>> WELCOME EVERYBODY, I'M
NINA SHORE AND I'M THE DEPUTY
DIRECTOR AND ACTING
SCIENTIFIC DIRECTOR OF NINDS AND
IT IS TRULY MY HONOR AND
PLEASURE TO WELCOME YOU TO THIS
WEDNESDAY AFTERNOON LECTURE.
THOSE OF YOU WHO ARE ON
VIDEOCAST, MAY NOT REALIZE THIS,
BUT THIS IS THE FIRST MAJOR NIH
EVENT THAT IS OCCURRING IN
PERSON HERE AT
LIPSETT AMPHITHEATRE.
WE HAVE A VERY SMALL AND VERY
SOCIALLY DISTANCED AUDIENCE AND
IT IS A REALLY EXCITING MOMENT
AFTER 2 YEARS OF EVERYTHING
BEING VIDEOCAST.
SO WELCOME WHETHER YOU ARE HERE
WITH US IN THE UDIANCY OR ON
VIDEOCAST TODAY.
I AM VERY EXCITED AND YOU WILL
HEAR IN A MOMENT WHY IT'S ALSO A
PERSONAL PLEASURE FOR ME TO
INTRODUCE TODAY'S SPEAKER
DR. ANNA HUTTENLOCHER,
DR. HUTTENLOCHER PUT ON THE MAP
THE ROLE OF CELL MIGRATION IN
TISSUE REPAIR AND IN THE REPAIR
OF INFLAMMATION
DR. ANNA HUTTENLOCHER IS A
PROFESSOR OF PEDIATRICS AND
MEDICAL MICROBIOLOGY AND
IMMUNOLOGY AT THE UNIVERSITY OF
WISCONSIN MADISON.
AS I SAID, HER RESEARCH REALLY
HAS FOCUSED FOR MANY YEARS ON
THE MOLECULAR MECHANISMS AND THE
MORE MORPHOLOGY OF CELL
MIGRATION IN RELATION TO THE
REPAIR OF INFLAMED AND INJURED
TISSUES.
SHE RECEIVED HER UNDERGRADUATE
DEGREE IN BIOLOGY FROM OBERLAND
IN COLLEGE AND HER MEDICAL
DEGREE FROM HARVARD MEDICAL
SCHOOL.
AS A YALEER AND OTHER THAN THAT
SHE HAD AN UNBLEMMISHED CAREER.
HE WENT ON TO DO HER RESIDENCY
AT PEDIATRICS AT UNIVERSITY OF
CALIFORNIA SAN FRANCISCO AND
POST DOCTORAL FELLOWSHIPS BOTH
AT UCSF AND AT UNIVERSITY OF
ILLINOIS URBANA CHAMPAGNE.
SHE MOVED TO THE UNIVERSITY OF
WISCONSIN MEDICAL SCHOOL AS AN
ASSISTANT PROFESSOR IN 1999 AND
HAS BEEN THERE EVER SINCE.
AND SHE NOW HAS AN ENDOWED
PROFESSORSHIP AND RUNS A VERY
ILLUSTRIOUS LABRATORY
ENTERPRISE.
SHE HAS WON MANY, MANY AWARDS
AND HAS MANY ACCOLADES INCLUDING
BEING A MEMBER OF THE NATIONAL
ACADEMY OF MEDICINE AND THE
ASSOCIATION OF AMERICAN
PHYSICIANS.
SHE IS A RECIPIENT OF THE BURROW
SIZE AMERICAN MEDICAL SCIENTIST
AWARD AND SHE HAS BEEN WITHIN
THE LAST YEAR, I BELIEVE, A
MEMBER OF THE BSC OF THE NIAMS.
SO SHE IS NO STRANGER TO NIH AND
IT'S INTRAMURAL PROGRAM AND JUST
ON A PERSONAL NOTE, I FIRST GOT
TO KNOW ANNA IN THE CONTEXT OF
BOTH OF US BEING ON THE STEERING
COMMITTEE OF SOMETHING CALLED
THE PEDIATRIC SCIENTIST
DEVELOPMENT PROGRAM, A PROGRAM
THAT'S FUNDED FOR MANY YEARS BY
NICHD.
AND BECAUSE OF THAT ASSOCIATION,
I KNOW HER TO REALLY BE
PASSIONATE ABOUT FUELING,
EDUCATING, MENTORING AND
LAUNCHING THE NEXT GENERATION OF
SCIENTISTS AND PHYSICIANS, AND
PHYSICIAN SCIENTISTS, AND SO
IT'S WITH GREAT PLEASURE THAT I
TURN THE PODIUM OVER TO
DR. HUTTENLOCHER.
>> THANK YOU VERY MUCH.
IT'S TRULY AN HONOR TO BE
INVITED TO DO THIS AND IT'S
TRULY AN HONOR TO BE GIVING THE
FIRST HYBRID IN-PERSON TALK AT
NIH IN OVER 2 YEARS AND TO BE
HONEST IT'S MY FIRST TRIP TO
SPEAK IN PERSON, SO THANK YOU
VERY MUCH.
THIS IS FANTASTIC, SO I WILL BE
TALKING TO YOU TODAY ABOUT OUR
LONG-TERM INTERESTS IN
UNDERSTANDING LEUCOCYTE MOTILITY
IN TISSUES AND WHAT THIS MEANS
FOR INFLAMMATION, IN PARTICULAR,
WORK FOCUSED ON THE RESOLUTION
OF INFLAMMATION AND THE CELL
TYPE THAT I WILL BE FOCUSING ON
TO A LARGE EXTENT IS THE
NUTRIFILL.
SO NUTRIFILLS ARE PRIMARY CELLS
OF THE INNATE IMMUNE RESPONSE
AND THEY'RE THE FIRST RESPONDERS
TO TISSUE DAMAGE AND IN THIS
VERY CLASSIC MOVIE OF A NEWT
FILL RESPONDING TO A BACTERIUM,
CAN YOU SEE THEIR ABILITY TO
MIGRATE AND GRATE AND CHANGE DIRECTION
CONVICTLY--CONTRIBUTES TO
DEVELOPMENT OF AUTOIMMUNITY SO
REALLY WE'VE BEEN INTERESTED IN
THIS QUESTION OF HOW NEUTROPHILS
GET SITES OF TISSUE DAMAGE
VERSUS INFECTION, YOU WANT THEM
AT INFECTED TISSUES BUT YOU
DON'T WANT THEM TO STAY AT SITES
OF TISSUE DAMAGE.
SO AT A REVIEW ARTICLE WE WROTE
A NUMBER OF YEARS AGO, THERE ARE
FACTORS INVOLVED IN THE
RECRUITMENT AND MANY RESEARCH
GROUP VS WORKED ON THIS IN TERMS
OF DAMPS, WOUNDS THAT MEDIATE
NEUTROPHIL IMPROVEMENT, CHEMO
KINE RECEPTOR SIGNALING AND THEN
THIS WHOLE INTERESTING
PHENOMENON OF NEUTROPHILS
RELEASING ATTRACT ANTS LIKE LTB4
WHICH LEAD TO SWARMING AND LEAD
TO INCREASED FORMATION AND
TISSUES AND THIS IS WHAT HAPPENS
IN THE KIND OF STERILE TISSUE
DAMAGE.
WITH INFECTION YOU ADD
ADDITIONAL COMPONENTS AND P A
MPs AND MICROORGANISM THAT
SAYS LEAD TO THE ACTIVATION OF
DIFFERENT SIGNALING PATHWAYS AND
WE WERE EXPWED IN HAD
DISTINGUISHED NEUTROPHIL
RESPONSE TO TISSUE DAMAGE AND
MICROBIAL SIGNALS AND IN
PARTICULAR WHAT SIGNALS PROMOTE
RESOLUTION OF NEUTROPHIL
INFLAMMATION AND FOR MUCH OF
THIS WORK, WE USE ZEBRA FISH AS
A MODEL SYSTEM, SO WHY DO WE USE
ZEBRA FISH?
BECAUSE YOU CAN WATCH MOTILITY
WITH HIGH ABILITY INVIVO BECAUSE
THE ZEBRA FISH ARE TRANSPARENT.
AND YOU CAN SEE HERE AT 72 HOURS
POST FERTILIZATION, CAN YOU SEE
THAT NUTRIFILL VS 2 POPULATIONS.
SO THESE RAPIDLY MODEL CITIZEN
TILE AMOEBOID NEUTROPHILS IN THE
HEAD REGION AND THEN MUCH MORE
STATIONARY NUTRIFILLS WITHIN THE
TISSUE AND TISSUE DAMAGE THEY'RE
RAPIDLY MOBILIZED AND LEAD THE
CHT AND THIS IS VERY DIFFERENT
FROM MACROPHAGES AND THEY
EXHIBIT THIS SURVEILLANCE
MIGRATION.
THEIR MYRRHIGRATION IS PROTEASE
INDEPENDENT AND THEY NIGREAT
MORE IN A MESSENTERIEN CHIMAL
TYPE MODE OF MIGRATION AND SO
WE'RE INTERESTED IN
UNDERSTANDING THESE DIFFERENT
MECHANISMS OF MIGRATION AND IN
ADDITION WITH NEUTROPHILS WHAT
MEDIATES THE RECRUITMENT TO
TISSUE DAMAGE.
