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>> GOOD AFTERNOON, EVERYBODY.
WELCOME TO THE WEDNESDAY
AFTERNOON LECTURE SERIES.
MY NAME IS BOB COLBERT.
IT'S A DISTINCT PLEASURE FOR ME
TO BE ABLE TO INTRODUCE TODAY'S
SPEAKER, WHO IS JENNY TING.
DR. KING IS THE WILLIAM KEENAN
PROFESSOR OF MICROBIOLOGY AND
IMMUNOLOGY AT THE UNIVERSITY OF
NORTH CAROLINA AT CHAPEL HILL,
WHERE SHE DIRECTS THE CENTER FOR
TRANSLATIONAL IMMUNOLOGY AND IS
CO-DIRECTOR OF THE INFLAMMATORY
DISEASES INSTITUTE ALSO AT UNC.
I FIRST GOT TO KNOW JENNY ABOUT
ALMOST 25 YEARS AGO, WHEN WE
COTAUGHT AN IMMUNOLOGY LAB FOR
MEDICAL STUDENTS AT THE
UNIVERSITY OF NORTH CAROLINA.
YOU CAN TELL IT'S A LONG TIME
AGO BECAUSE I SAID IMMUNOLOGY
LAB AND MEDICAL STUDENTS IN THE
SAME SENTENCE.
SO WE ALSO SHARE A LITTLE BIT OF
A COMMON ACADEMIC ANCESTRY
BECAUSE WE BOTH DID POSTDOCTORAL
WORK WITH JEFF FRELENGER,
ALTHOUGH AT DIFFERENT TIMES.
LET ME SAY A FEW THINGS ABOUT
JENNY AND SOME OF THE MAJOR
CONTRIBUTIONS THAT SHE'S MADE TO
IMMUNOLOGY.
SHE HAS A VERY LONG TRACK RECORD
OF REALLY OUTSTANDING WORK
CONTRIBUTING TO OUR OVERALL
UNDERSTANDING OF IMMUNE
REGULATION.
IN THE EARLY 90s, SHE
DISCOVERED HOW C2TA, WHICH IS
THE MHC CLASS 2 TRANSACTIVATOR
KNOWN TO BE MUTATED IN CAUSATIVE
BARE LYMPHOCYTE SYSTEM, HOW THIS
MEDIATES CYTOKINE CLASS
2 EXPRESSION WHICH IS CRITICALLY
IMPORTANT FOR ADAPTIVE IMMUNITY.
I THINK WHAT'S REALLY
INTERESTING IS IT WAS HER
INTEREST IN C2TA WHICH LED TO --
AND THE RECOGNITION THAT MUTANTS
IN PROTEINS THAT HAD A SIMILAR
DOMAIN STRUCTURE ACTUALLY CAUSED
INFLAMMATORY DISORDERS THAT LED
HER TO LOOK FOR GENES WITH
SIMILAR DOMAIN STRUCTURE AND
THAT RESULTED IN THE DISCOVERY
OF A LARGE MULTI-GENE FAMILY, I
THINK 22 MEMBERS TO DATE AT
LEAST IN HUMANS, THAT WE NOW
KNOW AS THE NOD-LIKE RECEPTORS
OR GENES THAT ENCODE THE
NOD-LIKE RECEPTORS OR NLRs.
SO MANY NLRs ARE INTERCELLULAR
PATTERN RECOGNITION RECEPTORS
INVOLVED IN INITIATING AND/OR
REGULATING THE INNATE IMMUNE
RESPONSE TO DANGER SIGNALS,
CREATED DURING CELLULAR INJURY
OR TOXINS OR INNOVATION BY
MICROORGANISMS.
AND OF COURSE WE KNOW THAT
MUTATIONS IN VARIOUS NLRs,
SUCH AS NLR P3 CAUSE A VARIETY
OF SYSTEMIC INFLAMMATORY
DISEASES SUCH AS THE PERIODIC
SYNDROMES OR CAPs.
SO WHILE MANY OF THESE DISEASES
ARE RELATIVELY RARE, UNLESS YOU
WORK AT THE NIH, THEY REALLY CAN
BE QUITE DEVASTATING DISEASES,
AND THEY REALLY HIGHLIGHT THE
IMPORTANCE OF THESE GENES TO
FUNDAMENTAL BIOLOGICAL AND
IMMUNOLOGICAL PROCESSES.
THIS HAS ALL BECOME INCREASINGLY
APPARENT OVER THE LAST DECADE
AND IT SEEMS TO BE GROWING
ALMOST ON A DAILY BASIS.
SO WHAT WE'RE GOING TO HEAR
ABOUT TODAY IS HOW NLRs
REGULATE DIVERSE CELL DEATH
RESPONSES SUCH AS AP PTOSIS,
NECROSIS AND AUTOPHAGY AS WELL
AS DIVERSE SIGNALING PATHWAYS
INCLUDING PATHWAYS INVOLVING
NF -- SO DR. TING WILL PRESENT
EVIDENCE FOR THE BROAD
BIOLOGICAL AND CLINICAL IMPACT
OF THE NLR FAMILY OF GENES.
SO JENNY, WELCOME, THANK YOU FOR
COMING, AND I WANT TO MENTION
THAT THERE WILL BE A RECEPTION
IMMEDIATELY FOLLOWING FOLLOWING
HER TALK IN THE LIBRARY.
>> I JUST WANT TO THANK YOU, SO
MUCH, BOB FOR INVITING ME AND
THANK ALL THE PEOPLE FOR COMING
DESPITE THE WEATHER REPORTS.
SO WHAT I'D LIKE TO TELL YOU
TODAY IS SOME OF THE THINGS THAT
WE'VE BEEN DOING RECENTLY, AND I
JUST WANT TO SAY THAT I'VE KNOWN
BOB FOR 25 YEARS AND AT THAT
TIME, I WAS ABOUT 10 YEARS OLD
AND BOB WAS ABOUT 6.
SO WE'RE NOT AS OLD AS WE LOOK.
SO I'D JUST LIKE TO GIVE A BIG
PICTURE BECAUSE I KNOW THIS
MIGHT BE A MORE HETEROGENEOUS
GROUP AND GIVE YOU A BACKGROUND
ABOUT THIS FIELD, THE NLR FIELD,
AND JUST TO PUT THINGS INTO
PERSPECTIVE, THE INNATE IMMUNE
SENSORS OR RECEPTOR FIELD HAS
REALLY EXPLODED IN THE LAST 15
YEARS.
THE FIRST ONE, OF COURSE -- WE
NOW KNOW THERE ARE RNA SENSORS,
THERE ARE A BUNCH OF DNA SENSORS
LISTED HERE AND THIS IS WHERE
OUR PROTEIN BELONGS, OUR FAMILY
OF PROTEINS BELONG.
THESE HAVE AT LEAST TWO NAMES.
ONE IS THE NAME.
-- TO BE HONEST,
I ACTUALLY PREFER THE MBDLR,
WHICH IS THE NAME THAT 22 OF US
PROPOSED YEARS AGO, AND THE
REASON IS BECAUSE WE REALLY
DON'T KNOW HOW THESE FUNCTION.
THIS IS ONE OF THE LEADING
ISSUES THAT I WILL DISCUSS IN
THIS TALK.
AND SO WE DON'T KNOW IF THESE
ARE TRULY RECEPTORS, THEY MIGHT
BE OR MIGHT NOT BE.
BUT THEY IMPACT A LOT OF THINGS
INCLUDING INFLAMMATION,
INFECTIOUS DISEASES, CANCER, ET
CETERA.
THE WAY WE CAME INTO THIS, BOTH
OF US HAVE WORKED A LONG TIME AS
COLLEAGUES AND FRIENDS, THIS
MOLECULE,CIITA, IT WAS
DISCOVERED -- MOST OF US WHO ARE
STUDYING MHC JUMPED ON IT WHEN
THIS PROTEIN WAS DISCOVERED
BECAUSE IT IS THE PROTEIN THAT'S
MUTATED IN A DISEASE THAT CANNOT
EXPRESS -- THIS IS A CLASS II
TRANSACTIVATOR.
WE FOUND THAT IT HAS THE
FUNCTIONAL ATP BINDING DOMAIN
CALLED -- A NUCLEOTIDE BINDING
DOMAIN, AND LEUCINE RICH REPEATS
AS WELL.
SO BY COMBINING THESE TWO, WE
WORKED FOR A LONG TIME ON CIITA,
AND AT THE END, I'LL COME BACK
TO THAT PROTEIN, BUT IT'S A
TRANSCRIPTION COACTIVATOR.
SO IT RECOGNIZES DNA THROUGH DNA
BINDING PROTEINS, AND THEN
ACTIVATES TRANSDESCRIPTION.
BUT TWO PEOPLE IN MY LAB DECIDED
THIS IS SUCH AN IMPORTANT
PROTEIN BECAUSE YOU CAN POP IT
ON ANY CELL AND IT WILL
EXPRESS -- THEY USE THESE TWO
DOMAINS AND SEARCH THE HUMAN
GENOME BEFORE IT WAS PUBLISHED
AND CAME UP WITH 22 GENES.
THEN WE NAMED IT CATERPILLAR FOR
A LONG REASON I WON'T TELL YOU
ABOUT IT, BUT NOW IT'S RENAMED
EITHER MBDLR OR NOD-LIKE
RECEPTORS.
FROM THAT, THE 22 GENES, THAT'S
IT, THERE'S NO MORE.
IF YOU SEARCH AND SEARCH, YOU
CANNOT FIND ANYMORE, THAT'S THE
END OF THE STORY.
