Effect of age on susceptibility to Salmonella Typhimurium infection in C57BL/6 mice

Abstract

Ageing is associated with a decline in immune function, which predisposes the elderly to a higher incidence of infections. Information on the mechanism of the age-related increase in susceptibility to Salmonella enterica serovar Typhimurium (S. Typhimurium) is limited. In particular, little is known regarding the involvement of the immune response in this age-related change. We employed streptomycin (Sm)-pretreated C57BL/6 mice to develop a mouse model that would demonstrate age-related differences in susceptibility and immune response to S. Typhimurium. In this model, old mice inoculated orally with doses of 3 x 10(8) or 1 x 10(6) c.f.u. S. Typhimurium had significantly greater S. Typhimurium colonization in the ileum, colon, Peyer's patches, spleen and liver than young mice. Old mice had significantly higher weight loss than young mice on days 1 and 2 post-infection. In response to S. Typhimurium infection, old mice failed to increase ex vivo production of IFN-gamma and TNF-alpha in the spleen and mesenteric lymph node cells to the same degree as observed in young mice; this was associated with their inability to maintain the presence of neutrophils and macrophages at a 'youthful' level. These results indicate that Sm-pretreated C57BL/6 old mice are more susceptible to S. Typhimurium infection than young mice, which might be due to impaired IFN-gamma and TNF-alpha production as well as a corresponding change in the number of neutrophils and macrophages in response to S. Typhimurium infection compared to young mice.

Fig. 1.

Kinetic analysis of S. Typhimurium colonization in different tissues of streptomycin-pretreated young and old mice. Data represent c.f.u. (g tissue)⁻¹ in ileum (a), PP (b), colon (c), and liver cells (d) of mice infected with low (1×10⁶ c.f.u.) or high doses (3.4×10⁸ c.f.u.) of S. Typhimurium for 1, 2, or 4 days. Grey circle, young mice; black triangle, old mice; level line, mean of each group; # indicates that the number of c.f.u. recovered from young mice was significantly different from c.f.u. recovered from old mice at P<0.05; * indicates a similar trend at P<0.1 by the non-parametric Kruskal–Wallis test (n=11 per group) from three independent experiments.

Fig. 2.

Effect of S. Typhimurium infection on weight loss in young and old mice. Percentage weight loss in streptomycin-pretreated young and old mice infected with 3.4×10⁸ or 1×10⁶ c.f.u. S. Typhimurium at days 1, 2, and 4 p.i. (n=11 per group; data from three independent experiments). Blank circle, old mice infected with the low dose; blank triangle, young mice infected with the low dose; solid circle, old mice infected with the high dose; solid triangle, young mice infected with the high dose; grey circle, old uninfected mice; grey triangle, young uninfected mice; * indicates significantly greater percentage weight loss than infected young mice of the same dose group at P<0.05.

Fig. 3.

Effect of S. Typhimurium infection on ex vivo cytokine production by splenocytes of streptomycin-pretreated young and old mice. IFN-γ (a, b, c) and TNF-α (d, e, f) production by splenocytes from young and old mice, uninfected or infected with 1×10⁶ c.f.u. S. Typhimurium at day 4 p.i. is shown. Splenocytes were stimulated with medium, HKS, ConA or LPS for 48 h. Supernatants were collected and cytokines were measured by ELISA. (a) Non-stimulated IFN-γ; (b) HKS-stimulated IFN-γ; (c) ConA-stimulated IFN-γ; (d) non-stimulated TNF-α; (e) HKS-stimulated TNF-α; (f) LPS-stimulated TNF-α. Solid bars, young mice; blank bars, old mice; * indicates significant age-related difference in uninfected or infected mice at P<0.05 using Student's t test; # and ## indicate significant differences between uninfected and infected mice in their respective age groups at P<0.05 and <0.01, respectively, using Student's t test. Data are means±sem (n=14−18 per group) from two independent experiments.

Fig. 4.

Effect of S. Typhimurium infection on ex vivo cytokine production in MLN cells. IFN-γ (a) and TNF-α (b) production in MLN cells from streptomycin-pretreated young and old mice, uninfected or infected with 1×10⁶ c.f.u. S. Typhimurium at day 4 p.i. is shown. Lymphocytes in MLN were stimulated with ConA or LPS for 48 h. Supernatants were collected and cytokines were measured by ELISA. (a) ConA-stimulated IFN-γ; (b) LPS-stimulated TNF-α. Solid bars, young mice; blank bars, old mice; * indicates significant age-related difference in uninfected or infected mice at P<0.05 using Student's t test; # and ## indicate significant differences between uninfected and infected mice in their respective age groups at P<0.05 and <0.01, respectively, using Student's t test. Data are means±sem (n=14−18 per group) from two independent experiments.