Functions and Mechanisms of NF-kB Factors and their Regulators

National Institute Of Allergy And Infectious Diseases

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Abstract

This project is focused on the identification of physiologically critical functions and mechanisms of action of NF-kB transcription factors and their regulators in health and disease. NF-kB is a family of related dimeric transcription factors that serve as primary intracellular mediators during innate and adaptive immune responses. In addition, and importantly, aberrant regulation of NF-kB plays a major role in inflammatory and autoimmune diseases as well as in numerous tumors. It is thus imperative to understand the functions and mechanisms of action of individual NF-kB factors and their regulators, as this will be required to devise appropriate strategies for therapeutic interventions aimed at curtailing aberrantly regulated NF-kB in a precisely targeted manner. To identify physiologic roles and mechanisms we make use of mouse models engineered to lack components of the NF-kB transcription factor family or their regulators, as well as models in which the NF-kB factors can be selectively activated. Our work is focused on so-called alternatively, but also classically activated NF-kB, but we are especially investigating the regulators Bcl-3 and IkBzeta. The alternative NF-kB activation pathway is normally initiated by a subset of TNF receptors. Bcl-3 and IkBzeta are atypical IkB family member that function as nuclear regulators of NF-kB activity. We previously discovered a critical role for Bcl-3 in the ability of dendritic cells to properly prime T cells to proliferate in response antigen, and thus to initiate a protective adaptive immune response to pathogens, such as to Toxoplasma gondii, a serious health risk in immune-compromised patients. In the absence of Bcl-3 in dendritic cells, mice succumb to this infection. Bcl-3 also has critical functions epithelial cells, such as in keratinocytes, where it helps to delimit hypersensitivity reactions. Of particular interest is our previous discovery that Bcl-3 is required in T cells to drive T-cell dependent autoimmune diseases, including experimental autoimmune encephalomyelitis, a model for Multiple Sclerosis, and T cell transfer-induced colitis, a model for Inflammatory Bowel Disease. In addition, we recently discovered that in contrast to its pro-tumorigenic role in B cells, it has a tumor-suppressive role in gut epithelial cells. Mice lacking Bcl-3 in these cells exhibited an increased tumor rate in a colitis inflammation-induced colon cancer model, highlighting the context-specific functions of the regulator. In FY2019 we have made significant progress towards our long-term goal to identify context-dependent functions of Bcl-3, its mechanisms of actions and its transcriptional targets. First, we collected wild-type and Bcl-3-deficient CD4 T cells from CD4-T cell transfer induced colitis mice and subjected them to bulk RNAseq analyses and are presently verifying potentially critical targets of Bcl-3. We also established an acute LCMV viral infection model to elucidate the role of Bcl-3. Using this model, we determined that Bcl-3 appears to have an important role in CD8 memory T cell development and we are in the process of analyzing single-cell RNAseq experiments. This opens the door towards understanding the overall functions, target genes and pathways controlled by Bcl-3 in a cell-type specific manner. Understanding what genes Bcl-3 controls in CD8 T cell memory formation is also vital in the context of tumor immunotherapy, where CD8 memory-like T cells, rather than effector T cells are thought to be specifically efficacious in long-term tumor suppression. In order to better understand the mechanisms of action of Bcl-3 we have made significant progress in identifying interacting proteins. In mice lacking Bcl3 globally or specifically in CD11c+ cells, we previously reported that Toxoplasma gondii infection is uniformly fatal and is associated with an impaired Th1 immune response. Since Bcl3 expression in dendritic cells (DC) is pivotal for antigen presentation and since classical DCs (cDC) are major antigen presenting cells, in FY23 we investigated the role of Bcl3 specifically in cDCs in vivo by crossing Zbtb46 cre mice with Bcl3flx/flx mice. Bcl3flx/flx Zbtb46 cre mice were as susceptible to lethal T. gondii infection as total Bcl3-/- mice and generated poor Th1 immune responses. Consistent with this, compared to wildtype controls, splenic Xcr1+ Bcl3-deficient cDC1 cells were defective in presenting Ova antigen to OT-I cells both for Ova257-264 peptide and after infection with Ovalbumin-expressing T. gondii. Moreover, splenic CD4+ and CD8+ T cells from infected Bcl3flx/flx Zbtb46 cre mice exhibited decreased T. gondii-specific priming as revealed by both reduced cytokine production and reduced T. gondii-specific tetramer staining. In vitro differentiation of cDCs from bone marrow progenitors also revealed Bcl3-dependent cDC-specific antigen-presentation activity. Consistent with this, splenocyte single cell RNA seq (scRNAseq) in infected mice revealed Bcl3-dependent expression of genes involved in antigen processing in cDCs. We also identified by scRNAseq, a unique Bcl3-dependent hybrid subpopulation of Zbtb46+ DCs co-expressing the monocyte/macrophage transcription factor Lysozyme M. This subpopulation exhibited Bcl3-dependent expansion after infection. Likewise, by flow cytometry we identified two T. gondii-induced hybrid subpopulations of Bcl3-dependent cDC1 and cDC2 cells both expressing monocyte/macrophage markers, designated as icDC1 and icDC2. Together, our results indicate that Bcl3 in classical DCs is a major determinant of protective T cell responses and survival in T. gondii-infection.