Mycobacterium tuberculosis impairs protective cytokine production via transcription factor MafB manipulation

Abstract

Although an increased expression of the transcription factor v-maf avian musculoaponeurotic fibrosarcoma oncogene homolog B (MAFB) has been reported in patients with active tuberculosis (TB), its potential role in Mycobacterium tuberculosis infection remains unknown. Herein, we report that MafB in macrophages is a regulator of the pro-inflammatory cytokines, TNF-α and IL-12p40, which are crucial for host defense against M. tuberculosis infection. Cell-based luciferase assays showed that MafB inhibited TNF-α and IL-12p40 transcriptional activity in a dose-dependent manner. At the molecular level, MafB interacted with IFN regulatory factor (IRF)-5 and PU.1 and inhibited IRF-5- and PU.1-mediated transactivation, via the basic-leucine zipper domain. Analysis using gene-deficient macrophages demonstrated that the suppressed pro-inflammatory cytokine production during M. tuberculosis infection depends on MafB expression. Finally, in vivo studies indicated that M. tuberculosis-mediated increase of MafB expression was responsible for the exacerbation of M. tuberculosis infection. Thus, our results provide a functional view of MafB as a cytokine regulator as well as novel insights into host factors involved in TB susceptibility.

Fig 1. Inhibition of TNF-α and IL-12p40 promoter activation by MafB.

(A, B) 293T cells were transfected with Tnf or Il12b promoter-driven luciferase reporter plasmid along with a combination of expression plasmids for p65 (-; 0 or +; 18 ng/well), IRF-5 (-; 0 or +; 91 ng/well), PU.1 (-; 0, + ; 2.3, or ++; 9.1 ng/well), and/or MafB (-; 0, + ; 5.7, or ++; 23 ng/well). After 24 h, cell lysates were measured using the luciferase assay kit. Luciferase activities are represented as a fold increase above background level of lysates prepared from mock-transfected cells. Data are representative of three independent experiments. ***P < 0.001; mean ± SD. (C, D) WT 293T cells or MAFB KO 293T cells were transfected with the Tnf or Il12b promoter-driven luciferase reporter plasmid along with a combination of expression plasmids for p65 (9 ng/well), IRF-5 (46 ng/well), and PU.1 (-; 0, + ; 2.3, or ++; 9.1 ng/well). After 24 h, cell lysates were measured using the luciferase assay kit. Luciferase activities are represented as a fold increase above background level of lysates prepared from PU.1 negative cell. Data are representative of three independent experiments. **P < 0.01; mean ± SD.

Fig 2. Interaction of MafB with IRF-5 and PU.1.

(A) 293T cells were transiently transfected with the indicated expression plasmids using Lipofectamine 2000. After 24 h, cell lysates were immunoprecipitated with the indicated antibodies, and tag-specific bands were subsequently detected using western blotting. Whole cell lysate (WCL) represents a positive control. Data are representative of three independent experiments. (B, C) 293T cells were transiently transfected with full-length Myc-tagged MafB (full), bZip domain deletion mutant (ΔbZip), and Flag-tagged IRF-5 (B) or HA-tagged PU.1 (C) expression plasmids using Lipofectamine 2000. After 24 h, cell lysates were immunoprecipitated with the indicated antibodies, and tag-specific bands were then detected using western blotting. Data are representative of three independent experiments. (D) 293T cells were transfected with Tnf or Il12b promoter-driven luciferase reporter plasmid along with a combination of expression plasmids for p65 (18 ng/well), IRF-5 (91 ng/well), PU.1 (9.1 ng/well), and/or MafB (-; 0, + ; 2.9, or ++; 5.7 ng/well). After 24 h, cell lysates were measured using the luciferase assay kit. Luciferase activities are represented as a fold increase above background level of lysates prepared from mock-transfected cells. Data are representative of two independent experiments. ***P < 0.001; n.s., not significant; mean ± SD. (E, F) 293T cells were transiently transfected with full-length Flag-tagged IRF-5 (full), IAD deletion mutant (ΔIAD), and Myc-tagged MafB (E) or full-length HA-tagged PU.1 (full), Ets domain deletion mutant (ΔEts), and Myc-tagged MafB (F) expression plasmids using Lipofectamine 2000. After 24 h, cell lysates were immunoprecipitated with the indicated antibodies, and tag-specific bands were then detected using western blotting. Asterisks represent nonspecific bands. Data are representative of three independent experiments. (G) 293T cells were transfected with Tnf or Il12b promoter-driven luciferase reporter plasmid along with a combination of expression plasmids for p65 (18 ng/well) and/or IRF-5 (-; 0, + ; 11, or ++; 45 ng/well). After 24 h, cell lysates were measured using the luciferase assay kit. Luciferase activities are represented as a fold increase above background level of lysates prepared from mock-transfected cells. Data are representative of two independent experiments. ***P < 0.001; n.s., not significant; mean ± SD. (H) 293T cells were transfected with Tnf or Il12b promoter-driven luciferase reporter plasmid along with a combination of expression plasmids for p65 (18 ng/well), IRF-5 (91 ng/well), and/or PU.1 (-; 0, + ; 2.3, or ++; 9.1 ng/well). After 24 h, cell lysates were measured using the luciferase assay kit. Luciferase activities are represented as a fold increase above background level of lysates prepared from mock-transfected cells. Data are representative of two independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; n.s., not significant; mean ± SD.