SO THIS KIND OF DEMONSTRATES THE
POWER OF THE FISH SYSTEM SO WE
CAN BOTH CUT OFF THE TAIL AND
INJECT BACTERIA INTO THE EAR
REGION OF THE ZEBRA FISH AND YOU
CAN SEE NEUTROPHILS ARE
RECRUITED BOTH TO THE INFECTION
AND TO THE SITE OF STERILE
INJURY AND IF YOU ADD AN
INHIBITOR TO NADPHOX DACE IN
THIS CASE, DPI, YOU BROCK
RECRUITMENT TO THE STERILE
INJURY SITE BUT YOU DO NOT BLOCK
IT TO THE SITE OF INFECTION AND
IN FACT, YOU SEE MORE NEUTROPHIL
RECRUITMENT.
SO THIS DEMONSTRATES NICELY THAT
DIFFERENT SIGNALS MEDIATE
NEUTROPHIL RECRUITMENT TO WOUNDS
AND INFECTION.
SO WE WERE INTERESTED IN TRYING
TO FIGURE OUT SOME OF THESE
DIFFERENTIAL SIGNALS.
SO I WOULD LIKE TO FIRST
HIGHLIGHT THOUGH THAT IN
ADDITION TO BEING A GREAT SYSTEM
TO LOOK AT MOTILITY, THE INNATE
SYSTEM IS HIGHLY CONSERVED SO
CAN YOU MODEL HUMAN DISEASES IN
ZEBRA FISH.
AN EXAMPLE FOR MANY YEARS AGO IS
AN INFANT BOY WE SAW AT THE
UNIVERSITY OF WISCONSIN WHO
PRESENTED WITH RECURRENT
BACTERIAL INFECTIONS AND WE USED
MICROFLUIDICS IN COLLABORATION
WITH THE BB LAB AND YOU CAN SEE
NICE CHEMO TAXIS OF CONTROL
NEUTROPHILS AND SIGNIFICANTLY
IMPAIRED MOTILITY OF PATIENT
NEUTROPHILS.
AND THIS PATIENT WAS FOUND TO
HAVE A FORNL OF LEUCOCYTE
ADHESION DEFICIENCY.
IN THIS CASE, IT WAS A DOMINANT
INHIBITORY MUTATION IN THE SMALL
DTPACE RAB 2, NOW RAC2 AND
HIGHLY CONSERVED IN ZEBRA FISH
SO WE CAN TAKE ADVANTAGE OF THIS
CONSERVATION TO INTRODUCE THE
MUTATION SPECIFICALLY INTO
NEUTROPHILS.
SO WHAT IS KNOWN ABOUT RAC2
SIGNALING AND THEIR OWN
MOTILITY.
SO DOWN STREAM OF CHEMO KINE
SIGNALING YOU GET ACTIVATION OF
G-PROTEIN COUPLED RECEPTORS,
GAPS THAT LEAD TO RAC, GTPACE
ACTIVATION OF THE LEADING EDGE
WHICH IS A CRITICAL REGULATOR OF
DYNAMIC ACTIN AT THE LEADING
EDGE.
IN ADDITION AT THE REAR OF THE
CELL, YOU'VE MORE STABILIZED AND
I WILL SHOW A MOVIE WHERE YOU
SEE MORE STABLE ACTIN KNOWN AS
UTROAPIN AND WHERE IT LOCALIZED
NEAR THE END OF THE CELL, FOR
ROW A ACTIVATION, SO THIS IS
JUST SORT OF A FUN MOVE FREE
RADICALS YEARS AGO THAT IF YOU
ACTIVATE RAC AT THE LEADING
EDGE, WHAT HAPPENS TO MODEL
CITIZEN TILL INVIVO.
SO IN THIS CASE, IF YOU
REPEATEDLY, PHOTO ACTIVATE RAC
AT THE LEADING EDGE, WHAT YOU
CAN DO IS CONTROL WHERE
NEUTROPHILS ARE MIGRATING INVIVO
AND IN THIS CASE SPELL THE WORD
RAC.
SO THIS SHOWS THAT RAC SIGNALING
IS SUFFICIENT TO INDUCE
NEUTROPHIL POLARIZATION AND
MOTILITY.
I WAS TALKING TO CLAIR WATER MAN
EARLIER TODAY ABOUT THIS WHOLE
IDEA OF CELLS BEING ABLE TO GET
THROUGH NARROW CHANNELS INVIVO
AND WITHIN TISSUES AND YOU CAN
SEE HERE WHEN WE REPEATEDLY
PHOTO ACTIVATE RAC, THE LEADING
EDGE, THIS NEUTROPHIL CAN
SQUEEZE THROUGH A VERY NARROW
SPACE.
SO WHAT HAPPENS WITH THE
DOMINANT INHIBITORY MUTATION OF
RAC 2?
SO THIS IS CONTROL NEUTROPHILS
WHICH YOU CAN SEE MIGRATE
RAPIDLY WITH A POLARIZATION OF
STABILIZED OF ACTIN TO THE REAR
AS INDICATED BY THE UTROPIN GFP
AND WHEN YOU HAVE THIS DOMINANT
INHIBITORY MUTATION OF RAC 2,
WHAT YOU SEE IS THE NEUTROPHILS
DON'T MIGRATE, THEY DON'T
POLARIZE AND WHAT'S INTERESTING
IS YOU HAVE STABILIZE OF ACTIN
THROUGHOUT THE CORTICAL REGION
OF THE NEUTROPHIL.
SO SIGNIFICANTLY IMPAIRED
MOTILITY.
AND THE CONSEQUENCE OF THIS IS
THAT THE ZEBRA FISH RAPIDLY DIE
FROM INFECTION AND NEUTROPHILS
ARE NOT RECRUITED TO PSEUDOMOAN
ISOTOPE INJECTED INTO THE EAR,
SO THIS RECAPITULATES THE HUMAN
DISEASE.
WHILE THERE ARE A NUMBER OF
VARIANTS IN RAC 2 THERE ARE
ASSOCIATED OTHER DISEASES, SO
THIS SAY VERY POWERFUL SYSTEM TO
LOOK AT SOME OF THOSE OTHER RAC
2 VARIANTS.
SO AN EXAMPLE WAS PUBLISHED BY
STEVE HOLLAND'S LAB, AND THIS IS
AN ACTIVATING FORM OF RAC 2,
WHICH IS ASSOCIATED WITH IMMUNE
DISREGULATION AND IN THIS CASE,
INFLAMMATION AND TISSUES, BRONCH
YECT SIS AND WHAT YOU CAN SEE IN
THIS CASE IS THAT CONTROL
NEUTROPHILS MIGRATE TO THE SITE
OF DAMAGE AND LEAVE WITH THE
ACTIVATED MUTATION IN RAC 2, YOU
SEE THAT THE NEUTROPHILS MIGRATE
FASTER, QUANTIFIED HERE, AND
MORE NEUTROPHILS ARE RECRUITED
AND THIS IS WHAT YOU WOULD
EXPECT WITH AN INFLAMED TISSUE,
YOU WOULD EXPECT MORE
NEUTROPHILS WOULD BE MIGRATING
THERE.
AND THAT THERE MOTILITY WOULD BE
INCREASED SO WE BECAME VERY
INTERESTED IN THIS CASE
QUESTION, BASED ON PATIENTS THAT
I SAW YEARS AGO, WITH NEONATAL
ONSET MULTISYSTEM INFLAMMATORY
DISEASE, SO I HAD A LOT OF FUN
TALKING TO COLLEAGUES AND
OBVIOUSLY SOME AMAZING WORK HAS
BEEN DONE HERE LOOKING AT
VARIOUS AUTOINFLAMMATORY
DISEASES.
THIS PATIENT AT THE TIME THAT I
SAW HER, WE DIDN'T KNOW WHAT HER
UNDERLYING GENETIC MUTATION WAS.
THAT SHE HAD A DISEASE
CALLEDNYONATAL ONSET MULTISYSTEM
INFLAMMATORY DISEASE AND LATER
WE FOUND SHE HAD AN NLR3 P
MUTATION.
AND SHE PRESENT WIDE FEVER, SKIN
RASH, JOINT SWELLING AND WHEN
YOU BIOPSY HER SKIN, YOU SAW
NEUTROPHIL INFILTRATION INTO THE
SKIN.
BUT WHAT WAS VERY INTERESTING IS
WE LOOKED AT HER NEUTROPHILS AND
WE LOOKEDDA THE NEUTROPHILS OF
SEVERAL PATIENTS WITH NOMID AND
IN ALL CASES THEIR NEUTROPHILS
SHOWED IMPAIRED CHEMO TAXIS TO
ALL THE CHEMO KINES WE STUDY.
SO WHY IS IT THAT SOMEONE WITH A
LOT OF INFLAMMATION AND TISSUES,
WHY WOULD THEIR NEUTROPHILS SHOW
IMPAIRED CHEMO TAXIS.
SO THIS RAISED THE QUESTION THAT
HAS REALLY BEEN A DRIVING FORCE
OF SOME OF OUR RESEARCH IS WHAT
THE RELATIONSHIP BETWEEN
NEUTROPHIL MIGRATION AND
INFLAMMATION?
