Objective
We used a unique approach to find new genes that might be involved in T1D pathogenesis or progression. The anti-diabetic and anti-inflammatory lipids, Palmitic-Acid-Hydroxy-Stearic-Acids (PAHSAs), markedly reduce the incidence and delay the onset of autoimmune T1D in non-obese diabetic (NOD) mice. PAHSAs attenuate autoimmune responses by lowering immune cell infiltration in the islets of NOD mice. PAHSAs also directly protect islets β-cells independent of immune modulation since they attenuate cytokine-induced apoptosis and necrosis, reduce endoplasmic reticulum stress and prevent and reverse metabolic-stress-induced senescence in human islets. PAHSAs also directly augment glucose-stimulated insulin secretion from human islets. Bulk RNA sequencing in islets from PAHSA-treated female NOD mice revealed several genes for which the human homologs strongly associate with T1D in human GWAS databases, including IER3 (Immediate Early Response-3). We chose to study the role of IER3 in T1D pathogenesis since it plays a critical role in pro-inflammatory signaling pathways. Furthermore, mice overexpressing IER3 in immune cells exhibit higher susceptibility to lupus-like autoimmune disease. In addition, in human islets from normal donors and NOD mouse-derived clonal pancreatic β-cells (NIT-1 cells), cytokines and thapsigargin (endoplasmic reticulum-stress inducer) treatment increased IER3 gene expression.
In this innovative proposal, we will identify the role of the novel gene, IER3, in T1D pathogenesis. Since IER3 is associated with the beneficial effects of PAHSAs to prevent T1D NOD mice, IER3 may improve β-cell survival and function, and could attenuate T1D in people at risk for T1D. We will study this under three aims: Specific Aim 1 and 2: To determine whether knocking down IER3 or inhibiting its function improves β-cell survival and function, and delays the onset of, or reduces diabetes incidence in NOD mice. Here, we will knockdown the IER3 gene in human islets from normal donors and NIT-1 cells using small interference RNA. After the knockdown, we will measure β-cell survival, function, and proliferation. To check if knocking down these genes protects β-cells under stress conditions, we will treat NIT-1 cells and human islets with high glucose, cytokines, or thapsigargin after knocking down these genes. We will measure β-cell survival and function. We will also use a small molecule inhibitor for IER3, Zunsemetinib, to study whether inhibitor treatment delays the onset or reduces diabetes incidence in NOD mice and improve β-cell survival and function. We will also treat human islets with Zunsemetinib and study β-cell survival and function under cytokine and endoplasmic reticulum stress. Specific Aim 3: To determine the effects of IER3 inhibition on diabetogenic T cell activation and immune function of antigen-presenting cells. Here, we will treat human peripheral blood mononuclear cells with IER3 inhibitor, Zunsemetinib, and activate them to study the activation profile of diabetogenic T cells. We will also measure if inhibiting IER3 gene function affects the immune function of antigen-presenting cells, and cytokine and chemokine release.
We anticipate that this approach will identify IER3 as a novel drug target that has not yet been studied, and could play a vital role in promoting β-cell survival and function in the setting of immune attack. If these studies are promising, in the future we will carry out similar studies in autoimmune NOD mice with a humanized immune system to investigate the effects of IER3 inhibition on survival of the transplanted human islets. Overall, the proposed studies will provide insights into how the knockdown of a novel uncharacterized gene, IER3, modulates islet β-cell function and promotes survival and proliferation to blunt the progression to diabetes or even reverse it in people. These studies may also reveal new previously unidentified mechanisms by which one can improve β-cell survival and function without depleting islet insulin content.
Background Rationale
Type 1 Diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin producing islet β cells. Agents currently used for prevention or treatment of recent onset T1D in humans provide only transient islet β cell preservation and have adverse effects. Recent data show that immune modulatory agents can delay the onset of T1D but do not permanently prevent it. Thus, agents which are safe, and can preserve or regenerate islet β cells, and enhance insulin production are needed. At present, combination drug therapy including low doses of immune drug modulators and agents that improve islet β cell survival and function are being tested to minimize potential side effects while retaining efficacy.
