECONDARY NECROSIS BUT
YOU HAVE TO HAVE OVERWHELMING
APOPTOSIS.
>> HI.
WHEN YOU THINK ABOUT LIKEth
THE SPEED OF THE PROCESS, LIKE
YOU SUGGEST THAT ONE, THERE'S AN
INACTIVE AND ACTIVE STRUCTURE,
THIS IS LIKE THE MECHANISM WHICH
RELEASES PORES, MEGA PORES, IF I
GET IT RIGHT.
LIKE WHAT HAPPENS, SO WHERE ARE
THOSE MOLECULES LOCALIZED?
ALWAYS THEY MEMBRANE AND THEN
THEY JUST GET BROUGHT TOGETHER
OR ARE THEY IN INTRACELLULAR
COMPARTMENTS TRANSFERRED TO
MEMBRANES?
>> VERY GOOD QUESTION.
SO, THERE'S A HUGE RESIDENT POOL
AT THE MEMBRANE.
BUT WHEN WE DO OUR STAINING, IT
IS CERTAINLY PRESENT IN WHAT
SEEMS TO BE THE SORT OF ER
GOLGI.
I DON'T KNOW WHETHER THAT'S JUST
NINJ1 ON ITS WAY OR IF THERE'S
NINJ1 RESIDENT THERE.
BUT I WOULD VENTURE TO SAY THAT
IT'S THE PLASMA MEMBRANE NINJ1
THAT IS RESPONSIBLE FOR THIS
MEGA PORE FORMATION THAT YOU
SEE.
NINJ1 IS NOT, FOR EXAMPLE, ON
MITOCHONDRIAL MEMBRANES.
>> DO YOU THINK THAT THERE IS
LIKE REQUIREMENT FOR THE
TRANSLOCATION FROM INTRACELLULAR
MEMBRANES TO FORM THESE PORES OR
DO YOU THINK IT'S SIMPLY LIKE
ARE THERE LOCATIONS OR
PSEUDOLOCATIONS IN MEMBRANES?
>> YEAH, NO, NO, I THINK IT'S
ALL IN THE PLASMA MEMBRANE.
THEY ARE MONITORING THE HEALTH.
THINK OF A MECHANISM, I DON'T
KNOW IF THIS IS THE CASE, BUT WE
KNOW THAT ALL DYING CELLS WILL
LOSE ATP, RIGHT?
AND WE KNOW TO MAINTAIN
ASYMMETRY ARE YOU HAVE TO EXPEND
ENERGY.
YOU CAN IMAGINE DYING CELLS ONE
THING THEY WILL HAVE IN COMMON
IS ALTERATION OF THEIR MEMBRANE
COMPOSITION.
RIGHT?
MAYBE IT MONITORS THAT.
BUT IT'S -- I DON'T THINK IT'S
THE ONE THAT IS TRANSLOCATED.
I THINK IT'S THE ONE THAT'S
ALREADY PRESENT ON THE PLASMA
MEMBRANE.
>> SO THEN DO YOU SEE
DEPOLARIZATION OF THE MEMBRANES?
DO YOU SEE LIKE PHOSPHOLIPIDS
SHIFT FROM ENTRY OUTSIDE OR DID
YOU SEE CHANGES IN THE KNOCKOUT
MICE?
>> NO, OH, YEAH, IT'S NOT AS
SIMPLE AS THAT.
BUT, YEAH, NO, NO, YOU'RE
GETTING AT THE RIGHT ANGLE.
I THINK THERE WILL BE A PAPER
COMING OUT SOON, NOT FROM US,
FROM SOMEBODY ELSE, SAYING THAT
IT'S CALCIUM FLUX THAT LEADS US.
I DON'T KNOW.
WE DON'T SEE THAT.
WHO KNOWS.
IT IS A CHANGE IN THE MEMBRANE.
YEAH?
>> FIRST, REALLY EXCITING WORK.
EXCELLENT TALK.
>> NO SINGLE CELL RNAseq?
NO.
[LAUGHTER]
DAMN!
>> I DID HAVE TWO QUESTIONS.
