12-Lipoxygenase inhibition suppresses islet immune and inflammatory responses and delays autoimmune diabetes in human gene replacement mice.

Type 1 diabetes (T1D) is characterized by the autoimmune destruction of insulin-producing ß cells and involves an interplay between ß cells and cells of the innate and adaptive immune systems. We investigated the therapeutic potential of targeting 12-lipoxygenase (12-LOX), an enzyme implicated in inflammatory pathways in ß cells and macrophages, using a mouse model in which the endogenous mouse Alox15 gene is replaced by the human ALOX12 gene. Our findings demonstrate that VLX-1005, a potent 12-LOX inhibitor, effectively delays the onset of autoimmune diabetes in human gene replacement non-obese diabetic (NOD) mice. By spatial proteomics analysis, VLX-1005 treatment resulted in marked reductions in infiltrating T and B cells and macrophages with accompanying increases in immune checkpoint molecules PD-L1 and PD-1, suggesting a shift towards an immune-suppressive microenvironment. RNA sequencing analysis of isolated islets from inhibitor-treated mice revealed significant alteration of cytokine-responsive pathways. RNA sequencing of polarized proinflammatory macrophages showed that VLX-1005 significantly reduced the interferon response. Our studies demonstrate that the ALOX12 human replacement gene mouse provides a platform for the preclinical evaluation of LOX inhibitors and supports VLX-1005 as an inhibitor of human 12-LOX that engages the enzymatic target and alters the inflammatory phenotypes of islets and macrophages to promote the delay of autoimmune diabetes.

Macrophage Specific Lipoxygenase Deletion Amplify Cardiac Repair Activating Tregs in Chronic Heart Failure.

Splenic leukocytes, particularly macrophage-expressed lipoxygenases, facilitate the biosynthesis of resolution mediators essential for cardiac repair. Next, we asked whether deletion of 12/15 lipoxygenase (12/15LOX) in macrophages impedes the resolution of inflammation following myocardial infarction (MI). Using 12/15flox/flox and LysMcre scheme, we generated macrophage-specific 12/15LOX (Mɸ-12/15LOX-/-) mice. Young C57BL/6J wild-type and Mɸ-12/15LOX-/- male mice were subjected to permanent coronary ligation micro-surgery. Mice were monitored at day (d)1-d5 (as acute HF; AHF) and to d56 (chronic HF; CHF) post-MI, maintaining no-MI as d0 naïve controls. Post-ligation, Mɸ-12/15LOX-/- mice showed increased survival (88%vs56%) and limited heart dysfunction compared with WT. In AHF, Mɸ-12/15LOX-/- mice have increased biosynthesis of epoxyeicosatrienoic acid (EETs) by 30%, with the decrease in D-series resolvins, protectin, and maresin by 70% in the infarcted heart. Overall, myeloid cell profiling from the heart and spleen indicated that Mɸ-12/15LOX-/- mice showed higher immune cells with reparative Ly6Clow macrophages during AHF. In addition, the detailed immune profiling revealed reparative macrophage phenotype (Ly6Clow) in Mɸ-12/15LOX-/- mice in a splenocardiac manner post-MI. Mɸ-12/15LOX-/- mice showed an increase in myeloid population that coordinated increase of Tregs (CD4+/Foxp3+) in the spleen and injured heart at CHF compared with WT. Thus, macrophage-specific deletion of 12/15LOX directs reparative macrophage phenotype to facilitate cardiac repair. The presented study outlines the complex role of 12/15LOX in macrophage plasticity, and Treg signaling that indicates resolution mediators are viable targets to facilitate cardiac repair in heart failure post-MI.

Macrophage-derived bioactive lipids promote the safe clearance of inflammation (resolution), thus modulating macrophage-specific 12/15 lipoxygenase restores structure, function, and survival after heart attack in mice.

Myeloid cell deficiency of the inflammatory transcription factor Stat4 protects long-term synaptic plasticity from the effects of a high-fat, high-cholesterol diet.

