UNC93B1 and nucleic acid-sensing Toll-like receptors mediate host resistance to infection with Leishmania major

Abstract

The mammalian homolog B1 of Unc-93 Caenorhabditis elegans known as UNC93B1 is a chaperone protein that mediates translocation of the nucleic acid-sensing Toll-like receptors (TLRs) from the endoplasmic reticulum to the endolysosomes. The triple deficient (UNC93B1 mutant) mice have a functional single point mutation in the UNC93B1 that results in non-functional TLR3, TLR7, and TLR9. Herein, we demonstrate that UNC93B1 mutant mice, in the C57BL/6 (resistant) genetic background, are highly susceptible to Leishmania major infection. Enhanced swelling of the footpad was associated with high levels of interleukin 10, decreased levels of interferon γ, and increased parasitism. None of the single TLR3, TLR7, and TLR9 knock-out (KO) mice resemble the UNC93B1 mutant phenotype upon infection with L. major. Whereas the double TLR7/TLR9 KO showed a partial phenotype, the triple TLR3/TLR7/TLR9 KO mice were as susceptible as the UNC93B1 mutant mice, when infected with Leishmania parasites. Finally, we demonstrate that treatment with either anti-interleukin 10 receptor monoclonal antibody or recombinant interleukin 12 restored a robust anti-parasite TH1 response and reverted the susceptible phenotype of UNC93B1 mutant mice. Altogether, our results indicate the redundant and essential role of nucleic acid-sensing TLR3, TLR7 and TLR9 in inducing interleukin 12, development of a TH1 response, and resistance to L. major infection in resistant C57BL/6 mice.

FIGURE 1.

UNC93B1 mutant mice show larger footpad lesions and higher parasite load after infection with L. major when compared with C57BL/6 wild type mice. A, mice were infected with 1 × 10⁶ promastigotes subcutaneously in the right hind footpad. The course of infection was monitored weekly and the diameter of the primary footpad lesions was measured. The mean size of lesions and S.E. are shown (five mice per group). Bar graphs correspond to the parasite load quantitation in lesions by limiting dilution analysis at week 10 post-infection. B, spleens were harvested from infected mice, and cells were cultured with no stimulation, under the stimuli of 1 × 10⁶ FTAg or Concanavalin A (10 μg/ml). Accumulation of IFN-γ and IL-10 in supernatants of cells cultured for 24 h was evaluated by ELISA assay as described under “Experimental Procedures.” Results are expressed as mean and S.E. of duplicate measurements of four animals per group. Asterisks indicate statistically significance (p < 0.05), when comparing triple deficient (3D) and WT mice by two-way ANOVA with Bonferroni's post-test. The data shown are representative of three independent experiments.

FIGURE 2.

Quantification of RNA expression after L. major infection. C57BL/6 (black circles) and UNC93B1 mutant (white squares) mice were infected with 1 × 10⁶ promastigotes and had their popliteal lymph nodes harvested after 4 weeks. Gene expression was quantified in total RNA by nCounter Analysis System. Data represent the mean ± S.E. (n = 3). Differences considered statistically significant between WT and UNC93B1 mutant mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001) after two-way ANOVA with Bonferroni's post-test, are indicated.

FIGURE 3.

UNC93B1 mutant mice show larger footpad lesions and higher parasite load when compared with TLR3, TLR7 and TLR9 single KOs infected with L. major. A, WT, UNC93B1 mutant, and single KO mice were infected subcutaneously in the right hind footpad with 1 × 10⁶ promastigotes. The course of infection was monitored weekly and the diameter of the primary footpad lesions was measured. The mean size of lesions and S.E. are shown (five mice per group). Bar graphs correspond to the parasite load quantification in lesions by limiting dilution analysis at week 10 post-infection. The mean counts of four footpads per group are shown. B, accumulation of IFN-γ in supernatants of splenocytes from infected WT, 3d, and single KO mice was measured by ELISA, 24 h after antigen stimulation, as described under “Experimental Procedures.” Data shown are mean ± S.E. and are representative of three independent experiments. Asterisks indicate statistically significant differences, after two-way ANOVA with Bonferroni's post-test (p < 0.01), when comparing infected 3d mice with infected WT (C57BL/6), TLR3 KO, TLR7 KO, and TLR9 KO mice. No significant difference was observed when comparing different single KOs and WT mice.

FIGURE 4.

UNC93B1 mutant mice are more susceptible to infection with L. major than TLR3/7, TLR7/9, and TLR3/9 double KO mice. A, animals were infected subcutaneously in the right hind footpad with 1 × 10⁶ promastigotes. The course of infection was monitored weekly, and the diameter of the primary footpad lesions was measured. The mean size of lesions and S.E. are shown (five mice per group). Bar graphs correspond to the parasite load quantification in lesions by limiting dilution analysis at week 10 post-infection. Data shown are mean ± S.E. and are representative of three independent experiments. B, spleens were harvested from infected mice, and cells were cultured with no stimulation, under the stimuli of 1 × 10⁶ FTAg or Concanavalin A. Accumulation of IFN-γ and IL-10 in culture supernatants, 48 h after antigen stimulation, was evaluated by ELISA assay as described under “Experimental Procedures.” Results are expressed as mean and S.E. of duplicate measurements of four animals per group. The presented data are representative of three independent experiments.

