GOOD AFTERNOON, WELCOME TO THE 24th ANNUAL MARK KELLER HONORARY LECTURE. THIS LECTURE SERIES COMMEMORATES MARK KELLER'S LEGACY, ONE OF THE GUIDING FORCES OF EARLY ALCOHOL RESARCH. MR.KELLER BEGAN HIS CAREER IN 1935, IN THE PSYCHIATRIC DIVISION OF NEW YORK'S BELLEVUE HOSPITAL WHILE STUDYING NUTRITION-RELATED DISEASE AT BELLEVUE, HE NOTICED THESE DISEASES AFFECT PATIENTS WITH ALCOHOL USE DISORDER. THE HOSPITAL ADMITTED AND READMITTED THOUSANDS OF PATIENTS WITH ALCOHOL USE DISORDER EACH YEAR, AND I SUSPECT THEY STILL DO. THESE OBSERVATIONS INSPIRED MR. KELLER TO TRY AND UNDERSTAND WHY SOME INDIVIDUALS CONTINUED TO DRINK ALCOHOL DESPITE LIFE-THREATENING CONSEQUENCES. ONE OF HIS PIONEERING ACHIEVEMENTS WAS ESTABLISH A CENTER IN THE '40s HOUSED AT YALE UNIVERSITY AND THEN MOVED ULTIMATELY TO RUTGERS. HE CONTRIBUTED MORE THAN 200 PUBLICATIONS INCLUDING HIS DICTIONARY OF WORDS ABOUT ALCOHOL AND INFLUENTIAL REFERENCE WORK THAT STANDARDIZED LANGUAGE USED BY ALCOHOL RESEARCHERS TODAY. AMONG HIS CROWNING ACHIEVEMENTS WAS HELPING TO LAUNCH NIAAA'S EXISTENCE, AS A NEW FEDERAL RESEARCH ORGANIZATION. IT WAS NIAAA THAT WAS SIGNED INTO LEGISLATION ON THE FINAL DAY OF 1970, SO IF YOU DO THE MATH THAT MAKES THIS NATIONAL INSTITUTE ON ALCOHOL ABUSE AND ALCOHOLISM'S 50th ANNIVERSARY. OUR STATUS AS AN INSTITUTE CAME ABOUT BECAUSE FOR YEARS MR. KELLER AND OTHERS WORKED TIRELESSLY WITH LAWMAKERS, FEDERAL HEALTH OFFICIALS AND OTHER STAKEHOLDERS WITH AN INTEREST IN ADVANCING ALCOHOL RESEARCH AND HE SERVED ON THE COMMITTEE THAT ULTIMATELY RECOMMENDED THE INSTITUTE'S FORMATION AND WENT ON TO GUIDE MUCH OF NIAAA'S EARLY RESEARCH. SO TO HONOR HIS PIONEERING CONTRIBUTIONS NIAAA ESTABLISHED TODAY'S LECTURE SERIES IN HIS NAME. KELLER AWARDEES ARE OUTSTANDING ALCOHOL INVESTIGATORS WHOSE LIFETIMES OF RESEARCH OFFER ANSWERS TO QUESTIONS ABOUT HOW ALCOHOL AFFECTS THE BODY AND THE MIND, ABOUT HOW WE PREVENT, DIAGNOSE AND TREAT ALCOHOL USE DISORDERS, AND ABOUT HOW TODAY'S SCIENTIFIC ADVANCES PROVIDE HOPE FOR TOMORROW. THIS YEAR'S HONOR REIS DR. LAURA NAGY, A LEADER IN THE FIELD OF RESEARCH ON ALCOHOL-INDUCED ORGAN DISEASE. DR. NAGY IS PROFESSOR OF MOLECULAR MEDICINE AT CLEVELAND CLINIC LERNER COLLEGE OF MEDICINE AT CASE WESTERN UNIVERSITY. AND SHE'S A STAFF MEMBER IN THE DEPARTMENTS OF INFLAMMATION AND IMMUNITY AND GASTROENTEROLOGY AND HEPATOLOGY AT THE CLEVELAND CLINIC. IN ADDITION, SHE'S AN ADJUNCT PROFESSOR OF NUTRITION AT CASE WESTERN. DR. NAGY MADE SIGNIFICANT CONTRIBUTIONS TO YOUR UNDERSTANDING OF THE INNATE IMMUNE SYSTEM ROLE IN THE PROGRESSION OF ALCOHOL-ASSOCIATED LIVER DISEASE, BEGAN HER CAREER BY CONDUCTING POSTDOCTORAL RESEARCH AT THE UNIVERSITY OF CALIFORNIA, SAN FRANCISCO WHERE SHE STUDIED EFFECTS OF ALCOHOL ON ADENOSINE RECEPTOR SIGNALING. AFTER JOINING CASE WESTERN RESERVE UNIVERSITY SHE CONTINUED TO MAKE MAJOR CONTRIBUTIONS TO UNDERSTANDING ABOUT ALCOHOL'S IMPACT ON ORGAN AND IMMUNE SYSTEM INTERACTIONS, ONE OF THOSE CONTRIBUTIONS WAS GROUND BREAKING OBSERVATION ALCOHOL IMPAIRS ACTION OF ANTI-INFLAMMATORY CYTOKINE AND ITS ROLE IN DAMPENING THE INFLAMMATORY RESPONSE IN THE LIVER. RECENTLY HER RESEARCH FOCUSES ON CONTRIBUTION OF INNATE IMMUNE SYSTEM IN INITIATION AND PROGRESSION OF ALCOHOL-INDUCED LIVER INJURY, SHE DEVELOPED A SPECIFIC MOUSE MODEL TO EXAMINE THE MECHANISMS THROUGH WHICH ALCOHOL ACCELERATES SCARRING IN THE LIVER AND IS MAKING PRO PROGRESS IN ELUCIDATING ROLE OF NECROPTOSIS, PROGRAMMED CELL DEATH WAY PATHWAY IN THE LIVER OF PATIENTS WITH SEVERE ALCOHOLIC HEPATITIS, AND WORKING TOWARD DEVELOPING BIOMARKERS. IN HER CAREER DR. NAGY CHAMPIONED COLLABORATIVE RESEARCH EFFORTS, INITIATED NORTHERN OHIO ALCOHOL CENTER AT THE CLEVELAND CLINIC INTERDISCIPLINARY RESEARCH GROUP, SPEARHEADED RESEARCH WITH NIAAAs ON GENETIC RISK. FOR 20 YEARS FUNDED BY NIAAA AND DEPARTMENT OF DEFENSE, RECEIVED MANY ACCOLADES FOR HER ACHIEVEMENTS AND SERVICE, PROMINENTLY LEADERSHIP ROLES, SHE HAS BEEN CHAIR OF THE NIAAA BOARD OF SCIENTIFIC COUNSELORS, ASSOCIATE EDITOR OF BOTH ALCOHOLISM CLINICAL AND EXPERIMENTAL RESEARCH, AND THE JOURNAL OF HEPATOLOGY, IN ADDITION TO BEING ON THE EDITORIAL BOARD OF SEVERAL OTHER SCIENTIFIC JOURNALS. SHE'S THE PAST PRESIDENT OF THE RESEARCH SOCIETY ON ALCOHOLISM, CO-DIRECTOR OF CELL BIOLOGY PROGRAM AT CASE PERSON WHERE SHE'S BEEN HONORED FOR MENTORING. SHE'S RECIPIENT OF NIH MERIT AWARD AND MARIE AND SAM MILLER AWARD FROM CLEVELAND CLINIC. DR. NAGY, NIAAA IS PROUD TO HONOR YOU WITH THE MARK KELLER AWARD. THANK YOU FOR YOUR CONTRIBUTIONS. IT'S MY PLEASURE TO TURN THE STAGE OVER TO YOU FOR YOUR PRESENTATION, WHICH IS ENTITLED -- FOR ADVANCING UNDERSTANDING OF ALCOHOL-ASSOCIATED LIVER DISEASE, THE TITLE IS "INFLAMMATION AND CELL DEATH IN ALCOHOL-ASSOCIATED LIVER DISEASE." CONGRATULATIONS. FIRST I'M GOING TO GIVE ACKNOWLEDGMENTS BECAUSE THIS IS AN AWARD. NO ONE GETS AN AWARD BY WORKING ALONE. I'M GOING TO GIVE BACKGROUND ON INNATE IMMUNITY AND ALCOHOL-ASSOCIATED DELIVER DISEASE AND TELL YOU TWO STORIES OF SURPRISING RESULTS, HYPOTHESES GONE WRONG. SOMETIMES WHEN PEOPLE GET AWARDS YOU THINK, OH, THEY MADE A HYPOTHESIS, IT WAS TRUE AND THEY WENT TO THE NEXT ONE AND SO FORTH. THEN IT WAS 20 YEARS LATER. THAT'S NOT HOW SCIENCE WORKS. I WAS GOING TO TELL YOU, MIXED IN HERE ARE TWO STORIES WHERE WE MADE GOOD HYPOTHESES BUT THEY WERE WARNING, AND WEAVE IN THINGS. WE DO BASIC RESEARCH, MECHANISTIC UNDERSTANDING LEAD TO TRANSLATIONAL INSIGHTS. THE OTHER PART I WANT TO WEAVE THROUGHOUT EVERYTHING IS TREMENDOUS VALUE OF COLLABORATION. SO FIRST ACKNOWLEDGMENTS, MY