Human Neutrophil Response to Trichomonas vaginalis

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PROJECT SUMMARY / ABSTRACT Trichomonas vaginalis (Tv) is a protozoan pathogen that causes the most common non-viral sexually transmitted infection in the United States and worldwide. Studies on Tv pathogenesis and immunity have been limited. However, the rise in drug resistant strains of Tv, and an emerging appreciation of the link between asymptomatic Tv infections with inflammation-driven pathologies demands better understanding of this prevalent human infection. Notably, Tv infection has been linked to reproductive complications, increased susceptibility to HIV, increased incidence of cervical cancer, and increased aggressiveness of prostate cancer. Clinical observation, previous work by others, and preliminary studies in our laboratory point to neutrophils (PMN) as the key player in anti-Tv immunity. PMN are extremely destructive cells, propagating a cascade of tissue damage and inflammation, which has been associated with inflammatory pathologies, initiation of new cancers, and exacerbation of existing neoplasms. If an infection is not cleared efficiently, the cycle of inflammation could persist chronically. While the importance of PMN in Tv infection is recognized, how they work to fight Tv infection is not characterized. Therefore, we aim to (i) determine how PMN kill Tv, and (ii) determine the role of PMN in establishing inflammation during Tv infection. Our preliminary data suggest that PMN kill Tv using trogocytosis (trogo= nibble), a process by which effector cells take small “bites” from target cells. We will isolate PMN from human blood and co-culture with Tv. We will then use flow cytometry- based cytolysis assays, live cell imaging microscopy and imaging flow cytometry to determine whether PMN trogocytosis leads to death of Tv. We will also determine whether PMN use neutrophil extracellular traps NETosis to kill Tv, a process by which PMN release nuclear contents externally to ensnare, immobilize and kill pathogens. To assess NETosis, we will use extracellular DNA quantification, imaging, and flow-cytometry based cytolysis assays. In addition, to determine the consequences of PMN-Tv interaction on inflammation, we will probe supernatants of PMN-Tv co-cultures to determine the cytokine program that Tv elicits. We will use live Tv versus heat-inactivated Tv to determine the contribution of Tv-mediated tissue destruction on inflammation, and we will also perform Tv-PMN co-cultures in the presence of vaginal epithelial cells to determine how destruction of the epithelial layer by Tv and PMN contributes to inflammation. The knowledge gained from this study on how acute Tv infection is cleared by PMN and what inflammatory signals are established, will help to suggest potential strategies to monitor inflammatory pathologies, cervical cancers and prostate cancers associated with Tv infection, and inform the design of potential immunotherapy interventions.