Neuroinflammation is associated with neurodegenerative diseases, including Alzheimer's and Parkinson's. The cytokine interleukin-12 activates signal transducer and activator of transcription 4 (Stat4), and consumption of a high-fat, high-cholesterol diet (HFD-C) and Stat4 activity are associated with inflammation, atherosclerosis, and a diabetic metabolic phenotype. In studies of in vitro hippocampal slices from control Stat4fl/flLdlr-/- mice fed a HFD-C diabetogenic diet, we show that Schaffer collateral-CA1 synapses exhibited larger reductions in activity-dependent, long-term potentiation (LTP) of synaptic transmission, compared to mice fed a standard diet. Glucose tolerance and insulin sensitivity shifts produced by HFD-C diet were reduced in Stat4ΔLysMLdlr-/- mice compared to Stat4fl/flLdlr-/- controls. Stat4ΔLysMLdlr-/- mice, which lack Stat4 under control of the LysMCre promoter, were resistant to HFD-C induced impairments in LTP. In contrast, Schaffer collateral-CA1 synapses in Stat4ΔLysMLdlr-/- mice fed the HFD-C diet showed larger LTP than control Stat4fl/flLdlr-/- mice. Expression of a number of neuroinflammatory and synaptic plasticity genes was reduced by HFD-C diet in control mice, and less affected by HFD-C diet in Stat4ΔLysMLdlr-/- mice. These data suggest that suppression of Stat4 activation may protect against effects of Western diet on cognition, type 2 diabetes, and reduce risk of Alzheimer's disease and other neurodegenerative disorders associated with neuroinflammation.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr-/- mice.

Background and aims: Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. Methods: We generated myeloid-specific Stat4ΔLysMLdlr-/-, neutrophil-specific Stat4ΔS100A8Ldlr-/-, and control Stat4fl/flLdlr-/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4ΔLysMLdlr-/- and Stat4fl/flLdlr-/- bone marrow cells into aged atherosclerotic Apoe-/- mice and detected by flow cytometry. Results: STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in HFD-C fed Stat4ΔLysMLdlr-/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Conclusions: Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

Neutrophil-specific STAT4 deficiency attenuates atherosclerotic burden and improves plaque stability via reduction in neutrophil activation and recruitment into aortas of Ldlr -/- mice.

Background and Aims: Neutrophils drive atheroprogression and directly contribute to plaque instability. We recently identified signal transducer and activator of transcription 4 (STAT4) as a critical component for bacterial host defense in neutrophils. The STAT4-dependent functions of neutrophils in atherogenesis are unknown. Therefore, we investigated a contributory role of STAT4 in neutrophils during advanced atherosclerosis. Methods: We generated myeloid-specific Stat4 ΔLysM Ldlr -/- , neutrophil-specific Stat4 ΔS100A8 Ldlr -/- , and control Stat4 fl/fl Ldlr -/- mice. All groups were fed a high-fat/cholesterol diet (HFD-C) for 28 weeks to establish advanced atherosclerosis. Aortic root plaque burden and stability were assessed histologically by Movat Pentachrome staining. Nanostring gene expression analysis was performed on isolated blood neutrophils. Flow cytometry was utilized to analyze hematopoiesis and blood neutrophil activation. In vivo homing of neutrophils to atherosclerotic plaques was performed by adoptively transferring prelabeled Stat4 ΔLysM Ldlr -/- and Stat4 fl/fl Ldlr -/- bone marrow cells into aged atherosclerotic Apoe -/- mice and detected by flow cytometry. Results: STAT4 deficiency in both myeloid-specific and neutrophil-specific mice provided similar reductions in aortic root plaque burden and improvements in plaque stability via reduction in necrotic core size, improved fibrous cap area, and increased vascular smooth muscle cell content within the fibrous cap. Myeloid-specific STAT4 deficiency resulted in decreased circulating neutrophils via reduced production of granulocyte-monocyte progenitors in the bone marrow. Neutrophil activation was dampened in Stat4 ΔLysM Ldlr -/- mice via reduced mitochondrial superoxide production, attenuated surface expression of degranulation marker CD63, and reduced frequency of neutrophil-platelet aggregates. Myeloid-specific STAT4 deficiency diminished expression of chemokine receptors CCR1 and CCR2 and impaired in vivo neutrophil trafficking to atherosclerotic aorta. Conclusions: Our work indicates a pro-atherogenic role for STAT4-dependent neutrophil activation and how it contributes to multiple factors of plaque instability during advanced atherosclerosis in mice.

The AAV-PCSK9 murine model of atherosclerosis and metabolic dysfunction.

