Aberrant inflammatory response to Streptococcus pyogenes in mice lacking myeloid differentiation factor 88

Abstract

Several in vitro studies have emphasized the importance of toll-like receptor/myeloid differentiation factor 88 (MyD88) signaling in the inflammatory response to Streptococcus pyogenes. Since the extent of inflammation has been implicated in the severity of streptococcal diseases, we have examined here the role of toll-like receptor/MyD88 signaling in the pathophysiology of experimental S. pyogenes infection. To this end, we compared the response of MyD88-knockout (MyD88(-/-)) after subcutaneous inoculation with S. pyogenes with that of C57BL/6 mice. Our results show that MyD88(-/-) mice harbored significantly more bacteria in the organs and succumbed to infection much earlier than C57BL/6 animals. Absence of MyD88 resulted in diminished production of inflammatory cytokines such as interleukin-12, interferon-gamma, and tumor necrosis factor-alpha as well as chemoattractants such as monocyte chemotactic protein-1 (MCP-1) and Keratinocyte-derived chemokine (KC), and hampered recruitment of effector cells involved in bacterial clearance (macrophages and neutrophils) to the infection site. Furthermore, MyD88(-/-) but not C57BL/6 mice exhibited a massive infiltration of eosinophils in infected organs, which can be explained by an impaired production of the regulatory chemokines, gamma interferon-induced monokine (MIG/CXCL9) and interferon-induced protein 10 (IP-10/CXCL10), which can inhibit transmigration of eosinophils. Our results indicate that MyD88 signaling targets effector cells to the site of streptococcal infection and prevents extravasation of cells that can induce tissue damage. Therefore, MyD88 signaling may be important for shaping the quality of the inflammatory response elicited during infection to ensure optimal effector functions.

Figure 1

Enhanced susceptibility of MyD88^−/− mice to infection with S. pyogenes. A: Survival curves of C57BL/6 (black symbols) and MyD88^−/− (white symbols) mice after subcutaneous infection with 5 × 10⁷ CFU of S. pyogenes. Groups of 10 MyD88^−/− or C57BL/6 mice were infected with 5 × 10⁷ CFU of S. pyogenes and survival was monitored over time. Comparison of survival curves was performed by using the Long-rank test. ***P < 0.001. Kinetics of bacterial growth in blood (B), liver (C), and lungs (D) of C57BL/6 (black symbols) and MyD88^−/− (white symbols) after subcutaneous infection with 5 × 10⁷ CFU of S. pyogenes are shown. Data are presented as mean ± SD of a minimum of 10 mice per time point and are the compilation of three independent experiments.

Figure 2

Serum levels of biochemical markers of organ damage in S. pyogenes-infected MyD88^−/− and C57BL/6 mice. The levels of alanine aminotransferase (ALT, A), aspartate aminotransferase (AST, B), creatin phosphokinase (CPK, C), and lactate dehydrogenase (LDH, D) were determined in the plasma of C57BL/6 (black bars) and MyD88^−/− (white bars) mice at 20 hours of infection. The plasma levels of these parameters in uninfected mice are included for comparison. Each bar represents the mean ± SD value of five mice. *P < 0.05; **P < 0.01. Similar results were obtained from three different experiments.

Figure 3

Impaired production of inflammatory cytokines in S. pyogenes-infected MyD88^−/− mice. C57BL/6 (black symbols) and MyD88^−/− (white symbols) mice were infected subcutaneously with 5 × 10⁷ CFU of S. pyogenes, and serum samples were obtained at increasing times of infection for the evaluation of the systemic production of IL-6 (A), TNF-α (B), IL-12 (C), and IL-1β (D). Each plot shows the mean ± SD value for 9 to 15 mice of the indicated genotype obtained from three separate experiments.

Figure 4

Phagocytosis and killing of S. pyogenes by macrophages and neutrophils isolated from C57BL/6 or MyD88^−/− mice. A: Kinetics of S. pyogenes killing by macrophages isolated from C57BL/6 (black symbols) or MyD88^−/− (white symbols) mice. Each point represents the mean ± SD value of three independent experiments. B: Kinetic of S. pyogenes killing by neutrophils isolated from bone marrow of C57BL/6 (black symbols) or MyD88^−/− (white symbols) mice. Each point represents the mean ± SD value of three independent experiments.

Figure 5

MyD88^−/− mice display impaired production of chemoattractants and recruitment of inflammatory macrophages and neutrophils into the site of S. pyogenes infection. A: Air pouches were generated in C57BL/6 (black bars) and MyD88^−/− (white bars) mice as described in Materials and Methods. The total number of recruited cells as well as the numbers of macrophages (Gr-1⁻/Mac-1⁺), neutrophils (Gr-1⁺/Mac-1⁺), dendritic cells (CD11c⁺), B cells (CD19⁺), and T cells (CD4⁺ and CD8⁺) recruited into the air pouches in response to S. pyogenes infection are shown. **P < 0.01; ***P < 0.001. Kinetics of KC (B) and MCP-1 (C) appearance in the serum of C57BL/6 (black symbols) and MyD88^−/− (white symbols) mice during the course of S. pyogenes infection. Each plot shows the mean ± SD value for 9 to 15 mice of the indicated genotype. Data are the compilation of three separate experiments.

Figure 6

Exacerbated development of pathology in S. pyogenes-infected MyD88^−/− mice. Formalin-fixed, paraffin-embedded tissue sections were prepared from C57BL/6 and MyD88^−/− animals at 20 hours after subcutaneous inoculation with S. pyogenes and stained with H&E. The representative sections shown are from uninfected MyD88^−/− lung tissue (A), uninfected C57BL/6 lung tissue (B), S. pyogenes-infected MyD88^−/− lung tissue (C and F), S. pyogenes-infected C57BL/6 lung tissue (D), and S. pyogenes-infected MyD88^−/− liver tissue (E). The sections were photographed with a ×10 objective for C and D, ×20 objective for A and B, ×40 objective for E, and ×60 for F. Arrows in E indicate areas of tissue destruction. G: Mean eosinophil numbers in lung tissue of infected wild-type or MyD88^−/− mice at 20 hours after subcutaneous inoculation with S. pyogenes. Quantification was performed as described in Materials and Methods. H: EPO activity in lung homogenates of C57BL/6 and MyD88^−/− mice at 20 hours after subcutaneous inoculation with S. pyogenes. Homogenates of uninfected lungs were used as controls. Each bar represents the mean ± SD of six mice. Data are the compilation of two separate experiments. **P < 0.01; ***P < 0.001; HPF = high-power field.

Figure 7

Decreased production of regulatory chemokines in MyD88^−/− mice during infection with S. pyogenes. C57BL/6 (black symbols) and MyD88^−/− (white symbols) mice were infected subcutaneously with 5 × 10⁷ CFU of S. pyogenes, and serum samples were obtained at increasing times of infection for the evaluation of the systemic production of MIG (A), IP-10 (B), and IFN-γ (C). Each plot shows the mean ± SD value for 9 to 15 mice and they are the compilation of three separate experiments.