PROJECT SUMMARY Herpes simplex virus-2 (HSV-2) is the major causative agent of genital herpes, an incurable sexually transmitted infection that disproportionately infects women and poses a global health burden. Genital herpes is associated with recurrent symptoms that lead to tissue damage in the genital tract, and this pathology has been proposed as a mechanism for increased susceptibility to other viral sexually transmitted infections such as human immunodeficiency virus (HIV)2,3. The cellular and molecular mechanisms that lead to inflammation and tissue damage within the female genital tract during HSV-2 infection are poorly understood. Neutrophils are highly destructive innate immune cells that patrol the vagina at the steady state4. The role of neutrophils during viral sexually transmitted infections is not well defined. Our data show that neutrophils drive a pathogenic response against HSV-2. Depletion of neutrophils prior to infection resulted in significantly attenuated disease severity and tissue damage without affecting viral titers, suggesting that immunopathology plays a key role in genital herpes. Neutrophils are capable of multiple effector functions, including cytokine secretion. After neutrophil depletion, there was a profound reduction in IL-1b levels in the vagina, and the majority of IL-1b expressing cells in the vagina after HSV-2 infection were neutrophils, suggesting that neutrophil-mediated disease during HSV-2 infection may be driven by this pleiotropic pro-inflammatory cytokine. A multitude of factors can activate the neutrophil response, including virus- and bacteria-derived products. Transient perturbation of the vaginal microbiota led to an increase in neutrophil numbers in the vaginal tissue, suggesting that vaginal bacteria may play a role in regulating neutrophil-dependent tissue damage during genital herpes. Together, this proposal aims to dissect the mechanisms of neutrophil activation and identify the key factors that drive neutrophil-dependent tissue damage and inflammation at the vaginal mucosa during HSV-2 infection. The requirement of IL-1b produced by neutrophils in driving genital inflammation will be assessed in vivo using knockout models and cytokine neutralization, and downstream effector mechanisms that may lead to mucosal permeability will be examined. The role of the vaginal microbiota in regulating pathogenic neutrophil responses will be tested by transient introduction of common vaginal bacteria into mice. Finally, CRISPR/cas9 will be used to delete candidate pattern recognition receptors in neutrophils derived from Hoxb8-transformed progenitor cells to identify pathways ...