FIGURE 5.

Severely impaired T_H1 responses and susceptibility of TLR3/7/9 triple KO mice to infection with L. major. A, WT, UNC93B1 mutant, MyD88, and TLR3/7/9 triple KO mice were infected with 1 × 10⁶ promastigotes subcutaneously in the right hind footpad. The course of infection was monitored weekly and the diameter of the primary footpad lesions was measured. The mean size of lesions and S.E. are shown (five mice per group). The letters a, b, and c indicate that lesion curve is significantly different when comparing UNC93B1 mutant and TLR3/7/9 KO mice to MyD88 KO and WT mice. Bar graphs correspond to the quantification of parasite load in the footpad lesions by limiting dilution analysis at week 10 post-infection. B, spleens were harvested from infected mice and cells cultured with no stimulation, under the stimuli of 1 × 10⁶ FTAg or Concanavalin A. Accumulation of IFN-γ and IL-10 in culture supernatants, 48 h after antigen stimulation, was evaluated by ELISA assay as described under “Experimental Procedures.” Results are expressed as mean and S.E. of duplicate measurements of four animals per group. The presented data are representative of three independent experiments. C, serum was obtained from five animals per group, and the levels of circulating L. major specific IgG1 and IgG2c determined by ELISA. Data shown are mean ± S.E. and are representative of three independent experiments. Asterisks or different letters (a, b, c) indicate statistically significant differences after two-way ANOVA with Bonferroni's post test (p < 0.01) when comparing results from UNC93B1 mutant and TLR3/7/9 KO mice with WT and TLR7/9 KO mice.

FIGURE 6.

Characterization of early cellular immune response upon infection with L. major. Wild type C57BL/6 mice were infected in the right footpad with 10⁶ metacyclic forms of the L. major and, at 12 days after inoculation, animals were sacrificed, and the draining lymph nodes of the infected footpad were obtained. Cells were grown in absence (medium) or presence of a total lysate of L. major metacyclic parasites (FTAg) 48 h, and supernatant was used for quantification of IFN-γ (A), IL-10 (B), and IL-12 (C) by ELISA. Analysis by flow cytometry of CD4⁺ and CD8⁺ cells from WT and TLR3/7/9^−/− mice 12 days after infection is also shown. B and C, T cells expressing CD4⁺ and CD8⁺ surface markers were stained for endogenous IFN-γ and IL-10, and cellular populations were separated by flow cytometry, as well CD11b⁺ and CD11c⁺ cells that were tested for IL-10 and IL-12 (please refer to “Experimental Procedures” for all cytokines tested). E, data represent the mean ± S.E. (n = 3). Differences are considered statistically significant (*, p < 0.05; **, p < 0.01; and ****, p < 0.0001) after two-way ANOVA with Bonferroni's post-test, are indicated.

FIGURE 7.

Quantification of RNA expression in UNC93B1 mutant mice treated with αIL-10R and rIL-12p70 after L. major infection. UNC93B1 mutant mice (white squares), UNC93B1 mutant+αIL-10R (black circles), and UNC93B1 mutant+rIL-12p70 (black triangles) mice were infected with 1 × 10⁶ promastigotes and had their popliteal lymph nodes harvested after 10 weeks (see “Experimental Procedures” for details). Quantification of gene expression was evaluated in total RNA by nCounter Analysis System. Data represent the mean ± S.E. (n = 3). Differences considered statistically significant between WT and UNC93B1 mutant mice (*, p < 0.05; **, p < 0.01; ***, p < 0.001; and ****, p < 0.0001) after two-way ANOVA with Bonferroni's post-test, are indicated.

FIGURE 8.

Administration of anti-IL-10R and rIL-12p70 restores T_H1 responses and reduces the development of footpad lesions in UNC93B1 mutant mice infected with L. major. A, C57BL/6 and UNC93B1 mutant mice were infected with 1 × 10⁶ promastigotes in the footpad and treated with either anti-IL-10R or rIL-12p70 (five mice per group). Splenocytes were cultured for 48 h in the presence or absence of antigen or Concanavalin A (10 μg/ml), and accumulation of IFN-γ and IL-10 in culture supernatants evaluated by ELISA, as described under “Experimental Procedures.” Results are expressed as mean and S.E. of duplicate measurements of four animals per group. The data shown are representative of three independent experiments. B, UNC93B1 mutant mice were infected with 1 × 10⁶ promastigotes promastigotes in the footpad and treated with anti-IL-10R or rIL-12p70 (five mice per group). The course of infection was monitored weekly, and the diameter of the primary footpad lesions was measured. The mean size of lesions and S.E. are shown. Lesion size progression was monitored with a metric caliper. Bar graphs correspond to the parasite load quantification in lesions by limiting dilution analysis. Data represent the mean ± S.E. from two independent experiments. Asterisks indicate statistically significant differences after two-way ANOVA with Bonferroni's post-test when comparing non-treated controls and mice receiving treatment with either anti-IL-10R or rIL-12p70 (p < 0.01 for C57BL/6 and p < 0.0001 for UNC93B1 mutant mice).