Interferon-gamma plays a critical role in intestinal immunity against Salmonella typhimurium infection

Abstract

Salmonella bacteria are a major cause of food-borne infectious diarrhoea and there is great interest in understanding the pathogenesis of Salmonella infection and in vaccine development. Potential vaccines include the aromatic mutants of S. typhimurium. Such non-lethal Aro mutants have also been useful for studying Salmonella infections in mouse models. Studies of systemic infection, using these Aro mutants, in both normal and cytokine gene knockout mice, indicate that interferon-gamma (IFN-gamma) plays a key role in the resolution of Salmonella infection. The present studies have investigated the outcome of oral infection in mice with attenuated Salmonella because this infection route mimics natural infection in humans. In IFN-gamma gene knockout (IFN-gamma-/-) mice, intestinal immunity was impaired and oral challenge resulted in disseminated septicaemia 2 weeks later. No dissemination of infection was seen in wild-type mice. In wild-type mice, both CD4 and CD8 cell numbers increased in the gut following Salmonella challenge, together with increased expression of major histocompatibility complex (MHC) II and vascular cell adhesion molecule-1 (VCAM-1). No such changes were seen in IFNgamma-/- mice. Following oral challenge, antilipopolysaccharide (LPS) and antiphosphoryl choline antibodies increased by more than 100-fold in both serum and faecal pellet extracts of IFNgamma-/- mice compared with wild-type mice. Our data show that IFN-gamma production is essential for resolution of enteric Salmonella infection and that antibody has little effect on this process.

Figure 1

Bacterial counts from (a) faecal pellets (♦, ◊) and intestinal washes (•, ○), and from (b) liver (▴, ▵) and spleen (▪, □) from interferon-γ gene knockout (IFN-γ^−/−) mice after oral challenge with viable Salmonella typhimurium at high (5 × 10⁸) and low (2·5 × 10⁷) doses. The filled symbols represent mice receiving high inoculum, open symbols represent mice receiving low inoculum. The data represent the means of observations from 10 mice per group; vertical bars depict the standard error of the mean. (a) F/h, faecal bacteria recovered from high inoculum; I/h, intestinal bacteria recovered from high inoculum; F/l, faecal bacteria recovered from low inoculum; I/l, intestinal bacteria recovered from low inoculum. (b) L/h, liver bacteria recovered from high inoculum; S/h, spleen bacteria recovered from high inoculum; L/l, liver bacteria recovered from low inoculum; S/l, spleen bacteria recovered from low inoculum.

Figure 2

Histopathological findings in interferon-γ gene knockout (IFN-γ^−/−) and wild-type mice inoculated with Salmonella typhimurium. At week 2 postinoculation, liver lesions in wild-type mice (a, × 200; b, × 400) were smaller and less prominent than those in IFN-γ^−/− mice (c, × 200; d, × 400). By 4 weeks, liver lesions in the wild-type mice had largely resolved (e, × 200; f, × 400) but continued to progress in IFN-γ^−/− mice (g, × 200) and were associated with thromboses in nearby blood vessels (arrow) (h, × 400). In the spleen, there were increased numbers of lymphocytes in the red pulp of wild-type mice at week 1 (i, × 200; j, × 400, inset × 800) but no significant changes thereafter. The spleens of IFN-γ^−/− mice were normal until week 3 when pronounced extramedullary haemopoiesis was present (o, × 200; p, × 400, inset × 800). At 4 weeks, the normal spleen of wild-type mice (m, × 200; n, × 400, inset × 800) contrasted markedly with that of IFN-γ^−/− mice in which there were numerous granulomas and intense haemopoiesis (o, × 200; p, × 400, inset × 800). The mesenteric lymph nodes of wild-type mice exhibited a sinusoidal accumulation of macrophages at week 2 (r, × 200, inset × 800) while severe pyogranulomatous lymphadenitis was present in IFN-γ^−/− mice from this time-point onwards (s, × 200, inset × 800). A mild, mixed leucocyte infiltrate was present in Peyer’s patches of wild-type mice at week 2 (t, × 200, inset × 800) in contrast to the severe pyogranulomatous inflammation in IFN-γ^−/− mice (u, × 200, inset × 800). All sections were stained with haematoxylin and eosin.

Figure 3

Total anti-lipopolysaccharide (LPS) antibody and immunoglobulin (Ig)A-, IgG- and IgM-specific anti-LPS antibody in faecal pellets and serum collected at weekly intervals following oral challenge with viable Salmonella typhimurium at high (5 × 10⁸) and low (2·5 × 10⁷) doses in wild-type (IFN-γ^+/+) and interferon-γ gene knockout (IFN-γ^−/−) mice. Data represent the means of observations from 10 mice per group expressed as enzyme-linked immunosorbent assay (ELISA) units, and vertical bars depict the standard error of the mean. HD, high dose; LD, low dose.

Figure 4

Total anti-phosphoryl choline (PC) antibody and immunoglobulin (Ig)A-, IgG- and IgM-specific anti-PC antibody in faecal pellets and serum collected at weekly intervals following oral challenge with viable Salmonella typhimurium at high (5 × 10⁸) and low (2·5 × 10⁷) doses in wild-type (IFN-γ^+/+) and interferon-γ gene knockout (IFN-γ^−/−) mice. Data represent the means of observations from 10 mice per group expressed as enzyme-linked immunosorbent assay (ELISA) units, and vertical bars depict the standard error of the mean. HD, high dose; LD, low dose.

Figure 5

Major histocompatibility complex (MHC) II (a) and vascular cell adhesion molecule-1 (VCAM-1) (b) expression in the gut from wild-type (IFN-γ^+/+) and interferon-γ gene knockout (IFN-γ^−/−) mice from week 0 to week 4 postchallenge. The y-axis is image analysis units. CD4⁺ (c) and CD8⁺ (d) cells in the gut from IFN-γ^+/+ and IFN-γ^−/− mice from week 0 to week 4 postchallenge. The y-axis is expressed as cells per high power field (× 40) CD8⁺ cells in the gut from IFN-γ^+/+ and IFN-γ^−/− mice from week 0 to week 4 postchallenge. Data represent the means of observations from 10 mice per group, and vertical bars depict the standard error of the mean