I ALWAYS JOKE WITH MY TLR
COLLEAGUES, TLR, THERE'S
TRICKLES OF TLRs THAT CAME
OUT, THERE'S ONLY THESE MANY
NLRs.
SO ONE OF THE REASONS WE'RE SO
INTERESTED IN THIS FAMILY IS
BECAUSE THEY'RE LINKED TO HUMAN
DISEASES, FOR EXAMPLE, CI.
TA IS LINKED TO THE BARE
LYMPHOSITE SYNDROME, PEOPLE NAME
THIS AS BARE LYMPHOCYTE.
THE WORK OF A NUMBER OF PEOPLE
HAVE SHOWN THAT THIS IS ONE OF
THE MOST IMPORTANT GENETIC
LINKAGE TO CROHN'S DISEASE AS
WELL AS THIS REUM TOE LOGIC
DISEASE.
NLRP1 IS ANOTHER ONE THAT I'LL
MENTION FOR A LITTLE BIT.
THIS IS GOING TO A SKIN DISEASE
AS WELL AS A VARIETY OF
AUTOIMMUNE DISEASES, AND OF
COURSE THE BIG ONE, NRP3, THAT
I'LL TELL YOU MORE ABOUT IN JUST
A LITTLE BIT.
I JUST WANT TO SUMMARIZE BECAUSE
MOST PEOPLE ONLY THINK ABOUT THE
INFLAMMASOME, AND THAT'S
ACTUALLY NOT THE CASE.
I WILL EXPLAIN IT TO YOU IN JUST
A LITTLE BIT.
SO WE STARTED, BECAUSE CIITA IS
A TRANSCRIPTION COACTIVATOR THAT
LEADS TO CLASS II MHC GENE
REGULATION, THE MAJOR
HISTOCOMPATIBILITY COMPLEX.
AND MORE RECENTLY, IT WAS FOUND
THAT NLRC5 IS THE MASTER
REGULATOR OF CLASS I MHC GEEP
AND MOSTLY LYMPHOCYTES.
SO THESE TWO ARE TRANSCRIPTIONAL
REGULATORS, AND THIS IS AGAIN
THE REASON CAN I PREFE I PREFER THE TERM
MBDLR BECAUSE IT DOESN'T LOOK
LIKE THESE TWO ARE TYPICAL --
RECEPTORS SO THEY'RE REALLY NOT
CONSIDERED AS -- RECEPTORS.
I THINK THIS IS VERY IMPORTANT
WHAT WE CALL THINGS, BECAUSE AS
WE CALL SOMETHING A SPECIFIC
TERM, THEN WE HAVE PRECONCEIVED
NOTION ABOUT WHAT THEY SHOULD
DO.
BUT IN THE FIELD OF NLR, I THINK
WE'RE STILL STRUGGLING IN TERMS
OF TRYING TO UNDERSTAND WHAT
THEY ALL DO.
THE OTHER ONE OF COURSE IS
INFLAMMASOME, AND I WILL TELL
YOU MORE ABOUT THIS, BUT
BASICALLY THIS IS A COMPLEX
FORMATION THAT LEADS TO THE
PROCESSING OF IL 1 AND IL 18
FROM THEIR PREMATURE FORM INTO
THEIR MATURE FORM, DUE TO
ACTIVATION OF -- 1.
NOW WE HAVE AT LEAST NINE NLRs
THAT HAVE EXHIBITED THIS
FUNCTION IN AT LEAST ONE PAPER.
SO THERE ARE SEVEN OF THESE --
I'M SORRY -- NINE OF THESE.
THEN THE GROUP THAT I'LL
CONCENTRATE ON THE MOST AT THE
LAST PART OF THE TALK IS
REGULATORS OF NFKB OR
INTERFERON, AND I'LL FOCUS MY
TALK ON THE INTERFERON SIGNATURE
THAT'S RELATED TO THESE NLR
PROTEINS.
OF COURSE INTERFERON IS REALLY
IMPORTANT FOR A LOT OF
INFLAMMATORY DISEASES AND
GENETIC DISEASES, AND TODAY I
HEARD MORE AND MORE ABOUT THAT
AND THESE ARE SEL -- THERE ARE
SEVERAL NLRs IN THAT PATHWAY.
ANOTHER ONE I WISH I HAD MORE
TIME TO TELL YOU ABOUT, MANY OF
THESE NLRs ARE ALSO PROCESS OF
CELL DEATH, NECROSIS, AUTOPHAGY,
AND REMEMBER THAT NLR ACTUALLY
ARE FOUND FROM PLANTS TO HUMANS,
AND THEY'RE PRESERVED ALL THE
WAY IN PLANTS, BUT THEY ALSO
LOOK LIKE A APOPTIC -- WHICH
MAKES SENSE IN TERMS OF THE ROLE
IN CELL DEATH.
SO THIS IS THE OVERALL PICTURE
OF THIS ENTIRE FAMILY OF
PROTEINS.
I WANT TO CHECK THROUGH ALL OF
THE FUNCTIONS AND SHOW YOU SOME
EVIDENCE OF WHAT THEY DO SO YOU
CAN HAVE A BIG PICTURE OF THIS
ENTIRE FAMILY OF PROTEINS.
THE FIRST ONE IS HOW WE CAME TO
THIS, AND I SHOULD SAY BY THE
TIME WE CAME TO ALL OF THE
GENES, THERE WERE AT LEAST FIVE
OF THESE PROTEINS THAT WERE
ALREADY FOUND BY SOMEBODY ELSE.
THAT INCLUDES CIITA, AND OF
COURSE WE AND OTHERS HAVE DONE A
LOT OF WORK ON, BUT THE LAST
PROTEIN WHICH FUNCTION WAS
ELUCIDATED WAS NLRC5.
MANY OF US HAVE DONE A LOT OF
EXPERIMENTS TO TRY TO FIGURE OUT
WHAT DOES THIS PROTEIN DO, AND A
YOUNG FACULTY WHO IS NOW AT
HARVARD WAS THE FIRST PERSON WHO
HAD A CLUE ABOUT WHAT IT MIGHT
BE DOING, AND MAY BE DOING OTHER
THINGS BUT THE MOST PROMINENT
FUNCTION IS IT REGULATES CLASS 1
MHD.
IT LOOKS LIKE IT ALSO BINDS
TO -- IT CAN BE FOUND BY CHIP
ASSAYS TO BE LOCATED TO THE
CLASS 1 MHC PROMOTER.
SO WE MADE A KNOCKOUT, AND WE
LOOK AT CLASS 1 IMAGE GENE
INCLUDING HT-K, BAY TO 2M, TLA,
AND EVERY -- IF YOU KNOCK OUT
NLRC5, THERE'S VERY LITTLE CLASS
I EXPRESSION ON B CELLS, T CELLS
OR MK CELLS.
THIS IS TRUE FOR H26789-K, B2M,
AS WELL AS TLA.
HOWEVER, IT HAS NO FUNCTION
ON -- AS SHOWN HERE, NOR DOES IT
HAVE ANY EFFECT ON CD1, WHICH IS
NOT ENCODED BY THE MHC DOMAIN.
SO THIS SHOWS THIS MAY BE A
MASTER REGULATOR OF THESE THREE
GENES, AND OTHER PEOPLE HAVE
SIMILAR DATA AND JUST TO SHOW
YOU THAT WE MADE A KNOCKOUT OF
C2TA A LONG TIME AGO AND
NLRC5 RIGHT HERE AND
NLRC5 KNOCKOUT MOUSE, REDUCED
CLASS #, THERE'S NO REDUCTION OF
CLASS 1, AND IF YOU LOOK OVER
HERE, HOWEVER, THERE'S C2TA
KNOCKOUTS HAVE REDUCED CLASS 2,
IN FACT CLASS 2 IS ALMOST GONE,
BUT NLRC5 KNOCKOUT MICE HAVE NO
REDUCTION OF CLASS 2.
SO IT'S RECIPROCAL IN THAT THESE
TWO PROTEINS REGULATE CLASS
1 AND CLASS 2 RECIPROCALLY.
SO WHEN YOU THINK ABOUT HOW A
PROTEIN WANTS TO REGULATE
ADAPTIVE IMMUNE RESPONSE, I
CAN'T THINK OF A BETTER PATHWAY
FOR THEM TO REGULATE, WHICH IS
BOTH CLASS 1 AND CLASS 2.
SO THIS CHANGES THE ENTIRE
LANDSCAPE FOR THE ADAPTIVE
IMMUNE RESPONSE.
SO I'VE COVERED THIS PRETTY
QUICKLY, THEN THE INFLAMMASOME.
OF COURSE THIS HAS RECEIVED A
TON OF ATTENTION, BUT I AGAIN
WANT TO EMPHASIZE INFLAM SOAPS
ARE JUST A SUBGROUP OF NLRs.
THEY'RE NLRs THAT DON'T HAVE
ANY INFLAMMASOME FUNCTION.
THE REASON WE CAME TO THIS IS
THE WORK OF SEVERAL
RHEUMATOLOGISTS, DAN CASS NER
AND HIS COLLEAGUE AND RAFAEL
MASSTEE, OF COURSE, ARE REALLY
GIANTS IN THE FIELD.
HOFFMAN WAS THE FIRST TO
RECOGNIZE THE CONNECTION OF THIS
FAMILY OF DISEASES WITH THIS
GENE CALLED NLR P 3, BECAUSE IT
WAS FOUND IN PATIENTS WHO HAVE
AN INFLAMMATORY RESPONSE TO A
DROP IN TEMPERATURE, SO HOW
HOFFMAN CALLED IT THE --
PROTEIN.
