GREETINGS AND SALUTATIONS!
I'M DAN KASTNER, THE SCIENTIFIC
DIRECTOR EMERITUS OF THE
NATIONAL HUMAN GENOME RESEARCH
INSTITUTE.
AND STILL ACTIVE PHYSICIAN,
SCIENTIST, IN THE INTRAMURAL
RESEARCH PROGRAM OF THE NIH.
IT IS MY ENORMOUS PLEASURE TO
WELCOME YOU TO THIS, THE 16th
INSTALLMENT IN THE 2025 SEASON
OF DEMYSTIFYING MEDICINE.
AN EXCITING COURSE THAT BRIDGES
DEVELOPMENT IN BIOLOGY,
ENGINEERING, COMPUTER SCIENCE,
WITH MEDICINE.
YOU CAN SEE IN THE UPPER
RIGHT-HAND CORNER OF THE SLIDE
THE ICONIC IMAGE OF THE
CONSTRUCTION OF THE BROOKLYN
BRIDGE, BACK IN THE LATE
1800s, UP ON THAT CATWALK YOU
CAN SEE A PHYSICIAN, PROBABLY
FROM MANHATTAN, AND A BASIC
SCIENTIST FROM BROOKLYN, HAVING
A DISCUSSION UP ON THE CATWALK
TALKING ABOUT THE LATEST
DEVELOPMENTS IN PHAGOCYTOSIS AND
THE ACTIONS OF ANTIBODIES.
AND AS IT WOULD HAPPEN, TODAY
WE'RE GOING TO BE TALKING ABOUT
A RELATED TOPIC, NEUTROPHIL
NETs, AND JUST BEFORE WE GET
INTO THAT LET ME REMIND YOU THAT
THIS SERIES IS ONE THAT OCCURS
TUESDAY AFTERNOONS FROM 3 TO 5
IN THE AFTERNOON JANUARY THROUGH
MAY.
YOU CAN WATCH ON VIDEOCAST AT
THE LINK SHOWN ON THE SCREEN,
FOR THOSE OF YOU WHO WANT
CONTINUING MEDICAL EDUCATION
CREDIT THE CODE IS 58795.
AGAIN, THAT'S 58795.
DURING THE TWO PRESENTATIONS,
AND WE'LL HAVE TWO PRESENTATIONS
AND THEN A LIVELY DISCUSSION
SESSION FOR THE LAST HALF HOUR
OR SO, BUT IF YOU HAVE QUESTIONS
THAT OCCUR TO YOU DURING THE
PRESENTATIONS, PLEASE USE THE
"SEND LIVE FEEDBACK" BUTTON ON
THE VIDEOCAST DISPLAY.
ALL THE PREVIOUS SESSIONS ARE
ARCHIVED AT THE LINK SHOWN ON
THE SCREEN AND FOR ADDITIONAL
INFORMATION YOU CAN USE THAT
DEMYSTIFYING MEDICINE E-MAIL
ADDRESS.
OF COURSE, HERE WE ARE BRIDGING
SILOS AT NIH SINCE 2003.
MOVING ON TO OUR FIRST SPEAKER,
WHO WILL BE TALKING TO US ABOUT
NEUTROPHIL EXTRACELLULAR TRAPS,
NETs, WE HAVE THE GOOD DR.
ARTURO ZYCHLINSKY, WHO IS THE
DIRECTOR OF THE DEPARTMENT FOR
CELLULAR MICROBIOLOGY AT THE MAX
PLANCK INSTITUTE IN BERLIN.
OBTAINED A BACHELOR'S DEGREE, I
DON'T SPEAK SPANISH, IN MEXICO
CITY.
AND THEN HE RECEIVED HIS Ph.D.
IN IMMUNOLOGY AT THE ROCKEFELLER
UNIVERSITY.
HE DID AN EMBO POSTDOCTORAL
FELLOW AND MOVED TO SKIRBALL
BEFORE ASSUMING HIS CURRENT
POSITION AT DIRECTOR AT THE MAX
PLANCK INSTITUTE.
HE'S RECIPIENT OF A NUMBER OF
PRESTIGIOUS AWARDS, INCLUDING
THE IRMA T HIRSCHL AWARD, THE
GROHE AWARD, A MEMBER OF THE
GERMAN NATIONAL ACADEMY OF
SCIENCES LEOPOLDINA AND EUROPEAN
ACADEMY OF MICROBIOLOGY, WORKING
ON THE ROLE OF NEUTROPHIL
EXTRACELLULAR TRAPS IN HEALTH
AND DISEASE, ON THE
ANTIMICROBIAL CAPACITY OF
HISTONES AND EVOLUTION AND THEIR
FUNCTION IN GENE REGULATION AND
DURING MULTI-CELLULAR
DEVELOPMENT, AND HE'S ALSO BEEN
INTERESTED IN THE INFLAMMASOME,
AND ITS ACTIVATION.
AND HOW THE IMMUNE SYSTEM
MEASURES THE DANGER AND ADAPTS
ITS RESPONSE.
OUR SECOND SPEAKER IS DR.
MARIANA KAPLAN, FROM HERE AT THE
NIH, WHO IS GOING TO BE TALKING
TO US ABOUT FRIENDLY FIRE, HOW
NETs CONTRIBUTE TO
INFLAMMATION AND AUTOIMMUNITY.
SHE'S AN NIH DISTINGUISHED
INVESTIGATOR, DEPUTY SCIENTIFIC
DIRECTOR, AND CHIEF OF THE
SYSTEMIC AUTOIMMUNITY BRANCH AT
THE NATIONAL INSTITUTE FOR
ARTHRITIS, MUSCULOSKELETAL AND
SKIN DISEASES.
SHE'S ALSO AN ADJUNCT PROFESSOR
OF INTERNAL MEDICINE, UNIVERSITY
OF MICHIGAN AND ADJUNCT
PROFESSOR IN THE GRADUATE
PROGRAM IN IMMUNOLOGY AT THE
UNIVERSITY OF PENNSYLVANIA.
SHE OBTAINED HER MEDICAL DEGREE
FROM THE NATIONAL AUTONOMOUS
UNIVERSITY OF MEXICO, GOT HER
INTERNAL MEDICINE TRAINING AT
THE NATIONAL INSTITUTE OF
MEDICAL SCIENCES AND NUTRITION
IN MEXICO CITY, RHEUMATOLOGY
TRAINING AT THE UNIVERSITY OF
MICHIGAN.
SHE TOO IS THE RECIPIENT OF A
NUMBER OF PRESTIGIOUS AWARDS
INCLUDING THE HENRY KUNKEL YOUNG
INVESTIGATOR AWARD, EDMUND
DUBOISE MEMORIAL LECTURESHIP,
BASIC AND TRANSLATIONAL
INVESTIGATOR AWARD AND EVELYN
HESS AWARD FROM LUPUS FOUNDATION
OF AMERICA.
SHE'S ALSO A MEMBER OF THE
AMERICAN SOCIETY FOR CLINICAL
INVESTIGATION, ASSOCIATION OF
AMERICAN PHYSICIANS, AND
NATIONAL ACADEMY OF MEDICINE.
HER RESEARCH GROUP WORKS ON HOW
THE INNATE IMMUNE SYSTEM
PROMOTES AUTOIMMUNITY AND END OR
G
AN DAMAGE IN NETs, SYSTEMIC
AUTOIMMUNE DISEASE, NOVEL
THERAPEUTIC TARGETS TO PREVENT
PREMATURE VASCULAR DAMAGE, AND
SHE IS INVOLVED IN CLINICAL
TRIALS TO REDUCE BLOOD VESSEL
DYSFUNCTION AND MITIGATE ORGAN
DAMAGE.
WELL, THAT'S BEEN A MOUTHFUL,
BUT NOW IT'S TIME TO TURN THINGS
OVER TO OUR SPEAKERS, AND WE'LL
START WITH THE GOOD DR.
ZYCHLINSKY TALKING ABOUT
NEUTROPHIL EXTRACELLULAR TRAPS.
ARTURO, PLEASE, TAKE IT AWAY.
>> THANK YOU, DAN, FOR SUCH A
NICE INTRODUCTION.
IT'S A PLEASURE TO TALK HERE,
ESPECIALLY AS A COMPANION OF
MARIANA.
I WANTED TO DOUBLECHECK YOU CAN
SEE MY SCREEN.
>> INDEED.
>> OKAY, GOOD.
SO, I'M GOING TO TALK ABOUT
NEUTROPHILS, THE MOST ABUNDANT
WHITE BLOOD CELLS IN HUMAN
BLOOD.
THEY MAKE AROUND 70% OF ALL THE
WHITE BLOOD CELLS AND THEY ARE
ESSENTIAL FOR IMMUNITY.
PEOPLE OR KIDS THAT ARE BORN
WITH DEFECT IN NEUTROPHIL
BIOLOGY OR PEOPLE BECAUSE OF
CHEMOTHERAPY HAVE A LOW NUMBER
AND ARE SUSCEPTIBLE TO
INFECTION, ALSO NEUTROPHILS ARE
THE FIRST RESPONDERS AFTER
INFECTION.
SO IN THIS SLIDE I TRIED TO
ILLUSTRATE HOW A MICROBE STARTS
AN INFECTION BY BREAKING
EPITHELIAL BARRIER, RIGHT UNDER
THE EPITHELIUM THERE ARE CELLS
LIKE DENDRITIC CELLS AND MICRO
PHAGES SIGNAL THERE'S AN
INFECTION, PRO-INFLAMMATORY
CYTOKINES AND AND EXTRAVASITESA
HUNDRED-FOLD MORE EFFECTIVE AT
KILLING PHAGOCYTES LIKE
MACROPHAGES, SET UP THE STAGE
FOR THE RECRUITMENT OF MORE
NEUTROPHILS IF INFECTION IS NOT
CONTROLLED.
THE NEUTROPHIL IS EASY TO
RECOGNIZE IN BLOOD, IT HAS A
NUCLEUS THAT'S LOBULATED AS
OPPOSED TO MOST CELLS WITH
CIRCLE OR ELLIPSOID NUCLEUS.
NOW I'M SHOWING AN IMAGE OF A
NEUTROPHIL ISOLATED FROM HUMAN
BLOOD BY DENSITY CENTRIFUGATION.
ONE OF THE JOBS OF THE
NEUTROPHIL IS EAT BACTERIA, THIS
IS A CULTURE OF NEUTROPHILS,
INFECTED WITH SHIGELLA, YOU CAN
SEE HOW NEUTROPHIL SEES THE
BACTERIA, ENGULFS IT AND
GRANULES THAT CONTAIN
ANTIMICROBIALS, THIS IS
PHAGOCYTOSIS DESCRIBED OVER A
HUNDRED YEARS AGO, ONE OF THE
VERY EFFECTIVE WAYS HOW
NEUTROPHILS DISPOSE OF BACTERIA.
MANY YEARS AGO WE DESCRIBED A
SECOND WAY OF HOW NEUTROPHILS
CAN ACTIVATE AND KILL MICROBES,
BY FORMATION OF NETs.
NETs STANDS FOR NEUTROPHIL
EXTRACELLULAR TRAPS, A CULTURE
OF HUMAN NEUTROPHILS ISOLATED
AND ACTIVATED IN VITRO AND
STAINED BY IMMUNOFLUORESCENCE
WITH MARKERS.
I'M SHOWING PROJECTIONS.
YOU CAN SEE THAT WE'LL START
WITH ELASTASE, A PROTEASE THAT'S
EXPRESSED IN THE GRANULE OF
NEUTROPHILS, YOU CAN SEE THESE
NEUTROPHILS, NOT ACTIVATED, IT'S
IN THE GRANULES AND ALSO IN THE
NETs YOU CAN SEE HERE.
HISTONES ARE THE PROTEINS THAT
COVER CHROMATIN, COVER DNA TO
FORM CHROMATIN, AND YOU CAN SEE
NON-ACTIVATED NEUTROPHIL ONLY
THE NUCLEUS IS ACTIVATED AND YOU
CAN SEE HERE HOW CLEARLY
LOBULATED THE NUCLEI OF
NEUTROPHILS ARE, AND ALSO USE
DNA STAINS AND HERE YOU CAN SEE
THE LOBULATED NUCLEUS OF THE
INTACT NEUTROPHILS BUT YOU CAN
SEE CHROMATIN THAT IS DNA ALSO
PRESENT IN NETs.
NETS ARE COMPOSED OF CHROMATIN
WITH ADDITION OF SPECIFIC
PROTEINS THAT COME MOSTLY FROM
NEUTROPHILS.
ONE OF THE QUESTIONS THAT HAS
INTERESTED MY LAB FOR THE LAST
15 YEARS IS HOW THESE NETs ARE
MADE.
BECAUSE NETs, WE THINK THAT
CELL DEATH OF A CELL IS REQUIRED
FOR THE EXTRAVASATION OF
CHROMATIN SO WE WANTED TO
UNDERSTAND HOW THE CHROMATIN IS
EXPELLED FROM THE CELL INTACT.
SO, THROUGHOUT MY TALK I'LL USE
MOSTLY EXPERIMENTS DONE IN MY
LAB BY ISOLATING NEUTROPHILS AND
ONE OF THE FIRST THINGS WE FOUND
OUT IS THAT THE CELL ACTUALLY
HAS TO DIE IN ORDER TO RELEASE
ITS CHROMATIN CONTENT.
THIS IS ANOTHER MOVIE.
THIS IS, AGAIN, A CULTURE OF
HUMAN NEUTROPHILS.
YOU CAN SEE THE LOBULATED
NUCLEUS WITH DNA DYE THAT IS
HERE IN PINK.
AND IT CONTAINS TWO DNA DYEs,
ONE PERMITTABLE IN PINK, YOU CAN
SEE THE NUCLEUS ALREADY PINK.
THE SECOND IS DNA IMPERMEABLE IN
GREEN.
YOU CAN SEE HOW THE NUCLEUS GOES
THROUGH A SPECIFIC PROCESS IT
GETS SMALLER, IT EXPANDS AND
FINALLY ALL CHROMATIN IS
RELEASED FOR NETs AGAIN, I'LL
PLAY THIS MOVIE AGAIN SO YOU CAN
SEE, FOR EXAMPLE, HERE HOW THE
NUCLEUS CONTRACTS, EXPANDS,
FORMS NETs.
SO, THIS IS SIMILAR EXPERIMENT
AS THE ONE I SHOWED, A DIFFERENT
WAY OF LOOKING AT THE
NEUTROPHILS.
IN THIS CASE USING TRANSITION
ELECTROMICROSCOPY, A NEUTROPHIL
HAS NOT BEEN ACTIVATED.
YOU CAN SEE TWO OF THE MANY FOUR
OR FIVE LOBULATED NEUTROPHILS
HAVE AND YOU CAN ALSO SEE THE
DIFFERENT GRANULES THAT COME IN
DIFFERENT FLAVORS.
NOW, ONE HOUR AFTER ACTIVATION
ALL THE LOBULATION HAS BEEN
LOST, THERE'S VERY SPECIFIC
ALTERATION ALSO STARTING TO
HAPPEN, FOR EXAMPLE, TO THE
NUCLEAR MEMBRANE.
TWO HOURS AFTER ACTIVATION, NOW
WE CAN SEE THIS IS THE
CHROMATIN, THE REMNANT, NOW THE
CHROMATIN IS IN DIRECT CONTACT
WITH THE CYTOPLASM OF THE CELL.
YOU CAN SEE VESICLES, I'M TRYING
TO POINT OUT WITH MY CURSOR, AND
THESE ARE THE REMNANTS.
WHEN WE DO MICROSCOPE WE WITH
MARKERS WE CAN SEE THIS.
THIS IS THE MOMENT WHERE THE
CHROMATIN GETS IN TOUCH WITH
CYTOPLASM, GRANULES ARE
DISSOLVED, WE BELIEVE THIS IS A
TIME WHERE SPECIFIC PROTEINS ARE
ATTACHED TO THE CHROMATIN IN
ORDER TO FORM THE NETs.
