Concurrent infection with an intestinal helminth parasite impairs host resistance to enteric Citrobacter rodentium and enhances Citrobacter-induced colitis in mice

Abstract

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To test the hypothesis that intestinal helminth infection may be a risk factor for enteric bacterial infection, a murine model was established by using the intestinal helminth Heligomosomoides polygyrus. To analyze the modulatory effect of a Th2-inducing helminth on the outcome of enteric bacterium Citrobacter rodentium infection, BALB/c and STAT 6 knockout (KO) mice were infected with H. polygyrus, C. rodentium, or both. We found that only BALB/c mice coinfected with H. polygyrus and C. rodentium displayed a marked morbidity and mortality. The enhanced susceptibility to C. rodentium and intestinal injury of coinfected BALB/c mice were shown to be associated with a significant increase in helminth-driven Th2 responses, mucosally and systemically, and correlated with a significant downregulation of protective gamma interferon and with a dramatic upregulation of the proinflammatory tumor necrosis factor alpha response. In addition, C. rodentium-associated colonic pathology in coinfected BALB/c mice was significantly enhanced, whereas bacterial burden was increased and clearance was delayed. In contrast, coinfection in STAT 6 KO mice failed to promote C. rodentium infection or to induce a more severe intestinal inflammation and tissue injury, demonstrating a mechanism by which helminth influences the development of host protective immunity and susceptibility to bacterial infections. We conclude that H. polygyrus coinfection can promote C. rodentium-associated disease and colitis through a STAT 6-mediated immune mechanism.

FIG. 1.

Coinfection with H. polygyrus and C. rodentium induces significant body weight loss (top panel) and mortality (bottom panel). BALB/c mice were infected with H. polygyrus (200L₃) and inoculated with C. rodentium (5 × 10⁸ CFU) 7 days later. (Top panel) Body weight changes of mice that were infected with C. rodentium (Cr; ○), H. polygyrus (Hp; ▪), and both H. polygyrus and C. rodentium (Hp + Cr; •) and of normal controls (□) during the course of the experiment (4 weeks). The body weights of C. rodentium-infected mice were significantly lower than those of noninfected controls. Mice with coinfection weighed significantly less than mice with C. rodentium infection alone, whereas H. polygyrus infection had no effect on host body weight gain. The data shown are pooled from three independent experiments and are expressed as the body weight change as a percentage of the individual mouse's initial body weight ± the standard error (SE). n = 12 to 15 for C. rodentium-infected mice, n = 9 to 12 for control mice, n = 9 to 12 for H. polygyrus-infected mice, and n = 18 for coinfected mice. (Bottom panel) Survival curve. There was no mortality in C. rodentium-infected mice.

FIG. 2.

Coinfection with the intestinal helminth parasite, H. polygyrus, promotes C. rodentium-mediated colonic pathological alterations. Colon tissue was removed from uninfected mice (A) or from mice infected with H. polygyrus (B), C. rodentium (C), or both (D) at 2 weeks after bacterial infection, frozen in Tissue Tek OCT compound, and stained with hematoxylin and eosin. (A) Uninfected colon. (B) Colon from H. polygyrus-infected mice. There were no significant changes observed. (C) C. rodentium-induced colon inflammatory response with epithelial hyperplasia, inflammatory cell infiltration, and thickening of the gut wall. (D) Mice infected with both pathogens developed disrupted epithelial surfaces and induced massive cellular infiltration into colon lamina propria, and the submucosae became edematous. Magnification, ×100. (E) Disease score of colonic inflammation in uninfected mice and in mice infected with H. polygyrus, C. rodentium, or both. The scores were assessed by determination of infiltration of inflammatory cells (score range, 0 to 4), together with the evaluation of colon tissue damage (score range, 0 to 4), with 0 as normal and 4 as the most diseased. The data shown are pooled from three independent experiments with total (n = 9 to 12 per group).

FIG. 3.

H. polygyrus coinfection prolonged C. rodentium-mediated colonic inflammation. Colon tissues were removed from uninfected mice (A) or from mice infected with H. polygyrus (B), C. rodentium (C), or both (D) at 4 weeks after bacterial infection; frozen in Tissue Tek OCT compound; and stained with hematoxylin and eosin. For a detailed description, see Fig. 2. Magnification, ×100.

FIG. 4.

Coinfection with H. polygyrus results in an enhanced C. rodentium output in the fecal pellets. The data shown are the numbers of bacteria recovered from fecal samples of C. rodentium-infected and coinfected mice at 1, 2, and 3 weeks postinfection. The data shown are log transformed and are represented as the mean ± the SEM (n = 5 to 10 mice at each time point). ✽, P < 0.05 for a comparison of coinfected versus C. rodentium-infected mice.

