Interplay among microbial communities, epithelial cells, and immune system in vaginal mucosa of women with high-risk Human Papillomavirus infection

Persistent infection with HR-HPVs is a primary cause of cervical cancer worldwide. Among HR-HPV, subtypes HPV-16 and HPV-18 are most associated with invasive cancers and are thought to cause approximately 65-75% of cases. Emerging evidence indicates that CVM plays a substantial role in the viral persistence and subsequent disease. Accordingly, a role of CVM composition in the regression of high grade of CIN lesions has been reported. Data from clinical studies show that Lactobacillus-dominated cervicovaginal microbiota and L. crispatus particularly was positively associated with the clearance of the virus and negatively associated with the neoplastic progression of intraepithelial lesions. In contrast, vaginal-dysbiosis associated bacteria were correlated with the persistence of HR-HPVs infection and with increasing cancer risk. The molecular mechanisms based on such effects are not elucidated yet. Aim of this study was to investigate whether each vaginal bacterial species, commonly found in cervicovaginal microbial communities, may affect the expression of viral oncogenes and accelerate the neoplastic transformation of HR-HPV transformed cells. For this purpose, the HPV-16 transformed SiHa epithelial cells (with ~1,2 copies of integrated viral genome) were selected as experimental model of CIN-1 and bacterial strains, representatives of the 5 CSTs, were evaluated for their effect on the expression of viral oncogenic proteins E6 and E7, the fate of cellular oncosuppressor protein p53, the effects on cell survival/growth of HPV-transformed SiHa cells, and the synthesis of matrix-degrading enzymes. Our data show that G. vaginalis and M. micronuciformis directly induce the expression of the viral E6 and E7 oncogenes and the synthesis of the respective products by SiHa cells. These bacteria also induce the expression of E6-AP, a ubiquitin protein ligase that binds to E6 and induce p53 degradation. As consequence of E6 activation and p53 degradation, cell proliferation is increased. L. crispatus never induces the expression of viral oncogenes or proteins, while L. iners and L. jensenii slightly induce the viral E7 gene -4- expression but not E6. In contrast to G. vaginalis or M. micronuciformis, vaginal Lactobacilli did not affect the cell cycle of cervical epithelial cells. G. vaginalis and M. micronuciformis also induce high expression of MMP-9, a cellular protein which overexpression has been observed in different malignant tumors. All these data were obtained by using live bacterial cells, supernatants, or bacterial lysates. Supernatants from bacterial culture are not effective in the system suggesting the responsivity of very labile or not secreted products. Although not all strains of G. vaginalis have the same property to induce the expression of viral oncogenes and oncoproteins, our data strongly suggest that this species together with M. micronuciformis may play an important role in neoplastic transformation of HR-HPV infected cell. According with data from clinical data, the presence of G. vaginalis or M. micronuciformis in the vaginal fluid of women with persistent HR-HPV infection or CIN-1 should direct these groups to a close follow-up or to surgical excision respectively. Abbreviations: HR-HPV (High-Risk Human Papillomavirus), CVM (cervicovaginal microbiota), CST (community state type), CIN (cervical intraepithelial neoplasia), CIN-1 (low grade of cervical intraepithelial neoplasia), E6-AP (E6-Associated Protein), L. (Lactobacillus), G. (Gardnerella), M. (Megasphaera), MMP-9 (Matrix Metalloproteinase-9).