SO MANY YEARS AGO WE WERE 1 OF
THE FIRST LABS TO GENERATE A
TRANSGENIC THAT LABELS
NEUTROPHILS AND WHEN WE WOUND
THE ZEBRA FISH, WHAT WE FOUND IS
THAT NEUTROPHILS CAME TO THE
SITE OF TISSUE DAMAGE AND THEN
VERY PRIZINGLY, THEY LEFT.
SO AT THE TIME, THE DOGMA WAS
THAT NEUTROPHILS GO INTO SITES
OF TISSUE DAMAGE AND THEY DIE
AND THEN THEY'RE CLEARED BY
MACROPHAGES AND THIS SUGGESTED
SOMETHING VERY DIFFERENT THAT
THEY CAN COME INTO SITES OF
TISSUE DAMAGE AND ACTUALLY
LEAVE.
SO ANOTHER TOOL THAT I AM GOING
TO BRING UP LATER IS 1 WHERE WE
CAN ACTUALLY PHOTO CONVERT
NEUTROPHILS THAT HAVE ARRIVED AT
THE WOUND IN THIS CASE, WE PHOTO
CONVERTED THE FIRST 5
NEUTROPHILS, THAT ARRIVED AT THE
WOUND, AND THEN WE CAN TRAP THE
FATE, AND WHAT WAS INTERESTING
TO LOOK AT THESE FIRST 5
NEUTROPHILS, WITHIN THE TISSUES
THEY MIGRATE RANDOMLY.
THEY ALMOST PONDER BEFORE
FINALLY GOING BACK INTO THE
CIRCULATION.
SO THEN FINALLY THEY WILL
UNDERGO REVERSE TRANSMIGRATION,
GO INTO THE CIRCULATION, AND WE
CAN FIND THEM IN TISSUES 2 DAYS
LATER.
AND SO, THEY WILL GO TO VARIOUS
TISSUES INCLUDING HEMATOPOIETIC
TISSUE.
SO, JUST TO ANSWER THIS
QUESTION, HOW DOES NEUTROPHIL
INFLAMMATION RESOLVE, AND SOME
TYPES OF TISSUE DAMAGE IN
PARTICULAR UNINFECTED TISSUE
DAMAGE, WE THINK THAT PROUDLY
THE PRIMARY MECHANISM RESOLUTION
CAN OFTEN BE THIS NEUTROPHIL
REVERSE MIGRATION, SO RATHER
THAN THIS TRADITIONAL IDEA OF
NEUTROPHILS GOING IN AND DYING
AND BEING CLEAVED BY
MACROPHAGES, THEY WILL GO IN AND
THEY WILL LEAVE BY REVERSE
MIGRATION.
SO THERE WAS A LOT OF SKEPTICISM
ABOUT THIS FOR A LONG TIME WITH
THE ZEBRA FISH SPECIFIC
PHENOMENON?
AND THEN AFTER A DECADE WHEN WE
FIRST MADE THIS OBSERVATION FROM
PAUL COOP'S LAB AM, SHOWED IF
YOU LOOK AT THOSE AND PHOTO
CONVERT THE NEUTROPHILS THEY
LEAVE THE SITE OF TISSUE DAMAGE
AND THEY TRAFFIC TO THE LUNG AND
THEY TRAFFIC TO THE BONE MARROW.
SUPPORTING THE IDEA THAT AGAIN,
NEUTROPHIL REVERSE MIGRATION IS
INVOLVED IN RESOLUTION OF
INFLAMMATION.
BUT THERE'S ALSO BEEN SOME VERY
EXCITING DATA SUGGESTING THAT
NEUTROPHIL REVERSE MIGRATION MAY
ALSO BE INVOLVED IN SYSTEMIC
INFLAMMATION, MAY HAVE DISEASE
IMPLICATIONS RELATED FOR EXAMPLE
TO PAN KRE TINSA TITIS AND THEN
ARDS OR IN THE CASE OF
METASTASIS WHERE TUMOR CELLS MAY
BE ASSOCIATE WIDE NEUTROPHILS IN
THE CIRCULATION.
SO THESE LOCATIONS MAY BE BROAD,
WE HAVE DONE A LITTLE BIT OF
WORK LOOKING AT ZEBRA FISH
MODELS OF INFLAMMATION, WHERE WE
FEED ZEBRA FISH NORMAL OR HIGH
FAT DIET AND THEN WE LOOK AT
NEUTROPHIL RECRUITMENT AND YOU
CAN SEE WHERE THE HIGH FAT DIET
IS KIND OF SCARE SKPE BAKUGAN
LOTS OF NEUTROPHILS ARE
RECRUITED TO THE WOUND AND THIS
WORK IS BEING DONE AND PURSUED
NOW IN THE LABORATORY OF A
FORMER POST DOC, SOFIA
DE OLIVEIRA, WHO IS A PROFESSOR
AT ALBERT EINSTEIN.
SO SOFIA ASKED THE QUESTION
WHERE ARE THEY COMING FROM?
OUT OF CIRCULATION?
HEMATOPOIETIC TISSUE?
SO SHE CONVERTED NEUTROPHILS
FROM THE LIVER AND FOUND THAT
THE--IN THE CASE OF A HIFAT DIET
MANY OF THESE NEUTROPHILS WERE
ACTUALLY COMING FROM THE LIVER.
THIS WAS NOT THE CASE WITH
CONTROL.
SO AGAIN, THIS SUPPORTS THE IDEA
THAT NEUTROPHIL WILL REVERSE
MIGRATION AND THAT CASE THAT THE
LIVER WILL CONTRIBUTE TO
SYSTEMIC INFLAMMATORY RESPONSE.
SO, WE AND OTHERS HAVE BEEN
INTERESTED IN WHAT REGULATES
NEUTROPHIL REVERSE MIGRATION?
SO WHEEF BEEN LOOKING AT CHEMO
KINES RECEPTOR SIGNALING, NOW WE
KNOW THAT CHEMO KINES ARE VERY
IMPORTANT FOR THE ONSET OF
INFLAMMATION, SO IF YOU BLOCK
IL8 SIGNALING, NEUTROPHIL
RECRUITMENT TO WOUND CAN BE
IMPAIRED.
BUT 1 THING THAT WAS INTERESTING
TO US IS WHEN WE CONTROLLER OF
BANKED OUT 1 OF THE ISOFORMS IN
ZEBRA FISH AND 1 OF THE
RECEPTORS CXCRC2 IN ZEBRA FISH,
WE FOUND THAT NEUTROPHILS WERE
MORE PERSISTENT AT THE WOUND.
THEY DID NOT HAVE THIS RANDOM
MOTILITY AND THEY HAD IMPAIRED
REVERSE MIGRATION.
NOW WORK DONE BY [INDISCERNIBLE]
GROUP AT CAMBRIDGE HAS GONE ON
ON TO SHOW NICELY THAT WHEN THE
RECEPTOR IS STAINED ON THE CELL
SURFACE, THIS IS ASSOCIATE WIDE
A ROLE IN THE VERMEN INFECTED
VERSE MIGRATION, WHEN IT GETS
INTERNALIZED THEN THE
NEUTROPHILS, THAT'S NOT AS
INVOLVED IN THE REVERSE
MIGRATION PROCESS.
SO SUGGESTED TO US THAT THIS
CHEMO KINESIS THAT'S INDUCED FOR
SIGNATURES SIGNALING FOR THE
RECEPTORS IS A STEP IN THE
CASCADE.
IT ALSO RAISES QUESTIONS ABOUT A
PATHWAY THAT IS TARGETED
THERAPEUTICALLY THAT MAY IN FACT
LEAD IN SOME CASES TO LESS
INFLAMMATION AND OTHERS
POTENTIALLY TO MORE INFLAMMATION
WHICH WE KNOW IS TRUE OF A
NUMBER OF DIFFERENT TARGETS.
SO THIS GETS BACK TO THIS IDEA,
WE'RE INTERESTED IN THIS WHOLE
BALANCE AND NEUTROPHIL
INFLAMMATION AND TISSUE REPAIR.
IF YOU HAVE TOO MUCH
INFLAMMATION, THEY DEPLETE.
SO IF WE DEPLETE THESE, WE FOUND
MANY MODELS TISSUE REPAIR FASTER
BUT NOT ALL NEUTROPHILS ARE THE
SAME.
A RECENT PAPER IN IMMUNOLOGY
FOUND THAT IF YOU DEPLETE
NEUTROPHILS THEY'RE REQUIRED FOR
EFFICIENT REPAIR BY REMODELING
THE MATRIX, SO, ALL NEUTROPHILS
ARE NOT ALIKE.
OBVIOUSLY IF YOU HAVE 2 FEW
NEUTROPHILS YOU END UP WITH
IMMUNE DEFICIENCY AND IN THAT
CASE, CHRONIC INFECTION AND
IMPAIRED HEALING.
SO WE WERE INTERESTED IN
IDENTIFYING NEW MECHANISMS THAT
MIGHT REGULATE INFLAMMATION
RESOLUTION.
AND FOR THIS, WE WANTED TO LOOK
FOR FACTORS THAT WERE
UPREGULATED AND STERILE
TRIGGERS, BUT NOT INFECTION.
AND TO DO THIS WORK, WE TOOK
ADVANTAGE OF 2 DIFFERENT
INFLAMMATION MODELS, ACUTE
WOUNDING OF THE ZEBRA FISH WHERE
YOU GET RECRUITMENT OF
NEUTROPHILS AND MACROPHAGES AND
THEN, CANCER AS KIND OF AN
UNHEALED WOUND.