Aims: Mouse models with genetic modifications are required to investigate atherogenesis and associated metabolic syndrome. Adeno-associated virus-8 (AAV8)-mediated overexpression of PCSK9 (AAV8-PCSK9) induces hyperlipidaemia and promotes atherosclerosis in C57BL/6 mice. We aimed to assess whether AAV8-PCSK9-injected C57BL/6 mice fed high-fat diet with added cholesterol (HFD-C) would serve as a model of combined metabolic syndrome and atherosclerosis. Methods and results: C57BL/6 mice received i.v. injection of AAV-PCSK9 and sex- and age-matched Ldlr-/- and C57BL/6 control mice were placed on HFD-C or chow diet for 20 weeks (B6-PCSK9-HFD-C, Ldlr-/- HFD-C, B6-HFD-C, and B6-Chow, respectively). High-fat diet with added cholesterol feeding led to insulin resistance and impaired glucose clearance in B6-PCSK9-HFD-C mice compared with B6-Chow controls. This decrease in metabolic health in B6-PCSK9-HFD-C mice as well as the development of atherosclerosis was similar to Ldlr-/- HFD-C mice. Importantly, HFD-C feeding induced pancreatic islet hyperplasia in B6-PCSK9-HFD-C and B6-HFD-C compared with B6-Chow controls. In line with alterations in the metabolic phenotype, there was an increase in the number of pro-inflammatory Ly6Chigh/med monocytes within the adipose tissues of B6-PCSK9-HFD-C and B6-HFD-C compared with B6-Chow controls. Conclusion: High-fat diet with added cholesterol-fed AAV-PCSK9-injected C57BL/6 mice can serve as a useful model of integrated metabolic syndrome and atherosclerosis that does not require genetic manipulations.

Proinflammatory signaling in islet ß cells propagates invasion of pathogenic immune cells in autoimmune diabetes.

Type 1 diabetes is a disorder of immune tolerance that leads to death of insulin-producing islet ß cells. We hypothesize that inflammatory signaling within ß cells promotes progression of autoimmunity within the islet microenvironment. To test this hypothesis, we deleted the proinflammatory gene encoding 12/15-lipoxygenase (Alox15) in ß cells of non-obese diabetic mice at a pre-diabetic time point when islet inflammation is a feature. Deletion of Alox15 leads to preservation of ß cell mass, reduces populations of infiltrating T cells, and protects against spontaneous autoimmune diabetes in both sexes. Mice lacking Alox15 in ß cells exhibit an increase in a population of ß cells expressing the gene encoding the protein programmed death ligand 1 (PD-L1), which engages receptors on immune cells to suppress autoimmunity. Delivery of a monoclonal antibody against PD-L1 recovers the diabetes phenotype in knockout animals. Our results support the contention that inflammatory signaling in ß cells promotes autoimmunity during type 1 diabetes progression.

Docking and mutagenesis studies lead to improved inhibitor development of ML355 for human platelet 12-lipoxygenase.

Human platelet 12-(S)-Lipoxygenase (12-LOX) is a fatty acid metabolizing oxygenase that plays an important role in platelet activation and cardiometabolic disease. ML355 is a specific 12-LOX inhibitor that has been shown to decrease thrombosis without prolonging hemostasis and protect human pancreatic islets from inflammatory injury. It has an amenable drug-like scaffold with nM potency and encouraging ADME and PK profiles, but its binding mode to the active site of 12-LOX remains unclear. In the current work, we combined computational modeling and experimental mutagenesis to propose a model in which ML355 conforms to the "U" shape of the 12-LOX active site, with the phenyl linker region wrapping around L407. The benzothiazole of ML355 extends into the bottom of the active site cavity, pointing towards residues A417 and V418. However, reducing the active site depth alone did not affect ML355 potency. In order to lower the potency of ML355, the cavity needed to be reduced in both length and width. In addition, H596 appears to position ML355 in the active site through an interaction with the 2-methoxy phenol moiety of ML355. Combined, this binding model suggested that the benzothiazole of ML355 could be enlarged. Therefore, a naphthyl-benzothiazole derivative of ML355, Lox12Slug001, was synthesized and shown to have 7.2-fold greater potency than ML355. This greater potency is proposed to be due to additional van der Waals interactions and pi-pi stacking with F414 and F352. Lox12Slug001 was also shown to be highly selective against 12-LOX relative to the other LOX isozymes and more importantly, it showed activity in rescuing human islets exposed to inflammatory cytokines with comparable potency to ML355. Further studies are currently being pursued to derivatize ML355 in order to optimize the additional space in the active site, while maintaining acceptable drug-like properties.

STAT4 is expressed in neutrophils and promotes antimicrobial immunity.