FIG. 5.

Viable C. rodentium recovered in the MLN (A and B) and spleen (C and D). Mice were infected with H. polygyrus or uninfected. At 1 week after H. polygyrus infection, some of the infected as well as uninfected mice were inoculated with C. rodentium (5 × 10⁸ CFU/mouse). At 1 and 2 weeks after bacterial infection, mice were killed, and the MLN and spleens were removed and homogenized. Aliquots of diluted homogenized tissue were plated on selective MacConkey agar plates and incubated overnight. Viable bacteria were counted. (A and C) Recovery of bacteria in MLN and spleen at 1 week after bacterial infection. (B and D) Bacterial recovery at 2 weeks after bacterial infection. The data presented are mean ± the SEM (n = 5 per group). ✽, P < 0.05 for a comparison of coinfected versus C. rodentium-infected mice.

FIG. 6.

Serum IgG1 and IgG2a responses to parasite antigen (A and B) and bacterial antigen (C and D). Serum samples were collected at 1 to 4 weeks after bacterial infection and assayed for antigen-specific antibody production by using ELISA. (A) H. polygyrus infection induced anti-parasite antigen specific IgG1 antibody (Th2 response). (B) None or a very low level of anti-parasite antigen-specific IgG2a (Th1) was detected in all mice. (C) Coinfection with H. polygyrus promotes antibacterial antigen-specific IgG1 response. (D) C. rodentium infection induced antibacterial antigen-specific IgG2a response, and coinfection with H. polygyrus enhanced this IgG2a response. The data shown are mean concentrations of serum antibody ± the SEM. The different lowercase letters represent significant differences (n = 5 to 10 mice at each time point; P < 0.05).

FIG. 7.

Th1 and Th2 cytokine profile of mice infected with C. rodentium, H. polygyrus, or both at 2 weeks after bacterial infection. MLN cells were cultured with surface-bound anti-CD3 MAb (10 μg/ml) (A), parasite antigen (B), or C. rodentium antigen (C). Culture supernatants were collected 48 h later. Cytokine secretion into the culture supernatants was determined by ELISA. The data show that H. polygyrus infection drove a Th2 response, whereas C. rodentium infection induced a Th1-type response. Coinfection with H. polygyrus significantly inhibited C. rodentium induced IFN-γ production. C. rodentium infection negatively influenced the parasite-induced IL-4 response in the MLN of coinfected hosts. The results are displayed as means ± the SEM and are representative of three independent experiments. Different letters represent significant difference (P < 0.05; n = 5 mice per group).

FIG. 8.

Cytokine mRNA expression in colon tissue as measured by real-time PCR at 2 weeks after bacterial infection. Values are mean fold increases compared to the baseline obtained from normal control animals. (A) IFN-γ; (B) IL-4; (C) TNF-α; (D) IL-12. The data shown are from one of two experiments performed, showing similar results. Different letters represent significant difference (P < 0.05, n = 3 to 5 mice per group).

FIG. 9.

Impact of H. polygyrus coinfection on STAT 6 KO mice exposed to C. rodentium. (A) Body weight changes of mice that were infected with C. rodentium, H. polygyrus, or both H. polygyrus and C. rodentium and normal controls at 2 weeks after C. rodentium infection. The data shown are pooled from two experiments and are the body weight change as a percentage of an individual mouse's initial body weight ± the SEM (n = 6 to 8 animals per group). (B and C) Th1 and Th2 cytokine profile of STAT 6 KO mice infected with C. rodentium, H. polygyrus, or both at 2 weeks after C. rodentium infection. MLN cells were cultured with surface-bound anti-CD3 MAb (10 μg/ml) (B) or to C. rodentium antigen (C). The results are displayed as the mean ± the SEM and are pooled from two independent experiments. Different letters represent significant differences within a mouse strain (P < 0.05, n = 6 to 8 mice per group).

FIG. 10.

Coinfection with H. polygyrus fails to promote C. rodentium-mediated colonic pathological alterations in STAT 6 KO mice. Colon tissue was removed from uninfected mice (A), from mice infected with H. polygyrus (B), from C. rodentium-infected STAT 6 KO mice (C), from coinfected STAT 6 KO mice (D), from C. rodentium-infected BALB/c mice (E), or from coinfected BALB/c mice (F) at 2 weeks after bacterial infection. (G) Disease score of colonic inflammation and injury. The scores were assessed as in Fig. 2. The data shown are measurements of individual mouse (pooled from two experiments with a total n = 6 to 8 mice per group). Magnification, ×100.