Interferon gamma-dependent intestinal pathology contributes to the lethality in bacterial superantigen-induced toxic shock syndrome

Abstract

Toxic shock syndrome (TSS) caused by the superantigen exotoxins of Staphylococcus aureus and Streptococcus pyogenes is characterized by robust T cell activation, profound elevation in systemic levels of multiple cytokines, including interferon-γ (IFN-γ), followed by multiple organ dysfunction and often death. As IFN-γ possesses pro- as well as anti-inflammatory properties, we delineated its role in the pathogenesis of TSS. Antibody-mediated in vivo neutralization of IFN-γ or targeted disruption of IFN-γ gene conferred significant protection from lethal TSS in HLA-DR3 transgenic mice. Following systemic high dose SEB challenge, whereas the HLA-DR3.IFN-γ(+/+) mice became sick and succumbed to TSS, HLA-DR3.IFN-γ(-/-) mice appeared healthy and were significantly protected from SEB-induced lethality. SEB-induced systemic cytokine storm was significantly blunted in HLA-DR3.IFN-γ(-/-) transgenic mice. Serum concentrations of several cytokines (IL-4, IL-10, IL-12p40 and IL-17) and chemokines (KC, rantes, eotaxin and MCP-1) were significantly lower in HLA-DR3.IFN-γ(-/-) transgenic mice. However, SEB-induced T cell expansion in the spleens was unaffected and expansion of SEB-reactive TCR Vβ8(+) CD4(+) and CD8(+) T cells was even more pronounced in HLA-DR3.IFN-γ(-/-) transgenic mice when compared to HLA-DR3.IFN-γ(+/+) mice. A systematic histopathological examination of several vital organs revealed that both HLA-DR3.IFN-γ(+/+) and HLA-DR3.IFN-γ(-/-) transgenic mice displayed comparable severe inflammatory changes in lungs, and liver during TSS. Remarkably, whereas the small intestines from HLA-DR3.IFN-γ(+/+) transgenic mice displayed significant pathological changes during TSS, the architecture of small intestines in HLA-DR3.IFN-γ(-/-) transgenic mice was preserved. In concordance with these histopathological changes, the gut permeability to macromolecules was dramatically increased in HLA-DR3.IFN-γ(+/+) but not HLA-DR3.IFN-γ(-/-) mice during TSS. Overall, IFN-γ seemed to play a lethal role in the immunopathogenesis of TSS by inflicting fatal small bowel pathology. Our study thus identifies the important role for IFN-γ in TSS.

Figure 1. In vivo antibody mediated neutralization of IFN-γ protects from lethal SEB-induced TSS.

Age-matched HLA-DR3 transgenic mice were challenged with SEB (50 µg) immediately followed by indicated amounts of monoclonal rat anti-mouse IFN-γ or isotype control. Mice were closely monitored for symptoms of TSS.

Figure 2. Effect of antibody mediated neutralization of IFN-γ on SEB-induced cytokine and chemokine responses in vivo.

Age-matched HLA-DR3 transgenic mice were challenged with SEB (5 µg) immediately followed by 100 µg of goat anti-mouse IFN-γ or control goat antibodies. Mice were bled 3 hrs later and serum cytokine levels were determined using a multiplex suspension array system (Bio-Plex, Bio-Rad). Each bar represents the mean±SE of 4–6 mice.

Figure 3. Effect of antibody mediated neutralization of IFN-γ on SEB-induced splenic T cell expansion and thymocyte deletion.

Age-matched HLA-DR3 transgenic mice were challenged with SEB (5 µg) immediately followed by 100 µg of goat anti-mouse IFN-γ or control goat antibodies. Mice were killed 3 days later and distribution of different T cell subsets in the spleen (A and B) and thymus (C and D) was determined by flow cytometry. Each bar represents the mean±SE of 4–6 mice.

Figure 4. Effect of IFN-γ deficiency on SEB-induced TSS.

Age-matched HLA-DR3.IFN-γ^+/+ and HLA-DR3.IFN-γ^−/− transgenic mice were challenged with SEB (50 µg) and monitored closely. Data from 6–13 mice in each group.

Figure 5. Effect of IFN-γ deficiency on SEB-induced cytokine responses in vivo.

Age-matched HLA-DR3.IFN-γ^+/+ and HLA-DR3.IFN-γ^−/− transgenic mice were challenged with SEB (10 µg). Mice were bled at 3 hrs and serum cytokine levels were determined using a multiplex suspension array system (Bio-Plex, Bio-Rad).

Figure 6. Effect of IFN-γ deficiency on SEB-induced chemokine responses in vivo.

Age-matched HLA-DR3.IFN-γ^+/+ and HLA-DR3.IFN-γ^−/− transgenic mice were challenged with SEB (10 µg). Mice were bled at 3 hrs and serum chemokine levels were determined using a multiplex suspension array system (Bio-Plex, Bio-Rad).

Figure 7. Effect of IFN-γ deficiency on SEB-induced splenic T cell expansion and thymocyte deletion.

Age-matched HLA-DR3.IFN-γ^+/+ and HLA-DR3.IFN-γ^−/− transgenic mice were challenged with SEB (10 µg). Mice were killed 3 days later and distribution of different T cell subsets in the spleen (A and B) and thymus (C and D) was determined by flow cytometry. Each bar represents the mean±SE of 4–6 mice.

Figure 8. Effect of IFN-γ deficiency on SEB-induced pulmonary immunopathology.

Age-matched HLA-DR3.IFN-γ^+/+ (A and C) and HLA-DR3.IFN-γ^−/− (B and D) transgenic mice were left untreated (A and B) or challenged with SEB (50 µg, C and D). Mice were killed 48 hrs later, lungs were collected in buffered formalin and processed for H&E. The bar corresponds to 100 µM.

Figure 9. Effect of IFN-γ deficiency on SEB-induced hepatic immunopathology.

Age-matched HLA-DR3.IFN-γ^+/+ (A to D) and HLA-DR3.IFN-γ^−/− (E and H) transgenic mice were challenged with PBS (A, B and E, F) or challenged with SEB (50 µg) (C, D and G, H). Mice were killed 48 hrs later, livers were collected in buffered formalin and processed for H&E. The bar corresponds to 100 µM.

Figure 10. Effect of IFN-γ deficiency on SEB-induced intestinal immunopathology.

Age-matched HLA-DR3.IFN-γ^+/+ (D, E and F) and HLA-DR3.IFN-γ^−/− (G, H and I) transgenic mice were challenged with SEB (50 µg). Mice were killed 48 hrs later, intestinal segments were collected in buffered formalin and processed for H&E. Panels A, B and C – sections from naïve HLA-DR3.IFN-γ^+/+ mice shown for comparison. The bar corresponds to 100 µM.

Figure 11. Effect of IFN-γ deficiency on SEB-induced duodenal immunopathology.

Age-matched HLA-DR3.IFN-γ^+/+ (A, C and E) and HLA-DR3.IFN-γ^−/− (B, D and F) transgenic mice were challenged with SEB (50 µg). Mice were killed at 24, 48 and 72 hrs, duodenal segments were collected in buffered formalin and processed for H&E. The bar corresponds to 100 µM.

Figure 12. Effect of IFN-γ deficiency on SEB-induced alteration in gut permeability.

Age-matched HLA-DR3.IFN-γ^+/+ and HLA-DR3.IFN-γ^−/− transgenic mice were challenged with SEB (50 µg). Mice were gavaged with FITC-dextran as indicated in methods. Three hours after gavage, mice were killed, sera collected and the fluorescence in the sera were determined at 490 nm excitation and 525 nm emission.