Dysbiotic bacteria drive suppressive neutrophil phenotypes and prolonged lifespan in mucosal tissues of HIV-infected individuals Nichole Klatt AIDS 2016, Drivers of HIV Progression July 19 th, 2016 Durban, South Africa

Inflammation (particularly associated with mucosal dysfunction) is a key predictor of morbidities and mortality

Epithelial Barrier Breaches and Microbial Translocation Estes et al., Plos Pathogens 2010 What are the mechanisms of mucosal dysfunction in HIV Infection? 3

Burgener, McGowan, Klatt; Curr Opinion Immunol 2015

Neutrophils have both phagocytic and suppressive functions Kolaczkowska, et al. Nat. Rev. Immun., 2013; Leliefield et al., Critical Care 2016 Tissue Damage T cell suppression 5 Anti-microbial functions

Neutrophils can be suppressive Neutrophils can be inflammatory

CD15 CD66b CD16 CD11b CD49d SSC-A CD15 CD11b CD66b CD49d CD16 CD49d CD16 SSC-A Neutrophils and Eosinophils Exclude eosinophils GranulocytesNeutrophils eosinophils HIV+ Blood HIV+ GI Neutrophils accumulate in GI tract of HIV-infected individuals What mechanisms underlie this accumulation during HIV infection?

Dysbiosis in HIV infection Zevin, McKinnon, Burgener, Klatt; Curr Opinion HIV/AIDS 2016

Question: Does altered microbiome in HIV infection affect neutrophil populations? Whole blood: 4 HIV- 2 HIV+ Incubate with HAMBs 20 hours Increased in HIV: Prevotella copri Prevotella stercorea Decreased in HIV: Rhumnicoccus bromii Lactobacullus plantarum Controls: No stim (-) Media alone (-) LPS (+) CD11b CD15 SSC-A (CD45+/CD3-/CD20-) Neutrophils PD-L1 CD16 CD62L Arginase-1 “Suppressive” “Phagocytic”

Microbiota increased in HIV induce “suppressive” neutrophil phenotype, but not “normal” neutrophils

Prevotella induce PD-L1 on neutrophils

PD-1 expression correlates with HIV reservoir PD-1 expression correlates with HIV load

Neutrophil PD-L1 expression associated with CD4+ T cell PD-1 expression

Potential mechanism of neutrophil accumulation in GI tract is enhanced survival by dysbiotic bacteria

Conclusions : Neutrophils and HIV Altered Mucosal Bacteria Dysbiotic bacteria induce suppressive phenotype of neutrophils in HIV infection Potential contributions to microbial translocation/inflammation in HIV infection Dysbiotic bacteria promote PD-L1 expression on neutrophils, which may drive CD4 PD-1 Implications for reservoir Dysbiotic bacteria in HIV may promote neutrophil survival, leading to accumulation of neutrophils in the GI tract Potential contribution to mucosal dysfunction

UW/WaNPRC/Pharmaceutics Klatt Lab Tiffany Hensley-McBain Alex Zevin, PhD Ryan Cheu Jennifer Manuzak, PhD Charlene Miller De’Neka Gary Ernesto Coronado Arina Wu Lydia Sweet Kevin Fogassey Toni Gott UC Denver Cara Wilson Steph Dillon Eric Lee Acknowledgements UW Harborview Hospital Ann Collier Michalina Montano Lindsay Legg Jai Lingappa Bob Harrington Northwestern University Tom Hope Michael Dixson University of Manitoba/National Microbiology Laboratory Adam Burgener UCSF SCOPE Peter Hunt NIH/NIAID 1K22AI NIH/NIDA 1DP1DA NIH/NIAID 1R01AI NSF Fellowship (Hensley-McBain) UW CFAR