The major function of the immune system is defense against pathogens. However, after the invading agent has been successfully neutralized, the immune responses must be suppressed. Even when initially protective, the activation of immune system during prolonged period may cause tissue damage and become harmful instead of beneficial for the host. The mechanisms involved in the down-modulation of effector responses are quite complex and in many cases still poorly understood. Toxoplasma gondii infection is an excellent model for studying both induction and regulation of Th1 cells since the impairment in either phase of the response is detrimental for the host. Our laboratory has demonstrated roles for three independent regulatory circuits required for downmodulation of Th1 responses during T. gondii infection in mice: IL-10 production, glucocorticoid receptor expression on T cells and lipoxins. In the case of IL-10, we have also shown that the most important sources of this anti-inflammatory cytokine are effector T-bet+IFNgamma+ Th1 cells that display dual function by simultaneously mediating IFN gamma dependent intracellular killing of the parasite and suppressing IL-12 production by APC thereby limiting Th1 expression. Interestingly, IL-10+IFN-gamma+ Th1 cells express IL-10 transiently, while IFN-gamma production is a stable property. The regulation of IL-10 at the molecular level is still poorly understood. In collaboration with Dr. Jeff Zhu (Laboratory of Immunology, NIAID), we have shown that the transcription factor Bhlhe40, also known as Bhlhb2, Dec1, and Stra13, which is up-regulated during T cell activation suppresses IL-10 production but is required for optimal production of IFN-gamma by Th1 cells. Bhlhe40-deficient CD4 T Bhlhe40 conditional knockout (cKO) mice are susceptible to T. gondii infection due to increased production of IL-10 and diminished secretion of IFN-gamma which tips the balance in favor of uncontrolled parasite proliferation. Blockade of IL-10 signaling in Bhlhe40 cKO mice during T. gondii infection promoted their survival. Therefore, Bhlhe40 serves as an important molecular switch for the development of both inflammatory IFN-gamma and anti-inflammatory IL-10+IFN-gamma+ Th1 cells. In previous work we studied the induction of thymic atrophy by T. gondii infection and showed that it is accompanied by a persistent decrease in the size of the naive CD4+ T lymphocyte pool leading to an immunocompromised state that both promotes chronic toxoplasma infection and leads to decreased resistance to challenge with an unrelated pathogen. Toxoplasma infection affects not only thymic function but also triggers changes in the composition and output of bone marrow. This is exemplified in an unexpected susceptibility of NOD1 deficient mice to T. gondii challenge. While infected NOD1-/- mice display normal serum levels of IL-12, indicated an impaired innate immune response, they failed to mount an efficient IFN-gamma response which was associated with the induction of a lymphopenic state. Moreover, we showed that the thymus, spleen and lymph nodes of naive NOD1-/- mice already display significantly reduced cellularity due to decreased numbers of lymphocytes (T, B and NK cells). In contrast to NOD2 that is expressed primarily in myeloid cells, NOD1 is expressed ubiquitously. Bone marrow (BM) reconstitution experiments demonstrated that this deficiency is due to lack of NOD1 in the hematopoetic compartment, while additional experiments excluded the contribution of the microbiota and the decreased serum levels of hematopoietic cytokines. The defect in hematopoietic cells of NOD1-/- mice was even more accentuated in competitive BM chimera experiments. Subsequently, we demonstrated that intrinsic expression of NOD1 is required for optimal renewal and differentiation of hematopoietic stem cells (HSC) and plays an essential role in determining the lymphopoietic potential of common lymphoid precursors (CLP). In addition to their impaired lymphopoietic potential, CD4+ T lymphocytes from NOD1-/- mice were hyporesponsive when stimulated in the presence of TCR and IL-2. Interestingly, these defects were amplified in a NOD1-/- mouse strain that expresses a truncated form of NOD1 lacking the CARD signaling domain that functions as a dominant negative variant. Moreover, we showed that NOD1-deficiency not only affects resistance to T. gondii infection but also anti-tumor immunity while preventing colitis. Taken together, our findings reveal a cell intrinsic role for NOD1 in lymphocyte homeostasis and CD4+ T cell function that we believe involves recognition of an endogenous, as opposed to microbial, ligand. Our previous work revealed that the mouse, although one of the natural hosts of T. gondii, may not accurately model the innate response to this pathogen in humans. In mice initiation of the innate immune response depends on expression of TLR11/12, however, humans lack functional TLR11 and the entire TLR12 gene, yet are still capable of mounting a protective immune response. We have addressed this shortcoming by systematically interrogating different human myeloid populations for their responsiveness to the parasite. We have identified non-classical CD16+, but not classical CD16neg CD14+, monocytes as the major cells in human peripheral blood that produce IL-12 and TNF in response to T. gondii. Moreover, whereas the innate cytokine response to T. gondii in the mouse involves stimulation of TLR11/12 receptors by a soluble parasite ligand, profilin, the response of human cells requires phagocytosis of the live pathogen. In this year's studies, we showed that priming of total monocytes with IFN-gamma increases the secretion of IL-12 and TNF in response to T. gondii. Surprisingly, this augmented responsiveness is entirely due to the stimulatory effect of IFN-gamma on the non-responder CD16neg CD14+ population and did not correlate with any significant metabolic shift. Despite comparable expression of IFN-gamma receptor, IFN-gamma failed to increase the cytokine response of CD16pos monocytes. In contrast, priming with IFN-alpha failed to promote the response of CD16neg monocytes and, in addition, inhibited the response of both CD16pos and IFN-gamma-primed CD16neg monocytes. Furthermore, after exposure to IFN-alpha, CD16neg monocytes become refractory to the stimulatory effects of IFN-gamma. Interestingly, and in agreement with our findings, monocytes from patients with the autoimmune syndrome, CANDLE, characterized by sustained production of type I IFN, display deceased responsiveness when stimulated with T. gondii tachyzoites and, moreover, cannot be primed with IFN-gamma. The inhibitory effect of type I IFN is IFN-gamma specific, since subsequent stimulation with Rapamycin augmented the secretion of IL-12 and TNF by monocytes from both healthy donors and CANDLE patients. In summary, our results demonstrate that IFN-gamma-priming selectively targets the human CD16neg subset and reveals antagonistic effects of IFN-gamma and IFN-alpha on IL-12 secretion by primary monocytes.
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