IN EUROPE, MICHAEL MCDERMOTT WAS
REALLY DOING MANY OF THE SAME
THINGS IN THE TRANSLATIONAL
IMMUNOLOGY.
SO IT WAS FOUND MUTATIONS IN
THIS PROTEIN THAT INITIALLY WAS
CALLED CRYOPIE REASON, WE SAW IT
IN A LOT OF SKIN RASHES,
STIFFNESS, THERE'S THREE
DIFFERENT DISEASES THAT ARE
LINKED TO THIS WITH A GRADATION
OF SEVERITY, AND THERE'S MORE
THAN 80 MUTATIONS NOW, I THINK,
IN THIS GENE THAT'S RELATED TO
INFLAMMATORY RESPONSE.
ONE OF THE TRIUMPHS OF THE FIELD
IS THAT THEY FOUND OUT IL-1 IS
INCREASED, YOU CAN TREAT THESE
PATIENTS WITH THE IL 1 RECEPTOR
ANTAGONIST AND WITHIN A DAY,
THEY ALL GOT BETTER.
SO THAT'S REALLY A REMARKABLE
TRIUMPH OF WHAT THESE GROUPS
HAVE DONE INCLUDING, OF COURSE,
THE GIANTS OF THE FIELD AT NIH.
SO I JUST WANT TO GIVE YOU A
LITTLE BIT OF BACKGROUND ON WHAT
THE INFLAMMASOME ACTIVATION
PROCESS IS.
WE NOW KNOW THAT WHAT YOU REALLY
WANT AT THE END IS PROCESSING OF
PRO IL 1 AND PRO IL 18 INTO
THEIR MATURE FORM.
WE HAVE TWO SIGNALS THAT ARE
REQUIRED IN MANY CELL TYPES, BUT
NOT ALL CELL TYPES.
SOME CELL TYPES ALREADY HAVE
SIGNAL 1 THAT'S ACTIVATED.
SO THESE SIGNALS CAN BE TLRs
OR TNF RECEPTOR.
ALAN SHEER HAS BEAUTIFUL WORK TO
SHOW THAT CARD 9 IS ALSO VERY
IMPORTANT FOR THE FIRST STEP OF
INDUCING PRO IL 1 PRODUCTION AND
WE'VE SHOWED A LONG TIME AGO
THAT LPS INDUCES NLRP3 INDUCTION
AND IL 18, SO YOU HAVE TO HAVE
THESE TO BE TRANSCRIPTIONALLY
INDUCED AND TRANSLATIONALLY
INDUCED, BUT THIS IS NOT ENOUGH.
YOU NEED SIGNAL 2, AND SIGNAL
2 I ALWAYS JOKE WITH THE PEOPLE
IN THE INFLAMMASOME FIELD,
BASICALLY YOU LOOK AT THE CELLS
WRONG AND THEY HAVE SIGNAL 2.
SO THIS COULD BE VERY EARLY AND
PROPHETIC WORK OF POLYAT NIH
THAT THERE'S DAMAGE ASSOCIATED
OR DANGER ASSOCIATED MOLECULAR
PATTERNS AND MANY OTHER TOXINS,
MAPS PROTEIN, ET CETERA, CAN
INDUCE A NUMBER OF FACTORS, WE
DON'T KNOW HOW COMMON ALL OF
THESE ARE.
THERE'S DEBATES OF HOW EACH ONE
IS IMPORTANT AND SOME WOULD
ARGUE THAT -- THE MOST IMPORTANT
FACTOR, OTHERS HAVE ALMOST
SEEN -- UPSTREAM OF INFLAMMASOME
ACTIVATION CALCIUM MITOCHONDRIAL
DNA RELEASE, ET CETERA, AND YOU
HAVE NEGATIVE REGULATORS AS
WELL.
AND TEASE REALLY WHAT THEY'RE
DOING IS LEADING TO THE COMPLEX
FORMATION OF THIS INFLAMMASOME
COMPLEX.
COMPRISING OF NLR PROTEIN
INDICATED HERE AS NLRP3, ADAPTOR
MOLECULE -- AND WHAT ALL THESE
THINGS DO IS -- THE ASSEMBLY OF
THIS INTO A MULTI-MERIC COMPLEX
SUCH AS THIS.
IT'S PREDICTED TO HAVE ABOUT SIX
OR SEVEN COPIES OF EACH ONE OF
THESE BASED ON -- STUDIES.
THIS COMPLEX THEN LEADS TO THE
PROXIMAL ACTIVATION -- WHICH
THEN CLEAVES IL 1 BETA AND IL 18
TO THE MATURE FORM.
THIS WHOLE CONCEPT IS THE
INFLAMMASOME.
AND AGAIN, FOR THOSE OF YOU WHO
WORK WITH HUMAN CELLS, I JUST
WANT TO SAY HUMAN MO MONO CYTES DO
NOT -- THEIR SPECIFICITY,
ALTHOUGH SOME HAVE VERY BROAD
SPECIFICITY, SOME HAVE VERY
REFINED, FOR EXAMPLE, NLR P 3,
LIKE I SAID, MANY THINGS CAN
ACTIVATE THIS.
NLRP1 IS HIGHLY SPECIFIC,
ESPECIALLY IN RODENTS, SO
ANTHRAX IS VERY IMPORTANT AND
THAT'S, AGAIN, BASED ON MUCH OF
THE WORK DONE HERE AT NIH, AND
MORE RECENTLY IT SOUND LIKE --
THAT THERE'S THE ALTERNATIVE --
INFLAMMASOME THAT ACTUALLY
ACTIVATES -- 11 AND -- SO LPS
CAN ACTIVATE THIS.
WE DON'T KNOW ALL THE PATHWAYS
THAT LEADS TO THIS.
AND THERE'S ALSO DSS COLITIS CAN
ACTIVATE NLRP6.
AGAIN, IT'S NOT ENTIRELY CLEAR
WHAT ACTIVATES THIS.
THERE'S PROBABLY MICROBIOME
CHANGES.
AND FINALLY, IN ADDITION TO NLR
PROTEINS, THERE ARE OTHER IMFLAN
SOMES SUCH AS -- 2 CAN BE
ACTIVATED BY DNA, CRYSTALLIZED
BY THE SHELL LAB HERE AT NIH.
SO OBVIOUSLY NIH HAS DONE A HUGE
AMOUNT OF WORK TO REALLY PROMOTE
THIS FIELD.
SO THE REASON MANY OF THESE
IMMUNE SENSORS OR RECEPTORS WERE
DISCOVERED WAS THE THINKING THAT
THESE ARE RELATED TOIN FEKTIOUS
DISEASES.
AND THAT IS INDEED THE CASE.
SO I JUST WANT TO SHOW YOU SOME
OF THE WORK WE DID KNOWING THAT
THIS IS THE SEA OF WORK THAT
OTHER PEOPLE HAVE CONTRIBUTED
TO, AND SO YEARS AGO, WE WANTED
TO KNOW IF THIS IS IMPORTANT FOR
INFECTION AND THEY PICK
INFLUENZA, THIS IS 2009, OF
COURSE, THE H1N1 FLU WAS GOING
CRAZY AT THAT TIME AND OF COURSE
OUR WORK WAS DONE WAY BEFORE THE
H1N1 FLU CAME BUT WE WERE
PUBLISHED IN THIS YEAR.
BEFORE WE SHOWED IT HAS A
DRAMATIC EFFECT IN COUNTERING
THE FLU INFECTION, SO THIS IS
NRP3 KNOCKOUT MICE.
THEY DON'T DO VERY WELL, THEY
DON'T SURVIVE VERY WELL.
WILD TYPE, THEY DO OKAY IN THIS
SYSTEM.
THEN WE LOOK AT VIRAL TITERS,
KNOCKOUTS HAVE MUCH HIGHER --
ABOUT A 2 OFF DIFFERENCE, SO WE
THINK THIS AN IMPORTANT PROTEIN
TO COMBAT INFLUENZA INFECTIONS.
THE LAB OF -- PUBLISHED A SIDE
BY SIDE PAPER.
BASED ON THAT, WE'RE NOT THE
FIRST ONE TO PROPOSE THIS BUT
MANY PEOPLE HAVE NOTICED THAT
MANY OF THESE STEPS LEADING TO
IL 1 ARE TARGETED BY BOTH
VIRUSES AS WELL AS BY BACTERIA,
SO CERTAINLY WE'VE WORKED A LOT
OF -- SHOWED MANY STEPS CAN
INHIBIT THIS -- 1 ACTIVATION.
WHAT WE DIDN'T SHOW, HOWEVER, IS
THAT THERE MAY BE A VIRAL
PROTEIN THAT CAN DIRECTLY
INFLUENCE INFLAMMASOME
ACTIVATION.
SO IN COLLABORATION ABOUT TWO OR
THREE YEARS AGO WITH THE
LABORATORY OF -- WE LOOKED AT
THIS VIRUS WHICH CAN CAUSE
CANCER, AND WE WERE LOOKING
THROUGH THIS GENOME AND WE WERE
ABLE TO SHOW THAT THE VIRAL
GENOME OF THIS VERY LARGE ***
VIRUS HAS A VIRAL NLR ENCODED BY
OPEN READING FRAME 63 THAT HAS
MBDNLR DOMAIN, AND IT MOST
LOOKED LIKE -- SO THE FACT
THERE'S A VIRAL NLR MADE US
WONDER IF THIS COULD INHIBIT
INFLAMMASOME ACTIVATION, AND
THAT IS WHAT THIS PAPER SHOWS,
AND I JUST WANT TO SHOW YOU THE
TWO FIGURES OUT OF A LARGE
NUMBER OF EXPERIMENTS IN THAT IF
WE OVEREXPRESS THIS PROTEIN,
THIS IS A HUMAN DISEASE ONLY, WE
CAN SHOW THAT OVEREXPRESSION OF
THIS PROTEIN REDUCES IL
1 PRODUCTION.
IF WE HAVE OVEREXPRESSION OF
THIS PROTEIN INDICATED HERE, WE
WERE ABLE TO SHOW THAT INSTEAD
OF GETTING PRO IL 1 PROCESS INTO
IL 1, WE WERE ABLE TO BLOCK THIS
WITH THE SARCOMA -- LIKE
MOLECULE.
