Induction of TGF-beta1 and TGF-beta1-dependent predominant Th17 differentiation by group A streptococcal infection

Abstract

Recurrent group A Streptococcus (GAS) tonsillitis and associated autoimmune diseases indicate that the immune response to this organism can be ineffective and pathological. TGF-beta1 is recognized as an essential signal for generation of regulatory T cells (Tregs) and T helper (Th) 17 cells. Here, the impact of TGF-beta1 induction on the T-cell response in mouse nasal-associated lymphoid tissue (NALT) following intranasal (i.n.) infections is investigated. ELISA and TGF-beta1-luciferase reporter assays indicated that persistent infection of mouse NALT with GAS sets the stage for TGF-beta1 and IL-6 production, signals required for promotion of a Th17 immune response. As predicted, IL-17, the Th17 signature cytokine, was induced in a TGF-beta1 signaling-dependent manner in single-cell suspensions of both human tonsils and NALT. Intracellular cytokine staining and flow cytometry demonstrated that CD4(+) IL-17(+) T cells are the dominant T cells induced in NALT by i.n. infections. Moreover, naive mice acquired the potential to clear GAS by adoptive transfer of CD4(+) T cells from immunized IL-17A(+)/(+) mice but not cells from IL-17A(-)/(-) mice. These experiments link specific induction of TGF-beta1 by a bacterial infection to an in vivo Th17 immune response and show that this cellular response is sufficient for protection against GAS. The association of a Th17 response with GAS infection reveals a potential mechanism for destructive autoimmune responses in humans.

Fig. 1.

Induction of TGF-β1 and TGF-β1 signaling in human tonsil cells by GAS. Single-cell suspensions from a fresh human tonsil specimen were seeded into tissue culture plates. (A) Culture supernatants were collected 48 hr after treatment with HK-90-226 at the indicated multiplicity of infection (MOI) and assayed for active TGF-β1 by a TGF-β1-luciferase reporter. Data are mean ± SEM of three independent determinations. Representative results from one of three independent experiments with different tonsils are shown. (B) Western blots of extracts from tonsil cells treated with HK-90-226 at the indicated MOI for 24 hr. Phosphorylated Smad2 (P-Smad 2) was detected on Western blots using anti-P-Smad2. Antiactin (control) was used to assess total protein loaded.

Fig. 2.

Multiple GAS infections amplify TGF-β1 expression in mouse secondary lymphoid tissues. Mice were infected i.n. four times with viable 90-226 (A) At 48 hr after the last infection, single-cell suspensions of NALT (0.5 × 10⁶ cells per well), CLN (2.5 × 10⁶ cells per well), and spleen (SP; 2.5 × 10⁶ cells per well) were prepared separately from each mouse (n = 5) and cultured for 48 hr without ex vivo exposure to GAS. Culture supernatants were assayed with the TGF-β1 reporter. The gray column in SP shows data from mice that received one i.p. infection alone with the last i.n. infection. (B) Blood specimens were taken 48 hr after the last of four infections. Serum samples (n = 5) were assayed for TGF-β1 as in A. Data are mean ± SEM of five mice per group. Representative data from one of three independent experiments are shown.

Fig. 3.

GAS infection induces IL-6. Mice were inoculated i.n. (1.5 × 10⁸ CFUs) and i.p. (1.5 × 10⁷ CFUs) once with viable 90-226 or PBS. At 48 hr after infection, single-cell suspensions of NALT and CLN cells pooled from three mice and splenocytes from individual mice were separately incubated with HK-90-226. (A–C) Cell culture supernatants (n = 3) were collected after 72 hr and assayed for IL-6 by ELISA. Data are mean ± SEM of three independent treatments of pooled cells.

Fig. 4.

IL-17 induction by GAS is dependent on TGF-β1 signaling. Mice were inoculated i.p. with viable 90-226 or PBS. At 48 hr after infection, splenocyte suspensions were prepared and incubated with HK-90-226 [multiplicity of infection (MOI) = 10] or buffer for 72 hr (A); splenocytes were first treated with TGF-β1 type I receptor inhibitor (SB 431542) or inhibitor solvent (DMSO) for 1 h before incubation with HK-90-226 at an MOI of 10 for 72 hr (B). Supernatants of splenocytes from mice (n = 3) were collected and assayed for IL-17 by ELISA. Data are mean ± SEM of three independent treatments from one of three experiments.

Fig. 5.

GAS induces Th17 cell differentiation in mouse NALT in vivo. NALT, CLN, and spleen were taken 3 and 7 days following four i.n. infections. Cell suspensions were stained with allophycocyanin (APC)-CD4, followed by intracellular staining with phycoerythrin (PE)-IL-17, and were analyzed by FACS (Materials and Methods). (A) Analysis of stained NALT cells gated on CD4 T cells on day 3 and day 7. The percentages of CD4⁺IL-17⁺ cells were expressed as a fraction of total CD4⁺ cells (Upper Right). (B) Percentages of CD4⁺IL-17⁺, CD4⁺IFN-γ⁺, and CD4⁺CD25⁺FoxP3⁺ cells among total CD4⁺ cells are plotted (7 days following infection). Data are mean ± SEM of three mice per group.

Fig. 6.

Th17 cell differentiation in mouse NALT paralleled TGF-β1 induction. Mice were infected i.n. one to four times. (A) Three days after the last infection, NALT cells from each group were stained and analyzed by FACS as described in Fig. 5. (B) Blood samples were collected, and serum TGF-β1 was measured by a TGF-β1-luciferase reporter. Data are mean ± SEM of three to four mice per group.

Fig. 7.

CD4⁺IL-17A⁺ T cells promote clearance of GAS from NALT. Immunized mice were euthanized, and single-cell suspensions of lymph nodes were pooled separately from each group. (A) Immunized IL-17A^−/− (I-IL17A^−/−) and WT B6 (I-B6) mice, and nonimmunized (NI-B6) mice (PBS sham controls) are shown. (B and C) CD4⁺ T cells were separately pooled from each group of immunized donor mice and then transferred to three groups of naive WT B6 mice (S7). At 24 hr after transfer, mice were challenged i.n., and they were euthanized 24 hr later. (B) Representative FACS data showing intracellular cytokine staining for IL-17A and IFNγ on enriched cells. (C) Plots shown were gated on CD4⁺ cells. CFU equals the number of viable bacteria per NALT. In A and C, the horizontal bars are geometrical means.