Functional bowel disorder patients can suffer from chronic abdominal pain, likely due to visceral hypersensitivity to mechanical stimuli. As there is only a limited understanding of the basis of chronic visceral hypersensitivity (CVH), drug-based management strategies are ill defined, vary considerably, and include NSAIDs, opioids, and even anticonvulsants. We previously reported that the 1.1 subtype of the voltage-gated sodium (NaV; NaV1.1) channel family regulates the excitability of sensory nerve fibers that transmit a mechanical pain message to the spinal cord. Herein, we investigated whether this channel subtype also underlies the abdominal pain that occurs with CVH. We demonstrate that NaV1.1 is functionally upregulated under CVH conditions and that inhibiting channel function reduces mechanical pain in 3 mechanistically distinct mouse models of chronic pain. In particular, we use a small molecule to show that selective NaV1.1 inhibition (a) decreases sodium currents in colon-innervating dorsal root ganglion neurons, (b) reduces colonic nociceptor mechanical responses, and (c) normalizes the enhanced visceromotor response to distension observed in 2 mouse models of irritable bowel syndrome. These results provide support for a relationship between NaV1.1 and chronic abdominal pain associated with functional bowel disorders.
Juan Salvatierra, Joel Castro, Andelain Erickson, Qian Li, Joao Braz, John Gilchrist, Luke Grundy, Grigori Y. Rychkov, Annemie Deiteren, Rana Rais, Glenn F. King, Barbara S. Slusher, Allan Basbaum, Pankaj J. Pasricha, Stuart M. Brierley, Frank Bosmans
BACKGROUND. Myeloid-derived suppressor cells (MDSCs) are a population of immature immune cells with several protumorigenic functions. CD38 is a transmembrane receptor–ectoenzyme expressed by MDSCs in murine models of esophageal cancer. We hypothesized that CD38 could be expressed on MDSCs in human colorectal cancer (CRC), which might allow for a new perspective on therapeutic targeting of human MDSCs with anti-CD38 monoclonal antibodies in this cancer. METHODS. Blood samples were collected from 41 CRC patients and 8 healthy donors, followed by peripheral blood mononuclear cell (PBMC) separation. Polymorphonuclear (PMN-) and monocytic (M-) MDSCs and CD38 expression levels were quantified by flow cytometry. The immunosuppressive capacity of M-MDSCs from 10 CRC patients was validated in a mixed lymphocyte reaction (MLR) assay. RESULTS. A significant expansion of CD38+ M-MDSCs and a trend of expansion of CD38+ PMN-MDSCs (accompanied by a trend of increased CD38 expression on both M- and PMN-MDSCs) were observed in PBMCs of CRC patients when compared with healthy donors. The CD38+ M-MDSCs from CRC patients were found to be immunosuppressive when compared with mature monocytes. CD38+ M- and PMN-MDSC frequencies were significantly higher in CRC patients who previously received treatment when compared with treatment-naive patients. CONCLUSIONS. This study provides a rationale for an attempt to target M-MDSCs with an anti-CD38 monoclonal antibody in metastatic CRC patients. FUNDING. NCI P01-CA14305603, the American Cancer Society, Scott and Suzi Lustgarten Family Colon Cancer Research Fund, Hansen Foundation, and Janssen Research and Development.
Tatiana A. Karakasheva, George A. Dominguez, Ayumi Hashimoto, Eric W. Lin, Christopher Chiu, Kate Sasser, Jae W. Lee, Gregory L. Beatty, Dmitry I. Gabrilovich, Anil K. Rustgi
Inflammatory bowel disease (IBD) is associated with enhanced levels of the IL-1 family cytokines IL-1β and IL-18, which are activated by the Nlrp3 inflammasome. Here, we investigated the role of inflammasome-driven cytokine release on T cell polarization and DC differentiation in steady state and T cell transfer colitis. In vitro and in vivo data showed that IL-1β induces Th17 polarization and increases GM‑CSF production by T cells. Reduced IL-1β levels in Nlrp3–/– mice correlated with enhanced FLT3L levels and increased frequency of tolerogenic CD103+ DC. In the T cell transfer colitis model, Nlrp3 deficiency resulted in lower IL‑1β levels, reduced Th17 immunity, and less severe colitis. Unaltered IL-18 levels in both mouse strains pointed toward Nlrp3-independent processing. Importantly, cohousing revealed that the gut microbiome had no impact on the observed Nlrp3–/– phenotype. This study demonstrates that NLRP3 acts as a molecular switch of intestinal homeostasis by shifting local immune cells toward an inflammatory phenotype via IL-1β.
