IFNγ-induction of TH1-like regulatory T cells controls antiviral responses.

Regulatory T (Treg) cells are an immunosuppressive population that are required to maintain peripheral tolerance and prevent tissue damage from immunopathology, via anti-inflammatory cytokines, inhibitor receptors and metabolic disruption. Here we show that Treg cells acquire an effector-like state, yet remain stable and functional, when exposed to interferon gamma (IFNγ) during infection with lymphocytic choriomeningitis and influenza A virus. Treg cell-restricted deletion of the IFNγ receptor (encoded by Ifngr1), but not the interleukin 12 (IL12) receptor (encoded by Il12rb2), prevented TH1-like polarization (decreased expression of T-bet, CXC motif chemokine receptor 3 and IFNγ) and promoted TH2-like polarization (increased expression of GATA-3, CCR4 and IL4). TH1-like Treg cells limited CD8+ T cell effector function, proliferation and memory formation during acute and chronic infection. These findings provide fundamental insights into how Treg cells sense inflammatory cues from the environment (such as IFNγ) during viral infection to provide guidance to the effector immune response. This regulatory circuit prevents prolonged immunoinflammatory responses and shapes the quality and quantity of the memory T cell response.

Environmental enteric dysfunction induces regulatory T cells that inhibit local CD4+ T cell responses and impair oral vaccine efficacy.

Environmental enteric dysfunction (EED) is a gastrointestinal inflammatory disease caused by malnutrition and chronic infection. EED is associated with stunting in children and reduced efficacy of oral vaccines. To study the mechanisms of oral vaccine failure during EED, we developed a microbiota- and diet-dependent mouse EED model. Analysis of E. coli-labile toxin vaccine-specific CD4+ T cells in these mice revealed impaired CD4+ T cell responses in the small intestine and but not the lymph nodes. EED mice exhibited increased frequencies of small intestine-resident RORγT+FOXP3+ regulatory T (Treg) cells. Targeted deletion of RORγT from Treg cells restored small intestinal vaccine-specific CD4 T cell responses and vaccine-mediated protection upon challenge. However, ablation of RORγT+FOXP3+ Treg cells made mice more susceptible to EED-induced stunting. Our findings provide insight into the poor efficacy of oral vaccines in EED and highlight how RORγT+FOXP3+ Treg cells can regulate intestinal immunity while leaving systemic responses intact.

Mammalian Target of Rapamycin Complex 1 Activation in Macrophages Contributes to Persistent Lung Inflammation following Respiratory Tract Viral Infection.

Respiratory tract virus infections cause millions of hospitalizations worldwide each year. Severe infections lead to lung damage that coincides with persistent inflammation and a lengthy repair period. Vaccination and antiviral therapy help to mitigate severe infections before or during the acute stage of disease, but there are currently limited specific treatment options available to individuals experiencing the long-term sequelae of respiratory viral infection. Herein, C57BL/6 mice were infected with influenza A/PR/8/34 as a model for severe viral lung infection and allowed to recover for 21 days. Mice were treated with rapamycin, a well-characterized mammalian target of rapamycin complex 1 (mTORC1) inhibitor, on days 12 to 20 after infection, a time period after viral clearance. Persistent inflammation following severe influenza infection in mice was primarily driven by macrophages and T cells. Uniform manifold approximation and projection analysis of flow cytometry data revealed that lung macrophages had high activation of mTORC1, an energy-sensing kinase involved in inflammatory immune cell effector functions. Rapamycin treatment reduced lung inflammation and the frequency of exudate macrophages, T cells, and B cells in the lung, while not impacting epithelial progenitor cells or adaptive immune memory. These data highlight mTORC1's role in sustaining persistent inflammation following clearance of a viral respiratory pathogen and suggest a possible intervention for post-viral chronic lung inflammation.

Sex-Based Differences in Persistent Lung Inflammation Following Influenza Infection of Juvenile Outbred Mice.