Fig 3. Regulation of TNF-α and IL-12p40 by MafB.

(A) Thioglycolate-elicited peritoneal macrophages were stimulated with or without 100 ng/mL Pam3CSK4. After 3 h, cells were stained with antibodies to PU.1, phalloidin, and nucleus (upper panels); IRF-5, MafB, and nucleus (middle panels); or p65, MafB, and nucleus (lower panels) and visualized using fluorescence microscopy. Scale bar represents 10 μm. (B) Thioglycolate-elicited peritoneal macrophages were stimulated with 100 ng/mL Pam3CSK4. Cells were harvested at the indicated time points and analyzed using quantitative RT–PCR. Relative gene expression is represented as a fold increase above background level of samples prepared from unstimulated cells. Data are representative of three independent experiments. (C) Thioglycolate-elicited peritoneal macrophages were treated with 5 μg/mL cycloheximide to inhibit protein synthesis, and cells were then stimulated with 100 ng/mL Pam3CSK4 after culture in the presence or absence of 100 μM chloroquine, 100 nM MG-132, or 100 nM bortezomib. Cells were harvested at the indicated time points, and MafB-specific bands were detected using western blotting. Anti-β-actin bands represent a positive control for the cell lysate. The intensity of MafB-specific bands is quantified using the ImageQuant TL analysis software. Data are representative of two independent experiments. (D) Thioglycolate-elicited peritoneal macrophages were treated with 0, 1, 5, or 10 μM RA. After 6 h, cells were harvested and analyzed using quantitative RT–PCR. Mafb expression was represented as a fold increase above background level of samples prepared from untreated cells. Data are representative of two independent experiments. **P < 0.01; ***P < 0.001; mean ± SD. (E) Thioglycolate-elicited peritoneal macrophages were stimulated with or without 100 ng/mL Pam3CSK4 after culture in the presence or absence of 30 μM RA. Cells were harvested after 6 h, and MafB-specific bands were detected using western blotting. Anti-β-actin bands represent a positive control for the cell lysate. The intensity of MafB-specific bands is quantified using the ImageQuant TL analysis software. Data are representative of three independent experiments. (F) Thioglycolate-elicited peritoneal macrophages were stimulated with or without 100 ng/mL Pam3CSK4 after priming with or without 30 μM RA. After 24 h, cytokine production was measured using ELISA. Data are representative of three independent experiments. ***P < 0.001; mean ± SD. (G) Thioglycolate-elicited peritoneal macrophages were stimulated with or without 100 ng/mL Pam3CSK4 after priming with or without 30 μM RA. Cells were harvested at the indicated time points and analyzed using quantitative RT–PCR. Relative gene expression is represented as a fold increase above background level of samples prepared from unstimulated cells. Data are representative of three independent experiments.

Fig 4. MafB-dependent cytokine restriction in M. tuberculosis infection.