Rachel Mak’Anyengo, Peter Duewell, Cornelia Reichl, Christine Hörth, Hans‑Anton Lehr, Sandra Fischer, Thomas Clavel, Gerald Denk, Simon Hohenester, Sebastian Kobold, Stefan Endres, Max Schnurr, Christian Bauer
Crohn’s disease (CD) is a chronic inflammatory disease of the gastrointestinal tract (GIT). Cigarette smoke (CS) exposure and chronic obstructive pulmonary disease (COPD) are risk factors for CD, although the mechanisms involved are poorly understood. We employed a mouse model of CS-induced experimental COPD and clinical studies to examine these mechanisms. Concurrent with the development of pulmonary pathology and impaired gas exchange, CS-exposed mice developed CD-associated pathology in the colon and ileum, including gut mucosal tissue hypoxia, HIF-2 stabilization, inflammation, increased microvasculature, epithelial cell turnover, and decreased intestinal barrier function. Subsequent smoking cessation reduced GIT pathology, particularly in the ileum. Dimethyloxaloylglycine, a pan-prolyl hydroxylase inhibitor, ameliorated CS-induced GIT pathology independently of pulmonary pathology. Prior smoke exposure exacerbated intestinal pathology in 2,4,6-trinitrobenzenesulfonic acid–induced (TNBS-induced) colitis. Circulating vascular endothelial growth factor, a marker of systemic hypoxia, correlated with CS exposure and CD in mice and humans. Increased mucosal vascularisation was evident in ileum biopsies from CD patients who smoke compared with nonsmokers, supporting our preclinical data. We provide strong evidence that chronic CS exposure and, for the first time to our knowledge, associated impaired gas exchange cause systemic and intestinal ischemia, driving angiogenesis and GIT epithelial barrier dysfunction, resulting in increased risk and severity of CD.
Michael Fricker, Bridie J. Goggins, Sean Mateer, Bernadette Jones, Richard Y. Kim, Shaan L. Gellatly, Andrew G. Jarnicki, Nicholas Powell, Brian G. Oliver, Graham Radford-Smith, Nicholas J. Talley, Marjorie M. Walker, Simon Keely, Philip M. Hansbro
Pancreatic cancer is characterized by nearly universal activating mutations in KRAS. Among other somatic mutations, TP53 is mutated in more than 75% of human pancreatic tumors. Genetically engineered mice have proven instrumental in studies of the contribution of individual genes to carcinogenesis. Oncogenic Kras mutations occur early during pancreatic carcinogenesis and are considered an initiating event. In contrast, mutations in p53 occur later during tumor progression. In our model, we recapitulated the order of mutations of the human disease, with p53 mutation following expression of oncogenic Kras. Further, using an inducible and reversible expression allele for mutant p53, we inactivated its expression at different stages of carcinogenesis. Notably, the function of mutant p53 changes at different stages of carcinogenesis. Our work establishes a requirement for mutant p53 for the formation and maintenance of pancreatic cancer precursor lesions. In tumors, mutant p53 becomes dispensable for growth. However, it maintains the altered metabolism that characterizes pancreatic cancer and mediates its malignant potential. Further, mutant p53 promotes epithelial-mesenchymal transition (EMT) and cancer cell invasion. This work generates new mouse models that mimic human pancreatic cancer and expands our understanding of the role of p53 mutation, common in the majority of human malignancies.