THIS IS IMAGED THREE HOURS AFTER
ACTIVATION, THESE ARE THE
PRE-NETs, MODIFIED CHROMATINS
WE BELIEVE THE CELL WILL EXPLODE
VERY SOON TO RELEASE THE NETs
OUT OF THE CYTOPLASMIC MEMBRANE.
THIS IS A SCHEME I TRIED TO
AGAINST AGAIN SHOWING HOW THE
NUCLEUS FIRST IS COMPACTED, THEN
EXPANDED, AND HOW THE DIFFERENT
GRANULAR PROTEINS GET INTO THE
NUCLEUS TO EVENTUALLY MAKE
NETs.
SO, VERY EARLY ON, WE REALIZE
THAT REACTIVE OXYGEN SPECIES
WERE RELEVANT FOR NET FORMATION.
REACTIVE OXYGEN SPECIES ARE
PRODUCED BY SPECIFIC ENZYMES,
WHERE MOW MOLECULAR OXYGEN IS
CONVERTED, NEUTROPHIL IS VERY
EFFECTIVE, THE MOST EFFECTIVE
CELL IN THE BODY TO PRODUCE
PEROXIDE.
PEROXIDE MUTATES INTO HYDROGEN
PEROXIDE, THIS IS A REACTION
THAT HAPPENS NATURALLY BUT THERE
ARE TWO ENZYMES TO PROCESS THESE
MUTATIONS THAT ALLOWS THIS
REACTION.
FINALLY MELA PEROXIDASE CONVERTS
HYDROGEN PEROXIDE INTO
HYPOCHLORIC ACID, YOU ARE
FAMILIAR, USED TO DISINFECT, AND
ONE IS BLEACH.
THEY IMMEDIATELY OXIDIZE
PROTEINS, LIPIDS, NUCLEIC ACIDS,
EVERYTHING BY THIS VERY ACTIVE
OXIDANT.
WE THINK THESE MOLECULES ACT AS
PROTEIN MODIFIERS THAT ALLOW
THIS FUNCTION OF NETs.
COMING BACK TO NET FORMATION
NEUTROPHILS WITHIN HALF AN HOUR,
USING INHIBITORS NADPH OXIDASE
WAS REQUIRED.
IT'S INTERESTING TO CONFIRM
GENETICALLY, AND WE STARTED TO
WORK WITH PATIENTS WITH CHRONIC
DISEASE.
MUTATIONS IN ADPH OXIDASE CANNOT
PRODUCE SUPEROXIDE.
WHEN WE TAKE NEUTROPHILS FROM
THESE PATIENTS, YOU CAN SEE
NEUTROPHILS LOOK PERFECTLY WELL.
AND THIS IS CONVENTIONAL
MICROSCOPY, GRANULAR MARK
NUCLEAR ELASTASE, WHEN WE
ACTIVATE THESE CELLS THE CELLS
LOOK MUCH FLATTER, LIKE IN THE
MOVIE I SHOWED AT THE START OF
THE TALK OF THE NEUTROPHIL
INFECTED WITH BACTERIA BUT THE
NUCLEUS ARE COMPLETELY LOBULATED
AND THESE CELLS CANNOT MAKE
NETs.
AND IN ORDER TO COMPLEMENT THE
MUTATION IN THESE PATIENTS, WE
DID IT GENETICALLY AS I'LL SHOW
IN A SMALL ANECDOTE TOWARDS THE
END OF THE TALK.
WE HAVE ADD HYDROGEN PEROXIDE
AND SHOW NOW WE CAN MAKE THE
CGD, NEUTROPHILS MAKE NETs
BECAUSE WE'RE GOING AFTER THE
NADPH OXIDASE, COMPLEMENTING THE
PHENOTYPE.
WE PROPOSE BASED ON THIS GENETIC
DATA AND PHARMACOLOGICAL DATA
THAT THE NADPH OXIDASE WAS
REQUIRED FOR NET FORMATION, THE
STIMULI WE USED FOR NET
FORMATION ARE MAINLY MICROBES,
EITHER BACTERIA OR FUNGI, AND
THIS IS PATHWAY I'M DESCRIBING
ALSO TRUE FOR, FOR EXAMPLE, FOR
NITROGEN, THERE ARE OTHER FORMS
OF NET FORMATION THAT USE
TENTATIVE PATHWAY NOT WELL
DESCRIBED THAT DOESN'T REQUIRE
THE FORMATION.
BACK TO THE REACTIVE OXYGEN
SPECIES, WE ASSUME THAT THESE
NADPH OXIDASE WAS REQUIRED, ALSO
WOULD NEED THE PEROXIDASE
MUTATIONS, DEFECTS IN MILA
PEROXIDASE FUNCTION ARE COMMON,
QUITE OFTEN, BUT THESE ARE ONLY
HYPERMARKS.
MPO-DEFICIENT PATIENTS ARE RARE,
PHENOTYPE REMINISCENT OF CGD
PATIENT SUSCEPTIBLE TO
INFECTION.
WE MANAGE TO COLLECT A FEW
MPO-DEFICIENT PATIENTS AND COULD
SHOW THEY ALSO CANNOT MAKE
NETs.
THIS IS A CONTROL OF A HEALTHY
DONOR.
I HOPE YOU ALREADY RECOGNIZE THE
STRANDS ARE THE NETs THAT ARE
STAYING BOTH WITH GRANULAR
MARKER LIKE NEUTROPHIL ELASTIC
AND CHROMATIN MARK, THIS
ANTIBODY, BUT MPO DEFICIENT
PATIENTS THE NEUTROPHILS ARE
UNABLE TO MAKE NETs.
THE NEUTROPHIL ELASTIC IS STILL
IN THE GRANULES, NUCLEI ARE
STILL LOBULATED.
WE THING THE FIRST STEP REQUIRES
FORMATION. -- FORMATION OF ROS.
WE TAKE NEUTROPHILS ISOLATED
FROM HEALTHY HUMANS, WE CAN SEE
BEFORE ACTIVATION THE NEUTROPHIL
ELASTIC IS IN THE GRANULES,
AFTER 90 MINUTES OF ACTIVATION
WE CAN SEE THAT ELASTIC IS
STARTING TO MIGRATE INTO THE
NUKE
NUCLEUS.
WE DON'T UNDERSTAND HOW THE
NEUTROPHIL ELASTIC GETS OUT OF
THE GRANULES OR HOW IT GETS INTO
THE NUCLEUS, IT'S A SMALL
PROTEIN SO COULD PASS THROUGH
THE NUCLEAR PORE BUT THIS IS AN
AREA OF INVESTIGATION THAT WE
REALLY HAVEN'T PURSUED.
SO, I'M SHOWING THAT THE
NEUTROPHIL ELASTASE
TRANSLOCATES, AND WE TURNED TO
MICE TO TELL IF IT WAS REQUIRED
FOR NET FORMATION.
I'M ONLY GOING TO SHOW ONE PIECE
OF DATA.
AND THIS IS A SCANNING ELECTRON
MICROGRAPH OF THE LUNG OF MICE
INFECTED WITH KLEBSIELLA, QUITE
A DANGEROUS BACTERIA THAT CAUSES
PNEUMONIA AND WE'RE LOOKING AT
THE HIGH MAGNIFICATION OF THE
ALVEOLI.
YOU CAN SEE BACTERIA ARE COVERED
IN FIBROUS MATERIAL WHICH ARE
THE NETs.
WHEN WE INFECT NEUTROPHIL
ELASTIC KNOCKOUT MICE YOU CAN
SEE THE BACTERIA, AND WHAT YOU
CANNOT SEE IN THE NET, BUT IN
PARALLEL EXPERIMENT I'M NOT
SHOWING INFILTRATION OF
NEUTROPHILS INTO THE LUNG OF THE
NEUTROPHIL ELASTICS ELASTICS MT
EFFECTIVE, THEY ARE THERE BUT
UNABLE TO MAKE NETs.
SECOND STEP AFTER FORMATION OF
ROS TRANSLOCATION FROM GRANULES
INTO NUCLEUS ALLOWING THE
FORMATION OF NETs.
SO, I SHOWED YOU THIS IMAGE
BEFORE.
AND I MENTIONED HERE THE
CHROMATIN IS IN DIRECT CONTACT
WITH CYTOPLASM AND NUCLEAR
MEMBRANE HAS BEEN COMPLETELY
VACULATED AND DISAPPEARED.
ANOTHER EXAMPLE IN BIOLOGY, THIS
IS MITOSIS THROUGH CLASSICAL
CELL DIVISIONS, I SHOULD MENTION
NEUTROPHILS ARE TERMINALLY
DIFFERENTIATED CELLS THAT WILL
NOT DIVIDE, SHORT HALF-LIFE
MEASURED IN HOURS, NOT DAYS.
SO, A POSTDOC IN MY LAB PROPOSED
THE IDEA THAT THERE WERE COMMON
MECHANISMS IN THE FORMATION OF
MITOSIS AND NETOSIS.
DURING THE CELL CYCLE AFTER
MITOSIS THERE'S A PHASE HISTONES
AND DNA SYNTHESIZE, GOES INTO A
PHASE AND CELLS DIVIDE.
NEUTROPHILS ARE HERE IN THE G
SERA, TERMINALLY DIFFERENTIATED,
ESCAPE CELL CYCLE.
SO, ONE OF THE FIRST EXPERIMENTS
THAT WE DID WAS TO SHOW THAT
ACTUALLY MITOGENS CAN INDUCE,
THIS IS AN IMAGE, IF WE ACTIVATE
THE NEUTROPHILS CAN MAKE NETs
EFFECTIVELY.
THIS IS THE FIRST COMMONALITY
BETWEEN NET FORMATION AND
MITOSIS, BOTH CAN BE ACTIVATED
BY MITOGENS.
THERE'S A MARKER KI-67 FOR
PROLIFERATION, NOT EXPRESSED BY
NEUTROPHILS, THEY ARE NOT
DIVIDING.
WE TAKE NAIVE NEUTROPHILS, THE
DNA STAIN, THEY DON'T EXPRESS
Ki67.
WE SEE THE CLEAR ACTIVATION WHEN
WE ACTIVATE, THIS IS THE SECOND
CLUE THAT THE NEUTROPHILS ARE
USING THE MITOTIC PATHWAY IN
ORDER TO MAKE NETs.
ACTUALLY, THE Ki67 POSITIVITY
HAPPENS ALSO IN VIVO, THIS IS A
SECTION OF PATIENT THAT DIED OF
FUNGAL ABSCESS IN THE BRAIN,
GOOD AT RECRUITING NEUTROPHILS,
HERE WE'RE LABELING IN RED, MANY
IN THE AREA, AND WE'RE USING
ALSO Ki67 TO IDENTIFY
NEUTROPHILS, SO THERE ARE
NEUTROPHILS LIKE THIS ONE OR
THIS ONE, YOU CAN SEE THE
LOBULATED NEUTROPHIL AND NOT
ACTIVATED YET BUT ARE
NEUTROPHILS Ki67 POSITIVE, WE
BELIEVE ABOUT TO MAKE NETs IN
VIVO.
MITOSIS REQUIRES ACTIVATION OF
CYCLIN DEPENDENT KINASE AND THIS
IS AN EXPERIMENT WITH MICE, NOT
HUMAN CELLS.
IF WE TAKE NEUTROPHILS FROM
WILDTYPE OR KNOCKOUT MICE THEY
DON'T MAKE NETs BUT IF WE
ACTIVATE EITHER WITH MITOGEN OR
MICROBE WITH CANDIDA ALBICANS
THE NEUTROPHIL FROM WILD TYPE
MAKE NETS, SEVERELY HAMPERED IN
ANIMALS CDK 6 IS KNOCKED OUT.
THE FINAL EXAMPLE I SHOWED HOW
NET FORMATION REQUIRES MITOSIS
WE CAN SEE DIFFERENT TUBULES
MADE IN CELL BUT IN ACTIVATED
CELLS MAKE CENTROSOME, GOING TO
THE TWO SIDES OF THE NUCLEUS OF
THE CELL.
WE'VE NEVER SEEN MITOTIC
STIMULUS.
SOMEHOW THIS IS THE FORMATION OF
CENTROSOMES, WE'VE NOT FIGURED
OUT.
THIS IS A SUMMARY OF THIS PART.
WHEN WE ACTIVATE NEUTROPHILS TO
MAKE NETs, THEY EXPRESS
Ki67, THEY REQUIRE ACTIVITY OF
CYCLIN DEPENDENT KINASE AND
ESSENTIAL FOR MIGRATION BEFORE
THE FORMATION OF NETs.
IN ORDER TO SEE THE THE ACTUAL
FINAL EFFECTOR OF NETs WE DID
A LARGE SCREEN, LOOKED FOR
INHIBITORS OF NET FORMATION BY
ISOLATING NEUTROPHILS FROM
HUMANS AND THEN ACTIVATE TO MAKE
NETs WITH A MITOGEN IN
PRESENCE OR ABSENCE OF
INHIBITORS, SCREEN FOR
COMPOUNDS, CLASSICAL SCREEN,
SCREENED IN COLLABORATION WITH
INSTITUTE ALSO MAX PLANCK, AND A
COMPANY THAT DEPENDS ON THEM,
OVER 250,000 COMPOUNDS, WHICH IF
YOU THINK THE CELLS THAT WERE
ISOLATED FOR HUMANS AND THEY
ONLY LIVE FOR SIX HOURS, IT WAS
REALLY A VERY HEROIC EFFORT OF
THE PEOPLE WHO ACTUALLY MADE
THESE ASSAYS, I MENTIONED THEM.
WE FOUND SEVERAL INHIBITORS,
WORKING THROUGH THEM.
ONE OF THE INHIBITORS WE
IDENTIFY IS INHIBITOR OF
GASDERMIN THAT ALLOWS RELEASE OF
NETs, CONTROL CELL ACTIVATED
IN ABSENCE OF INHIBITORS,
SIMILAR TO ONE I SHOWED EARLIER.
IN THE PRESENCE OF GASDERMIN
INHIBITORS CELLS ARE STUCK AND
THERE IS SOME EXPANSION OF THE
NUCLEUS BUT VERY LITTLE NET
FORMATION.
SO, THIS IS WHERE WE ARE IN THE
PROCESS OF NET FORMATION.
AND WE SEE THAT THERE IS
REQUIREMENT OF ROS.
NEUTROPHIL ELASTICS MAKE IT TO
THE NUCLEUS, ALSO REQUIRED,
GASDERMIN-D IS ACTIVATED WHICH
ALLOWS NET FORMATION, AND THIS
IS HOW IT GOES.
THERE'S A RECEPTOR ACTIVATED,
FORMATION OF ROS, NPO,
NEUTROPHIL ELASTIC MIGRATES TO
THE NEUTROPHIL, CELL EXPANDS,
ACTIVATION OF GASDERMIN, FINALLY
NET FORMATION.
SO, NETs HAVE THREE
INDEPENDENT FUNCTION.
ONE OF THEM IS ANTIMICROBIAL
CELLS, VERY IMPORTANT IN KILLING
MICROBES.
SECOND ONE IS THEY ARE
PRO-INFLAMMATORY.
I'LL MENTION A LITTLE BUILT ON
THIS BECAUSE THIS IS WHERE I
THINK MARIANA IS GOING TO EXPAND
IN THE SECOND PART OF THIS
SEMINAR.
AND NETs ALSO HAVE A POWERFUL
PRO COAGULANT ACTIVITY, THE WORK
OF DENISE WAGNER, AND I WILL NOT
MENTION THAT FURTHER.
SO, THE ANTIMICROBIAL ACTIVITY
OF NETs HAS DIFFERENT EFFECTS,
THIS IS A SCANNING EM OF
NEUTROPHILS INFECTED WITH
SHIGELLA, WE MENTIONED GRAM
NEGATIVE THAT CAUSES DYSENTERY,
OR STAPHYLOCOCCUS AURA, GRAM
POSITIVE THAT CAUSES ALL KINDS
OF DISEASE LIKE IMPETIGO AND OH
OTITIS MEDIA, ET CETERA.