THE FIRST ONE, ON THE MOUSE
MODEL OF THE LIVER, AND LOWER
ALT AND ASC DID YOU ALSO MEASURE
LIVER FUNCTION OR DEATH?
ASC AND ALT WILL BE RELEASED AS
CELL EXPLODES, YOU'RE PREVENTING
THAT, COULD THEY JUST HAVE BAD
LIVES?
>> WE'VE RUN A SURVIVAL ASSAY
AND WE'VE SEEN A SURVIVAL
BENEFIT IN THESE EXPERIMENTS
IT'S DIFFICULT TO SEE ONE
BECAUSE THE STIMULUS IS, WITH
TIME, JUST LETHAL.
I THINK WHEN YOU HAVE SUCH
LYSIS, INTENSE LYSIS, LIKE IN
THESE MODELS, YOU GET MASSIVE
ELECTROLYTE IMBALANCES, PROBABLY
LIKE HYPER KULEMUA, OTHER
ASSOCIATED FACTORS, WE DO SEE A
SURVIVAL BENEFIT.
>> I'VE SEEN ONES LOOKING AT THE
RECOVERY RATE OF LIKE IF YOU
HAVE ACUTE NECROSIS OF THE
KIDNEY FIT GOES THROUGH
APOPTOSIS IF THEY PREVENTED PARA
APOPTOSIS KIDNEY CELLS WERE ABLE
TO RECOVER FAST IR, HAVE YOU
LOOKED AT YOUR MOUSE MODEL, IF
THEY ARE ABLE TO RECOVER FASTER
FROM LIVER INJURY, FROM --
>> NO, WE REALLY HAVEN'T DONE
ANYTHING MORE THAN THE LIVER
MODELS.
I THINK IT'S A VERY INTERESTING
QUESTION.
WE'VE BEEN -- WE'VE GONE TO THE
RAT.
WE'VE MADE A KNOCKOUT OF NINJ1
IN THE RAT BECAUSE THAT'S
PROBABLY BETTER FOR ISCHEMIA
REPERFUSION.
THEY ARE ONGOING EXPERIMENTS.
I THINK THE RAT IS A BETTER
MODEL SO WE WENT THE EXTRA MILE
THERE FOR THAT.
>> AND THE LAST QUESTION, DOES
IT ALSO -- IS THIS SOMETHING YOU
COULD USE FOR RED BLOOD CELLS,
FOR DEFICIENCY, LYSIS OF RED
BLOOD CELLS?
>> I HAVE TO CONFESS I DON'T
KNOW.
YEAH.
>> THANK YOU.
>> IT'S A GOOD QUESTION.
YOU SHOULD COME TO GENENTECH.
>> WONDERFUL TALK, DR. DIXIT.
TWO QUESTIONS BASICALLY.
DO YOU THINK THAT THERE'S ANY
ASSOCIATION BETWEEN LIKE
ACTIVATION OF THE NINJ WITH
ACUTE INFLAMMATION?
>> I THINK IT'S ACUTE INJURY.
I THINK YOU HAVE TO OVERWHELM
THE EFFEROCYTOSIS MECHANISMS SO
DYING CELLS LICE.
>> ACTIVITY OF NINJ1, DO YOU
THINK THAT?
>> WELL, IN ACUTE INJURY, DYING
CELLS ARE NOT TAKEN UP THEY WILL
GO VERY RAPID NINJ1 MEDIATED
LYSIS.
I THINK THE QUESTION WE'RE
STRUGGLING WITH IS WHY DO YOU
GET FROM MONOMER TO POLYMER IN
DYING CELLS.
>> THE OTHER QUESTION, CISPLATIN
ASSOCIATED WITH NINJ1-ASSOCIATED
DEATH, SO AS WE KNOW THAT
CISPLATIN IS TOXIC TOWARDS THE
CELLS, TOXICITY IS A MAJOR CAUSE
FOR NINJ1 ACTIVATION OR NOT?
>> NO, I DON'T THINK IT WILL
PROTECT FROM CISPLATIN TOXICITY,
WE HAVEN'T DONE THOSE STUDIES
OURSELVES.
REMEMBER WITH NINJ1 THE CELLS
ARE DEAD.
RIGHT?