THE MECHANISM TURNS OUT THAT
THIS VIRAL NLR CAN BLOCK THE
ASSEMBLY OF THE INFLAMMASOME
COMPLEX THAT I INDICATED TO YOU
EARLIER.
SO -- WHAT WE HAVE DONE AND MANY
OTHERS LOOKED AT BACTERIAL
DISEASES, VIRAL DISEASES, FUNGAL
DISEASES, ET CETERA, IN THE ROLE
OF INFLAM SEWS.
APPRECIATED THAT THE ROLE OF
THESE PROTEINS ARE NOT JUST
LIMITED TO INFECTIOUS DISEASES,
AND THEY HAVE A LARGE ROLE IN
MANY OTHER FUNCTIONS, AND I'LL
JUST LIST SOME OF THE ONES THAT
WE HAVE CONTRIBUTED TO AND THE
FIRST ONE IS THIS ROLE IN
ADABTIVE IMMUNITY, THE SECOND
ONE IS JUST GENERAL INFLAMMATORY
DISORDERS AND THIRDLY, THIS IS A
HUGE GROUP OF DISEASES THAT WE
ALL BECAME MUCH MORE AWARE OF
BECAUSE OF THE PUBLIC HEALTH
HAZARD THAT IS METABOLIC
DISEASES AND COLITIS AND COLITIS
ASSOCIATED COLON CANCER.
I FOR GOAT TO MENTION THE
INFLAMMASOME -- THE CONCEPT IS
REALLY, WOFT LATE -- WHO DID
MANY OF THE WORK THAT LEADS --
OTHER PEOPLE HAVE ALSO FOUND
THESE PROTEINS AND OTHER
DISEASES SUCH AS MACULAR
DEGENERATION, OTHER NEUROLOGICAL
DISEASES, TOXICITY, YOU NAME IT.
SO WHEN I THINK ABOUT THIS,
INFECTIOUS DISEASE, ADAPTIVE
IMMUNITY, INFLAMMATION,
METABOLIC DISEASES AND CANCER,
THAT PRETTY MUCH COVERS A LOT OF
THE PROBLEMS THAT WE ARE
ENCOUNTERS RIGHT NOW.
AND JUST TO GO THROUGH SOME OF
THESE DISCOVERIES THAT WE HAVE
FOUND IS WE LOOK AT ADAPTIVE
IMMUNITY THINKING WE SHOULD
INFLUENCE ADAPTIVE IMMUNITY
BECAUSE IL 1 HAS A PROFOUND
EFFECT ON TH17, SO THIS IS JUST
ONE OF THE TYPICAL SLIDES THAT
WE HAVE DONE.
WE LOOK AT NLRP3, INNATE IMMUNE
MOLECULE, INCREASED DURING --
AND IF WE HAVE A KNOCKOUT MOUSE,
KNOCKOUTS HAVE MUCH REDUCED --
INDICATED HERE, SO THIS HAS A
REALLY STRONG EFFECT ON ADAPTIVE
IMMUNITY, THEN WE ALSO LOOKED AT
AN INFLAMMATORY DISEASE THAT HAS
NOTHING TO DO WITH THE T CELL
SYSTEM OR ACTUALLY MANY OF THE
THINGS WE THINK ABOUT IN TERMS
OF THE -- BUT IT DOES RESULT
IN -- MICRO GLIA ARE ALL THE RED
CELLS, AND IN THE KNOCKOUT,
THERE'S VERY LITTLE INFLAMMATORY
RESPONSE IN THIS MODEL SYSTEM.
THE BOTTOM PART, WE WERE LOOKING
AT THE LEFT HEMISPHERE AND THE
RIGHT HEMISPHERE, AND THIS IS
MAYBE -- THEY DECIDED NOT ONLY
SHOULD WE LOOK AT THE LEFT
HEMISPHERE, BECAUSE -- BUT WE
SHOULD ALSO LOOK AT THE RIGHT
HEMISPHERE, SO WE OBLIGE BECAUSE
IT'S SUCH AN EASY EXPERIMENT,
AND THERE'S NO DIFFERENCE
BETWEEN THE TWO.
SO THIS OF COURSE SHOWS THAT IT
HAS VERY IMPORTANT ROLE IN MANY
GENERAL INFORMATION INCLUDING
LUNG, CNS, SKIN AND BONE, AND
THEN SELF OF US ALSO STARTED
LOOKING AT METABOLIC DISEASES.
-- THE FIRST ONE TO LOOK AT
GOUT, THE FIRST ONE TO LOOK AT
ARTHRITIS, AND MANY OF US LOOKED
AT DIABETES, AND THIS IS OUR
CONTRIBUTION AND WE'RE STILL
LOOKING MORE ON THIS WHICH IS
THE IDEA THAT SATURATED FATTY
ACID IS NOT GOOD FOR YOU, BUT
COULD INDUCE THE INFLAMMASOME.
WE KNOW THAT MANY OF THE OBESITY
RELATED DISEASE ARE LINKED TO --
HAVE AN INFLAMMATORY COMPONENT,
SO WE USED -- ACID AND BECAUSE
IT'S INSOLUBLE, WE HAD TO LINK
IT -- TO GET IT SOLUBLIZED, AND
IF WE HAD SIGNAL 1 AND 2, WE
WERE ABLE TO PRODUCE IL
1 PRODUCTION.
SO SATURATED FATTY ACID ALSO
GOES TO THIS -- AND WE SHOW THAT
THIS IS THROUGH THE
NLRP3 PATHWAY BECAUSE IF WE HAVE
A KNOCKOUT CELL, WE HAVE VERY
LITTLE IL 1 PRODUCTION.
THIS WAS PUBLISHED MORE THAN TWO
YEARS AGO.
TO MAKE A VERY LONG STORY SHORT,
WE WERE ABLE TO SHOW THAT PAL
MEDIC ACID TURNS OFF -- AND THIS
IN TURN TURNS OFF AUTOPHAGY, AND
MANY LINK AUTOPHAGY AS A
NEGATIVE -- OF THIS
INFLAMMASOME.
SO THESE TWO, WHEN THEY'RE
RELIEVED, THIS TURNS ON
INFLAMMASOME WHICH MAKES IL
1 WHICH THEN GOES TO INSULIN
TARGETING TISSUES SUCH AS LIVER,
ADIPOSE TISSUES, MUSCLE CELLS,
TO INHIBIT THE INFLUENCE
SENSITIVITY PATHWAYS, SO THIS IS
WHAT WE HAVE FOUND.
OTHER PEOPLE HAVE FOUND MANY
OTHER THINGS SUCH AS -- CRYSTAL
ASSOCIATED WITH ATHEROSCLEROSIS,
MEURK ACID ASSOCIATED WITH GOUT,
BETA AM LIED PROTEIN ASSOCIATED
WITH ALZHEIMER'S, CAN ACTIVATE
THE INFLAMMASOME PATHWAYS.
MANY OF US HAVE GONE IN VIVO TO
SHOW THAT WHEN YOU DON'T HAVE
THE INFLAMMASOME PATHWAY --
WHICH IS WHY MANY COMPANIES ARE
TARGETING THIS FOR METABOLIC
DISEASES.
SO I'VE QUICKLY GONE THROUGH
THESE THREE AND I'LL JUST GO
QUICKLY TO THE CANCER FIELD, AND
THIS IS SOME STUDIES WE DID
LOOKING AT ITS RELEVANCE IN
COLITIS ASSOCIATED CANCER MODEL,
AND WE HAVE FOUND THAT ACTUALLY
INFLAMMASOME IN CONTRAST, ONE
CAN ANTICIPATE, WHEN YOU DON'T
HAVE THE INFLAMMASOME, YOU HAVE
INCREASED -- FORMATION IN THIS
MODEL SYSTEM.
SO ACTUALLY THE INFLAMMASOME IN
THE G.I. IS PROTECTIVE, AND
THAT'S BEEN FOUND IN MANY CASES
THAT THE TLR MOLECULES AND NLR
IN THE COLON PATHWAY ARE
PROTECTED BECAUSE THEY'RE TO
PROVIDE A HOMEOSTATIC PATHWAY.
SO INSTEAD OF INFLAMMATION BEING
BAD IN THIS CASE, INNATE IMMUNE
RECEPTORS ARE PROTECTED.
-- REALLY PROVIDED SOME OF THE
VERY IMPORTANT MECHANISMS AS TO
WHY THIS CAN BE DONE, AND WHAT
HE SHOWED IS DOWNSTREAM OF THE
INFLAMMASOME, OF COURSE, THERE'S
IL 1 AND IL 18, AND IL 1 DOESN'T
HAVE ANY EFFECT ON POLYP FOR
MAIG, BUT IL 18 HAS A PROFOUND
ROLE IN THAT IF YOU TAKE WAY IL
18, POLYPS ACTUALLY GO UP IN THE
COLON.
