Immune recognition of Streptococcus pyogenes by dendritic cells

Abstract

Streptococcus pyogenes is one of the most frequent human pathogens. Recent studies have identified dendritic cells (DCs) as important contributors to host defense against S. pyogenes. The objective of this study was to identify the receptors involved in immune recognition of S. pyogenes by DCs. To determine whether Toll-like receptors (TLRs) were involved in DC sensing of S. pyogenes, we evaluated the response of bone marrow-derived DCs obtained from mice deficient in MyD88, an adapter molecule used by almost all TLRs, following S. pyogenes stimulation. Despite the fact that MyD88(-/-) DCs did not differ from wild-type DCs in the ability to internalize and kill S. pyogenes, the up-regulation of maturation markers, such as CD40, CD80, and CD86, and the production of inflammatory cytokines, such as interleukin-12 (IL-12), IL-6, and tumor necrosis factor alpha, were dramatically impaired in S. pyogenes-stimulated MyD88(-/-) DCs. These results suggest that signaling through TLRs is the principal pathway by which DCs sense S. pyogenes and become activated. Surprisingly, DCs deficient in signaling through each of the TLRs reported as potential receptors for gram-positive cell components, such as TLR1, TLR2, TLR4, TLR9, and TLR2/6, were not impaired in the secretion of proinflammatory cytokines and the up-regulation of costimulatory molecules after S. pyogenes stimulation. In conclusion, our results exclude a major involvement of a single TLR or the heterodimer TLR2/6 in S. pyogenes sensing by DCs and argue for a multimodal recognition in which a combination of several different TLR-mediated signals is essential for a rapid and effective response to the pathogen.

FIG. 1.

S. pyogenes-induced maturation of DCs is MyD88 dependent. Bone marrow-derived DCs from C57BL/6 (left histograms) and MyD88^−/− (right histograms) mice were infected for 2 h with S. pyogenes and further incubated for 48 h. Uninfected DCs were used as a control. DCs were collected, stained with PE-labeled monoclonal anti-mouse CD11c antibody and FITC-labeled monoclonal anti-mouse CD40, CD80, or CD86 antibody, and subjected to flow cytometry analysis. CD11c⁺ cell populations were gated and analyzed for CD40, CD80, and CD86 expression. Thin lines represent uninfected DCs; thick lines represent DCs infected with S. pyogenes. Results are representative of four independent experiments.

FIG. 2.

DCs from MyD88^−/− mice show impaired S. pyogenes-induced IL-12 responses in vitro. Purified bone marrow-derived DCs from C57BL/6 or MyD88^−/− mice were infected in vitro with S. pyogenes for 2 h and further incubated in the presence of antibiotics. (A) IL-12p40 was measured in the supernatant of S. pyogenes-infected (hatched bars) or uninfected (white bars) DCs by ELISA 48 h after infection. DCs stimulated with 2 μg/ml of LPS were used as a control (black bars). Bars show means plus standard deviations (SD) for triplicates. Similar experiments were performed at least three times. (B) RT-PCR amplification of IL-12p40 cDNA from unstimulated, LPS-stimulated, and S. pyogenes-infected DCs isolated from wild-type C57BL/6 (left) or MyD88^−/− (right) mice. β-actin expression served as a loading control. Plasmids containing the sequences encoding IL-12p40 or β-actin were used as positive controls (CTR), and reactions in the absence of cDNA were used as negative controls. (C) Changes (x-fold) in the ratio of IL-12p40 mRNA to β-actin mRNA in S. pyogenes-infected or LPS-stimulated DCs compared to those for unstimulated control DCs isolated from either wild-type C57BL/6 mice (black bars) or MyD88^−/− mice (white bars).

FIG. 3.

Production of TNF-α (A) and IL-6 (B) in response to S. pyogenes in wild-type and MyD88^−/− DCs. DCs from wild-type C57BL/6 or MyD88^−/− mice were infected with S. pyogenes or stimulated with 2 μg/ml LPS and further cultured for 48 h. Concentrations of TNF-α and IL-6 in the culture supernatants were measured by ELISA. The results are shown as means plus SD for three experiments. *, P < 0.05 (by F test).

FIG. 4.

In vitro phagocytosis and killing of S. pyogenes by DCs isolated from wild-type C57BL/6 or MyD88^−/− mice. (A and B) Double-immunofluorescence staining of S. pyogenes-infected DCs isolated from wild-type C57BL/6 (A) or MyD88^−/− (B) mice showing the intracellular or extracellular location of S. pyogenes within the DCs. Extracellular bacteria are shown in yellow-green, intracellular bacteria are shown in red, and DNA in the nucleus is stained in blue. (C) Kinetics of S. pyogenes killing by DCs isolated from wild-type C57BL/6 (squares) or MyD88^−/− (triangles) mice. DCs from the indicated mice were infected with S. pyogenes at an MOI of 1:10 for 2 h and further incubated in the presence of gentamicin (100 μg/ml) at 37°C in 5% CO₂ to kill extracellular bacteria. The numbers of viable intracellular S. pyogenes cells were determined after different periods by disrupting infected DCs with distilled H₂O and plating them onto blood agar. Each point represents the mean value ± SD for four independent experiments.

FIG. 5.

S. pyogenes-induced maturation and production of cytokines in DCs deficient in expression of TLR2. (A) Flow cytometry analysis for detection of CD40 expression on DCs derived from bone marrows of wild-type C57BL/6 (left histograms) or TLR2^−/− (right histograms) mice (uninfected [thin lines] or after infection with S. pyogenes [thick lines]). DCs were infected with S. pyogenes for 2 h and further cultured in the presence of antibiotics for 48 h. The DCs were then stained with PE-conjugated anti-CD11c and FITC-conjugated anti-CD40 antibodies and analyzed by fluorescence-activated cell sorting. (B and C) Induction of IL-12p40 (B) and IL-6 (C) in DCs derived from bone marrows of C57BL/6 or TLR2^−/− mice by wild-type S. pyogenes (black bars) or the isogenic S. pyogenes mutant strain deficient in the expression of M1 protein (hatched bars). Cytokines in the supernatants of uninfected and infected DCs were determined after 48 h of culture. Bars represent the means plus SD for three experiments. *, P < 0.05 (by F test).

FIG. 6.

Secretion of IL-12p40 by DCs derived from bone marrows of wild-type C57BL/6 or TLR2/6^−/− mice after infection with S. pyogenes (hatched bars) or stimulation with LPS (black bars). DCs were infected with S. pyogenes for 2 h and further cultured in the presence of antibiotics for 48 h. IL-12p40 levels in the supernatants of untreated and treated DCs were determined at 48 h of culture. Bars represent means plus SD for three experiments. *, P < 0.05 (by F test).

FIG. 7.

S. pyogenes DNA stimulation of IL-12p40 secretion by DCs is dependent on TLR9, but stimulation of DC IL-12p40 secretion by the whole S. pyogenes microorganism is TLR9 independent. (A) DCs isolated from bone marrows of C57BL/6 mice were pretreated or not with the TLR9 inhibitor ODN 2088 for 2 h, followed by stimulation with 50 μg/ml of purified S. pyogenes DNA for 48 h and analysis of IL-12p40 by ELISA. Data are means plus SD for triplicates. *, P < 0.05 (F test). (B) DCs were pretreated or not with the TLR9 inhibitor ODN 2088 for 2 h prior to stimulation with the TLR9 agonist CpG ODN 1826 or infection with S. pyogenes and were further cultured for 48 h. IL-12p40 levels in the supernatants were determined by ELISA. Data are means plus SD for triplicates. *, P < 0.05 (F test).