WHAT WE'RE PREVENTING IS THEIR
ACUTE LYSIS AND SUBSEQUENT
INFLAMMATORY RESPONSE.
>> OKAY.
THANK YOU.
>> I'LL FOLLOW UP SINCE -- YEAH,
I WAS WONDERING ABOUT
COMPENSATORY MECHANISMS IN
ABSENCE OF NINJ1, SURPRISED HOW
SOMETHING SO CONSERVED WHEN
DELETED IN THE MOUSE STILL
DOESN'T SEEM TO HAVE A MEASURE.
>> YEAH.
>> SO, YEAH --
>> I THINK TWO POSSIBILITIES
THERE.
ONE IS THAT NORMALLY DYING CELLS
ARE TAKEN UP BY EFFEROCYTOSIS,
THERE'S NO DEFECT IN THE ABSENCE
OF NINJ1 SO IT'S ONLY WHEN YOU
STRESS THE MODEL WITH
OVERWHELMING INJURY YOU SEE A
PHENOTYPE.
THE OTHER POSSIBILITY, RELATED,
I DIDN'T GO INTO NINJ2 IN
MAMMALS.
SO THAT'S A WHOLE BAG OF WORMS
THAT I DIDN'T WANT TO OPEN UP.
SO WHAT THAT'S DOING, WHETHER
THERE'S REDUNDANCY WITH THAT, IS
A POSSIBILITY.
BUT I PERSONALLY FAVOR THAT
EFFEROCYTOSIS MECHANISMS ARE SO
EFFICIENT UNLESS YOU HAVE
OVERWHELMING INJURY, DYING CELLS
ACCUMULATE YOU'RE NOT GOING TO
SEE A NINJ1 PHENOTYPE.
>> I GUESS DURING LIKE THE AGING
PROCESS, THESE PROCESSES, WOULD
ACCUMULATE.
>> YEAH.
>> SO WHETHER THE AGED -- YOU
KNOW, WHETHER AGED MICE BEGIN TO
SEE GREAT -- YOU KNOW, THERE'S
GOING TO BE MORE DAMAGE.
>> YEAH, NO, IT'S A GOOD -- I
THINK WE'RE AGING A COHORT OF
MICE SO WE'LL SEE.
>> OKAY.
ONE MORE.
SO, HAVE YOU HAD THE CHANCE TO
LOOK FOR EXAMPLE AT ALZHEIMER'S
DISEASE WHERE YOU HAVE LIKE
ACCUMULATION OF PHAGOCYTIC
CELLS, HAVE A MAJOR ROLE, AND
THEY WOULD BE SOMEHOW
OVERWHELMED, OR DO YOU SEE SOME
CELLS BEING MORE RESISTANT TO
THIS MECHANISM?
>> TO THE NINJ1?
>> UH-HUH.
>> NO, NO, WE HAVEN'T LOOKED.
YOU KNOW, THIS IS WONDERFUL.
THIS IS A NEW DISCOVERY.
THIS IS WHY I ENJOY SCIENCE.
IT'S LIKE NEW NINJ1, RIGHT?
IT'S NOT LIKE I'M STUDYING MYC.
[LAUGHTER]
YOU KNOW.
IT'S NEW!
I DON'T KNOW!
I WILL MAKE THE REAGENTS
AVAILABLE.
EVERYBODY SHOULD -- JUST LIKE
WITH GASDERMIN-D IN 2015, IT
WAS.
NEW WE MADE THE REAGENTS
AVAILABLE.
IT'S NEW!
I DON'T KNOW.
THERE ARE HUNDREDS OF QUESTIONS
YOU COULD ASK.
MORE PEOPLE, BETTER.
AND REMEMBER WE MAKE REAGENTS
AVAILABLE.
>> I THINK I'M GOING TO ASK ALSO
A QUESTION THAT YOU MAY NOT
KNOW.
BUT, YOU KNOW, YOU HAVE BEEN
SAYING THAT INHIBITING NINJ1
INHIBITS STERILE INFLAMMATION
BUT THE SAME TIME YOU SHOWED US
ACTUALLY GASDERMIN IS THERE AND
RELEASING EVERYTHING THAT YOU'VE
MEASURED.
RIGHT?
>> YES.
