CD4+ T cells are not essential for control of early acute Cryptosporidium parvum infection in neonatal mice

Abstract

Cryptosporidiosis is an important diarrheal disease of humans and neonatal livestock caused by Cryptosporidium spp. that infect epithelial cells. Recovery from Cryptosporidium parvum infection in adult hosts involves CD4(+) T cells with a strong Th1 component, but mechanisms of immunity in neonates are not well characterized. In the present investigation with newborn mice, similar acute patterns of infection were obtained in C57BL/6 wild-type (WT) and T and B cell-deficient Rag2(-/-) mice. In comparison with uninfected controls, the proportion of intestinal CD4(+) or CD8(+) T cells did not increase in infected WT mice during recovery from infection. Furthermore, infection in neonatal WT mice depleted of CD4(+) T cells was not exacerbated. Ten weeks after WT and Rag2(-/-) mice had been infected as neonates, no patent infections could be detected. Treatment at this stage with the immunosuppressive drug dexamethasone produced patent infections in Rag2(-/-) mice but not WT mice. Expression of inflammatory markers, including gamma interferon (IFN-γ) and interleukin-12p40 (IL-12p40), was higher in neonatal WT mice than in Rag2(-/-) mice around the peak of infection, but IL-10 expression was also higher in WT mice. These results suggest that although CD4(+) T cells may be important for elimination of C. parvum, these cells are dispensable for controlling the early acute phase of infection in neonates.

FIG. 1.

Course of C. parvum infection in neonatal C57BL/6 and Rag2^−/− mice. C57BL/6 and Rag2^−/− animals were infected by oral gavage with C. parvum oocysts at 7 days of age, and microscopic measurements of oocyst shedding in Ziehl-Neelsen acid-fast stained fecal smears from mice at different times of infection were made. The values represent the mean numbers of oocysts per 50 fields ± standard errors for 6 to 10 animals per group.

FIG. 2.

Measurement of excretion of C. parvum oocysts in C57BL/6 and Rag2^−/− mice immunosuppressed with dexamethasone (DXM) following recovery from neonatal infection. C57BL/6 and Rag2^−/− mice were infected by oral gavage with C. parvum oocysts at 7 days of age. Ten weeks after recovery from acute infection, mice were immunosuppressed by i.p. injection with 1 mg dexamethasone on three consecutive days, and oocyst excretion was monitored for 6 days after the first injection. The percentage of mice that developed patent infection is shown. The values represent the mean numbers of oocysts per 50 fields ± standard errors for 9 animals per group.

FIG. 3.

Frequency of intestinal T cells in neonatal C57BL/6 mice at the peak of C. parvum infection and effect of CD4⁺ T cell depletion on the course of infection. (A) Neonatal C57BL/6 mice were infected by oral gavage with C. parvum oocysts, and the surface expression of CD3, CD4, and CD8 on CD45⁺ lamina propria cells (top) and on cells of the intestinal epithelium (bottom) on day 5 postinfection was analyzed by flow cytometry. Representative plots show the frequencies of CD4⁺ and CD8⁺ T cells among the CD3⁺ CD45⁺ cells. (B and C) Effect of CD4⁺ T cell depletion. (B) Neonatal C57BL/6 mice were infected by oral gavage with C. parvum oocysts. Animals were administered anti-CD4 antibody or control IgG on days 0, 2, and 4 postinfection. The efficiency of the ablation of CD4⁺ CD3⁺ CD45⁺ T cells in the spleen, mesenteric lymph nodes, lamina propria, and intestinal epithelium was assessed by flow cytometry on day 5 postinfection. Representative plots are shown. (C) The infection was followed by microscopic examination of fecal smears from mice at different times of infection and by quantification of oocyst shedding. The values shown represent the mean numbers of oocysts per 50 fields ± standard errors for at least 7 animals per group.

FIG. 4.

Effect of NK cell depletion on the course of C. parvum infection in neonatal C57BL/6 mice. Neonatal C57BL/6 animals were infected by oral gavage with C. parvum oocysts and administered either anti-NK1.1 antibodies or control IgG. The infection was followed by microscopic examination of fecal smears from mice at different times of infection and by quantification of oocyst shedding. The values represent the mean numbers of oocysts per 50 fields ± standard errors for 6 to 8 animals per group. Significant differences between mean values for control and antibody-treated mice are marked (*, P = 0.0286; #, P = 0.0476).

FIG. 5.

Intestinal cytokine expression in neonatal C57BL/6 and Rag2^−/− mice during acute C. parvum infection (C.p.). Neonatal C57BL/6 and Rag2^−/− animals were infected by oral gavage with C. parvum oocysts, and intestinal tissue samples from the small intestine were collected on day 5 postinfection. The amounts of intestinal IFN-γ (A), IL-12p40 (B), and IL-10 (C) mRNA were quantified by real-time quantitative PCR using the threshold cycle (ΔΔC_T) method, with β-2-microglobulin as the reference gene and samples collected from uninfected animals as calibrators. The examples shown represent at least 6 animals per group (*, P < 0.05; **, P ≤ 0.01; ***, P < 0.001).

FIG. 6.

Intestinal expression of NK cell and T cell markers in neonatal C57BL/6 and Rag2^−/− mice during acute C. parvum infection. Neonatal C57BL/6 and Rag2^−/− animals were infected by oral gavage with C. parvum oocysts, and intestinal tissue samples from the small intestine were collected 5 days postinfection. The amounts of intestinal IL-2Rβ (A), Klrb1b (B), CD69 (C), and granzyme B (D) mRNA were quantified by real-time quantitative PCR using the ΔΔC_T method, with β-2-microglobulin as the reference gene and samples collected from uninfected animals as calibrators. The examples shown represent at least 6 animals per group (*, P < 0.05; **, P ≤ 0.01; ***, P < 0.001).