| Toxoplasma gondii (T. gondii) is an opportunistic intracellular parasite which is highly prevalent in humans and warm-blooded animals throughout the world. The host immune response to T. gondii infection is complex and can influence the development and progression of toxoplasmosis. In an immunocompetent host, infection is often asymptomatic. However, if the host becomes immunocompromised, T. gondii may cause widespread tissue damage, which can be fatal if not appropriately treated. Maternal infection during pregnancy is associated with increased adverse outcomes of pregnancy, including abortion, congenital defects in the fetus, etc. Recently, with the increased incidence of cancer and acquired immunodeficiency syndrome (AIDS), the threat to human health caused by T. gondii is increasing, and to date, there is no safe and effective drug available for the treatment of toxoplasmosis. Therefore, to further investigate the mechanisms of immune response to T. gondii is important for prevention and control of toxoplasmosis.In mouse models of T. gondii infection, NK cells represent an important early source of IFN-y, and play a pivotal role in innate defense against T. gondii. Previous studies have demonstrated that IL-12 secreted by antigen-presenting cells (APCs) is required for triggering IFN-y production by NK cells during T. gondii Infection. MyD88, a key adaptor for signaling by almost all Toll-like receptors (TLRs), has been proven to be required for T. gondii-induced IL-12 production and play an essential role in IFN-y-dependent resistance to this parasite. It is generally believed that the acute susceptibility of MyD88-/- mice to T. gondii is associated with diminished IL-12 responses. A more recent study has shown that, just like MyD88-/- mice, mice lacking MyD88 in DCs (DC-MyD88-/- mice) also exhibit a nearly complete defect in IL-12 production following T. gondii infection. However, treatment of mice with IL-12 after infection could only prevent acute mortality in DC-MyD88-/- mice but not in MyD88-/- mice, associated with the correction of defective induction of IFN-y in NK cells in DC-MyD88-/- mice. These results suggest that APC-extrinsic MyD88, especially the TLR/MyD88 signals in NK cells, might be required for IL-12-dependent early IFN-y production during T. gondii infection. To verify this hypothesis, we first investigated whether NK cells could be directly activated by T. gondii antigens in combination with IL-12 and then produced IFN-y in vitro. Then we assessed the role of TLR/MyD88 signals in T. gondii antigen-induced IFN-y production by NK cells. Multiple signaling pathways downstream of MyD88 adaptor molecule were also analyzed. Furthermore, the effector molecules in T. gondii antigens which can stimulate NK cells to produce IFN-y were identified. Finally, we used in vivo adoptive transfer experiments to further confirm our hypothesis.The main results we got are as follows:1. T. gondii antigens and IL-12 synergistically induce NK cell activation and production of IFN-γ in vitro. Purified NK cells were stimulated with T. gondii excreted-secreted antigens (ESA) or soluble tachyzoite antigens (STAg), in the absence or presence of exogenous IL-12. The results showed that both T. gondii ESA and STAg induced NK cells to produce low levels of IFN-y. There was a strong synergistic effect of IL-12 on T. gondii antigen-induced IFN-y production, and the secretion of IFN-y increased in a dose-dependent manner with increasing concentrations of T. gondii antigens. The synergistic effect was also confirmed by analysis of IFN-γ mRNA in NK cells. Unlike IFN-y, the production of TNF-a by NK cells stimulated with T. gondii antigens was at low levels regardless the presence of IL-12. In addition to cytokine secretion, combination of T. gondii antigens and IL-12 induced prominent increase in the expression of both CD69 and CD25 activation markers on NK cells.2. T. gondii antigens do not enhance NK cell-mediated cytotoxicity. Unstimulated NK cells slightly killed YAC-1 cells, and we were unable to detect higher cytotoxicity of NK cells after stimulation with T. gondii antigens. When NK cells were stimulated with IL-12 alone, a slightly higher cytolytic activity was detected. However, combination of T. gondii antigens and IL-12 did not further increase the cytolytic activity of NK cells. In accordance with the results of cytotoxicity assay, NK cells did not show any increased expression of NKG2D receptor after stimulation with T. gondii antigens and IL-12.3. Requirement of TLR/MyD88 signaling for IFN-7 production by NK cells. Addition of MyD88 