>> SO --
>> YES, SO THE GASDERMIN
REMEMBER HAS A MOLECULAR WEIGHT
CUTOFF.
>> YEAH, SO BASICALLY YOU'RE
SAYING THAT THIS, YOU KNOW, AT
THE SAME TIME AT LEAST THE DATA
THAT YOU HAVE SHOWN DON'T
REALLY -- SO YOU'RE SAYING THAT,
YES, THERE ARE THESE LARGER
MOLECULES THAT MUST BE RELEASED
FOR THE ENTIRE INFLAMMATORY
CASCADE TO REALLY HAPPEN, RIGHT?
>> YEAH, YOU KNOW, I THINK YOU
MAKE A VERY GOOD POINT.
I THINK THAT WITH THE
GASDERMIN-D INJURY, I NEVER
THOUGHT I WOULD SAY THAT BUT I
THINK THAT IS POTENTIALLY
REVERSIBLE.
SO THERE'S SOME DATA IN THE
LITERATURE, AND OUR OWN DATA, WE
MADE HUNDREDS OF ANTIBODIES TO
GASDERMIN-D, MANY THOUSANDS,
SHEEP-LOOKING CREATURES, LLAMAS
OR SOMETHING.
AND THEN THE TECHNICIAN CAME IN
MY LAB, FAIRLY SENIOR SCIENTIST,
I WONDER IF ANY INHIBIT
GASDERMIN-D FUNCTION?
THAT'S A REALLY, REALLY BAD
IDEA, BECAUSE GASDERMIN-D'S
INSIDE THE CELLS, AND IT'S
GETTING ACTIVATED.
YOU'RE ADDING THE ANTIBODY
OUTSIDE THE CELLS, AND WE HAVE A
MEMBRANE.
NOT GOING TO WORK.
DON'T DO EXPERIMENT.
SHE DID IT ANYWAY.
[ LAUGHTER ]
AND THERE IS A NANOBODY, SERIES
OF NANOBODIES THAT WE'VE
DISCOVERED BUT ALSO BEEN
REPORTED BY OTHERS, THAT WILL
ACTUALLY REVERSE THAT INJURY.
SO THERE IS A CAPACITY.
SO WHAT THAT SAYS, FOR MEMBRANE
DAMAGE YOU HAVE TO EXCEED A
THRESHOLD FOR IT TO BECOME
IRREVERSIBLE, RIGHT?
I DON'T THINK THERE'S ANY GOING
BACK FROM NINJ1 THOUGH.
ONCE THAT'S ACTIVATED, KABOOM.
>> THANK YOU.
>> A PHILOSOPHICAL --
>> THANK YOU FOR THE TALK.
MAYBE THE LAST TWO.
ACTUALLY VERY BEGINNING OF THE
NINJ1 FINDINGS, IN ACTIVATION,
REQUIRED.
AND THEN SO SEEMS LIKE THERE'S
WHY ACTIVATED CANNOT BE SITUATED
THROUGH THE NINJ1 1
(INDISCERNIBLE).
>> YEAH, I WOULD SAY THAT IF YOU
DON'T HAVE GASDERMIN-D, YOU'RE
NOT GOING TO RELEASE IL-1 BETA.
BUT EVENTUALLY THE CELL WILL
LYSE AND YOU'LL RELEASE IL-1
BETA.
SINCE YOU'VE NOT ACTIVATED OR IF
YOU HAVEN'T ACTIVATED THE
CASPASES, IT WILL BE UNPROCESSED
IL-1 BETA.
IF YOU'VE ACTIVATED CASPASES
YOU'LL RELEASE WITH GREATLY
DELAYED PAN KINETICS,
INDEPENDENT MANNER, BUT I DON'T
THINK THAT'S PHYSIOLOGICALLY
RELEVANT BECAUSE OF ALL THE
STUDIES WE'VE DONE IN VIVO
CHALLENGES.
SO AS YOU SAW THAT WE'RE ABLE TO
COMPLETELY SUPPRESS IL-1 BETA
RELEASE IN A GASDERMIN-D
KNOCKOUT.
>> OKAY.
THANK YOU.
[APPLAUSE]
[END OF PROGRAM]