TLR2 and endosomal TLR-mediated secretion of IL-10 and immune suppression in response to phagosome-confinedListeria monocytogenes

Listeria monocytogenesis a facultative intracellular bacterial pathogen that escapes from phagosomes and induces a robust adaptive immune response in mice, while mutants unable to escape phagosomes fail to induce a robust adaptive immune response and suppress the immunity to wildtype bacteria when co-administered. The capacity to suppress immunity can be reversed by blocking IL-10. In this study, we sought to understand the host receptors that lead to secretion of IL-10 in response to phagosome-confinedL.monocytogenes(Delta hly), with the ultimate goal of generating strains that fail to induce IL-10. We conducted a transposon screen to identify Delta hly L.monocytogenesmutants that induced significantly more or less IL-10 secretion in bone marrow-derived macrophages (BMMs). A transposon insertion inlgt, which encodes phosphatidylglycerol-prolipoprotein diacylglyceryl transferase and is essential for the formation of lipoproteins, induced significantly reduced IL-10 secretion. Mutants with transposon insertions inpgdAandoatA, which encode peptidoglycan N-acetylglucosamine deacetylase and O-acetyltransferase, are sensitive to lysozyme and induced enhanced IL-10 secretion. A Delta hly Delta pgdA Delta oatAstrain was killed in BMMs and induced enhanced IL-10 secretion that was dependent on Unc93b1, a trafficking molecule required for signaling of nucleic acid-sensing TLRs. These data revealed that nucleic acids released by bacteriolysis triggered endosomal TLR-mediated IL-10 secretion. Secretion of IL-10 in response to infection with the parental strain was mostly TLR2-dependent, while IL-10 secretion in response to lysozyme-sensitive strains was dependent on TLR2 and Unc93b1. In mice, the IL-10 response to vacuole-confinedL.monocytogeneswas also dependent on TLR2 and Unc93b1. Co-administration of Delta hlyand Delta actAresulted in suppressed immunity in WT mice, but not in mice with mutations in Unc93b1. These data revealed that secretion of IL-10 in response toL.monocytogenesinfectionin vitrois mostly TLR2-dependent and immune suppression by phagosome-confined bacteriain vivois mostly dependent on endosomal TLRs. Author summary Listeria monocytogenesis a Gram-positive bacterial pathogen that has shown promise as a vaccine-delivery vector because of its ability to stimulate a robust T-cell response. The efficacy of a vaccine is in part tied to how well it avoids inducing the immunosuppressive cytokine IL-10. In this work, we investigated the bacterial and host factors that contribute to secretion of IL-10 and immunosuppression following infection with a strain ofL.monocytogenesthat cannot escape from host cell vacuoles. We identified TLR2 and Unc93b1-dependent nucleic-acid-sensing Toll-like receptors as the primary host mediators of IL-10 secretion and immunosuppression. Unc93b1-dependent TLRs likely recognize nucleic acids released upon bacterial lysis in phagosomes. Strategies that reduce lytic death of bacteria and subsequent recognition of nucleic acids by endosomal TLRs could be used to improve bacteria-based vaccines.