Epigenetically active chromatin in neonatal iWAT reveals GABPα as a potential regulator of beige adipogenesis.

Background: Thermogenic beige adipocytes, which dissipate energy as heat, are found in neonates and adults. Recent studies show that neonatal beige adipocytes are highly plastic and contribute to >50% of beige adipocytes in adults. Neonatal beige adipocytes are distinct from recruited beige adipocytes in that they develop independently of temperature and sympathetic innervation through poorly defined mechanisms. Methods: We characterized the neonatal beige adipocytes in the inguinal white adipose tissue (iWAT) of C57BL6 postnatal day 3 and 20 mice (P3 and P20) by imaging, genome-wide RNA-seq analysis, ChIP-seq analysis, qRT-PCR validation, and biochemical assays. Results: We found an increase in acetylated histone 3 lysine 27 (H3K27ac) on the promoter and enhancer regions of beige-specific gene UCP1 in iWAT of P20 mice. Furthermore, H3K27ac ChIP-seq analysis in the iWAT of P3 and P20 mice revealed strong H3K27ac signals at beige adipocyte-associated genes in the iWAT of P20 mice. The integration of H3K27ac ChIP-seq and RNA-seq analysis in the iWAT of P20 mice reveal epigenetically active signatures of beige adipocytes, including oxidative phosphorylation and mitochondrial metabolism. We identify the enrichment of GA-binding protein alpha (GABPα) binding regions in the epigenetically active chromatin regions of the P20 iWAT, particularly on beige genes, and demonstrate that GABPα is required for beige adipocyte differentiation. Moreover, transcriptomic analysis and glucose oxidation assays revealed increased glycolytic activity in the neonatal iWAT from P20. Conclusions: Our findings demonstrate that epigenetic mechanisms regulate the development of peri-weaning beige adipocytes via GABPα. Further studies to better understand the upstream mechanisms that regulate epigenetic activation of GABPα and characterization of the metabolic identity of neonatal beige adipocytes will help us harness their therapeutic potential in metabolic diseases.

Emerging Role of Hepatic Ketogenesis in Fatty Liver Disease.

Non-alcoholic fatty liver disease (NAFLD), the most common chronic liver diseases, arise from non-alcoholic fatty liver (NAFL) characterized by excessive fat accumulation as triglycerides. Although NAFL is benign, it could progress to non-alcoholic steatohepatitis (NASH) manifested with inflammation, hepatocyte damage and fibrosis. A subset of NASH patients develops end-stage liver diseases such as cirrhosis and hepatocellular carcinoma. The pathogenesis of NAFLD is highly complex and strongly associated with perturbations in lipid and glucose metabolism. Lipid disposal pathways, in particular, impairment in condensation of acetyl-CoA derived from ß-oxidation into ketogenic pathway strongly influence the hepatic lipid loads and glucose metabolism. Current evidence suggests that ketogenesis dispose up to two-thirds of the lipids entering the liver, and its dysregulation significantly contribute to the NAFLD pathogenesis. Moreover, ketone body administration in mice and humans shows a significant improvement in NAFLD. This review focuses on hepatic ketogenesis and its role in NAFLD pathogenesis. We review the possible mechanisms through which impaired hepatic ketogenesis may promote NAFLD progression. Finally, the review sheds light on the therapeutic implications of a ketogenic diet in NAFLD.

Oxidative Stress and Redox Signaling in the Pathophysiology of Liver Diseases.

The increased production of derivatives of molecular oxygen and nitrogen in the form of reactive oxygen species (ROS) and reactive nitrogen species (RNS) lead to molecular damage called oxidative stress. Under normal physiological conditions, the ROS generation is tightly regulated in different cells and cellular compartments. Any disturbance in the balance between the cellular generation of ROS and antioxidant balance leads to oxidative stress. In this article, we discuss the sources of ROS (endogenous and exogenous) and antioxidant mechanisms. We also focus on the pathophysiological significance of oxidative stress in various cell types of the liver. Oxidative stress is implicated in the development and progression of various liver diseases. We narrate the master regulators of ROS-mediated signaling and their contribution to liver diseases. Nonalcoholic fatty liver diseases (NAFLD) are influenced by a "multiple parallel-hit model" in which oxidative stress plays a central role. We highlight the recent findings on the role of oxidative stress in the spectrum of NAFLD, including fibrosis and liver cancer. Finally, we provide a brief overview of oxidative stress biomarkers and their therapeutic applications in various liver-related disorders. Overall, the article sheds light on the significance of oxidative stress in the pathophysiology of the liver. © 2022 American Physiological Society. Compr Physiol 12:3167-3192, 2022.