THEY PRESENT THE BACTERIA
USUALLY AN IONIC, NETS ARE
CATIONIC, AND PREVENT
DISSEMINATIONS SHOWN BY SEVERAL
LABS THAT MANY PATHOGENS HAVE
DEVELOPED THAT ALLOW THEM TO
ESCAPE.
THE SECOND FUNCTION IS KILL
MICROBES, WE ACTIVATE
NEUTROPHILS, BY FOUR HOURS NO
NEUTROPHILS ARE LEFT ALIVE.
WE INFECT CULTURES, WAIT 20
MINUTES, SEE HOW MANY BACTERIA
ARE ACTUALLY KILLED.
WHEN WE TAKE THESE EXPERIMENTS
AND DO IT IN THE PRESENCE OF
NETs, WE SEE THAT IN 20
MINUTES AROUND 25% OR 35% OF
CULTURE OF SHIGELLA OR
STAPHYLOCOCCUS ARE KILLED.
IF WE DO CONTROLLED EXPERIMENT
WHERE WE DESTROY THE NETs BY
DIGESTING WE DON'T SEE KILLING.
ALTERNATIVE WAY IS TO USE
ANTI-HISTONE ANTIBODIES.
SO HERE WE ALLOW THE NEUTROPHILS
TO MAKE NETs.
BEFORE INFECTING THEM WE USE
ANTI-HISTONE ANTIBODIES.
OR CONTROL ANTIBODY.
IF WE USE CONTROL ANTIBODY WE
STILL SEE KILLING WITH SOME OF
THE EXAMPLES THAT WE USE BUT
WHEN WE USE ANTI-HISTONE
ANTIBODY WE COMPLETELY BLOCK THE
ANTIMICROBIAL ACTIVITY OF
NETs.
I THINK THIS EXPERIMENT HAS TWO
POSSIBLE EXPLANATIONS, NOT
NECESSARILY MUTUALLY EXCLUSIVE.
ONE IS THAT THE ANTI-HISTONE
ANTIBODIES PREVENT BINDING OF
THE BACTERIA TO NETs, BINDING
IS REQUIRED.
ALSO SHOWS ANTI-HISTONES ARE
VERY IMPORTANT ANTIMICROBIALS,
ACTUALLY A DISCOVERY FROM THE
LATE 50s, THE ACTIVITY WAS
FIRST DESCRIBED BY HIRSCH, AND
THEY ARE PROBABLY THE MOST
POTENT ANTIMICROBIALS THAT
EUKARYOTES HAVE.
THEY KILL MICROBES, CALCIUM
MAGNESIUM.
SO I THINK IT'S INTERESTING TO
THINK WHY IN EVOLUTION HISTONES
EVOLVING EUKARYOTES AS
ANTIMICROBIALS AND IN ORDER TO
PROTECT LARGE EUKARYOTES.
SO ONE OF THE QUESTIONS THAT I
THINK IS RELEVANT TO US WHETHER
NETs ARE RELEVANT IN
INFECTION.
AND I WILL USE ONE EXAMPLE WITH
AN N-of-1, BUT I THINK IT'S A
VERY DRAMATIC VERSION.
THIS IS A SCANNING ELECTRON
MICROSCOPE.
WHAT YOU SEE HERE IS
ASPERGILLUS, HIGH C IS LONG,
NEUTROPHIL TRYING TO
PHAGOCYTOSE.
THE NEUTROPHIL WILL NOT BE
EFFECTIVE.
SO PHAGOCYTOSIS IS NOT EFFECTIVE
IN FUNGAL INFECTIONS.
THIS IS THE N-of-1.
THIS IS A PATIENT, CGD PATIENT,
I ALREADY MENTIONED CGD PATIENTS
HAVE MUTATIONS IN NADPA OXIDASE.
THIS KID WAS SUFFERING FROM A
SEVERE ASPERGILLUS INFECTION, HE
WAS IN A HOSPITAL IN SWITZERLAND
AND THE PHYSICIAN AND SCIENTIST
TAKING CARE OF THIS KID REACHED
OUT TO US.
THIS IS THE -- THE KID WAS VERY
SICK, THE DECISION WAS THAT THE
KID WOULD NOT SURVIVE BONE
MARROW TRANSPLANTATION, ONE OF
THE WAYS THEY ARE TREATED, AND
INSTEAD THE TEAM DECIDED TO DO
GENE THERAPY.
SO, THIS IS ANALYSIS OF THE
NADPH OXIDASE SUBUNIT WHERE THE
KID ARE MUTATION OR ROS, AND
CONTROLLED DONOR HAS NADPA
OXIDASE PRODUCES ROS, AFTER GENE
THERAPY THERE WAS A CHIMERISM,
30% OF CELLS STARTED EXPRESSING
SUBUNIT, AND MADE ROS.
NOW, WE ALSO ISOLATED THE
NEUTROPHILS FROM THIS PATIENT.
AGAIN, THE CONTROL DISEASE ONLY
A DNA STAIN, YOU CAN SEE THE
CONTROL MAKES EFFECTIVE NETs.
BEFORE GENE THERAPY THE PATIENT,
NUKE LEE
NUCLEI ARE LOBULATED, 30% OF NET
FORMATION AFTER GENE THERAPY OF
THIS KID.
AND WE ALSO ISOLATED THE
ASPERGILLUS STRAIN FROM THE LUNG
OF THIS PATIENT AND THEN WE
TESTED WHETHER THE NEUTROPHILS
WERE ABLE TO KILL THE
ASPERGILLUS.
THE CONTROL DONOR OF THE
NEUTROPHILS IS VERY EFFECTIVE AT
KILLING ASPERGILLUS IN THE
PRESENCE OF NETs IF WE DESTROY
THE NETs THEN IT'S MUCH LESS
EFFECTIVE AS KILLING
ASPERGILLUS.
BEFORE GENE THERAPY THIS PATIENT
WAS NOT ABLE , BUT AFTER ACQUIRD
ABILITY TO KILL ASPERGILLUS.
THESE ARE SITES OF INFECTION
WITH ASPERGILLUS, THIS IS BEFORE
GENE THERAPY AND TWO WEEKS AFTER
GENE THERAPY, YOU CAN SEE THE
INFECTION IS SOLVED.
SO THIS IS AN EXAMPLE OF HOW
RELEVANT OUR NETs ARE DURING
INFECTION.
AGAIN, STEPPING INTO WHAT
MARIANA IS GOING TO TALK ABOUT
NETs SEEM TO HAVE EVOLVED TO
CONTROL INFECTIONS, AND THEY ARE
IMPORTANT IN VIRAL BACTERIAL,
PARASITIC, FUNGAL INFECTIONS,
BUT AS MOST ASPECTS OF
IMMUNOLOGY THERE'S A DARK SIDE
OF AUTOIMMUNITY.
THIS IS PROBABLY BECAUSE NETs
ARE PRO-INFLAMMATORY.
THIS IS A MOVIE OF A CULTURE
WHERE WE MAKE MONOCYTES AND
NEUTROPHILS, SORRY, MACROPHAGES
AND NEUTROPHILS, THE NUCLEUS IS
BLUE, NEUTROPHILS ARE IN PINK.
CAN YOU SEE HOW WHEN WE ACTIVATE
THIS CULTURE THE MACROPHAGES
EATS THE NETs.
SO YOU CAN FOLLOW THE MACROPHAE
THAT WILL EAT THE CELL, WHEN IT
MAKES NETs.
WE'VE SHOWN THEN NET INSIDE THE
CELL ACTIVATE FORMATION OF THE
TRANSCRIPTION OF CYTOKINES,
IMPORTANTLY INTERFERON, USING
CRISPR/Cas COULD SHOW THAT
MACROPHAGES WHERE WE KNOCKED OUT
EITHER CGAS OR STING, ONE OF THE
PATHWAYS THAT NEUTROPHILS
INTRACELLULAR AACTIVATE IS
THROUGH CGAS AND STING.
PATHOLOGY OF NETs IS ALSO VERY
IMPORTANT, IT'S IMPORTANT IN
THROMBOSIS, MANY AUTOIMMUNE
DISEASES, AS MARIANA WILL TALK,
ATHEROSCLEROSIS AND CANCER
METASTASIS.
I'LL FINISH THIS PART OF THE
PRESENTATION BY REMINDING THAT
YOU NEUTROPHILS WHEN THEY
RESPOND TO MICROBES OR OTHER
MITOGENS GO THROUGH A PROGRAMMED
CELL DEATH, NETosis, ALLOWS
RELEASE OF NETs, NETS CAN KILL
MICROBES AND PROBABLY THEY
EVOLVE, PRO-INFLAMMATORY AND PRO
COAGULANT AND IF NETs ARE NOT
MADE AT THE RIGHT TIME AND BY
THE RIGHT CELLS OR THEY ARE NOT
DESTROYED EFFICIENTLY THEY CAN
CAUSE IMMUNITY, THERE'S MANY
PEOPLE'S -- I PRESENTED HERE THE
WORK OF MANY PEOPLE THAT I WILL
LEAVE THIS SLIDE FOR A MINUTE,
AND I WOULD LIKE TO THANK THE
MAX PLANCK SOCIETY WHO SUPPORTED
THIS WORK FROM THE START.
THANK YOU VERY MUCH.
>> THANK YOU.
WE'LL NOW MOVE TO MARIANA.
>> GOOD AFTERNOON.
I HOPE YOU CAN SEE MY SLIDES.
>> INDEED.
OKAY.
WONDERFUL.
THANK YOU.
THANK YOU VERY MUCH, DAN, ALSO
FOR THE KIND INTRODUCTION AND
INVITING US TO GIVE THIS TALK, A
PLEASURE TO BE ABLE TO GIVE THE
PRESENTATION FOLLOWING ARTURO.
I'M GOING TO FOCUS ON THE DARK
SIDE OF NETs.
AND TRY TO DISCUSS HOW WHEN
THESE DRAMATIC AND IMPORTANT
BIOLOGIC PROCESS OF NET
FORMATION GOES AWRY, IT MAY
CONTRIBUTE TO INITIATE CHRONIC
INFLAMMATORY CONDITIONS AND I
WILL PRIMARILY FOCUS ON SYSTEMIC
AUTOIMMUNE DISEASES, TOWARD THE
END OF PRESENTATION WILL TALK ON
THE ROLE OF NETs IN PROMOTING
VASCULAR DAMAGE, WHICH IS
COMPLICATION THAT IS FREQUENT IN
AUTOIMMUNE DISEASE AND OTHER
CHRONIC INFLAMMATORY CONDITIONS.
AND SO THIS IS A LIST OF WHAT WE
CALL SYSTEMIC AUTOIMMUNE
DISEASES, THEY ARE FASCINATING
CONDITIONS, VERY CHALLENGING TO
DIAGNOSE AND TO TREAT.
AND ONE SHARED OBSERVATION ABOUT
MANY SYSTEMIC AUTOIMMUNE DISEASE
THEY ARE MUCH MORE PREVALENT IN
WOMEN.
HERE I'M SHOWING SOME OF THE
ONES THAT INCLUDING SYSTEMIC
MORE SPECIFIC ONES WHERE CAN YOU
SEE WOMEN ARE MUCH MORE AFFECTED
THAN MEN.
THIS IS PROBABLY A COMBINATION
OF DIFFERENCE IN MODULATION,
IMMUNITY, ALSO DIFFERENCES IN
SEX CHROMOSOMES HAVING TWO X
CHROMOSOMES WHICH CARRY MANY
IMMUNE-ASSOCIATED GENES MAY ALSO
PREDISPOSE WOMEN TO HAVE MORE
AUTOIMMUNITY.
AND THEY ARE ALSO LONG ENCODING
RNAs IMPLICATED IN CONDITIONS
BY WOMEN.
A LOT IS KNOWN HOW SEX
DIFFERENCES MODULATE HOW VARIOUS
IMMUNE CELLS BEHAVE.
WE KNEW LESS UNTIL RECENTLY
ABOUT WHETHER THERE ARE
SIGNIFICANT SEX DIFFERENCES IN
NEUTROPHIL BIOLOGY, AND THIS IS
IMPORTANT IN NET FORMATION.
SOME YEARS AGO WE FOUND THAT
NEUTROPHILS HAVE FEMALES AND
MALES ARE DIFFERENT, AND WHEN
YOU LOOK AT GENE EXPRESSION OF
PERIPHERAL BLOOD IMMUNE CELLS
AND COMPARE YOUNG ADULT FEMALES
AND YOUNG ADULT MALES THE MAIN
DIFFERENCE WE SEE IS ACTUALLY IN
GENES ASSOCIATED TO NEUTROPHIL
BIOLOGY AND MORE SPECIFICALLY TO
MATURATION.
AND LONG STORY SHORT WHAT WE
FOUND IS THAT NEUTROPHILS IN
WOMEN ARE MORE MATURE THAN
NEUTROPHILS IN MEN, WHEN WE LOOK
AT YOUNG HEALTHY ADULTS.
AND JUST TO SUMMARIZE THE MAIN
FINDINGS OF THESE, WOMEN HAVE
MORE ACTIVATED NEUTROPHILS.
THEY ACTUALLY RESPOND MORE
ROBUSTLY TO DANGER SIGNALS.
FOR EXAMPLE, MORE RESPONSIVE TO
TYPE I INTERFERONS, IMPORTANT
ANTI-VIRAL PROTEINS, ALSO HAVING
IMPLICATION IN PATHOGENESIS OF
AUTOIMMUNE DISEASES AND THEY ARE
MORE LIKELY TO MAKE NETs.
AND SO NEUTROPHILS FROM WOMEN
UPON EXPOSURE TO INFECTIOUS
STIMULI OR STERILE INFLAMMATORY
STIMULI MORE ROBUSTLY FORM
NETs, AND MAY HAVE IMPORTANT
IMPLICATIONS FOR INFECTIONS, AS
WE KNOW IT'S BEEN WELL DESCRIBED
WOMEN TEND TO FIGHT INFECTIONS
BETTER THAN MEN, BUT ALSO AS I
MENTIONED THAT WOMEN ARE MORE
PRONE TO AUTOIMMUNITY, ONE
POTENTIAL FACTOR IS ACTIVATION
STAT
STATUS OF NEUTROPHIL AND ABILITY
TO FORM NETs.
CHANGES IN MATURATION LEVEL OF
NEUTROPHILS IN WOMEN APPEAR TO
VARY DURING PREGNANCY WHERE
DURING THE SECOND AND THIRD
TRIMESTER OF PREGNANCY
NEUTROPHILS OF WOMEN BECOME LESS
MATURE AND RESEMBLE NEUTROPHILS
FROM MEN, FOLLOWING POSTPARTUM
PERIOD THEY GO BACK TO THE STAGE
OF ENHANCED ACTIVATION AND
MATURATION.
SO WE ARE HYPOTHESIZING, THIS IS
WORK ONGOING, THAT SEX HORMONES
ARE PROBABLY PLAYING AN
IMPORTANT ROLE IN THE NEUTROPHIL
BIOLOGY, FOR INSTANCE.
THIS LEADS ME TO THE NEXT PART
OF THE TALK, WHICH IS I WANTED
TO START WITH THIS PREMISE THAT
AUTOIMMUNE DISEASE IS MORE
COMMON IN WOMEN.
WOMEN NEUTROPHIL APPEARS TO FORM
MORE NETs, WHAT IS THE ROLE IN
AUTOIMMUNITY?
AUTOIMMUNITY DISEASES ARE
HETEROGENEOUS, THERE ARE
COMMONALITIES, AS MANY CHRONIC
CONDITIONS WE CONSIDER THEM
MULTI-STEP PROCESSES WHERE BOTH
GENETIC AND ENVIRONMENTAL
FACTORS PLAY KEY ROLES IN
INDUCING CLINICAL STATES WHERE
BOTH INNATE AND ADAPTIVE IMMUNE
RESPONSES BECOME VERY ABNORMAL.