THIS IS DUE BECAUSE IL 18 CAN
REDUCE IL 6 WHICH IS PRO
INFLAMMATORY CYTOKINE, IL 18 CAN
ALSO DOWNREGULATE STAT3, WHICH
IS VERY IMPORTANT FOR COLON
CANCER, AND IL 18 CAN REPAIR THE
EPITHELIUM IN THE EXPERIMENTAL
COLITIS AND OTHER GROUPS HAVE
ALSO FOUND THIS.
SO THIS IS JUST SHOWING THAT
INFLAMMASOME HAS AT LEAST TWO
TARGETS, BETA AND IL 18 AND THEY
MAY HAVE DIVERGENT ROLES IN
DIFFERENT TISSUES. SO I'VE
QUICKLY GONE THROUGH THIS VERY
HUGE FIELD OF INFLAMMASOMES, AND
NOW I JUST WANT TO TOUCH ON THIS
AND SHOW YOU SOME UNPUBLISHED
WORK THAT WE HAVE BEEN WORKING
ON IN TERMS OF THE NLR PROTEINS,
AND ANOTHER VERY IMPORTANT
PATHWAY, INTERFERON.
SO AGAIN, IN THE FIRST DISCOVERY
OF NOD 1 AND NOD 2, SEVERAL OF
THE EARLY WORK OF NOD 1 AND NOD
2 SHOW THEY'RE RELATED TO --
HAVE POINTED TO THIS, AND SO WE
WERE INTERESTED TO SEE IF THERE
ARE OTHER NLRs THAT CAN
REGULATE NFKB, ADDITIONALLY WE
HAVE FOUND A LINK TO INTERFERON,
AND I'LL JUST TELL YOU SOME OF
THE WORK THAT WE HAVE DONE IN
THIS.
WHAT'S SURPRISING IS WE FOUND
MOST OF THESE CASES, THESE
PROTEINS ARE NEGATIVE
INHIBITORS, ARE INHIBITORS OF
THE INTERFERON PATHWAY.
SO -- OR NF COMPANY KB PATHWAY.
THE FIRST ONE WE FOUND IS ONE WE
ISOLATED IN EARLY 2000, AND THIS
ONE WAS NAMED DIFFERENT THINGS
INCLUDING MONARCH, NOW IT'S
NAMED NLRP12.
WE HAVE A PAPER THAT CAME OUT
ABOUT A YEAR OR TWO AGO SHOWING
NLRP12 CAN REGULATE THE NFKB
PATHWAY.
NOW THERE ARE AT LEAST FOUR
PAPERS INDICATING THIS IS THE
CASE, AND THESE ARE SHOWN HERE.
IN OUR MODEL SYSTEM, WE LOOK AT
A COLON CANCER COLITIS MODEL AND
FOUND THE NLRP12 NEGATIVELY
REGULATES NICS, WHICH IS THE
INDUCING KINASE WHICH CAUSES --
AND NLRP12, JUST TO SHOW YOU AN
OVEREXPRESSION SYSTEM, THIS IS A
VERY OLD PAPER, REDUCES THE
AMOUNT OF NIC.
I WON'T GET INTO ALL OF THE
MECHANISMS BUT BECAUSE OF THIS,
IT DOWNREGULATES -- PATHWAY AND
INFLUENCES NON-COULD NON-CAL
PATHWAY HEMOKIEN WHICH ARE NOW
ALL FOUND TO BE REALLY IMPORTANT
FOR SOLID TUMORS.
A SECOND NLR THAT WE FOUND TO BE
NEGATIVELY REGULATING -- I WON'T
HAVE TOO MUCH TIME TO TALK TO
YOU ABOUT THIS EXCEPT TO SAY
THIS IS A VERY INTERESTING
MITOCHONDRIAL NLR, WE FOUND IT
TO BE A NEGATIVE REGULATOR --
AND INTERFERON, AND IN OUR
HANDS, WE FOUND THIS INTERFERES
WITH THIS PROTEIN CALLED
MITOCHONDRIA ANTIVIRAL SIGNALING
PROTEIN MAP, AND THEREFORE,
REDUCING BOTH INTERFERON AS WELL
AS THESE I INFLAMMATORY CYTOKINES.
THE LABORATORY OF -- WONG IN OUR
LAB ALSO SHOWED THIS IS A
NEGATIVE REGULATOR OF THE -- IT
REDUCES LPS SIGNALS.
MACROPHAGES ACTUALLY DOESN'T
REDUCE INTERFERON.
OTHER PEOPLE HAVE SHOWN IT'S A
POSITIVE REGULATOR OF AUTOPHAGY.
NLRX1 BINDS TO THIS MASS PROTEIN
WHICH IS REALLY IMPORTANT FOR
TNF IL 6 AND INTERFERON, AND
THIS IS ALL OCCURRING IN THE
MITOCHONDRIAL.
NOW, MORE RECENTLY, THE
LABORATORY OF -- WAIT, I DON'T
HAVE THAT YET.
ANOTHER LAB HAS SHOWN -- HAD
SHOWN THAT NOD 2 CAN ALSO
INTERACT WITH MAP SO THIS CAN BE
A PRETTY COMMON ASSOCIATION OF
NLRs WITH MAVS.
MORE RECENTLY THE LABORATORY OF
ROGER MAIN HAS SHOWN THAT MAVS
INTERACTS WITH NLRT3 TO IP -- IN
OUR CASE WE USE ENDOGENOUS -- TO
SHOW THIS INTERACTION.
SO THESE CELLS SHOW THAT THESE
NLRs -- SO WE WERE BE LOOKING
AT ANOTHER MOLECULE CALLED
NLRC3 AND THIS IS THE ONE I'LL
SPEND MOST OF THE TIME ON.
THIS IS A PROTEIN WITH A CARD
DOMAIN ENCODED ON HUMAN ON
CHROMOSOME 16 AND WE SHOWED IN A
NATURE IMMUNOLOGY PAPER
PUBLISHED OVER A YEAR AGO THAT
IF WE OVEREXPRESS NLRC3, WE HAVE
A REDUCTION OF TNF AS WELL AS IL
6 RESPONSE IN RESPONSE TO -- SO
WE THINK THERE'S A LINK BETWEEN
THE TLR PATHWAY AND NFT NLR
PATHWAY.
IF WE KNOCK DOWN -- WE SEE
INCREASED TRANSCRIPTION OF THESE
GENES.
SUGGESTING THAT IT IS AN
INHIBITOR OF TRANSDESCRIPTION OF
THESE GENES.
SO THEN THE MECHANISM WE TRIED
TO LOOK AT, WE NARROWED IT DOWN
TO A TRAP AND WE FOUND NLRC3 CAN
ENTER -- DURING LPS TREATMENT
AND THIS IS SHOWN HERE, WHICH WE
SHOWED THAT NLRC3 INTERACTS WITH
TRAP.
IF WE MUTATE A TRAP FINDING
SITE, WE KNOCK DOWN THIS
INTERACTION.
THROUGH MANY DIFFERENT STUDIES,
WE WERE ABLE TO SHOW THAT
NLRC3 KNOCKOUT MICE CELLS,
THERE'S A REDUCTION IN THE
ACTIVATING E UBIQUINATION OF TRAP
6, ACTIVATED BY THE
K63 UBIQUINATION OF TRAP 6.
IN NLR KNOCKOUT MICE, THERE'S
INCREASED -- SO THIS WOULD LEAD
TO TRAP 6 ACTIVATION.
SO TO SUMMARIZE, WE THINK WHAT
HAPPENS WITH NLRC3 INTERACTS
WITH -- TO PREVENT IT NORMALLY
FROM BEING ACTIVATED BY
UBIQUINATION.
IF WE REMOVE NLRC3, WE NOW HAVE
ENHANCED K63 UBIQUINATION.
SO DOES THIS DO ANYTHING IN VIVO
IN RESPONSE TO LPS?
WE SHOWED THAT NLRC3 KNOCKOUT
MICE DO HAVE INCREASED TNF,
INCREASED IL 6 IN RESPONSE TO
LPS TREATMENT.
SO THIS JUST INDICATES THAT THIS
MOLECULE HAS AN IB HIB TRI ROLE
IN INFLAMMATORY RESPONSE.
SO I'LL LEAD YOU NOW TO A SECOND
FUNCTION THAT WE HAVE RECENTLY
FOUND, AND I JUST WANT TO
PRESS -- MANY NLRs ACTUALLY
HAVE MULTIPLE FUNCTION.
FOR EXAMPLE, NLRP1 IS KNOWN TO
BE REALLY IMPORTANT IN APOP
PTOSIS, ALSO IMPORTANT IN
INFLAMMASOMES AND ALSO IMPORTANT
IN HEMO POE HE EE CYST.
SO IT'S KNOWN TO BE IMPORTANT
FOR ALL THESE FUNCTIONS AND WE
REALLY DON'T KNOW HOW WE CAN TIE
ALL OF THESE THREE TOGETHER.
SO IT'S NOT SURPRISING THAT
NLRC3 HAS DIFFERENT FUNCTIONS
DEPENDING ON WHAT YOU'RE LOOKING
AT, SO ONE OF THE STUDENTS IN MY
LAB REALLY WANTED TO SEE IF SHE
COULD FIND INTERFERE ON CHANGES,
SO WE SCREENED A NUMBER OF NLR
KNOCKOUTS THAT WE MADE AND WHAT
SHE FOUND WAS THIS, SHOWING
NLRC3 REDUCES DNA-INDUCED
INTERFERON RESPONSE.
THIS IS SHOWN HERE.
