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. 2017 Aug 23;17(1):185.
doi: 10.1186/s12866-017-1095-2.

IRAK-M alters the polarity of macrophages to facilitate the survival of Mycobacterium tuberculosis

Pei Shen  1   2 , Quan Li  3 , Jilei Ma  1 , Maopeng Tian  1 , Fei Hong  1 , Xinjie Zhai  1 , Jianrong Li  1 , Hanju Huang  1 , Chunwei Shi  4
Affiliations

IRAK-M alters the polarity of macrophages to facilitate the survival of Mycobacterium tuberculosis

Pei Shen et al. BMC Microbiol. .

Abstract

Background: Intracellular bacterium, Mycobacterium tuberculosis (M. tb), infects specifically macrophages as host cells. IRAK-M, a member of IRAK family, is a negative regulator in TLR signaling and specifically expresses in monocytes and macrophages. The role of IRAK-M in intracellular growth of M. tb and macrophage polarization was explored, for deeply understanding the pathogenesis of M. tb, the significance of IRAK-M to innate immunity and pathogen-host interaction.

Methods: IRAK-M expression was detected in M. tb infected macrophages and in human lung tissue of pulmonary tuberculosis with immunofluorescence staining, Western blot and immunohistochemistry. IRAK-M knock-down and over-expressing cell strains were constructed and intracellular survival of M. tb was investigated by acid-fast staining and colony forming units. Molecular markers of M1-type (pSTAT1 and iNOS) and M2-type (pSTAT6 and Arg-1) macrophages were detected using Western blot in IRAK-M knockdown U937 cells infected with M. tb H37Rv. U937 cells were stimulated with immunostimulant CpG7909 into M1 status and then infected with M. tb H37Rv. Expression of IRAK-M, IRAK-4 and iNOS was detected with immunofluorescence staining and Western blot, to evaluate the effect of IRAK-M to CpG directed M1-type polarization of macrophages during M. tb infection. Molecules related with macrophage's bactericidal ability such as Hif-1 and phosphorylated ERK1/2 were detected with immunohistochemistry and Western blot.

Results: IRAK-M increased in M. tb infected macrophage cells and also in human lung tissue of pulmonary tuberculosis. IRAK-M over-expression resulted in higher bacterial load, while IRAK-M interference resulted in lower bacterial load in M. tb infected cells. During M. tb infection, IRAK-M knockdown induced M1-type, while inhibited M2-type polarization of macrophage. M1-type polarization of U937 cells induced by CpG7909 was inhibited by M. tb infection, which was reversed by IRAK-M knockdown in U937 cells. IRAK-M affected Hif-1 and MAPK signaling cascade during M. tb infection.

Conclusions: Conclusively, IRAK-M might alter the polarity of macrophages, to facilitate intracellular survival of M. tb and affect Th1-type immunity of the host, which is helpful to understanding the pathogenesis of M. tb.

Keywords: IRAK-M; Intracellular survival; Macrophage; Mycobacterium tuberculosis; Polarization.

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Figures

Fig. 1
Fig. 1
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. 2
Fig. 2
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)
Fig. 3
Fig. 3
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. 4
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
Fig. 5
IRAK-M affected Hif-1 and MAPK signaling cascade during M. tb infection. a Human samples from pulmonary tuberculosis (n = 6) were collected for Hif-1α 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 (400 ×, scale bar 10 μm). b U937 cells were infected with M. tb (MOI 10) at indicated time and expression of Hif-1α was evaluated by Western blot. c Lentivirus infected U937 cells (NC and KD) were infected with M. tb (MOI 10) for 24 h and Western blot was performed to detect Hif-1α, VEGF and phosphorylated ERK1/2. Densitometric analysis was performed using pooled data from three such experiments. Data were mean ± SD (**, p < 0.01; ***, p < 0.001)
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