AND THROUGH A VARIETY OF
PROGRAMS THERE'S ENVIRONMENTAL
FACTORS AND STOCHASTIC EVENTS,
RECEIVING VARIOUS TIPS FROM THE
ENVIRONMENT THAT WOULD LEAD
EVENTUALLY TO PRODUCTION OF
AUTOANTIBODIES THAT TARGET
CELLULAR AND EXTRACELLULAR
AUTOANTIGENS.
INDIVIDUALS AT RISK MAY DEVELOP
THESE AUTOANTIBODIES MANY YEARS
BEFORE CLINICAL DISEASE DEVELOPS
BUT EVENTUALLY AFTER ADDITIONAL
HITS FROM THE ENVIRONMENT AN
INDIVIDUAL WILL DEVELOP
CHARACTERISTICS OF SYSTEMIC
AUTOIMMUNE DISEASE, GO THROUGH
REMISSION AND FLARES, AS THEY
LIVE LONGER NOW WE KNOW THEY
HAVE A HIGHER RISK TO ACQUIRE
CONDITIONS THAT ARE KNOWN TO BE
SECONDARY TO PROLONGED EXPOSURE
TO OXIDATIVE STRESS, SUCH AS
CARDIOVASCULAR DISEASE, BONE
DAMAGE, FIBROSIS, AND CANCER.
AND SO FOR US STUDYING
AUTOIMMUNITY ONE KEY QUESTION
HAS BEEN HOW DO THESE
INTRACELLULAR ANTIGENS BECOME
IMMUNOGENS, THE DOGMA FOR MANY
YEARS HAS BEEN THERE'S A
DYSREGULATION IN CELL DEATH
FOCUSED ON APOPTOSIS, INCREASE
THE APOPTOSIS OR INCREASED CELL
DEATH OR CLEARANCE OF THE DEAD
CELLS MAY PROMOTE TO INCREASING
HALF-LIFE OF AUTOANTIGENS AND
MODIFYING AUTOANTIGENS IN A WAY
THAT PRE-DISPOSED INDIVIDUALS
MAY LEAD TO AUTOANTIBODY
FORMATION AND OUR DOWNSTREAM
PROCESSES.
WE FOCUSED ON APOPTOSIS BECAUSE
WE KNOW LESS ABOUT OTHER TIMES
OF CELL DEATH, WE NOW KNOW CELLS
CAN DIE IN MANY DIFFERENT WAYS.
NOT ONLY WHETHER A CELL WILL DIE
OR NOT DIE BUT HOW, WHICH TYPE,
WHAT IS THE MICROENVIRONMENT, TO
DAMPEN INFLAMMATORY RESPONSE.
AND SO WHEN ARTURO PUBLISHED HIS
INITIAL WORK ON NETs, WE
BECAME EXCITED BECAUSE THIS WAS
A NOVEL CELL DEATH MECHANISM
THAT MADE A LOT OF SENSE, COULD
PLAY POTENTIAL ROLE IN
AUTOIMMUNITY.
AND SO WE NOW KNOW THAT FROM THE
INITIAL DESCRIPTION THAT
MICROBES ARE MAIN INDUCERS OF
NET FORMATION THERE ARE A NUMBER
OF OTHER STERILE INFLAMMATORY
STOMACH LIE -- STIMULI WITH
ABILITY TO INDUCE NET FORMATION
INCLUDING PLATELETS, CYTOKINES,
COMPLEMENT COMPONENTS, VARIOUS
CRYSTALS, URIC ACID CRYSTALS AND
COMPONENTS THAT ARE IMPORTANT IN
AUTOIMMUNE DISEASES SUCH AS
IMMUNE COMPLEXES AND
AUTOANTIBODIES, ALL OF THESE MAY
LED TO FORMATION OF NETs, VERY
IMPORTANTLY AS ARTURO MENTIONED
DURING HIS TALK THAT NETs
CARRY OR EXTERNALIZE MANY
AUTOANTIGENS KNOWN TO BE KEY
TARGETS.
IN THE CASE OF LUPUS, FOR
EXAMPLE, IN OTHER AUTOIMMUNE
CONDITIONS
IN THE KALES OF RHEUMATOID
ARTHRITIS MODIFIED PROTEINS
EXTERNALIZED WILL BE KEY
AUTOANTIGENS IN THE DISEASE.
AND WE NOW KNOW THAT FOR MANY
AUTOIMMUNE DISEASES THERE'S A
PROFOUND IMBALANCE BETWEEN NET
FORMATION INCREASED AND NET
DEGRADATION THAT IS IMPAIRED,
STARTING WITH ARTURO'S GROUPS
AND VARIOUS GROUPS HAVE SHOWN A
THIRD OF LUPUS PATIENTS HAVE
IMPAIRMENT IN NET DEGRADATION,
HALF-LIFE MAY BE INCREASED IN
CIRCULATION AND ALSO IN TISSUES.
WE HAVE FOUND THAT THIS
PHENOMENON IS NOT ONLY IN VITRO
BUT OPERATIONALLY IN VIVO
BECAUSE IN MANY AUTOIMMUNE
DISEASES WE FOUND EVIDENCE OF
NETTING NEUTROPHILS IN THE
TISSUES OF THESE PATIENTS.
AND SO I'M GOING TO TELL BUT
THREE AUTOIMMUNE CONDITIONS AND
THE WORK WE'VE DONE TO SHOW
EVIDENCE THAT NETs MAY BE
IMPLICATED IN THE DEVELOPMENT
AND PERPETRATION OF AUTOIMMUNE
DISEASES.
ONE WHERE WE CAN FOCUSED MOST OF
THE RESOURCE IS SYSTEMIC LUPUS
,
THE POSTER CHILD OF SYSTEMIC
AUTOIMMUNITY.
AFFECTS PRIMARILY WOMEN, IT IS
PROFOUNDLY HETEROGENEOUS
CONDITION, MORE A SYNDROME THAN
A DISEASE.
THERE ARE PROBLEM MANY WAYS BY
WHICH WE CAN -- AN INDIVIDUAL
CAN DEVELOP LUPUS.
CHARACTERISTICSALLY THE SINE QUA
NON IS DEVELOPMENT OF
ANTIBODIES, TARGETS NUCLEIC ACID
OR PROTEINS BINDING SUCH AS
HISTONES, SO TO THE NUCLEOSOME.
IF WE DO NOT SEE THESE WE DOUBT
A PATIENT WOULD HAVE LUPUS.
THESE PATIENTS HAVE PROFOUND
DYSREGULATION IN THE TYPE I
INTERFERON PATHWAY THAT I
MENTIONED TO YOU, HOST DEFENSE,
ALSO IN AUTOIMMUNITY
DEVELOPMENT, SIGNIFICANT
PROPORTION OF LUPUS PATIENTS
HAVE ENHANCED RISK TO DEVELOP
HEART ATTACKS OR STROKE AND WE
KNOW THIS IS PRIMARILY DRIVEN BY
IMMUNE DYSREGULATION
CHARACTERISTICS OF LUPUS.
AND SO ALMOST 15 YEARS AGO WE
FOUND THAT WHEN WE WERE TRYING
TO PURIFY MONO NUCLEAR CELLS
FOLLOWING GRADIENT, EVEN THE
INCREASED DENSITY IN REGULAR
NEUTROPHILS TYPICALLY WHEN WE DO
THIS GO MORE TO THE BOTTOM NEXT
TO RED BLOOD CELLS AND THEN MONO
NUCLEAR CELLS ARE ISOLATED
HIGHER UP IN THESE WHAT WE CALL
MONO NUCLEAR CELL ENTER PHASE.
IN THE CASE OF LUPUS WHEN TRYING
TO PURIFY WERE ALWAYS FINDING
SIGNIFICANT PROPORTION OF
NEUTROPHILS PRESENT IN THESE
MONONUCLEAR CELL INTERFACE.
WHAT WE FOUND LUPUS PATIENTS HAD
A SUBSET OF NEUTROPHILS THAT WE
NAMED LOW DENSITY GRANULOCYTES
BECAUSE THEY SEEM TO FLOAT SUCH
AS THEY HAD LOWER THAN REGULAR
NEUTROPHILS, AND WE FOUND ONE OF
THE KEY FEATURES OF THESE LOW
DENSITY GRANULOCYTES OR
NEUTROPHILS THAT WERE PURIFIED
FROM LUPUS PATIENTS IN SOME
CASES REPRESENTING UP TO 50%,
THEY CAME OUT OF CIRCULATION
PRIMED TO FORM NETs.
THERE WAS NOTHING WE COULD
FURTHER DO IN VITRO TO STIMULATE
NET FORMATION.
THEY WERE ALREADY MAXED OUT IN
THEIR ABILITY TO FORM NETs.
AND WE HAVE STUDIED THESE CELLS
OVER MANY YEARS TO TRY TO FIGURE
OUT FIRST WHERE ARE THEY COMING
FROM, SECOND WHAT IS THEIR ROLE.
ARE THEY JUST A PHENOMENON,
INNOCENT BYSTANDERS?
DO THEY REPRESENT MERELY THE
GRANULATED CELLS?
AND WE HAVE COME AFTER MANY
YEARS OF STUDY THESE APPEAR TO
REPRESENT PRO-INFLAMMATORY
SUBSET IN LUPUS PATIENTS ENDOWED
WITH PATHOGENIC CAPACITY.
AMONG THEM LISTING HERE SOME OF
THE ONES WE'VE FOUND.
THEY HAVE A HIGHER ABILITY TO BE
PRO-INFLAMMATORY, TO SYNTHESIZE
FROM CYTOKINES, A KEY FEATURE IS
THAT THEY CAN MAKE NETs
WITHOUT ANY IN VITRO
STIMULATION.
SOMETHING HAS HAPPENED IN VIVO
THAT MADE THEM VERY PRONE TO
FORM NETs.
NETs HAVE ABILITY TO DAMAGE
ENDOTHELIAL CELLS, A KEY FEATURE
OF THESE GRANULOCYTES, THEY ARE
NOT MYELOID SUPPRESSOR CELLS.
THEY HAVE STRONG ABILITY TO
ACTIVATE T CELLS, FURTHER
PROMOTING INFLAMMATION.
AND SOMETHING QUITE UNIQUE ABOUT
THE CELLS THEIR BIOMECHANICAL
PROPERTIES APPEAR TO BE
DIFFERENT THAN REGULAR
NEUTROPHILS, IF WE MAKE THEM
TRAVEL TO SOME SORT OF SMALL
BLOOD VESSEL IN EPIC THEY GET
TRAPPED IN THESE SMALL BLOOD
VESSELS COMPARED TO NORMAL
DENSITY NEUTROPHILS SUGGESTING
SEEM TO BE STIFFER, ADAPT LESS
WELL TO TRAVELING IN SMALL BLOOD
VESSELS AND MAY PROMOTE FOR
EXAMPLE TISSUE HYPERPERFUSION
AND ORGAN DAMAGE AND SEEM TO
HAVE DIFFERENT BIOENERGETICS
THAN REGULAR NEUTROPHILS.
FURTHERMORE AS WE HAVE STUDIED
OTHER AUTOIMMUNE DISEASES AND
OTHER AUTO-INFLAMMATORY DISEASES
WE'VE BEEN ABLE TO STUDY HERE AT
THE NIH WE HAVE FOUND THAT IN
OTHER AUTOIMMUNE INFLAMMATORY
CONDITIONS THERE'S ALSO THIS
PRESENCE OF LOW DENSITY
GRANULOCYTES AND INCREASED
NUMBERS THAT HAVE AN ENHANCED
CAPACITY TO FORM THESE NETs.
AND SOMETHING THAT'S PERHAPS
SHAREDDED BY MANY OF THESE
AUTOIMMUNE AND AUTO-INFLAMMATORY
CONDITIONS IS ABILITY TO FORM
DYSREGULATED NETs, MAY LEAD A
SHARED PHENOTYPE TO ENHANCE
BLOOD VESSEL DAMAGE TO PROMOTE
END ORGAN COMPLICATIONS AND
PREMATURE DISEASE.
SO, THIS IS THE WORK SUMMARIZING
OF OTHER GROUPS IT APPEARS WHEN
THE PROCESS OF NET FORMATION
GOES AWRY IT CONTRIBUTES TO AN
OF A BE IN -- A NUMBER OF
SYNDROMES.
WE KNOW NETs ARE IMPLICATED IN
DEVELOPMENT OF ATHEROSCLEROSIS
AND THROMBOSIS, CANCER AND
METASTASES, METABOLIC SYNDROME,
DIABETES, THINGS AS MUNDANE OR
COMMON AS GALLBLADDER STONES AND
MORE RECENTLY THEY WERE ALSO
IMPLICATED IN COMPLICATIONS IN
THE COVID-19 PANDEMIC.
FORMING NETs IN CONTEXT OF
IMMUNITY LEAD TO VICIOUS CYCLE
PROMOTING INFLAMMATION IN ORGAN
DAMAGE SUCH AS, FOR EXAMPLE,
INDUCING INFLAMMASOME ACTIVATION
IN MACROPHAGES, PROMOTING
ACTIVATION OF T CELLS AND B
CELLS WITH AUTOANTIBODY
PROTECTION, LEADING THE KEY
CELLS PRODUCING TYPE I
INTERFERONS TO MAKE MORE OF IT
WHICH IS IMPORTANT IMPLICATION
IN AUTOIMMUNITY.
NETs ARE MORE PRONE TO PROMOTE
VASCULAR DAMAGE AND THROMBOSIS.
AND SO WHY THE KEY QUESTION,
SOMETHING WE HAVE ASKED FOR A
NUMBER OF YEARS, ARE ALL NETs
CREATED EQUAL?
AND WHAT WE THINK IS ACTUALLY
NOT ALL NETs ARE CREATED THE
SAME.
AFTER VARIOUS WAYS BY WHICH WE
HAVE LOOKED AT THIS, FOR EXAMPLE
WE KNOW THAT IF NETs ARE
INDUCED THROUGH LUPUS RELEVANT
STIMULI SUCH AS IMMUNE
COMPLEXES, ENHANCED FORMATIONS
NOT ONLY IT'S IMPORTANT IN
GENOMIC DNA BUT MAY ALSO LEAD TO
ENHANCED OXIDATION OF
MITOCHONDRIAL DNA.
AND SO IN AUTOIMMUNE DISEASES WE
HAVE FOUND THAT THE NETs ARE
EXCLUDED IN OXIDATION OF NUCLEIC
ACID PARTICULARLY MITOCHONDRIAL
DNA BUT ALSO GENOMIC DNA.
AND AS MENTIONED BY ARTURO WE
HAVE FOUND ALSO THAT WHEN THESE
NETs ARE INTERNALIZED BY
TARGET CELLS, THEIR NUCLEIC ACID
IS LESS LIKELY TO BE DEGRADED
BECAUSE IT'S OXIDIZED, SO
EXTRACELLULARLY AND
INTRACELLULARLY HAVE A LONGER
HALF-LIFE, TRIGGERING CGAS
PATHWAY RELEVANT IN
AUTOIMMUNITY.
NOT ONLY NUCLEIC ACID, GENOMIC
DNA, MITOCHONDRIAL AND RNA ARE
OXIDIZED AND PRO IMMUNOGENIC BUT
THE PROTEIN CONTENT, THE CARGO
AND POST-TRANSLATIONAL
MODIFICATIONS PRESENT IN NETs
MAY VARY DEPENDING ON THE
STIMULUS AND EXAMINE
IMMUNOGENICITY.
I'LL TELL YOU MORE ABOUT THIS
LATER ON DURING MY TALK.
BOTH PROTEINS AND NUCLEIC ACID
ARE DIFFERENT DEPENDING HOW
NETs ARE FORMED AND THIS IS
WHY NOT JUST EVERYONE IS FORMING
NETs MAY BE CONDUCIVE TO
DEVELOP AUTOIMMUNITY.
