Toll-Like Receptor 4 Protects Against Clostridium perfringens Infection in Mice

Abstract

Toll-like receptor 4 (TLR4) has been reported to protect against Gram-negative bacteria by acting as a pathogen recognition receptor that senses mainly lipopolysaccharide (LPS) from Gram-negative bacteria. However, the role of TLR4 in Gram-positive bacterial infection is less well understood. Clostridium perfringens type A is a Gram-positive bacterium that causes gas gangrene characterized by severe myonecrosis. It was previously demonstrated that C. perfringens θ-toxin is a TLR4 agonist, but the role of TLR4 in C. perfringens infection is unclear. Here, TLR4-defective C3H/HeJ mice infected with C. perfringens showed a remarkable decrease in survival rate, an increase in viable bacterial counts, and accelerated destruction of myofibrils at the infection site compared with wild-type C3H/HeN mice. These results demonstrate that TLR4 plays an important role in the elimination of C. perfringens. Remarkable increases in levels of inflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6), and granulocyte colony-stimulating factor (G-CSF), were observed in C. perfringens-infected C3H/HeN mice, whereas the increases were limited in C3H/HeJ mice. Generally, increased G-CSF accelerates granulopoiesis in the bone marrow and the spleen to exacerbate neutrophil production, resulting in elimination of bacteria. The number of neutrophils in the spleen was increased in C. perfringens-infected C3H/HeN mice compared with non-infected mice, while the increase was lower in C. perfringens-infected C3H/HeJ mice. Furthermore, DNA microarray analysis revealed that the mutation in TLR4 partially affects host gene expression during C. perfringens infection. Together, our results illustrate that TLR4 is crucial for the innate ability to eliminate C. perfringens.

Keywords: Clostridium (C.) perfringens; G-CSF; Granulopoiesis; TLR4; innate immunity.

Figure 1

TLR4 plays an important role in effective host defense against C. perfringens. C3H/HeN and C3H/HeJ mice were intramuscularly injected with 5 × 10⁷ (A) or 1 × 10⁷ (B, C) colony-forming units (CFU) of C. perfringens Strain 13 (Cp-infected), or TGY medium as a control (Uninfected). (A) The survival of mice was monitored, and Kaplan–Meier survival curves are shown. (B) C. perfringens-infected femoral muscles were dissociated 72 h after infection, and C. perfringens CFU counts were determined. (C) Muscles were isolated 24 h after infection. Representative H&E-stained sections are shown, and 100 muscle fibers were measured. Log-rank test (A), two-tailed Student’s t-test (B), and one-way ANOVA (C) were employed to assess significance. Values are mean ± standard error.

Figure 2

TLR4 signaling accelerates expression of inflammatory cytokines during C. perfringens infection. C3H/HeN and C3H/HeJ mice were intramuscularly injected with 1 × 10⁷ CFU of C. perfringens Strain 13 (Cp-infected), or TGY medium as a control (Uninfected). At 24 h after the infection, IL-1β and IL-6 levels in the muscle were determined (A), or the muscle was subjected to immunohistochemical analysis with antibodies against CD31 and IL-1β (B). One-way ANOVA was employed to assess significance. Values are mean ± standard error.

Figure 3

TLR4 signaling accelerates production of neutrophils. C3H/HeN and C3H/HeJ mice were intramuscularly injected with 1 × 10⁷ CFU of C. perfringens Strain 13 (Cp-infected), or TGY medium as a control (Uninfected). (A) At 24 h after infection, G-CSF levels in the muscle were determined. (B) At 72 h after the infection, spleen cells were isolated from the mice, and flow cytometry analysis was performed using a Guava easyCyte. The proportions of CD11b⁺Ly-6G⁺ neutrophils, NK1.1⁻CD3⁺ T cells and NK1.1⁺CD3⁻ natural killer cells are shown. (C) Model of accelerated production of neutrophils in C. perfringens-infected host through TLR4 signaling activation. One-way ANOVA was employed to assess significance. Values are mean ± standard error.

Figure 4

Differentially expressed genes between C3H/HeN and C3H/HeJ mice during C. perfringens infection. C3H/HeN and C3H/HeJ mice were intramuscularly injected with 1 × 10⁷ CFU of C. perfringens Strain 13 (Cp-infected), or TGY medium as a control (Uninfected). At 1.5 h after infection, muscles were isolated from the mice, and total RNAs from the tissues were subjected to DNA microarray analysis as described in Methods. (A) Volcano plot depicting information about differentially expressed genes between C. perfringens-infected and uninfected muscles from C3H/HeN mice. (B) Volcano plot depicting information about differentially expressed genes in C. perfringens-infected muscles between C3H/HeN and C3H/HeJ mice. (C) Venn diagrams for microarray data.