Children are susceptible to influenza infections and can experience severe disease presentation due to a lack of or limited pre-existing immunity. Despite the disproportionate impact influenza has on this population, there is a lack of focus on pediatric influenza research, particularly when it comes to identifying the pathogenesis of long-term outcomes that persist beyond the point of viral clearance. In this study, juvenile outbred male and female mice were infected with influenza and analyzed following viral clearance to determine how sex impacts the persistent inflammatory responses to influenza. It was found that females maintained a broader cytokine response in the lung following clearance of influenza, with innate, type I and type II cytokine signatures in almost all mice. Males, on the other hand, had higher levels of IL-6 and other macrophage-related cytokines, but no evidence of a type I or type II response. The immune landscape was similar in the lungs between males and females post-infection, but males had a higher regulatory T cell to TH1 ratio compared to female mice. Cytokine production positively correlated with the frequency of TH1 cells and exudate macrophages, as well as the number of cells in the bronchoalveolar lavage fluid. Furthermore, female lungs were enriched for metabolites involved in the glycolytic pathway, suggesting glycolysis is higher in female lungs compared to males after viral clearance. These data suggest juvenile female mice have persistent and excessive lung inflammation beyond the point of viral clearance, while juvenile males had a more immunosuppressive phenotype.

Heterotypic Influenza Infections Mitigate Susceptibility to Secondary Bacterial Infection.

Influenza-associated bacterial superinfections have devastating impacts on the lung and can result in increased risk of mortality. New strains of influenza circulate throughout the population yearly, promoting the establishment of immune memory. Nearly all individuals have some degree of influenza memory before adulthood. Due to this, we sought to understand the role of immune memory during bacterial superinfections. An influenza heterotypic immunity model was established using influenza A/Puerto Rico/8/34 and influenza A/X31. We report in this article that influenza-experienced mice are more resistant to secondary bacterial infection with methicillin-resistant Staphylococcus aureus as determined by wasting, bacterial burden, pulmonary inflammation, and lung leak, despite significant ongoing lung remodeling. Multidimensional flow cytometry and lung transcriptomics revealed significant alterations in the lung environment in influenza-experienced mice compared with naive animals. These include changes in the lung monocyte and T cell compartments, characterized by increased expansion of influenza tetramer-specific CD8+ T cells. The protection that was seen in the memory-experienced mouse model is associated with the reduction in inflammatory mechanisms, making the lung less susceptible to damage and subsequent bacterial colonization. These findings provide insight into how influenza heterotypic immunity reshapes the lung environment and the immune response to a rechallenge event, which is highly relevant to the context of human infection.

A Fbxo48 inhibitor prevents pAMPKα degradation and ameliorates insulin resistance.

The adenosine monophosphate (AMP)-activated protein kinase (Ampk) is a central regulator of metabolic pathways, and increasing Ampk activity has been considered to be an attractive therapeutic target. Here, we have identified an orphan ubiquitin E3 ligase subunit protein, Fbxo48, that targets the active, phosphorylated Ampkα (pAmpkα) for polyubiquitylation and proteasomal degradation. We have generated a novel Fbxo48 inhibitory compound, BC1618, whose potency in stimulating Ampk-dependent signaling greatly exceeds 5-aminoimidazole-4-carboxamide-1-ß-ribofuranoside (AICAR) or metformin. This compound increases the biological activity of Ampk not by stimulating the activation of Ampk, but rather by preventing activated pAmpkα from Fbxo48-mediated degradation. We demonstrate that, consistent with augmenting Ampk activity, BC1618 promotes mitochondrial fission, facilitates autophagy and improves hepatic insulin sensitivity in high-fat-diet-induced obese mice. Hence, we provide a unique bioactive compound that inhibits pAmpkα disposal. Together, these results define a new pathway regulating Ampk biological activity and demonstrate the potential utility of modulating this pathway for therapeutic benefit.

Influenza sequelae: from immune modulation to persistent alveolitis.

