Critical role of IL-36β for the in vivo Th1 response to an intracellular bacterium. WT and IL-36R^−/− mice were infected intravenously with 10⁷ living M bovis BCG. On day 28, spleen cells of individual mice (n = 5 per genotype) were isolated, and cultured in the absence (Unstim) or presence of antigens derived from (A) M bovis BCG (BCG-Ag) or (B-D) viable M bovis BCG (10³ CFU/well). Culture supernatants were harvested after 3 days of incubation and assayed by ELISA for (A) IFN-γ production, or by multiplex analysis for (B) IFN-γ, (C) IL-6, and (D) TNF-α production. (E) Accumulation of nitrite was measured using the Griess reagent. Values are the mean ± SD of 5 mice in each group. *P < .05 (Student t test), BCG-Ag, or BCG restimulation in WT cells significantly differs from the same stimulation in IL-36R^−/− cells. Data are shown from one of 2 independent experiments with similar results. (F) Lung histopathologies at 4 weeks postinfection of IL-36R^−/− (right panels) and WT (left panels) mice showing more extensive lesions in IL-36R^−/− (original magnifications: ×40 top panel, and ×200 bottom panel). These results are representative of 2 independent experiments (n = 5 mice per group). (G) Evaluation of cellular content in lung sections from 4-week BCG-infected mice. Data are represented as the mean number of cells/field ± SD in 5 mice per group with at least 3 lobes analyzed per mouse. *P < .0005 (Student t test). (H) Determination of lung tissue/free space on lung sections. Data are represented as the mean of occupied/free space ± SD in 10 mice per group with at least 3 lobes analyzed per mouse. *P < .02 (Student t test). (I) Lung bacterial loads at 4 weeks postinfection are represented mean CFU per lung ± SD (n = 6-8 mice per group).