CURRENT LAB IS LISTED HERE, SOME OF THEM I THINK ARE WATCHING ON THE VIDEOCAST FROM CLEVELAND. THE STORIES I'LL TELL YOU TODAY WERE PRIMARILY DONE BY PREVIOUS POSTDOCS, I HAVE TO GET USED TO THIS. OKAY. HERE WE GO. AND THEN CURRENT WORK BY THESE. THE COLLABORATORS ARE CAROL, DOSHA. I WANT TO THANK THE NIAAA, USUALLY IN TALKS WE ACKNOWLEDGE THE NIH AND NIAAA, THINKING ABOUT THE MONEY THEY GIVE US, BUT I'M ALSO GRATEFUL FOR THE SUPPORT THAT'S PROVIDED PARTICULARLY FROM THE INTRAMURAL PEOPLE, SOME OF WHOM WERE IN THE AUDIENCE NOW AS WELL AS COLLABORATIONS IN THE EXTRAMURAL -- WITH THE EXTRAMURAL SCIENTISTS. IT'S A GREAT INSTITUTE AND IT'S BEEN A PLEASURE TO WORK WITH EVERYONE OVER THE YEARS. I WANT TO SAY THAT WE REALLY WORK COLLABORATIVELY IN CLEVELAND, FACILITATED BY P50-FUNDED ALCOHOL CENTER. IT TAKES A VILLAGE TO WORK ON THIS BECAUSE WE KNOW THAT ALCOHOL IMPACTS A NUMBER OF CELLS AND TISSUES EVENTUALLY LEADING TO INJURY. WITHIN OUR COLLABORATIVE GROUP, WE HAVE PEOPLE WHO ARE WORKIN ON INNATE IMMUNITY, HEPATOCYTE INJURY, WE HAVE A COUPLE NEUROSCIENTISTS MAKING TREMENDOUS INSIGHTS, BRINGING NEW PERSPECTIVE INTO ALCOHOL-INDUCED INJURY IN THE BRAIN. WE ALSO HAVE A VERY LARGE GROUP THAT'S INTERESTED IN THE GUT AND MICROBIOME. AND THEN WE HAVE DR. SASARATHIU HOW ALCOHOL AFFECTS SKELETAL MUSCLE. IT TAKES MORE THAN A VILLAGE. IT GOES BEYOND A VILLAGE. GEORGE SPOKE TO THIS WHERE WE HAVE MULTIPLE COLLABORATIONS WITH PEOPLE OUTSIDE OUR LOCAL AREA. GEORGE MENTIONED WE HAVE THIS VERY EXCITING INTRAMURAL/EXTRAMURAL U01 GRANT WORKING WITH DAVID GOLDMAN'S GROUP LOOKING FOR GENETIC SIGNATURES OF ALCOHOL-ASSOCIATED LIVER DISEASE AS WELL AS HEAVY DRINKING. WE JUST COMPLETED AN INTERNATIONAL U01 WITH A GROUP IN SPAIN AND ITALY, AND I'LL SHOW YOU SOME -- USING SOME DATA AT THE VERY END USING SOME OF THE SAMPLES THAT THEY WERE ABLE TO SHARE WITH US. AND THEN WE'RE ALSO TREMENDOUSLY LUCKY TO WORK WITH ALCOHOL HEPATITIS NETWORK, ORIGINALLY PART OF THE DASH CONSORTIUM, WE'RE PART OF THE GREATER NETWORK. WE'VE HAD TREMENDOUS COLLABORATIVE ENVIRONMENT TO MAKE INROADS INTO UNDERSTANDING ALCOHOL-ASSOCIATE LIVER DISEASE. NOW THE BACKGROUND. REALLY THE NATURAL HISTORY IS SIMILAR TO OTHER FORMS OF NON-ALCOHOL FATTY LIVER AND ALCOHOLIC HEPATITIS. THE INITIAL PRESENTATION STEATOSIS, PROGRESSING. NOT ALL HEAVY DRINKERS PROGRESS ALONG THIS TIME LINE. LIKELY THERE'S AN ASSOCIATION BETWEEN GENETICS AND ENVIRONMENT IN THE PROGRESSION OF THE DISEASE. THIS IS TYPICALLY A SLOW PROGRESSION OVER 20 TO 30 YEARS OF HEAVY DRINKING. ONE SPECIAL ASPECT OF ALCOHOL-INDUCED LIVER INJURY IS ALCOHOLIC HEPATITIS, SEVERE FORM YOU CAN HAVE MORTALITY ON THE ORDER OF 40 TO 60% OF PATIENTS WITHIN 30 DAYS. THIS IS REALLY THE FOCUS OF THE ALCOHOLIC HEPATITIS NETWORK TO TRY TO IDENTIFY INTERVENTIONS THAT CAN PREVENT THE DISEASE. AT EACH STAGE OF THE DISEASE WE HAVE CHARACTERISTICS, STEATOSIS, MEASURE HEPATIC ENZYMES, LOCALIZED INFLAMMATION. ALL THE CELL TYPES IN THE LIVER ARE CHANGED IN THEIR FUNCTION AND PHENOTYPE IN RESPONSE OR DURING THE PROGRESSION OF LIVER DISEASE. THE HEPATOCYTES ARE BASICALLY THE WORKHORSE OF THE LIVER. THAT'S WHAT WE LEARN ABOUT IN GRADUATE SCHOOL AND MEDICAL SCHOOL, BUT WITHIN THE LIVER THERE'S A NUMBER OF OTHER CELL TYPES THAT PLAY IMPORTANT ROLES BOTH IN HOMEOSTASIS AND DISEASE. THERE'S SPECIALIZED SINUSOIDAL EPITHELIAL CELL, SMALL WINDOWS WHERE MACRONUTRIENTS AND TOXINS CAN FLOW BACK AND FORTH READILY. A NUMBER OF CELLS ARE RESIDENT. WE HAVE NATURAL KILLER AND NATURAL KILLER T CELLS AND RESIDENT HEPATIC MACROPHAGE, THE KUPFER CELL. WE HAVE THE FAMOUS STELLATE CELL, WHICH IS THE CELL THAT WHEN TRANSFORMED DURING FIBROSIS GENERATES EXTRACELLULAR MATRIX. WE SEE CHANGES OF THE CELLS, KUPFFER CELLS BECOME ACTIVATED, WE SEE NEUTROPHILS INFILTRATING MONOCYTES, THE LIVER SINUSOIDAL ENDOTHELIAL CELLS LEAVE STRUCTURE AND FUNCTION. THE HEPATOCYTES THEMSELVES UNDERGO WHAT I TYPICALLY CALL ORGANELLE STRESS, CHANGES IN A NUMBER OF FUNCTION OF ORGANELLES IN THE HEPATOCYTES, THESE CHANGES ARE COORDINATED TO LEAD TO LIVER INJURY. IF WE THINK ABOUT HOW THIS IS HAPPENING, SOME OF THE EFFECTS OF ALCOHOL ARE DIRECT EFFECTS OF ALCOHOL AND HEPATOCYTES, LEADING TO ORGANELLE STRESS. AND THIS IS JUST AN OLD ELECTRON MICROGRAPH WHICH I THINK NICELY ILLUSTRATES -- I CAN POINT TO THE SCREEN. VERY NICE. WE'VE KNOWN FOR A LONG TIME ALCOHOL IMPAIRS MITOCHONDRIAL FUNCTION, CAUSES ENDOPLASMIC RETICULAR STRESS, CHANGE IN TRAFFICKING, ENDOCYTOSIS, MICROTUBULE DYSFUNCTION. ALCOHOL IMPAIRS THE PROTEASOMAL ACTIVITY, AND CAUSES CHANGES IN HISTONE ACETYLATION AND DNA METHYLATION. YOU CAN SEE WITH ALL THESE IMPAIRED FUNCTIONS WITHIN HEPATOCYTES IT CAN GENERATE A POTENTIALLY INJURIOUS ENVIRONMENT. SOME OF THESE EFFECTS ARE DIRECT EFFECTS OF ALCOHOL METABOLISM ON HEPATOCYTES. SO ETHANOL IS METABOLIZED AND CONVERTED TO ACETATE. WE SEE THE INDUCTION OF CYTOCHROME P 452E 1 WHICH CAN METABOLIZE ALCOHOL TO ACETALDEHYDE AND GENERATE REACTIVE OXYGEN SPECIES, CREATING PERFECT STORM LEADING TO ACCUMULATION OF DAMAGED MACROMOLECULES, WHICH IN TURN BECAUSE THE CELL CANNOT HANDLE THE ACCUMULATED DAMAGED MACROMOLECULES CAN CONTRIBUTE TO ORGANELLE STRESS AND HEPATOCYTE INJURY. BUT THERE ARE ALSO INDIRECT EFFECTS OF ALCOHOL ON HEPATOCYTES, THIS IS WHERE THE INNATE IMMUNE SYSTEM COMES IN. IN MY LABORATORY WE'VE STUDIED EFFECTS OF ALCOHOL ON THE CELLULAR COMPONENTS, IN PARTICULAR KUPFFER CELL, ONE OF MY STORIES IS ABOUT THE KUPFFER CELL. WE STUDIED SOLUBLE COMPONENTS, INCLUDING SPECIALIZED CYTOKINE CHEMOKINES LIKE