Project Grant R21AA031725

ALCOHOL IMPAIRS THE HSPC RESPONSE TO SEPTIC INFECTION - PROJECT SUMMARY/ABSTRACT ALCOHOL ABUSE INCREASES THE HOST RISK OF DEVELOPING SERIOUS INFECTIONS, PARTICULARLY SEPTICEMIA/SEPSIS. NEUTROPHIL GRANULOCYTES CONSTITUTE THE FIRST LINE OF PHAGOCYTIC DEFENSE AGAINST BACTERIAL PATHOGENS. OUR RECENT STUDIES HAVE DEMONSTRATED THAT ACTIVATION OF HEMATOPOIETIC STEM CELLS (HSCS) IS CRUCIAL FOR ENHANCING GRANULOCYTE PRODUCTION BY BONE MARROW IN RESPONSE TO BACTERIAL INFECTION. ALCOHOL IMPAIRS THIS CRITICAL STEP OF THE GRANULOPOIETIC RESPONSE. CURRENTLY, KNOWLEDGE ABOUT THE UNDERLYING MECHANISMS REMAINS LIMITED. GUANYLATE-BINDING PROTEIN 7 (GBP7) IS A MEMBER OF THE 65KDA LARGE CYTOKINE INDUCIBLE GTPASE FAMILY. LITTLE IS KNOWN ABOUT THE FUNCTION OF GBP7 IN HEMATOPOIETIC STEM/PROGENITOR CELLS (HSPCS). OUR PILOT STUDIES REVEALED THAT GBP7 EXPRESSION BY HSPCS WAS MARKEDLY UPREGULATED AT BOTH MRNA AND PROTEIN LEVELS IN MICE WITH E. COLI SEPTICEMIA. THIS INCREASE IN GBP7 EXPRESSION WAS ACCOMPANIED BY ACTIVATION OF HSPCS. CHRONIC PLUS BINGE ALCOHOL ADMINISTRATION SUPPRESSED THE UPREGULATION OF GBP7 EXPRESSION ALONG WITH INHIBITION OF HSPC ACTIVATION DURING E. COLI SEPTICEMIA. SEARCHING PROMOTER REGIONS OF MURINE AND HUMAN GBP7 GENES SHOWED MULTIPLE FOS/JUN BINDING SITES. ALCOHOL SUPPRESSED ACTIVATION OF C-JUN N-TERMINAL KINASES (JNK1/2) IN MARROW CELLS DURING SEPTICEMIA. INHIBITION OF JNK1/2 ACTIVATION WITH SPECIFIC INHIBITOR SUPPRESSED LIPOPOLYSACCHARIDE- INDUCED UP-REGULATION OF GBP7 EXPRESSION BY HSPCS. THE PREDOMINANT DISTRIBUTION OF GBP7 WAS DETECTED IN NUCLEI OF HSPCS FROM BOTH MURINE AND HUMAN ORIGINS. SEPTIC STIMULI CAUSED A MARKED ENHANCEMENT OF GBP7 EXPRESSION IN THE NUCLEI OF HSPCS, WHICH IMPLIED A CLOSE ASSOCIATION OF GBP7 WITH THE CHANGE IN NUCLEAR ACTIVITY. GBP ACTIVATION LEADS TO THEIR CONFORMATIONAL CHANGES FACILITATING INTERACTION OF THESE LARGE GTPASES WITH DIFFERENT MACROMOLECULES TO FORM COMPLEXES FOR CARRYING OUT DIFFERENT FUNCTIONS IN CELLS. CYCLIN D1-CYCLIN- DEPENDENT KINASE (CDK) 4/6 SIGNALING DRIVES HSPC PROLIFERATION. SEPTICEMIA CAUSED A MARKED INCREASE IN CYCLIN D1 EXPRESSION AND ENHANCEMENT OF PROLIFERATION IN HSPCS, BOTH OF WHICH WERE SUPPRESSED BY ALCOHOL OR GBP7 KNOCKOUT (KO). GBP7 KO IMPAIRED MARROW GRANULOPOIETIC RESPONSE, WHICH WAS SIMILAR TO WHAT CAUSED BY ALCOHOL INTOXICATION. OUR CURRENT PROJECT PROPOSES TO EXPLORE THE ROLE OF GBP7 IN THE REGULATION OF HSPC ACTIVATION AND INVESTIGATE HOW ALCOHOL IMPAIRS IT. THE CENTRAL HYPOTHESIS IS THAT ALCOHOL INHIBITS GBP7 SIGNALING FOR HSPC ACTIVATION DURING THE GRANULOPOIETIC RESPONSE TO SEPTIC INFECTION. TWO SPECIFIC AIMS ARE: 1) TO EXAMINE IF ALCOHOL SUPPRESSES UP-REGULATION OF GBP7 EXPRESSION BY HSPCS VIA INHIBITING ACTIVATION OF THE TOLL- LIKE RECEPTOR (TLR) 4-JNK1/2-FOS/JUN PATHWAY IN RESPONSE TO SEPTIC INFECTION; 2) TO DETERMINE IF ALCOHOL INHIBITS HSPC ACTIVATION DURING THE GRANULOPOIETIC RESPONSE VIA IMPAIRING GBP7-CYCLIN D1-CDK4/6 SIGNALING. RESULTS OBTAINED FROM THIS EXPLORATORY INVESTIGATION WILL GREATLY ADVANCE OUR KNOWLEDGE ABOUT THE DEFECT OF IMMUNE DEFENSE AGAINST SERIOUS INFECTION IN ALCOHOL ABUSERS. IT WILL ALSO IDENTIFY KEY TARGETS FOR FURTHER DEVELOPING NOVEL THERAPEUTIC INTERVENTIONS TO TREAT FATAL INFECTION AND SEPSIS IN THESE IMMUNOCOMPROMISED HOSTS.