Barbara Nikolajczyk - US grants

DESCRIPTION (provided by applicant): One of the first steps in successful B cell development is establishing an accessible, or open, immunoglobulin mu (IgM) chromatin structure. Increased accessibility is critical for converting the quiescent IgM locus of the hematopoietic precursor into a biologically functional, or activated, locus in the first committed member of the B lineage. However, the mechanisms regulating IgM accessibility are unknown. The proposed work will focus on determining how an inaccessible, or closed, mu chromatin structure becomes accessible during B cell development then remains accessible in the mature B cell. This work will answer the question: how is the mu enhancer activated in the context of chromatin? Completing the proposed work represents a first step towards the long-term goal of characterizing mechanisms driving tissue-specific regulation of IgM transcription. Work from multiple investigators spanning 20 years suggests that alterations in chromatin packaging regulate mu enhancer activation during B cell development. Increased mu enhancer accessibility, a prelude to full activation, correlates with transcription factor binding and modification of histone proteins packaging the mu enhancer. However, a direct "cause and effect" relationship showing transcription factors directing changes in chromatin structure has not been established. Overall, we will test the hypothesis that the mu enhancer, and hence B cell development is regulated by transcription factor-directed histone modifications through three approaches: 1. We will define the combination of proteins required for establishing maximal accessibility from a naturally chromatinized mu locus by ectopically expressing transcription factors in cells; 2. We will destroy DNA binding activity of the accessibility factors then measure stability of the open mu chromatin structure; 3. We will test how transcription factors that induce mu accessibility affect acetylation and methylation of the histones packaging the mu enhancer by chromatin immunoprecipitation. Understanding the details of mu locus accessibility during B cell development will provide logical targets for pharmaceutically controlling B cell generation in disease states by either blocking or enhancing this developmentally critical process. In addition understanding the rules for tissue-specific gene expression will allow exquisitely regulated delivery of treatments for a variety of single tissue syndromes from cancer to diabetes.

DESCRIPTION (provided by applicant): Type 2 diabetes (T2D) is associated with chronic inflammation that predominantly originates from immune system cells. These cells are chronically activated by the in vivo milieu of T2D patients, which includes elevated plasma levels of free fatty acids (FFAs) and endotoxin. Both of FFAs and endotoxin are ligands for toll-like receptors (TLRs), and both generally activate pro-inflammatory cytokine production upon TLR engagement. These cytokines likely play important roles in the chronic systemic inflammation characterizing T2D and other metabolic syndromes. Multiple studies show that controlling inflammation in obese mice by blocking TLR function decreases the incidence of insulin resistance, a hallmark of T2D. Although many cell types are sources of inflammatory cytokines, immune cell cytokines are required for insulin resistance. Taken together, these analyses indicate that TLR-activated inflammation from immune system cells is a key process that promotes T2D and other metabolic diseases. Although monocytes are generally considered the most important immune cell type for pro-inflammatory cytokine production, B cells also produce significant levels of cytokines. Our new data on B cells from T2D patients demonstrate that B cell TLR2 and TLR4 activation generally results in production of pro-inflammatory cytokines. However, TLR4 ligand decreases TLR2- mediated production of TNF-1. This anti-inflammatory effect may be balanced by the complete inability of B cells from T2D patients to secrete IL-10, a critical anti-inflammatory cytokine. The discoveries of an elevated percentage of TLR4-positive B cells in T2D patients and the mixed inflammatory responses of these B cells to TLR ligands lead to the hypothesis that B cells and B cell TLRs play important roles in T2D by regulating inflammation thus insulin resistance and glucose tolerance. Because the general endotoxemia and elevated free fatty acids in T2D patients provide innumerable TLR ligands, our preliminary data predict that B cell TLRs influence T2D through cytokine regulation in vivo. Although additional studies on human B cell TLR function are likely to be mechanistically and clinically important, the next critical step in this line of investigation is to unequivocally demonstrate B cells and B cell TLRs play roles in T2D inflammation and pathogenesis. These studies will be undertaken in a diet-induced obesity mouse model of T2D that takes advantage of available genetically altered mice. This project will test the effects of 1. the absence of B cells;2. TLR function only on B cells;and 3. B cell-specific TLR inactivation on inflammatory and metabolic outcomes of high fat diet in vivo. These studies will define the role of B cells in metabolic imbalance, and predict the likely efficacy of using approved B cell ablation therapies as new treatments for metabolic syndrome and T2D. PUBLIC HEALTH RELEVANCE: Type 2 diabetes (T2D) is increasing at epidemic rates world-wide, and the treatment arsenal for this disease is limited. Inflammation plays an important role in the etiology and complications of T2D. Inflammation stemming from immune cell cytokine production plays a demonstrated role in insulin resistance, one of the key features of T2D. The role of myeloid cell cytokines in T2D is relatively well understood. Our new work in T2D patients demonstrates that B cells may be another major source of cytokines in T2D. However, our human subjects research, by its nature, cannot definitively link B cells to disease pathogenesis. The project proposes to test the role of B cells and their surface receptors in T2D using model organisms. Importantly, this work will determine whether existing B cell ablation therapies can be used as fundamentally new treatments for T2D.