Vertical sleeve gastrectomy increases duodenal Lactobacillus spp. richness associated with the activation of intestinal HIF2α signaling and metabolic benefits.

OBJECTIVE: Vertical Sleeve Gastrectomy (VSG) is one of the most efficacious treatments for obesity and its comorbidities. Although a range of evidence suggests that alterations of the microbiota in the distal gut following VSG are pivotal to these metabolic improvements, the effect of surgery to alter the microbiota of the proximal intestine and its effect on host physiology remain largely unknown. As the main bacteria in the upper small intestine, Lactobacillus subspecies have been appreciated as important regulators of gut function. These bacteria also regulate intestinal Hypoxia- Inducible Factor 2α (HIF2α) signaling that plays an integral role in gut physiology and iron absorption. In the present study, we sought to determine the impact of VSG on Lactobacillus spp. in the small intestine and potential downstream impacts of Lactobacillus spp. on HIF2α, specifically in the duodenum. METHODS: To determine the effects of VSG on the microbiota and HIF2α signaling in the duodenum, VSG surgeries were performed on diet-induced obese mice. To further probe the relationship between Lactobacillus spp. and HIF2α signaling in the duodenum, we applied a customized high-fat but iron-deficient diet on mice to increase duodenal HIF2α signaling and determined alterations of gut bacteria. To explore the causal role of Lactobacillus spp. in duodenal HIF2α signaling activation, we chronically administered probiotics containing Lactobacillus spp. to high-fat-fed obese mice. Lastly, we studied the effect of lactate, the major metabolite of Lactobacilli, on HIF2α in ex vivo duodenal organoids. RESULTS: There were pronounced increases in the abundance of Lactobacillus spp. in samples isolated from duodenal epithelium in VSG-operated mice as compared to sham-operated mice. This was accompanied by an increase in the expression of genes that are targets of HIF2α in the duodenum of VSG-treated mice. Activating HIF2α signaling with a high-fat but iron-deficient diet resulted in weight loss, improvements in glucose regulation, and increased Lactobacillus spp. richness in the duodenum as compared to mice on an iron-replete diet. Chronic administration of probiotics containing Lactobacillus spp. not only increased HIF2α signaling in the duodenum such as occurs after VSG but also resulted in reduced weight gain and improved glucose tolerance in high-fat-fed mice. Furthermore, lactate was able to activate HIF2α in ex vivo duodenal organoids. CONCLUSIONS: These results support a model whereby VSG increases duodenal Lactobacillus richness and potentially stimulates intestinal HIF2α signaling via increased lactate production.

Gut HIF2α signaling is increased after VSG, and gut activation of HIF2α decreases weight, improves glucose, and increases GLP-1 secretion.

Gastric bypass and vertical sleeve gastrectomy (VSG) remain the most potent and durable treatments for obesity and type 2 diabetes but are also associated with iron deficiency. The transcription factor HIF2α, which regulates iron absorption in the duodenum, increases following these surgeries. Increasing iron levels by means of dietary supplementation or hepatic hepcidin knockdown does not undermine the effects of VSG, indicating that metabolic improvements following VSG are not secondary to lower iron levels. Gut-specific deletion of Vhl results in increased constitutive duodenal HIF2α signaling and produces a profound lean, glucose-tolerant phenotype that mimics key effects of VSG. Interestingly, intestinal Vhl deletion also results in increased intestinal secretion of GLP-1, which is essential for these metabolic benefits. These data demonstrate a role for increased duodenal HIF2α signaling in regulating crosstalk between iron-regulatory systems and other aspects of systemic physiology important for metabolic regulation.

Membrane Bound Peroxiredoxin-1 Serves as a Biomarker for In Vivo Detection of Sessile Serrated Adenomas.