Signal transducer and activator of transcription 4 (STAT4) is expressed in hematopoietic cells and plays a key role in the differentiation of T helper 1 cells. Although STAT4 is required for immunity to intracellular pathogens, the T cell-independent protective mechanisms of STAT4 are not clearly defined. In this report, we demonstrate that STAT4-deficient mice were acutely sensitive to methicillin-resistant Staphylococcus aureus (MRSA) infection. We show that STAT4 was expressed in neutrophils and activated by IL-12 via a JAK2-dependent pathway. We demonstrate that STAT4 was required for multiple neutrophil functions, including IL-12-induced ROS production, chemotaxis, and production of the neutrophil extracellular traps. Importantly, myeloid-specific and neutrophil-specific deletion of STAT4 resulted in enhanced susceptibility to MRSA, demonstrating the key role of STAT4 in the in vivo function of these cells. Thus, these studies identify STAT4 as an essential regulator of neutrophil functions and a component of innate immune responses in vivo.

Regulation of Tissue Inflammation by 12-Lipoxygenases.

Lipoxygenases (LOXs) are lipid metabolizing enzymes that catalyze the di-oxygenation of polyunsaturated fatty acids to generate active eicosanoid products. 12-lipoxygenases (12-LOXs) primarily oxygenate the 12th carbon of its substrates. Many studies have demonstrated that 12-LOXs and their eicosanoid metabolite 12-hydroxyeicosatetraenoate (12-HETE), have significant pathological implications in inflammatory diseases. Increased level of 12-LOX activity promotes stress (both oxidative and endoplasmic reticulum)-mediated inflammation, leading to damage in these tissues. 12-LOXs are also associated with enhanced cellular migration of immune cells-a characteristic of several metabolic and autoimmune disorders. Genetic depletion or pharmacological inhibition of the enzyme in animal models of various diseases has shown to be protective against disease development and/or progression in animal models in the setting of diabetes, pulmonary, cardiovascular, and metabolic disease, suggesting a translational potential of targeting the enzyme for the treatment of several disorders. In this article, we review the role of 12-LOXs in the pathogenesis of several diseases in which chronic inflammation plays an underlying role.

12-Lipoxygenase governs the innate immune pathogenesis of islet inflammation and autoimmune diabetes.

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 ß 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 ß 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 ß 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.

Loop-Mediated Isothermal Amplification Detection of SARS-CoV-2 and Myriad Other Applications.

As the second year of the COVID-19 pandemic begins, it remains clear that a massive increase in the ability to test for SARS-CoV-2 infections in a myriad of settings is critical to controlling the pandemic and to preparing for future outbreaks. The current gold standard for molecular diagnostics is the polymerase chain reaction (PCR), but the extraordinary and unmet demand for testing in a variety of environments means that both complementary and supplementary testing solutions are still needed. This review highlights the role that loop-mediated isothermal amplification (LAMP) has had in filling this global testing need, providing a faster and easier means of testing, and what it can do for future applications, pathogens, and the preparation for future outbreaks. This review describes the current state of the art for research of LAMP-based SARS-CoV-2 testing, as well as its implications for other pathogens and testing. The authors represent the global LAMP (gLAMP) Consortium, an international research collective, which has regularly met to share their experiences on LAMP deployment and best practices; sections are devoted to all aspects of LAMP testing, including preanalytic sample processing, target amplification, and amplicon detection, then the hardware and software required for deployment are discussed, and finally, a summary of the current regulatory landscape is provided. Included as well are a series of first-person accounts of LAMP method development and deployment. The final discussion section provides the reader with a distillation of the most validated testing methods and their paths to implementation. This review also aims to provide practical information and insight for a range of audiences: for a research audience, to help accelerate research through sharing of best practices; for an implementation audience, to help get testing up and running quickly; and for a public health, clinical, and policy audience, to help convey the breadth of the effect that LAMP methods have to offer.

Proteomic and Transcriptional Profiles of Human Stem Cell-Derived ß Cells Following Enteroviral Challenge.

