Anti-Inflammatory and Therapeutic Effects of a Novel Small-Molecule Inhibitor of Inflammation in a Male C57BL/6J Mouse Model of Obesity-Induced NAFLD/MAFLD.

Purpose: Non-alcoholic fatty liver disease (NAFLD), recently renamed metabolic (dysfunction) associated fatty liver disease (MAFLD), is the most common chronic liver disease in the United States. Presently, there is an intense and ongoing effort to identify and develop novel therapeutics for this disease. In this study, we explored the anti-inflammatory activity of a new compound, termed IOI-214, and its therapeutic potential to ameliorate NAFLD/MAFLD in male C57BL/6J mice fed a high fat (HF) diet. Methods: Murine macrophages and hepatocytes in culture were treated with lipopolysaccharide (LPS) ± IOI-214 or DMSO (vehicle), and RT-qPCR analyses of inflammatory cytokine gene expression were used to assess IOI-214's anti-inflammatory properties in vitro. Male C57BL/6J mice were also placed on a HF diet and treated once daily with IOI-214 or DMSO for 16 weeks. Tissues were collected and analyzed to determine the effects of IOI-214 on HF diet-induced NAFL D/MAFLD. Measurements such as weight, blood glucose, serum cholesterol, liver/serum triglyceride, insulin, and glucose tolerance tests, ELISAs, metabolomics, Western blots, histology, gut microbiome, and serum LPS binding protein analyses were conducted. Results: IOI-214 inhibited LPS-induced inflammation in macrophages and hepatocytes in culture and abrogated HF diet-induced mesenteric fat accumulation, hepatic inflammation and steatosis/hepatocellular ballooning, as well as fasting hyperglycemia without affecting insulin resistance or fasting insulin, cholesterol or TG levels despite overall obesity in vivo in male C57BL/6J mice. IOI-214 also decreased systemic inflammation in vivo and improved gut microbiota dysbiosis and leaky gut. Conclusion: Combined, these data indicate that IOI-214 works at multiple levels in parallel to inhibit the inflammation that drives HF diet-induced NAFLD/MAFLD, suggesting that it may have therapeutic potential for NAFLD/MAFLD.

Toll-Like Receptor 3 Is Critical to the Pancreatic Islet Milieu That Is Required for Coxsackievirus B4-Induced Type 1 Diabetes in Female Nonobese Diabetic Mice.

OBJECTIVE: Genetic and environmental influences play a role as triggers of type 1 diabetes mellitus (T1DM). Female nonobese diabetic (NOD) mice are useful for studying T1DM as they spontaneously develop T1DM, which can be accelerated by some viruses. Toll-like receptor 3 (TLR3) is believed to play a critical role in viral-induced T1DM and ß-cell destruction, because female Tlr3 knockout (Tlr3-/-) NOD mice are protected from Coxsackievirus B4 (CVB4)-induced acceleration of T1DM. However, the exact role(s) TLR3 plays in the pathogenesis of CVB4-induced T1DM remain unknown. METHODS: This longitudinal study used immunostaining, laser capture microdissection, and reverse transcription real-time polymerase chain reaction of islets from female uninfected and CVB4-infected Tlr3+/+ and Tlr3-/- NOD mice. RESULTS: Islets isolated from female Tlr3+/+ NOD mice 4 to 8 weeks of age had higher amounts of insulitis, Cxcl10, Il1b, Tnfa, and Tgfb1 expression compared with Tlr3-/- NOD mice. After CVB4 infection, Tlr3+/+ NOD mice had higher amounts of insulitis and T-cell infiltration at 3 days after infection compared with Tlr3-/- CVB4-infected NOD mice. CONCLUSIONS: Toll-like receptor 3 is necessary for establishment of a pancreatic islet inflammatory microenvironment by increasing insulitis and cytokine expression that facilitates CVB4-induced T1DM in female NOD mice.

Consequences of Both Coxsackievirus B4 and Type 1 Diabetes on Female Non-Obese Diabetic Mouse Kidneys.

