Cell-programmed nutrient partitioning in the tumour microenvironment.

Cancer cells characteristically consume glucose through Warburg metabolism1, a process that forms the basis of tumour imaging by positron emission tomography (PET). Tumour-infiltrating immune cells also rely on glucose, and impaired immune cell metabolism in the tumour microenvironment (TME) contributes to immune evasion by tumour cells2-4. However, whether the metabolism of immune cells is dysregulated in the TME by cell-intrinsic programs or by competition with cancer cells for limited nutrients remains unclear. Here we used PET tracers to measure the access to and uptake of glucose and glutamine by specific cell subsets in the TME. Notably, myeloid cells had the greatest capacity to take up intratumoral glucose, followed by T cells and cancer cells, across a range of cancer models. By contrast, cancer cells showed the highest uptake of glutamine. This distinct nutrient partitioning was programmed in a cell-intrinsic manner through mTORC1 signalling and the expression of genes related to the metabolism of glucose and glutamine. Inhibiting glutamine uptake enhanced glucose uptake across tumour-resident cell types, showing that glutamine metabolism suppresses glucose uptake without glucose being a limiting factor in the TME. Thus, cell-intrinsic programs drive the preferential acquisition of glucose and glutamine by immune and cancer cells, respectively. Cell-selective partitioning of these nutrients could be exploited to develop therapies and imaging strategies to enhance or monitor the metabolic programs and activities of specific cell populations in the TME.

A synthesis and subgroup analysis of the eosinophilic esophagitis tissue transcriptome.

BACKGROUND: Eosinophilic esophagitis (EoE) is a chronic immune mediated inflammatory disorder of the esophagus. It is still unknown why children and adults present differently, and there is little evidence about why it is more common in men than women. OBJECTIVE: Our aim was to synthesize published and unpublished esophageal bulk RNA-sequencing (RNA-seq) data to gain novel insights into the pathobiology of EoE and examine the differences in EoE transcriptome by sex and age group. METHODS: Esophageal bulk RNA-seq data from 5 published and 2 unpublished studies resulting in 137 subjects (EoE: N = 76; controls: N = 61) were analyzed. For overall analysis, combined RNA-seq data of patients with EoE were compared with those of controls and subgroup analysis was conducted in patients with EoE by age of the patient (children [<18 years] vs adults [≥18 years]) and sex (female vs male). Gene-set enrichment analysis, ingenuity pathway analysis (IPA), cell-type analysis, immunohistochemistry, and T-cell or B-cell receptor analysis were performed. RESULTS: Overall analysis identified dysregulation of new genes in EoE compared with controls. IPA revealed that EoE is characterized by a mixed inflammatory response compared with controls. Cell-type analysis showed that cell composition varied with age: children had more mast cells, whereas adults had more macrophages. Finally, gene-set enrichment analysis and IPA revealed pathways that were differentially regulated in adults versus children and male versus female patients with EoE. CONCLUSIONS: Using a unique approach to analyze bulk RNA-seq data, we found that EoE is characterized by a mixed inflammatory response, and the EoE transcriptome may be influenced by age and sex. These findings enhance insights into the molecular mechanisms of EoE.

MCC is a centrosomal protein that relocalizes to non-centrosomal apical sites during intestinal cell differentiation.

The gene mutated in colorectal cancer (MCC) encodes a coiled-coil protein implicated, as its name suggests, in the pathogenesis of hereditary human colon cancer. To date, however, the contributions of MCC to intestinal homeostasis and disease remain unclear. Here, we examine the subcellular localization of MCC, both at the mRNA and protein levels, in the adult intestinal epithelium. Our findings reveal that Mcc transcripts are restricted to proliferating crypt cells, including Lgr5+ stem cells, where the Mcc protein is distinctly associated with the centrosome. Upon intestinal cellular differentiation, Mcc is redeployed to the apical domain of polarized villus cells where non-centrosomal microtubule organizing centers (ncMTOCs) are positioned. Using intestinal organoids, we show that the shuttling of the Mcc protein depends on phosphorylation by casein kinases 1δ and ε, which are critical modulators of WNT signaling. Together, our findings support a role for MCC in establishing and maintaining the cellular architecture of the intestinal epithelium as a component of both the centrosome and ncMTOC.

Colonic Epithelial-Derived Selenoprotein P Is the Source for Antioxidant-Mediated Protection in Colitis-Associated Cancer.