MACROPHAGE INHIBITOR FACTOR. AND BASICALLY THE IMPACT OF ALCOHOL ON THE FUNCTION OF THESE INNATE IMMUNE CELLS ALSO LEADS TO INJURY WITHIN THE HEPATOCYTE. SO, ONE OF MY FAVORITE CELLS IS THE KUPFFER CELL. I WANTED TO START OFF TELLING YOU A KUPFFER STORY, AS PART OF THIS LECTURE. SO THE KUPFFER CELL AS I MENTIONED IS THE RESIDENT MACROPHAGE IN THE LIVER. IT'S INTERESTING BECAUSE IN A HEALTHY LIVER IT'S PROTECTIVE, FIRST LINE OF DEFENSE, POISED FOR PHAGOCYTOSIS, YOU CAN SEE IN THE SCANNING EM PICTURE, LOOKS LIKE IT'S READY TO POUNCE. I ACTUALLY SHOW THIS PICTURE ALL THE TIME BECAUSE YOU CAN SEE THE FINESTRA. THE KUPFFER CELLS VERY ACTIVE IN CLEARANCE OF LPS OR ENDOTOXIN, SO BACTERIAL BYPRODUCTS COMING IN FROM THE GUT. AND IN TURN IT'S SECRETES INFLAMMATORY MEDIATORS AN BIOLOGICALLY ACTIVE PRODUCTS. WHAT'S BECOME MORE OBVIOUS IS THAT THESE TISSUE MACROPHAGES EXHIBIT TREMENDOUS PLASTICITY AND THEIR PHENOTYPE IS DEPENDENT ON THE METABOLIC AND IMMUNE MICROENVIRONMENT. SO OVER MANY DECADES NOW, THE ROLE OF THE KUPFFER CELL IN ALCOHOLIC LIVER DISEASE IS IN MY GRAPHICS ALWAYS PLACED VERY CENTRALLY. IT MAY NOT BE CENTRAL IN EVERYONE'S GRAPH BUT DOES PLAY AN IMPORTANT ROLE. SO THE WORKING HYPOTHESIS GOES THAT ALCOHOL IMPAIRS THE BARRIER FUNCTION OF THE INTESTINE, ALLOWING FOR MICROBIAL PRODUCTS LIKE LPS OR BETA GLUCANS TO COME IN PORTAL CIRCULATION, INTERACT OR CLEARED OR ACTIVATE CELL SURFACE RECEPTORS SUCH AS TLR 4 OR DECTIN-1, ALCOHOL WITH ACTIVATE THE COMPLEMENT SYSTEM, COMPLEMENT IS PART OF THE INNATE IMMUNE SYSTEM. IN THE INITIAL STAGES CAN GENERATE C3A AND C5A, ACTIVATING COGNATE RECEPTORS ON THE KUPFFER CELL, THE COMBINATION LEADS TO THE INCREASED GENERATION OF INFLAMMATORY MEDIATORS. FOR THIS TALK I'M GOING TO USE TNF ALPHA AS MY CYTOKINE. WE SEE GLOBAL CHANGES IN A NUMBER, BUT I'M GOES TO USE TNF ALPHA TODAY. THE TNF CAN INTERACT WITH RECEPTORS ON HEPATOCYTE LEADING TO STEATOSIS, CELLULAR DEATH, ET CETERA. IT'S INTERESTING BECAUSE WHEN I FIRST CAME TO THE LABORATORY I HAD DONE MY POSTDOCTORAL FELLOWSHIP WITH DIAMOND AND GORDON AT UCSF. THEY ARE STUDYING HOW ALCOHOL OR THE LAB WAS STUDYING HOW ALCOHOL AFFECTS TRANSDUCTION, CAUSE INTOXICATION, DEPENDENCE, TOLERANCE. WHEN I STARTED MY OWN LABORATORY I THOUGHT WE COULD ASK WHETHER ALCOHOL NOT ONLY INCREASED EXPOSURE OF THESE PATHOGEN MOLECULAR PATTERN RECEPTORS BUT ALSO DID IT ALTER SIGNALING LIKE IT DOES NEUROTRANSMITTER SIGNALING. WE DEVELOPED A PRIMARY CULTURE SYSTEM FOR HEPATIC KUPFFER CELLS, THESE ARE FROM RATS, ORIGINALLY. THE RATS WERE EITHER FED CONTROL DIET OR ANOTHER DIET FOR FOUR WEEKS CAUSING EARLY STAGES OF LIVER DISEASE. WHAT WE WERE ABLE TO SHOW WHEN THE HEPATIC MACROPHAGES WERE ISOLATED AND STIMULATED, WITH LPS EX VIVO, NOW IN A CULTURE DISH, WE STIMULATE WITH LPS, AND WE CAN SEE AS YOU WOULD EXPECT IN CONTROL WE SEE DOSE-DEPENDENT INCREASE, THIS IS TNF ALPHA BY ELISA. AFTER THE ETHANOL FEEDING, WE SEE THE SIGNALING PATHWAY. 20 POSTDOC YEARS LATER, MORE LIKE 40 POSTDOC YEARS LATER, WE KNOW IN GREAT DETAIL SOME MECHANISMS FOR THIS ACTIVATION OR THIS SENSITIZATION. WE KNOW THAT ALCOHOL CAN ENHANCE THE MYD88 INDEPENDENT AND PATH ARMS OF SIGNALING. ON THE MYD88 SEE ENHANCED REGULATION OF P38 AND ERK AND NF-kappaB. A LOT IS DEPENDENT ON THE PRODUCTION OF REACTIVE OXYGEN SPECIES. KUPFFER CELL INDUCES AND SHOWS ENHANCED ACTIVATION. THERE'S MULTIPLE WAYS TO ENHANCE TLR 4 SIGNALING. THESE SIGNALS HAVE AN EFFECT ON BOTH TRANSCRIPTION AND mRNA STABILITY OF A NUMBER OF CYTOKINES AND CHEMOKINES. WE ALSO KNOW THAT ALCOHOL AFFECTS THE PATHWAY LEADING TO ENHANCED PRODUCTION OF INTERFERON AND INTERFERON-DEPENDENT GENES. SO THIS IS KIND OF SPOILER ALERT. THIS IS THE FIRST HYPOTHESIS THAT WINDS UP GOING WRONG, WITH SURPRISING AND GOOD RESULTS. WE SPENT SO MANY YEARS TRYING TO UNDERSTAND TRL4 SIGNALING, HOW ALCOHOL AFFECTS THAT. WE HYPOTHESIZED THAT IN THE PROCESS OF -- AS LIVER INJURY DEVELOPED, WE ALSO ARE GOING TO SEE THE RELEASE OF DANGER ASSOCIATED MOLECULAR PATTERNS, DAMPs. AS HEPATOCYTES ARE INJURED AND DYING, THEY ARE RELEASING DAMPs WHICH IN COMBINATION WITH PAMPs WOULD ACTIVATE THE KUPFFER CELL. HYPOTHESIS STARTED BECAUSE I HAD ATTENDED A LECTURE BY MICHAEL LUTZ AT UNIVERSITY OF PITTSBURGH SHOWING NOT REALLY ALCOHOL BUT THAT PAMPs AND DAMPs HAVE DIFFERENT microRNA SIGNATURES. A POSTDOC SET OUT TO ASK DOES ALCOHOL AFFECT DAMP SIGNALING AND IS THERE A DIFFERENT microRNA SIGNATURE IN RESPONSE TO PAMP OR DAMP? AND WE FIRST THOUGHT ABOUT USING HYALURONIC ACID, HYALURONIC ACID IS A CARBOHYDRATE, A MAJOR COMPONENT OF EXTRACELLULAR MATRIX, REPEATING DISACCHARIDE OF GLUCOSAMINE, AND HA IS PRESENT IN LARGE CABLES BUT CAN BE FRAGMENTS DURING TISSUE INJURY. AND THEN DEPENDENT ON THE SIZE IT CAN EITHER HAVE PRO OR ANTI-INFLAMMATORY EFFECTS. WE PUT THIS PICTURE OF THE ROOSTER UP HERE BECAUSE YOU PROBABLY HEARD ABOUT HYALURONIC ACID, PEOPLE HAVE INJECTED INTO KNEES, TO DECREASE PRESSURE, ACTS AS A CUSHION, ALSO WHAT KEEPS THE COMB FIRM ON THE ROOSTER. SO HYALURONIC ACID HAS SIZE DEPENDENT FUNCTION, SYNTHESIZED BY THREE ENZYMES, THE HYALURONIC ACID SYNTHETASES, AND DEGRADED. THESE HIGH CABLES, AND THEN ALSO LOWER MOLECULAR WEIGHT CARBOHYDRATES. WHAT WE SET OUT TO DO WAS -- SORRY. ONE REASON WE THOUGHT HYALURONIC ACID MIGHT BE POTENTIAL DAMP IN ALCOHOLIC LIVER DISEASE BECAUSE OF OLDER STUDIES FROM JAPAN, SERUM INCREASES AS SEVERITY OF ALCOHOL-INDUCED FIBROSIS INCREASES. THIS IS BASICALLY A MODIFIED ELISA ASSAY, AND WITH MILD, MODERATE AND SEVERE OR LIVER CANCER DUE TO ALCOHOL. SO WHAT PARMA SET OUT