ANOTHER IMPORTANT FACTOR THAT
MAY BE IMPORTANT IN DEVELOPMENT
OF AUTOIMMUNE DISEASES AND ORGAN
DAMAGE IS NETs ARE CARRIERS OF
A NUMBER OF MESSENGERS THAT
DEPENDING ON THE CELL IN THE
TISSUE BEING AN IMMUNE CELL OR
STROMAL CELL, MAY INDUCE
DOWNSTREAM RESPONSES THAT MAY BE
PRO-INFLAMMATORY.
AN EXAMPLE HERE IS THAT NETs
CARRY SMALL RNAs, AND WE HAVE
FOUND THESE MAY ACTIVATE IN THE
CELLS TO HAVE PRO-INFLAMMATORY
RESPONSES.
NETs HAVE ALSO BEEN FOUND
THROUGH MODIFIED HISTONES TO
INDUCE DIRECT DAMAGE TO SKELETAL
MUSCLE, IMPLICATED IN
PATHOGENESIS OF ANOTHER
AUTOIMMUNE DISEASE CALLED
IDIOPATHIC INFLAMMATORY
MYOPATHIES.
I'LL TELL YOU ABOUT OTHER
EFFECTS MOLECULES PRESENT MAY
HAVE IN STROMAL CELLS.
AND SO AS ARTURO MENTIONED, THE
PROCESS OF NETs ONCE
NEUTROPHILS ENCOUNTER DANGEROUS
SIGNAL IS QUITE DRAMATIC AND
COMPLEX.
AND ONE OBSERVATION THAT HAS
BEEN MADE IS THAT DURING NET
FORMATION IN MANY CASES THERE'S
ACTIVATION OF A GROUP OF
ENZYMES, PADs, SPECIFICALLY
ONE OF THEM PAD 4 IS PRESENT
QUITE CHARACTERISTICALLY AND
SPECIFICALLY IN NEUTROPHILS.
AND WHAT THIS ENZYME DOES,
HISTONES PROMOTE THE ABILITY OF
CHROMATIN AMONG OTHER FACTORS
THAT ARTURO MENTIONED SUCH AS
NPO AND NEUTROPHIL ASSAYS, THIS
IS A KEY FACTOR IN THE FORMATION
OF NETs AND EXTRUSION INTO THE
EXTRACELLULAR SPACE.
GROUPS INCLUDING OURS HAVE BEEN
INTERESTED IN FINDING WHETHER
BLOCKING PAD ACTIVATION BECAUSE
IN MANY ANIMAL MODELS THEY HAVE
LOCATED PAD 4, EITHER WITH
CHEMICAL INHIBITORS OR THROUGH
GENETIC KNOCKOUTS WILL LIMIT
ABILITY OF NEUTROPHIL TO FORM
NETs AND EVEN IF NETs FORM
THE QUALITY OF THE NETs MAY BE
DIFFERENT.
AND SO JUST TO SHOW YOU THAT IN
A NUMBER OF ANIMAL MODELS WE
HAVE FOUND IF WE BLOCK NETs
CHEMICALLY, WE TRY CHEMICALLY
INHIBITORS OF PADs, WE'RE ABLE
TO INHIBIT ORGAN DAMAGE IN LUPUS
MODELS SEEING LESS RENAL
INFLAMMATION, LESS FORMATION OF
THE POSITION OF IMMUNE COMPLEX
IN THE KIDNEY, PROMINENT EFFECTS
ON IMPROVING VASCULAR FUNCTION
IN THE MICE.
WHEN WE BLOCK NETs THROUGH
BLOCKING PADS BLOOD VESSELS
DILATE BETTER, TIME TO CLOT UPON
STIMULATION IS PROLONGED MEANING
IT TAKES LONGER TO CLOT AND THIS
MAY PREVENT PATHOGENIC
THROMBOSIS.
THIS IS AN AVENUE THAT IS NOW
BEING EXPLORED AT THE
PRE-CLINICAL LEVEL BLOCKING
COULD HAVE THERAPEUTIC ROLES.
IN ANIMAL MODELS OF
ATHEROSCLEROSIS, APO-E PROTEIN,
IF WE BLOCK NET FORM FORMATIONR
LIMIT PADs WE SEE LESS PLAQUE
FORMATION, HERE SIGNIFICANTLY
DEPRESSED.
AND THERE ARE LESS LIPIDS RICH
PLAQUE IN THE AORTA, IT TAKES
MICE LONGER TO CLOT UPON A
STIMULUS THAT LEADS TO CLOT
FORMATION, PROTECT IVE EFFECT IN
VASCULAR DAMAGE.
IF WE KNOCK OUT THE ENZYME WE
SEE SIGNIFICANT DECREASES IN
ATHEROSCLEROSIS IN THESE PRONE
MICE, JUST TO START SEE HOW
INHIBITING NETs MAY HAVE
THERAPEUTIC ROLES IN THESE
CONDITIONS.
I'LL GO TO THE MOST PREVALENT,
1% OF THE POPULATION, SIMILAR TO
LUPUS THERE'S A CONTINUUM, AN
AREA OF PRE-CLINICAL IMMUNE
DYSREGULATION THAT INTERESTINGLY
MAY START AT THE LEVEL ALSO OF
MUCOSA IN THE LUNG AND OTHER
MUCOSAS, PEOPLE AT RISK TO
DEVELOP RHEUMATOID ARTHRITIS,
WORK BY THE UNIVERSITY OF
COLORADO AND WE WERE VERY LUCKY
TO COLLABORATE WITH HIM, WE
FOUND ACTUALLY IN LUNG MUCOSA
THERE'S SIGNIFICANT INCREASED
LEVELS OF NETs.
AND WHAT IS INTERESTING IS THAT
THESE NETs WHICH CARRY
INCREASED LEVELS OF ANTIGENS
BECAUSE PATHS ARE ACTIVATED MAY
EXPOSE IMMUNE SYSTEM, ENHANCED
EVEN BEFORE RHEUMATOID ARTHRITIS
BECOME EVIDENT.
AND KEY AUTOANTIBODY THAT IS
PRESENT IN RHEUMATOID ARTHRITIS,
PATHOGNOMONIC, ANTIBODIES THAT
TARGETS, USED IN DIAGNOSIS OF
DISEASE.
WE THINK EARLY ON DURING THE
DISEASE THERE MAY BE ENHANCED,
EVEN BEFORE DISEASE DEVELOPS,
NETs ARE HAPPENING IN THE
LUNGS.
AND THIS LEADS TO EXPANSION AND
DEVELOPMENT EVENTUALLY OF
RHEUMATOID ARTHRITIS THAT,
AGAIN, THE DISEASE PROGRESSES
CAN LEAD TO VERY SIGNIFICANT
DAMAGE AND DISABILITY.
AND SEVERAL YEARS AGO WE
HYPOTHESIZED ANTIGENS MAY BE
GENERATED, PERHAPS NETs WERE A
KEY INITIATOR IN THE DISEASE.
AND JUST TO SUMMARIZE THIS STORY
WHAT WE FOUND IS THAT THE
RHEUMATOID ARTHRITIS
MICROENVIRONMENT,
PRO-INFLAMMATORY, BOTH IN THE
SYSTEMIC BUT ALSO INSIDE THE
JOINTS WAS A CHARACTERIZED BY
ENHANCED FORMATION OF NETs
THAT MANY NEUTROPHILS ARE FOUND
IN THE SYNOVIAL FLUID OF
RHEUMATOID ARTHRITIS PATIENTS
AND THESE HAVE ENHANCED CAPACITY
TO FORM NETs, EXTERNALIZE,
REACH LEVELS OF ANTIGEN, LED TO
THE INDUCTION OF PHOTOANTIBODIES
THAT WERE DIRECTED AGAINST THE
PEPTIDE AND IN TURN
AUTOANTIBODIES CAN LEAD TO MORE
NET FORMATION CREATING A VICIOUS
CYCLE.
THESE AUTOANTIGENS HAD THE
ABILITY TO STIMULATE A KEY CELL
PRESENT IN THE RHEUMATOID -- IN
THE JOINT KNOWN TO BE IMPORTANT
IN THE PATHOGENESIS OF
RHEUMATOID ARTHRITIS,
SYNOVIAL -- FIBROBLAST IN
SYNOVIUM, AND THAT WHEN NETs
STIMULATED THE FIBROBLASTS THEY
BECAME PRO-INFLAMMATORY AND
AGGRESSIVE.
SO WE FOLLOW WITH THIS STORY AND
THIS LED US TO FIND THAT IN FACT
WHEN SYNOVIAL CYTO BLASTS
ENCOUNTER NETs, THEY HAVE THE
ABILITY TO INTERNALIZE, LEADING
TO ACTIVATION, AND THAT WHEN
THEY BECAME ACTIVATED THEY
ACQUIRE ANTIGEN PRESENTING CELL
CAPABILITIES, WENT TO ADAPTIVE
IMMUNE SYSTEM AND PRESENT
AUTOANTIGENS TO T CELLS WHICH
PROMOTE FORMATION OF THESE KEY
AUTOANTIBODIES PRESENT IN
RHEUMATOID ARTHRITIS, THE ONES
THAT TARGET SUGGESTING AGAIN
NETs MAY PLAY IMPORTANT ROLES
IN THE INITIATION.
NOT ONLY THAT WE KNOW THERE ARE
OTHER POST-TRANSLATIONAL
MODIFICATIONS PRESENT IN
RHEUMATOID ARTHRITIS, THE OTHER
ONE IS CARBAMYLATION.
AND WHAT WE FOUND YEARS AGO IS
THAT CARBAMYLATION PROTEINS ARE
ALSO PRESENT IN NETs, AND
SPECIFICALLY HISTONES CAN BE
FOUND IN NETs, AND IT IS
INTERESTING IN RHEUMATOID
ARTHRITIS PRESENCE TARGETING TS
ASSOCIATED TO BONE DAMAGE.
PEOPLE THAT CARRY ANTIBODIES
HAVE ENHANCED RISK TO DEVELOP
BONE DAMAGE, ENHANCED BONE
RESORPTION, AND A FEW YEARS AGO
WE DESCRIBED POTENTIAL
MECHANISM, IN THAT NETs
CARRYING THIS HAVE ABILITY TO
ACTIVATE THROUGH ENGAGEMENT,
ALLOWING MONOCYTES TO UNDERGO
ACCELERATED PROGRAM WHERE THEY
BECOME ACTIVATED OSTEOBLASTS
THAT RESORB BONE AND ALSO
ACTIVATE T CELLS TO PRODUCE
OTHER PRO OSTEOGENIC OR BONE
ABSORPTION MOLECULES LEADING TO
NET FORMATION IMPORTANCE IN
INDUCING ARTHRITIS DEVELOPMENT
OR ACTIVATION OF SYNOVIAL
FIBROBLASTS BUT LEAD TO
END-ORGAN DAMAGE, TO BONE
DAMAGE.
AND FINALLY MY THIRD EXAMPLE OF
AUTOIMMUNITY DEVELOPMENT LINKED
TO NETs IS INDUCED MODEL OF
AUTOIMMUNITY WHICH IS
VASCULITIS, ANOTHER FASCINATING
CONDITION WHERE LEVAMISOLE IS
UNIT IN MEDICINE, AN AGONIST,
MOST COCAINE CONSUMED IN THE
U.S. IS CONTAMINATED WITH
LEVAMISOLE, AND THERE ARE CASES
ASSOCIATED WITH PATIENTS THAT
USE COCAINE AND DEVELOP
CHARACTERISTICS TYPE OF
VASCULITIS THAT WAS INDUCED BY
CONTAMINATION.
TYPICALLY THESE PATIENTS DEVELOP
FEVER, PURPURIC RASHES, IN THE
NOSE, EXTREMITIES, A MIX OF
ANTIBODIES TARGET AUTOANTIGENS,
PRESENT IN NETs.
MANY DEVELOP LOW LEVELS OF
NEUTROFIRST SUGGESTING
NEUTROPHILS MAY BE DYING.
WHEN THE PATIENT STOP THE
COCAINE AND EXPOSURE TO
LEVAMISOLE THIS VASCULITIS
RESULTS.
IN COLLABORATION WE FOUND A
PATIENT THAT HAD DEVELOPED
LEVAMISOLE INDUCED VASCULITIS,
FOUND THIS PATIENT HAD
SIGNIFICANT LEVELS OF LOW
DENSITY GRANULOCYTES FORMING
PROFOUND LEVELS OF NETs, AND
WE ALSO HAD ABILITY TO STUDY
TISSUES OF PATIENTS WITH
LEVAMISOLE INDUCED VASCULITIS,
TISSUES WERE INFILTRATED BY
NETTING NEUTROPHILS.
WHAT WE FOUND WAS A LINK BETWEEN
NEURAL IMMUNOMODULATION IN THAT
AS I MENTIONED LEVAMISOLE
STIMULATES RECEPTORS, USED WITH
MEDICINE, SO WE ASKED HERE IN
VITRO YOU SEE WHAT HAPPENS IF WE
ADD LEVAMISOLE WE SEE PROFOUND
INDUCTION OF NETs.
AND WE HYPOTHESIZE PERHAPS IT
WAS THROUGH INDUCTION OF
RECEPTORS NETs WERE BEING
INDUCED.
IF YOU ADD ANOTHER MUSCARINIC
ACID, INDUCED NETs.
AND ATROPINE, WE WERE ABLE TO
BLOCK ABILITY OF LEVAMISOLE TO
INDUCE NETs.
QUANTITY VERSUS QUALITY WE FOUND
IF WE INDUCE NETs WITH
LEVAMISOLE, THEY HAVE AN
ENHANCED ABILITY TO DAMAGE BLOOD
VESSELS WHEN COMPARED TO NETs
INDUCED BY OTHER TYPES OF
STIMULATION, SUGGESTING AGAIN
HOW NETs ARE INDUCED MAY BE
KEY IN WHETHER THEY ARE
PRO-INFLAMMATORY AND HARM
VASCULATURE.
AND SO JUST IN MY LAST MINUTES
HOW CAN WE TARGET NET FORMATION?
COULD THIS BE POTENTIALLY USED
TO MITIGATE AUTOIMMUNITY AND
VASCULAR DAMAGE?
INHIBITION IS BEING
INVESTIGATED, MODULATING ROS
PRODUCTION IS BEING INVESTIGATED
AS A WAY TO TARGET RESPONSE OF
NEUTROPHILS TO STIMULUS THAT
COULD DAMPEN NET FORMATION AND
RECENTLY FOCUSED ON A -- THIS IS
JUST TO ALSO MENTION THERE ARE
DRUGS THAT WE'RE ALREADY USING
AND HAVE USED FOR MANY YEARS TO
TREAT AUTOIMMUNITY THAT HAVE
BEEN UNCLEAR WHAT MECHANISM OF
ACTION IS, ONE IS
ANTI-MALARIALS, QUITE BENEFICIAL
FOR MANY LUPUS PATIENTS AND
PATIENTS WITH OTHER AUTOIMMUNE
DISEASES, THEY CAN INHIBIT NET
FORMATION AND EXTRUSION OF
NETs AND CYTOTOXIC MOLECULES
PRESENT BUT ALSO ABILITY OF
NETs TO BE INTERNALIZED BY
OTHER CELLS CAN BE INHIBITED
WITH ANTI-MALARIALS, DRUGS FOR
MANY YEARS WOULD THE KNOWING HOW
THEY WORK IN PART, IN PART
INHIBIT NET FORMATION.
THE OTHER WAY BY WHICH WE'VE
FOCUSED ON WHETHER INHIBITING
NETs MAY BE BENEFICIAL IS CAN
WE INHIBIT THE ABILITY OF NETs
TO HARM THE VASCULATURE BECAUSE
AT THE END WE THINK THIS IS A
KEY MECHANISM BY WHICH NETs
ARE DELETERIOUS IN AUTOIMMUNITY.
A KEY PATHWAY CYTOKINE SIGNAL
MAY BE CONDUCIVE TO NET
FORMATION IS JAK/STAT PATHWAY,
ACTIVE IN NEUTROPHILS, WE FOUND
WHEN TRIGGERED MAY LEAD TO
INDUCTION OF NETs.