Enteroviral infections are implicated in islet autoimmunity and type 1 diabetes (T1D) pathogenesis. Significant ß-cell stress and damage occur with viral infection, leading to cells that are dysfunctional and vulnerable to destruction. Human stem cell-derived ß (SC-ß) cells are insulin-producing cell clusters that closely resemble native ß cells. To better understand the events precipitated by enteroviral infection of ß cells, we investigated transcriptional and proteomic changes in SC-ß cells challenged with coxsackie B virus (CVB). We confirmed infection by demonstrating that viral protein colocalized with insulin-positive SC-ß cells by immunostaining. Transcriptome analysis showed a decrease in insulin gene expression following infection, and combined transcriptional and proteomic analysis revealed activation of innate immune pathways, including type I interferon (IFN), IFN-stimulated genes, nuclear factor-kappa B (NF-κB) and downstream inflammatory cytokines, and major histocompatibility complex (MHC) class I. Finally, insulin release by CVB4-infected SC-ß cells was impaired. These transcriptional, proteomic, and functional findings are in agreement with responses in primary human islets infected with CVB ex vivo. Human SC-ß cells may serve as a surrogate for primary human islets in virus-induced diabetes models. Because human SC-ß cells are more genetically tractable and accessible than primary islets, they may provide a preferred platform for investigating T1D pathogenesis and developing new treatments.

Activation of the 12/15 lipoxygenase pathway accompanies metabolic decline in db/db pre-diabetic mice.

The 12-lipoxygenase (12LO) pathway is a promising target to reduce islet dysfunction, adipose tissue (AT) inflammation and insulin resistance. Optimal pre-clinical models for the investigation of selective12LO inhibitors in this context have not yet been identified. The objective of this study was to characterize the time course of 12LO isoform expression and metabolite production in pancreatic islets and AT of C57BLKS/J-db/db obese diabetic mouse in a pre-diabetic state in order to establish a suitable therapeutic window for intervention with selective lipoxygenase inhibitors. Mice have 2 major 12LO isoforms -the leukocyte type (12/15LO) and the platelet type (p12LO) and both are expressed in islets and AT. We found a sharp increase in protein expression of 12/15LO in the pancreatic islets of 10-week old db-/- mice compared to 8- week old counterparts. Immunohistochemistry showed that the increase in islet 12/15LO parallels a decline in islet number. Analysis of 12- and 15-hydroperoxytetraeicosanoid acids (HETE)s showed a 2-3 fold increase especially in 12(S)-HETE that mirrored the increase in 12/15LO expression in islets. Analysis of AT and stromal vascular fraction (SVF) showed a significant increase of platelet 12LO gene expression along with 12- and 15- HETEs. The data demonstrate that the db/db mouse is a suitable model for investigation of 12/15LO inhibitors in the development of inflammatory mediated type 2 diabetes, with a narrow window of therapeutic intervention prior to 8 weeks of age.

Coxsackievirus-Induced Proteomic Alterations in Primary Human Islets Provide Insights for the Etiology of 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.

STAT4 Regulates the CD8+ Regulatory T Cell/T Follicular Helper Cell Axis and Promotes Atherogenesis in Insulin-Resistant Ldlr-/- Mice.

The metabolic syndrome and diabetic conditions support atherosclerosis, but the exact mechanisms for accelerated atherogenesis remain unclear. Although the proinflammatory role of STAT4 in atherosclerosis and diet-induced insulin resistance (IR) was recently established, the impact of STAT4 on atherogenesis in conditions of IR is not known. In this study, 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) cells and plasma B cells but a sharp elevation in CD8+ regulatory T cells (Tregs) in spleens and aortas of Stat4-/-Ldlr-/- mice compared with Ldlr-/- mice. Similarly, STAT4 deficiency supported CD8+ Treg differentiation in vitro. STAT4-deficient CD8+ Tregs suppressed Tfh cell and germinal center B cell development upon immunization with keyhole limpet hemocyanin, indicating an important role for STAT4 in CD8+ Treg functions in vivo. Furthermore, adoptive transfer of Stat4-/-Ldlr-/- CD8+ Tregs versus Ldlr-/- CD8+ Tregs resulted in a significant reduction in plaque burden and suppression of Tfh cell and germinal center B cells in DDC-fed Ldlr-/- recipients. STAT4 expression in macrophages (MΦs) also affected the Tfh/CD8+ Treg axis, because conditioned media from Stat4-/-Ldlr-/- MΦs supported CD8+ Treg differentiation, but not Tfh cell differentiation, in a TGF-ß-dependent manner. These findings suggest a novel mechanism by which STAT4 supports atherosclerosis in IR Ldlr-/- mice via STAT4-dependent MΦs, as well as cell-intrinsic suppression of CD8+ Treg generation and functions and maintenance of Tfh cell generation and the accompanying humoral immune response.

Comparative quantitative proteomic analysis of disease stratified laser captured microdissected human islets identifies proteins and pathways potentially related to type 1 diabetes.

