Immune Regulation In Toxoplasmosis

National Institute Of Allergy And Infectious Diseases

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Abstract

In FY 2021, we followed up our earlier work on characterization of the IL-12 response by human primary monocyte stimulated with T. gondii and our more recent study on how type I interferon and IFN-gamma (type II) effect cytokine secretion by the CD16+CD14low (non-classical) and CD16negCD14+ (classical) monocyte subsets. Having established that the CD16+CD14low but not CD16negCD14+ subset of human monocytes displays an IFN-induced gene signature at baseline which is associated with their ability to produce IL-12 in response to T. gondii challenge, we sought to investigate the involvement of type I IFN and IFN-gamma in this functional dichotomy of the two major monocyte subsets. To this end we have continued our collaboration with Dr. Michail Lionakis, who provided monocytes from patients that have mutations in IFN-gamma receptor as well as patients with loss (LOF) or gain of function (GOF) mutations in the STAT1 gene. Our preliminary results indicate that while lack of INF-gamma signaling does not dramatically affect the frequency of CD16+ monocytes, their ability to produce IL-12 in response to microbial stimulation is compromised. In addition, in collaboration with Dr. Dusan Bogunovic (Icahn School of Medicine at Mount Sinai, NY) who is studying inborne mutations that cause interferonopathies, we tested whether our observations using healthy donor monocytes can be applied to specific immunopathologic conditions in which levels of type I interferon are aberrantly increased. One example is autosomal recessive USP18 deficiency. As predicted based on our findings in healthy donors, monocytes isolated from patients with USP18 deficiency display decreased lL-12 production after microbial stimulation. The latter observation may explain, at least in part, the increased (and often life threatening) susceptibility to mycobacterial infections exhibited by these patients. IFN-gamma is a critical cytokine in host defense against T. gondii. Multiple cell types: ILC1, NK, CD4+ and CD8+ T cells can serve as sources of this cytokine. Production of IFN-gamma depends on expression of the transcriptional factor T-bet. In collaboration with Dr. Jeff Zhu, we showed that IL-18-mediated Runx3 induction controls upregulation of T-bet in NK cells, while in CD4+ Th1 effectors upregulation of T-bet is controlled by regulatory elements that bind STATs. These finding extend the conclusions of previous work performed in collaboration with Dr. John OShea's lab (NIAMS) and enhance our understanding of the molecular mechanisms that control IFN-gamma gene regulation and its transcriptional landscape in activated NK cells vs. Th1 effector lymphocytes. In the last annual report, we showed that a subset of CD4 T lymphocytes, pathogen-independent memory-phenotype (MP) cells, are poised to secrete IFN-gamma and represent an additional innate source of this cytokine (beside NK and ILC1 cells) that could contribute to Th1 host-defense mechanisms. We further showed that MP cells are a heterogeneous population with T-bet-hi MP cells rapidly producing IFN-gamma and only requiring stimulation with innate Th1-associated inflammatory cytokines in vitro and in a strictly IL-12-dependent manner in response to toxoplasma infection in vivo. We have now extended these observations and demonstrated that the MP subpopulation that exerts the highest responsiveness to Th1-differentiating cytokines possesses a CD127hi Sca1hi and Bcl2low phenotype and represents the most mature and cell division-experienced fraction of MP CD4+ lymphocytes. We have previously shown that T. gondii infection induces an irreversible impairment of thymic function (Kugler et al., 2017), raising the question of its potential impact on the bone marrow (BM), the second of the primary lymphoid organs. In FY 2021, we performed a detailed flow cytometric analysis of BM from T. gondii infected mice by examining monocytic, granulocytic, erythropoietic and B cell lineages at 2-day intervals during the first week of infection and at 1-week intervals thereafter. While T. gondii infection triggers a rapid and significant loss of essentially all BM cell types during the acute phase (resulting in a 25% drop in total cellularity on day 7 post-infection), the recovery of bone marrow functionality occurred in a sequential and temporally ordered manner: myelopoiesis was first-re-established at 2 weeks, followed by increased erythropoiesis at week 3 and, finally, B cell lymphopoiesis at 5-6 weeks post infection. Interestingly the two most important host-protective cytokines during T. gondii infection, IL-12 and IFN-gamma have opposing effects on hematopoiesis by promoting and suppressing bone marrow function, respectively. Indeed, the loss of BM cellularity correlated with the peak in IFN-gamma levels detected in serum and was rapidly down-modulated to pre-infection levels by week 2. Interestingly, systemic IL-12p40, but not p70, levels remain increased throughout the entire bone marrow hematopoietic recovery period. In this regard, the effects of T. gondii infection on bone marrow function are still poorly understood. The decreased bone marrow output during acute T. gondii infection generates, together with the dramatic thymic atrophy occurring at the same time, a profound state of immunosuppression that favors parasite evasion and dissemination setting up ideal conditions for the establishment of chronic infection. In addition, since the balance between monocytes and granulocyte is known to play a critical role in host-resistance against T. gondii infection, the rate at which bone marrow replenishes their pools can play and important role in host-resistance together with the production of chemoattractants that control myeloid cell recruitment at the site of infection.