SHE USED POLLLY DADTs, WHICH
IS AN ANALOG OF DOUBLE STRANDED
DNA, AND USED IT EXTRA
CELLULARLY, WHICH WOULD BE
ACTIVATING THROUGH EXTRA
CELLULAR MAIN -- RECEPTORS OR
PUT IT INTERCELLULARLY IN
SENSORS OR RECEPTORS.
I SHOULD POINT OUT THAT ALL
NLRs ARE CONSIDERED
INTRACELLULAR RECEPTORS OR
SENSORS.
WHILE TLRs ARE CONSIDERED
AS -- SO SHE FOUND THAT IF SHE
PUT DOUBLE STRANDED DNA ANALOG
INTO CELLS, THAT THE WILD TYPE
HAS A PRETTY GOOD RESPONSE IN
TERMS OF INTERFERON BUT THE
KNOCKOUT HAS AN EVEN MORE
PROFOUND RESPONSE WHEN IT COMES
TO INTERFERON.
THIS IS JUST SHOWING THE SAME
PATTERN THAT INTRACELLULAR
DOUBLE STRANDED DNA SUGGESTING
DNA INVOLVED IN DNA SENSING BUT
AS A NEGATIVE MOLECULE REGULATOR
OF DNA SENSING, SO THE KNOCKOUTS
HAVE A HIGHER RESPONSE TO
INTRACELLULAR DNA.
SO SINCE INTRACELLULAR DNA IS A
ME ME TICK ESSENTIALLY OF DNA
VIRUSES, WE WANTED TO SEE IF
THIS HAS ANY FUNCTION -- ***
SIMPLEX VIRUS ONE, AND WE WERE
ABLE TO INDUCE INTERFERON
INDUCTION AND THE KNOCKOUTS HAVE
HIGHER LEVELS OF INTERFERON
AGAIN.
AND WE WERE ABLE TO SHOW THIS.
NOW IN OUR PREVIOUS PAPER WITH
LPS, WE SHOW THAT THAT INVOLVED
TRAF 6, SO WE WANTED TO SEE IF
THIS WAS -- WE DID A KNOCKOUT
STUDY PROVIDED BY -- AND WE WERE
ABLE TO SHOW THAT TRAF
6 KNOCKOUT CELLS DO NOT SHOW ANY
DIFFERENCE IN TERMS OF
INTERFERON REDUCTION, SO WE
THINK WITH HSC RESPONSE, THIS IS
SEPARATE FROM THE TRAF
6 PATHWAY.
SO WE THINK THIS IS A DIFFERENT
PATHWAY THAN THE TRAF 6 THAT I
WAS TELLING YOU ABOUT.
SO WE LOOKED AT MORE CLUES IN
TERMS OF WHAT THE PATHWAY MIGHT
BE, AND MOSTLY THE LABORATORY
OF -- AND HIS COLLEAGUES AS WELL
AS OTHERS HAVE FOUND THAT SIGH
CLICK -- CAN IMDUES TYPE
1 INTERFERON WHEN IT'S PRODUCED
BY BACTERIA.
AND THE BACTERIA THAT PRODUCES
THIS INCLUDES WISTERIA.
SO WE WANTED TO SEE IF NLRC3 CAN
REGULATE -- INDUCED TYPE
1 INTERFERON.
THIS IS SHOWN HERE, SO AGAIN,
WILD TYPE MAF -- THIS IS WHAT
THE -- IN EVERY CASE, THE
NLRC3 KNOCKOUTS PRODUCED MORE
INTERFERON.
SO AGAIN, SUGGESTING IT'S AN
INHIBITOR OF THESE PATHWAYS.
IT ALSO DID THIS WITH THE WHOLE
BACTERIA, WISTERIA.
SO AT THIS POINT, THESE TWO
CLUES WERE ENOUGH TO MAKE US
THINK ABOUT A SPECIFIC PATHWAY
THAT HAS GAINED A LOT OF
ATTENTION, ESPECIALLY LAST YEAR,
AND THAT'S THE STING PATHWAY.
THERE'S MANY STUDIES TRYING TO
FIGURE OUT WHAT ARE THE PATHWAYS
IMPORTANT FOR DNA SENSING BY
INTRACELLULAR MOLECULES.
THE STING HAS BECOME THE MAJOR
CENTRAL ADAPTER OF MANY OF THESE
DIFFERENT PATHWAYS.
SO DNA CAN FEED IN, FOR EXAMPLE,
INTO IFI16 OR P200, THIS PIECE
IN THE STING PATHWAY, DNA CAN BE
CONVERTED BY -- 3, ACTIVATE --
GO TO THE STING PATHWAY, BUT ONE
OF THE THINGS ABOUT STING IS IT
ACTIVATES -- NFKB TO INDUCE
INTERFEAR ON. SO THIS IS A --
SENSING MOLECULE BECAUSE IT'S
BEEN CRYSTALLIZED AND FOUND TO
BIND TO -- BUT ALSO THE
LABORATORY GLEN BARBER HAS FOUND
THAT THIS CAN BIEND DIRECTL BIND DIRECTLY TO
DNA.
SO THIS CAN EITHER SENSE DNA
OR --, ACTIVATING PDK.
SO WE WANTED TO SEE IF NLRC3 CAN
BE PLACED IN THIS PATHWAY.
SO AGAIN WE HAVE STING HERE, WE
WANTED TO SEE IF IF
OVEREXPRESSED NLRC3 CAN REDUCE
INTERFERON PROMOTER APPLICATION
BY STING, AND IT DID, AND BY --
AND IT DID AND BECAUSE THE STALE
IS SO LOW, THIS IS PROBABLY
ABOUT 20 FOLD,
IRF3 AND NLRC3 HAVE NO EFFECT ON
IRF3.
THAT'S BECAUSE IT ACTS OVER HERE
AND SO IF YOU HAVE
IR3 ACTIVATION, IT'S TOO HIGH
AND CANNOT AFFECT DOWNSTREAM --
SO THIS WHERE WE'RE PLACING NLRC
P 3 RIGHT UPSTREAM AND THE SING
TBK PATHWAY, THESE TWO.
THEN WE WANTED TO KNOW, HOW DOES
NLRC3 INHIBIT STING AND TBK,
DOES IT DIRECTLY BIND TO THESE
TWO?
SO WE STARTED WITH
OVEREXPRESSION SYSTEM, BY
OVEREXPRESSING NLRC3 AND LOOKING
AT ITS INTERACTION WITH STING
AND WE WERE ABLE TO SEE PRETTY
STRONG INTERACTION WITH STING,
AS WELL AS SOME INTERACTION WITH
TBK RIGHT OVER HERE.
THEN WE WANTED TO KNOW IF THIS
COULD HAPPEN IN A HEMIDODGE NUSS
PATHWAY BECAUSE THIS WAS ALL
OVEREXPRESSED SO WE WANTED TO
SEE IF THIS COULD HAPPEN WITH
ENDOGENOUS STING THAT'S NOT
OVEREXPRESSED, AND WE WERE NOT
ABLE TO SHOW THAT OVEREXPRESSED
NLRC3 WOULD -- THEY
COPRECIPITATE WITH EACH OTHER.
BUT NLRC3 CAN ALSO PRECIPITATE
WITH ENDOGENOUS -- 1.
WE DID A LOT OF MAPPING, THIS
PAPER IS NOT PUBLISHED BUT --
AND WE WERE ABLE TO SHOW THAT
THE FRAGMENT THAT IT INTERACTS
WITH AND STING AND -- ARE
EXACTLY THE SAME DOMAIN, THAT
THESE TWO MOLECULES USE TO
INTERACT WITH ONE ANOTHER.
SO THE PROPOSAL IS NLRC3 CAN
INTERFERE WITH INTERACTIONS OF
STING AND TBK.
SO THEN THE REVIEWERS ASKED US,
CAN WE SHOW THIS WITH PURIFIED
PROTEIN, AND AS ALL YOU HAVE
KNOW, PURIFIED NLRs ARE A
NIGHTMARE, BUT WE WERE VERY
HAPPY THAT WE HAVE A COLLEAGUE
WHO CAN PRODUCE THESE PRO DEANS,
AND WE WERE ABLE TO GET PURIFIED
NLRC3 AND WE USED TWO DIFFERENT
STING, USED FOR CRYSTALLIZATION
BY THE -- LAB AND WE WERE ABLE
TO SHOW THAT STING AND
NLRP3 PURIFIED CAN INTERACT WITH
EACH OTHER RIGHT OVER HERE, AND
THE REVERSE DIRECTION, WE WERE
ABLE TO SHOW THAT IF WE
PRECIPITATE NLRC3, IT'S ALSO
COPRECIPITATED STING.
SO I THINK THIS PRETTY CLEARLY
SHOWS HA STING CAN INTERACT WITH
NLRC3.
THEN THE QUESTION IS, WHAT IS
THE FUNCTIONAL CONSEQUENCE OF
THIS.
WE KNOW ONE OF THE FUNCTIONAL
CONSEQUENCES OF STING
ACTIVATION, TPK IS THE --
KINASE -- AND IF YOU ACTIVATE
STING, YOU CAN SEE --
PHOSPHORYLATION INDICATED HERE,
SO THIS IS WILD TYPE CELLS,
ACTIVATED WITH DOUBLE STRANDED
DNA, AND TBK IS NOT THAT MUCH
PHOSPHORYLATED UNTIL SIX HOURS
AFTER TREATMENT, IF WE LOOK AT
NLRC3 KNOCKOUT CELLS, WE SEE
INCREASED TBK PHOSPHORYLATION
VERY EARLY ON, SO THERE'S A
KINETIC SWITCH TO A VERY EARLY
TIME POINT OF TBK
PHOSPHORYLATION.