Aim: Sessile serrated adenomas (SSAs) are premalignant lesions driven by the BRAFV600E mutation to give rise to colorectal cancers (CRCs). They are often missed during white light colonoscopy because of their subtle appearance. Previously, a fluorescently labeled 7mer peptide KCCFPAQ was shown to detect SSAs in vivo. We aim to identify the target of this peptide. Results: Peroxiredoxin-1 (Prdx1) was identified as the binding partner of the peptide ligand. In vitro binding assays and immunofluorescence staining of human colon specimens ex vivo supported this result. Prdx1 was overexpressed on the membrane of cells with the BRAFV600E mutation, and this effect was dependent on oxidative stress. RKO cells harboring the BRAFV600E mutation and human SSA specimens showed higher oxidative stress as well as elevated levels of Prdx1 on the cell membrane. Innovation and Conclusion: These results suggest that Prdx1 is overexpressed on the cell surface in the presence of oxidative stress and can serve as an imaging biomarker for in vivo detection of SSAs. Antioxid. Redox Signal. 36, 39-56.

Intestinal HIF-2α Regulates GLP-1 Secretion via Lipid Sensing in L-Cells.

BACKGROUND & AIMS: Compelling evidence shows that glucagon-like peptide-1 (GLP-1) has a profound effect in restoring normoglycemia in type 2 diabetic patients by increasing pancreatic insulin secretion. Although L-cells are the primary source of circulating GLP-1, the current therapies do not target L-cells to increase GLP-1 levels. Our study aimed to determine the molecular underpinnings of GLP-1 secretion as an impetus to identify new interventions to target endogenous L-cells. METHODS: We used genetic mouse models of intestine-specific overexpression of hypoxia-inducible factor (HIF)-1α and HIF-2α (VhlΔIE), conditional overexpression of intestinal HIF-2α (Hif-2αLSL;Vilin-Cre/ERT2), and intestine-specific HIF-2α knockout mice (Hif-2αΔIE) to show that HIF signaling, especially HIF-2α, regulates GLP-1 secretion. RESULTS: Our data show that intestinal HIF signaling improved glucose homeostasis in a GLP-1-dependent manner. Intestinal HIF potentiated GLP-1 secretion via the lipid sensor G-protein-coupled receptor (GPR)40 enriched in L-cells. We show that HIF-2α regulates GPR40 in L-cells and potentiates fatty acid-induced GLP-1 secretion via extracellular regulated kinase (ERK). Using a genetic model of intestine-specific overexpression of HIF-2α, we show that HIF-2α is sufficient to increase GLP-1 levels and attenuate diet-induced metabolic perturbations such as visceral adiposity, glucose intolerance, and hepatic steatosis. Lastly, we show that intestinal HIF-2α signaling acts as a priming mechanism crucial for postprandial lipid-mediated GLP-1 secretion. Thus, disruption of intestinal HIF-2α decreases GLP-1 secretion. CONCLUSIONS: In summary, we show that intestinal HIF signaling, particularly HIF-2α, regulates the lipid sensor GPR40, which is crucial for the lipid-mediated GLP-1 secretion, and suggest that HIF-2α is a potential target to induce endogenous GLP-1 secretion.

Hypoxia via ERK Signaling Inhibits Hepatic PPARα to Promote Fatty Liver.

BACKGROUND & AIMS: Fatty liver or nonalcoholic fatty liver disease (NAFLD) is the most common liver disease associated with comorbidities such as insulin resistance and cardiovascular and metabolic diseases. Chronic activation of hypoxic signaling, in particular, hypoxia-inducible factor (HIF)2α, promotes NAFLD progression by repressing genes involved in fatty acid ß-oxidation through unclear mechanisms. Therefore, we assessed the precise mechanism by which HIF2α promotes fatty liver and its physiological relevance in metabolic homeostasis. METHODS: Primary hepatocytes from VHL (VhlΔHep) and PPARα (Ppara-null) knockout mice that were loaded with fatty acids, murine dietary protocols to induce hepatic steatosis, and fasting-refeeding dietary regimen approaches were used to test our hypothesis. RESULTS: Inhibiting autophagy using chloroquine did not decrease lipid contents in VhlΔHep primary hepatocytes. Inhibition of ERK using MEK inhibitor decreased lipid contents in primary hepatocytes from a genetic model of constitutive HIF activation and primary hepatocytes loaded with free fatty acids. Moreover, MEK-ERK inhibition potentiated ligand-dependent activation of PPARα. We also show that MEK-ERK inhibition improved diet-induced hepatic steatosis, which is associated with the induction of PPARα target genes. During fasting, fatty acid ß-oxidation is induced by PPARα, and refeeding inhibits ß-oxidation. Our data show that ERK is involved in the post-prandial repression of hepatic PPARα signaling. CONCLUSIONS: Overall, our results demonstrate that ERK activated by hypoxia signaling plays a crucial role in fatty acid ß-oxidation genes by repressing hepatocyte PPARα signaling.

