Abstract
Leishmaniasis is caused by protozoan parasites of the genus Leishmania In mammalians, these parasites survive and replicate in macrophages and parasite elimination by macrophages is critical for host resistance. Endosomal Toll-like receptors (TLRs) have been shown to be crucial for resistance to Leishmania major in vivo For example, mice in the resistant C57BL/6 genetic background that are triple-deficient for TLR3, -7, and -9 (Tlr3/7/9-/-) are highly susceptible to L. major infection. Tlr3/7/9-/- mice are as susceptible as mice deficient in MyD88 or UNC93B1, a chaperone required for appropriate localization of endosomal TLRs, but the mechanisms are unknown. Here we found that macrophages infected with L. major undergo autophagy, which effectively accounted for restriction of parasite replication. Signaling via endosomal TLRs was required for autophagy because macrophages deficient for TLR3, -7, and 9, UNC93B1, or MyD88 failed to undergo L. major-induced autophagy. We also confirmed that Myd88-/-, Tlr3/7/9-/-, and Unc93b1-/- cells were highly permissive to L. major replication. Accordingly, shRNA-mediated suppression of Atg5, an E3 ubiquitin ligase essential for autophagosome elongation, in macrophages impaired the restriction of L. major replication in C57BL/6, but did not affect parasite replication in Myd88-/- or Unc93b1-/- macrophages. Rapamycin treatment reduced inflammatory lesions formed in the ears of Leishmania-infected C57BL/6 and Tlr3/7/9-/- mice, indicating that autophagy operates downstream of TLR signaling and is relevant for disease development in vivo Collectively, our results indicate that autophagy contributes to macrophage resistance to L. major replication, and mechanistically explain the previously described endosomal TLR-mediated resistance to L. major infection.
Highlights
Because it has been previously demonstrated that endosomal Toll-like receptors (TLRs) signaling is essential to protect the host against infection by L. major [16], we sought to investigate whether the engagement of endosomal TLRs by L. major triggers autophagy and if autophagy triggered by endosomal TLRs contributes to resistance to L. major infection
Because we found that autophagy is an important mechanism controlling intracellular replication of L. major in bone marrow-derived macrophages (BMDMs), and BMDMs defective in multiple endosomal TLRs fail to trigger autophagy during L. major infection, we tested whether macrophages deficient in multiple endosomal TLRs signaling were more permissible to L. major replication
It was demonstrated that the combined signaling of endosomal TLR3, -7, and -9, but not individual receptors, accounts for the production of IL-12 by L. major-infected macrophages and host resistance to Leishmaniasis in a murine model of L. major infection [16]
Summary
Autophagy is the major self-catabolic pathway for lysosomal degradation of cytoplasmic macromolecules and organelles, being essential in periods of stress and nutrient deprivation. Because we found that autophagy is an important mechanism controlling intracellular replication of L. major in BMDMs, and BMDMs defective in multiple endosomal TLRs fail to trigger autophagy during L. major infection, we tested whether macrophages deficient in multiple endosomal TLRs signaling were more permissible to L. major replication To address this question, we first evaluated the replication of L. major in Unc93b1Ϫ/Ϫ and Myd88Ϫ/Ϫ BMDMs. We found a higher percentage of infected cells and an increased number of intracellular amastigotes in Myd88Ϫ/Ϫ and Unc93b1Ϫ/Ϫ BMDMs as compared with cells obtained from C57BL/6 mice (Fig. 3, B and C). The susceptibility of UNC93B1 mutant BMDMs was comparable with Myd88Ϫ/Ϫ cells, supporting the hypothesis that endosomal TLRs signaling mediate the MyD88-dependent resistance to L. major infection, as previously shown [16] Both Unc93b1Ϫ/Ϫ and Myd88Ϫ/Ϫ BMDMs had a higher percentage of cells harboring from 3 to 5 or 6 to 10 intracellular parasites (Fig. 3D). Our data suggest that endosomal TLR signaling favors the induction of autophagy, which represents a critical process for restriction of L. major replication in macrophages
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