(A) Thioglycolate-elicited peritoneal macrophages were stimulated with 0, 100, 200, 400, or 500 ng/mL Pam3CSK4. After 6 h, cells were harvested and analyzed using quantitative RT–PCR. Mafb expression is represented as a fold increase above background level of samples prepared from untreated cells. Data are representative of two independent experiments. (B) Thioglycolate-elicited peritoneal macrophages were stimulated with 0, 100, 200, 400, or 500 ng/mL Pam3CSK4. Cells were harvested after 6 h, and MafB-specific bands were detected using western blotting. Anti-β-actin bands represent a positive control for the cell lysate. The intensity of MafB-specific bands is quantified using the ImageQuant TL analysis software. Data are representative of three independent experiments. (C) Thioglycolate-elicited peritoneal macrophages were stimulated with 0, 100, 200, 400, or 500 ng/mL Pam3CSK4. After 6 h, cytokine production was measured using ELISA. Data are representative of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001; ND, not detected; n.s., not significant; mean ± SD. (D) Thioglycolate-elicited peritoneal macrophages were infected with M. tuberculosis H37Rv (MOI of 0, 0.5, 1, 2, or 5). After 6 h, cells were harvested and analyzed using quantitative RT–PCR. Mafb expression is represented as a fold increase above background level of samples prepared from uninfected cells. Data are representative of three independent experiments. (E) Thioglycolate-elicited peritoneal macrophages were infected with M. tuberculosis H37Rv (MOI of 0, 1, 2, 3, or 4). Cells were harvested after 6 h, and MafB-specific bands were detected using western blotting. Anti-β-actin bands represent a positive control for the cell lysate. The intensity of MafB-specific bands is quantified using the ImageQuant TL analysis software. Data are representative of two independent experiments. (F) Thioglycolate-elicited peritoneal macrophages were infected with M. tuberculosis H37Rv (MOI of 0, 1, 2, 3, or 4). After 24 h, cytokine production was measured using ELISA. Data are representative of three independent experiments. ***P < 0.001; ND, not detected; n.s., not significant; mean ± SD. (G) Thioglycolate-elicited peritoneal macrophages were infected with or without M. tuberculosis H37Rv (MOI of 5). After 6 h, cells were harvested and analyzed using quantitative RT–PCR. Relative gene expression is represented as a fold increase above background level of samples prepared from uninfected control cells. Data are representative of three independent experiments. ***P < 0.001; mean ± SD. (H) Thioglycolate-elicited peritoneal macrophages were infected with M. tuberculosis H37Rv (MOI of 0, 1, 2, 3, or 4). After 24 h, cytokine production was measured using ELISA. Data are representative of three independent experiments. ***P < 0.001; ND, not detected; mean ± SD. (I) Thioglycolate-elicited peritoneal macrophages were infected with M. tuberculosis H37Rv (MOI of 5). At the indicated time points after infection, cells were harvested and plated onto 7H10-OADC agar. Data are representative of three independent experiments. *P < 0.05; n.s., not significant; mean ± SD. (J) Thioglycolate-elicited peritoneal macrophages were infected with or without M. tuberculosis H37Rv (MOI of 5). After 6 h, cells were harvested and analyzed using quantitative RT–PCR. Nos2 expression is represented as a fold increase above background level of samples prepared from uninfected cells. Data are representative of three independent experiments. ***P < 0.001; mean ± SD. (K) Thioglycolate-elicited peritoneal macrophages were infected with or without M. tuberculosis H37Rv (MOI of 5). After 24 h, NO levels in the culture supernatants were measured. Data are representative of three independent experiments. ***P < 0.001; ND, not detected; mean ± SD. (L) Thioglycolate-elicited peritoneal macrophages were stimulated with 0, 10, 20, 50, or 100 ng/mL recombinant IL-10. After 6 h, cells were harvested and analyzed using quantitative RT–PCR. Mafb expression is represented as a fold increase above background level of samples prepared from untreated cells. Data are representative of two independent experiments. (M) Thioglycolate-elicited peritoneal macrophages were infected with M. tuberculosis H37Rv (MOI of 0, 0.5, 1, 2, or 5). After 6 h, cells were harvested and analyzed using quantitative RT–PCR. Il10 expression is represented as a fold increase above background level of samples prepared from uninfected cells. Data are representative of two independent experiments. (N) Thioglycolate-elicited peritoneal macrophages were infected with M. tuberculosis H37Rv (MOI of 0, 0.5, 1, 2, or 5) after priming with 100 μg/mL anti-mouse IL-10R. After 6 h, cells were harvested and analyzed using quantitative RT–PCR. Mafb expression is represented as a fold increase above background level of samples prepared from untreated cells. Data are representative of two independent experiments.