homodimerization inhibitory peptide to NK cells prior to T. gondii antigen stimulation led to an inhibition of the IFN-y mRNA expression. Based on the results of our inhibition experiment, NK cells were further prepared from MyD88"’" mice, and then stimulated with T. gondii antigens and IL-12. As expected, the IFN-y expression at both the protein and mRNA levels was significantly decreased in MyD88-/- NK cells when compared with that in wild-type (WT) NK cells. MyD88 is an adaptor protein necessary for signal transduction of both TLRs and the IL-1R/IL-18R family. To evaluate whether the secretion of IFN-γ by NK cells was mediated by IL-1 or IL-18 which was possibly produced by a few contaminating cells in purified NK cells, neutralizing antibodies against mouse IL-1α、IL-1β and IL-18 were used. Results showed that neutralization of IL-1α、IL-1β and IL-18 did not cause any change in IFN-y production, suggesting a role for TLRs but not for IL-1R/IL-18R in MyD88-dependent IFN-y production by NK cells.4. P38 MAPK, ERK, JNK and NF-κB multiple pathways contribute to IFN-y production by NK cells. MyD88-mediated signaling activates multiple protein kinases and transcription factors. To evaluate which of these pathways mediates the IFN-γ secretion by NK cells, we assessed the effect of a panel of pharmacological inhibitors on the IFN-y production of NK cells. The results showed that, inhibition of p38 MAPK, ERK, JNK and NF-κB all significantly suppressed the IFN-y secretion by NK cells in a concentration-dependent manner. Furthermore, the NF-κB inhibitor showed the strongest suppression effect on IFN-y production. A relative low concentration brought almost complete suppression.5. T. gondii profilin and HSP70 in combination with IL-12 induce IFN-y production by NK cells. To identify the effector molecules in T. gondii antigens, we first investigated NK cell expression of TLRs. Except for TLR5, splenic NK cells isolated from C57BL/6 mice expressed all TLR1～4, TLR6-9 and TLR11～13 mRNAs. Studies have shown that, in order to regulate the effector functions, TLRs may change their expression levels when exerting biological effects. However, no changes on the expression of all TLR mRNAs in NK cells before and after stimulation with T. gondii antigens could be detected. The roles of TLRs in other immune cells, especially in macrophages and DCs, have been widely studied during T. gondii infection, and several TLR agonists derived from T. gondii have been identified. To figure out whether these TLR agonists contributed to T. gondii antigen-induced IFN-y secretion by NK cells, we stimulated NK cells with recombinant T. gondii profilin, HSP70, or purified glycosylphosphatidylinositols (GPIs). Results showed that T. gondii profilin and HSP70 could evoke a similar IFN-γ secretory response to that evoked by T. gondii antigens, while purified GPIs could not. Stimulation of NK cells prepared from MyD88-/- mice showed that induction of IFN-y in NK cells by T. gondii profilin and HSP70 was also dependent on TLR/MyD88 signaling. Finally, By Western blot using purified antibodies against profilin or HSP70 prepared in rabbits, we found profilin was only present in STAg while HSP70 existed in both ESA and STAg.6. NK cell expression of MyD88 is required for innate resistance to T. gondii. We used adoptive transfer experiments to further confirm NK cell-intrinsic TLR-MyD88 signaling is required for IL-12-dependent early IFN-y production during T. gondii infection. The results showed that, upon challenge with T. gondii, NOD/SCID-β2m-/- mice (NK-as well as T- and B-cell development and functions are disrupted) transferred with MyD88-/- NK cells had reduced serum IFN-y levels and increased splenic tachyzoite burdens, and were more susceptible than that transferred with wild-type NK cells. These data indicate that NK cell expression of MyD88 is required for IL-12-dependent early IFN-y production during T. gondii infection. Based on our current data and previous findings that IFN-y-dependent host resistance to T. gondii is independent of IL-1R and IL-18R signaling, we conclude that NK cell-intrinsic TLR/MyD88 signaling is required for NK cell IFN-y production and host innate resistance to T. gondii.In conclusion, we have established a role for NK cell-intrinsic TLR/MyD88 signaling in IL-12-dependent early IFN-y production and innate defense to T. gondii. Since production of IFN-y by NK cells is similarly essential for defense against other pathogens, these data provide further insight into the innate immune responses to these pathogens. |
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