Acute influenza virus infections are a global public health concern accounting for millions of illnesses worldwide ranging from mild to severe with, at time, severe complications. Once an individual is infected, the immune system is triggered in response to the pathogen. This immune response can be beneficial ultimately leading to the clearance of the viral infection and establishment of immune memory mechanisms. However, it can be detrimental by increasing susceptibility to secondary bacterial infections and resulting in permanent changes to the lung architecture, in the form of fibrotic sequelae. Here, we review influenza associated bacterial super-infection, the formation of T-cell memory, and persistent lung injury resulting from influenza infection.

Adropin treatment restores cardiac glucose oxidation in pre-diabetic obese mice.

Exposure to a high fat (HF) diet promotes increased fatty acid uptake, fatty acid oxidation and lipid accumulation in the heart. These maladaptive changes impact cellular energy metabolism and may promote the development of cardiac dysfunction. Attempts to increase cardiac glucose utilization have been proposed as a way to reverse cardiomyopathy in obese and diabetic individuals. Adropin is a nutrient-regulated metabolic hormone shown to promote glucose oxidation over fatty acid oxidation in skeletal muscle homogenates in vitro. The focus of the current study was to investigate whether adropin can regulate substrate metabolism in the heart following prolonged exposure to a HF diet in vivo. Mice on a long-term HF diet received serial intraperitoneal injections of vehicle or adropin over three days. Cardiac glucose oxidation was significantly reduced in HF animals, which was rescued by acute adropin treatment. Significant decreases in cardiac pyruvate dehydrogenase activity were observed in HF animals, which were also reversed by adropin treatment. In contrast to previous studies, this change was unrelated to Pdk4 expression, which remained elevated in both vehicle- and adropin-treated HF mice. Instead, we show that adropin modulated the expression of the mitochondrial acetyltransferase enzyme GCN5L1, which altered the acetylation status and activity of fuel metabolism enzymes to favor glucose utilization. Our findings indicate that adropin exposure leads to increased cardiac glucose oxidation under HF conditions, and may provide a future therapeutic avenue in the treatment of diabetic cardiomyopathy.

Reduced intestinal lipid absorption and body weight-independent improvements in insulin sensitivity in high-fat diet-fed Park2 knockout mice.

Mitochondrial dysfunction is associated with many human diseases and results from mismatch of damage and repair over the life of the organelle. PARK2 is a ubiquitin E3 ligase that regulates mitophagy, a repair mechanism that selectively degrades damaged mitochondria. Deletion of PARK2 in multiple in vivo models results in susceptibility to stress-induced mitochondrial and cellular dysfunction. Surprisingly, Park2 knockout (KO) mice are protected from nutritional stress and do not develop obesity, hepatic steatosis or insulin resistance when fed a high-fat diet (HFD). However, these phenomena are casually related and the physiological basis for this phenotype is unknown. We therefore undertook a series of acute HFD studies to more completely understand the physiology of Park2 KO during nutritional stress. We find that intestinal lipid absorption is impaired in Park2 KO mice as evidenced by increased fecal lipids and reduced plasma triglycerides after intragastric fat challenge. Park2 KO mice developed hepatic steatosis in response to intravenous lipid infusion as well as during incubation of primary hepatocytes with fatty acids, suggesting that hepatic protection from nutritional stress was secondary to changes in energy balance due to altered intestinal triglyceride absorption. Park2 KO mice showed reduced adiposity after 1-wk HFD, as well as improved hepatic and peripheral insulin sensitivity. These studies suggest that changes in intestinal lipid absorption may play a primary role in protection from nutritional stress in Park2 KO mice by preventing HFD-induced weight gain and highlight the need for tissue-specific models to address the role of PARK2 during metabolic stress.

Empagliflozin restores cardiac metabolic flexibility in diet-induced obese C57BL6/J mice.