Tregs facilitate obesity and insulin resistance via a Blimp-1/IL-10 axis.

Interleukin-10 (IL-10) is a critical cytokine used by immune cells to suppress inflammation. Paradoxically, immune cell-derived IL-10 can drive insulin resistance in obesity by suppressing adipocyte energy expenditure and thermogenesis. However, the source of IL-10 necessary for the suppression of adipocyte thermogenesis is unknown. We show here that CD4+Foxp3+ regulatory T cells (Tregs) are a substantial source of IL-10 and that Treg-derived IL-10 can suppress adipocyte beiging. Unexpectedly, Treg-specific loss of IL-10 resulted in increased insulin sensitivity and reduced obesity in high-fat diet-fed male mice. Mechanistically, we determined that Treg-specific loss of the transcription factor Blimp-1, a driver of IL-10 expression by Tregs, phenocopied the Treg-specific IL-10-deficient mice. Loss of Blimp-1 expression in Tregs resulted in reduced ST2+KLRG1+, IL-10-secreting Tregs, particularly in the white adipose tissue. Blimp-1-deficient mice were protected from glucose intolerance, insulin resistance, and diet-induced obesity, through increased white adipose tissue browning. Taken together, our data show that Blimp-1-regulated IL-10 secretion by Tregs represses white adipose tissue beiging to maintain adipose tissue homeostasis.

An indispensable role for dynamin-related protein 1 in beige and brown adipogenesis.

Emerging evidence indicates that proper mitochondrial dynamics are critical for adipocyte differentiation and functional thermogenic capacity. We found that the mitochondrial fission protein dynamin-related protein 1 (DRP1, also known as DNML1) is highly expressed in brown adipose tissue compared to expression in white adipose tissue, and these expression levels increase during brown adipocyte differentiation. Our results reveal that the inhibition of DRP1 using mdivi-1 mitigates beige adipocyte differentiation and differentiation-associated mitochondrial biogenesis. We found that DRP1 is essential for the induction of the early-phase beige adipogenic transcriptional program. Intriguingly, inhibition of DRP1 is dispensable following the induction of beige adipogenesis and adipogenesis-associated mitochondrial biogenesis. Altogether, we demonstrate that DRP1 in preadipocytes plays an essential role in beige and brown adipogenesis.This article has an associated First Person interview with the first author of the paper.

Sustained mitochondrial biogenesis is essential to maintain caloric restriction-induced beige adipocytes.

BACKGROUND: Caloric restriction (CR) delays the onset of metabolic and age-related disorders. Recent studies have demonstrated that formation of beige adipocytes induced by CR is strongly associated with extracellular remodeling in adipose tissue, decrease in adipose tissue inflammation, and improved systemic metabolic homeostasis. However, beige adipocytes rapidly transition to white upon CR withdrawal through unclear mechanisms. MATERIALS AND METHODS: Six-week old C57BL6 mice were fed with 40% CR chow diet for 6 weeks. Subsequently, one group of mice was switched back to ad libitum chow diet, which was continued for additional 2 weeks. Adipose tissues were assessed histologically and biochemically for beige adipocytes. RESULTS: Beige adipocytes induced by CR rapidly transition to white adipocytes when CR is withdrawn independent of parkin-mediated mitophagy. We demonstrate that the involution of mitochondria during CR withdrawal is strongly linked with a decrease in mitochondrial biogenesis. We further demonstrate that beige-to-white fat transition upon ß3-AR agonist-withdrawal could be attenuated by CR, partly via maintenance of mitochondrial biogenesis. CONCLUSION: In the model of CR, our study highlights the dominant role of mitochondrial biogenesis in the maintenance of beige adipocytes. We propose that loss of beige adipocytes upon ß3-AR agonist withdrawal could be attenuated by CR.

Loss of Ceacam1 promotes prostate cancer progression in Pten haploinsufficient male mice.