TO DO IS GO BACK TO PRIMARY CULTURE SYSTEM OF KUPFFER CELLS AND ASK WHETHER HYALURONICS OF MOLECULAR WEIGHT WOULD HAVE DAMP ACTIVITY OR ACCELERATED DAMP ACTIVITY IN ALCOHOL-INDUCED -- IN THE KUPFFER CELLS AFTER ALCOHOL FEEDING. THIS IS REALLY -- WE GO BACK TO OUR TYPICAL CULTURE SYSTEM, JUST THE CONTROL STIMULATED WITH LPS, NOW WE'RE SEEING TNF ALPHA, WHEN WE ADD FRAGMENTS SMALLER SIZES, 7 AND 12, 59 AND 74, REALLY THE SMALL SIZES THERE WAS ABSOLUTELY NO DIFFERENCE TO THE CONTROL, A LITTLE BIT OF DAMP ACTIVITY, JUST AT THE HIGHER MOLECULAR WEIGHT THINGS. WE WERE DISAPPOINTED WITH THIS AND WERE SITTING IN A SEMINAR LIKE THIS IN ONE OF OUR COLLABORATORS, WHO WAS PART OF -- WE HAD A CENTER OF EXCELLENCE FOR GLYCOBIOLOGY AT THE CLEVELAND CLINIC FOR A WHILE, A LOT OF EXPERTS AROUND US, HOW WE GOT STARTED WITH THIS. CAROL SAID, LAURA, YOU'RE MISSING THE IMPORTANT POINT HERE. WE'VE GOT A HYALRON THAT NORMALIZED YOUR RESPONSE TO LPS. CAROL WAS ALERT TO THOSE EFFECTS OF HA-35 BECAUSE HER LASH STUDIES HOW HA-35 CAN PROMOTE INTESTINAL HEALTH AND INCREASE ANTIMICROBIAL RESPONSE, PRIMARILY IN MODELS OF INFLAMMATORY BOWEL DISEASE. SO, IT'S KIND OF OTHER HYPOTHESIS WAS WRONG BUT HAD FOUND SPECIFIC SIZE HYALURONIC ACID THAT ACTUALLY COULD ACT AS ANTI-INFLAMMATORY AGENT, DECREASING THE LPS-STIMULATED TNF ALPHA EXPRESSION. PARMA VALIDATED THIS WITH OTHER CYTOKINES, AND THESE PATTERNS ARE THE SAME. HERE WE HAVE LPS STIMULATION, WE HAVE TNF ALPHA, IL-6 IS ALSO INCREASED AFTER ALCOHOL, AND THEN BY TREATING CELLS EX VIVO WE NORMALIZED EACH OF THOSE RESPONSES. INTERESTINGLY, WE HAVE ACCESS TO PERIPHERAL BLOOD MONOCYTIC CELLS THROUGH OUR P50 CLINICAL CORE. CRAIG McCLAIN HAD SHOWN PBMC WITH ALCOHOLIC HEPATITIS, THEY ALSO SHOW ENHANCED TLR 4 SIGNALING. WHEN WE TREATED THE PBMCs WITH HA-35, FROM THE PATIENTS, YOU CAN SEE THAT TLR4 RESPONSES WERE REDUCED. SO, WE WENT BACK THEN TO THE ORIGINAL IDEA, WE WERE INTERESTED IN LOOKING AT microRNAs. PARMA DID NEXT GENERATION SEQUENCING OF microRNAs FROM KUPFFER CELLS, BASICALLY BASE LINE AND ETHANOL, WITH AND WITHOUT HA. IT'S EXCITING BECAUSE WE'VE NOW -- IT'S A VERY RICH DATASET. NOW WE HAVE MORE THAN THREE PAPERS THAT ARE COMING OUT OF THE SINGLE DATASET. I'LL TELL YOU ABOUT ONE OF THE SIMPLEST WAYS THAT WE ANALYZED THE DATA. AND THAT'S BY LOOKING VERY SIMPLY AND SAYING, OKAY, IF ALCOHOL IS AFFECTING LPS SIGNALING AND HA-35 IS NORMALIZING CAN WE LOOK AT microRNA DECREASED WITH ETHANOL AND THOSE RESTORED WITH HA-35 AND REMARKABLY WHEN WE ASKED THREE microRNAs THAT FOLLOW THAT PATTERN. SO DECREASED BUT THEN RESTORED. IN A MORE INTERESTINGLY, THESE THREE TURN OUT TO REGULATE EXPRESSION OF mRNA PROTEINS THAT CONTROL NUCLEAR CYTOPLASMIC TRANSPORT, AND PARMA FOCUSED ON THIS ONE, microRNA 181B3P, PARTLY BECAUSE IT WAS THE MOST HIGHLY DIFFERENTIATED, SO WE WENT IN THEN AND CONFIRMED THAT INDEED 181B3P WAS REDUCED IN ETHANOL-FED KUPFFER CELLS AND TREATED WITH HA-35 WERE RESTORED. THESE ARE THE SAME PATTERNS YOU'VE SEEN BEFORE. HERE WE JUST HAVE OUR CONTROL SITUATION WHERE LPS INCREASES TNF ALPHA. THESE ARE KUPFFER CELLS TRANSFECTED WITH BASICALLY A CONTROL microRNA. OR THESE ON THE RIGHT-HAND SIDE ARE TRANSFECTED WITH A MIMIC FOR 181B3P, OVEREXPRESSING A MIMIC SHOULD RESTORE THE AMOUNT OF 181B3P AND INDEED ACTED JUST LIKE HA-35 AND NORMALIZED THAT ACTIVITY. SO, THIS THIS CONTROLS -- ANOTHER NAME IS IMPORTANT ALPHA 5, WHICH IS MORE COMMON IN THE LITERATURE TO SEE. AND WHAT THESE IMPORTIN ALPHA FAMILYDO IS MEDIATE. THEY WILL BIND TO MACROMOLECULES WITH NUCLEAR LOCALIZATION SIGNALS, INTERACTING WITH OTHER MACHINERY INCLUDING SMALL GTPaseS, TRANSPORTING THESE, RELEASE CARGO, CYCLED BACK OUT. THE FIRST QUESTION SINCE THE microRNA SHOULD BE REGULATING IMPORTANT ALPHA 5, DID IT ACTUALLY DO THAT IN OUR SYSTEM. AND SO BY WESTERN BLOT ANALYSIS JUST LOOKING, THIS IS THE IMPORTANT ALPHA 5 PROTEIN IS INCREASED, AND WHEN TREATED IT'S NORMALIZED. WHEN WE PUT IN THE MIMIC NOT AS UPDATED, THERE WAS A SIGNIFICANT DECREASE IN THE EXPRESSION OF IMPORTIN ALPHA 5 SUGGESTING INTERPLAY BETWEEN THE TWO. THE IMPORTIN FAMILY MEMBERS ARE PROMISCUOUS TRANSPORTING CARGO. ONE OF THE CARGO IS P 65 SUBUNIT OF NF-kappaB, IMPORTANT FOR LPS TLR4 SIGNALING LEADING TO CYTOKINE EXPRESSION. I'M GOING TO POINT YOU TO THE BLOWUPS HERE. THIS IS BLUE, P 65 SUBUNIT IN GREEN. WHEN STIMULATED YOU GET GREEN COMING INTO THE NUCLEUS, STIMULATING TRANSCRIPTION OF CYTOKINES. AFTER ETHANOL FEEDING YOU SEE LARGER AMOUNTS OF P 65 IN THE NUCLEUS, THAT'S THAT TEAL COLOR. WHEN THE CELLS ARE TREATED WITH HA-35 I THINK YOU CAN APPRECIATE THERE'S LOTS OF P65 AROUND BUT IT'S NOT GETTING INTO THE NUCLEUS, SUGGESTING THAT ITS TRANSPORT INTO THE NUCLEUS IS IMPAIRED. WE THINK THAT ALCOHOL FEEDING WILL DECREASE THE AMOUNT OF THIS microRNA 181B3P, WHICH IN TURN LIMIT OR INCREASES THE EXPRESSION OF THE IMPORTIN ALPHA 5 ALLOWING FOR MORE P 65 TO GO INTO THE NUCLEUS, HA-35 NOW ACTING VIA CD44 TO HAVE THIS EFFECT, BUT I WON'T SHOW YOU THAT DATA, WILL RESTORE THE 181B-3P AND LIMIT EXPRESSION OF THE IMPORTIN ALPHA 5 AND LIMIT P65 INTO THE NUCLEUS. WE THINK IN A HEALTHY CELL THE IMPORTIN ALPHA 5 IS NOT THE RATE-LIMITING STEP IN TLR 4 SIGNALING BUT WE KNOW UPSTREAM FACTORS ARE INCREASED, IT'S LIKELY IT'S MORE RATE LIMITING. SO IF IN OUR SCHEME OF ALCOHOL-INDUCED LIVER INJURY WE THINK THE KUPFFER CELL AND INCREASED PRODUCTION OF TLR4 STIMULATED CYTOKINE EXPRESSION IS IMPORTANT FOR PROGRESSION OF DISEASE CAN HA-35 PROTECT FROM DISEASE IN IN THE MURINE MODEL? WE MADE USE OF A SHORT-TERM EXPOSURE MODEL