Table 1.
Nucleotide sequences of primers used for RT-qPCR.
Fig 1.
Elevated lung IL-36γ mRNA levels following LPS instillation.
Lung total RNA was extracted 4 h after intra-tracheal instillation of LPS (n = 5 mice) or saline (n = 5 mice). The results represent the levels of Il36g or Il36r mRNA determined by RT-qPCR normalized to Gapdh mRNA. **P<0.01, as assessed by Mann-Whitney test.
Fig 2.
Kinetic of IL-36 cytokines, IL-36R, IL-1β and TNF-α mRNA expression after M. bovis BCG infection.
mRNA levels of Il36a, Il36b, Il36g, Il36r, Il1b, and Tnfa measured by RT-qPCR and normalized to Gapdh mRNA levels from the lungs of C57BL/6 wild-type mice 2 weeks (n = 4) and 4 weeks (n = 5) after M. bovis BCG infection. Three naïve mice were included as controls (A). In situ hybridization was performed using a digoxigenin-labeled riboprobe complementary to Il36g mRNA (B). The micrographs are representative of lungs from one naïve mice (upper panel) and one infected mice 2 weeks after M. bovis BCG inoculation (lower panel). *P<0.05, as assessed by Kruskal-Wallis, followed by Mann-Whitney test.
Fig 3.
Role of IL-36R signaling in M. bovis BCG infection.
Wild-type and IL-36R deficient mice were inoculated intravenously with M. bovis BCG (n = 12 per group). Six mice from each genotype were sacrificed at 28 days and the other mice were followed for up to 180 days. The percentage of survival (A) and evolution of body weight (B) for WT and IL-36R deficient mice are depicted. (C) Total lung weight and absolute lung cell numbers at 28 days after infection are represented in left and right panels, respectively. No significant differences were observed between WT and IL-36R KO mice, as assessed by log-rank (Mantel-Cox) test (A) or two-way ANOVA for repeated measures, followed by Sidak’s multiple comparison test (B). *P<0.05, as assessed by Kruskal-Wallis, followed by Mann-Whitney test (C). Black and grey symbols represent WT and IL-36R deficient mice, respectively.
Fig 4.
Changes in lung cell populations following M. bovis BCG inoculation.
Lung cells were isolated from 3 naive WT and IL-36R deficient mice, and from 6 WT and 6 IL-36R deficient mice 4 weeks after intravenous M. bovis BCG inoculation. The results represent CD11b+, CD4+, CD8+, and B220+ cell numbers as assessed by flow cytometric analysis. *P<0.05, as assessed by Kruskal-Wallis, followed by Mann-Whitney test.
Fig 5.
Histopathology and bacterial loads in wild-type and IL-36R deficient mice.
Hematoxylin eosin staining of lung sections 4 weeks after M. bovis BCG infection of WT (left panels) and IL-36R deficient (right panels) mice (original magnifications X2, upper panel, and X40, lower panel) (A). Determination of free alveolar space on lung sections. Data are represented as the mean ± SD of percentage of lung lesion free/total lung tissue in 5 mice per group with 1 or 2 lobes analyzed per mouse (B). Lung bacterial loads at 4 weeks post-infection are represented as CFU per lung (n = 6 mice per group) (C). **P<0.01 as assessed by Mann Whitney test.
Fig 6.
CXCL1 expression in response to M. bovis infection in IL-36R deficient and wild-type mice.
A. Total RNA was prepared from the lungs of naïve (open symbols) and M. bovis BCG infected (closed symbols) WT (black circles) and IL-36R deficient (grey squares) mice. The results represent Cxcl1, Cxcl2, and Il6 mRNA levels determined by RT-qPCR normalized to Gapdh mRNA levels in individual mice. *P<0.05, as assessed by Kruskal-Wallis, followed by Mann-Whitney test. B. Serum levels of Cxcl1 were measured by ELISA in WT (black symbols) and IL-36R deficient (grey symbols) mice. The results represent means ± SEM of 6 mice per group. Two-way ANOVA for repeated measures, followed by Sidak’s multiple comparison test was used to compare IL-36R deficient and WT mice. ***P<0.001 only at week 2.
