Project Grant R01AI184122

ROLE OF INTESTINAL MICROFOLD (M) CELLS IN CREATING A HOTSPOT ENVIRONMENT FOR HIV RESERVOIR PERSISTENCE AND REACTIVATION - ABSTRACT DESPITE THE EFFECTIVENESS OF ANTIRETROVIRAL TREATMENT (ART) AT TREATING HIV, IT FAILS TO COMPLETELY ERADICATE THE VIRAL RESERVOIR. HIV REACTIVATION OCCURS NATURALLY, AS EVIDENCED BY OCCASIONAL LOW-LEVEL VIREMIA ("BLIPS") OBSERVED IN PATIENTS ON ART. STUDYING THE ANATOMICAL SITES OF HIV REACTIVATION AND THE MOLECULAR MECHANISMS DRIVING THESE BLIPS CAN PROVIDE ESSENTIAL INFORMATION FOR THE DEVELOPMENT OF HIV CURE STRATEGIES. NON-HUMAN PRIMATE STUDIES, AUTOPSIES, AND CLINICAL TRIALS INDICATE THAT THE GASTROINTESTINAL (GI) TRACT CONTAINS UP TO 98% OF THE HIV RESERVOIR AND THAT ITS IMMUNOPHYSIOLOGY COULD BE ESPECIALLY CONDUCIVE FOR HIV REACTIVATION. IN OUR PREVIOUS WORK, WE FOUND THAT THE EPITHELIUM OF THE INTESTINAL MUCOSA CONTAINS A POPULATION OF CELLS PRODUCING EXTREMELY HIGH LEVELS OF TYPE I/III INTERFERON-STIMULATED PROTEINS, AMONG THEM, IFN-STIMULATED GENE 15 (ISG15). THESE SPECIALIZED ENTEROCYTES IN THE GUT EXPRESS GLYCOPROTEIN 2, SUGGESTING THAT SOME OF THESE EPITHELIAL CELLS WITH HEIGHTENED IMMUNITY ARE MICROFOLD CELLS (M CELLS). TYPE I IFN STIMULATION DRIVES BYSTANDER T CELL PROLIFERATION IN VIVO, WHICH LIKELY INCLUDE LATENTLY INFECTED CELLS, THUS CONTRIBUTING TO RESERVOIR PERSISTENCE. THERE IS ALSO EVIDENCE THAT TYPE I IFN EFFICIENTLY REACTIVATES HIV-1 IN VITRO AND EX VIVO. FURTHERMORE, MOUSE STUDIES INDICATE THAT BLOCKING TYPE I INTERFERON SIGNALING DIMINISHES IMMUNE ACTIVATION, RESTORES T CELL FUNCTION, AND REDUCES THE SIZE OF THE HIV RESERVOIR. TAKEN TOGETHER, THE DATA SUGGEST THAT THE GI TRACT, AND PARTICULARLY M CELLS, MAY CONTRIBUTE TO HIV PERSISTENCE AND REACTIVATION. OUR OVERARCHING HYPOTHESIS IS THAT IMMUNOLOGICALLY HYPERACTIVE MICROFOLD ("M") CELLS IN THE GUT CREATE A MICROENVIRONMENT THAT FOSTERS PROLIFERATION OF LATENTLY INFECTED BYSTANDER CD4+ T CELLS, SPORADIC HIV REACTIVATION FROM THESE CELLS, OR BOTH. WE WILL INVESTIGATE OUR HYPOTHESIS BY EX VIVO STUDIES OF MUCOSAL GI TISSUES, INCLUDING FROM PEOPLE LIVING WITH HIV (PLH), AND IN VITRO M CELL CULTURES TO MODEL AND MANIPULATE THEIR EFFECT ON HIV LATENCY. IN AIM 1, WE WILL INVESTIGATE WHETHER THE INTERACTION WITH M CELLS (ISG15HIGH ENTEROCYTES) INCREASES IMMUNE ACTIVATION OF BYSTANDER CD4+ T CELLS AND MACROPHAGES USING A SINGLE-CELL SPATIAL TRANSCRIPTOMICS/PROTEOMICS APPROACH. IN AIM 2, WE WILL TEST THE HYPOTHESIS THAT T CELLS/MACROPHAGES CONTAINING HIV-1 PROVIRAL DNA/MRNA ARE FOUND IN HIGH NUMBER IN THE VICINITY OF M CELLS USING DUODENAL AND RECTAL BIOPSIES FROM A CLINICAL STUDY OF 40 PLH ON STABLE ART. WE WILL QUANTIFY HIV-1 DNA AND MRNA COPIES BY DIGITAL PCR, DETERMINE T CELL CLONALITY BY SPATIAL TRANSCRIPTOMICS AND ANALYZE THE SPATIAL RELATIONSHIP OF HIV- 1 DNA+ AND MRNA+ CELLS BY DNA/RNASCOPE. IN AIM 3, WE WILL USE IN VITRO MODELS TO TEST THE HYPOTHESIS THAT M CELLS DRIVE T CELL PROLIFERATION AND HIV REACTIVATION VIA THEIR INCREASED ACTIVITY OF TYPE I/III IFN PATHWAYS. THIS STUDY WILL PROVIDE KEY INFORMATION ABOUT THE INTESTINAL MICROARCHITECTURE OF THE HIV RESERVOIR THAT IS NECESSARY FOR THE DEVELOPMENT OF NOVEL LATENCY REVERSAL AGENTS OR STRATEGIES TO TARGET LATENTLY INFECTED CELLS VIA M CELLS.