Sodium-glucose co-transporter type 2 (SGLT2) inhibitor therapy to treat type 2 diabetes unexpectedly reduced all-cause mortality and hospitalization due to heart failure in several large-scale clinical trials, and has since been shown to produce similar cardiovascular disease-protective effects in patients without diabetes. How SGLT2 inhibitor therapy improves cardiovascular disease outcomes remains incompletely understood. Metabolic flexibility refers to the ability of a cell or organ to adjust its use of metabolic substrates, such as glucose or fatty acids, in response to physiological or pathophysiological conditions, and is a feature of a healthy heart that may be lost during diabetic cardiomyopathy and in the failing heart. We therefore undertook studies to determine the effects of SGLT2 inhibitor therapy on cardiac metabolic flexibility in vivo in obese, insulin resistant mice using a [U13C]-glucose infusion during fasting and hyperinsulinemic euglycemic clamp. Relative rates of cardiac glucose versus fatty acid use during fasting were unaffected by EMPA, whereas insulin-stimulated rates of glucose use were significantly increased by EMPA, alongside significant improvements in cardiac insulin signaling. These metabolic effects of EMPA were associated with reduced cardiac hypertrophy and protection from ischemia. These observations suggest that the cardiovascular disease-protective effects of SGLT2 inhibitors may in part be explained by beneficial effects on cardiac metabolic substrate selection.

Liver-specific Prkn knockout mice are more susceptible to diet-induced hepatic steatosis and insulin resistance.

OBJECTIVE: PARKIN is an E3 ubiquitin ligase that regulates mitochondrial quality control through a process called mitophagy. Recent human and rodent studies suggest that loss of hepatic mitophagy may occur during the pathogenesis of obesity-associated fatty liver and contribute to changes in mitochondrial metabolism associated with this disease. Whole-body Prkn knockout mice are paradoxically protected against diet-induced hepatic steatosis; however, liver-specific effects of Prkn deficiency cannot be discerned in this model due to pleotropic effects of germline Prkn deletion on energy balance and subsequent protection against diet-induced obesity. We therefore generated the first liver-specific Prkn knockout mouse strain (LKO) to directly address the role of hepatic Prkn. METHODS: Littermate control (WT) and LKO mice were fed regular chow (RC) or high-fat diet (HFD) and changes in body weight and composition were measured over time. Liver mitochondrial content was assessed using multiple, complementary techniques, and mitochondrial respiratory capacity was assessed using Oroboros O2K platform. Liver fat was measured biochemically and assessed histologically, while global changes in hepatic gene expression were measured by RNA-seq. Whole-body and tissue-specific insulin resistance were assessed by hyperinsulinemic-euglycemic clamp with isotopic tracers. RESULTS: Liver-specific deletion of Prkn had no effect on body weight or adiposity during RC or HFD feeding; however, hepatic steatosis was increased by 45% in HFD-fed LKO compared with WT mice (P < 0.05). While there were no differences in mitochondrial content between genotypes on either diet, mitochondrial respiratory capacity and efficiency in the liver were significantly reduced in LKO mice. Gene enrichment analyses from liver RNA-seq results suggested significant changes in pathways related to lipid metabolism and fibrosis in HFD-fed Prkn knockout mice. Finally, whole-body insulin sensitivity was reduced by 35% in HFD-fed LKO mice (P < 0.05), which was primarily due to increased hepatic insulin resistance (60% of whole-body effect; P = 0.11). CONCLUSIONS: These data demonstrate that PARKIN contributes to mitochondrial homeostasis in the liver and plays a protective role against the pathogenesis of hepatic steatosis and insulin resistance.

Hepatic insulin sensitivity is improved in high-fat diet-fed Park2 knockout mice in association with increased hepatic AMPK activation and reduced steatosis.