Fig 7.
Kinetics of pulmonary cytokine expression after M. tuberculosis infection.
Total RNA was prepared from the lungs of naïve WT (black symbols) and TNF-α deficient mice (grey symbols) and at days 7, 14, 21 and 27 after M. tuberculosis infection in WT (black symbols) and at day 27 in TNF-α deficient mice (grey symbols). The results represent the levels of Il36g, Il36r, Tnfa, Il1b, and Ifng mRNA determined by RT-qPCR normalized to L32 mRNA (n = 5 per group). *P<0.05, **P<0.01, as assessed by Kruskal-Wallis, followed by Mann-Whitney test (C).
Fig 8.
IL-36R is dispensable to control acute M. tuberculosis infection.
Mice deficient for IL-36R, IL-1R1 or TNF-α, and WT mice were exposed to an aerogenic dose of M. tuberculosis H37Rv and monitored for survival (A) and body weight. The results represent mean body weight ± SEM values of 8–14 mice per group, pooled from 2 independent experiments. Lung weight (left panels) and bacterial loads (right panels) were determined in TNF-α deficient, IL-36R deficient, IL-1R1 deficient, and WT mice at 1 month (C) and 4 months (D) post infection. Results are expressed as individual values and mean (n = 4–9 for lung weight, n = 3–9 for CFU) from 1 experiment at 1 month and (n = 5–12 for lung weight, n = 5–10 for CFU) from 2 experiments at 4 months. *P<0.05, **P<0.01, ***P<0.001 as compared to the wild-type controls (assessed by Kruskal-Wallis, followed by Mann-Whitney test).
Fig 9.
Controlled lung pathology after M. tuberculosis infection in the absence of IL-36R pathway.
Macroscopic lung lesions of IL-36R deficient, IL-1R1 deficient, TNF-α deficient, and WT mice at 1 month, and of IL-36R deficient and WT mice at 4 months post M. tuberculosis infection (A). Histological studies were performed at 1 month in IL-36R deficient, IL-1R1 deficient, TNF-α deficient, and WT mice (B), and at 4 months in IL-36R deficient and WT mice (C). Representative lung H&E (1x and 40x magnifications) and Ziehl-Neelsen (60x magnification) staining at 1 and 4 months post M. tuberculosis infection. Free alveolar space quantification is represented as the mean ± SD of percentage lung lesion free/total lung tissue in 4–7 mice per group with 1–2 lobes analyzed per mouse (D). Statistical analysis was one-way ANOVA, no statistically significantly differences were observed between animal groups.
Fig 10.
Lung IFN-γ and Cxcl-1 levels in M. tuberculosis infection.
The levels of IFN-γ and Cxcl-1 protein were determined in lung homogenates 1 month post aerogenic M. tuberculosis infection in TNF-α deficient, IL-36R deficient, IL-1R1 deficient, and wild-type mice, and 4 months post-infection in IL-36R deficient and WT mice. Results are expressed as mean +/- SEM of n = 3–9 from 1 experiment at 1 month, n = 5–10 from 2 experiments at 4 months. P<0.05 *; P<0.01 ** as compared to the WT mice.
Fig 11.
Unaffected lung inflammatory cell infiltration in the absence of IL-36R pathway after M. tuberculosis infection.
Infiltrating cells in the lungs of IL-36R deficient (right panels) and wild-type mice (left panels) were isolated on day 28 after M. tuberculosis H37Rv infection, and analyzed by flow cytometry for the expression of CD11b, Gr1 (Ly6G/6C) and CD3. Gating strategy and representative dot blots are shown (A). Results expressed as the absolute number of CD11b+Gr1high, CD11b+Gr1low and Gr1low CD3ε+ cells per lung are from 3 individual mice with mean values as horizontal bars (B).