A MyD88-JAK1-STAT1 complex directly induces SOCS-1 expression in macrophages infected with Group A Streptococcus

Abstract

Some pathogens can use host suppressor of cytokine signaling 1 (SOCS-1), an important negative-feedback molecule, as the main mode of immune evasion. Here we found that group A Streptococcus (GAS) is capable of inducing SOCS-1 expression in RAW264.7 and BMDM macrophages. IFN-β plays a role in GAS-induced SOCS-1 expression in macrophages following the induction of cytokine expression by GAS, representing the classical pathway of SOCS-1 expression. However, GAS also induced STAT1 activation and SOCS-1 expression when GAS-infected cells were incubated with anti-IFN-β monoclonal antibody in this study. Moreover, upon comparing TLR4(-/-) BMDM macrophages with wild-type (WT) cells, we found that TLR4 also plays an essential role in the induction of SOCS-1. MyD88, which is an adaptor protein for TLR4, contributes to STAT1 activation and phosphorylation by forming a complex with Janus kinase 1 (JAK1) and signal transducer and activator of transcription 1 (STAT1) in macrophages. GAS-stimulated expression of STAT1 was severely impaired in MyD88(-/-) macrophages, whereas expression of JAK1 was unaffected, suggesting that MyD88 was involved in STAT1 expression and phosphorylation. Together, these data demonstrated that in addition to IFN-β signaling and MyD88 complex formation, JAK1 and STAT1 act in a novel pathway to directly induce SOCS-1 expression in GAS-infected macrophages, which may be more conducive to rapid bacterial infection.

Figure 1

Expression of SOCS-1 and STAT-1 phosphorylation in GAS-induced macrophages. RAW 264.7 macrophages and BMDMs from wild-type mice were infected with GAS or non-viable GAS at an MOI of 100∶1 for 1 h. At different post-infection times, the levels of SOCS-1 mRNAs were determined by RT-PCR in RAW 264.7 cells (a) or BMDMs (b), and SOCS-1 protein expression were detected by western blot respectively in RAW 264.7 cells (d) or BMDMs (f). Under the same condition, the levels of STAT-1 phosphorylation were determined by western blot analysis in RAW264.7 cells (c) or BMDMs (e). *P<0.05 vs. nonviable GAS group. Each experiment was repeated three times and similar results were obtained. GAS, group A Streptococcus; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.

Figure 2

GAS induced IFN-β, SOCS-1 and p-STAT1 expression in RAW 264.7 macrophages in the presence of neutralizing anti-IFN-β or cycloheximide. RAW 264.7 macrophages were infected with GAS or non-viable GAS at an MOI of 100∶1 for 1 h. At different time points post-infection, IFN-β mRNA expression was determined by real-time PCR (a). After RAW 264.7 macrophages were infected with GAS in the presence of neutralizing anti-IFN-β (10 µg/ml) for 6 h of incubation, SOCS-1 mRNA expression was detected by real-time PCR (b) and p-STAT1 expression was analyzed by western blot (c). Following 10 h of incubation as described above, SOCS-1 expressions were analyzed by western blot (d). RAW 264.7 macrophages were incubated with cycloheximide (10 µM) for 30 min prior to GAS infection and then the expressions of SOCS-1 were determined by RT-PCR (e) and the expressions of p-STAT1 were detected by western blot following 6 h of infection (f). *P<0.05 vs. GAS group. ⁺P<0.05 vs. nonviable GAS group. ^#P>0.05 vs. GAS group. Each experiment was repeated three times and similar results were obtained. GAS, group A Streptococcus; MOI, multiplicity of infection; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.

Figure 3

Activation of TLR4 and TLR2 expression by RAW 264.7 macrophages and SOCS-1 expression in BMDMs from TLR4^−/− knockout mice infected by GAS. RAW 264.7 cells were infected with GAS or non-viable GAS at an MOI of 100∶1. At different time points, the mRNA levels of TLR2 (a) and TLR4 (b) were assayed by real-time PCR. BMDMs from TLR4^−/− knockout mice were stimulated with GAS at the same MOI for 1 h, and then washed GAS with PBS. Six hours later, treated cells were lysed, and equal amounts of cell lysate were blotted with anti-pSTAT-1, anti-STAT-1 and β-actin (c), and 10 h later, the treated cells were collected and determined by western blot using antibody targeted to SOCS-1 (d). *P<0.05 vs. WT group. ⁺P<0.05 vs. nonviable GAS group. ^#P>0.05 vs. nonviable GAS group. Each experiment was repeated three times and similar results were obtained. GAS, group A Streptococcus; MOI, multiplicity of infection; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; TLR, Toll-like receptor; WT, wild-type.

Figure 4

JAK1 and STAT1 interaction with MyD88 in RAW 264.7 macrophages with/without cycloheximide treatment following GAS infection. The interaction of MyD88 (red) with JAK1 (green) or STAT1 (green) was detected by immunofluorescence assay (magnification, ×100) (a) and IP (b) in RAW 264.7 cells. And cells were infected with GAS at an MOI 100∶1. Two hours post-infection or 4 h post-infection, Expressions of MyD88, p-JAK1(green) (c) or p-STAT1 (green) (d) were detected by immunofluorescence assay (magnification, ×100) and following 1–4 h GAS post- infection, MyD88, JAK1, STAT1, p-JAK1 and p-STAT1 levels were determined by IP (e). Representative cells from the same field for each group are shown. Blue represents DAPI staining. (f) The cycloheximide-treated RAW 264.7 cells were infected by GAS, and 4 h post-infection, the expression levels of MyD88, JAK1, STAT1 and p-STAT1 in these cells was detected by IP. *P<0.05 vs. control group, ⁺P<0.05 vs. GAS group. Each experiment was repeated for three times and similar results were obtained. GAS, group A Streptococcus; IP, immunoprecipitation; MOI, multiplicity of infection; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription.

Figure 5

GAS-induced SOCS-1 and p-STAT1 expressions in macrophages knocked out MyD88 or treated with JSH23. BMDMs from WT or MyD88^−/− mice were stimulated with GAS, 6 h or 10 h later, the mRNA or protein expressions of SOCS-1 were detected by RT-PCR (a) or western blot (b), and 6 h later, the expressions of p-STAT1, STAT1 or JAK1 were detected by western blot (c). BMDMs from WT mice with/without NF-κB inhibitor JSH-23 (20 µM) were infected with GAS. At post-infection times of 1 h, 2 h, 4 h and 6 h, the treated cells were lysed, and equal amounts were blotted with NF-kB (p-p65), STAT-1 and β-actin Ab (d), at 6 h of post infection, p-STAT-1, STAT-1 and β-actin were detected by western blot (e), and at 10 h of post-infection, SOCS-1 were detected by western blot (f). *P<0.05 vs. WT group; ⁺P<0.05 vs. control group, **P<0.05 vs. GAS group. Each experiment was repeated for three times and similar results were obtained. Ab, antibody; GAS, group A Streptococcus; SOCS, suppressor of cytokine signaling; STAT, signal transducer and activator of transcription; WT, wild-type.

Figure 6

Schematic model of SOCS-1 induction in a TLR4/MyD88-dependent and IFN-β-dependent signaling pathway. SOCS, suppressor of cytokine signaling; TLR, Toll-like receptor.