DESCRIPTION (provided by applicant): Periodontal disease (PD) pathogenesis is characterized by a chronically elevated immune response to the oral flora. Multiple immune cell types play roles in PD, with lymphocytes infiltrating the site of infection relatively late in disease progression. In patients with the most severe PD, the majority of cells in the gingival lesions are B cells. These findings suggest that B cells play a dominant role relatively late in PD pathogenesis. However, the function of B cells in PD has not been rigorously established in vivo. Our recent demonstration that B cells from PD patients constitutively secrete cytokines was accompanied by the discovery of an elevated percentage of Toll-like receptor (TLR) 2 and TLR4-positive B cells in human periodontal lesions and PD patient blood. Functional analyses demonstrate that B cell TLR2 and TLR4 activation generally results in production of pro- inflammatory and osteoclastogenic cytokines. Additionally, TLR4 ligand decreases TLR2-mediated production of IL-10, an important anti-inflammatory cytokine. The discoveries of an elevated percentage of TLR-positive B cells in PD patients, the overall pro-inflammatory responses of these B cells to TLR ligands, and the prevalence of B cells in PD lesions indicate that B cell TLRs play important roles in PD by regulating inflammation thus bone loss. Because the oral flora provides innumerable TLR ligands, our preliminary data predict that B cell TLRs promote inflammation directly through cytokine activation in vivo. Although additional studies on human B cell TLR function are likely to be mechanistically and clinically important, the next critical step in this line of investigation is to unequivocally demonstrate B cells and B cell TLRs play a role in PD. We hypothesize that B cell TLRs promote inflammation and bone loss thus exacerbate PD. We will test this hypothesis in the oral gavage mouse model of PD to determine how 1. absence of B cells; and 2. TLR2 or TLR4 expression only on B cells affect PD pathogenesis. These studies will unequivocally demonstrate roles of B cells in PD to significantly expand the understanding of basic B cell biology in mucosal inflammatory diseases. PUBLIC HEALTH RELEVANCE: Periodontal Disease (PD) is a common infection linked to serious complications including cardiovascular disease. Novel PD treatments are needed to supplement current regimens, which have limited effectiveness in many patients. Although the immune system plays an important role in PD, the importance of one immune cell type, B cells, is poorly understood. New evidence from PD patients suggests that B cells play important roles in PD, but human subjects research, by its nature, cannot definitively link B cells to disease pathogenesis. The project proposes to test the role of B cells and their surface receptors in PD using model organisms with a long-term goal of identifying new treatments for PD.

DESCRIPTION (provided by applicant): The appreciation of insulin resistance (IR) and type 2 diabetes (T2D) as chronic inflammatory diseases has necessitated development of the new field of immunometabolism, which will define the role of the immune system in metabolic disease. Recent animal studies have shown that inflammatory T cells and B cells play key roles in the development of IR in T2D. For example, T cell skewing towards the pro-inflammatory Th17 and Th1 subsets and elevated Th17/Th1 numbers in the expanding diet-induced obesity (DIO) mouse adipose tissue (AT) promotes inflammation, thus IR (1, 5). Importantly, Th17 and Th1 cells are major sources of IL-17 and IFN-¿, respectively, two pro-inflammatory, anti-adipogenic, IR-linked cytokines. Complementary studies showed loss of anti-inflammatory regulatory T cells (Tregs) increases AT inflammation and serum insulin levels, while gain of Treg function decreases AT inflammation and rescues glucose sensitivity. Together, these studies show a pro-inflammatory T cell balance plays important roles in IR. Similar approaches also show B cells promote inflammation and IR. Like these murine studies, our pioneering human immunometabolism work identified a pro-inflammatory T cell subset balance in blood from T2D patients: pro-inflammatory Th17 and Th1 cells are elevated, and anti-inflammatory Tregs are reduced. Furthermore, we found that blood B cells from T2D patients are pro-inflammatory. Importantly, new data show T cell/B cell interaction is critical for elevated Th17 function in T2D; thus we have initiated some of the first mechanistic immunometabolism studies in human samples. Taken together, our work indicates that detailed human immune cell analysis will identify new mechanisms of IR/T2D thus introduce new concepts in assessment and treatment strategies. Furthermore, detailed analysis of the human immune system in IR/T2D individuals is absolutely essential to justify attempts to harness the power of the immune system as a biomarker and/or treatment for T2D. We hypothesize that the elevated pro-inflammatory T cell function we have found in T2D 1. is similar in blood and AT from a single individual; 2. Predicts IR and/or AT function in obesity patients; and 3. Requires support from B cells, which can be safely ablated with FDA-approved drugs. Testing these hypotheses would assess the innovative possibility that a simple blood test (T cell subset analysis) can identify risk of transition from insulin-sensitive to IR. The availability of such a test would be a major advance with immediate clinical impact. In addition to preliminarily identifying a biomarker for the IS/IR transition, outcomes will address the novel possibility that existing B cell depletion drugs may prevent the IS/IR transition.