OBJECTIVE: PTEN haploinsufficiency plays an important role in prostate cancer development in men. However, monoallelic deletion of Pten gene failed to induce high prostate intraepithelial neoplasia (PIN) until Pten+/- mice aged or fed a high-calorie diet. Because CEACAM1, a cell adhesion molecule with a potential tumor suppression activity, is induced in Pten+/- prostates, the study aimed at examining whether the rise of CEACAM1 limited neoplastic progression in Pten+/- prostates. METHODS: Pten+/- were crossbred with Cc1-/- mice harboring a null deletion of Ceacam1 gene to produce Pten+/-/Cc1-/- double mutants. Prostates from 7-month old male mice were analyzed histologically and biochemically for PIN progression. RESULTS: Deleting Ceacam1 in Pten+/- mice caused an early development of high-grade PIN in parallel to hyperactivation of PI3 kinase/Akt and Ras/MAP kinase pathways, with an increase in cell proliferation, epithelial-to-mesenchymal transition, angiogenesis and inflammation relative to Pten+/- and Cc1-/- individual mutants. It also caused a remarkable increase in lipogenesis in prostate despite maintaining insulin sensitivity. Concomitant Ceacam1 deletion with Pten+/- activated the IL-6/STAT3 signaling pathways to suppress Irf-8 transcription that in turn, led to a decrease in the expression level of promyelocytic leukemia gene, a well characterized tumor suppressor in prostate. CONCLUSIONS: Ceacam1 deletion accelerated high-grade prostate intraepithelial neoplasia in Pten haploinsufficient mice while preserving insulin sensitivity. This demonstrated that the combined loss of Ceacam1 and Pten advanced prostate cancer by increasing lipogenesis and modifying the STAT3-dependent inflammatory microenvironment of prostate.

Temporal induction of intestinal epithelial hypoxia-inducible factor-2α is sufficient to drive colitis.

Hypoxia is a notable feature of inflammatory bowel disease and chronic induction of hypoxia-inducible factor (HIF)-1α and HIF-2α (endothelial PAS domain protein 1, EPAS1) play important, but opposing, roles in its pathogenesis. While activation of HIF-1α decreases intestinal inflammation and is beneficial in colitis, activation of HIF-2α exacerbates colitis and increases colon carcinogenesis in animal models, primarily due to the role of epithelial HIF-2α in mounting a potent inflammatory response. Previous work from our laboratory showed that mice overexpressing intestinal epithelial HIF-2α led to massive intestinal inflammation and decreased survival. As oxygen homeostasis and HIFs are critical in embryonic development, it is not clear whether the observed intestinal inflammatory response was secondary to developmental defects. To address this question, the present study used a mouse model to temporally modulate expression of intestinal epithelial HIF-2α to assess its role in mediating inflammatory response. Remarkably, activation of HIF-2α in intestinal epithelial cells in adult mice increased expression of proinflammatory mediators; however, no decrease in survival was observed. Furthermore, in an acute model of colitis, activation of HIF-2α was sufficient to exacerbate colitis. These data confirm our previous finding that epithelial HIF-2α mediates inflammatory response and demonstrates that activation of HIF-2α is sufficient to exacerbate colitis.NEW & NOTEWORTHY Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory disease of the intestinal tract. Hypoxia and activation of its downstream transcription factors hypoxia-inducible factor (HIF)-1α and HIF-2α are notable features of IBD. HIF-1α has well-characterized protective roles in IBD; however, the role of HIF-2α has been less studied. Using novel HIF-2α mouse models, we show that activation of HIF-2α in intestinal epithelial cells is sufficient to exacerbate colitis.

Intestinal non-canonical NFκB signaling shapes the local and systemic immune response.

Microfold cells (M-cells) are specialized cells of the intestine that sample luminal microbiota and dietary antigens to educate the immune cells of the intestinal lymphoid follicles. The function of M-cells in systemic inflammatory responses are still unclear. Here we show that epithelial non-canonical NFkB signaling mediated by NFkB-inducing kinase (NIK) is highly active in intestinal lymphoid follicles, and is required for M-cell maintenance. Intestinal NIK signaling modulates M-cell differentiation and elicits both local and systemic IL-17A and IgA production. Importantly, intestinal NIK signaling is active in mouse models of colitis and patients with inflammatory bowel diseases; meanwhile, constitutive NIK signaling increases the susceptibility to inflammatory injury by inducing ectopic M-cell differentiation and a chronic increase of IL-17A. Our work thus defines an important function of non-canonical NFkB and M-cells in immune homeostasis, inflammation and polymicrobial sepsis.

Hypoxia-Inducible Factor (HIF)-1α Promotes Inflammation and Injury Following Aspiration-Induced Lung Injury in Mice.