Specific classes of lipids including short chain fatty acids and omega (ɷ)-3 polyunsaturated fatty acids reduce inflammation and attenuate T1D in an autoimmune non-obese diabetic (NOD) mouse model. The anti-diabetic and anti-inflammatory lipids, palmitic acid hydroxy stearic acids (PAHSAs) which we discovered, markedly reduce the incidence and delay the onset of T1D in NOD mice. PAHSAs attenuate autoimmune responses by lowering mature T helper cell activation and B cell number, and increasing regulatory T cell (Treg) activity the pancreatic infiltrates of NOD mice. PAHSAs also directly protect β-cells independent of immune modulation since they attenuate cytokine-induced apoptosis and necrosis, reduce endoplasmic reticulum stress and prevent and reverse metabolic-stress-induced senescence in human islets. PAHSAs also directly augment glucose-stimulated insulin secretion from human islets. Many Fatty Acid Hydroxy Fatty Acids (FAHFAs) including PAHSAs and ɷ-3-FAHFAs are anti-inflammatory and lower LPS-induced dendritic cell activation and pro-inflammatory cytokine production. In a mouse model of colitis, PAHSAs lower colonic inflammation and delay the onset and reduce the severity of colitis. The fact that PAHSAs modulate immune cell responses and have direct protective effects on β-cells independent of the immune system makes them unique compared to other conventional approaches to prevent or treat early T1D which focus primarily on immune cell modulation. Based on the published and preliminary data, we hypothesize that additional genes are likely to be involved in the beneficial effects of PAHSAs on islet β-cells since bulk RNA seq analysis revealed genes that are associated with T1D in Genome-Wide Association Studies (GWAS) data. The overall goal is to determine distinct PAHSA-regulated genes that could be targeted to promote islet β-cell survival and function, and prevent T1D progression. In this context, we have identified the PAHSA-regulated novel gene, Immediate Early Response-3 (IER3) gene to be involved in T1D pathogenesis. Targeting IER3 gene and the pathways it regulate may prevent the immune system from attacking healthy β-cells and thereby prevent T1D. Therefore, identifying anti-inflammatory agents or their targets that are safe and effective could lead to new therapeutic agents to attenuate T1D in people at risk for T1D.
Description of Project
Type 1 diabetes (T1D) results from destruction of insulin-producing islets β-cells by one`s own immune system. There is no known way to prevent T1D. Recent data show that immune modulatory agents can delay the onset of T1D but do not permanently prevent it. Many clinical trials resulted in only transient β cell preservation and/or adverse effects due to whole body suppression of the immune system. Also, immune modulation with some agents has proven heterogeneous in efficacy in new-onset T1D humans. Islet transplantation has also been pursued, but it has several limitations including a shortage of donors, fibrosis of transplanted islets, and side effects of immunosuppressive agents. Therefore, agents are needed that are safe, and can preserve β cells and enhance endogenous insulin production in humans at risk for or with overt T1D.
We discovered a new class of lipids called Fatty Acid Hydroxy Fatty Acids (FAHFAs) with potent anti-inflammatory and anti-diabetic activity. One type of FAHFA, Palmitic Acid Hydroxy Stearic Acids (PAHSAs) protects against T1D in mice and increases human islet β-cell survival and function. Daily oral PAHSA administration to mice with autoimmune T1D, delayed T1D onset and markedly reduced T1D incidence when PAHSA treatment was started either before or after immune attack of β-cells. PAHSAs also directly protect β-cells independent of immune modulation since they attenuate several types of cell death induced by immune modulators, reduce endoplasmic reticulum stress, and prevent and reverse metabolic-stress-induced senescence (pathologic aging) in human islets. PAHSAs also directly augment glucose-stimulated insulin secretion from human islets. Analysis of thousands of genes in islets from PAHSA-treated female NOD mice identified several genes for which the human homologs strongly associate with T1D in human Genome Wide Association Studies. One gene is IER3 (Immediate Early Response-3). We chose to study the role of IER3 in T1D pathogenesis since it plays a critical role in pro-inflammatory signaling pathways. Mice overexpressing IER3 in immune cells exhibit higher susceptibility to lupus-like autoimmune disease. In addition, our preliminary data shows that in human islets from normal donors and in T1D- mouse-derived clonal pancreatic β-cells, cytokines and a compound that induces endoplasmic reticulum-stress increased IER3 gene expression.
Therefore, we hypothesize that the protein encoded by the IER3 gene may be involved in T1D pathogenesis. This grant aims to determine whether a gene that is newly found to be linked to T1D, IER3, augments beta cell immune attack and whether inhibiting IER3 may prevent the immune system from attacking healthy β-cells and thereby prevent T1D. In this innovative proposal, we will determine whether knocking down the IER3 gene or inhibiting its function with small molecule inhibitors attenuates immune responses and improves β-cell survival. We will use human islets from normal donors and diabetic mouse β-cells to study the role of IER3 in islet β-cells under stress conditions. The investigational small molecule inhibitor for IER3, Zunsemetinib (ATI-450; CDD-450), is an orally active, selective inhibitor that attenuates pro-inflammatory immune function. This inhibitor is currently being studied in clinical trials in auto-inflammatory diseases, including Rheumatoid Arthritis and inflammatory bowel disease. We hypothesize that Zunsemetinib might also attenuate immune cell activation and promote β-cell survival and function in people at risk for, or with early, T1D. We will determine whether chronic Zunsemetinib treatment delays the onset or reduces diabetes incidence in NOD mice. We will also study whether Zunsemetinib treatment attenuates diabetogenic T cell activation and immune function of antigen-presenting cells in human peripheral blood mononuclear cells. The proposed studies will determine whether this novel islet gene could be targeted as an innovative approach to prevent T1D and/or potentially reverse early-stage T1D in people.
