Immunomodulatory roles of autophagic flux and IFIT in human ectocervical cells upon Trichomonas vaginalis infection

Trichomonas vaginalis (Tv) is the causative agent of trichomoniasis, the most common non-viral sexually transmitted infection worldwide. Despite its high prevalence, the mechanisms underlying Tv-induced inflammatory responses remain poorly understood. Herein, we investigated the signaling pathways mediating Tv-induced inflammation in ectocervical cells (Ects). We initially measured the production of various cytokines using a multiplex immunoassay, revealing a significant increase in IL-6, IL-8, IP-10, and CXCL1 secretion in Ects upon Tv infection. We then assessed the role of autophagy in regulating Tv-induced inflammation in Ects by using autophagy inhibitors and small interfering RNA targeting LC3B (si-LC3B) to block different stages of autophagy. Our findings indicated that Tv-induced autophagic flux mediates the secretion of proinflammatory cytokines in Ects. Additionally, blocking autophagosome formation via si-LC3B increases IL-6 and IP-10 levels while reducing IL-8 secretion. To further identify novel pathways involved in Tv-induced inflammation in Ects, we conducted a time-series proteomic analysis using 2D-LC-MS/MS. Intriguingly, we noticed robust activation of antiviral-related pathways in Ects after 8 h of Tv stimulation. Specifically, the most enriched proteins in these pathways were tetratricopeptide repeats (IFIT) family proteins (IFIT1, IFIT2, and IFIT3). Functional validation revealed that IFIT3 positively regulates downstream IL-8 and IP-10 secretion. Furthermore, we proved that si-LC3B enhanced IFIT expression in Ects upon Tv infection, suggesting that autophagy negatively regulates IFIT expression. Collectively, this study demonstrates that Tv infection induces autophagic flux and IFIT overexpression to modulate inflammatory responses in Ects, providing novel insights into the inflammatory mechanisms governing trichomoniasis.