Acid aspiration-induced lung injury is a common disease in the intensive care unit (ICU) and acute respiratory distress syndrome (ARDS). Hypoxia-inducible factor (HIF)-1α is a major transcription factor responsible for regulating the cellular response to changes in oxygen tension. A clear understanding of the function of HIF-1α in lung inflammatory response is currently lacking. Here, we sought to determine the role of HIF-1α in type 2 alveolar epithelial cells (AEC) in the generation of the acute inflammatory response following gastric aspiration (GA). GA led to profound hypoxia at very early time points following GA. This correlated to a robust increase in HIF-1α, tissue albumin and pro-inflammatory mediators following GA in AECs. The extent of lung injury and the release of pro/anti-inflammatory cytokines were significantly reduced in HIF-1α (-/-) mice. Finally, we report that HIF-1α upregulation of the acute inflammatory response is dependent on NF-κB following GA.

Hepatic hepcidin/intestinal HIF-2α axis maintains iron absorption during iron deficiency and overload.

Iron-related disorders are among the most prevalent diseases worldwide. Systemic iron homeostasis requires hepcidin, a liver-derived hormone that controls iron mobilization through its molecular target ferroportin (FPN), the only known mammalian iron exporter. This pathway is perturbed in diseases that cause iron overload. Additionally, intestinal HIF-2α is essential for the local absorptive response to systemic iron deficiency and iron overload. Our data demonstrate a hetero-tissue crosstalk mechanism, whereby hepatic hepcidin regulated intestinal HIF-2α in iron deficiency, anemia, and iron overload. We show that FPN controlled cell-autonomous iron efflux to stabilize and activate HIF-2α by regulating the activity of iron-dependent intestinal prolyl hydroxylase domain enzymes. Pharmacological blockade of HIF-2α using a clinically relevant and highly specific inhibitor successfully treated iron overload in a mouse model. These findings demonstrate a molecular link between hepatic hepcidin and intestinal HIF-2α that controls physiological iron uptake and drives iron hyperabsorption during iron overload.

Neutrophils Restrict Tumor-Associated Microbiota to Reduce Growth and Invasion of Colon Tumors in Mice.

BACKGROUND & AIMS: Neutrophils are among the most prevalent immune cells in the microenvironment of colon tumors; they are believed to promote growth of colon tumors, and their numbers correlate with outcomes of patients with colon cancer. Trials of inhibitors of neutrophil trafficking are underway in patients with cancer, but it is not clear how neutrophils contribute to colon tumorigenesis. METHODS: Colitis-associated colon cancer was induced in mice with conditional deletion of neutrophils (LysMCre;Mcl1fl/fl) and wild-type littermates (LysMCre;Mcl1wt/wt, control mice) by administration of azoxythmethane and/or dextran sulfate sodium. Sporadic colon tumorigenesis was assessed in neutrophil-deficient and neutrophil-replete mice with conditional deletion of colon epithelial Apc (Cdx2-CreERT2;Apcfl/fl). Primary colon tumor tissues from these mice were assessed by histology, RNA sequencing, quantitative polymerase chain reaction, and fluorescence in situ hybridization analyses. Fecal and tumor-associated microbiota were assessed by 16s ribosomal RNA sequencing. RESULTS: In mice with inflammation-induced and sporadic colon tumors, depletion of neutrophils increased the growth, proliferation, and invasiveness of the tumors. RNA sequencing analysis identified genes that regulate antimicrobial and inflammatory processes that were dysregulated in neutrophil-deficient colon tumors compared with colon tumors from control mice. Neutrophil depletion correlated with increased numbers of bacteria in tumors and proliferation of tumor cells, tumor-cell DNA damage, and an inflammatory response mediated by interleukin 17 (IL17). The 16s ribosomal RNA sequencing identified significant differences in the composition of the microbiota between colon tumors from neutrophil-deficient vs control mice. Administration of antibiotics or a neutralizing antibody against IL17 to neutrophil-deficient mice resulted in development of less-invasive tumors compared with mice given vehicle. We found bacteria in tumors to induce production of IL17, which promotes influx of intratumor B cells that promote tumor growth and progression. CONCLUSIONS: In comparisons of mice with vs without neutrophils, we found neutrophils to slow colon tumor growth and progression by restricting numbers of bacteria and tumor-associated inflammatory responses.

Natural Secretory Immunoglobulins Promote Enteric Viral Infections.