SO WE PUT MICE ON A LOW CONCENTRATION, ABOUT 5.5% CALORIES OF ALCOHOL, 1% BY VOLUME, FOR TWO DAYS TO GET THEM ACCLIMATIZED AND GIVE TWO DAYS AT 6% A DAY, WHICH IS EQUIVALENT OF SIX BEERS A DAY. WE MEASURE THE BLOOD ALCOHOL CONCENTRATION TWO HOURS INTO THE DARK CYCLE SO WHEN THEY ARE ACTUALLY ACTIVELY DRINKING AND WE SEE CONCENTRATIONS ON THE ORDER OF 55 MILLIMOLAR. WE PROVIDED HA-35 TO THE MICE DAILY BY GAVAGE, A FEW HOURS BEFORE WE REFILLED THE ALCOHOL BOTTLES. SO THIS WORK WAS DONE BY DAMIAN BELLOWS. HE FOUND SIMILAR TO WHAT WE'D SEEN IN THE ISOLATED CELLS, IF WE LOOKED IN THE LIVER, THE ALCOHOL FEEDING TO THE MICE DECREASED EXPRESSION OF 181B-3P AND WHEN TREATED WITH HA-35 THAT WAS PREVENTED. PREVENTED. WHEN WE ISOLATED NON-PARENCHYMAL CELLS, ALL IMMUNE CELLS FROM THE LIVER, YOU CAN SEE THE IMPORTIN ALPHA 5 WAS INCREASED IN EXPRESSION, AND MICE WITH THE HA-35 WERE PROTECTED. WITH NF-kappaB ACTIVATION HERE WE USED PHOSPHO-P-65, I'LL POINT YOU TO THE ZOOMED IMAGES, BLACK POINT TO P-65 IN THE NUCLEUS, WE ALSO SEE SOME ALCOHOL INCREASE, P-65 IN THE NUCLEUS OF HEPATOCYTES AS WELL. YOU CAN APPRECIATE HA-35-TREATED MICE THAT WAS BASICALLY PREVENTED. USING MORE TYPICAL MEASURES OF INJURY WE CAN SEE THAT SHORT-TERM MODEL, INCREASES, AND TREATMENT WITH HA-35 BASICALLY PREVENTED ALL THOSE CHANGES. BECAUSE OF OUR WORK WITH CAROL, INTERESTED IN HA 35 EFFECTS ON THE GUT, AND BECAUSE WE KNOW ALCOHOL AFFECTS THE GUT AS WELL, WE ALSO LOOKED AT EFFECTS OF HA 35 ON MARKERS OF INTESTINAL PERMEABILITY USING CIRCULATING ENDOTOXIN AND THEN ALSO LOOKING FOR CO-LOCALIZATION OF TIGHT JUNCTION PROTEINS ZO-1 AND OCCLUDIN. THIS MODEL DOES INCREASE THE PLASMA ENDOTOXINS, MICE TREATED WITH HA 35 DID NOT SO THAT INCREASE. AND THAT PROTECTION WITH WAS ASSOCIATED WITH BETTER GUT INTEGRITY IN TERMS OF ZO-1 AND OCCLUDIN. WE HAVE BLOWUPS AT THE -- ACTUALLY THE BLOWUPS ARE TO THE RIGHT. BUT WE'RE LOOKING AT ZO-1 AND OCCLUDIN, IF THEY ARE CO-LOCALIZED, THAT THE IMAGE IS YELLOW. THAT MEANS THE TIGHT JUNCTION IS INTACT. AND SO AFTER ETHANOL FEEDING WE SEE A LOT OF YELLOW, PROXIMAL COLON, SOME AREAS ARE PERFECTLY FINE BUT OTHER AREAS SHOW A LOSS OF TIGHT JUNCTION. AND THAT'S IN A BLOWUP HERE. AFTER HA 35 IT'S INTERESTING, WE CAN SEE ENHANCED YELLOW COLOR, THIS IS CONSISTENT WITH CAROL'S DATA THAT HA 35 JUST HAS PROTECTIVE EFFECTS OR HEALTH-PROMOTING EFFECTS ON THE GUT. ETHANOL-FED WHEN TREATED WITH HA 35 WE SEE A REALLY NICE EVEN TIGHT JUNCTION LAYER ALL ACROSS THE PROXIMAL COLON. SO IT LOOKS LIKE THAT WE SEE A PROTECTIVE EFFECT OF HA 35 IN THIS MODEL OF ETHANOL INDUCED INJURY. IT'S CLEAR THAT IT'S PROTECTING THE GUT INTEGRITY. AND IT ALSO IS DECREASING CYTOKINES. AT THIS POINT WE DON'T KNOW WHETHER THE DECREASE IN CYTOKINES ARE BECAUSE THE LPS ISN'T COMING IN, THERE'S NOT A SENSITIZATION OF THE KUPFFER CELLS TO FIX OR WHETHER IT'S A DULY ACTING AGENT, WE'RE WORKING ON PROTOCOLS TO TRY TO DISTINGUISH WHAT THE TARGET IS MAKING USE OF CELL SPECIFIC KNOCKOUTS FOR SOME HA 35 RECEPTORS. WHAT'S EXCITING, I WANT TO BRING THIS MECHANISTIC UNDERSTANDING TO TRANSLATIONAL INSIGHT, WE THINK THERE'S POTENTIAL THERAPEUTIC VALUE FOR HA 35 IN HEAVY DRINKERS. CAROL WORKING WITH MYSELF COMPLETED A SAFETY AND TOLERABILITY STUDY WITH HA 35 IN 20 HEALTHY ADULT SUBJECTS, IT WAS SAFE AND TOLERABLE, WE'RE READY FOR THE NEXT PHASE OF THINGS. WE'RE IN A PLANNING STAGE FOR DOING A PILOT CLINICAL TRIAL, TO LOOK TO SEE WHETHER BASICALLY PROVIDING HA 35 TO HEALTHY ADULTS BEFORE A SINGLE ALCOHOL DRINK CAN PROTECT THE GUT. THERE'S CERTAIN MODELS, A GROUP AT UMass USES THEM, BASICALLY PROVIDING A SINGLE DRINK IN THE EVENING, THE NEXT DAY YOU CAN SEE MORE LPS IN THE CIRCULATION. SO THE IDEA IS IF WE TREAT -- PROVIDE HA 35 CAN WE PROTECT THE GUT FROM THAT INITIAL INSULT. THAT'S JUST BASICALLY A BABY STEP FORWARD. WE'RE VERY EXCITED BECAUSE THE HYALURONIC ACID IS PHARMACEUTICALLY AVAILABLE IN PHARMACEUTICAL GRADE, BASICALLY APPROVED IN A NUMBER OF DIFFERENT COUNTRIES FOR USE. SO THE SECOND PART OF MY STORY GOES TO THE CELL DEATH SIDE OF THIS. WE NOW NOTICE A LOT MORE TNF ALPHA, PROTOTYPICAL CYTOKINE, BEING EXPRESSED. WHY IS THAT BAD FOR HEPATOCYTES? SO TNF SIGNALING IN HEPATOCYTE, IN A HEALTHY HEPATOCYTES GOES TOWARDS HEPATOPROTECTION. BASICALLY TNF INTERACTING WITH RECEPTOR DOWNSTREAM LEADS TO NF-kappaB AND HEPATOCYTES, PROTECTIVE, COMPARED TO PRO-INFLAMMATORY IN IMMUNE CELLS. SOMETHING HAPPENED SO TNF IS NO LONGER PROTECTIVE IN HEPATOCYTES AND SHIFTS IT MORE TOWARDS CELL-DEATH PATHWAYS. THERE'S TWO DIRECTIONS THAT IT CAN GO. IT CAN BE HEPATOPROTECTIVE OR CONTRIBUTE TO DEATH. HEPATOCYTES CAN DIE IN MULTIPLE WAYS. REALLY INITIALLY CHARACTERIZED IS THE NON-PROGRAMMED CELL-DEATH PATHWAY OF NECROSIS WHERE THERE'S CELL SWELLING, PLASMA MEMBRANE RUPTURES, THERE'S A RELEASE OF DAMP. NEXT THOSE DESCRIBE THE REGULATED PROCESS OF APOPTOSIS WHERE THERE'S CELL SHRINKAGE, NUCLEAR FRAGMENTATION, THOUGHT TO BE LESS PRO-INFLAMMATORY BUT DEPENDING ON THE SITUATION. MORE RECENTLY, THERE'S A PROGRAMMED PATHWAY OF CELL DEATH, NECROPTOSIS, SIMILAR TO NECROSIS, YET HIGHLY REGULATED. EVEN MORE RECENTLY WE HAVE DATA ABOUT PYROPTOSIS, AS WELL AS FERROPTOSI ASSOCIATED WITH IRON OVERLOAD. FOCUSING TODAY ON APOPTOSIS VERSUS NECROPTOSIS. WE'VE KNOWN THERE'S ACCUMULATED APOPTOTIC CELLS WITHIN THE LIVER OF PATIENTS WITH ALCOHOL-INDUCED LIVER INJURY. FIRST THOUGHT WAS IF WE BLOCK CASPASES WOULD WE PREVENT ALCOHOL INDUCED INJURY? AND WE MADE USE OF A NUMBER OF MOUSE MODELS. THIS IS A SLIGHTLY LONGER -- WELL, VERY MUCH LONGER MODEL OF