T cell expression of MyD88 is required for resistance to Toxoplasma gondii

Abstract

Resistance to Toxoplasma gondii depends on dendritic cells to recognize this pathogen and secrete IL-12, in turn promoting IFN-gamma production from responding T cells. The adaptor protein, myeloid differentiation primary-response gene 88 (MyD88), is important for most Toll-like receptor (TLR) signaling, as well as IL-1R/IL-18R signals. There is considerable evidence that MyD88 is required for the innate sensing of T. gondii and IL-12 responses. Although Myd88(-/-) mice challenged with T. gondii have defective IL-12 and Th1 effector responses and succumb to disease, administration of IL-12 to Myd88(-/-) mice partially restores the Th1 response and yet fails to prolong survival. This finding suggested that MyD88 may mediate signals within T cells important for resistance to this pathogen. To evaluate the role of MyD88 in T cells under noncompetitive conditions, bone marrow chimeras were generated, in which the T cells lacked MyD88, but MyD88-dependent innate immune responses were intact. Upon challenge with T. gondii, these chimeric mice were more susceptible to disease, developing severe toxoplasmic encephalitis and succumbing within 30 days. Splenocytes and brain mononuclear cells isolated from infected chimeric mice produced less IFN-gamma when cultured with a T. gondii-derived antigen. The increase in susceptibility observed was independent of signals via the IL-1R and IL-18R, suggesting a role for TLRs in MyD88-mediated T cell responses to T. gondii. These observations show that, in addition to a role for MyD88 in innate responses, T cell expression of MyD88 is necessary for prolonged resistance to a pathogen.

Fig. 1.

A mouse model in which T cells are MyD88-deficient, but MyD88-dependent innate responses are intact. (A) Scheme representing the hematopoietic reconstitution strategy. (B) Twelve weeks after reconstitution, T cells were isolated from mice (>90% purity), and an immunoblot was performed to assess MyD88 expression. (C) Chimeric mice were generated as illustrated in A, except that the Myd88^−/− mice were CD45.1+, whereas the other marrow donors and the recipients were CD45.2+. After 12 weeks, flow cytometry was performed on PBMCs; data represent two individually reconstituted cohorts. (D) Splenocytes were cultured for 24 h in the presence or absence of CpG DNA and then analyzed by flow cytometry for B cell expression of CD86. Open histogram, CpG DNA stimulated; shaded histogram, unstimulated. These data are representative of three experiments. (E) Mice from each cohort were injected with STAg, and serum was obtained after 3 h for ELISA; these data are representative of three experiments; N.D., none detected. For all experiments, unless otherwise indicated, data represent at least three experiments.

Fig. 2.

T cell expression of MyD88 is required for resistance to the pathogen T. gondii independent of the innate response. (A) Mice were inoculated i.p. with T. gondii and monitored for survival. Data are pooled from two experiments (n = 13 each cohort). (B) PECs isolated from mice on day 7 after infection was used to prepare cytospin smears. Data are the mean of three pooled experiments (n = 6). (C) Sera obtained from mice on day 5 of infection were evaluated by ELISA. Data are representative of three experiments. (D) Cells were isolated from draining lymph nodes and spleen on day 8 of infection and analyzed by flow cytometry. Data are representative of two experiments (n = 6 total for each cohort).

Fig. 3.

TMyd88^−/− chimeric mice have increased susceptibility to toxoplasmic encephalitis. (A) Brain histopathology of TMyD88^−/− chimeric mice is shown near median survival days 25–27 after infection. Parenchymal regions of necrotic encephalitis are evident in the center of the pictogram. (B) Brain histopathology of TMyD88^+/+ control mice is shown near median survival days 60–62 after infection. Focus of cellular infiltrate is in the center of the pictogram; arrows indicate bradyzoite tissue cysts. The data are representative of mice in two experiments (n = 6 total). (Magnification: ×100.) (C) BMNCs were prepared from mice on day 23 of infection, counted, and analyzed by flow cytometry to enumerate T cells. Data are representative of three experiments. (D) Quantitative real-time PCR of parasite DNA isolated from the brains of mice on day 23 of infection. Pooled data are from three experiments, with at least two mice in each group per experiment.

Fig. 4.

TMyd88^−/− chimeric mice have less IFN-γ-producing responder T cells. (A) Sera obtained from mice on day 8 (Upper) and day 23 (Lower) of T. gondii infection were analyzed by ELISA. (B) Splenocytes were isolated from mice on day 8 (Upper) and day 23 (Lower) of infection and cultured in the presence or absence of STAg. After 72 h, supernatants were analyzed by ELISA. (C) BMNCs prepared from mice on day 23 of infection were cultured in the presence or absence of STAg, and after 72 h cell-free supernatants were assessed by ELISA. (D) Splenocytes were isolated from mice on day 23 of infection, cultured in the presence or absence of STAg for 48 h, and then processed for intracellular IFN-γ. (E) Splenocytes were isolated from mice on day 23 after infection, cultured in the presence or absence of STAg for 72 h, and then analyzed by flow cytometry. Gate is on CD4⁺ T cells. (F) Mice were pulsed with BrdU on days 5 and 6 of T. gondii infection, and splenocytes were analyzed by flow cytometry on day 8. Gate is on CD4⁺ T cells. Data represent two experiments (n = 6 mice total). N.D., none detected. For all experiments, unless otherwise indicated, data are representative of three experiments.

Fig. 5.

Chimeric mice with simultaneous disruption of both IL-1R- and IL-18R-mediated signals are less susceptible to T. gondii than TMyd88^−/− chimeric mice. (A) Chimeric mice and controls were infected with T. gondii, and the indicated groups were given 500 mg/kg IL-1Ra daily. Mice were killed on day 23 of infection, and quantitative real-time PCR was performed on DNA isolated from brains to assess parasite burden. Data are from one experiment with seven to eight mice per group. (B) Splenocytes were isolated from mice on day 23 of infection and cultured in the presence or absence of STAg. After 72 h, supernatants were analyzed by ELISA. (C) Splenocytes were isolated from mice on day 23 of infection, cultured in the presence or absence of STAg for 48 h, and then processed for intracellular IFN-γ. Gate is on CD4⁺ T cells.