Noroviruses are enteric pathogens causing significant morbidity, mortality, and economic losses worldwide. Secretory immunoglobulins (sIg) are a first line of mucosal defense against enteric pathogens. They are secreted into the intestinal lumen via the polymeric immunoglobulin receptor (pIgR), where they bind to antigens. However, whether natural sIg protect against norovirus infection remains unknown. To determine if natural sIg alter murine norovirus (MNV) pathogenesis, we infected pIgR knockout (KO) mice, which lack sIg in mucosal secretions. Acute MNV infection was significantly reduced in pIgR KO mice compared to controls, despite increased MNV target cells in the Peyer's patch. Natural sIg did not alter MNV binding to the follicle-associated epithelium (FAE) or crossing of the FAE into the lymphoid follicle. Instead, naive pIgR KO mice had enhanced levels of the antiviral inflammatory molecules interferon gamma (IFN-γ) and inducible nitric oxide synthase (iNOS) in the ileum compared to controls. Strikingly, depletion of the intestinal microbiota in pIgR KO and control mice resulted in comparable IFN-γ and iNOS levels, as well as MNV infectious titers. IFN-γ treatment of wild-type (WT) mice and neutralization of IFN-γ in pIgR KO mice modulated MNV titers, implicating the antiviral cytokine in the phenotype. Reduced gastrointestinal infection in pIgR KO mice was also observed with another enteric virus, reovirus. Collectively, our findings suggest that natural sIg are not protective during enteric virus infection, but rather, that sIg promote enteric viral infection through alterations in microbial immune responses.IMPORTANCE Enteric virus, such as norovirus, infections cause significant morbidity and mortality worldwide. However, direct antiviral infection prevention strategies are limited. Blocking host entry and initiation of infection provides an established avenue for intervention. Here, we investigated the role of the polymeric immunoglobulin receptor (pIgR)-secretory immunoglobulin (sIg) cycle during enteric virus infections. The innate immune functions of sIg (agglutination, immune exclusion, neutralization, and expulsion) were not required during control of acute murine norovirus (MNV) infection. Instead, lack of pIgR resulted in increased IFN-γ levels, which contributed to reduced MNV titers. Another enteric virus, reovirus, also showed decreased infection in pIgR KO mice. Collectively, our data point to a model in which sIg-mediated microbial sensing promotes norovirus and reovirus infection. These data provide the first evidence of the proviral role of natural sIg during enteric virus infections and provide another example of how intestinal bacterial communities indirectly influence MNV pathogenesis.

Pancreatic HIF2α Stabilization Leads to Chronic Pancreatitis and Predisposes to Mucinous Cystic Neoplasm.

BACKGROUND & AIMS: Tissue hypoxia controls cell differentiation in the embryonic pancreas, and promotes tumor growth in pancreatic cancer. The cellular response to hypoxia is controlled by the hypoxia-inducible factor (HIF) proteins, including HIF2α. Previous studies of HIF action in the pancreas have relied on loss-of-function mouse models, and the effects of HIF2α expression in the pancreas have remained undefined. METHODS: We developed several transgenic mouse models based on the expression of an oxygen-stable form of HIF2α, or indirect stabilization of HIF proteins though deletion of von Hippel-Lindau, thus preventing HIF degradation. Furthermore, we crossed both sets of animals into mice expressing oncogenic KrasG12D in the pancreas. RESULTS: We show that HIF2α is not expressed in the normal human pancreas, however, it is up-regulated in human chronic pancreatitis. Deletion of von Hippel-Lindau or stabilization of HIF2α in mouse pancreata led to the development of chronic pancreatitis. Importantly, pancreatic HIF1α stabilization did not disrupt the pancreatic parenchyma, indicating that the chronic pancreatitis phenotype is specific to HIF2α. In the presence of oncogenic Kras, HIF2α stabilization drove the formation of cysts resembling mucinous cystic neoplasm (MCN) in humans. Mechanistically, we show that the pancreatitis phenotype is linked to expression of multiple inflammatory cytokines and activation of the unfolded protein response. Conversely, MCN formation is linked to activation of Wnt signaling, a feature of human MCN. CONCLUSIONS: We show that pancreatic HIF2α stabilization disrupts pancreatic homeostasis, leading to chronic pancreatitis, and, in the context of oncogenic Kras, MCN formation. These findings provide new mouse models of both chronic pancreatitis and MCN, as well as illustrate the importance of hypoxia signaling in the pancreas.