WE HAVE CHRONIC ASSOCIATION OF
NEUTROPHILS AND MACK RAPHAGES
WITH TRANSFORM CELLS.
AND WHAT WE DID IS WE DID
SOMETHING CALLED TRAP SEQ,
RNA-SEQ AND THEN TARGETED
PATHWAYSA BY CRISPR.
SO TO DO THIS, WE GENERATED
ZEBRA FISH LINES THAT EXPRESS
THE RIBOSOMAL L10 A PROTEIN FUSE
TO GFP AND WE MADE LINES AND
NEUTROPHILS, MACROPHAGES AND
EPITHELIAL CELLS.
AND THEN, IN THIS CASE, WORKED
ON BY RUTH HOUSRIGHT A FORMER
GRADUATE STUDENT IN THE LAB, SHE
INJURED THE FISH IN MULTIPLE
PLACES AND THEN 3 HOURS POST
WOUND COLLECTED THE SAMPLES FOR
THIS TRAP-SEQ AND THERE WAS A
GENE THAT WAS UPREGULATED BOTH
IN NEUTROPHILS AND MACROPHAGES,
1 THING TO NOTE IS SOMETHING
THAT WAS INTERESTING IS VERY FEW
GENES WERE COMMONLY UPREGULATED
IN A DIFFERENT CELL TYPES BUT
THERE WAS 1 THAT WAS UPRIGGING
BOTH NEUTROPHILS AND MACROPHAGES
WITH STERILE INJURY AND THAT WAS
A GENE CALLED MYELOID DERIVED
GROWTH FACTOR AND AT THAT TIME
THERE WAS NOT MUCH OF IT UNKNOWN
ABOUT IT IN THE LITERATURE, SO
MYELOID DRIVE GROWTH FACTOR IS A
PERIKRIN 17 KILODALTON SECRETED
PROTEIN, IT WAS REFERRED TO AS A
SPECIFIC ORF THAT WAS FOUND TO
BE UPREGULATED CAN IN FLUM
INFLAMMATION, SYNOVIAL FLUID
FROM OUR RA PATIENTS AND
HEPATITIS EATIC CARCINOMA THAT
WAS ASSOCIATE WIDE POOR
PROCEEDINGINOSEIS SO THEN IT WAS
NAMED MYELOID-DERIVED GROWTH
FACTOR, THAT WAS SECRETED BY
MONOCYTES AND MACROPHAGES AND
TISSUE REPAIR IN A MURINE MODEL
OF MY O CARDIAL INFARCTION.
AND THIS KNOCK OUT MOUSE WAS
PERFECTLY FINE BUT THEY DID MUCH
LESS WELL WHEN THEY HAD AN MI.
SO IT'S AN INTERESTING PROTEIN
THAT WE DON'T KNOW MUCH ABOUT
AND I WILL TELL YOU ABOUT HOW
WE'RE TRYING TO GET AT HOW THIS
IN FACT, WORKS BUT WE STILL HAVE
MANY QUESTIONS REMAINING.
IT CAN BE A RESIDENT ER PROTEIN,
IT CAN ALSO BE SECRETED AND IT
BINDS TO KDEL RECEPTORS, SO IT
HAS A KDEL MOTIF AND IT'S
INCREASED HYPOXIA AND IT'S
CONTINUATION OF DIABETIC KIDNEY
DISEASE AND VASCULAR
INFLAMMATION.
SO WE KNOCKED IT OUT, OF ZEBRA
FISH AND YOU CAN SEE HERE, YOU
GENERATED AN ANTIBODY, AND MADE
A CRISPR MUTANT AND YOU CAN SEE
THAT THE PROTEIN IS GONE.
I JUST WANT TO HIGHLIGHT THAT
THE ZEBRA FISH GROWTH FACTOR IS
HIGHLY CONSERVED BOTH
STRUCTURALLY AND BASED ON AMINO
ACID SIMILARITY.
AND WHAT HAPPENS WHEN WE KNOCK
IT OUT?
SO, THE MODEL THAT I'M GOING TO
BE USING QUITE OFTEN IN THIS
TALK THAT I WANT TO JUST TELL
YOU ABOUT IS A DIFFERENT TYPE OF
INJURY MODEL, IT'S A MORE
SIGNIFICANT INJURY WHERE WE USE
A HEAT SOURCE, A CAUTERY WIRE
AND WE BURN THE TAIL AND THEN WE
LOOK AT INFLAMMATORY RESPONSE
WHICH IS VERY DIFFERENT THAN IF
WE CUT OFF THE TAIL AND I WILL
GET INTO THAT A BIT MORE LATER.
SO LAWN MOWER WE FOUND IN BOTH
THE TAIL TRANSSECTION, WHERE WE
CUT OFF THE TAIL OR WE BIRN THE
ZEBRA FISH TAIL, WE FOUND THAT
DEPLETION OF MYELOID GROWTH
FACTOR LED TO INCREASED
NEUTROPHILS AT THE WOUNDS
QUANTIFIED HERE EVEN 24 HOURS,
POST BURN.
YOU CAN SEE THE NEUTROPHILS
HERE.
IF YOU LOOK AT A MOVIE, WHAT YOU
CAN SEE, IS THERE'S A ROBUST
RESPONSE OF BOTH NEUTROPHILS AND
MACROPHAGES TO A BURN WOUND AND
IF YOU LOOK AT THE SIDE VIEW
HERE, CAN YOU SEE EVEN MORE THAT
WHEN YOU KNOCK OUT MILEN GROWTH
FACTOR, THERE ARE EVEN MORE
NEUTROPHILS RECRUITED TO A BURN
WOUND.
BUT WE DID NOT SEE A DIFFERENCE
IN MACROPHAGE RECRUITMENT.
SO THIS SUGGESTS THAT MYELOID
DRIVE GROWTH FACTOR IS
NEGATIVELY REGULATING NEUTROPHIL
INFLAMMATION, SO IS THE PROTEIN
SUFFICIENT TO ACTUALLY INHIBIT
NEUTROPHIL INFLAMMATION?
SO WHAT WE DID IS WE TOOK
MYELOID DRIVE GROWTH FACTOR AND
WE INJEKED INTO INTO THE EAR OF
THE ZEBRA FISH, AND WE USED
EITHER CONTROL OR MYELOID
DERIVED GROWTH FACTOR AND WHAT
WE FOUND IS JUST WHEN YOU HIT
THE EAR WITH THE NEEDLE, YOU SEE
NEUTROPHILS COME TO THE EAR SITE
AS A WOUND AND THEN MYELOID
DERIVED GROWTH FACTOR
DRAMATICALLY INHIBIT THE
NEUTROPHIL RECRUITMENT, YOU CAN
SEE THIS QUANTIFIED HERE AND
WHILE WE LOOK AT THE MUTANT AS
COMPARED TO THE NONMUTANT, CAN
YOU SEE, MANY MORE NEUTROPHILS
ARE RECRUITED TO THIS WOUND, AND
THIS IS RESCUED BY INJECTING THE
MYELOID DRIVE GROWTH FACTOR.
SO THIS SUGGESTS THAT THIS IS A
NEW INHIBITOR OF NEUTROPHIL
INFLAMMATION DISP WE'RE REALLY
EXCITED ABOUT IT BECAUSE THERE
AREN'T A LOT OF THINGS THAT
INHIBIT NEUTROPHIL INFLAMMATION
IN RESPONSE TO STERILE INJURY
BUT NOT INFECTION AND THAT'S
WHAT WE FOUND.
SO THIS IS PRETTY COOL.
IT'S SUGGESTS IT MIGHT BE AN
INTERESTING THERAPEUTIC TARGET.
SO HOW DOES MYELOID DRIVE GROWTH
FACTOR AFFECT WOUND HEALING, WE
KNOW IT PLAYS A ROLE IN MI
RESPONSE AND WHAT WE FOUND IS IF
YOU DO A BURN WOUND IN ZEBRA
FISH OR A TAIL TRANSSECTION, YOU
HAVE IMPAIRED, REPAIR AT ALL
TIME POINTS IN THE MYELOID
DERIVED GROWTH FACTOR MUTANT BUT
THE THIN IS GROWING JUST FINE
NONWOUNDED ZEBRA FISH.
AND THEN THE QUESTION IS LOKAY,
SO WE HAVE MORE NEUTROPHILS
THERE, THERE'S IMPAIRED HEALING
OF THE WOUND, ARE NEUTROPHILS
INVOLVED IN THIS PHENOTYPE?
SO WHAT WE DID IS WE LOOKED AT
THE WOUND HEALING DEFECT AND
NEUTROPHIL DEFICIENT ZEBRA FISH.
SO WE USE THAT RAC 2 D57 INLINE
I DESCRIBED BEFORE AND WE DID
MORPHOLIN O OR MYELOID DRIVEN
AND WE SAW THE TRANSIENT GROWTH
MYELOID GROWTH FACTOR BUT THIS
DOES NOT OCCUR IN THE RAC
DEFICIENT ZEBRA FISH.
SO IT SUGGESTS THAT THE WOUND
HEALING DEFECT IS NEUTROPHIL
DEPENDENT IN MYELOID DEFICIENT
LARVA.