ALCOHOL EXPOSURE. RATHER THAN 2 DAYS AT 6% WE RAMP UP, A MORE SEVERE MODEL OF CHRONIC INJURY. WE MADE USE OF BOTH GENETIC AND PHARMACOLOGIC MANIPULATION AND USED A PAN CASPASE INHIBITOR, TO SHOW IN ETHANOL FEEDING WE CAN SEE ACCUMULATION OF APOPTOTIC HEPATOCYTES. HERE WE'RE USING -- THIS IS AN IMAGE OF CLEAVAGE PRODUCT, IN THE LIVER HEPATOCYTES ARE THE ONLY CELLS TO MAKE IT WHEN IT'S CLEAVED, IN CASPASE DEPENDENT WAY MAKES A FRAGMENT IDENTIFIED WITH SPECIFIC ANTIBODIES. WE LOOKED FOR TUNNEL POSITIVE CELLS. YOU CAN SEE. SO QUANTIFIED HERE THE ALCOHOL FEEDING PROTOCOL INCREASES TUNNEL POSITIVE CELLS, AS WELL AS THESE CK-18 AND POSITIVE CELLS. YOU DIDN'T SEE THE APOPTOSIS IN THE KNOCKOUT MICE. IT'S GONE. SAME WITH VERTEX CASPASE INHIBITOR, INHIBITED APOPTOSIS. OUR STRATEGIES TO INHIBIT APOPTOSIS WERE EFFECTIVE BUT DID NOT CHANGE INJURY IN THIS PARTICULAR MODEL. SO AGAIN WE HAVE ALT, AST, AND TRIGLYCERIDES, CYTOKINES FOLLOW THE SAME TREND. BID KNOCKOUTS WERE NOT PROTECTED. VERTEX PANCASE INHIT YOSHES -- INHIBITORS WERE NOT PROTECTED. WE MADE USE OF CASPASE 8 KNOCKOUTS, AND THEY ARE NOT PROTECTED. ANOTHER SIDE TO THE STORY IS THAT THE APOPTOSIS ACTUALLY CONTRIBUTES TO FIBROSIS BUT NOT TO THE INFLAMMATORY RESPONSE SO IT'S A WAY TO DISTINGUISH MODES OF CELL DEATH WITH TIMES -- TYPES OF INJURY. I'M NOT GOING TO TELL YOU THAT SIDE OF THE STORY. IF IT'S NOT APOPTOSIS MAYBE IT'S NECROPTOSIS, AN INTERESTING PATHWAY REGULATED VIA THE SHIFT IN THE INTERACTION BETWEEN A NUMBER OF DIFFERENT PROTEINS. SO THIS FAMILY OF PROTEINS, RECEPTOR INTERACTING PROTEINS, INTERACTS WITH FADD OR CASPASE 8, WE SEE A SHIFT FROM APOPTOSOME TO THE NECROSOME. SO THE RECEPTOR KINASE, RIP 1 AND RIP 3 HAVE KINASE DOMAINS, AS WELL AS RIM DOMAIN, ALLOWS FOR PROTEIN-PROTEIN INTERACTION BETWEEN THE FAMILY MEMBERS. SO WE WERE LUCKY ENOUGH TO BE ABLE TO GET RIP3 DEFICIENT MICE FROM GENENTECH AND BASICALLY SANJAY CHOWDHURY IN THE LAB SET OUT TO ASK IF THE RIP3 WAS INVOLVED IN ETHANOL INDUCED LIVER INJURY. THE FIRST CASE WE'RE LOOKING HERE AT RIP3 EXPRESSION. IT'S INTERESTING BECAUSE IN A HEALTHY LIVER, YOU'LL SEE REPORTS ABOUT THIS IN A NUMBER OF PLACES, IT'S NOT VERY MUCH RIP3 THAT'S EXPRESSED. BUT THEN IN RESPONSE TO ALCOHOL, EITHER -- SORRY, THIS IS A LOW DOSE ALCOHOL, 2% PER DAY, NOT MUCH GOING ON. THEN WHEN WE GO TO THE MODEL I SHOWED YOU EARLIER WHERE WE HAVE THE 6% FOR TWO DAYS WE SEE INDUCTION OF THE RIP3 PROTEIN, ALSO OVER THE LONGER TERM CHRONIC EXPRESSION. YOU CAN SEE THE EXPRESSION IS IN PRIMARILY INCREASED IN HEPATOCYTES. WE ALSO SAW SIMILAR EXPRESSION IN BIOPSY SAMPLES FROM PATIENTS WITH ALCOHOL-ASSOCIATED LIVER DECEMBER. DISEASE. THEY GAVE IT THE TITLE, THE LIVER GETS RIPPED BY ETHANOL, WHICH IS INTERESTING. BUT YOU CAN SEE AGAIN JUST LIKE IN THE MOUSE LIVES, IN HUMAN RED IS RIP3 EXPRESSION, HIGHLY INDUCED. SO THEN RIP3 KNOCKOUT MICE, WHAT HAPPENS TO THEM? TURNS OUT THEY WERE PROTECTED FROM ETHANOL-INDUCED LIVER INJURY. I'M SHOWING YOU TYPICAL MARKERS IN THE PAPERS WHERE WE HAVE THESE 30 PANELS OF DIFFERENT INJURY MARKERS BUT THEY TREND IN THE SAME WAY. SO AGAIN WE HAVE TYPICAL INJURY OF ALT, AST, TRIGLYCERIDES. CYTOKINE EXPRESSION BY mRNA WAS NORMALIZED IN THE RIP3 KNOCKOUT MICE AS WELL AS PRODUCTION OF THE TNF ALPHA PROTEIN. SO IN CONTRAST THEN TO THE INHIBITORS OF APOPTOSIS, THE RIP3 KNOCKOUT MOUSE WAS PROTECTED, NECROPTOSIS MIGHT BE AN IMPORTANT PATHWAY. I'LL TELL YOU A FEW AREAS WE'RE WORKING ON. WHAT SHIFTS THIS TNF RECEPTOR SIGNALING FROM HEPATOPROTECTION TO DEATH. WHEN WE LOOK AT PATHWAYS, I THINK AS YOU CAN SEE THERE'S A BUNCH OF UBIQUITINNATION GOING ON. ONE THING THAT HAPPENS WHEN THE RIP1 PROTEIN IS UBIQUITINNATED, PARTICULARLY WITH K 63 UBIQUITINS, IT SHIFTS THE THING OR KEEPS TOWARDS HEPATOPROTECTION. WHEN THE RIP1 BECOMES DEUBIQUITINNATED, SHIFTS TOWARDS APOPTOTIC OR NECROPTOTIC PATHWAYS OVER HERE. WE THOUGHT PERHAPS ALCOHOL WAS AFFECTING UBIQUITINNATION. WHEN THEY DO LIVER TRANSPLANTS IN PATIENTS WITH ALCOHOL HEPATITIS, ALCOHOLIC LIVER DISEASE, THAT THEY HAVE SAMPLE FROM HEALTHY CONTROLS, A GREAT RESOURCE FOR MOVING FROM MOUSE TO HUMAN SAMPLES. WHAT WE DID WITH UBIQUITIN ARRAY, SO ESSENTIALLY SEMI QUANTITATIVE, LOOKS AT A NUMBER OF PROTEINS UBIQUITINNATED ASKING WHAT'S GOING ON, BLUES ARE HEALTHY CONTROL, SET THEM AT 1, IT'S N OF 3, AND WHAT YOU CAN SEE IS THAT A NUMBER OF PROTEINS ARE INCREASED IN THEIR UBIQUITINNATION OR DECREASED IN UBIQUITINNATION SUGGESTING THIS MIGHT BE IMPORTANT PATHWAY IN THIS PARTICULAR SYSTEM. AS MENTIONED THIS K 63 LINKED RIP1 IS CRITICAL FOR MAINTAINING HEPATOPROTECTION. A POSTDOC MADE USE OF AN INTERESTING TOOL THAT'S CALLED A K63 TUBE. THIS IS A TANDEM UBIQUITIN-BINDING ELEMENT. SO RATHER THAN DOING A PULLDOWN WITH AN ANTIBODY, YOU CAN GET HIGH SPECIFICITY PULLDOWN OF SPECIFIC TYPES OF UBIQUITINNATION, K63 OR M1 LINKAGE SIMILAR TO PULLDOWN ASSAY. FIRST LOOKED TO SEE WHETHER ALCOHOL WAS AFFECTING RIP1 EXPRESSION, IT DOES NOT AFFECT EXPRESSION, WE HAVE CONTROLS HERE SO THESE ARE HEPATOCYTES TRANSFECTING AGAINST RIP1, IMPORTANT TO USE NEGATIVE CONTROLS BECAUSE ANTIBODIES ARE NOT PERFECT. IT DIDN'T CHANGE EXPRESSION. WHEN WE DO THE K 63 PULLDOWN, REPLICATES, AFTER ETHANOL, THESE ARE MICE NOW, WE SEE DRAMATIC REDUCTION IN AMOUNT OF UBIQUITINNATION SUGGESTING THAT DECREASE IN UBIQUITINNATION IS SHIFTING TOWARDS CELL DEATH FROM HEPATOPROTECTION. SO WE KNOW THAT RIP3 SEEMS TO BE INVOLVED IN ETHANOL-INDUCED LIVER INJURY. WE HAVE A BEGIN SENSE OF MECHANISMS BY WHICH THIS MIGHT BE HAPPENING. SECOND HYPOTHESIS AGAIN SPOILER ALERT