Role of interleukin-17A in cell-mediated protection against Staphylococcus aureus infection in mice immunized with the fibrinogen-binding domain of clumping factor A

Abstract

Clumping factor A (ClfA) is a fibrinogen-binding cell wall-attached protein and an important virulence factor of Staphylococcus aureus. Previous studies reported that an immunization with the fibrinogen-binding domain of ClfA (ClfA(40-559)) protected animals against S. aureus infection. It was reported that some cytokines are involved in the pathogenesis of staphylococcal diseases and in host defense against S. aureus infection. However, the role of cytokines in the protective effect of ClfA(40-559) as a vaccine has not been elucidated. In this study, we demonstrated that the spleen cells of ClfA(40-559)-immunized mice produced a large amount of interleukin-17A (IL-17A). The protective effect of immunization was exerted in wild-type mice but not in IL-17A-deficient mice. IL-17A mRNA expression was increased in the spleens and kidneys of immunized mice after infection. CXCL2 and CCL2 mRNA expression was increased in the spleens and kidneys, respectively. Consistent with upregulation of the mRNA expression, neutrophils infiltrated into the spleens extensively and macrophage infiltration was observed in the kidneys of immunized mice. These results suggest that immunization with ClfA(40-559) induces the IL-17A-producing cells and that IL-17-mediated cellular immunity is involved in the protective effect induced by immunization with ClfA(40-559) against S. aureus infection.

FIG. 1.

Protection against S. aureus infection by immunization with ClfA_40-559. BALB/c mice were immunized with ClfA_40-559 and alum adjuvant. Control mice were treated with antigen-free PBS and alum adjuvant or with antigen-free PBS only. Immunized and control BALB/c mice were infected with S. aureus. (A) Survival rates of both groups were observed for 14 days after infection. Each group of mice included 6 to 10 mice. An asterisk represents a statistically significant difference from the control group administered PBS plus alum (P < 0.05). (B) Bacterial numbers in the spleens and kidneys of immunized and control mice were determined at 1 and 3 days after infection. Data are presented as box plots, with the boxes representing the interquartile range. The median value is represented by the line across each box. An asterisk represents a statistically significant difference (P < 0.05).

FIG. 2.

Cytokine responses in the spleen cells of naïve mice and mice immunized with ClfA_40-559 after stimulation with ClfA_40-559. Spleen cells of naïve BALB/c mice were incubated in the absence or presence of 10 μg/ml ClfA_40-559. (A) IL-6 production in the spleen cell culture supernatants was determined at 48 h after stimulation. (B) The relative mRNA expression of TGF-β in the spleen cells was determined by real-time quantitative RT-PCR at 4 h after stimulation. BALB/c mice were immunized with ClfA_40-559 and alum adjuvant, and control mice were injected with antigen-free PBS and alum adjuvant. Spleen cells of both groups were taken at 7 days after the last booster immunization and incubated in the absence or presence of 10 μg/ml ClfA_40-559 for 48 h. (C to E) IFN-γ (C) and IL-17 (D) titers in the cell culture supernatants were determined. Simultaneously, the relative mRNA expression of IL-17A was determined by real-time quantitative RT-PCR after 4 h of incubation (E). Data are expressed as the medians ± interquartile range for a group of six to eight mice. ND, not detectable. An asterisk represents a statistically significant difference from the control group (P < 0.05).

FIG. 3.

Survival rates of naïve IL-17A-deficient (IL-17A KO) mice and wild-type (WT) C57BL/6 mice. Both groups of mice were infected with S. aureus intravenously. (A) Survival rates were observed for 7 days after infection. Each result represents data for a group of 10 mice. Defective protection against S. aureus infection was observed in IL-17A-deficient mice immunized with ClfA_40-559. IL-17A KO mice and WT mice were immunized with ClfA_40-559 plus alum adjuvant or injected with antigen-free PBS and alum adjuvant. Both groups of mice were infected with S. aureus. (B) Survival rates were observed for 14 days after infection. Each result represents data for a group of 12 mice. (C) Bacterial numbers in the spleens and kidneys of immunized and control mice were determined at 3 days after infection. Each group included six to eight mice. Data are presented as box plots, with the boxes representing the interquartile range. The median value is represented by the line across each box. An asterisk represents a statistically significant difference (P < 0.05).

FIG. 4.

Antibody production by active immunization with ClfA_40-559 and effect of passive immunization with anti-ClfA_40-559 antibody. Mice were immunized with ClfA_40-559 as described in Materials and Methods. Control mice were injected with antigen-free PBS and alum adjuvant. (A and B) Sera from immunized and control mice were collected at day 7 after the last booster immunization. Titers of anti-ClfA_40-559 antibody in the sera of BALB/c mice (A) and IL-17A deficient (KO) mice and wild-type (WT) C57BL/6 mice (B) were determined by ELISA. Data are expressed as median ± interquartile range for a group of six to eight mice. (C and D) Bacterial numbers in the spleens and kidneys of mice passively immunized with 10 mg (C) or 0.3 mg (D) of anti-ClfA_40-559 antibody were determined at 3 days after infection. Each group included six or seven mice. Data are presented as box plots, with the boxes representing the interquartile range. The median value is represented by the line across each box.

FIG. 5.

Inflammatory responses caused by S. aureus infection in the spleens of mice immunized with ClfA_40-559. BALB/c mice were immunized with ClfA_40-559 or injected with antigen-free PBS and alum adjuvant. Immunized and control BALB/c mice were infected with S. aureus. The spleens of mice were obtained at 1, 2, and 3 days after infection. (A to C) The mRNA expression of IL-17 (A), CXCL2 (B), and IL-6 (C) in the spleens was determined by real-time quantitative RT-PCR. Data are expressed as the medians ± interquartile range for a group of five to seven mice. (D) MPO activity in the spleens of immunized and control mice was determined at 2 and 3 days after infection. Data are expressed as the medians ± interquartile range for a group of six mice. An asterisk represents a statistically significant difference from the control group (P < 0.05). (E) Histology of the spleens of immunized and nonimmunized mice was observed at 2 and 3 days after infection. Spleen sections were stained with antineutrophil antibody. Magnification, ×40.

FIG. 6.

Inflammatory responses in the kidneys of mice immunized with ClfA_40-559. BALB/c mice were immunized with ClfA_40-559 or injected with antigen-free PBS and alum adjuvant. Immunized and control BALB/c mice were infected with S. aureus. The kidneys of mice were obtained at 1, 2, and 3 days after S. aureus infection. (A to C) IL-17A mRNA expression (A) in the kidneys was determined at 1 day after infection, and the mRNA expression of CXCL2 (B) and CCL2 (C) was determined at 2 days after infection by real-time quantitative RT-PCR. Data are expressed as the medians ± interquartile range for a group of six to seven mice. (D) MPO activities in the kidneys of immunized and control mice were determined at 2 and 3 days after infection. (E) Quantitative analysis of macrophages in the kidneys of the immunized and control mice was carried out. Macrophage density was determined as described in Materials and Methods. Each result represents the median ± interquartile range for 10 random fields of each section. An asterisk represents a statistically significant difference from the control (P < 0.05). (F and G) The histology of the kidney sections of immunized and control mice was observed 3 days after infection. The sections were stained with antineutrophil antibody Ly-6G (F) (magnification, ×40) or antimacrophage antibody F4/80 (G) (magnification, ×100). Arrows indicate F4/80-positive cells.