Park2 is an E3 ubiquitin ligase known for its role in mitochondrial quality control via the mitophagy pathway. Park2 KO mice are protected from diet-induced obesity and hepatic insulin sensitivity is improved in high-fat diet (HFD)-fed Park2 KO mice even under body weight-matched conditions. In order to better understand the cellular mechanism by which Park2 KO mice are protected from diet-induced hepatic insulin resistance, we determined changes in multiple pathways commonly associated with the pathogenesis of insulin resistance, namely levels of bioactive lipid species, activation of the endoplasmic reticulum (ER) stress response and changes in cytokine levels and signaling. We report for the first time that whole-body insulin sensitivity is unchanged in regular chow (RC)-fed Park2 KO mice, and that liver diacylglycerol levels are reduced and very-long-chain ceramides are increased in Park2 KO mice fed HFD for 1 week. Hepatic transcriptional markers of the ER stress response were reduced and plasma tumor necrosis factor-α (TNFα), interleukin-6 and -10 (IL6, IL10) were significantly increased in HFD-fed Park2 KO mice; however, there were no detectable differences in hepatic inflammatory signaling pathways between groups. Interestingly, hepatic adenylate charge was reduced in HFD-fed Park2 KO liver and was associated increased activation of AMPK. These data suggest that negative energy balance that contributed to protection from obesity during chronic HFD manifested at the level of the hepatocyte during short-term HFD feeding and contributed to the improved hepatic insulin sensitivity.

Adropin reduces blood glucose levels in mice by limiting hepatic glucose production.

Adropin is a liver- and brain-secreted peptide hormone with striking effects on fuel metabolism regulation in a number of tissues. Previous studies demonstrated that adropin secretion is decreased in obese mice subjected to a long-term high-fat diet (HFD), and that whole-body loss of adropin expression resulted in systemic insulin resistance. Treatment of obese mice with adropin improves glucose tolerance, which has been linked to increased glucose oxidation and inhibition of fatty acid utilization in isolated skeletal muscle homogenates. In this study, we used in vivo physiological measurements to determine how treatment of obese mice with adropin affects whole-body glucose metabolism. Treatment with adropin reduced fasting blood glucose and, as shown previously, increased glucose tolerance in HFD mice during standard glucose tolerance tests. Under hyperinsulinemic-euglycemic clamp conditions, adropin treatment led to a nonsignificant increase in whole-body insulin sensitivity, and a significant reduction in whole-body glucose uptake. Finally, we show that adropin treatment suppressed hepatic glucose production and improved hepatic insulin sensitivity. This correlated with reduced expression of fatty acid import proteins and gluconeogenic regulatory enzymes in the liver, suggesting that adropin treatment may impact the pathways that drive vital aspects of hepatic glucose metabolism.

Hepatocyte-Specific Ablation or Whole-Body Inhibition of Xanthine Oxidoreductase in Mice Corrects Obesity-Induced Systemic Hyperuricemia Without Improving Metabolic Abnormalities.

Systemic hyperuricemia (HyUA) in obesity/type 2 diabetes facilitated by elevated activity of xanthine oxidoreductase (XOR), which is the sole source of uric acid (UA) in mammals, has been proposed to contribute to the pathogenesis of insulin resistance/dyslipidemia in obesity. Here, the effects of hepatocyte-specific ablation of Xdh, the gene encoding XOR (HXO), and whole-body pharmacologic inhibition of XOR (febuxostat) on obesity-induced insulin resistance/dyslipidemia were assessed. Deletion of hepatocyte Xdh substantially lowered liver and plasma UA concentration. When exposed to an obesogenic diet, HXO and control floxed (FLX) mice became equally obese, but systemic HyUA was absent in HXO mice. Despite this, obese HXO mice became as insulin resistant and dyslipidemic as obese FLX mice. Similarly, febuxostat dramatically lowered plasma and tissue UA and XOR activity in obese wild-type mice without altering obesity-associated insulin resistance/dyslipidemia. These data demonstrate that hepatocyte XOR activity is a critical determinant of systemic UA homeostasis, that deletion of hepatocyte Xdh is sufficient to prevent systemic HyUA of obesity, and that neither prevention nor correction of HyUA improves insulin resistance/dyslipidemia in obesity. Thus, systemic HyUA, although clearly a biomarker of the metabolic abnormalities of obesity, does not appear to be causative.