SO THEN WE'RE INTERESTED IN THE
QUESTION, WHAT IS THE MECHANISM
OF INCREASED NEUTROPHIL
INFLAMMATION IN MUTANTS, MORE
RECRUITMENT, DIFFERENT IN
REVERSE MIGRATION AND FOR THIS
WE DID THAT PHOTO CONVERSION
EXPERIMENT THAT I SHOWED YOU
EARLIER, WHERE WE PHOTO CONVERT
NEUTROPHILS AT 2 HOURS POST
WOUND AND A TAIL TRANSSECTION,
AND IMAGE AGAIN AT 6 HOURS POST
WOUND, AND YOU CAN SEE HERE THAT
YOU HAVE MANY MORE NEUTROPHILS
IN THE MYELOID DEFICIENT LARVA
AND THAT THE PHOTO CONVERTED
NEUTROPHILS ARE PERSISTENT AND
THIS IS SHOWN HERE.
THERE'S IMPAIRED RESOLUTION OF
THE NEUTROPHILS FROM THIS SITE
OF DAMAGE.
NOW THIS IS ALSO TRUE WHEN WE
LOOK AT THERMAL OR BURN INJURY.
WE THROW A PROTOCONVERTED LATER
BECAUSE THERE'S MUCH MORE
INFLAMMATION AT 4 HOURS AND
IMAGE AT 24 HOURS AND YOU CAN
SEE AGAIN RESOLUTION IS IMPAIRED
IN THE MYELOID DERIVED GROWTH
FACTOR MUTANT.
SO WHAT ELSE IS KNOWN ABOUT
NEUTROPHIL REVERSE MIGRATION AND
SIGNALING PATHWAYS THAT ARE
INVOLVED WITH IT?
AND 1 THAT HAS BEEN IMPLICATED
IS THE HIP 1 ALPHA SO AS I
MENTIONED BOTH GROWTH FACTOR AND
HIP 1 ALPHA ARE UPREGULATED WITH
HYPOXIA.
SO WORK FROM SPHEEF RENSHAH'S
GROUP HAS SHOWN THAT IN HYPOXIA
YOU GET AN UPREGULATION OF HIP 1
ALPHA AND THIS LEADS TO
NEUTROPHIL PERSISTENCE AT THE
WOUND AND THEY DON'T REVERSE
MIGRATE AS MUCH, THEY DON'T DIE
AS MUCH AND TO READ OUT HIP 1
ALPHA SIGNALING, WE CAN LOOK AT
Ph.D. EXPRESSION BY RTPC R,
AND WE FOUND IT WAS INCREASED IN
BOTH TAIL SECTION AND BURN AND
THE MYELOID DRIVE DEFICIENT
FISH.
SO THEN THE QUESTION IS CAN WE
ACTUALLY RESCUE THIS PHENOTYPE
BY KNOCKING DOWN THE HIF 1 HALFA
PATHWAY, SO WE DID THIS, BY
DEPLETING ART 1 USING A
MORPHOLIN O IN EITHER CONTROL OR
THE MYELOID DERIVED DEFICIENT
ZEBRA FISH AND YOU CAN SEE THAT
YOU RESCUE THIS NEUTROPHIL
INFLAMMATION PHENOTYPE AND THIS
AFFORDS THE IDEA THAT MYELOID
GROWTH FACTOR IS WORKING THROUGH
HIP1 ALPHA PATHWAY SO JUST TO
SUMMARIZE MYELOID DRIVE DPROAGHT
FACTOR EFFECTS ON WOUND HEALING.
SO IT'S INDUCED BY STERILE
INJURY BUT NOT INFECTION, IT
LIMITS NEUTROPHIL INFLAMMATION,
AND IT'S A DAMAGE SIGNAL THAT
ACTS VIA HIF1 ALPHA PATHWAY AND
SEEMS TO PROMOTE NEUTROPHIL
REVERSE MIGRATION AND
INFLAMMATION RESOLUTION.
SO A QUESTION IS, HOW DOES IT
WORK?
WHAT IS IT'S RECEPTOR, WHAT'S
THE SOURCES AND TISSUES?
AND ARE THERE--IS THERE A ROLE
FOR MACROPHAGES BECAUSE IN THE
MOUSE STUDIES, IT SEEMS LIKE
MYELOID DRIVE GROWTH FACTOR IN
MACROPHAGES WAS PARTICULARLY
IMPORTANT SO WHAT DO WE KNOW
ABOUT MACROPHAGES IN NEUTROPHILS
AND HOW THEY INTERACT AT SITES
OF TISSUE DAMAGE.
SO IN EARLIER WORK, WE HAD SHOWN
THAT WHEN NEUTROPHILS COME TO
THE WOUND FIRST AND MACROPHAGES
APPEAR, THE NEUTROPHILS LEAVE.
THIS IS THE CASE WITH
MACROPHAGES AND STERILE
INFLAMMATION, NOT IN INFECTED
DAMAGE, SO THESE ARE M2
MACROPHAGES.
AND IF YOU DEPLETE MACROPHAGES,
USING IRF8 MORPHOLIN O, THEN
MORE NEUTROPHILS ARE
PRESCRIBINGENT AT THE WOUND AND
THEY HAVE IMPAIRED REVERSE
MIGRATION SO IT'S 1 OF THE
PATHWAYS THAT'S INVOLVED TBH
THIS NEUTROPHIL REVERSE
MIGRATION AND NICE WORK DONE BY
TD GERMANE LAB IN MOUSE MODELS,
THEY FOUND THAT MACROPHAGES CAN
CLOAK A WOUND AND THAT THIS
INHIB THORS THES NEUTROPHIL
INFLAMMATION.
SO IT SUGGESTS THAT THERE'S SOME
MACROPHAGES ARE VERY INHIBITORY
TO NEUTROPHIL BEHAVIOR.
SO THIS RAISES THE WHOLE
QUESTION LIKE WE ALWAYS TALK
ABOUT M1, M2 MACROPHAGES, WITHIN
TISSUES AND SO, A VERY TALENTED
POST DOC IN THE LAB, VERONICA
MISKOLCI, WAS INTERESTED IN
TRYING TO UNDERSTAND MACROPHAGE
ACTIVATION PHENOTYPES WITHIN
TISSUES AND SHE DID THIS BY
TAKING CARE OF ENDOGENOUS
FLUORESCENCE IN COLLABORATION
WITH MELISSA SKALA, SO CURRENTLY
WHAT WE DO IN ZEBRA FISH TO READ
OUT IN 1 MACROPHAGE IS WE USE A
TNF ALPHA REPORTER.
FOR M2 THERE REALLY HASN'T BEEN
MUCH THAT'S BEEN USED, BUT THE
RGENERATEDAISE REPORTER JUST
CAME OUT BUT I SHOULD SAY WHEN
YOU HAVE THIS TNF ALPHA REPORTER
TURNED ON, THERE TENDS TO BE
MORE PERSISTENT NEUTROPHIL
INFLATION, THOSE DON'T SEEM TO
CARE THE NEUTROPHILS AWAY.
SO WHAT VERONICA USED WAS 1, SO
FLUORESCENCE LIFETIME IMAGING
MICROSCOPY IS A TOOL THAT YOU
CAN USE TO MEASURE INTRA
CELLULAR METABOLISM OF WOUND
MACROPHAGES WITHIN TISSUES.
NOW SLEE DID A LOT OF WORK, THIS
TOOK OVER A YEAR OF REVISIONS,
AND IN PART BECAUSE IT WENT
AGAINST THE DOGMA OF WHAT WE
KNOW INVITRO.
SO WHAT SHE FOUND WAS THAT IN
WOUNDED TISSUES THEY WERE
MACROPHAGE PRESENT, THEY WERE
NOT TNP ALPHA POSITIVE, BUT WITH
WE BURNED THE TISSUE INITIALLY,
WE HADOXIDATIVE TISSUES
RECRUITED AND I AM JUST
SUMMARIZING WHAT I WILL SHOW YOU
AND THESE HAVE BEEN TURNED
NIGGATIVE AS INFLAMMATION
RESOLVES.
SO LET ME SHOW YOU SOME OF THIS
DATA.
SO THE FIRST MODEL I'M SHOWING
YOU IS 1 WHERE WE HAVE INFECTED
A WOUND.
SO THIS TURNS ON TNF ALPHA AND
ALL OF THE MACROPHAGES YOU GET
THIS ROBUST INFLAMMATORY
RESPONSE AND YOU DON'T GET ANY
HEALING AND HERE'S YOU CAN SEE
TNF STAYS ON.
SO WHAT HAPPENS TO THE
METABOLISM OF THESE MACROPHAGES
USING FLEM?
SO WHAT SHE FOUND IS THAT
MACROPHAGES ARE MORE OXIDIZED
WITH THE INCREASED NADPH
LIFETIME AND WE THINK NADPH
LIFETIME IS A PARTICULARLY
USEFUL TOOL TO USE AND A REDUCED
REDOCKS RATIO AT THE INFECTED
WOUND AND YOU CAN SEE THIS HERE.
SO AS I SAID, THIS GOES AGAINST
WHAT WAS--HAD BEEN PUBLISHED
INVITRO.
