Exacerbated susceptibility to infection-stimulated immunopathology in CD1d-deficient mice

Abstract

Mice lacking functional CD1d genes were used to study mechanisms of resistance to the protozoan parasite Toxoplasma gondii. Wild-type (WT) BALB/c mice, CD1d-deficient BALB/c mice, and WT C57BL/6 mice all survived an acute oral infection with a low dose of mildly virulent strain ME49 T. gondii cysts. In contrast, most CD1d-deficient C57BL/6 mice died within 2 wk of infection. Despite having parasite burdens that were only slightly higher than WT mice, CD1d-deficient C57BL/6 mice displayed greater weight loss and intestinal pathology. In C57BL/6 mice, CD4(+) cells can cause intestinal pathology during T. gondii infection. Compared with WT mice, infected CD1d-deficient C57BL/6 mice had higher frequencies and numbers of activated (CD44(high)) CD4(+) cells in mesenteric lymph nodes. Depletion of CD4(+) cells from CD1d-deficient mice reduced weight loss and prolonged survival, demonstrating a functional role for CD4(+) cells in their increased susceptibility to T. gondii infection. CD1d-deficient mice are deficient in Valpha14(+) T cells, a major population of NKT cells. Involvement of these cells in resistance to T. gondii was investigated using gene-targeted Jalpha18-deficient C57BL/6 mice, which are deficient in Valpha14(+) T cells. These mice did not succumb to acute infection, but experienced greater weight loss and more deaths than B6 mice during chronic infection, indicating that Valpha14(+) cells contribute to resistance to T. gondii. The data identify CD4(+) cells as a significant component of the marked susceptibility to T. gondii infection observed in CD1d-deficient C57BL/6 mice, and establish T. gondii as a valuable tool for deciphering CD1d-dependent protective mechanisms.

Figure 1. CD1d-deficient B6 mice, but not CD1d-deficient BALB/c mice, are acutely susceptible to oral infection with T. gondii

Groups of 5 mice were infected orally with 10 ME49 cysts or sham-infected. (A) BALB/c mice. Body weights and survival of WT BALB/cJ and CD1d-deficient BALB/cJ mice did not differ significantly. (B) B6 mice. Loss of weight was significantly greater (P<0.05) in infected CD1d-deficient B6 mice than in infected WT controls. (C) Survival data pooled from 3 experiments. The difference in survival between WT B6 (N=10) and CD1d-deficient B6 mice (N=15) is significant (P<0.01).

Figure 2. Intestinal pathology in T. gondii-infected WT B6 and CD1d-deficient B6 mice

(A) Small intestine from a WT B6 mouse killed on day 8 post infection showing mild to moderate infiltration of mononuclear cells in the lamina propria, occasional sloughing of individual epithelial cells at the tips of intestinal villi (arrowheads), and the accumulation of necrotic cellular debris in the cryptic lumens (arrows). (B) Small intestine from a CD1d-deficient B6 mouse killed on day 8 post infection showing severe and extensive infiltration of mononuclear cells throughout the entire intestinal lamina propria, focal ulceration of the tips of villi (arrowheads), and focal necrosis in the submucosal regions (*). H&E, bar = 40m.

Figure 3. Serum cytokines and ileal mRNA levels in WT B6 and CD1d-deficient B6 mice infected with T. gondii

(A) Sera were harvested on day 8 post infection. Values depict means +/− SD. The difference in IFN-γ was significant (P<0.05, N= 5 mice per group). (B) mRNA was isolated from ileums of infected mice on day 8 post infection. Values depict log₁₀ mean +/− SD increase above that of sham-infected controls of the same strain. The difference in IFN-γ between WT B6 and CD1d-deficient B6 mice is significant (P<0.05, N= 5 mice per group). Significant differences are denoted by *.

Figure 4. Elevated numbers of activated CD4⁺ T cells in mesenteric lymph nodes of T. gondii-infected CD1d-deficient B6 mice

Spleens and mesenteric lymph nodes were harvested from WT B6 and CD1d-deficient B6 mice on day 8 post infection. (A) Percentage of CD4⁺ cells with a CD44^hi phenotype. (B) Numbers of CD4⁺ cells with a CD44^hi phenotype in mesenteric lymph nodes. Numbers of cells in the different populations were obtained by multiplying counts of cells per organ by the percentages obtained by flow cytometry. Bars denote mean +/− SD from 3 or 4 mice per group. * denotes significantly (P<0.05) elevated compared with infected WT B6 mice.

Figure 5. Changes in survival and weight loss of T. gondii-infected WT B6 and CD1d-deficient B6 mice treated with antibodies to deplete CD4⁺ cells

(A) Weight loss. Values are mean +/− SD per cent initial body weights of mice given anti-CD4 or an isotype-matched control mAb (1 mg i.p. on days 0 and 2 relative to infection). The difference in body weight between anti-CD4-treated and control mAb-treated CD1d-deficient mice was significant (P<0.05) by day 12, but could not be tested beyond that time owing to the deaths of CD1d-deficient mice in the control mAb-treated group. (B) Survival. Anti-CD4-treated CD1d-deficient mice survived significantly (P<0.05) longer than control mAb-treated CD1d-deficient mice. In each experiment, there were 5 mice per treatment group.

Figure 6. Parasite burdens in the ileums of T. gondii-infected WT B6 and CD1d-deficient B6 mice treated with anti-CD4 or a control mAb

Parasites numbers in ileums of mice infected for 9 days were determined by a real-time PCR-based method (Materials and Methods). Values are log₁₀ mean number +/− SD of parasites per mg tissue. N=4 mice per group.

Figure 7. Survival and body weights of T. gondii-infected WT B6 and Vα14-deficient B6 mice

(A) Survival of groups of 10 WT B6 and Vα14-deficient (Jα18-deficient) B6 mice infected orally with 10 ME49 cysts. Data pooled from two experiments. Significantly (P = 0.013) greater mortality was evident in Vα14-deficient mice. (B) Body weights of groups of 5 mice orally infected with 10 ME cysts. Differences in body weight are significant (P<0.01) from day 20 onward. Asterisks (*) denote significant differences. (C) Body weights of groups of 5 mice orally infected with 40 ME49 cysts. Differences in weight were significant (P<0.05) from day 12 onward. Not shown are weights beyond day 16, when there were some deaths in the Vα14-deficient group.