Fig 2: IRAK-M knockdown induced M1-type, while inhibited M2-type polarization of macrophage, during M. tb infection. U937 cells were infected with NC or KD lentivirus for 96 h, challenged with H37Rv (MOI 10) for 24 h. Cells were resuspended in lysis buffer and Western blot was performed to detect STAT1, phosphorylated STAT1 (pSTAT1), iNOS (a), STAT6, phosphorylated STAT6 (pSTAT6) and Arg-1 (b). Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (**, p < 0.01; ***, p < 0.001)

Fig 3: IRAK-M increased in M. tb infected macrophage cells and also in M. tb infected lung tissue. M. tb induced increased expression of IRAK-M in U937 cells. U937 cells were infected with M. tb H37Rv (MOI 10) for 24 h after seeding. a Cells grown on the coverslips were stained with anti-IRAK-M by immunocytochemistry. Representative images from three experiments were shown as merged results of IRAK-M (Cy3, red) and DAPI (blue) (1000×, scale bar 10 µm). b Expression of IRAK-M in U937 cells infected by inactivated or living M. tb H37Rv (MOI 10) was evaluated by Western blot. Densitometric analysis of IRAK-M expression was performed using pooled data from three such experiments. Results were expressed as Mean ± SD, ***: P < 0.001. c IRAK-M expression was increased in tissue of pulmonary tuberculosis. Human samples from pulmonary tuberculosis (n = 6) were collected for IRAK-M detection by immunohistochemistry (400×, scale bar 50 µm). Para-carcinoma tissues of human lung cancer (n = 6), were used as negative control. Representative images were shown

Fig 4: M1-type polarization of U937 cells induced by CpG7909 was inhibited by M. tb infection, which can be reversed by IRAK-M knockdown in U937 cells. a 2 µg/ml CpG7909 induced M1-type polarization of U937 cells. U937 cells were stimulated with CpG7909 (0, 0.5 µg/ml and 2 µg/ml) for 24 h and 50 µg of cell lysates was analysed by Western blot to detect the expression of phosphorylated ERK1/2 and iNOS. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (**, p < 0.01; ***, p < 0.001). b M. tb infection inhibited M1-type polarization of U937 cells induced by CpG7909. U937 cells were stimulated with CpG7909 (2 µg/ml) for 24 h, then challenged with H37Rv (MOI 10) for 24 h. Western blot was performed to detect IRAK4 and iNOS. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (**, p < 0.01; ***, p < 0.001). c IRAK-M knockdown rescued M1-type polarization of U937 cells induced by CpG7909, which was inhibited by M. tb infection. U937 cells were infected with IRAK-M KD lentivirus for 96 h, stimulated with CpG7909 (2 µg/ml) for 24 h, challenged with H37Rv (MOI 10) for 24 h. Fifty microgram of cell lysates was analysed by Western blot to detect IRAK4 and iNOS. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (**, p < 0.01; ***, p < 0.001). d IRAK-4 and IRAK-M expression in CpG-stimulated and M. tb-infected U937 cells. U937 cells grown on the coverslips were stimulated with CpG7909 (2 µg/ml) for 24 h, then infected with H37Rv (MOI 10) for 24 h, co-stained with anti-IRAK4 (Cy3) or anti-IRAK-M (Cy3) and DAPI (blue) by immunocytochemistry (400×, scale bar 50 µm)

Fig 5: IRAK-M over-expression resulted in higher bacterial load, while IRAK-M interference resulted in lower bacterial load in M. tb infected cells. Lentiviruses that over-expresses (IRAK-M-GFP-Lentivirus, OE) or interferes with (IRAK-M-RNAi-GFP-Lentivirus, KD) IRAK-M expression, were constructed. a Lentiviruses of negative control (NC) and OE were introduced into Jurkat cells. Cells were examined by light microscope and fluorescent microscope at 96 h post-transfection. More than 80% of Jurkat cells expressed GFP (200×, scale bar 50 µm). b Lentiviruses of NC and KD were introduced in U937 cells. Cells were examined by light microscope and fluorescent microscope at 96 h post-transfection. More than 90% of U937 cells expressed GFP (200×, scale bar 100 µm). c Expression of IRAK1-4 in Jurkat and U937 cells was evaluated by Western blot. d-g Bacterial load in Jurkat (d, e) and U937 cells (f, g) challenged with virulent M. tuberculosis H37Rv strain (MOI 10) were analyzed by acid-fast staining and colony forming units (CFU). Cells were infected with lentivirus for 96 h, challenged with H37Rv for 5 h and washed three times with PBS. d, f At 24 h post-infection, 1 × 106 cells were resuspended in 20 µl PBS and fixed in 4% para-formaldehyde for 15 min on the slides. Acid-fast staining (AF) was performed and arrows indicated AF-positive bacteria in the cells (1000×, scale bar 10 µm). e, g For determination of CFU, at 5 and 24 h post-infection, 1 × 105 cells were washed aseptically, homogenized and plated at 10-fold serial dilutions on Middlebrook 7H11 agar. CFU numbers per plate were counted to evaluate the bacterial load 3 to 4 weeks later. The bacterial load in cells of different groups was expressed as Log10 CFU ± SEM (**, p < 0.01; ***, p < 0.001)