SO WHAT HAPPENS IN A WOUND WHERE
YOU HAVE THE DYNAMIC CHANGES IN
MACROPHAGES, FIRST 13 ARE
POSITIVE THEN TNF NEGATIVE
BEFORE YOU ACTUALLY GET HEALING
AND YOU CAN SEE HERE, YOU GET
IMPAIRED HEALING BUT EVENTUALLY,
A BURN WOUND DOES HEAL.
WHAT YOU CAN SEE HERE WITH TNF,
ASTERISKS FIRST IT'S ON, THEN
IT'S OFF PRIOR TO HEALING AND
THEN CAN YOU SEE HERE, AGAIN,
NEUTROPHIL INFLAMMATION'S HIGH
WHEN THE MACROPHAGES ARE 13 AT
POSITIVE AND IT RESOLVES AS THEY
TURN TNF NEGATIVE AND THEN THE
TISSUE HEALS.
SO WHEN SHE LOOKED AT THE
METABOLT-ONTIC PROFILE HERE,
MACROPHAGES THAT WERE BURNED
WERE MORE OXIDIZED EARLIER WITH
RESOLUTION BY 72 HOURS SO AGAIN,
TNF ALPHA POSITIVE HIGH
MACROPHAGES HAVE LOW NADPH
LIFETIME AND LOW REDOCKS RATIOS.
AND THIS IS SUMMARIZED HERE, I
GUESS I WENT THROUGH THIS SO I
WON'T GO THROUGH IT IN DETAIL
BUT JUST THIS WHOLE IDEA THAT
THIS IS A DYNAMIC CHANGE OF
ACTIVATION AND TISSUES THAT CAN
BE READ OUT BY LOOKINGA THE
LIFETIMES.
SO CAN WE MODULATE MACROPHAGE
ACTIVATION AND DETECT
CORRESPONDING MET A--BITS OLDER
PEOPLIC CHANGES WITH FLIM, WE
FOUND A REDUCED REDOCKS RATIO
NADPH LIFETIME, MORE NEUTROPHILS
IN THE TISSUES AND ON THE OTHER
EXTREME SHE TREATED WITH MET
FORMIN WHICH LEADS TO N2
MACROPHAGES AND REDUCED POSITIVE
CELLS AND FEWER NEUTROPHILS AND
ELECTRIC TO SEE WHAT LAPPED TO
WOUND HEALING.
AND THAT WAS A METABOLIC SWITCH
IN MACROPHAGES THAT CORRELATES
TO NEUTROPHIL INFLAMMATION AND
WOUND HEALING.
SO WHAT YOU CAN SEE HERE IS WITH
MET FORMIN, THE WOUNDS HEAL
BETTER WITH THE STAT 6 MUTANT,
THE WOUNDS HEALED POORER.
SO THIS SUPPORTS THE IDEA THAT
WE CAN ACTUALLY READ OUT DYNAMIC
CHANGES IN MACROPHAGE PHENOTYPE
BY USING FLIM.
SO HOW DOES MYELOID DRIVE GROWTH
FACTOR EFFECT MACROPHAGE
ACTIVATION?
THIS IS VERY PRELIMINARY DATA.
ONE THING WE FOUND IS THAT TNF
ALPHA IS NOT INCREASED IN
MACROPHAGES AND MYELOID FACTOR
MUTANT SO WE THINK THEY'RE MORE
ACTIVATED BUT WE CAN'T DETECT
THIS WITH THE TNF ALPHA REPORTER
BUT IN VERY PRELIMINARY DATA
WHERE SHE DID THE FLEM ANALYSIS,
SHE FOUND THAT NADPH LIFETIME
WAS REDUCED EVEN THOUGH WE
COULDN'T PICK THIS UP WITH THE
TNF ALPHA REPORTER AND THAT
THERE WAS A DECREASE ALSO--SORRY
THE REDOCKS RATIO WAS REDUCED
AND THE NADPH LIFETIME, SO THESE
MACROPHAGES ARE MORE OXIDIZED
WITH RECREASED LIFETIME.
SO, WHAT OTHER MARKERS CAN WE
USE TO TRY TO READ OUT
MACROPHAGE PHENOTYPES AND WHAT
DOES THIS MEAN FOR INFLAMMATION?
SO 1 THATY WOO WERE INTERESTED
IN WAS IMMUNO RESPONSIVE GENE 1,
IRG 1 SO IT'S AN ENZYME THAT
LEADS TO GENERATION, AND
ITACONNATE AND IT CAN BE
ANTIMICROBIAL AND INFLAMMATORY
AND IN ZEBRA FISH CHRIS HALL'S
LAB REPORTED ZEBRA FISH GFP 1
REPORTER LINE THAT GOES ON WITH
INFECTION.
SO WHAT HAPPENS WITH STERILE
INJURY?
SO WHAT HAPPENS WITH STERILE
INJURY IS WHAT VERONICA FOUND IS
THAT A SUBSET OF MACROPHAGES
TURN ON IRG 1.
SO YOU CAN SEE THAT WITH
THE--WITH THE MACROPHAGES THAT
TURN WHITE HERE ARE THE
MACROPHAGES THAT HAVE TURNED ON
IRG 1.
NOT ALL OF THEM, JUST A SUBSET
OF THEM.
HOW DOES MYELOID DRIVE GROWTH
FACTOR AFFECT THIS PHENOTYPE.
WHEN YOU LOOK AT CONTROL MORPH O
LINE O VERSUS MYELOID DRIVE
GROWTH FACTOR, WHAT YOU SLEEP
APNEA AND OBESITYY IS THESIS
MACROPHAGES TURN ON IRG 1, SO
THERE'S MUCH MORE IRG 1 REPORTER
ACTIVITY WHEN YOU DEPLOAT
MYELOID DRIVE GROWTH FACTOR SO
THIS IS A DIFFERENT TYPE OF
ACTIVATED MACK RAPHAGE.
SO JUST--MACROPHAGE SO JUST AS A
HUMBLING WAY OF SAYING THESE
THINGS ARE VERY COMPLICATED,
MYELOID GROWTH FACTOR ACTIVATES
AND DRIVES METABOLISM SO ARE
THERE NEUTROPHIL CHANGES DUE TO
THE CHANGES IN MACROPHAGES OR IS
THERE SOME NEUTROPHIL INTRINSIC
ASPECT OF THIS AND WE DON'T KNOW
THE ANSWER TO THAT.
SO--WELL I GIVE A BIT OF DATA I
THINK IS INTRIGUING.
SO MACK RAPHAGE ACTIVATION
ASSOCIATE WIDE METABOLIC
REPROGRAMMING INVIVO, WE ARE
INTERESTED IN THE CROSS TALK
BETWEEN THESE DIFFERENT TYPES OF
MACROPHAGES AND NEUTROPHILS AND
HOW IT PLAYS A ROLE IN
INFLAMMATION, SO HOW CAN WE
FURTHER GO AFTER THIS PATHWAY.
WELL, 1 WAY IS TO LOOK AT
DIFFERENT TYPES OF TISSUE DAMAGE
AND THIS IS IN THE CASE OF
MELANOMA.
RICHARD WHITE'S LAB SOME
BIOARCHIVES PAPER AND WE
COLLABORATED WITH THEM TO LOOK
AT WHAT HAPPENS TO MYELOID
DRIIVE GROWTH FACTOR IN THE
TUMOR MICROENVIRONMENT AND YOU
CAN SEE THAT MYELOID DERIVED
GROWTH FACTOR IS DRAMATICALLY
INCREASED.
SO GAYATHRI, YOU A POST DOC IN
THE LAB AND TRYING FIGURE OUT
WHAT THIS MEANS AND THE ISHT
ACTION BETWEEN NEUTROPHILS AND
MACROPHAGES.
TO ALSO GO AFTER THIS QUESTION,
WE HAVE GENERATED HIMAVEN IPS
CELLS THAT ARE DEFICIENT IN
MYELOID DERIVED GROWTH FACTOR
AND IN COLLABORATION WITH
IGOR WE HAVE GENERATED HEPATITIS
EAT O FILLS THAT ARE INTERESTING
FOR PURPOSES SO GENERATING
NEUTROPHILS THAT MIGHT HAVE
FUNCTIONS THAT WILL BE USEFUL
LIKE ENGINEERING, NEUTROPHILS TO
TREAT VARIOUS HUMAN DISEASES,
AND SHIP THEM TOWARDS AN
ANTITUMOR PHENOTYPE FOR EXAMPLE,
CAR NEUTROPHILS VERSUS A PRO
TUMOR PHENOTYPE.
AND THEN GOING FORWARD, TO TRY
TO GET MORE AT THIS PATHWAY, IN
ADDITION TO UNDERSTANDING OTHER
MECHANISMS THAT ARE INVOLVED IN
NEUTROPHIL BEHAVIOR AND
MACROPAGE BEHAVIOR AND TISSUE,
WERE INTERESTED IN UNDERSTANDING
THE DIVERSITY OF MACROPHAGES AND
INTERSTITIAL TISSUES USING LIECH
IMAGES AND MORE SINGLE CELL
ANALYSIS AND AS WELL AS SINGLE
CELL TRANSCRIPT OHMICS AND THIS
IS DONE IN COLLABORATION WITH
[INDISCERNIBLE] AN ASSIST ABT
PROFESSOR AT UW MADISON.
