Immune Regulation In Toxoplasmosis

National Institute Of Allergy And Infectious Diseases

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Abstract

In FY 2022, based on our findings that type I interferon and IFN-gamma (type II) have opposite effects on microbial triggered IL-12 production by human monocytes (Muglia Amancio et al. 2021, JI) and in collaboration with Dr. Dusan Bogunovic (Icahn School of Medicine at Mount Sinai, NY) who is studying inborne mutations that cause interferonopathies, we demonstrated that our observations using healthy donor monocytes can be applied to specific immunopathologic conditions in which systemic levels of type I interferon are aberrantly increased. One such example is autosomal recessive USP18 deficiency. As predicted based on our results on monocytes from healthy individuals treated with type I interferon, monocytes isolated from patients with USP18 deficiency display decreased lL-12 production after microbial stimulation (Martin-Fernandez et al. 2022, JEM). The latter observation may explain, at least in part, the increased (and often life threatening) predisposition to mycobacterial infections exhibited by this group of patients. IFN-gamma is a critical cytokine in host defense against T. gondii. Multiple cell types: innate lymphoid 1 (ILC1), natural killer (NK), CD4+ and CD8+ T cells can serve as sources of this cytokine. Moreover, they utilize an identical set of transcription factors (TFs) for their differentiation and functions. In collaboration with Dr. Jeff Zhu (NIAID), we showed that CD4 T helper 1 (Th1) and NK cells displayed distinct epigenomes at the Tbx21 locus, which encodes T-bet, a critical TF for regulating type 1 immune responses. The initial induction of T-bet in NK precursors was dependent on the NK-specific DNase I hypersensitive site Tbx21-CNS-3 that binds RUNX3 in response to signals downstream of IL-18 receptor. In contrast, signal transducer and activator of transcription (STAT)-binding motifs within Tbx21-CNS-12 were critical for IL-12-induced T-bet expression during Th1 cell differentiation of CD4 T lymphocytes both in vitro and in vivo. Thus, type 1 innate and adaptive lymphocytes utilize distinct enhancer elements of the same TF in their development and differentiation. A similar differential pattern in expression of IFN-gamma in adaptive and innate immunity was observed when 3D chromatin structure was perturbed by deleting the boundary around the topological associated domain in vicinity of IFN-gamma and IL-22 gene. While the latter manipulation results in disbalance of Th1 and Th17 immunity upon T. gondii infection, the same element is dispensable for cytokine regulation in innate and innate-like lymphocytes (collaboration with Dr. Han-Yu Shih (NEI). In addition to the microbicidal activity of IFN-gamma, host resistance to T. gondii depends on the ability of hematopoietic system to timely replenish myeloid and lymphoid cell populations. In related work, we demonstrated that T. gondii infection impairs the ability of the thymus to restore the pool of nave T lymphocytes which in turn promotes a state of immune hypo-responsiveness that facilitates the establishment of chronic infection (Kugler et al., 2017). By uncovering a critical ligand-independent role for NOD1 in the STAT-5-dependent cytokine signaling pathway that is essential for optimal lymphopoiesis, we have now documented the importance of adult bone marrow hematopoietic function in the maintenance of host resistance to T. gondii infection. To formally demonstrate NOD1/STAT5 interaction in primary cells (as opposed to cell lines), we have developed both a new mAb specific for murine NOD1 and through the use of CRISPR technology, generate mice that express NOD1 with an HA-tag. Using these new reagents we are in process of examining the NOD1/STAT5 interaction in both hematopoietic and lymphoid cells. 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, 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. To address the role of IL-12-dependent innate IFN-gamma in these changes, we compared the BM composition of wild-type with that of TLR11/12-/- mice that display defect in NK-dependent IFN-gamma response. Our results showed that in addition to controlling parasite replication, early IFN-gamma response stimulated by T. gondii infection triggers the mobilization of myeloid and lymphoid precursors causing a state of emergency hematopoiesis in secondary lymphoid organs that is necessary to compensate for the loss of the bone marrow function. In parallel, we are investigating the cellular targets of the early IFN-gamma secreted by NK cells that regulates the balance between granulopoiesis vs. monopoiesis. By employing epigenomic and transcriptomic analyses we plan to track changes in specific populations of immature and mature monocytes to better understand the narrow time window of NK-IFN-gamma mediated action on hematopoietic cells.