MyD88-dependent pathways mediate resistance to Cryptosporidium parvum infection in mice

Abstract

Cryptosporidium spp. cause diarrheal disease worldwide. Innate immune responses mediating resistance to this parasite are not completely understood. To determine whether MyD88-dependent pathways play a role in resistance to Cryptosporidium parvum, we compared the course of infection in MyD88(-/-) mice to that in their wild-type (WT) littermate controls. Three- to 4-week-old mice were infected with C. parvum, and infection was monitored by quantifying fecal oocyst shedding. Twelve days postinfection, the histology of the intestines was examined to quantify intestinal parasite burden and to determine if there were any pathological changes. Fecal oocyst shedding and intestinal parasite burden were significantly greater in MyD88(-/-) mice than in littermate controls. Nonetheless, both WT and MyD88(-/-) mice cleared the infection within 3 weeks. These results indicate that MyD88-dependent pathways are involved in mediating initial resistance to C. parvum. Since gamma interferon (IFN-gamma) is known to mediate resistance to C. parvum, we also studied infection in MyD88(-/-) mice and WT controls in which this cytokine was temporarily neutralized. Fecal oocyst shedding, as well as intestinal parasite burden, intestinal inflammation, and mortality, was significantly greater in MyD88(-/-) mice in which IFN-gamma was neutralized than in IFN-gamma-neutralized WT mice or in MyD88(-/-) mice in which this cytokine was active. These results suggest that MyD88 and IFN-gamma had an additive effect in conferring protection from C. parvum infection. While this study confirms the importance of IFN-gamma in conferring resistance to infection with C. parvum, it suggests that MyD88-mediated pathways also play a role in innate immunity to this parasite.

FIG. 1.

C. parvum fecal oocyst shedding and parasite burden in the intestines of WT and MyD88^−/− mice during infection. (a) Time course of shedding from day 1 to day 21 postinfection by WT and MyD88^−/− mice. Data from the second experiment are shown. (b) Intracellular stages of C. parvum in representative WT (a) and MyD88^−/− (b) mice. Mice were euthanized 12 days postinfection and the intestines processed for histological analysis. Arrows point to examples of intracellular forms of the parasite. Scale bars = 10 μm.

FIG. 2.

Histopathologies of the ileum and cecum of WT and MyD88^−/− mice infected with C. parvum. Mice were sacrificed 12 days postinfection and the intestines processed for histological analysis. Representative images at 10× magnification; scale bars = 100 μm; insets at 40× magnification to show greater cellular detail. (a) Uninfected MyD88^−/− ileum (EI = 0). (b) Infected WT ileum (EI = 0.5). (c) Infected MyD88^−/− ileum (EI = 2.5). (d) Uninfected MyD88^−/− cecum (EI = 0). (e) Infected WT cecum (EI = 1.0). (f) Infected MyD88^−/− cecum (EI = 3.0).

FIG. 3.

C. parvum fecal oocyst shedding and burden of parasites in the intestines of IFN-γ-neutralized WT and MyD88^−/− mice. (a) Time course of shedding from day 1 to day 21 postinfection by WT and MyD88^−/− mice pretreated with anti-IFN-γ mAb 2 hours prior to infection. Data from the second experiment are shown. (b) Intracellular stages of C. parvum in WT mice (left panel) and MyD88^−/− mice (right panel) pretreated with anti-IFN-γ antibody. Mice were sacrificed 12 days postinfection and the intestines processed for histological analysis. Arrows point to examples of intracellular forms of the parasite. Scale bars = 10 μm.

FIG. 4.

Histopathologies of the ileum and cecum of IFN-γ neutralized WT and MyD88^−/− mice infected with C. parvum. Mice were sacrificed 12 days postinfection and the intestines processed for histological analysis. Representative images at 10× magnification; scale bars = 200 μm; insets at 40× magnification. Arrows indicate crypt abscesses. (a) Uninfected IFN-γ-neutralized MyD88^−/− ileum (EI = 0). (b) Infected IFN-γ-neutralized WT ileum (EI = 6.0). (c) Infected IFN-γ-neutralized MyD88^−/− ileum (EI = 8.5). (d) Uninfected MyD88^−/− cecum (EI = 0). (e) Infected IFN-γ-neutralized WT cecum (EI = 2.5). (f) Infected IFN-γ neutralized MyD88^−/− cecum (EI = 5.0).

FIG. 5.

Survival of C. parvum-infected WT and MyD88^−/− mice with and without IFN-γ neutralization. Mice from both experiments which died spontaneously or were moribund and had to be euthanized were included in the analysis. P = 0.006.