IS THAT WE EXPECTED THAT THE CANONICAL RIP3 SIGNALING WOULD CONTRIBUTE TO ALD IN MURINE MODELS BUT ALSO NAFL AND NASH. WHAT WE DID WAS TO COMPARE ETHANOL FEEDING TO HIGH FAT FEEDING IN THE RIP3 KNOCKOUT MICE. THIS IS THE DATA I SHOWED YOU EARLIER. RIP3 KNOCKOUT MICE ARE PROTECTED FROM ALT, AST, AND THIS IS MCT1, TYPICALLY INFLAMMATORY CYTOKINES ARE REDUCED. IN THE HIGH FAT DIET, WE'VE USED TWO MODELS, THIS IS THE FFC DIET, HIGH FAT, HIGH FRUCTOSE, AND CHOLESTEROL. AS YOU WOULD EXPECT IN THE WILDTYPE MICE YOU GET ALT, AST, MCP1 INCREASING BUT THERE'S ABSOLUTELY NO PROTECTION IN THE RIP3 KNOCKOUT MICE. SO THEY ARE BEING INJURED IN DIFFERENT WAYS. BECAUSE MLKO IS -- LET ME GO BACK . ONCE THE RIP3 IS PHOSPHORYLATED, FINDS A PORE IN THE PLASMA MEMBRANE, CELL CONTENTS ARE RELEASED. YOU WHO EXPECT THE RIP3 KNOCKOUT IS PROTECTED DOWNSTREAM MLKL KNOCKOUT WOULD BE PROTECTED. BUT NO. IT WASN'T. SO, THIS IS THE CHRONIC 25-DAY MODEL. THIS IS THE ACUTE MODEL. YOU CAN SEE THERE'S NO PROTECTION IN THE MLKL KNOCKOUTS. IT'S NOT THAT THE MLKL KNOCKOUTS ARE BAD. IN THE HIGH FAT DIET THEY ARE PROTECTED. ONE OF THE STRONGEST PROTECTIONS WE'VE EVER SEEN REALLY. MLKL KNOCKOUTS ARE PROTECTED. WHAT WE SEE IS THERE'S REALLY NON-CANONICAL ACTIVATION OF MLKL IN THE MURINE MODEL, AND NON-CANONICAL ROLE OF RIP3 IN TERMS OF INJURY IN THE ALCOHOL MODEL. WE'RE WORKING NOW ON SOME OF THE KIND OF DETAILED MECHANISTIC FEATURES OF THIS AND BECAUSE OF THE TIME I'M NOT GOING TO TELL YOU ABOUT THAT BUT GIVE HINTS. FOR THE ALCOHOL, ONE OF THE NON-CANONICAL FUNCTIONS OF THE RIP3 IS THAT IT CAN INTERACT OR CONTRIBUTE TO THE PRODUCTION OF IL-1 BETA IN THE INFLAMMASOME. THERE'S SO MUCH DATA ABOUT THE IMPORTANCE OF THE INFLAMMASOME IN ALCOHOL-ASSOCIATED LIVER DISEASE, WE'RE PURSUING THIS AS POTENTIAL NON-CANONICAL FUNCTION. THE SECOND SIDE IS WITH THE HIGH FAT DIETS, WE HAVE DATA TO SUGGEST THAT MLKL ACTUALLY CONTRIBUTES TO IMPAIRED AUTOPHAGIC FLUX WHICH CONTRIBUTES TO INJURY. SO AGAIN WE'VE KIND OF HYPOTHESIZED WRONG, NON-CANONICAL FUNCTIONS ARE INTERESTING BUT COMPLEX. WHAT WE THOUGHT, MAYBE WE CAN USE THESE MECHANISTIC INSIGHTS INTO SOMETHING THAT HAS TRANSLATIONAL VALUE. AND CAN WE USE THIS DIFFERENCE TO DEVELOP BIOMARKERS TO DISTINGUISH ALCOHOL USED LIVER DISEASE FROM NAFL/NASH. WE DEVELOPED WESTERN BLOT ASSAYS FOR ACTIVATION MARKERS FOR RIP1, RIP3, MLKL PATHWAY, PHOSPHORYLATION HERE INCREASED OVER THE HEALTHY CONTROLS, THERE SEEMS TO BE SOME SHIFT, ANTIBODY DIDN'T RECOGNIZE PHOSPHORYLATED PROTEIN. RIP3 INDUCED AS WE EXPECT FROM PREVIOUS STUDIES. WE SEE SOME PHOSPHORYLATION OF MLKL. AND SMALL INCREASE IN MLKL EXPRESSION. BUT WHAT WE'VE NEVER DONE BEFORE TO PUT THE LIVER FROM ALCOHOL VERSUS NAFL/NASH ON THE SAME BLOT. WE WERE ABLE -- OUR GROUP HAS NASH BIOPSY SAMPLES FROM YEARS AGO, WE PUT THEM ON THE SAME BLOT AND IT REVEALS DIFFERENCES IN THE EXPRESSION. SO IF YOU REMEMBER THE ALCOHOL HEPATITIS WE HAD MORE OF THE PHOSPHO-RIP1, NONE OF THAT HAPPENING HERE IN THE NASH PATIENTS. AND THE EXPRESSION LEVEL OF RIP1 IS QUITE DIFFERENT BETWEEN THE TWO. RIP3 IS EXPRESSED IN BOTH SITUATIONS. AND BUT IN THE MLKL IT'S ONLY -- PHOSPHORYLATION OF THE MLKL IN THE NON-ALCOHOLIC HEPATITIS IS MUCH HIGHER THAN IN THE ALCOHOLIC HEPATITIS. AND THEN MLKL CONTENT IS DIFFERENT. THIS IS A LONGER EXPOSURE. THIS IS SUGGESTING DIFFERENT ACTIVATION PATHWAYS IN THE TWO DISEASES. IF WE TAKE THIS ONE STEP FURTHER, WE COULD POTENTIALLY USE IT AS A BIOMARKER TO DISTINGUISH ALCOHOLIC LIVER DISEASE, ASSOCIATED LIVER DISEASE FROM NASH. WE MADE USE OF TWO SETS OF SERUMS, SAMPLES, ONE FROM P50 CLINICAL CORE FOR HEALTHY CONTROLS AND THEN ALSO NASH PATIENTS. AND THEN I MENTIONED WE HAD THIS INTERNATIONAL COLLABORATION WITH THE BARCELONA GROUP, WE USED SOME OF THEIR ALCOHOLIC HEPATITIS PATIENTS FOR THIS. SO THERE ARE COMMERCIALLY AVAILABLE ELISA ASSAYS FOR RIP1. RIP1, RIP3, MLKL. A VISITING GRADUATE STUDENT IN THE LAB RAN THE ELISAs AS WELL AS ANALYSIS. YOU CAN SEE THAT RIP1 IN HEALTHY CONTROLS, THERE'S RIP1 IN CIRCULATION. WHERE IT COMES FROM WE DON'T KNOW. BUT THAT IN ALCOHOLIC HEPATITIS THAT'S SIGNIFICANTLY LOWER, IN NASH IT'S BASICALLY THE SAME AS CONTROLS. IT'S THE FLIP SIDE WITH THE RIP3. IT'S VERY LOW IN HEALTHY CONTROLS AND NASH PATIENTS, WHILE ELEVATED IN ALCOHOL-ASSOCIATED HEPATITIS. AND MLKL DOESN'T SEEM TO VARY WITH DISEASE. SHE RAN THE CURVES TO SEE WHETHER THESE COULD BE SENSITIVE IN SPECIFIC MARKERS FOR AH VERSUS HEALTHY CONTROL, AND AS YOU MIGHT EXPECT, YES, RIP3 IN PARTICULAR IS VERY SENSITIVE AND RIP1 AND MLKL BASICALLY HAS NO EFFECT. HE COULD SAY THAT THESE MARKERS COULD DISTINGUISH AH FROM NASH. THINKING ABOUT IT WE THOUGHT IN OUR ASH POPULATION WE HAD MODERATE AND SEVERE ASH PATIENTS, MAYBE THIS IS ONLY RELATED TO SEVERITY OF THE DISEASE. SO WE PULLED OUT ALCOHOLIC HEPATITIS PATIENTS WITH LOW MELD SCORES, BETWEEN 9 AND 15 FOR BOTH ALCOHOL AND NON-ALCOHOL. SO THE DISEASE SEVERITY IS QUITE SIMILAR HERE. AND YOU CAN STILL SEE THAT THERE'S DIFFERENCES BOTH RIP1 AND RIP3, THOSE DIFFERENCES ARE STILL PRESENT EVEN WITH MODERATE DISEASE. AGAIN, THE AEC CURVES LIKE NICE IN TERMS OF SPECIFICITIES. TO SUMMARIZE, I THINK I'VE TOLD YOU TWO STORIES WITH SURPRISING RESULTS THAT ARE MORE INTERESTING THAN ANYTHING WE COULD HAVE THOUGHT OF ORIGINALLY. AND I THINK THAT BOTH THESE STORIES GO FROM BASIC MECHANISMS TO SOME TRANSLATIONAL INSIGHTS, JUST TO SUMMARIZE THE STORIES THAT ONE IS THAT HA 35 PROTECTS THE GUT AND LIVER FROM ETHANOL IN MURINE MODELS AND WE'VE COMPLETED A SAFETY AND TOLERANCE TRIAL AND THERE'S