Anticipated Outcome
We anticipate that the approaches employed in Specific Aim 1 will identify Immediate Early Response-3 (IER3) as a novel candidate gene that has not yet been studied to play a vital role in promoting β-cell survival and function in the settings of Type 1 Diabetes (T1D). These studies will indicate whether knockdown of IER3 gene using small interference RNA or inhibiting IER3 gene function by a small molecule inhibitor in human islets and NIT-1 cells improve β-cell survival and function under stress conditions. We also expect IER3 inhibitor to improve islet β cell survival and function in human islets from normal donors.
In Specific Aim 2, we expect IER3 inhibitor studies to delay the onset of and attenuate T1D incidence in an autoimmune non-obese diabetic (NOD) mice. We also expect to identify IER3-mediated distinct pathways or targets responsible for β cell survival and function. If these studies are promising, in the future we will carry out similar studies in autoimmune NOD mice with a humanized immune system to investigate the effects of IER3 inhibitor on survival and rejection of the transplanted human islets. This information can be used to develop targets or the pathways they regulate as a therapeutic strategy to treat T1D in people.
In Specific Aim 3, we expect that IER3 inhibitor treatment will lower diabetogenic immune cell activation and function in human peripheral blood mononuclear cells to attenuate diabetes incidence. We anticipate that specific types of immune cells are affected with inhibitor treatment. If indicated, we will study these immune cells isolated from normal humans to determine whether these effects are primary or secondary. It is also possible that inhibitor treatment will target unknown immune cells in mediating their beneficial effects in the settings of T1D. Overall, the proposed studies will provide insights into how the knockdown of a novel uncharacterized gene, IER3, modulates islet β-cell function and promotes survival and proliferation to blunt the progression to diabetes or even reverse it. These studies may also reveal new previously unidentified mechanisms by which one can improve β-cell survival and function without depleting islet insulin content.
Relevance to T1D
Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β cells. The loss of β cells results in insulin deficiency and impaired glucose homeostasis manifested by hyperglycemia. Both genetic and environmental factors play important roles in the development of islet autoimmunity and subsequent progression to T1D. New cases of T1D are increasing worldwide and compromising patients’ quality and length of life. Despite major advances in glucose monitoring and insulin delivery techniques, many patients with T1D develop long-term complications. Abrogating autoimmunity and increasing β cell regeneration, proliferation, and islet β cell transplantation in the pursuit of a cure for T1D in humans remain major challenges. Many clinical trials have been performed but resulted in only transient β cell preservation and/or adverse effects due to generalized immune suppression. Recent data show that immune modulatory agents can delay the onset of T1D but do not permanently prevent it. Immune modulation with few agents have proven heterogeneous in their efficacy in new-onset T1D humans. Islet transplantation has also been pursued, but it has several limitations including a shortage of donors, fibrosis of transplanted islets, and side effects of immunosuppressive agents. The generation of stem cell–derived β cells and efforts to enhance insulin production is underway. In addition, although markers to predict T1D have improved, there is still a dire need for novel approaches to halt the progression of the autoimmune response to prevent T1D in islet antibody–positive individuals who have not yet developed overt diabetes. Therefore, agents are needed that are safe, preserve β cells and enhance endogenous insulin production in humans at risk for or with overt T1D.
In this context, the anti-diabetic and anti-inflammatory lipids our lab has discovered modulate immune cell responses and also have direct protective effect on β-cells, this makes them unique compared to other agents to prevent and/or treat early T1D which are primarily immune-modulators and are used for only short periods due to adverse effects. The proposed studies will identify novel PAHSA-regulated islet genes that could be targeted as therapeutic strategies for preventing T1D and/or potentially reversing early-stage T1D in people. Another favorable aspect for the development of this small molecule inhibitor as a potential pharmaceutical agent to treat T1D is the fact that this inhibitor is currently being studied in clinical trials in other auto-inflammatory diseases including Rheumatoid Arthritis and inflammatory bowel disease. Studies proposed here will contribute to a larger translational goal of eventually testing a small molecule IER3 inhibitor, Zunsemetinib, in humans to reduce the risk of developing T1D. Zunsemetinib may also 1) promote β-cell survival and enhance insulin secretion in newly diagnosed T1D people, 2) prolong the survival of transplanted islets or human pluripotent stem cells-derived β-cells, and 3) allow reduction of immunosuppressive drugs which have side effects in T1D people after islet transplantation.