THEN WE WANT TO LOOK AT A SECOND
FUNCTION OF STING WHEN --
CONTAINING CELLS ARE TREATED
WITH DOUBLE STRANDED RNA, TWO
THINGS HAPPEN.
FIRST OF ALL, STING ASSUMES A
VERY UNIQUE LOCALIZATION AS
SHOWN BY GLEN BARBER AND THAT
BECOMES PERINUCLEAR OR HAS A
PUNCTATED APPEARANCE.
IF WE OVEREXPRESS NLR3, THE
PERINUCLEAR APPEARANCE ALMOST
DISAPPEARED FROM 10% ABOUT TO
1%, AND WE CANNOT FIND ANY CELLS
WITH THIS PUNCTATED APPEARANCE.
SO IT CHANGED THE LOCALIZATION
OF STING.
SO IT CHANGED TBK WHICH IS
DOWNSTREAM OF STING, IT ALSO
CHANGED THE LOCALIZATION OF
STING FROM WHERE IT SHOULD BE
ASSOCIATED WITH ACTIVATION TO A
NON-ACTIVATION FORM.
THE THIRD WAY WE LOOKED AT THIS
IS CAN WE CHANGE THE
COLOCALIZATION OF TBK INDICATED
HERE, THIS IS TBK, AND THE
SECOND TWO ROLES ARE STING,
THEY'RE COLOCALIZATION, SO WE
ARE LOOKING AT DIFFERENT
FRACTIONS ON AN FPLC COLUMN FROM
WILD TYPE CELLS AND
NLRC3 KNOCKOUT CELLS, LOOKING AT
COFRACTIONATION -- VERSUS STING,
AND YOU CAN SEE THE WILD TYPE,
WE CAN SEE IT UP TO HERE, BUT
THERE'S N MORE STING COLOCALLIZED
IN NON-TREATED CELLS -- BUT IF
WE TREAT THESE CELLS -- INFECT
THESE CELLS WITH HSV, THERE'S A
MORE DRAMATIC COLOCALIZATION OF
STING IN THE KNOCKOUT CELLS, AND
THIS IS TBK, THIS IS STING, SO
THERE'S MORE COLOCALIZATION OF
THESE BANDS IN THE KNOCKOUT
CELLS THAN IN THE WILD TYPE
CELLS.
TO QUANTITATE THIS, WE
QUANTITATED IT BASED ON THE
FIRST FRACTION AND DID IT AS A
RATIO OF THE FIRST FRACTION, AND
IT'S PROBABLY CLEAREST HERE,
WHICH IS THE HSB TREATED CELLS,
AN THESE ARE LOOKING AT
FRACTIONS THAT CONTAIN BOTH TBK
AND STING -- IN THE KNOCKOUT, WE
HAVE A LOT MORE FRACTIONS THAT
CONTAINS FRACTIONS WITH BOTH TBK
AND STING.
SO THIS IS USING A BIOCHEMICAL
APPROACH KIND OF CONFIRMING THAT
WHEN YOU DON'T HAVE NLRC3, STING
AND TBK BECAME MORE COLOCALLIZED
TO THE SAME BIOCHEMICAL
FRACTION.
THEN WE WANTED TO BIOLOGICALLY
ASK IF NLRC3 IS A NEGATIVE -- IS
AN INHIBITOR OF STING, WE WOULD
EXPECT IN NLRC3 KNOCKOUT, WE
WOULD HAVE MORE STING ACTIVATION
AND THEREFORE MORE ANTIVIRAL
RESPONSES AND WE SHOULD SEE LESS
VIRUSES.
THAT IS INDEED WHAT WE SAW IN
VITRO AND CELL CULTURE, IF YOU
INFECT NLRC3 KNOCKOUTS WITH HSB,
WE SAW LESS HSB GENOMIC COPY AS
INDICATED HERE.
WE DON'T SEE AN EFFECT OF
NLRC3 IN POLY -- BUT WE ACTUALLY
LATER NOW REDUCED THE EFFECT ON
SOME VIRAL RNA VIRUSES, WE STEL
DON'T UNDERSTAND HOW THAT IS
ACHIEVED BUT IT'S MUCH MORE
PREFERENTIAL FOR -- BUT IN VIVO,
WE WENT ALL THE WAY FROM
OVEREXPRESSION TO ENDOGENOUS
NLRC3 KNOCKOUT TO BIOCHEMISTRY,
TO FUNCTION OF STING, NOW WE
WANT TO SEE AN OVERALL MOUSE,
WHAT HAPPENS WHEN WE INFECT
THESE MICE WITH HSB1.
WHAT WE EXPECT AGAIN IS NLRC3 IS
AN INHIBITOR OF THE STING
PATHWAY, WE WOULD EXPECT IF WE
KNOCK OUT NLRC3, WE WOULD HAVE
MORE OF A STING FUNCTION AND
THEREFORE MORE OF AN ANTIVIRAL
EFFECT, SO WE WOULD EXPECT THESE
NLRC3 MICE TO DO BETTER WHEN
THEY'RE INFECTED WITH THE HSB1,
AND INDEED THEY ALL DID BETTER,
AND THE WILD TYPE MICE DID VERY
POORLY IN THAT -- IN
INFORECASTTION THAT'S I.V., WE
SAW MUCH LESS SURVIVAL BECAUSE
THESE MICE HAVE A DRAMATIC DROP
IN WEIGHT.
OF OVER 20%.
IN THESE KNOCKOUT MICE, WE DO
SEE ENHANCED INTERFERON, WE SEE
ENHANCED IL 6, ENHANCED TLF AND
LOWER GENOMIC COPY.
SO IN OUR PAROLES A PROPOSAL, WE WOULD SAY
NLRC3 WAS THERE TO NATURALLY
PREVENT STING FROM ACTIVATING
WHEN THERE'S NO VIRUS, BUT THE
QUESTION IS, HOW DOES THIS BREAK
BECOME -- WE HAVEN'T DONE A
WHOLE LOT, BUT WHAT WE SAW IS
THAT IF YOU HAVE DNA OR DNA
INFECTION, WITH DNA VIRUSES,
THERE'S A DROP OF
NLRC3 EXPRESSION.
I COULDN'T MAKE MY STUDENT COME
IN AFTER SIX HOURS TO 16 BECAUSE
THAT'S I THINK AFTER 12 HOURS
WORKING IN THE LAB, BUT WE DON'T
KNOW WHAT HAPPENS IN BETWEEN,
BUT THERE'S A SIGNIFICANT DROP
OF THIS EXPRESSION.
SO IN OUR MODEL SYSTEM, WE WOULD
PROPOSE AS FOLLOWS THAT IF IN A
STUDY STATE, NLRC3 CAN INTERACT
WITH STING BECAUSE WE SAW THIS
INTERACTION WITHOUT DOING
ANYTHING.
SO THIS PREVENTS STING FROM
ACTIVATING TB K1 FROM
ASSOCIATING WITH TB K1 AND
ACTIVATING -- HOWEVER, WHEN YOU
HAVE A VIRAL IB FEKTION,
NLRC3 SE REMOVED, WE WOULD
SUGGEST ONE OF THE WAY IT IS
REMOVED, ITS EXPRESSION WENT
DOWN AND THIS ALLOWS THEN THE
STING MOLECULE TO INTERACT WITH
TBK.
THIS IS A POSSIBLE WAY IT DOES
THIS, AND THEN STING INTERACTS
WITH TBK AND ACTIVATES THE
ENTIRE PATHWAY.
SO WE THEN COME TO THE OVERALL
PICTURE THAT IF WE HAVE NLR
PROTEINS THAT CAN REGULATE
TRANSCRIPTION ACTIVATION OF
CLASS 1 AND CLASS 2, WE HAVE NLR
PROTEINS THAT ACT AS
INFLAMMASOME, WE HAVE NLR
PROTEINS THAT CHANGE INTERFERON
IN MY TALK, AND ALSO THERE ARE
SOME THAT CHANGES IN NFKB AND SO
FORTH, HOW DO WE HAVE AN OVERALL
PICTURE OF THIS?
I'M NOT SURE I CAN INTRODUCE AN
OVERALL PICTURE BUT I CAN THINK
OF SOME CONSENSUS IN TERMS OF
WHAT THIS MIGHT BE DOING.
SO I COME TO -- WE KNOW -- BASED
ON THE WORK OF MANY THAT THE C
T2 A PROMOTER IS OF COURSE THE
DNA, IT BINDS TO DNA BINDING
PROTEINS AND C2TA IS FOUND AT
THE PROMOTER BY CHROMATIN I.P.,
SO CHT IS VERY GOOD AT
RECOGNIZING DNA, WHETHER IT'S
PROTEIN COMPLEX OR NOT ENTIRELY
CLEAR, BUT IT CERTAINLY CAN
RECOGNIZE DNA.
OUR PREVIOUS WORK WITH
NLRX1 SUGGESTS THAT THIS
MOLECULE CAN INTERFERE WITH THIS
PATHWAY, WHICH IS RNA SENSING BY
RIG-I, WHICH BINDS TO RNA
MOLECULE, SO THIS MOLECULE CAN
ALSO BE PART OF THE RNA SENSING
PATHWAY.