Fig 5. High resistance to M. tuberculosis infection in Mafb-deficient mice.

(A) Mafb^f/f (n = 3 mice per group) and Mafb^f/f::LysM-Cre (n = 3 mice per group) mice were intratracheally infected with M. tuberculosis H37Rv (5 × 10³ CFU). Lungs were homogenized at the indicated time points and analyzed using quantitative RT–PCR. Mafb expression is represented as a fold increase above background level of samples prepared from uninfected control mice. Data are representative of two independent experiments. ***P < 0.001; mean ± SD. (B) Mafb^f/f (n = 3 mice per group) and Mafb^f/f::LysM-Cre (n = 3 mice per group) mice were intratracheally infected with M. tuberculosis H37Rv (5 × 10³ CFU). At 4 weeks after infection, lungs were homogenized and analyzed using quantitative RT–PCR. mRNA expression is represented as a fold increase above background level of samples prepared from control mice. Data are representative of two independent experiments. **P < 0.01; ***P < 0.001; mean ± SD. (C) Mafb^f/f (n = 5 mice per group) and Mafb^f/f::LysM-Cre (n = 5 mice per group) mice were intratracheally infected with M. tuberculosis H37Rv (5 × 10³ CFU). Blood samples were collected at 4 weeks post infection, and cytokine production was measured using ELISA. Data are representative of two independent experiments. *P < 0.05; n.s., not significant; mean ± SD. (D) Mafb^f/f (n = 10) and Mafb^f/f::LysM-Cre (n = 10) mice were intratracheally infected with M. tuberculosis H37Rv (5 × 10³ CFU), and their survival was monitored for 10 weeks. Data are representative of three independent experiments. *P < 0.05. (E) Mafb^f/f (n = 10) and Mafb^f/f::LysM-Cre (n = 10) mice were intratracheally infected with M. tuberculosis H37Rv (5 × 10³ CFU). At 0, 1, 2, or 4 weeks after infection, lungs were digitized by micro-CT scan. Red arrows represent several pulmonary lesions. H, heart; V, vertebral column; L, left lung; R, right lung. (F) Mafb^f/f (n = 3) and Mafb^f/f::LysM-Cre (n = 3) mice were intratracheally infected with M. tuberculosis H37Rv (5 × 10³ CFU). Three weeks after infection, lungs were embedded in paraffin, and lung tissue sections were used for hematoxylin-eosin staining (Scale bar represents 1 mm or 50 μm), Iba1 immunohistochemical staining (Scale bar represents 50 μm), and Ziehl-Neelsen staining (Scale bar represents 50 μm). Red arrows and frames represent several pulmonary granulomatous lesions. (G) Mafb^f/f (n = 7) and Mafb^f/f::LysM-Cre (n = 7) mice were intratracheally infected with M. tuberculosis H37Rv (5 × 10³ CFU). Three weeks after infection, homogenates of lungs, mediastinal lymph nodes, spleens, and livers were plated on 7H10-OADC agar, and the CFU titers were counted. Data are representative of three independent experiments. ***P < 0.001; mean ± SD. (H) Mafb^f/f (n = 5 mice per group) and Mafb^f/f::LysM-Cre (n = 5 mice per group) mice were intratracheally infected with M. tuberculosis H37Rv (5 × 10³ CFU) and their survival was monitored for 10 weeks. Neutralizing monoclonal antibodies (200 μg/ 250 μL/ mouse) were inoculated into the abdominal cavity of mice every other day until 4 weeks of infection. Data are representative of two independent experiments, and significant differences are compared between control and cKO mice. **P < 0.01; n.s., not significant.

Fig 6. MafB-dependent cytokine regulation in macrophages.

Schematic model of pro-inflammatory cytokine regulation by the transcription factor MafB. Under steady-state conditions or M. tuberculosis infection, MafB abrogates the induction of pro-inflammatory cytokines by interfering with IRF-5 and PU.1, which are key components essential for transcriptional activity. Upon stimulation by TLR ligands, the transcription and protein expression levels of MafB are sequentially reduced in macrophages, thereby leading to an abundance of cytokine production.