The chemokine CCL2 is required for control of murine gastric Salmonella enterica infection

Abstract

Salmonella enterica is a gram-negative intracellular pathogen that can cause a variety of diseases ranging from gastroenteritis to typhoid fever. The Typhimurium serotype causes gastroenteritis in humans; however, infection of mice results in an enteric fever that resembles human typhoid fever and has been used as a model for typhoid fever. The present study examined the role of the chemokine CCL2 in the control of Salmonella infection. Upon infection with salmonellae, mucosal expression of CCL2 is rapidly up-regulated, followed by systemic expression in the spleen. CCL2(-/-) mice became moribund earlier and had a higher rate of mortality compared to wild-type C57BL/6 mice. Moreover, CCL2(-/-) mice had significantly higher levels of bacteria in the liver compared to wild-type controls. Mucosal and serum interleukin-6 and tumor necrosis factor alpha levels were elevated in CCL2(-/-) mice compared to wild-type mice. In vitro analysis demonstrated that CCL2(-/-) macrophages infected with salmonellae resulted in dysregulated cytokine production compared to macrophages derived from wild-type mice. These data are the first to directly demonstrate CCL2 as a critical factor for immune responses and survival following S. enterica infection.

FIG. 1.

CCL2 expression in mucosal and systemic tissues after Salmonella infection. C57BL/6 mice were infected intragastrically with 10⁸ salmonellae. At different time points after infection, CCL2 levels in the mucosa (black bars), spleen (diagonal stripes), and serum (hatched bars) were measured by ELISA.

FIG. 2.

Survival following Salmonella infection. (A) C57BL/6 and CCL2^−/− mice were infected intragastrically with increasing doses of salmonellae, and the percent survival was determined 5 days after infection. If the mice became moribund, they were euthanized and considered to have succumbed to infection. Shown are survival percentages for infecting doses of 10⁸ (B) and 10⁴ (C) bacteria as a function of time postinfection. An asterisk denotes P < 0.05 when comparing the percent survival of CCL2^−/− mice to that of C57BL/6 mice for each day.

FIG. 3.

Systemic bacterial burdens following gastric Salmonella infection. C57BL/6 and CCL2^−/− mice were fed 10⁸ virulent salmonellae. At 2 and 5 days postinfection, four mice were euthanized and the GALT (A and B), spleens (C and D), and livers (E and F) were homogenized and plated on LB agar plates to determine numbers of CFU. The horizontal bar shows the average number of CFU per group. The data are representative of three separate experiments. An asterisk denotes P < 0.05 comparing the bacterial counts of CCL2^−/− mice to those of wild-type mice.

FIG. 4.

Cytokine expression after infection with salmonellae. C57BL/6 and CCL2^−/− mice were infected with 10⁴ virulent salmonellae. At days 1, 3, and 6 following infection, three mice were euthanized per group and TNF-α (A and B), IL-6 (C and D), IL-10 (E and F), and IL-12p70 (G and H) were measured in the serum (A, C, E, and G) and GALT (B, D, F, and H). The data are expressed as the mean cytokine production of three mice and are representative of three separate experiments. An asterisk denotes P < 0.05 comparing the mean CCL2^−/− cytokine production to that of the wild type for that day.

FIG. 5.

Survival following Salmonella LPS challenge. (A) C57BL/6 and CCL2^−/− mice were treated with 50 μg Salmonella LPS intraperitoneally (i.p.). Mice were evaluated twice daily for signs of illness and euthanized if they became moribund. These data are representative of three identical experiments. (B) Serum samples were collected from three mice per time point and pooled. CCL2 levels were evaluated in the pooled samples by cytokine bead array. An asterisk denotes P < 0.05 for CCL2^−/− mouse percent survival compared to that of C57BL/6 mice for that day.

FIG. 6.

Cytokine expression after Salmonella LPS challenge. At 3 and 5 days after Salmonella LPS challenge, C57BL/6 and CCL2^−/− mice were evaluated for proinflammatory cytokine secretion in the serum. TNF-α (A), IL-6 (B), IL-12 (C), and IL-10 (D) were measured by cytokine bead matrix. The data are the mean cytokine production from three individual mice per time point. These data are representative of three identical experiments. An asterisk denotes P < 0.05 when comparing CCL2^−/− mouse cytokine levels to those of C57BL/6 mice on the same day.

FIG. 7.

Macrophage cytokine response to Salmonella infection and LPS challenge. Wild-type (Wt) and CCL2^−/− mouse peritoneal macrophages were isolated, plated at a density of 5 × 10⁶/well, and incubated with S. enterica serovar Typhimurium SL1334 (at a multiplicity of infection of 10) or with 1 μg/ml Salmonella LPS. Medium was collected at various time points, and the supernatants were analyzed for production of TNF-α (A) and IL-6 (B). These data are representative of three identical experiments. An asterisk denotes P < 0.05 for CCL2^−/− mouse macrophages infected with salmonellae compared to wild-type macrophages infected with salmonellae. A double asterisk denotes P < 0.05 comparing CCL2^−/− mouse macrophages to wild-type mouse macrophages incubated with Salmonella LPS.