Heather K. Schofield, Jörg Zeller, Carlos Espinoza, Christopher J. Halbrook, Annachiara del Vecchio, Brian Magnuson, Tania Fabo, Ayse Ece Cali Daylan, Ilya Kovalenko, Ho-Joon Lee, Wei Yan, Ying Feng, Saadia A. Karim, Daniel M. Kremer, Chandan Kumar-Sinha, Costas A. Lyssiotis, Mats Ljungman, Jennifer P. Morton, Stefanie Galbán, Eric R. Fearon, Marina Pasca di Magliano
Eosinophilic esophagitis (EoE) is an allergic inflammatory disease of the esophagus mediated by an IL-13–driven epithelial cell transcriptional program. Herein, we show that the cytoskeletal protein synaptopodin (SYNPO), previously associated with podocytes, is constitutively expressed in esophageal epithelium and induced during allergic inflammation. In addition, we show that the SYNPO gene is transcriptionally and epigenetically regulated by IL-13 in esophageal epithelial cells. SYNPO was expressed in the basal layer of homeostatic esophageal epithelium, colocalized with actin filaments, and expanded into the suprabasal epithelium in EoE patients, where expression was elevated 25-fold compared with control individuals. The expression level of SYNPO in esophageal biopsies correlated with esophageal eosinophil density and was improved following anti–IL-13 treatment in EoE patients. In esophageal epithelial cells, SYNPO gene silencing reduced epithelial motility in a wound healing model, whereas SYNPO overexpression impaired epithelial barrier integrity and reduced esophageal differentiation. Taken together, we demonstrate that SYNPO is induced by IL-13 in vitro and in vivo, is a nonredundant regulator of epithelial cell barrier function and motility, and is likely involved in EoE pathogenesis.
Mark Rochman, Jared Travers, J. Pablo Abonia, Julie M. Caldwell, Marc E. Rothenberg
Cystic fibrosis (CF) is a genetic disorder in which epithelium-generated fluid flow from the lung, intestine, and pancreas is impaired due to mutations disrupting CF transmembrane conductance regulator (CFTR) channel function. CF manifestations of the pancreas and lung are present in the vast majority of CF patients, and 15% of CF infants are born with obstructed gut or meconium ileus. However, constipation is a significantly underreported outcome of CF disease, affecting 47% of the CF patients, and management becomes critical in the wake of increasing life span of CF patients. In this study, we unraveled a potentially novel molecular role of a membrane-bound cyclic guanosine monophosphate–synthesizing (cGMP-synthesizing) intestinal enzyme, guanylate cyclase 2C (GCC) that could be targeted to ameliorate CF-associated intestinal fluid deficit. We demonstrated that GCC agonism results in functional rescue of murine F508del/F508del and R117H/R117H Cftr and CFTR mutants in CF patient–derived intestinal spheres. GCC coexpression and activation facilitated processing and ER exit of F508del CFTR and presented a potentially novel rescue modality in the intestine, similar to the CF corrector VX-809. Our findings identify GCC as a biological CFTR corrector and potentiator in the intestine.
Kavisha Arora, Yunjie Huang, Kyushik Mun, Sunitha Yarlagadda, Nambirajan Sundaram, Marco M. Kessler, Gerhard Hannig, Caroline B. Kurtz, Inmaculada Silos-Santiago, Michael Helmrath, Joseph J. Palermo, John P. Clancy, Kris A. Steinbrecher, Anjaparavanda P. Naren
MTG16 is a member of the myeloid translocation gene (MTG) family of transcriptional corepressors. While MTGs were originally identified in chromosomal translocations in acute myeloid leukemia, recent studies have uncovered a role in intestinal biology. For example, Mtg16–/– mice have increased intestinal proliferation and are more sensitive to intestinal injury in colitis models. MTG16 is also underexpressed in patients with moderate/severe ulcerative colitis. Based on these findings, we postulated that MTG16 might protect against colitis-associated carcinogenesis. MTG16 was downregulated at the protein and RNA levels in patients with inflammatory bowel disease and in those with colitis-associated carcinoma. Mtg16–/– mice subjected to inflammatory carcinogenesis modeling exhibited worse colitis and increased tumor multiplicity and size. Loss of MTG16 also increased severity of dysplasia, apoptosis, proliferation, DNA damage, and WNT signaling. Moreover, transplantation of WT marrow into Mtg16–/– mice failed to rescue the Mtg16–/– protumorigenic phenotypes, indicating an epithelium-specific role for MTG16. While MTG dysfunction is widely appreciated in hematopoietic malignancies, the role of this gene family in epithelial homeostasis, and in colon cancer, was unrealized. This report identifies MTG16 as an important modulator of colitis and tumor development in inflammatory carcinogenesis.
