Background The periodontal pathogen Porphyromonas gingivalis (Pg) has been linked to esophageal and other cancers through epidemiology studies. Pg’s protease virulence factors known as gingipains have been identified in esophageal cancer tissue and correlate with worse disease prognosis. Anti-PD-1 antibodies have shown some success in esophageal cancer treatment, but further understanding of the induction of PD-L1 in esophageal cells is needed to identify potential treatment modalities. Pg has been shown to induce PD-L1 on the surface of infected cells, suggesting that the presence of Pg in esophageal cancer cells may contribute to PD-L1 expression and immune escape. One of the pathways known to induce PD-L1 is wnt pathway activation resulting in b-catenin translocation to the nucleus. Prior studies have demonstrated that Pg activates the wnt pathway by a non-canonical mechanism, leading to b-catenin nuclear localization.
Methods An immortalized non-transformed esophageal cell line, Het-1A, was used to investigate the level of PD-L1 induction by Pg infection using quantitative immunofluorescence. PD-L1 expression was measured using irreversible gingipain inhibitors against lysine-gingipain (Kgp) and arginine-gingipain (Rgp). Pg-induced PD-L1 expression pathways were investigated by Western blot and qPCR. PD-L1 induction by Pg was characterized in cancer cell lines that have an endogenous level of PD-L1 expression, including tongue squamous cell carcinoma (SCC25) and neuroblastoma (SH-SY5Y). PD-L1 induction by Pg was assessed in a murine derived RAW macrophage cell line that is critical for anti-PD-1 responses.
Results Pg infection increased PD-L1 expression on Het-1A cells within 24 hours of infection and increased PD-L1 mRNA within 4 hours of infection. PD-L1 expression level correlated with cellular bacterial burden on the cells in a dose-dependent manner. PD-L1 expression was decreased by the Kgp inhibitor, atuzaginstat, or an Rgp inhibitor, COR613, and PD-L1 expression was completely blocked when both gingipain inhibitors were used together (figure 1). Pg also induced expression of PD-L1 on the surface of infected SCC-25, SH-SY5Y, and RAW cell lines. Western blot analysis and qPCR revealed that Kgp inhibition, but not Rgp inhibition, was able to inhibit the non-canonical activation of b-catenin and down regulation of classical wnt pathway effectors at both the mRNA and protein level.
Conclusions In host cells infected with Pg, gingipains mediate the induction of PD-L1 as a mechanism of immune evasion through the non-canonical activation of the wnt pathway. Further studies to elucidate induction mechanisms are in progress. In esophageal cancer and other cancers infected with Pg, combining gingipain inhibitors with anti-PD-1 therapy may improve treatment outcomes.
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