POTENTIAL THERAPEUTIC VALUE THERE, MOST LIKELY IN MODERATE DISEASE SITUATIONS. IN TERMS OF RIP1, RIP 3, MLK STORY WE IDENTIFIED NON-CANONICAL FUNCTION FOR RIP3 AND MLKL IN MURINE MODELS. IT'S INTERESTING BECAUSE I THINK THE STRENGTH OF THESE STUDIES WE'VE DONE, THESE ARE THE SAME MICE, THE SAME LITTERMATE CONTROLS IN THE SAME VIVARIUM, YOU CAN'T EXPLAIN BY DIFFERENCES IN THE ENVIRONMENT OR GENETICS. SO I THINK THESE ARE REAL DIFFERENCES BETWEEN THE TWO MODELS. AND ALSO KIND OF BUILDING ON THAT DIFFERENCE IN THE MURINE MODELS, WE'VE POTENTIALLY IDENTIFIED RIP1 AND RIP3 AS BIOMARKERS TO DISTINGUISH MODERATE AGE FROM NASH. AND WITH THAT, THANK YOU FOR YOUR ATTENTION. AGAIN, JUST TO THANK THE PEOPLE IN MY LAB, IT'S A GREAT GROUP RIGHT NOW. WE'RE HAVING A LOT OF FUN. THANK YOU. [APPLAUSE] >> WE HAVE TIME FOR QUESTIONS, IF ANYONE WANTS TO STEP UP TO THE MICROPHONE. >> YEAH, I HAVE A COUPLE QUESTIONS. IN ALCOHOLIC HEPATITIS, SO THE NEUTROPHIL INFILTRATION IS IMPORTANT. YOU NEVER MENTIONED NEUTROPHILS, WHETHER IT'S H 35 EFFECTS NEUTROPHILS. >> YEAH, WE HAVEN'T DONE -- MODELS WE'RE USING ARE LIBRA DECARLY, WE DON'T SEE NEUTROPHILS. HA 35 WE HAVEN'T DONE ACUTE AND CHRONIC MODEL YET. IN THE RIP3 AND MLKL, SO IT WAS PUBLISHED RIP3 KNOCKOUTS WERE PROTECTED IN YOUR MODEL, ACUTE ON CHRONIC, MLKLs ARE NOT PROTECTED, WE DID THAT IN THERE. WE HAVEN'T SPECIFICALLY LOOKED FOR A ROLE FOR NEUTROPHILS FOR EITHER OF THESE. >> THE SECOND QUESTION REGARDING THIS RIP3 ML IN THE DIFFERENT IN ALCOHOL MODEL AND NASH MODEL, I THINK THE NASH MODEL YOU ARE USING IS NOT MUCH IN NEUTROPHIL INFILTRATION, NOT MUCH OXIDATIVE STRESS. SO WE HAVE A SEMINAL STUDY USES LIVER SPECIFIC P38 KNOCKOUT, IN ALCOHOL MODEL, YOU KNOW, KNOCKOUT, UNLESS INJURY, IN HIGH FAT MODEL IT GET WORSE. >> OH REALLY? >> BUT WHEN WE USE OUR NEW MODEL THIS HIGH FAT PLUS OVEREXPRESSION CXC1 IN MODEL OF NEUTROPHILS, SIMILAR RESULT AS ALCOHOL MODELS. >> IT'S GOING TO BE CONTEXT DEPENDENT THERE, DEPENDING IF YOU HAVE NEUTROPHILS COMING IN YOU MIGHT ONE RESPONSE OR ONE ROLE OF THESE PATHWAYS VERSUS IF YOU HAVE NEUTROPHILS THERE. WHICH I THINK IS AN IMPORTANT DISTINCTION BECAUSE NEUTROPHILS -- IN ALCOHOLIC HEPATITIS THEY ARE KEY PLAYERS BUT IN OTHER FORMS OF INJURY THEY MIGHT NOT CONTRIBUTE AS MUCH. AS MUCH. >> OKAY. THANK YOU. >> I HAVE A QUESTION. AS YOU MIGHT EXPECT, MY QUESTIONS WILL BE NAIVE. WHAT IS THE PURPOSE OF NECROPTOSIS IN A NORMAL CELL? WHY DO WE HAVE THAT MECHANISM WITH THOSE? >> VIROLOGISTS THINK IT'S BECAUSE IT'S A WAY TO EVADE VIRUSES THAT HAVE -- CAN AVOID APOPTOSIS MACHINERY. IF NECROPTOSIS YOU CAN KILL THE CELL IN THAT WAY. I THINK THERE'S SOME -- YEAH, THERE'S NOT SO MUCH A ROLE IN DEVELOPMENT PER SE, LIKE WITH APOPTOSIS WE THINK OF REMODELING TISSUES, AS THE ORGANISM DEVELOPS. BUT I THINK THERE'S NOT AS MUCH ROLE OR NOT AS WELL STUDIED WITH NECROPTOSIS. SO IT'S QUITE DIFFERENT. >> DOES IT INCREASE IF THE CELL IS INJURED IN SOME WAY? >> WELL, YEAH, SO THE IDEA -- >> KILLING THE CELL? >> SO ORIGINALLY PEOPLE THOUGHT APOPTOSIS WAS A WAY TO DIE AND NOT SET OFF INFLAMMATORY SIGNALS. AND THAT NECROSIS WAS THEN MORE DAMAGING BECAUSE DAMPS WOULD BE RELEASED. BUT IT'S INTERESTING NOW BECAUSE BOTH APOPTOTIC CELLS AND CELLS DYING BY NECROSIS OR NECROPTOSIS CAN STILL CONTINUE TO LIKE RELEASE CYTOKINES EVEN AS THEY DIE, BECAUSE THAT MACHINERY IS STILL THERE. SO IT MIGHT BE A WAY TO ENHANCE AN INFLAMMATORY RESPONSE WHICH IN MANY CASES IS A GOOD IDEA. IT'S JUST WHEN YOU CAN'T RESOLVE IT THAT IT BECOMES A BAD IDEA. >> GEORGE? >> BEAUTIFUL RESULTS. PARTICULARLY IMPRESSIVE IS THE MLKL BIOMARKER STORY, VERY DRAMATIC THE DIFFERENCE. I WAS WONDERING WHETHER YOUR PATIENT, DID YOU EXCLUDE ALCOHOLICS WHO WERE OBESE AND WHAT HAPPENS TO THAT SUBSET? >> YEAH, IN OUR COHORT WE DON'T HAVE ANY ALCOHOLIC HEPATITIS PATIENTS WHO ARE OBESE. BUT IN OUR NASH COHORT, IN OUR HEALTHY CONTROLS, WE CAN CONTROL FOR -- WE HAVE OBESE HEALTHY CONTROLS VERSUS NASH AND CAN START TO TEASE THAT APART. WE'RE ALSO WORKING TO BASICALLY COLLECT ADDITIONAL NASH. WE HAVE AGREEMENT FROM THE NASH CRN TO GET A LARGER COHORT OF NASH PATIENTS TO BUILD THIS OUT. SO WE'LL HOPEFULLY BE ABLE TO THAT DETAILED ANALYSIS. >> ONE PARTICULARLY INTERESTING GROUP COULD BE MORBIDLY OBESE PATIENTS WHO UNDERGO BARIATRIC SURGERY. >> YES. >> BUT THEY HAD PREVIOUS HISTORY OF OBESITY AND WHAT HAPPENS TO THOSE. >> YES. >> DO WE KNOW HOW HA 35 IMPACTS BRAIN, NEURAL INFLAMMATION ON MICROGLIA, ANY DATA ON THAT? >> NO, BUT ONE OF MY COLLABORATORS, WE'RE STARTING TO START TO DO THOSE EXPERIMENTS BECAUSE HE'S GOT A VERY NICE REPORTER SYSTEM FOR MICROGLIA ACTIVATION SO WE'LL START TO LOOK AT THAT AS WELL. I DON'T HAVE DATA YET. >> OKAY. >> HI, LAURA. WHAT A REALLY FASCINATING STORY ABOUT YOUR WORK. AT THE BEGINNING YOU MENTIONED ABOUT PARAPOPTOSIS, THERE'S BEEN PUBLICATION IN TERMS OF MOUSE MODEL, WONDERING IF YOU COULD COMPARE NECROPTOSIS IN BOTH MOUSE MODEL AND HUMAN PATIENT. >> YES. YEAH, THE PYRPPTOSIS LINKS TO THE INFLAMMASOME. TOOLS TO STUDY IT ARE DIFFICULT. WE WORKED WITH ANTIBODIES TO GET CONDITIONS APPROPRIATE FOR USING IT BUT WITH THE CLEAVED, YOU HAVE TO BE REALLY CAREFUL TO SEE THINGS. SO WE HAVEN'T REALLY EXPLORED IF THERE'S AN INTERACTION BETWEEN THE PATHWAYS YET BUT I DO THINK IT'S SOMETHING IN THE FUTURE WE NEED TO THINK ABOUT PARTICULARLY BECAUSE OF THIS IL-1 BETA NON-CANONICAL ROLE OF ERK 3 BUT WE DON'T KNOW YET. >> I'M SURE PEOPLE WANT TO TALK WITH LAURA AFTERWARDS. LET'S GIVE HER A GREAT ROUND OF APPLAUSE FOR A REALLY OUTSTANDING LECTURE. [APPLAUSE]