One of the primary goals of our study is to understand the roles played by the transcription factor, IRF8 in hematopoietic cells. In the mouse, studies have focused on dendritic cells, B cells and macrophages, which are complemented by analyses of human B cell lines and normal B cells. We have studied expression of IRF8 during B cell development in the bone marrow (BM) from hematopoietic stem cells (HSC) to immature B cells. These studies demonstrated that the gene is expressed at low levels in HSC but at greatly increased levels in common lymphoid progenitors and the earliest cells committed to the B cell pathway (pre-pro-B), and then at reduced levels in more mature cells. IRF8 was shown to regulate expression of genes encoding PU.1 and EBF1, thereby promoting B lineage choice by HSC. This places IRF8 in the transcriptional regulatory network that includes PU.1, IKAROS, E2A, EBF and PAX5 and which is responsible for B cell lineage specification, commitment and differentiation. We showed previously that in peripheral B cells, expression of IRF8 increases to highest levels in FOL B cell-derived germinal center (GC) B cells and then is strikingly downregulated in plasma cells. IRF8 contributes to the transcriptional regulation of two genes critically involved in GC B cell differentiation and function, BCL6 and AID. AID is the essential regulator of somatic hypermutation (SHM) and class witch recombination (CSR). Studies of AID-expressing cell lines with GC B cell features demonstrated that Igk V region genes were inducibly mutated at high rates, with a focus on A:T mutations that occur preferentially during phase 2 of SHM. Unexpectedly the mutation patterns resembled those seen with Poleta and MSH2 deficiencies suggesting an additional pathway for A:T mutations conserved in mice and humans. More recently, we found that IRF8 is physically associated with BCOR, a suppressor of BCL6 gene expression, providing a mechanistic link between IRF8 and BCL6 regulatory loops. SHM and CSR result in the formation of DNA strand breaks that in other cells could lead to cell cycle arrest driven by p21 and p53-d3pendent apoptosis. IRF8 also appears to collaborate with BCL6 in suppressing the activities of p53 and, secondarily, p21. Recently, we showed that IRF8 also activates the transcription of MDM2, a protein that normally induces degradation of p53. Suppression of p21, p53 and activation of MDM2 thus makes it possible for B cells to tolerate double stranded DNA breaks that occur physiologically during SHM and CSR. This feature of GC B cells might have unfortunate consequence of allowing escape of self-reactive cells that could contribute to autoimmune diseases. More recently, we identified a novel partner of IRF8, cyclin D3, an important component of the cell cycle machinery. This new partner of IRF8 allows us to understand the mechanisms of IRF8 regulation of proliferation in pre-B and germinal center B cells. IRF8 has also been shown to be an important contributor to the development of the Th17 T cell subset that plays important roles in autoimmunity and inflammation. In a collaboration with Dr. Xiongs group (Mt. Sinai Schood of medicine), we found that through interactions with RORgt, IRF8 functions as a transcriptional inhibitor of Th17 differentiation and plays role in suppression of colitis progression. Autoantibodies are also a prominent feature of the BXSB-Yaa mouse model of systemic lupus erythematosus. Previous studies showed that these mice express high levels of IL-21, a cytokine produced by follicular T helper cells known to drive GC reactions. To determine if IL-21 has a role in disease pathogenesis, the mice were made deficient in expression of the IL21 receptor (IL-21R). Remarkably, IL-21R-deficient BXSB-Yaa mice were essentially normal with none of the autoimmune or other features that normally characterize that strain. The development of severe SLE-like autoimmunity in this strain was found to be normally restrained by the activity of CD8 T cell-NK cell regulatory axis that appears to restrict the activity of T follicular helper cells. ENPP1 is an ectonucleotide pyrophosphatase phosphodiesterase that is broadly expressed in many tissues including the hematopoietic system. ENPP1 plays a role in phosphate metabolism by hydrolyzing NTP to generate pyrophosphate, an important inhibitor of calcification and bone formation. In addition, ENPP1 has been shown to modulate insulin receptor signal transduction and purinorector signaling such that overexpression of ENPP1 is associated with obesity and insulin resistance. Finally, ENPP1 Is involved in conversion of extracellular ATP to AMP, which is then converted to adenosine, an immunosuppressive factor. While this molecule has been discovered for four decades, mechanisms governing its expression and its functional role in B lineage cells have not been investigated. We have identified an antibody that recognizes ENPP1 on the surface of cells from all inbred strains of mice making it possible for the first time to study the biology of this protein in any mouse system. We found that ENPP1 is expressed at high levels on plasma cells and germinal center B cells compared with nave B cells. Studies on ENPP1-null mice led to the discovery that ENPP1 is required for plasma cell survival in the bone marrow microenvironment. Our results suggest that the long-lived plasma cells rely on ENPP1 to establish a survival niche in the bone marrow in a cell intrinsic manner. Therefore, inhibition of ENPP1 could serve as a novel therapeutic strategy for plasma cell-related diseases such as autoimmune diseases and multiple myeloma. More recently, by using the anti-ENPP1 monoclonal antibody, we have identified and characterized two novel B1a B cell subpopulations in mice. The two subsets express different immunoglobulin gene repertoires and exhibit different patterns in migration, IL-10 production, IgA secretion and immune responses to pneumococcal polysaccharide type 3. This latter finding has important implications in clinical immunization routines in young children with pneumococcal polysaccharide vaccines because we found that young mice have very few cells of the responsive subset and may thus be immunologically refractory to certain types of bacterial polysaccharide antigens. Identification of B1a B cell subsets in all mice reveals not only new aspects of the layered immune system during development but also divided labors among these important innate-like immune effectors. Strikingly, studies with ENPP1 have led to identification of a novel disease-associated B1a population unique to aged NZB and NZB/W F1 mice. Further characterization of this novel population in other autoimmune strains and humans will provide novel opportunities for understanding the pathogenesis of B1 cells in autoimmunity and help to design new strategies to target pathogenic B cell populations. The IgM antibodies produced by B1a cells play important roles in immune regulation and innate immunity. By investigating the mice deficient for the high affinity receptor for IgM, the FCMR, we have found that FCMR-deficient mice have reduced numbers of developing B cells, splenic follicular and peritoneal B-2 cells, but increased levels of peritoneal B-1a cells and autoantibodies. After immunization, germinal center B cell and plasma cell numbers are increased. FCMR-deficient B cells are sensitive to apoptosis induced by BCR ligation. Our studies demonstrate that FCMR is required for B cell differentiation and homeostasis, the prevention of autoreactive B cells, and responsiveness to antigenic challenge.

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Project End
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Support Year
7
Fiscal Year
2013
Total Cost
$1,170,431
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Wang, Hongsheng; Yan, Ming; Sun, Jiafang et al. (2014) A reporter mouse reveals lineage-specific and heterogeneous expression of IRF8 during lymphoid and myeloid cell differentiation. J Immunol 193:1766-77
Gaidamakov, Sergei; Maximova, Olga A; Chon, Hyongi et al. (2014) Targeted deletion of the gene encoding the La autoantigen (Sjogren's syndrome antigen B) in B cells or the frontal brain causes extensive tissue loss. Mol Cell Biol 34:123-31
Yoshida, Yuko; Yoshimi, Ryusuke; Yoshii, Hiroaki et al. (2014) The transcription factor IRF8 activates integrin-mediated TGF-? signaling and promotes neuroinflammation. Immunity 40:187-98
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