Elizabeth M. McDonough, Caitlyn W. Barrett, Bobak Parang, Mukul K. Mittal, J. Joshua Smith, Amber M. Bradley, Yash A. Choksi, Lori A. Coburn, Sarah P. Short, Joshua J. Thompson, Baolin Zhang, Shenika V. Poindexter, Melissa A. Fischer, Xi Chen, Jiang Li, Frank L. Revetta, Rishi Naik, M. Kay Washington, Michael J. Rosen, Scott W. Hiebert, Keith T. Wilson, Christopher S. Williams
Familial hemophagocytic lymphohistiocytosis 5 (FHL5) is an autosomal recessive disease caused by mutations in STXBP2, coding for Munc18-2, which is required for SNARE-mediated membrane fusion. FHL5 causes hematologic and gastrointestinal symptoms characterized by chronic enteropathy that is reminiscent of microvillus inclusion disease (MVID). However, the molecular pathophysiology of FHL5-associated diarrhea is poorly understood. Five FHL5 patients, including four previously unreported patients, were studied. Morphology of duodenal sections was analyzed by electron and fluorescence microscopy. Small intestinal enterocytes and organoid-derived monolayers displayed the subcellular characteristics of MVID. For the analyses of Munc18-2–dependent SNARE-protein interactions, a Munc18-2 CaCo2–KO model cell line was generated by applying CRISPR/Cas9 technology. Munc18-2 is required for Slp4a/Stx3 interaction in fusion of cargo vesicles with the apical plasma membrane. Cargo trafficking was investigated in patient biopsies, patient-derived organoids, and the genome-edited model cell line. Loss of Munc18-2 selectively disrupts trafficking of certain apical brush-border proteins (NHE3 and GLUT5), while transport of DPPIV remained unaffected. Here, we describe the molecular mechanism how the loss of function of Munc18-2 leads to cargo-selective mislocalization of brush-border components and a subapical accumulation of cargo vesicles, as it is known from the loss of polarity phenotype in MVID.
Georg F. Vogel, Jorik M. van Rijn, Iris M. Krainer, Andreas R. Janecke, Carsten Posovzsky, Marta Cohen, Claire Searle, Prevost Jantchou, Johanna C. Escher, Natalie Patey, Ernest Cutz, Thomas Müller, Sabine Middendorp, Michael W. Hess, Lukas A. Huber
Nonalcoholic fatty liver disease prevalence is soaring with the obesity pandemic, but the pathogenic mechanisms leading to the progression toward active nonalcoholic steatohepatitis (NASH) and fibrosis, major causes of liver-related death, are poorly defined. To identify key components during the progression toward NASH and fibrosis, we investigated the liver transcriptome in a human cohort of NASH patients. The transition from histologically proven fatty liver to NASH and fibrosis was characterized by gene expression patterns that successively reflected altered functions in metabolism, inflammation, and epithelial-mesenchymal transition. A meta-analysis combining our and public human transcriptomic datasets with murine models of NASH and fibrosis defined a molecular signature characterizing NASH and fibrosis and evidencing abnormal inflammation and extracellular matrix (ECM) homeostasis. Dermatopontin expression was found increased in fibrosis, and reversal of fibrosis after gastric bypass correlated with decreased dermatopontin expression. Functional studies in mice identified an active role for dermatopontin in collagen deposition and fibrosis. PPARα activation lowered dermatopontin expression through a transrepressive mechanism affecting the Klf6/TGFβ1 pathway. Liver fibrotic histological damages are thus characterized by the deregulated expression of a restricted set of inflammation- and ECM-related genes. Among them, dermatopontin may be a valuable target to reverse the hepatic fibrotic process.
Philippe Lefebvre, Fanny Lalloyer, Eric Baugé, Michal Pawlak, Céline Gheeraert, Hélène Dehondt, Jonathan Vanhoutte, Eloise Woitrain, Nathalie Hennuyer, Claire Mazuy, Marie Bobowski-Gérard, Francesco Paolo Zummo, Bruno Derudas, Ann Driessen, Guy Hubens, Luisa Vonghia, Wilhelmus J. Kwanten, Peter Michielsen, Thomas Vanwolleghem, Jérôme Eeckhoute, An Verrijken, Luc Van Gaal, Sven Francque, Bart Staels
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