Ryan K. Cheu1,2,3, Avid Mohammadi4, Tiffany Hensley-Mcbain1,2, Jennifer Manuzak1,2,3, Alexander S. Zevin1,2, Charlene Miller3, Mark Yudin5, Rupert Kaul4, Nichole R. Klatt1,2,3
1Department of Pharmaceutics, University of Washington, Seattle, WA, USA
2Washington National Primate Research Center, University of Washington, Seattle, WA, USA
3Department of Pediatrics, University of Miami Miller School of Medicine, Miami, FL, USA
4Department of Immunology, University of Toronto, Toronto, Canada
5Core Obstetrics & Gynecology, University of Toronto, Toronto, Canada
Background: Bacterial vaginosis (BV) is associated with an increased HIV transmission rate. Inflammatory cells such as neutrophils are critical for innate immune responses but can also contribute to barrier damage and inflammation. Currently, the role of neutrophils in HIV transmission and BV status is unknown. Here, we hypothesize that BV-associated bacteria increase HIV transmission rates by inducing activation and accumulation of neutrophils within the female reproductive tract (FRT), resulting in epithelial barrier damage.
Methods: In order to elucidate the mechanisms for the negative outcomes of BV, we collected cervicovaginal cytobrushes from 11 women with BV and 10 women without BV. We used flow cytometry to assess phenotype and functionality of neutrophils and performed in vitro whole blood co-cultures with bacteria associated with BV (G. vaginalis), healthy commensals such as L. iners and L. crispatus, media alone (negative control) and lipopolysaccharide and peptidoglycan (positive controls). To determine the impact neutrophils have on epithelial integrity, we isolated neutrophils from healthy human whole blood to co-culture with FRT bacteria across a vaginal epithelial cellular monolayer and used a transepithelial electrical resistance (TEER) set-up to assess barrier damage.
Results: We demonstrated increased neutrophil activation (p=0.0022), prolonged lifespan (p=0.0022), and more total neutrophils (p=0.0022) in the cytobrushes from women with non-Lactobacillus dominant (nLD) communities. Similarly, our co-culture experiments showed more neutrophil activation (p<0.0001), prolonged lifespan (p=0.008), and more total neutrophils (p<0.0001) in cultures with G. vaginalis compared with our negative controls and Lactobacillus cultures. Our 16S rRNA analysis identified two distinct community groups one dominated by Lactobacillus and the other a more diverse community consisting of G. vaginalis. Our TEER assays demonstrated significant barrier damage in cultures with neutrophils and G. vaginalis when compared with neutrophils alone (p<0.0001) or with neutrophils and Lactobacillus spp. (p<0.0001).
Conclusions: Here, we demonstrate that BVAB induce neutrophil activation, delay apoptosis leading to accumulation within the FRT, and jeopardize epithelial integrity. This study provides potential mechanistic insights into how BV may lead to FRT inflammation and increased HIV transmission.