Autophagy downstream of endosomal Toll-like receptor signaling in macrophages is a key mechanism for resistance to Leishmania major infection

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.

Keywords: Leishmania; autophagy; infection; macrophage; toll-like receptor (TLR).

Figure 1.

L. major induces autophagy in murine BMDMs. A, BMDMs obtained from C57BL/6 mice were transduced with lentivirus vector encoding GFP-LC3 and infected with L. major (at a multiplicity of infection of 10) for 24 h or treated with rapamycin (1 μm) for 5 h and fixed. Infected cells were stained with rabbit polyclonal antibody anti-Leishmania. GFP-LC3 dots (green) and L. major (red) are shown in the fluorescence micrographs. Scale bar: 10 μm. B, GFP-LC3-transduced BMDMs were infected with L. major at the indicated time points or treated with rapamycin for 5 h. The number of GFP-LC3 dots per cell was scored by fluorescence microscopy. C, BMDMs obtained from C57BL/6 mice were infected with L. major as described in A and stained with a monoclonal antibody against endogenous LC3. The number of autophagosomes per cell was scored by fluorescence microscopy. For B and C, bars represent mean ± S.E. of triplicate samples (100 infected cells were scored per sample). D, BMDMs were infected with L. major for 24 h and the expression of endogenous LC3-I and LC3-II was assessed by immunobloting. Actin immunoblotting was used as a loading control. Autophagic flux was assessed by treating the cells with bafilomycin A1 (100 nm) for 24 h. Data are from one representative experiment repeated three times. *, p < 0.05; t test.

Figure 2.

Autophagy contributes to resistance to L. major infection in BMDMs. BMDMs obtained from C57BL/6 mice were transduced with lentivirus vectors encoding two distinct shRNA sequences for Atg5 (Atg5#1 or Atg5#2), or shRNA control (scramble) followed by infection with L. major for 48 h at a multiplicity of infection of 10 parasites per cell. A, expression of Atg5 was evaluated by immunoblotting. B–D, number and frequency of intracellular parasites evaluated by Giemsa staining. E, representative micrography of L. major-infected BMDMs stained with Giemsa. F, BMDMs transduced with shRNA for Atg5 were infected with L. major stably expressing GFP (L. major-GFP) (10 parasites per cell) for 48 h. Parasite multiplication was evaluated by flow cytometry. G, representative histogram of the mean of GFP fluorescence intensity (MFI) from L. major-GFP-infected BMDMs. For B–D and F, bars represent mean ± S.E. of triplicate samples (100 infected cells per sample). Data are from of one representative experiment repeated three times. *, p < 0.05; t test (B, C, and F) and two-way ANOVA (D).

Figure 3.

BMDMs deficient for endosomal TLRs have defective autophagy response and are more susceptible to L. major infection. A, BMDMs were transduced with lentivirus vector encoding GFP-LC3 and infected with L. major (10 parasites per cell) or treated with rapamycin (1 μm) for 5 h. The number of GFP-LC3 dots per cell was scored by fluorescence microscopy. B–G, BMDMs were infected with L. major (10 parasites per cell) and 48 h after infection the number and frequency of intracellular parasites were evaluated by Giemsa staining. Bars represent mean ± S.E. of triplicate samples (100 infected cells per sample). Data are from one representative experiment repeated three (A–D) or two (E–G) times. *, p < 0.05; t test (B, C, E, and F), and two-way ANOVA (A, D, and G).

Figure 4.

Autophagy triggered by endosomal TLRs accounts for BMDMs resistance to L. major. BMDMs were transduced with lentivirus vector encoding a shRNA sequence specific for Atg5 or shRNA control (scramble) followed by infection with L. major for 48 h (10 parasites per cell). A and B, percentage of infected cells (A) and the average number of intracellular parasites (B) evaluated by Giemsa staining. Bars represent mean ± S.E. of triplicate samples (100 infected cells per sample). Data are representative of one independent experiment repeated three times. *, p < 0.05; two-way ANOVA.

Figure 5.

Rapamycin induces autophagy in BMDMs and facilitates the restriction of L. major replication. A, BMDMs obtained from C57BL/6 mice were left untreated or treated with rapamycin R-5000 with the indicated concentrations for 24 h and cell lysates were assayed for immunoblotting. B and C, BMDMs obtained from C57BL/6 or Tlr3/7/9^−/− mice were infected with L. major at a multiplicity of infection of 5 parasites per cell. After 12 h infection, cells were incubated with rapamycin at the indicated concentrations and the percentage of infected cells (B) and the average numbers of intracellular parasites (C) were evaluated at 48 h after infection by Giemsa staining. Bars represent mean ± S.E. of triplicate samples (at least 100 infected cells per sample). Data are representative of one independent experiment repeated two times. *, p < 0.05; two-way ANOVA.

Figure 6.

Rapamycin treatment in vivo contributes to reduce the inflammatory lesions induced by L. major. A, BMDMs were left untreated (control), treated with rapamycin R-5000 (30 μm) for 1 h (rapam.), infected with L. major (10 parasites per cell) (L.m.) for 24 h, or infected with L. major for 24 h and treated with rapamycin for 1 h (L.m.+rapam.). Cell lysates were collected and subjected to immunoblotting. B, mice were infected with L. major (10⁶ parasites per mouse) by intradermal inoculation in ear. At 3 weeks post-infection, mice were treated daily with one dose of rapamycin R-5000 (5 mg/kg) (rapamycin) or with vehicle control (vehicle) by intraperitoneal route during 10 days. Ear thickness was measured weekly. C, representative image of ear lesions at the moment of euthanasia. D, at 5 weeks post-infection, mice were euthanized and ears and retromaxillary lymph nodes (LN) were collected and parasite burden was evaluated. For B and D, data represent mean ± S.E. from 4–5 animals per group. In D, each dot represents a single animal. Data are representative of one independent experiment repeated three times. *, p < 0.05; t test.