? DESCRIPTION (provided by applicant): Inflammation is generally required for the development of insulin resistance in obese animals, consistent with demonstrations that inflammation predisposes obese people to be metabolically unhealthy. In contrast, obese people with negligible inflammation are often metabolically healthy. The role of inflammation as an accelerator of obesity-associated metabolic decline indicates that an inflammatory signature can predict the transition from obese/metabolically healthy to obese/insulin resistant/type 2 diabetes (T2D). Our proposed work will comprehensively define inflammatory signature(s) during T2D etiology and pathogenesis to fill critical gaps in 1. Identifying people most likely to transition to T2D in response to obesity; 2. Pinpointing treatments to new targets thus improve the currently modest efficacy of anti-inflammatory drugs in T2D; and 3. Using targeted therapies to uncouple obesity from inflammatory-mediated complications such as T2D and cardiovascular disease. We have used a multivariate mathematical approach to identify a previously unappreciated inflammatory signature that differentiates obese/non-T2D, obese/pre-T2D and obese/T2D subjects. This signature includes multiple T cell cytokines, many of which are preferentially produced by the Th17 or Th1 T cell subsets. Our identification of a human T cell inflammatory signature coupled with the proposed extention of the analyses to additional putative sources of diabetogenic inflammation provide unique opportunities to address important outstanding questions in the relationship between inflammation and T2D pathogenesis. We will test the hypothesis that a T cell signature that distinguishes T2D from non-T2D subjects is a predictive biomarker for T2D pathogenesis through a longitudinal multiple-PI project involving experts in human immunometabolism, cytokine network modeling and clinical research in obesity. The possibility that a cytokine signature validated by the proposed work will identify people who require more intensive monitoring, intervention or new anti-inflammatory drugs to delay or prevent obesity-associated T2D has tremendous potential to change clinical practice, emphasizing both the impact and the urgency of the project.

Periodontitis fuels the inflammation of obesity-associated type 2 diabetes (T2D), associates with poor glycemic control, and increases T2D morbidity. New strategies are critically needed to counter sources of periodontal infection and the resulting inflammation, both of which are refractory to standard treatments in people with T2D. However, mechanisms underlying the relationship between periodontitis and T2D remain poorly understood, impeding clinical progress. One unifying link between periodontitis and T2D is altered B cell function, and recent collaborative work between Drs. Barbara Nikolajczyk and Thomas Van Dyke showed that T2D-associated changes in B cells promote periodontitis. Our work further indicates that T cells cannot drive chronic periodontitis in T2D hosts in the absence of B cells, despite evidence that T cells promote periodontitis in lean hosts. Taken together, these findings support a model in which B cells support T2D-potentiated periodontitis, while T cells dominate periodontitis in leans. Both B cells and T cells are major sources of receptor activator of nuclear factor kappa-B ligand (RANKL), a key driver of osteoclastogenesis and periodontal bone loss. Obesity/T2D increases hematopoietic cell production of RANKL, most likely through increasing concentrations of a number of cytokines (TNF?, IL-1? and IL-6) known to drive RANKL production. These data, together with our demonstration that B cells are required for T2D-potentiated osteoclastogenesis and periodontitis, support our central hypothesis: T2D cytokines specifically up regulate B cell RANKL function, which uniquely potentiates periodontal complications of T2D. Definitive analyses are needed to fill the critical gaps in knowledge of how obesity-associated T2D impacts lymphocyte RANKL induction and function, and whether cellular sources of osteoclastogenic RANKL differ in T2D compared to lean hosts. We will use loss- and gain-of-function approaches in a standard mouse model of T2D, coupled with a standard model of chronic periodontitis, to study development of T2D-potentiated periodontal disease. We will complement the disease etiology work in mice with analysis of gingiva from people with T2D to query mechanistic underpinnings of chronic periodontitis. This strategy will identify drivers of both early and chronic phases of T2D-potentiated periodontitis to meet our long-term objective: to identify key factors that promote periodontitis in T2D compared to non-T2D subjects, and thereby pinpoint drug targets for future studies.