WE NOW HAVE EVIDENCE THAT
NLRC3 CAN REGULATE THIS DNA
SENSING PATHWAY -- SO IN MANY OF
THESE CASES, WE'RE SUGGESTING
NLR MAY BE CONNECTED TO DNA AND
RNA AND -- WE CALL THIS DNA AND
RNA SENSING, IN TRANSCRIPTION,
WE CALL THIS TRANSCRIPTIONAL
ACTIVATION, AND I SHOULD JUST
POINT OUT THERE ARE PAPERS
SHOWING THAT NLRP3 CAN RECOGNIZE
MOI TOE CON DREEL DNA, SO COMING
TO A FULL PICTURE, WHETHER
THAT'S SOMETHING WE SHOULD ALL
BE THINKING ABOUT, THAT IS MAYBE
THERE IS AN UNDERLYING RULE AND
IT'S CONNECTING NLRs WITH --
ACID.
SO I JUST WANT TO THEN COME BACK
AND THANK ALL THE PEOPLE WHO
WERE INVOLVED IN THE NLR FIELD
IN MY LAB.
THESE ARE COLLABORATORS I IN
HOUSE.
MAKING KNOCKOUTS IN THE VERY
EARLY DAYS, ALEX AND HIS --
PROVIDED MANY OF THE REAGENTS
THAT I MENTIONED.
I JUST WANT TO THANK YOU VERY
MUCH FOR YOUR ATTENTION.
[APPLAUSE]
>> HEY, JENNY, THAT WAS GREAT.
I WASN'T REVIEWER NUMBER THREE
WHO COMMENTED ON THE EXPRESSION,
BUT I DID HAVE A QUESTION --
>> YOU'RE REVIEWER NUMBER TWO.
>> I WAS THE GOOD REVIEWER.
NO.
REGARDING TISSUE SPECIFIC
EXPRESSION I NOTICED IT'S HIGH
IN --
>> AND D CELLS.
>> OKAY.
SO DO YOU IMAGINE THAT'S
SPECIFICALLY THERE TO MIMIC
PRODUCTION OF TYPE 1 INTERFERONS
IN THOSE CELLS --
>> I DON'T KNOW IF IT'S TYPE
1 INTERFERON.
WE'RE OBVIOUSLY WORKING ON
THOSE, AND THAT'S BEEN THE BANE
OF OUR EXISTENCE, TRYING TO FIND
A FUNCTION FOR THOSE.
IT'S NOT VERY SIMPLE.
IF YOU JUST TAKE NLRC3 KNOCKOUT
MOUSE AND JUST TREAT THEM WITH
WHATEVER T CELL ACTIVATORS OR --
YOU SEE NOTHING, THERE'S NO
DIFFERENCE.
YOU HAVE TO REALLY, REALLY DIG,
AND WE DUG AND HOPEFULLY THIS
YEAR WE'LL HAVE SOME ANSWERS
ABOUT THEIR FUNCTION.
THEY ALL SEEM TO BE NEGATIVELY
REGULATING T CELL FUNCTIONS OR B
CELL FUNCTIONS, SO THAT, WE'RE
PRETTY CLEAR ABOUT.
>> DO YOU SEE I GUESS A BARON
INTERFERON PRODUCTION IN --
>> WE HAVE IN ONE OF THE
SYSTEMS, YES.
YEAH.
>> SO GOING BACK TO THE
NLRC5 WORK, WHICH IS MAYBE LESS
INTERESTING THAN WHAT YOU'RE
TALKING ABOUT, BUT IT SEEMS TO
ME FROM THE LITERATURE THERE WAS
SOME EVIDENCE THAT IT MIGHT BE A
PATTERN RECOGNITION RECEPTOR BUT
IT'S CLEARLY IMPORTANT FOR CLASS
1.
WHAT'S THE CONSENSUS ON THAT?
>> SO IT'S A VERY CONFUSING
FIELD, NLRC5.
SO WE LOOKED IN HUMAN CELLS AND
RECENTLY THERE'S A PAPER THAT
LOOKS ALMOST LIKE OUR PAPER.
SO I'M PRETTY SURE IF YOU DO THE
SAME EXPERIMENT IN HUMAN CELLS T
LOOKS LIKE AN INFLAMMASOME
PROTEIN.
AND IT ALMOST MIM MICKED NLRP3.
SO WE THOUGHT AND WE COULD SEE
THAT IT FORMS -- SO WHEN THE
PAPER CAME OUT WHEN WE MADE THE
KNOCKOUT AND WE COULDN'T SEE
THAT, WE THOUGHT WE'RE REALLY
WRONG AND RECENTLY THERE'S A
PAPER LAST YEAR AND, THEY HAVE
PANEL BY PANEL -- PRIMARY CELLS,
DID EVERYTHING YOU CAN POSSIBLY
THINK OF.
LOOKS LIKE AN INFLAMMASOME
MOLECULE.
THERE ARE THREE GROUPS THAT SAYS
THERE'S ALSO INHIBITOR OF --
ACTUALLY EITHER INHIBITOR OR
ACTIVATOR, THAT'S WHERE THE
CONFUSION IS OF WHETHER
CYTOKINES OR INTERFERON AND I
DON'T REALLY KNOW, I THINK THERE
MAY BE COMPLEXITY OF THIS.
REMEMBER NLRs HAVE A LOT OF
ISOFORMS AND WE DON'T EVEN WANT
TO TALK ABOUT THIS BECAUSE WHO
CAN ANSWER THAT?
NLRP3 HAS 30 ISOFORMS.
SO WE DON'T KNOW WHAT THEY DO.
AND THERE MAY BE ISOFORMS THAT
ARE INHIBITING, EYE SO FORMS
THAT WORK ON OTHER THINGS.
ALL OF THESE HAVE MULTIPLE,
MEULT PEL ISOFORMS.
SO I THINK THE FIELD WILL BE
VERY COMPLEX SINCE THERE ARE
SOME GENES, SOME ISOFORMS,
POTENTIALLY LOTS OF FUNCTION.
SO ONE OF THE REVIEWERS DID SAY
THAT REGARDING -- AND REVIEWER
HOO HERE, I THINK YOU WHAT SAID
IS -- YOU ALREADY FOUND ONE
FUNCTION FOR THIS, HOW CAN THIS
HAVE A DIFFERENT FUNCTION?
I DON'T KNOW WHAT TOY PAUSE MOST
NLRs HAVE MULTIPLE FUNCTIONS.
BUT IF YOU'RE HERE, YOU'RE
GREAT.
BUT IT'S JUST -- NLRs JUST
HAVE MULTIPLE FUNCTIONS BECAUSE
THEY'RE SO COMPLEX.
>> WAS IT MORE FOREIGN IN TERMS
OF CLASS 1 FOR BASAL EXPRESSION
OR INDUCIBLE OR --
>> SO BOTH, AND I DIDN'T SHOW
YOU, BUT WHAT I SHOWED YOU IS
C2TA IS AMAZING BECAUSE YOU TAKE
A C2TA AWAY, THERE'S NO CLASS
II.
BUT THIS ONE, YOU TAKE IT AWAY,
THERE'S 30% RESIDUAL CLASS I.
SO ONE OF THE THINGS WE'VE BEEN
WONDERING IS WHAT IS THE OTHER
MOLECULE?
OR WHAT ARE THE OTHER MOLECULES?
AND THERE MAY BE OTHER NLRs IN
THAT PATHWAY.
WE ALSO FOUND OTHER THINGS WHICH
WORKS IN EVERY TISSUE,
NLRC5 DOESN'T WORK IN EVERY
TISSUE.
SO THERE'S MORE COMPLEXITY ABOUT
THIS.
>> VERY NICE.
CAN I ASK YOU A QUESTION ABOUT
THE NLRC3 KNOCKOUT?
>> CERTAINLY.
>> DID YOU ACTUALLY SEE ANY
METABOLIC PHENOTYPE, ANY ISSUES
WITH -- DO YOU ACTUALLY SEE --
>> WE'RE LOOKING A LOT AT THOSE
ISSUES.
I THINK I TOLD YOU THAT WE MADE
SOME DOUBLE KNOCKOUTS AND THEY
LOOK HORRIBLE.
WE HAVE SOME MICE THAT LOOK
REALLY FAT, SO CERTAINLY
METABOLISM, AND WE HAVE SOME
PATHWAYS WHERE THE FAT MIGHT BE.
SO THE NLR FIELD, TO ME IT'S
REALLY AMAZING, BECAUSE OF ALL
THE THINGS IT IMPACTS OF.
SO DEFINITELY IN METABOLISM.
>> DO YOU KNOW IF NLRP3 BINDING
INFLUENCES -- WITH LIE AGAINS?
>> I'M SORRY.
>> DOES IT INFLUENCE --
BINDING --
>> YOU KNOW, WE HAVEN'T REALLY
LOOKED AT -- WE JUST LOOK AT
TBK, BUT -- BECAUSE ONE OF OUR
COLLABORATOR IS THE PERSON WHO
DISCOVERED STING, HE'S THE GUY
WHO DISCOVERED THE CRYSTAL --
MADE THIS CRYSTAL STRUCTURE AND
SHOWED IT CAN ALSO BIND TO SIGH
CLICK KNEW CLEO SIDE TO WE'RE
WORKING ON THAT ASPECT.
SO WE HAVE HIS PROTEIN AND WE
SHOWED INTERACTION, SO WE'RE
KIND OF GOING DOWN THAT PATHWAY.
[APPLAUSE]
>> SO I JUST WANTED OH TO SAY
AGAIN, THANK YOU FOR COMING, AND
THANK YOU FOR GIVING A GREAT
TALK.
AND THERE IS A RECEPTION NOW,
JENNY HAS TO LEAVE PROMPTLY AT
4:30.
SO IF YOU WANT TO TALK TO HER,
PLEASE TRY TO GRAB HER.