Aims/hypothesis Islet inflammation leads to loss of functional pancreatic beta cell mass. Increasing evidence suggests that activation of 12-lipoxygenase leads to inflammatory beta cell loss. This study evaluates new specific small-molecule inhibitors of 12-lipoxygenase for protecting rodent and human beta cells from inflammatory damage. Methods Mouse beta cell lines and mouse and human islets were treated with inflammatory cytokines IL-1β, TNFα and IFNγ in the absence or presence of novel selective 12-lipoxygenase inhibitors. Glucose-stimulated insulin secretion (GSIS), gene expression, cell survival and 12-Shydroxyeicosatetraenoic acid (12-S-HETE) levels were evaluated using established methods. Pharmacokinetic analysis was performed with the lead inhibitor in CD1 mice. Results Inflammatory cytokines led to the loss of human beta cell function, elevated cell death, increased inflammatory gene expression and upregulation of 12-lipoxygenase expression and activity (measured by 12-S-HETE generation). Two 12-lipoxygenase inhibitors, Compounds 5 and 9, produced a concentration-dependent reduction of stimulated 12-S-HETE levels. GSIS was preserved in the presence of the 12-lipoxygenase inhibitors. 12-Lipoxygenase inhibition preserved survival of primary mouse and human islets. When administered orally, Compound 5 reduced plasma 12-S-HETE in CD1 mice. Compounds 5 and 9 preserved the function and survival of human donor islets exposed to inflammatory cytokines. Conclusions/interpretation Selective inhibition of 12-lipoxygenase activity confers protection to beta cells during exposure to inflammatory cytokines. These concept validation studies identify 12-lipoxygenase as a promising target in the prevention of loss of functional beta cells in diabetes.

Enteroviral infections have been associated with the development of type 1 diabetes (T1D), a chronic inflammatory disease characterized by autoimmune destruction of insulin-producing pancreatic beta cells. Cultured human islets, including the insulin-producing beta cells, can be infected with coxsackievirus B4 (CVB4) and thus are useful for understanding cellular responses to infection. We performed quantitative mass spectrometry analysis on cultured primary human islets infected with CVB4 to identify molecules and pathways altered upon infection. Corresponding uninfected controls were included in the study for comparative protein expression analyses. Proteins were significantly and differentially regulated in human islets challenged with virus compared with their uninfected counterparts. Complementary analyses of gene transcripts in CVB4-infected primary islets over a time course validated the induction of RNA transcripts for many of the proteins that were increased in the proteomics studies. Notably, infection with CVB4 results in a considerable decrease in insulin. Genes/proteins modulated during CVB4 infection also include those involved in activation of immune responses, including type I interferon pathways linked to T1D pathogenesis and with antiviral, cell repair, and inflammatory properties. Our study applies proteomics analyses to cultured human islets challenged with virus and identifies target proteins that could be useful in T1D interventions.

ML355 was efficacious in improving human islet function after cytokine treatment and in T2D islets in vitro. The study suggests that the blockade of the 12-LO pathway may serve as a target for both form of diabetes and provides the basis for further study of this small molecule inhibitor in vivo.

The metabolic syndrome and diabetic conditions support atherosclerosis, but the exact mechanisms for accelerated atherogenesis remain unclear. While the pro-inflammatory role of signal transducer and activator of transcription 4 (STAT4) in atherosclerosis and diet-induced insulin resistance (IR) was recently established, an impact of STAT4 on atherogenesis in conditions of IR is not known. Here we generated Stat4−/−Ldlr−/− mice that were fed a diabetogenic diet with added cholesterol (DDC). DDC fed Stat4−/−Ldlr−/− mice demonstrated improved glucose tolerance, insulin sensitivity, and a 36% reduction in atherosclerosis compared with Ldlr−/− controls. Interestingly, we detected a reduction in T follicular helper (Tfh) and plasma B cells, but a sharp elevation in CD8+ Tregs in spleens and aortas of Stat4−/−Ldlr−/− versus Ldlr−/− mice. Similarly, STAT4 deficiency supported CD8+ Treg differentiation in vitro. Stat4-deficient CD8+ Tregs suppressed Tfh and germinal center B cell development upon immunization with KLH indicating an important role for STAT4 in CD8+ Treg functions in vivo. Furthermore, adoptive transfer of Stat4−/−Ldlr−/− CD8+ Tregs vs Ldlr−/− CD8+ Tregs resulted in a significant reduction of plaque burden, suppression of Tfh and germinal center B cells in DDC fed Ldlr−/− recipients. STAT4 expression in macrophages also affected the Tfh/CD8+Treg axis, as conditioned media from Stat4−/−Ldlr−/− MΦs supported CD8+ Treg but not Tfh cell differentiation in TGFβ-dependent manner. These findings suggest a novel mechanism by which STAT4 supports atherosclerosis in IR Ldlr−/− mice via STAT4-dependent macrophage as well as cell-intrinsic suppression of CD8+ Treg generation and functions and maintenance of Tfh cell generation and accompanied humoral immune response.

Macrophages and related myeloid cells are innate immune cells that participate in the early islet inflammation of type 1 diabetes (T1D). The enzyme 12-lipoxygenase (12-LOX) catalyzes the formation of proinflammatory eicosanoids, but its role and mechanisms in myeloid cells in the pathogenesis of islet inflammation have not been elucidated. Leveraging a model of islet inflammation in zebrafish, we show here that macrophages contribute significantly to the loss of β cells and the subsequent development of hyperglycemia. The depletion or inhibition of 12-LOX in this model resulted in reduced macrophage infiltration into islets and the preservation of β cell mass. In NOD mice, the deletion of the gene encoding 12-LOX in the myeloid lineage resulted in reduced insulitis with reductions in proinflammatory macrophages, a suppressed T cell response, preserved β cell mass, and almost complete protection from the development of T1D. 12-LOX depletion caused a defect in myeloid cell migration, a function required for immune surveillance and tissue injury responses. This effect on migration resulted from the loss of the chemokine receptor CXCR3. Transgenic expression of the gene encoding CXCR3 rescued the migratory defect in zebrafish 12-LOX morphants. Taken together, our results reveal a formative role for innate immune cells in the early pathogenesis of T1D and identify 12-LOX as an enzyme required to promote their prodiabetogenic phenotype in the context of autoimmunity.