AND SO, WHEN YOU LOOK AT THE
BEHAVIOR OF NEUTROPHILS AND
MACROPHAGES AND RESPONSE TO A
BURN WOUND.
YOU CAN SEE THAT THE MACROPHAGES
AND NEUTROPHILS BEHAVIOR
DIFFERENTLY, MACROPHAGES DON'T
LIKE TO GO INTO THE BURN TISSUE
WHEREAS NEUTROPHILS MIGRATE VERY
RAPIDLY WITHIN THIS BURN TISSUE.
SO WHAT'S GOING ON HERE, WHEN
YOU BURN THE TAIL OF A ZEBRA
FISH AND CONTROL, WHEN YOU CUT
OFF THE TAIL, YOU SEE THIS NICE
COLLAGEN ORGANIZATION BY SECOND
HARMONIC IMAGING BUT WHEN YOU
BURN THE TAIL, YOU LOSE THIS
ORGANIZED COLLAGEN.
SO AS I MENTIONED TO YOU
EARLIER, NEUTROPHILS ARE CLASSIC
AMOEBOID CELLS THEY MIGRATE
INDEPENDENT OF INDEPENDENT
MEDIATED HESION AND AND MIGRATE
IN THIS COLLAGEN FREE ZONE WHERE
THESE ARE MORE MEASEN CHIMAL AND
MIGRATION IS SIGNIFICANTLY
IMPAIRED SO THIS IS THE SORT OF
SINGLE CELL BEHAVIOR, GROUP
BEHAVIOR THAT WE'RE DOING MORE
OF, BUT IN ADDITION IN THIS SAME
BURN MODEL WHAT WE'VE DONE S&P A
SINGLE CELL RNA SEQ ANALYSIS OF
NEUTROPHILS AND MACROPHAGES AND
THIS IS VERY PRELIMINARY NEW
DATA THAT'S COME OUT BUT IT'S
VERY INTERESTING THAT WHEN YOU
LOOK 24 HOURS AFTER BURN, YOU
CAN SEE THAT THEIR DISTINCT
POPULATIONS OF NEUTROPHILS, AND
DISTINCT POPULATIONS OF
MACROPHAGES THAT ARE SEPARABLE,
WHAT WAS ALSO INTERESTING IS
THERE'S SHIFT IN THE POPULATIONS
WITH A BURN WOUND AND IN
PARTICULAR ALL THE NEUTROPHIL
SUBSETS WERE INCREASED WITH THE
BURN WOUND BUT THE MACROPHAGE
SUBSETS MANY OF THEM WERE
DECREASED, NOT ALL OF THEM.
SO CAN WE USE THIS TO TRY TO
UNDERSTAND MORE OF WHAT'S GOING
ON WITH NEUTROPHIL BEHAVIOR
WITHIN TISSUES AND SO, AN
EXAMPLE OF LOOKING AT A RECEPTOR
LIGAND INTERACTION IS IL6 AND
IL6 RECEPTOR.
SO, IN CONTROL WHAT YOU CAN SEE
IS THAT THE MACROPHAGES EXPRESS
IL6 AND THE NEUTROPHILS EXPRESS
THE IL6 RECEPTOR.
SO, SO--SORRY HERE'S THE
NEUTROPHILS EXPRESS THE IL6
RECEPTOR SO THE LIGANDS BEING
PRODUCED BY MACROPHAGES AND THEN
THE NEUTROPHILS EXPRESS THE
RECEPTOR AND WITH BURN, THE
RECEPTOR IS SIGNIFICANTLY
UPREGULATE IN THE NEUTROPHIL
POPULATION AND THERE'S SOME DOWN
REGULATION OF IL6, IN THE
MACROPHAGES, 24 HOURS AFTER A
BURN.
SO WHAT DOES IL6 RECEPTOR
DEPLETION DO TO NEUTROPHIL
RESPONSES TO A BURN WOUND?
SO YOU MIGHT PREDICT THAT THE
NEUTROPHIL MACROPHAGE PENE O
TYPE WOULD BE VERY DIFFERENT
BECAUSE THE MACROPHAGES DO NOT
EXPRESS THE IL6 RECEPTOR.
INDEED THIS IS WHAT WE FOUND.
SO IN CONTROL, YOU SEE NICE
RECRUITMENT OF BOTH NEUTROPHILS
AND MACROPHAGES.
WHEN WE USE AN IL6 RECEPTOR
MUTANT, YOU CAN SEE THAT
NEUTROPHILS ARE NO LONGER
RECRUITED TO THE WOUND SITE
ALTHOUGH MACROPHAGES ARE
RECRUITED.
AND THIS IS QUANTIFIED EAR WHERE
YOU CAN SEE THAT NEUTROPHILS AND
MACROPHAGES ARE BOTH RECRUITED.
WHEREAS IN IL6 RECEPTOR MUTANT,
NEUTROPHIL RECRUITMENT IS
SIGNIFICANTLY IMPAIRED.
SO THIS JUST SUPPORTS THE POWER
OF THE SYSTEM TO TRY TO START
UNDERSTANDING SOME OF THESE
RECEPTOR LIGAND INTERACTIONS.
SO WHAT HAPPENS WITH MYELOID
DRIVE GROWTH FACTOR AND AGAIN
THIS IS DATA WE JUST PULLED OFF
SO IT'S VERY PRELIMINARY 1 OF
THE PUTATIVE RECEPTORS WOULD BE
THEICATE O RECEPTORS AND WE
LOOKEDDA THE ALL THEICATE O
RECEPTORS VERY INTERESTINGLY WAS
UP REGULATED IN THE SAME
NEUTROPHIL POPULATIONS THAT
UPREGULATED MYELOID DRIVE GROWTH
FACTOR WITH BURN.
SO IT'S INTERESTING TO THINK
ABOUT WHAT THIS MEANS?
SO DOES IT MAKE THIS RECEPTOR
AND SIGNAL TO THE RECEPTOR IN AN
AUTOKRIEN SIGNALING PATHWAY AND
IN MACROPHAGES ONLY 1 EXPRESSED
GROWTH FACTOR, THEY ALSO
EXPRESSED IRG 1 AND THEN AND
THEN WITH A BURN WOUND THEY ALSO
EXPRESSED THE KADEL RECEPTOR BUT
IT WAS NOT CHANGED WITH BURNS.
SO THESE ARE THE QUESTIONS WE
ARE GOING AFTER NOW.
WE CAN USE BOTH THE INVITRO
SYSTEM AND THE ZEBRA FISH SYSTEM
TO UNDERSTAND THESE
INTERACTIONS.
SO IT'S MORE OF A QUESTION OF
FUTURE RESEARCH TO UNCOVER
WHAT'S GOING ON HERE.
SO JUST TO END I WOULD LIKE TO
SAY THAT WE'RE VERY INTERESTED
IN THE BALANCE BETWEEN
NEUTROPHIL INFLAMMATION AND
REPAIR, THE FACTORS LIKE MYELOID
DERIVED GROWTH FACTOR THAT
IMPACT WHAT'S GOING ON AT
STERILE INJURY SITES TISSUE
DAMAGE, BUT NOT IN THE CASE OF
PATHOGEN OR INFECTED TISSUE.
AND THIS MAY BE A USEFUL TOOL TO
LIMIT INFLAMMATION, BUT WE STILL
DON'T KNOW WHETHER THIS IS
SECONDAR TOW THE MACROPHAGES
THAT ARE MAKING IT OR IS IT IN
FACT, SOME OF IT INTRINSIC TO
THE NEUTROPHIL, SO THESE ARE THE
SORTS OF QUESTIONS WE'RE
INTERESTED IN GOING AFTER SO I
WOULD LIKE TO JUST END AND
ACKNOWLEDGE MY RESEARCH GROUP
AND I WORK WITH A FANTASTIC
GROUP OF PEOPLE, MY LONG-TERM
MEMBERS, DAVID AND JULIE,
DAVID'S BEEN IN THE LAB FOR OVER
20 YEARS AND JULIE FOR OVER 12
YEARS HAVE BEEN AMAZING TO WORK
WITH, AND THEN THE WHOLE TEAM OF
GRADUATE STUDENTS AND POST DOCS,
MANY OF WHOM I ACKNOWLEDGED
DURING THE TALK AND HERE ARE
FORMER POST DOCS WHO NOW HAVE
THEIR OWN RESEARCH GROUPS.
AND COLLABORATORS WHO I
ACKNOWLEDGED DURING THE TALK.
I'D LIKE TO END THERE AND ASK IF
THERE ARE ANY QUESTIONS.
SO WHICH NADPHOX INHIBITOR
DID YOU USE IN THE ZEBRA FISH
WOUND/INFECTION MODEL THAT COMES
FROM AMELIA?
>> WE USED DPI SO THIS IS A VERY
COMMON IN NADPHOX DACE
INHIBITOR.
IF YOU DEPLETE P22 FOX YOU COULD
END UP GETTING MORE NEUTROPHIL
INFLAMMATION, AND SO, IT'S--IT'S
COMPLICATED HOW--WHAT THE SOURCE
IS OF THE NADPH AND THE IMPACT
ON INFLAMMATION.
>> THIS WAS REALLY WONDERFUL, I
HAVE 1 QUESTION REGARDS TO THE
MILEIN DERIVED GROWTH FACTOR.