Type 1 diabetes (T1D) is a chronic inflammatory disease that is characterized by autoimmune destruction of insulin-producing pancreatic beta cells. The goal of this study was to identify novel protein signatures that distinguish Islets from patients with T1D, patients who are autoantibody positive without symptoms of diabetes, and from individuals with no evidence of disease. High resolution high mass accuracy label free quantitative mass spectrometry analysis was applied to islets isolated by laser capture microdissection from disease stratified human pancreata from the Network for Pancreatic Organ Donors with Diabetes (nPOD), these included donors without diabetes, donors with T1D-associated autoantibodies in the absence of diabetes, and donors with T1D. Thirty-nine proteins were found to be differentially regulated in autoantibody positive cases compared to the no-disease group, with 25 upregulated and 14 downregulated proteins. For the T1D cases, 63 proteins were differentially expressed, with 24 upregulated and 39 downregulated, compared to the no disease controls. We have identified functional annotated enriched gene families and multiple protein-protein interaction clusters of proteins are involved in biological and molecular processes that may have a role in T1D. The proteins that are upregulated in T1D cases include S100A9, S100A8, REG1B, REG3A and C9 amongst others. These proteins have important biological functions, such as inflammation, metabolic regulation, and autoimmunity, all of which are pathways linked to the pathogenesis of T1D. The identified proteins may be involved in T1D development and pathogenesis. Our findings of novel proteins uniquely upregulated in T1D pancreas provides impetus for further investigations focusing on their expression profiles in beta cells/ islets to evaluate their role in the disease pathogenesis. Some of these molecules may be novel therapeutic targets T1D.

Inhibition of 12/15-Lipoxygenase Protects Against ß-Cell Oxidative Stress and Glycemic Deterioration in Mouse Models of Type 1 Diabetes.

Islet ß-cell dysfunction and aggressive macrophage activity are early features in the pathogenesis of type 1 diabetes (T1D). 12/15-Lipoxygenase (12/15-LOX) is induced in ß-cells and macrophages during T1D and produces proinflammatory lipids and lipid peroxides that exacerbate ß-cell dysfunction and macrophage activity. Inhibition of 12/15-LOX provides a potential therapeutic approach to prevent glycemic deterioration in T1D. Two inhibitors recently identified by our groups through screening efforts, ML127 and ML351, have been shown to selectively target 12/15-LOX with high potency. Only ML351 exhibited no apparent toxicity across a range of concentrations in mouse islets, and molecular modeling has suggested reduced promiscuity of ML351 compared with ML127. In mouse islets, incubation with ML351 improved glucose-stimulated insulin secretion in the presence of proinflammatory cytokines and triggered gene expression pathways responsive to oxidative stress and cell death. Consistent with a role for 12/15-LOX in promoting oxidative stress, its chemical inhibition reduced production of reactive oxygen species in both mouse and human islets in vitro. In a streptozotocin-induced model of T1D in mice, ML351 prevented the development of diabetes, with coincident enhancement of nuclear Nrf2 in islet cells, reduced ß-cell oxidative stress, and preservation of ß-cell mass. In the nonobese diabetic mouse model of T1D, administration of ML351 during the prediabetic phase prevented dysglycemia, reduced ß-cell oxidative stress, and increased the proportion of anti-inflammatory macrophages in insulitis. The data provide the first evidence to date that small molecules that target 12/15-LOX can prevent progression of ß-cell dysfunction and glycemic deterioration in models of T1D.

Enzymatic lipid oxidation by eosinophils propagates coagulation, hemostasis, and thrombotic disease.

Blood coagulation is essential for physiological hemostasis but simultaneously contributes to thrombotic disease. However, molecular and cellular events controlling initiation and propagation of coagulation are still incompletely understood. In this study, we demonstrate an unexpected role of eosinophils during plasmatic coagulation, hemostasis, and thrombosis. Using a large-scale epidemiological approach, we identified eosinophil cationic protein as an independent and predictive risk factor for thrombotic events in humans. Concurrent experiments showed that eosinophils contributed to intravascular thrombosis by exhibiting a strong endogenous thrombin-generation capacity that relied on the enzymatic generation and active provision of a procoagulant phospholipid surface enriched in 12/15-lipoxygenase-derived hydroxyeicosatetraenoic acid-phosphatidylethanolamines. Our findings reveal a previously unrecognized role of eosinophils and enzymatic lipid oxidation as regulatory elements that facilitate both hemostasis and thrombosis in response to vascular injury, thus identifying promising new targets for the treatment of thrombotic disease.