IN FACT, IN CLINICAL TRIALS IN
LUPUS PATIENTS WE FOUND THAT
WHEN THE PATIENTS TOOK THE JAK
INHIBITORS COMPARED TO PLACEBO,
ASSOCIATED WITH DECREASED LEVELS
OF NET FORMATION IN THESE
PATIENTS EVEN DURING SHORT
PERIOD OF TIME THEY TOOK THE
DRUG.
AND TRANSLATED INTO FINDINGS NOT
ONLY IMMUNE DYSREGULATION WAS
IMPROVED BUT THAT LEVEL OF
VASCULAR DAMAGE OR VASCULAR
DYSFUNCTION THAT WE FOUND IN THE
LUPUS PATIENTS WENT DOWN WHEN WE
DECREASED -- INHIBITS JAK AND
LOW DENSITY NETs SUGGESTING
THERE MAY BE WAYS TO REGULATE
NET FORMATION THAT MAY BE
BENEFICIAL TO PATIENTS IN THE
FUTURE.
SO I WILL END HERE JUST TO
SUMMARIZE SOME KEY ASPECTS OF MY
TALK.
ONE IS THAT SEX DIFFERENCES IN
NEUTROPHIL BIOLOGY MAY
CONTRIBUTE TO AUTOIMMUNITY AND
FOR INFECTIONS THAT WE BELIEVE
NETs WHEN PROCESS GOES AWRY
MAY SERVE AS IMPORTANT SOURCE OF
MODIFIED AUTOANTIGENS, NUCLEIC
ACID AND, AND CHARACTERIZING
NEUTROPHIL SUBSETS, NOT ALL, AND
ABILITY IN NET FORMATION MAY
CONTRIBUTE TO MITIGATE END-ORGAN
DAMAGE IN LUPUS AND OTHER
AUTOIMMUNE DISEASE.
I MENTION MANY FORMER AROUND
CURRENT MEMBERS OF NIGH OF MY
LAB, COLLABORATORS AND GREAT
SUPPORT FROM NIAMS AND THE NIH
IN GENERAL.
THANK YOU VERY MUCH FOR YOUR
ATTENTION.
>> WELL, THANK YOU VERY MUCH,
MARIANA AND ARTURO, FOR THIS
FASCINATING TOUR DE FORCE
TELLING US ABOUT BASIC SCIENCE
AND TRANSLATIONAL ASPECTS OF NET
OSIS.
WE HAVE AN ENORMOUS NUMBER OF
QUESTIONS BEFORE US.
NOT THAT THAT'S INDICATING IN A
THERE'S ANY LACK OF CLARITY OF
THE PRESENTATIONS BUT RATHER
EXCITEMENT ABOUT THE IDEAS THAT
HAVE BEEN PUT FORTH.
SO, WE'LL START OUT WITH ONE
FROM OUR AUDIENCE ASKING FOR A
LITTLE BIT MORE DETAIL IN TERMS
OF HOW HISTONES HAVE THEIR
ANTIMICROBIAL EFFECT AND PERHAPS
WE CAN START WITH ARTURO, WITH
THAT, BUT MARIANA MAY HAVE
SOMETHING TO ADD AS WELL.
ARTURO?
>> WE DON'T KNOW, THE SHORT
ANSWER.
HISTONES ARE CATIONIC, THAT
MIGHT BE ONE OF THE WAYS, BUT IT
IS NOT THE ONLY THING.
THEY ARE EQUALLY CATIONIC BUT
NOT AS STRONG AS ANTIMICROBIALS.
HISTONE H-1 WE KNOW THAT IN
STAPH AUREUS IT IS RECRUITED
INTO THE SURFACE OF THE CELL,
THEN BREAKS THE FORCE BETWEEN
THE MEMBRANES.
AND WE'VE SEEN BINDING EVER H1
TO PROKARYOTIC DNA BUT DON'T
KNOW IF THAT'S SIGNIFICANT.
>> SO, THAT'S REALLY
FASCINATING.
AND YOU MENTIONED AT LEAST
HYPOTHESIZED THAT THERE COULD BE
SOME SORT OF EVOLUTIONARY THING
GOING ON HERE, WHERE EUKARYOTES
BASICALLY HAVE EVOLVED HISTONES
AT ABOUT THE TIME THAT THEY
DEVELOPED NUCLEUS AND AT ABOUT
THE TIME THAT BACTERIA WOULD BE
A THREAT TO THEM.
DO CANDIDA YEASTS HAVE HISTONES?
>> YES.
ALL EUKARYOTES HAVE HISTONES,
THEY ARE EXTRAORDINARILY
CONSERVED THROUGHOUT EUKARYOTIC
EVOLUTION.
HISTONE-LIKE PROTEINS ARE MUCH
MORE SIMILAR TO EUKARYOTIC
HISTONES THAN PROTEINS THAT BIND
TO PROKARYOTIC DNA.
GREAT TO RECRUIT SOMEBODY
WORKING TO SEE WHAT IS THE
FUNCTION OF HISTONES.
>> YEAH, THAT WOULD BE
FASCINATING.
NOW PERHAPS A QUESTION FOR
MARIANA WITH REGARD TO LOW
DENSITY NEUTROPHILS, AND THEIR
PROCLIVITY TO PRODUCE NETs.
AND SO THE QUESTION HAS TO DO
WITH THE ISSUE OF WHETHER
NETOSIS IS SORT OF A FATAL
SUICIDAL ACTION BY NEUTROPHILS,
WHETHER THESE LOW DENSITY
NEUTROPHILS, WHETHER THEY ARE
PRECURSORS EVER CELLS THAT GO ON
TO MAKE NETs, YOU'RE CATCHING
THEM IN THE ACT OF, YOU KNOW,
MAKING NETs IN THE
CIRCULATION, WHAT'S GOING ON
WITH THOSE LOW DENSITY
NEUTROPHILS?
>> YEAH, SO GREAT QUESTION.
AND WE DON'T HAVE A FULL ANSWER.
WE'RE SPENDING A LOT OF TIME
TRYING TO FIGURE OUT.
WE KNOW IF YOU LOOK
TRANSCRIPTIONALLY AT PROTEOMIC
LEVEL THEY ARE DIFFERENT FROM
CELLS NORMALLY THEY EXTEND FROM
THE SAME PATIENT BUT ALSO FROM
HEALTHY VOLUNTEERS.
AND THEY LOOK SLIGHTLY LESS
MATURE BUT STILL HAVE THE FULL
MACHINERY OF MATURE NEUTROPHIL,
SO I WOULD SAY MAJORITY OF THESE
CELLS ARE MATURE.
SOME HETEROGENEITY, SOME APPEAR
TO BE LESS MATURE, BUT
ORIGINALLY THE CELLS WERE FULLY
A MATURE PHENOTYPE, WE HAVEN'T
BEEN ABLE TO RECAPITULATE, THAT
GOES WITH ENHANCED ABILITY TO
FORM NETs, TYPICALLY HAVE TO
BE MATURE TO FORM.
WHERE DO THEY COME FROM, ARE
THEY DIFFERENT FROM GET-GO IN
BONE MARROW OR SHAPED BY
ENCOUNTER IN VARIOUS ORGANS OR
AS THEY GO THROUGH CIRCULATION
DO THEY HAVE ENHANCED LIFE, MORE
SENESCENT, WE'VE BEEN TRYING TO
ADDRESS BUT PROVING TO TAKE
LONGER THAN WE WERE HOPING.
ONE ISSUE THAT WE HAVEN'T FOUND
A MOUSE COUNTERPART, SO THAT
MAKES IT HARDER.
MOUSE NEUTROPHILS ARE REALLY
QUITE DIFFERENT FROM HUMAN
NEUTROPHILS SO WE HAVE TO DO THE
STUDIES IN HUMANS, SO THAT'S
WHAT'S BEEN FASCINATING BUT
TAKING LONGER THAN WE HOPED TO
IDENTIFY WHAT THE CELLS ARE.
>> WELL, IT SHOWS THERE'S REALLY
SOME SUBTLETY TO THE WHOLE
THING.
DO HEALTHY INDIVIDUALS HAVE
LOWER NUMBERS PERHAPS OF THESE
LOW DENSITY NEUTROPHILS?
>> THEY DO.
SO WHAT WE HAVE FOUND IS THAT
THEY HAVE VERY SMALL AMOUNTS, SO
IT'S SOMETHING THAT EVERY HUMAN
APPEARS TO HAVE, BUT CELLS
BECOME MUCH MORE EXPANDED IN
CERTAIN DISEASES SUCH AS
AUTOIMMUNE DISEASES.
THEY WERE DESCRIBING COVID, BUT
IN COVID WE FOUND IT TO BE
DIFFERENT PHENOTYPICALLY SO THEY
WERE MORE SUPPRESSOR PHENOTYPE
SITES THAN WHAT WE SAW WITH
AUTOIMMUNITY, NOT SEE
SUPPRESSION, IF ANYTHING SEEING
MORE INFLAMMATION.
>> I'LL BITE MY TONGUE AND
BROKEN THIS UP BUT YOU DID
MENTION HYDROXYCHLOROQUINE,
NETs, NOW COVID,
NET-FORMING -- WELL, ANYWAY, I'M
NOT GOING ANY FURTHER WITH THAT.
BUT IN ANY CASE THERE'S ANOTHER
DETAIL ORIENTED MEMBER OF OUR
AUDIENCE WHO WANTS TO KNOW
WHETHER BASOPHILS AND
EOSINOPHILS CAN UNDERGO NET-OSIS
OR JUST NEUTROPHILS THAT DO?
>> THEY CAN, ALL GRANULOCYTES
CAN MAKE NETs.
>> OKAY.
>> THEY BOTH CAN.
>> UH-HUH, OKAY.
ALL RIGHT.
SO THEN HERE'S ANOTHER QUESTION.
THE AUDIENCE MEMBER WANTS TO
KNOW CAN CGD PATIENTS THAT DO
NOT MAKE NETs DEVELOP
AUTOIMMUNE DISEASES?
>> YES.
>> YES, THEY CAN --
>> I THINK THIS IS AN
INTERESTING QUESTION.
MOTHERS OF CGD KIDS ARE MORE
PRONE TO AUTOIMMUNITY, IS THAT
TRUE?
>> YES, WE'VE SEEN PATIENTS THAT
DEVELOP A VARIETY OF AUTOIMMUNE
INFLAMMATORY, LUPUS, COLITIS, SO
IT'S NOT UNCOMMON TO SEE
AUTOIMMUNITY DEVELOPING.
WHAT I MAY SAY IN OUR HANDS
WE'VE FOUND PRESENCE IN SOME OF
THESE PATIENTS THAT HAVE
COEXISTING CGD AND AUTOIMMUNITY,
THEY MAKE EVEN A -- OXIDASE IS
IMPAIRED, SEEM TO HAVE AN
ABILITY TO SYNTHESIZE
MITOCHONDRIAL ROS, AND WE'VE
HYPOTHESIZED THIS COULD BE
ALTERNATIVE MECHANISM THAT
PERHAPS IS COMPENSATORY IN THE
ABSENCE OF THE MACHINERY, BUT,
YEAH, IT'S A HYPOTHESIS.
>> I THINK THERE'S A VERY
INTERESTING PAPER IN NEW ENGLAND
JOURNAL MANY YEARS AGO BY A TEAM
AT NIH, A COHORT OF HUNDREDS OF
CGD PATIENTS EVEN A SMALL
PERCENTAGE OF DIFFERENCE IN
RESIDUAL ACTIVITY OF THE ENZYME,
BIG EFFECT ON PHENOTYPE.
COULD BE INTERESTING TO
RECLASSIFY THE CGD PATIENTS WITH
AUTOIMMUNITY VIS-A-VIS RESIDUAL
ACTIVITY.
>> ONE COULD SAY THERE COULD BE
MORE THAN ONE PATHWAY TO GET
AUTOIMMUNE DISEASE.
>> ABSOLUTELY.
>> NOT AS IF THE FACT YOU CAN
GET AUTOIMMUNE DISEASE WITH CGD
THAT SOMEHOW DISPROVES THAT
NET-OSIS ISN'T IMPORTANT IN SOME
PATIENTS WITH AUTOIMMUNE DISEASE
ANYWAY.
OKAY.
SO, NOW SOME QUESTIONS WITH
REGARD TO THE MECHANISMS OF
NET-OSIS.
SO ONE OF THE MEMBERS OF THE
AUDIENCE WAS INTRIGUED BY THE
CONNECTION WITH GASDERMIN-D, AND
OF COURSE THAT UNDER SOME
CIRCUMSTANCES CAN BE ACTIVATED
BY THE INFLAMMASOME, CASPASE 1
ACTIVATION CAN LEAD TO
GASDERMIN-D CLEAVAGE AND
ACTIVATION OF GASDERMIN-D.
THE VIEWER WANTS TO KNOW WHETHER
OR NOT INFLAMMASOME ACTIVATION
MIGHT BE RELATED TO NET-OSIS IN
SOME WAY.
>> YES, THERE IS WORK BY KATE
STRUTTER IN AUSTRALIA,
INFLAMMASOMIVATION LEADS TO
NET-OSIS I THINK IN MICE AND
SOME HUMANS IN HER WORK.
TO BE COMPLETELY TRANSPARENT THE
LABORATORY HAD CONTRADICTORY
RESULTS TO OURS, AND THAT ISSUE
WAS NEVER COMPLETE RESULT.
WE HAD THAT INHIBITOR, ALSO USED
GASDERMIN-D KNOCKOUT MICE AND
SHOWED THEY CAN'T -- THE LAB
BELIEVES IT'S INHIBITING AN
ENZYME IN A PATHWAY THAT'S
IMPORTANT FOR THE NADPH,
PROBABLY TOO DETAILED FOR THIS
DISCUSSION.
THE ANSWER IN SOME CIRCUMSTANCES
YES, THE INFLAMMASOME CAN BE
ACTIVATED.
GASDERMIN TO MAKE NET.
WE THOUGHT ACTIVATION OF
GASDERMIN THAT HAS TO BE CLEAVED
IS THROUGH ELASTASE.
>> YOU MENTIONED THAT.
OF COURSE, THIS GETTING BACK TO
POSSIBILITY INFLAMMASOME IS
SOMEHOW INVOLVED UNDER SOME
CIRCUMSTANCES REACTIVE OXYGEN
SPECIES CAN INDUCE INFLAMMASOME
ACTIVATION.
AND, YOU KNOW, AT LEAST IN THE
SENSE THAT REACTIVE OXYGEN
SPECIES SEEM TO BE INVOLVED IN
THE PROCESS, EARLY IN THE
PROCESS OF NETosis.
THERE COULD BE DEFINITELY A
CONNECTION.
AND CERTAINLY AN AREA FOR
FURTHER INVESTIGATION.
SO ONE OF THE MEMBERS OF THE
AUDIENCE I THINK THIS QUESTION
YOU'VE ALREADY ADDRESSED IS AT
WHAT STAGE IN DIFFERENTIATION DO
NEUTROPHILS ACQUIRE ABILITY TO
UNDERGO NET-OSIS?
AND I THINK THAT MARIANA MAY
HAVE SAID SOMETHING ABOUT THERE
NEEDING TO BE RELATIVELY MATURE
BUT I'LL POSE THE QUESTION FOR
THE SAKE OF OUR AUDIENCE.
>> ARTURO, DO YOU WANT TO
ANSWER?
>> MARIANA, WHY DON'T YOU
COMMENT.
