M Protein of Group a Streptococcus Plays an Essential Role in Inducing High Expression of A20 in Macrophages Resulting in the Downregulation of Inflammatory Response in Lung Tissue

Abstract

Group A streptococcus (GAS), a common pathogen, is able to escape host immune attack and thus survive for longer periods of time. One of the mechanisms used by GAS is the upregulated expression of immunosuppressive molecules, which leads to a reduction in the production of inflammatory cytokines in immune cells. In the present study, we found that macrophages produced lower levels of proinflammatory cytokines (IL-1β, TNF-α, IL-6) when challenged with GAS than they did when challenged with Escherichia coli (E. coli). Simultaneously, in a mouse model of lung infection, GAS appeared to induce a weaker inflammatory response compared to E. coli. Our data also indicated that the expression of the A20 transcriptional regulator was higher in GAS-infected macrophages than that in macrophages infected with E. coli, and that high expression of A20 correlated with a reduction in the production of TRAF6. SiRNA targeting of A20 led to the increased production of TRAF6, IL-1β, TNF-α, and IL-6, suggesting that A20 inhibits synthesis of these key proinflammatory cytokines. We also investigated the pathway underlying A20 production and found that the synthesis of A20 depends on My88, and to a lower extent on TNFR1. Finally, we showed a significant reduction in the expression of A20 in macrophages stimulated by M protein-mutant GAS, however, a speB-GAS mutant, which is unable to degrade M protein, induced a greater level of A20 production than wild type GAS. Collectively, our data suggested that M protein of GAS was responsible for inducing A20 expression in macrophages, which in turn down-regulates the inflammatory cytokine response in order to facilitate GAS in evading immune surveillance and thus prolong survival in the host.

Keywords: A20; Group A Streptococcus (GAS); M protein; macrophages; negative regulation.

Figure 1

Evaluation of proinflammatory cytokines in RAW cells stimulated with GAS or E. coli. RAW264.7 cells were seeded at a concentration of 1 × 10⁶/well in a 6-well plate containing medium without antibiotics overnight and then infected with GAS, or E. coli as a control, at an MOI of 10. We then collected supernatant and cells from each group at different time points. Total RNA was isolated from cells using RNeasy, and qPCR analysis was performed to detect the mRNA level of IL-1β (A), TNF-α (B), and IL-6 (C). Simultaneously, ELISA was performed to detect the protein expression of IL-1β (A′), TNF-α (B′), and IL-6 (C′) in the cell supernatant. Each experiment was performed at least three times. Results are presented as mean ± SD. P < 0.05 vs. E. coli group.

Figure 2

Histological evaluation of inflammatory response in mouse lung tissue following GAS or E. coli infection. The three experimental groups of mice were separately prepared for histopathological analysis. (A) Representative views of lung sections treated with PBS, GAS, or E. coli. In the left panels, H&E staining shows the overall outline of lung slices, with a magnified image. Inflammatory cells are identified by red circles in the middle panels (H&E staining, × 10 and ×40). Periodic acid–Schiff (PAS) staining showed bronchiolar mucus production in each group (right panels, objective ×20). (B) Quantitative analysis of recruited inflammatory cells per square mm. (C) Inflammatory cells were counted in lung lavage fluid (per ml). Each experiment was repeated three times, and similar results were obtained on each occasion.

Figure 3

Expression of A20 and its target protein, TRAF6, in GAS-induced macrophages. Macrophages from wild-type mice were infected with GAS or E. coli at an MOI of 10. At different post-infection times, the protein levels of A20 and TRAF6 were detected by Western blot in RAW 264.7 cells (A) or BMDMs (B). Each experiment was repeated three times, and similar results were obtained on each occasion.

Figure 4

Expression of A20 and the phosphorylation of p65 in RAW 264.7 macrophages with and without A20 SiRNA following GAS infection. RAW 264.7 cells were incubated with A20 SiRNA for 30 min, and then infected with GAS at an MOI of 10: 1. Six hours post-infection, an immunofluorescence assay (magnification, 3100) was performed to detect the expression of A20 (Green) (A) and p-P65 (Green) (B), which was verified by Western blotting (C). qPCR (for mRNA expression) or ELISA (for protein expression) were also used to detect the levels of proinflammatory cytokines in RAW cells or in the supernatant (Figure 5). The blue color represents DAPI staining. Representative cells from the same field for each group are shown. Each experiment was repeated three times, and similar results were obtained on each occasion.

Figure 5

Expression of proinflammatory cytokines in RAW 264.7 macrophages with and without A20 SiRNA following GAS infection. RAW 264.7 cells were incubated with A20 SiRNA for 30 min, and then infected with GAS at an MOI of 10. And 6 h post-infection, ELISA and RT-PCR were performed to analyze the expression of proinflammatory cytokines. Each experiment was repeated three times, and similar results were obtained on each occasion. Results are presented as mean ± SD.

Figure 6

Western blot analysis of A20 expression in wild type or MyD88-/- BMDMs stimulated with GAS, and the detection of A20 expression following blockade with anti-TNFR1. BMDMs from MyD88^−/− knockout mice were stimulated with GAS at an MOI of 10. Six hours later, the treated cells were collected and determined by Western blotting using antibodies targeted to A20, p-P-65, and β-actin (A). ^*P < 0.05 and ^# P < 0.001 vs. MyD88^−/− group. RAW cells were blocked with anti-TNFR1 monoclonal antibody for 30 min and then stimulated with GAS at an MOI of 10 for 6 h, following collection of the treated cells, A20 expression was detected by Western blotting (B). Each experiment was repeated three times, and similar results were obtained on each occasion.

Figure 7

WT RAW cells induced by gene mutant strain M^−/− or SpeB^−/− GAS, or WT GAS, or purified M protein. RAW cells were stimulated by WT or M^−/− (A) or SpeB^−/−GAS (B) at an MOI of 10. Six hours later, the treated cells were collected and A20 expression was determined by Western blotting. ^*P < 0.05 vs. the M^−/− GAS group. RAW cells were stimulated by recombinant M-GST protein, GST-tag alone, and PBS as a control (C), or recombinant purified M protein and PBS as a control (D). Six hours later, the treated cells were collected and A20 expression was determined by Western blotting. Each experiment was repeated three times, and similar results were obtained on each occasion.

Figure 8

Schematic diagram of the signaling pathways that are postulated to upregulate the expression of A20 and then inhibit inflammatory mediators in macrophages induced by M protein following GAS infection. Upon induction of macrophages by M protein of GAS, NF-κB is activated through the MyD88 pathway, which induces expression of genes associated with the proinflammatory cytokines IL-1β, IL-6, and TNF-α. A20 expression depends on activation of NF-κB and partly on the interaction of TNF-αand TNFR. In turn, A20 targets TRAF-6 to inhibit inflammatory signal pathways, which leads to a reduction in the production of proinflammatory cytokines.