Caspase-1 independent IL-1beta production is critical for host resistance to mycobacterium tuberculosis and does not require TLR signaling in vivo

Abstract

To investigate the respective contributions of TLR versus IL-1R mediated signals in MyD88 dependent control of Mycobacterium tuberculosis, we compared the outcome of M. tuberculosis infection in MyD88, TRIF/MyD88, IL-1R1, and IL-1beta-deficient mice. All four strains displayed acute mortality with highly increased pulmonary bacterial burden suggesting a major role for IL-1beta signaling in determining the MyD88 dependent phenotype. Unexpectedly, the infected MyD88 and TRIF/MyD88-deficient mice, rather than being defective in IL-1beta expression, displayed increased cytokine levels relative to wild-type animals. Similarly, infected mice deficient in caspase-1 and ASC, which have critical functions in inflammasome-mediated IL-1beta maturation, showed unimpaired IL-1beta production and importantly, were considerably less susceptible to infection than IL-1beta deficient mice. Together our findings reveal a major role for IL-1beta in host resistance to M. tuberculosis and indicate that during this infection the cytokine can be generated by a mechanism that does not require TLR signaling or caspase-1.

FIGURE 1

M. tuberculosis infected IL-1β^−/− mice display acute mortality and elevated bacterial loads. Survival of TLR2/TLR9^−/− (dark gray), TRIF/MyD88^−/− (green), MyD88^−/− (blue), IL-18^−/− (purple), IL-1β^−/− (red), IL-1R1^−/− (black), and C57BL6 WT (dotted black line, white circle) mice after low dose aerosol exposure to H37Rv. Vertical dotted line in lower panel represents survival of IL-1β^−/− mice (A). Data are representative of at least two independent experiments each involving 5–12 mice per group. At 4 wk p.i. bacterial loads were measured in BALF and lung suspensions from the strains indicated (B). Data are pooled from 3–6 independent experiments. C, Representative H&E-stained sections from lungs of C57BL6 WT, MyD88^−/− and IL-1β^−/− mice are shown at 4 wk p.i. Original magnifications ×50 and ×400. D, Presents % lung affected, mean granuloma sizes, as well as the relative number of acid fast bacilli and degree of necrosis scored blindly.

FIGURE 2

Effect of IL-1β deficiency on cytokine and total nitrite levels in lungs of M. tuberculosis-infected mice. Indicated cytokines were measured by ELISA in BALF and cell-free lung suspensions of uninfected C57BL6 WT (light gray) as well as 4 wk infected C57BL6 WT (white) and IL-1β^−/− (dark gray) mice (A–D). Total nitrite was measured in the same samples by Griess assay after nitrate reduction (A). Dotted lines indicate limits of detection of the respective ELISA. Statistical comparisons were performed by Student t test and p values are depicted. Data shown are pooled from at least three independent experiments.

FIGURE 3

Neither TRIF/MyD88 nor IL-1R1 are required for IL-1β induction in vivo. IL-1β was measured by ELISA in BALF and cell-free lung suspensions of the indicated mouse strains at 4 wk p.i. (A). Dotted line indicates limit of detection. Data shown are pooled from at least four independent experiments. IL-1β mRNA levels measured by RT-PCR in lungs of 4 wk infected C57BL6 WT (white), MyD88^−/− (dark gray) and IL-1R1^−/− (black) mice (B). Data shown represent the mean 6 SD-fold increase over uninfected WT controls (dotted line) for two independent experiments with a minimum of five mice per group. IL-1β measured by ELISA in supernatants of BMDM from mouse strains listed (B) with and without exposure to live H37Rv (MOI:1) for 48 h (C). Data shown are means ± SD and are representative of two independent experiments.

FIGURE 4

Host resistance and pulmonary IL-1β levels in M. tuberculosis infected casp1^−/− and ASC^−/− mice. IL-1β measured by ELISA in supernatant of BMDM from WT, casp1^−/− and ASC^−/− mice with and without exposure to live H37Rv (MOI:1) for 48 h (A). Data shown are the means 6 SD and are representative of two independent experiments. B, IL-1β levels in BALF and cell-free lung suspensions of the 4 wk infected mouse strains indicated are depicted. Dotted lines indicate the limit of detection of the ELISA assay. Data shown are pooled from at least three independent experiments. C57BL6 WT, casp1^−/− and ASC^−/− mice were infected with H37Rv by aerosol exposure and their survival monitored (C). Vertical dotted line represents survival of IL-1β^−/− mice. Data are pooled from two independent experiments each with 8-12 mice per group. Bacterial loads in BALF and lung suspension at 4 wks p.i. of the same mouse strains (D). Data are pooled from four independent experiments. E, Western blot detection of IL-1β in cell-free lung suspensions at 4 wk p.i. Each lane represents an individual mouse. In (B) and (D), differences that were statistically significant (p < 0.05) are indicated.