Despite the 2019 Executive Order on Advancing American Kidney Health Initiative, kidney disease has moved up in rank from the 9th to the 8th leading cause of death in the United States. A recent push in the field of nephrology has been to identify molecular markers and/or molecular profiles involved in kidney disease process or injury that can help identify the cause of injury and predict patient outcomes. While these studies have had moderate success, they have not yet considered that many of the health conditions that cause kidney disease (diabetes, hypertension, etc.) can also be caused by environmental factors (such as viruses), which in and of themselves can cause kidney disease. Thus, the goal of this study was to identify molecular and phenotypic profiles that can differentiate kidney injury caused by diabetes (a health condition resulting in kidney disease) and coxsackievirus B4 (CVB4) exposure (which can cause diabetes and/or kidney disease), both alone and together. Non-obese diabetic (NOD) mice were used for this study due to their susceptibility to both type 1 diabetes (T1D)- and CVB4-mediated kidney injury, in order to glean a better understanding of how hyperglycemia and viral exposure, when occurring on their own and in combination, may alter the kidneys' molecular and phenotypic profiles. While no changes in kidney function were observed, molecular biomarkers of kidney injury were significantly up- and downregulated based on T1D and CVB4 exposure, both alone and together, but not in a predictable pattern. By combining individual biomarkers with function and phenotypic measurements (i.e., urinary albumin creatinine ratio, serum creatinine, kidney weight, and body weight), we were able to perform an unbiased separation of injury group based on the type of injury. This study provides evidence that unique kidney injury profiles within a kidney disease health condition are identifiable, and will help us to identify the causes of kidney injury in the future.

Coxsackievirus B4 Exposure Results in Variable Pattern Recognition Response in the Kidneys of Female Non-Obese Diabetic Mice Before Establishment of Diabetes.

End-stage renal disease (ESRD) is described by four primary diagnoses, diabetes, hypertension, glomerulonephritis, and cystic kidney disease, all of which have viruses implicated as causative agents. Enteroviruses, such as coxsackievirus (CV), are a common genus of viruses that have been implicated in both diabetes and cystic kidney disease; however, little is known about how CVs cause kidney injury and ESRD or predispose individuals with a genetic susceptibility to type 1 diabetes (T1D) to kidney injury. This study evaluated kidney injury resulting from coxsackievirus B4 (CVB4) inoculation of non-obese diabetic (NOD) mice to glean a better understanding of how viral exposure may predispose individuals with a genetic susceptibility to T1D to kidney injury. The objectives were to assess acute and chronic kidney damage in CVB4-inoculated NOD mice without diabetes. Results indicated the presence of CVB4 RNA in the kidney for at least 14 days post-CVB4 inoculation and a coordinated pattern recognition receptor response, but the absence of an immune response or cytotoxicity. CVB4-inoculated NOD mice also had a higher propensity to develop an increase in mesangial area 17 weeks post-CVB4 inoculation. These studies identified initial gene expression changes in the kidney resulting from CVB4 exposure that may predispose to ESRD. Thus, this study provides an initial characterization of kidney injury resulting from CVB4 inoculation of mice that are genetically susceptible to developing T1D that may one day provide better therapeutic options and predictive measures for patients who are at risk for developing kidney disease from T1D.

Toll-like receptor 3 is critical for coxsackievirus B4-induced type 1 diabetes in female NOD mice.