>> YEAH, THERE HAVE BEEN STUDIES
BY SEVERAL GROUPS THAT HAVE
SHOWN THAT IMMATURE NEUTROPHILS
ARE LESS ABLE TO FORM NETs,
AND INDEED IN OUR CASE WHAT I
MENTIONED ABOUT THE DIFFERENCES
BETWEEN WOMEN AND MEN AND
ABILITY TO FORM NETs WE THINK
IS IN PART DRIVEN BY THE FACT
THAT WOMEN'S NEUTROPHILS ARE
MORE MATURE, THAT MAY BE ONE
REASON FOR EXAMPLE JAK/STAT
SIGNALING APPEARS TO BE MORE
ROBUST IN FEMALE NEUTROPHILS,
MAY BE MORE CONDUCIVE TO
ENHANCED NET FORMATION.
>> NOW, ANOTHER MEMBER OF THE
AUDIENCE WAS INTRIGUED BY A
COMMENT THAT ARTURO HAD MADE IN
HIS DESCRIPTION OF THE EARLY
PROCESS OF NETosis AND Ki67
IS EXPRESSED.
WHAT IS Ki67 FROM A PROTEIN
LEVEL, WHAT'S IT DOING, AND
WOULD THAT HAVE ANY MECHANISTIC
IMPLICATIONS FOR WHAT'S GOING ON
IN NETosis?
>> GREAT QUESTION.
EMBARRASSED TO SAY FOR YEARS
Ki67 WAS THE MARKER OF
NETosis WITHOUT KNOWING THE
FUNCTION OF THE PROTEIN.
IT WAS DESCRIBED A COUPLE YEARS
AGO BUT I'M EMBARRASSED TO SAY I
DON'T REMEMBER.
HAVE THE EXCUSE IT'S VERY LATE
HERE.
[LAUGHTER]
BUT WE NEVER THOUGHT THERE MIGHT
BE A LINK TO NETosis.
WE THINK IT'S A MARKER BUT WE
HAVEN'T DONE THE EXPERIMENTS OF
THE DELETING Ki67 IN
NEUTROPHILS TO SEE IF IT AFFECTS
NET FORMATION.
GOOD QUESTION.
>> THERE ARE A LOT OF THINGS,
THERE ARE MARKERS, WE USE THEM
AS SUCH IN VARIOUS EXPERIMENTS
OR EVEN LOOKING AT PATIENTS, BUT
DON'T EXACTLY KNOW THE BIOLOGIC
EFFECTS.
SO THEN DR. KAPLAN WAS TALKING
ABOUT VARIOUS MUSCARINIC
RECEPTORS AND THEIR POSSIBLE
ROLE IN NETosis.
AND THERE'S A VIEWER WHO IS
INTERESTED IN THE ROAM OF THE
VAGUS NERVE IN INFLAMMATION AND
OF COURSE THERE'S THE WORK OF
DR. TRACY UP IN NEW YORK HAVING
TO DO WITH VAGUS NERVE
STIMULATION AND ITS EFFECT ON
INFLAMMATION.
DO YOU WANT TO MAKE ANY COMMENTS
WITH REGARD TO THAT WORK AND HOW
IT MIGHT CONNECT TO NETosis OR
NOT?
>> YEAH, ALL THIS FIELD OF
NEUROIMMUNE MODULATION, RIGHT,
HOW RECEPTORS COULD BE PRESENT
ON IMMUNE CELLS WE'RE FINDING
NOW NOT ONLY THEY ARE PRESENT
BUT SEEM TO BE FUNCTIONAL AND
IMPORTANT, IT'S FASCINATING.
I MEAN, WE HAVE PRIMARILY
FOCUSED ON MUSCARINIC RECEPTORS
AND DID SOME WORK WITH THEM AT
OF ON ADENOSINE AND BUT WHAT
ROLE ADENOSINE PLAYS IN
PROMOTING NET FORMATION SO WE
ARE COMING OUT WITH MORE AND
MORE NEUROIMMUNOMODULATORS
RELEVANT TO MYELOID CELL
BIOLOGY, EITHER INHIBITING OR
ENGAGING RECEPTORS MAY HAVE
POTENTIALLY PRO
ANTI-INFLAMMATORY, BUT ALSO
IMPORTANT ROLES, STILL A LOT WE
DON'T KNOW BUT THIS IS A WAY TO
SHOW HOW STUDYING PATIENTS IS
ALSO HELPING US GO BACK AND
UNDERSTAND THE BIOLOGY OF HOW
THESE CELLS WORK.
>> YEAH, YEAH.
OKAY.
SO THEN ANOTHER MECHANISTIC
COMMENT BUT SORT OF GETTING US
INTO THE WORLD OF -- REAL WORLD
SITUATIONS IS THE WHOLE QUESTION
OF PAD 4 VARIANTS, AND THE
CONNECTION WITH PREDELICTION TO
NETosis.
AND THE QUESTIONER IS RECALLING
CONNECTION BETWEEN PAD 4 AND
CIGARETTE SMOKING, AND THE IDEA
THAT PERHAPS VARIANTS IN PAD 4
MIGHT BE ASSOCIATED WITH
RHEUMATOID ARTHRITIS IN SMOKERS.
SO THE QUESTION IS WHETHER
PERHAPS THE NETosis MIGHT BE
THE MECHANISM BY WHICH THAT IS
HAPPENING.
>> YEAH, SO THIS IS A REALLY
FASCINATING STORY.
I THINK ON ONE HAND IT'S BEEN
SHOWN THAT SMOKING BY ITSELF CAN
LEAD TO SITRILINATION OF
TISSUES, THE LINK MAY BE PERHAPS
IN PART DRIVEN BY THE FACT IF
YOU SMOKE THAN IF YOU HAVE
GENETIC PREDISPOSITION MAY BE
ONE MECHANISM.
IT'S SHOWN SMOKING MAY TRIGGER
NET FORMATION, PERFECT STORM,
THINGS HAPPENING IN MUCOSA OF
THE LUNGS, GENETICALLY DRIVEN OR
IF SOMEONE SMOKES, SITRILINASE,
MAY GET AMPLIFIED.
THERE ARE A NUMBER OF GENETIC
POLYMORPHISMS ASSOCIATED TO
IMMUNITY THAT HAVE BEEN FOUND TO
LEAD TO ENHANCE THE NET
FORMATION EVEN FORM AUTOIMMUNITY
DEVELOPS, AND ONE IS RELATED TO
PRODUCTIVITY BUT THERE ARE
OTHERS WHICH MAKE IT FASCINATING
HOW ENVIRONMENT AND GENETICS MAY
WORK TOGETHER TO INDUCE
DYSREGULATION EVEN BEFORE
AUTOIMMUNITY DEVELOPS.
>> FROM THE OTHER SIDE OF THE
GENETIC WORLD, ONE OF OUR
AUDIENCE WAS INTERESTED IN
LEARNING A LITTLE BIT MORE ABOUT
HOW MICROING MICROORGANISMS MAE
EVOLVED STRATEGIES TO SABOTAGE
NETosis, TO PREVENT OR
COUNTERACT IT.
I SUPPOSE NUCLEASES BEING
PRODUCED BY BACTERIA WOULD BE
ONE SUCH MECHANISM, BUT CAN YOU
MAYBE TALK ABOUT ANY OTHERS THAT
MAY HAVE BEEN LOOKED AT?
>> WELL, CATALASE CONSUMES
HYDROGEN PEROXIDE, PREVENTS NET
FORMATION, THAT'S ANOTHER.
NUCLEASES ARE ALSO FOR EXAMPLE
PRODUCED BY PARASITES.
THOSE ARE THE TWO THAT COME TO
MIND.
I'LL THINK -- I THINK WE WROTE A
REVIEW ABOUT THIS AT SOME POINT
A FEW YEARS AGO SO I'LL HAVE TO
RE-READ IT.
>> BUT --
>> MARIANA, DO YOU REMEMBER?
>> NETosis IS A BIG ONE.
>> THAT MAKES SENSE, IN FACT I
GUESS THAT'S EVEN PART OF THE
REASON WHY IN BACTERIOLOGY WE
HAVE CATALASE POSITIVE AND
CATALASE NEGATIVE AND SO FORTH
BACTERIA, SORT OF DENOTING THEM
BASED ON WHETHER THEY HAVE
EVOLVED THOSE MECHANISMS OR NOT.
OKAY.
SO, LET'S SEE.
HERE'S SOMEONE, A HOPEFUL MEMBER
OF THE AUDIENCE, FOR THE
NEUTROPHILS I GUESS, IS THERE
ANY WAY THAT NEUTROPHILS COULD
FORM NETs WITHOUT DYING?
>> SO THIS HAS BEEN AN AREA OF A
LOT OF CONTROVERSY, I THINK.
THERE HAVE BEEN GROUPS THAT HAVE
SUGGESTED UNDER CERTAIN
CIRCUMSTANCES UPON EXPOSURE TO
CERTAIN MICROBES NEUTROPHIL MAY
BE ABLE TO FORM NETs WITHOUT
DYING.
AND KIND OF EXTRUDE THE NUCLEUS
BUT CONTINUE TO FUNCTION AND
PERFORM VARIOUS ACTIVITIES.
IN OUR HANDS, NETs FORM DURING
AUTOIMMUNE DISEASES, WHAT WE
CALL SUICIDAL.
WE ALWAYS SEE THAT THE CELL
DIES, NO DISRUPTION OF CELL
MEMBRANE AND IT'S A FINAL EVENT
BUT ARTURO I'M SURE CAN TALK
ABOUT THIS.
>> I'LL SAY THE SAME THING.
THERE ARE REPORTS IT WOULD BE
VERY INTERESTING TO FIND IF
THERE'S A MECHANISM SO WE CAN
INTERRUPT IT AND SEE HOW IT
WORKS.
SO, THAT'S WHY IT'S IMPORTANT IN
WORK ON MECHANISMS TO SEE, TO
IDENTIFY FUNCTION AND TO
INTERRUPT AND I WILL SEE WHEN
THOSE APPEAR AND DECIDE.
>> IF IT DID HAPPEN, THERE WOULD
HAVE TO BE SOME SORT OF GHOST
CELLS OR WHATEVER THAT WOULD BE
THE SORT OF THE REMNANTS OF THE
NEUTROPHIL THAT'S EXTRUDED ITS
NUCLEIC ACIDS BUT STILL HAS SOME
FUNCTION OR AT LEAST THERE'S THE
CARCASS OF IT GOING AROUND IN
THE CIRCULATION.
SO, WELL, AN INTERESTING THING
TO LOOK FOR.
AND WE'LL AWAIT THE NEXT
PRESENTATION IN DEMYSTIFYING
MEDICINE TO HEAR ABOUT THAT I
GUESS.
SO, ANOTHER QUESTION, HOW IS
NETosis REGULATED?
ARE THERE SPECIFIC CYTOKINES
THAT POTENTIATE OR INHIBIT
NETosis?
I THINK THAT IN ONE OF THE
MARIANA'S DIAGRAMS THERE WERE
SOME CYTOKINES THAT WERE
INDICATED THERE BUT MAYBE YOU
COULD REVIEW THEM FOR THE
VIEWER.
>> YES, I MEAN, SOME THAT COME
TO MIND TNF WE'VE FOUND IS A
STRONG INDUCER OF NETs AND WE
WEEK IN RHEUMATOID SYNOVIUM.
IL-17 APPEARS TO HAVE SOME
EFFECT ON NET FORMATION, IT'S
BEEN DESCRIBED IN OUR HANDS IT'S
PRETTY WEAK BUT INDUCER OF
NETs.
CYTOKINES THAT INHIBIT I DON'T
THINK THERE'S ONE THAT COMES TO
MIND THAT'S BEEN FOUND TO HAVE
INHIBITORY EFFECTS ON ABILITY TO
FORM NETs.
MANY ARE TARGETS GOING TO THE
JAK/STAT PATHWAY BUT NOT ONLY,
RIGHT?
BUT CNS IS A BIG ONE.
>> THEN THAT WOULD ALSO PROVIDE
AT LEAST ONE OF THE MECHANISMS
BY WHICH TNF INHIBITORS FOR
EXAMPLE WORK IN CERTAIN
AUTOIMMUNE DISEASES ANYWAY.
SO THAT WOULD BE SATISFYING IN
TERMS OF KNOWING ABOUT THAT.
OKAY.
SO, ANOTHER MEMBER OF THE
AUDIENCE WANTS TO KNOW ABOUT
NEUTROPHIL NETs AND WHETHER
THEY PLAY A ROLE IN GOUT.
>> YES.
YOU GO AHEAD.
IT'S AUTOIMMUNE,
AUTO-INFLAMMATORY DISEASE.
YES, THERE'S VERY GOOD DATA FROM
GERMANY.
>> YES, IT WAS FOUND THAT IT'S
ONE OF THE MOST NEUTROPHILIC
CONDITIONS WE CAN FIND IN THE
SYNOVIUM AND THERE ARE'S SO MANY
NEUTROPHILS GOING IN WHEN GOUT
OCCURS IN RESPONSE TO URIC ACID,
INDUCERS OF NETs, BUT THERE'S
SO MANY NEUTROPHILS THERE THAT
THE NETS BECOME AGGREGATED AND
SO THESE NETs ACTUALLY NOT
ONLY HAVE INFLAMMATORY EFFECT
BUT EVENTUALLY LEAD TO
RESOLUTION OF THE INFLAMMATION
WHICH WE KNOW IS A PICTURE OF
GOUT.
GOUT WILL GO AWAY EVEN IF YOU
DON'T TREAT IT.
IT'S BELIEVES AGGREGATED NETs
ARE CHOKING CYTOKINES AND
CHEMOKINES AND MAKING THE --
DAMPENING OR LEADING TO
RESOLUTION OF INFLAMMATION SO
IT'S A STORY OF HOW THE NET CAN
HAVE A VERY PRO-INFLAMMATORY
EFFECT BUT IN BIG QUANTITY CAN
LEAD TO RESOLUTION OF
INFLAMMATION.
>> WELL, OF COURSE, JUST
BRINGING UP THE QUESTION OF
GOUT, YOU KNOW, THERE ARE ALL OF
THE DATA WITH REGARD TO DIETARY
FACTORS AND HOW THEY CAN
SOMETIMES HAVE AN EFFECT ON
ACUTE GOUT ATTACKS.
MAYBE JUST TO RIFF ON THAT, ARE
THERE DIETARY FACTORS THAT IN
SOME WAY INCREASE OR DECREASE
ONE'S PREDISPOSITION FOR
NETosis?
>> NOT THAT I KNOW OF.
MARIANA?
>> GREAT QUESTION.
NOTHING THAT COMES TO MIND, YOU
KNOW, THAT'S A WELL-KNOWN
TRIGGER.
I WANT TO MENTION WITH GOUT IT
HAS BEEN SHOWING COALCHISENE,
BECAUSE OF MITOSIS, IT CAN
INHIBIT NETs, COULD BE
ADDITIONAL MECHANISM BY WHICH IT
IS EFFECTIVE IN GOUT.
>> ANOTHER PHARMACOLOGIC
INTERVENTION THAT ONE COULD
THINK ABOUT THAT I GUESS ARTURO
AT LEAST TOUCHED UPON IS THE
QUESTION OF STING, AND I BELIEVE
THAT THERE'S QUITE A GREAT
INTEREST NOW IN THE DEVELOPMENT
OF STING INHIBITORS IN THE
BIOTECH SECTOR.
ANY WORDS ON THAT WITH REGARD TO
POSSIBLE APPLICATION IN THESE
CONDITIONS?
>> I DON'T HAVE ANY INSIGHT
INTO THAT.
I THINK IT'S A VERY GOOD IDEA
BUT I HAVEN'T HEARD ANYTHING.
>> YEAH.
>> I MEAN, WHAT WE HAD SEEN
PATIENTS THAT RECEIVE
AMIPROMYRAB WHICH BLOCKS TYPE 1
INTERFERONS, NOT DIRECTLY LINKED
TO STING BUT TO THE TYPE 1
PATHWAY DOWNSTREAM OF STING
APPEARS TO BE DECREASE IN NETs
IN THOSE PATIENTS WITH
INHIBITION.
>> I SEE DR. ARIAS TURNED ON HIS
CAMERA WHICH MAY INDICATE THAT
HE HAS A QUESTION THAT HE WOULD
LIKE TO POSE.
