In vitro infection of M. bovis BCG was performed as described previously101. Briefly, BMDMs (1 × 105) were incubated in a 96-well plate with 1 × 106 (MOI 10:1) of live M. bovis BCG for 4 h at 37 °C. After the infection, uninfected bacteria were washed-out with RPMI-10 and the infected cells were further cultured for 24 h in RPMI-10. The supernatant was collected and he nitrite concentration was measured by Griess assay. For CFU determination, the cells were disrupted by water and plated on Middlebrook 7H10 agar (Difco #262710) and number of BCG colonies was counted after 3-week culture.

Lung M. bovis BCG infection was performed as described previously102. Briefly, mice were intratracheally inoculated with 7.5 × 106 CFU of M. bovis BCG. The lung tissues were collected at days 3 and 14 after infection, and homogenized by gentleMACS. The homogenates were serially diluted in water and plated on Middlebrook 7H10 agars and colonies were counted, as described above. Total RNA was extracted from the lung homogenates and gene expression was analyzed by qRT-PCR, as described above.

For T-cell recall response to mycobacterial antigens, mediastinal lymph node cells were collected at day 14 after intratracheal infection and stimulated with 5 μg/ml of tuberculin PPD (Japan BCG laboratory #114015) for 3 days. Concentration of IFN-γ and IL-17 in the culture were measured by ELISA

Intraperitoneal M. bovis BCG infection was performed, as described previously103. Briefly, WT and Trem2−/− mice were infected intraperitoneally with 5 × 106 CFU of M. bovis BCG. The peritoneal lavages were collected at 4 h, day 1, and day 3 after infection by washing the peritoneal cavity with 1 ml of RPMI-10. The concentration of cytokines in the lavage was analyzed by ELISA. The peritoneal cells were analyzed by flow cytometry. CFU was counted as described above.

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