Group B coxsackieviruses (CVBs) are involved in triggering some cases of type 1 diabetes mellitus (T1DM). However, the molecular mechanism(s) responsible for this remain elusive. Toll-like receptor 3 (TLR3), a receptor that recognizes viral double-stranded RNA, is hypothesized to play a role in virus-induced T1DM, although this hypothesis is yet to be substantiated. The objective of this study was to directly investigate the role of TLR3 in CVB-triggered T1DM in nonobese diabetic (NOD) mice, a mouse model of human T1DM that is widely used to study both spontaneous autoimmune and viral-induced T1DM. As such, we infected female wild-type (TLR3(+/+)) and TLR3 knockout (TLR3(-/-)) NOD mice with CVB4 and compared the incidence of diabetes in CVB4-infected mice with that of uninfected counterparts. We also evaluated the islets of uninfected and CVB4-infected wild-type and TLR3 knockout NOD mice by immunohistochemistry and insulitis scoring. TLR3 knockout mice were markedly protected from CVB4-induced diabetes compared with CVB4-infected wild-type mice. CVB4-induced T-lymphocyte-mediated insulitis was also significantly less severe in TLR3 knockout mice compared with wild-type mice. No differences in insulitis were observed between uninfected animals, either wild-type or TLR3 knockout mice. These data demonstrate for the first time that TLR3 is 1) critical for CVB4-induced T1DM, and 2) modulates CVB4-induced insulitis in genetically prone NOD mice.

Phenylmethimazole suppresses dsRNA-induced cytotoxicity and inflammatory cytokines in murine pancreatic beta cells and blocks viral acceleration of type 1 diabetes in NOD mice.

Accumulating evidence supports a role for viruses in the pathogenesis of type 1 diabetes mellitus (T1DM). Activation of dsRNA-sensing pathways by viral dsRNA induces the production of inflammatory cytokines and chemokines that trigger beta cell apoptosis, insulitis, and autoimmune-mediated beta cell destruction. This study was designed to evaluate and describe potential protective effects of phenylmethimazole (C10), a small molecule which blocks dsRNA-mediated signaling, on preventing dsRNA activation of beta cell apoptosis and the inflammatory pathways important in the pathogenesis of T1DM. We first investigated the biological effects of C10, on dsRNA-treated pancreatic beta cells in culture. Cell viability assays, quantitative real-time PCR, and ELISAs were utilized to evaluate the effects of C10 on dsRNA-induced beta cell cytotoxicity and cytokine/chemokine production in murine pancreatic beta cells in culture. We found that C10 significantly impairs dsRNA-induced beta cell cytotoxicity and up-regulation of cytokines and chemokines involved in the pathogenesis of T1DM, which prompted us to evaluate C10 effects on viral acceleration of T1DM in NOD mice. C10 significantly inhibited viral acceleration of T1DM in NOD mice. These findings demonstrate that C10 (1) possesses novel beta cell protective activity which may have potential clinical relevance in T1DM and (2) may be a useful tool in achieving a better understanding of the role that dsRNA-mediated responses play in the pathogenesis of T1DM.

Luteinizing hormone pulsatility is disrupted at a threshold of energy availability in regularly menstruating women.

To investigate the dependence of LH pulsatility on energy availability (dietary energy intake minus exercise energy expenditure), we measured LH pulsatility after manipulating the energy availability of 29 regularly menstruating, habitually sedentary, young women of normal body composition for 5 d in the early follicular phase. Subjects expended 15 kcal/kg of lean body mass (LBM) per day in supervised exercise at 70% of aerobic capacity while consuming a clinical dietary product to set energy availability at 45 and either 10, 20, or 30 kcal/kg LBM.d in two randomized trials separated by at least 2 months. Blood was sampled daily during treatments and at 10-min intervals for the next 24 h. Samples were assayed for LH, FSH, estradiol (E2), glucose, beta-hydroxybutyrate, insulin, cortisol, GH, IGF-I, IGF-I binding protein (IGFBP)-1, IGFBP-3, leptin, and T3. LH pulsatility was unaffected by an energy availability of 30 kcal/kg LBM.d (P > 0.3), but below this threshold LH pulse frequency decreased, whereas LH pulse amplitude increased (all P < 0.04). This disruption was more extreme in women with short luteal phases (P < 0.01). These incremental effects most closely resembled the effects of energy availability on plasma glucose, beta-hydroxybutyrate, GH, and cortisol and contrasted with the dependencies displayed by the other metabolic hormones (simultaneously P < 0.05). These results demonstrate that LH pulsatility is disrupted only below a threshold of energy availability deep into negative energy balance and suggest priorities for future investigations into the mechanism that mediates the nonlinear dependence of LH pulsatility on energy availability.