WIN, DO YOU HAVE A QUESTION FOR
OUR SPEAKERS?
>> SURE.
FIRST OF ALL, THANK YOU VERY
MUCH FOR THIS VERY SUCCESSFUL
PRESENTATION OF BRIDGING SCIENCE
AND MEDICINE.
SO, I HAVE TWO QUESTIONS.
ONE IS STEROIDS,
CORTICOSTEROIDS, WIDELY USED IN
AUTOIMMUNE DISEASES WEREN'T
MENTIONED IN YOUR PRESENTATIONS.
DO CORTICOSTEROIDS PLAY ANY ROLE
IN NETs FORMATION?
AND THE SECOND QUESTION I HAVE
IS I'M A LITTLE CONFUSED BY SOME
OF THE DIAGRAMS.
ARE NETs STRICTLY
EXTRACELLULAR OR IN SOME
DIAGRAMS LOOKS LIKE THEY ARE AT
LEAST INITIALLY COVERED BY A
PLASMA MEMBRANE.
IS MUCH KNOWN ABOUT SORT OF THE
BASIC CELL BIOLOGY REGARDING THE
FORMATION OF NETs AND IF IT
INVOLVES EXTENSION OF PLASMA
MEMBRANE, MY GOSH, THERE MUST BE
AN ENORMOUS AMOUNT OF CHANGES
TAKING PLACE WITHIN THE CELL
OTHER THAN JUST PHENOMENA THAT
YOU DESCRIBED.
I'M CURIOUS WHETHER CELL
BIOLOGISTS HAVE JUMPED INTO
THIS.
YOU MENTIONED COALCHISENE.
AT ANY RATE, THIS HAS BEEN AN
EXTREMELY EXCITING AND WE
APPRECIATE YOUR PARTICIPATION
IMMENSELY.
>> MAYBE I CAN ANSWER, AND YOU
ADDRESS THE SECOND ONE.
>> YOU GO.
>> IT'S REALLY FASCINATING.
AND THERE WAS A RECENT PAPER BY
SOMEONE WHO WORKS ON NETs IN
CANCER.
THERE IS THIS OBSERVATION THAT
OF A DRIVER OF METASTASIS IN A
POORLY UNDERSTOOD AWAY BUT SHE
MADE A CONNECTION
CORTICOSTEROIDS PRODUCED FOR
EXAMPLE UNDER CONDITIONS OF
STRESS ARE ENHANCING NET
FORMATION AND THESE MAY BE A
MECHANISM BECAUSE KNITS -- NETE
BEEN IMPLICATED IN METASTASIS,
STRESS MAY BE AN ENHANCER.
AT LEAST IN THE CONTEXT OF
CANCER, HER OBSERVATIONS
STEROIDS IF ANYTHING INCREASE
NETs.
NOW, THIS COULD BE -- THERE'S
MORE TO IT, DEPENDS ON THE
DOSES, CHRONICITY OF EXPOSURE,
BUT AT LEAST IN THE CONTEXT OF
CANCER IF ANYTHING IT'S THE
OTHER WAY OF IMMUNOSUPPRESSIVE
IN AUTOIMMUNITY.
>> JUST THINKING ABOUT THAT FOR
A MOMENT, IF I CAN JUST COMMENT
ON YOUR COMMENT FOR A SECOND,
AND THAT IS THAT MAYBE
GLUCOCORTICOIDS COULD BE
MOBILIZING SUBSET OF NEUTROPHILS
PRONE TO MAKE NETs, YOU KNOW,
DEMARGINNATING A SUBPOPULATION
OR WHATEVER THAT'S NET PRONE.
>> YEAH, ABSOLUTELY.
I THINK, YEAH, MORE NUANCE, YES,
ABSOLUTELY, I AGREE.
>> AND TO THE FIRST PART OF THE
QUESTION, I AGREE WE SHOULD --
WE HOPE THAT MORE CELL
BIOLOGISTS GET INTERESTED IN
NETs.
THE PROBLEM OF WORKING IN NETs
IS THAT THERE IS NOT CELL LINES
THAT MIMICS ACTIVITY OF NETs.
THERE'S NO CELL LINE THAT MAKES
NETs.
ALL THE WORK THAT HAS TO BE DONE
WITH REAL NEUTROPHILS AS MARIANA
MENTIONED THE MOUSE IS A LOUSY
MODEL FOR NEUTROPHIL BIOLOGY.
THE RATS ARE BETTER.
RABBITS ARE BETTER.
THEIR NEUTROPHILS EXPRESS MORE
OF THE PROTEINS WE KNOW HUMAN
NEUTROPHILS WORK, SO A CELL THAT
THERE IS NO MODEL FOR AND THAT
LEAVES ONLY SIX HOURS AFTER
TAKING IT OUT OF BLOOD IS KIND
OF NOT AN EASY MODEL, RIGHT?
ONE HAS TO REINVENT BIOLOGY
WITHOUT THE BENEFITS OF
MOLECULAR BIOLOGY.
IT WOULD BE GREAT TO RECRUIT
MORE BIOLOGISTS TO THE FIELD.
DO YOU AGREE, MARIANA?
>> TOTALLY.
>> ALL RIGHT.
NOW WE HAVE A QUESTION FROM
ANOTHER MEMBER OF THE AUDIENCE
WITH REGARD TO A DIFFERENT FORM
OF CELL DEATH.
SO, THE QUESTIONER WANTS TO KNOW
DO NETs HAVE A ROLE IN
APOPTOSIS?
>> WELL, WHAT I HAVE READ A
NEUTROPHIL BY APOPTOSIS OR
NETs, AND THE AKT PATHWAY WAS
SORT OF REGULATOR ON WHICH WAY
IT WAS GOING TO GO, BUT AS
ARTURO SHOWED WE SEE AT THE
BEGINNING, IT'S THE OPPOSITE, IN
APOPTOSIS SEE CHROMATIN, IN
NECROSIS SEE CONDENSATION SO I'M
NOT AWARE THAT BOTH -- I ASSUME
UPON SPECIFIC SIMILAR WILL DIE
BY NETosis, A MORE SPECIFIC
PROCESS THAN THE REGULAR WAY
BOTH NEUTROPHILS WILL DIE.
>> I AGREE.
IF THE QUESTION WAS WHETHER YOU
CAN EXPOSE ANOTHER DEVELOP TO
NETs AND INDUCE APOPS SO --
APOPTOSIS I'M NOT AWARE, IN
BYSTANDER CELLS, NOT NEUTROPHILS
BUT OTHER TYPES OF CELLS.
>> WE DO KNOW THEY CAN INDUCE
ENDOTHELIAL CELL DEATH,
ESPECIALLY IN CONTEXT OF NETs
AUTOIMMUNE PATIENTS, GOOD AT
INDUCING, DO HAVE ABILITY TO
DAMAGE OTHER CELLS.
WE THINK THAT'S ONE OF THE
MECHANISMS.
>> NOW JUST AS YOU SAID THAT A
QUESTION CAME IN WITH REGARD TO
NETs AND KIDNEY DISEASE.
AND OF COURSE ONE COULD THINK OF
THAT CONNECTION I GUESS THROUGH
THE GLOMERULUS.
ARE THERE VASCULAR BEDS THAT ARE
MORE OR LESS PRONE TO THE
EFFECTS OF NETosis AND WHAT IS
THE MECHANISM BY WHICH NETs
PERHAPS CAN INCREASE THE RISK OF
CHRONIC KIDNEY DISEASE IF THEY
DO?
>> SO, THIS IS ACTUALLY ONGOING
WORK IN OUR LAB.
MARIO, A POSTDOCTORAL FELLOW,
WORKING VERY -- FOCUSING ON
THAT.
WHAT WE'RE SEEING PRELIMINARY IS
WE'RE SEEING EVIDENCE OF
NEUTROPHILS INFILTRATING
PARTICULARLY GLOMERULAR REGIONS,
ONE MECHANISM THAT LEADS THE
FINDING BESIDES POTENTIAL EFFECT
ON VASCULAR HEALTH MAY STIMULATE
CELLS, A MECHANISM COULD LEAD TO
ENHANCED VASCULAR DAMAGE.
WORK ON NETs AND FIBROSIS,
CERTAIN CIRCUMSTANCES NETs CAN
BE STRONG INDUCERS OF FIBROSIS,
IN THE CONTEXT OF THE KIDNEY ONE
COULD SPECULATE THIS COULD BE
ANOTHER POTENTIAL MECHANISM.
WE'RE SEEING INFILTRATING NETs
IN BIOPSIES FROM LUPUS PATIENTS,
APPEARS TO BE HAPPENING IN VIVO
AS WELL.
>> YOU RAISED QUESTION OF
FIBROSIS, SO THEN THAT WOULD
LEAD SOME TO THINK ABOUT
SCLERODERMA AS AN AUTOIMMUNE
DISEASE WITH FIBROSIS BUT ONE
COULD THINK OF OTHER FORMS OF
FIBROSIS, INTERSTITIAL
PULMONARY,ER IS A ROWS -- --
CIRRHOSIS OF THE LIVER.
>> THERE HAVE BEEN MAYBE MORE
INDIRECT BUT I'VE SEEN THINGS SO
THERE IS SOME ON NEUTROPHILS IN
THE LIVER AND ACTUALLY TISSUE
RESIDENT NEUTROPHILS IN THE
LIVER, ONE OF THE ONES YOU CAN
FIND NEUTROPHILS EVEN IN ABSENCE
OF INFECTION, SO CERTAINLY NOT
ONLY RELATED TO LUPUS AND LUNGS,
BUT MORE WORK IS NEEDED
REGARDING SPECIFICITY AND
PATHWAYS THAT ARE TRIGGERED BY
THESE NETs.
>> ALL RIGHT.
NOW ANOTHER OF THE EVOLUTIONARY
BIOLOGISTS BACK WITH US, WANTING
TO KNOW THIS MAY HAVE IN SOME
WAY BEEN COVERED BY ARTURO BUT
I'LL POSE THE QUESTION.
THAT IS ARE THERE PLANT CELLS
THAT CAN UNDERGO NETosis?
>> YES.
THERE'S A BEAUTIFUL WORK OF --
I'M BLOCKING THE NAME, SORRY.
SHE PASSED AWAY RECENTLY
UNFORTUNATELY, SHE SHOWED THERE
ARE SPECIFIC CELLS IN THE ROOTS
OF PLANTS THAT RE-FORM THEIR
CHROMATIN AND EXPEL INTO THE
SOIL.
WHAT IS INTERESTING IS THAT SHE
DID A VERY SIMPLE EXPERIMENT OF
INFECTING A PLANT WITH A PLANT
FUNGAL PATHOGEN AND SHOWED IF
SHE DESTROYED LET'S CALL THEM
PLANT NETs, PLANT BECAME FAR
MORE SUSCEPTIBLE TO THE FUNGAL
INFECTION.
SO IT IS MODIFIED CHROMATIN THAT
HAS THE FUNCTION OF IMMUNE
DEFENSE, OF COURSE IT DOESN'T
HAVE -- I DON'T THINK ANYBODY
HAS WORKED ON THE COMPOSITION OF
THESE PLANT NETs TO SEE THE
EQUIVALENT OF THE PEROXIDASE, ET
CETERA, AS FAR AS I KNOW, NO
WORK ON THE FORMATION OF NETs
PLANT FORMATION.
>> THERE ARE STUDIES
DEMONSTRATING -- THEY GO BACK A
LOT FURTHER IN PLANTS WITH
REGARD TO INNATE IMMUNITY, AND
VARIOUS MECHANISMS OF INNATE
IMMUNITY BUT I GUESS PLANTS
DON'T REALLY HAVE THE EQUIVALENT
OF NEUTROPHILS, FOR EXAMPLE,
THERE ISN'T A, I DON'T THINK,
BUT I'M ASKING, THEY DON'T HAVE
AN EQUIVALENT OF A NEUTROPHIL
BUT IT MAY BE THAT THIS IS AN
EXAMPLE OF CONVERGENT EVOLUTION
OR WHAT HAVE YOU IN WHICH THIS
MECHANISM IS VERY IMPORTANT FOR
ANTIMICROBIAL DEFENSE, AND THERE
HAVE BEEN MORE THAN ONE PATHWAY
FOR IT TO EVOLVE, I GUESS ONE
MIGHT THINK.
THAT'S FASCINATING.
WE'RE CLOSE TO THE END OF OUR
SESSION.
I GUESS I MIGHT START WRAPPING
THINGS UP BY ASKING THE TWO OF
YOU WHAT YOU THINK ARE THE MOST
IMPORTANT UNRESOLVED ISSUES IN
THE WORLD OF NETosis, AT
LEAST, YOU KNOW, FROM YOUR
PERSPECTIVE.
ARTURO, DOUGH WANT TO START OUT
MAYBE WITH REGARD TO THE BASIC
SCIENCE OF NETosis AND WHAT
ARE THE BIG UNRESOLVED QUESTIONS
THAT YOUNG INVESTIGATORS MIGHT
WANT TO ATTACK?
>> WELL, I THINK UNDERSTANDING
MECHANISM WHAT IS IS BEING
OXIDIZED IN NET FORMATION IS A
GOOD QUESTION, HOW IS CHROMATIN
MODIFIED IN ORDER TO ALLOW THE
UNWRAPPING AND FORMATION OF
NETs.
THAT'S TWO QUESTIONS THAT COME
TO MIND.
I STILL THINK THAT THE
ANTIMICROBIAL ACTIVITY OF
HISTONES IS EXTREMELY
INTERESTING.
AND I WOULD START A LAB ON THAT
IF I WERE STARTING A LAB AGAIN.
>> WELL, THOSE ARE REALLY ALL
FASCINATING QUESTIONS.
MARIANA, WHAT ABOUT FROM YOUR
VANTAGE POINT?
>> I THINK ANOTHER QUESTION I
HAVE THIS CONCEPT OF NEUTROPHIL
HETEROGENEITY.
WHEN YOU STIMULATE, ONLY A
CERTAIN PERCENTAGE UNDERGO
NETs, THE REST DON'T, RIGHT?
WHY IS THAT?
AND WE REALLY HAVE JUST STARTED
TO UNDERSTAND THAT NOT ALL
NEUTROPHILS ARE THE SAME.
I THINK THAT POTENTIALLY WE WANT
TO TARGET ABNORMAL NET FORMATION
WOULD BE SO USEFUL BECAUSE WE --
A MORE TARGETED APPROACH IS
NEEDED, TO ME THAT'S ONE OF THE
KEY QUESTIONS AND SOMETHING THAT
KEEPS ME UP WHEN I THINK ABOUT
INTERESTING THINGS TO DO IN THE
FUTURE.
I HOPE OTHERS WILL KEEP WORKING
ON MOVING FORWARD.
>> IT'S BEEN A FASCINATING
SESSION.
I THINK AT THIS POINT WE CAN
CALL IT TO A CLOSE.
THANK YOU SO, SO MUCH FOR REALLY
A WONDERFUL, WONDERFUL SET OF
PRESENTATIONS, AND A VERY
ENGAGING DISCUSSION HERE IN THE
LAST HALF HOUR OR SO.
AGAIN, WE REALLY APPRECIATE IT.
GOOD LUCK TO BOTH OF YOU ON
SOLVING THESE QUESTIONS THAT YOU
HAVE YOU NOW POSED AND WE'LL
LOOK FORWARD TO SEEING YOU BACK
IN THE NOT TOO DISTANT FUTURE
WITH PERHAPS THE NEXT
INSTALLMENT IN THE FIELD OF
NETosis.
SO THANK YOU VERY MUCH.
GOOD EVENING TO ARTURO AND GOOD
AFTERNOON TO MARIANA.
AND THANK EVERYONE IN OUR
AUDIENCE, ALL OVER THE WORLD,
FOR JOINING US TODAY.
ANYWAY, THANK YOU AGAIN AND SEE
YOU NEXT WEEK.
BYE-BYE.
>> THANKS FOR INVITING US.
[END OF PROGRAM]