YOU SHOWED THIS IS IMPORTANT FOR
HEALING OF THE BURN WOUNDS YOU
DID MENTION BRIEFLY THAT IT IS
NOT AS IMPORTANT FOR THE
INFECTIOUS WOUNDS.
HOW DOES IT ACTUALLY--WHAT ROLE
DOES IT PLAY OR HAVE YOU LOOKED
AT ITS ROLE OF OTHER STERILE
FORMS OF INFLAMMATION THAT ARE
NOT WOUNDS?
>> WITH REG TAIL TRANSSECTION,
IT'S ALSO IMPORTANT FOR HEALING.
>> SO IT'S THE CUTTING AND THE
BURN.
>> THE CUT AND THE BURN.
WE HAVEN'T LOOKEDDA THE OTHER
DAMAGE MODELS YET.
>> OR INFLAMMATORY STERILE
INFLAMMATION,.
>> YEAH WE HAVEN'T LOOKED AT
OTHER MODELS BUT I'M PREETY
EXCITED ABOUT IT AS A POTENTIAL
TARGET AND THERE'S OBVIOUSLY
GROWING INTEREST IN IT FROM ITS
IMPACT ON MY O CARDIAL
INFARCTION.
>> SO ANNA, THAT WAS REALLY
TERRIFIC.
YOU TALKED A LITTLE BIT ABOUT
THE ROLE OF VARIOUS CYTOKINES,
TNF AND DENDRITIC CELL ROUGH
ATOM LUKEIN 6 IN SOME OF THESE
PROCESSES, NEUTROPHIL AND
MACROPHAGE MEDIATED WOUND
HEALING FOR EXAMPLE, AND WE USE
NOW, CLINICALLY, A LOT OF
INHIBITORS OF TNF AND IL6 AND
OTHER CYTOKINES AND SO I JUST
WONDERED IF YOU HAD GIVEN ANY
THOUGHT TO OR LOOKED AT THE ROLE
OF SOME OF THESE INHIBITORS WE
USE CLINICALLY, AND THINKING
ABOUT WHAT EFFECT THEY MIGHT
HAVE ON WOUND HEALING IN
PATIENTS.
>> YEAH, IF IT'S A TRICKY THING
WITH ZEBRA FISH BECAUSE
SOMETIMES YOU DON'T NECESSARILY
GET CROSS REACTIVITY OF THESE
ANTIBODIES, BUT THE ZEBRA FISH
FORM OF THE CYTOKINES.
THE INTERESTING THING WITH IL6,
INHIBITION IS IT TOTALLY
INHIBITS NEUTROPHILS BUT IT DID
NOT INHIBIT WOUND HEALING WHICH
IS INTERESTING.
IN THE CASE OF TNF, IT IS ALSO
COMPLICATED, THERE'S A LOT OF
ISSUES WITH TOO MUCH OR TOO
LITTLE TNF SO IT'S NOT SUCH AS
EASY EXAMPLE OF SAYING, YOU KNOW
INHIBITING TNF IS NECESSARILY
BAD FOR WOUND HEALING.
SO THIS IS THE THING THAT'S
REALLY HARD IS THAT THERE'S
THESE SPECIFIC CELL TYPES THAT
MAKE THESE PRODUCTS BUT THEY CAN
FLIP EITHER WAY IN TERMS OF
THEIR IMPORTANCE WHICH MAKE ITS
SO CHALLENGING.
NOW WE KNOW TNF INHIBITION IS
INCREDIBLY EFFECTIVE FOR
TREATING OUR PATIENTS, AND IT
DOESN'T HAVE MANY SIDE EFFECTS,
RIGHT?
>> YEAH, YEAH.
>> ALL RIGHT, WELL, ANYWAY, IT'S
REALLY INTERESTING.
>> THANKS.
>> FASCINATING YES.
>> ANY OTHER QUESTIONS?
>> I HAD A QUESTION.
>> WOBDERFUL TALK BY THE WAY,
HICURIOSITY RELATED TO WHAT YOU
MENTIONED ABOUT MACROPHAGES
VERSUS NEUTROPHIL AND THE
COLLAGEN AND THE ISSUE, HOW IS
IT DIFFERENT WHEN YOU HAVE
INFECTION?
BECAUSE INITIALLY YOU SHOWED AN
EAR INFECTION WHERE A LOT OF
NEUTROPHIL WAS MIGRATING BUT ARE
THERE ECM CHANGES WHICH ARE
SIMILAR OR?
>> SO WE HAVE LOOKED AT INFECTED
WOUNDS AND YOU STILL HAVE
ORGANIZED COLLAGEN IN AN
INFECTED WOUND AND IT'S A GOOD
QUESTION BECAUSE YOU HAVE TONS
OF MMPs AND THINGS THAT ARE
PRODUCED SO YOU MIGHT THINK THAT
YOU ACTUALLY WOULD LOSE THAT
ORGANIZED COLLAGEN, IT CHANGES
BUT YOU STILL HAVE ORGANIZED
COLA SHEN.
>> BUT NEUTROPHILS DON'T REALLY
CARE ABOUT IT.
>> THEY DON'T SEEM TO REALLY
CARE AND MACROPHAGES SEEM TO
CARE MORE.
>> THANK YOU SO MUCH.
>> YES.
>> I HAVE A QUESTION, SO--YOU
KNOW, CLINICALLY, WE HEAR A LOT
THESE DAYS, PROBABLY A LOT MORE
THAN IN THE PAST ABOUT
MACROPHAGE ACTIVATION SYNDROME
AND IN A WHOLE VARIETY OF
DISORDERS THAT ARE--I MEAN
THEY'RE ALL IMMUNE SYSTEM
DISORDERS BUT THEY'RE OTHERWISE
NOT PARTICULARLY MECHANISTICALLY
RELATED TO 1 ANOTHER, DO YOU
THINK--I MEAN, I GUESS I'M
HAVING TROUBLE DECIDING IF THE
NEUTROPHIL ACTIVATES THE
MACROPHAGE AND THE MACROPHAGE
EFFECTS ALL OF THE SYMPTOMS OR
IS IT THE OTHER WAY AROUND IS
THAT THE MACROPHAGE GETS
ACTIVATED AND THE BAD ACTOR IN
THESE DISORDER SYSTEM ACTUALLY
THE NEUTROPHIL?
>> YEAH, THAT'S A VERY GOOD
QUESTION.
I THINK OFTEN THE BAD ACTOR IS
THE NEUTROPHIL BUT OFTEN THE
MACROPHAGE IS ENCOURAGING THAT
BAD BEHAVIOR, SO THE PARTNERS IN
CRIME, AND ESSENCE, BUT I THINK
THERE'S NO QUESTION THAT
THERE'S--THIS IS CIRCULAR,
RIGHT?
AND THE THING THAT'S SO
COMPLICATED IS THAT NEUTROPHILS
ARE NOT ALL THE SAME, RIGHT?
THEY CAN BE GOOD OR BAD FOR
WOUND HEALING, CANCER,
MACROPHAGES ARE THE SAME WAY.
SO WHAT IT IS IS THE MORE WE'VE
LEARNED, THE MORE COMPLICATED,
YOU KNOW IT'S MORE HUMBLING,
DOES THAT MAKE SENSE?
THAT'S WHAT I'M FINDING BUT
YEAH.
>> YOU SHOWED THE STRUCTURALLY
ARE OF MIDGIFT, NO?
WHICH IS A C-TERMINAL AND IF IT
IS DEPLETED OR REMOVED IT DOES
NOT, IT HAS ITS EFFECT MY
QUESTION IS WHICH OF THESE
PARTNERS INTERACKING WITH THE
C-TERMINAL AND HAVE YOU HAD ANY
OF THEM?
FOR THE MYELOID DERIVED FACTOR.
>> SO YES, THEY BIND TO THE KDEL
ON THE C-TERMINUS OF THE MYELOID
GROWTH FACTOR SO, YOU KNOW WE
WILL HAVE TO SEE, WE THOUGHT OF
KDEL RECEPTOR AS BEING ER REDENT
RECEPTOR, AND THEN WHEN WE GOT
THE SEQ DATA AND THEY WERE IN
THE SAME EXACT NEUTROPHIL
SUBSETS AND UPREGULATED, I WENT
HUH.
MAYBE THIS IS ACTING AS A
RECEPTOR MORE BROADLY BECAUSE
THIS IS SHOWING THAT THE SECTORS
CAN TRAFFIC TO THE CELL SURFACE
SO THEN IT WOULD BE POSSIBLE
THAT THEY RECOGNIZE SECRETED
MYELOID DRIVE GROWTH FACTOR AND
THAT COULD DAMPEN NEUTROPHIL
RESPONSES DIRECTLY SO
IT'S--YEAH.
INTERESTING.
>> TELL BE NICE TO HAVE THIS
COMPLEX ISOLATED AND THEN STUDY
IT.
>> RIGHT.
RIGHT.
AND THAT'S--YEAH.
LOTS OF WORK TO DO.
>> VERY GOOD.
WELL, I THINK WE SHOULD THANK
DR. HUTTENLOCHER AGAIN FOR A
WONDERFUL SEMINAR AND A GREAT
DISCUSSION, THANK YOU ALL FOR
COMING.
>> THANK YOU FOR COME NOTHING
PERSON, GUYS.
>> VERY COOL TO SEE EVERYBODY.