BACKGROUND & AIMS: Patients with inflammatory bowel disease (IBD) demonstrate nutritional selenium deficiencies and are at greater risk of developing colon cancer. Previously, we determined that global reduction of the secreted antioxidant selenium-containing protein, selenoprotein P (SELENOP), substantially increased tumor development in an experimental colitis-associated cancer (CAC) model. We next sought to delineate tissue-specific contributions of SELENOP to intestinal inflammatory carcinogenesis and define clinical context. METHODS: Selenop floxed mice crossed with Cre driver lines to delete Selenop from the liver, myeloid lineages, or intestinal epithelium were placed on an azoxymethane/dextran sodium sulfate experimental CAC protocol. SELENOP loss was assessed in human ulcerative colitis (UC) organoids, and expression was queried in human and adult UC samples. RESULTS: Although large sources of SELENOP, both liver- and myeloid-specific Selenop deletion failed to modify azoxymethane/dextran sodium sulfate-mediated tumorigenesis. Instead, epithelial-specific deletion increased CAC tumorigenesis, likely due to elevated oxidative stress with a resulting increase in genomic instability and augmented tumor initiation. SELENOP was down-regulated in UC colon biopsies and levels were inversely correlated with endoscopic disease severity and tissue S100A8 (calprotectin) gene expression. CONCLUSIONS: Although global selenium status is typically assessed by measuring liver-derived plasma SELENOP levels, our results indicate that the peripheral SELENOP pool is dispensable for CAC. Colonic epithelial SELENOP is the main contributor to local antioxidant capabilities. Thus, colonic SELENOP is the most informative means to assess selenium levels and activity in IBD patients and may serve as a novel biomarker for UC disease severity and identify patients most predisposed to CAC development.

Impedance and Histologic Characteristics of the Sub-Laryngeal Esophagus Distinguish Eosinophilic Esophagitis From Other Esophageal Disorders.

BACKGROUND & AIMS: The region of the esophagus 15-17 cm below the incisors, called the sub-upper esophageal sphincter (sub-UES), has not been characterized in adults with eosinophilic esophagitis (EoE) but appears different during endoscopy. We investigated how the sub-UES differs from the remaining esophagus in patients with EoE and aimed to determine whether these differences be used to distinguish patients with EoE from those with lichen planus. METHODS: We performed a prospective study of 14 patients with EoE, 7 patients with lichen planus (based on presence of Civatte bodies, dysphagia, and/or narrow esophagus with thin esophageal mucosa without signs of EoE), and 20 patients undergoing upper endoscopy for upper gastrointestinal or with dysphagia but without features of EoE (controls) at a single medical center from 2015 through 2018. Biopsies from the distal, middle, and sub-UES regions of the esophagus were analyzed by histology, quantitative PCR, and immunohistochemistry. We measured mucosal impedance (MI) in all subjects at the sub-UES and 2 cm, 5 cm, and 10 cm from the gastro-esophageal junction. RESULTS: Patients with EoE had significantly fewer eosinophils (median, 2 eosinophils/high-powered field [HPF]; range, 0-8 eosinophils/HPF) in sub-UES tissues compared with distal esophagus (median, 50 eosinophils/HPF; range, 22.5-60.8 eosinophils/HPF; P < .0001) or middle esophagus (median, 32 eosinophils/HPF; range, 19.3-60; P < .0001). Sub-UES tissues from patients with EoE had significantly less basal cell hyperplasia (P < .01), papillary elongation (P < .01), and dilated intercellular spaces (P < .01) than middle or and distal esophagus. MI in the sub-UES did not differ significantly between patients with EoE vs controls (P = .24), but was significantly lower in patients with lichen planus (median, 1344 ohms; range, 1046-1488) than patients with EoE (median, 2880 ohms; range, 2149-4858) (P < .001). mRNA and protein expression patterns did not differ significantly in the sub-UES of patients with EoE vs controls, except for expression of desmoglein-1, which was increased in sub-UES tissues from patients with EoE. CONCLUSIONS: Sub-UES tissues from patients with EoE differ in numbers of eosinophils, histologic features, and MI compared to controls or patients with lichen planus. These features might help to distinguish these 2 diseases.

Blood vessel epicardial substance reduces LRP6 receptor and cytoplasmic ß-catenin levels to modulate Wnt signaling and intestinal homeostasis.

Blood vessel epicardial substance (BVES, otherwise known as POPDC1) is an integral membrane protein known to regulate tight junction formation and epithelial-mesenchymal transition. BVES is underexpressed in a number of malignancies, including colorectal cancer. BVES loss leads to activation of the Wnt pathway, suggesting that decreased BVES expression functionally contributes to tumorigenesis. However, the mechanism by which BVES modulates Wnt signaling is unknown. Here, we confirm that BVES loss increases ß-catenin protein levels, leads to Wnt pathway activation in a ligand-independent fashion and coordinates with Wnt ligand to further increase Wnt signaling. We show that BVES loss increases levels and activation of the Wnt co-receptor, LRP6, in cell lines, murine adenoma tumoroids and human-derived colonoids. We also demonstrate that BVES interacts with LRP6. Finally, murine tumor modeling using a Wnt-driven genetic model and a chemically induced model of colorectal carcinogenesis demonstrate that BVES loss increases tumor multiplicity and dysplasia. Together, these results implicate BVES as an inhibitor of Wnt signaling, provide one of the first examples of a tight junction-associated protein regulating Wnt receptor levels, and expand the number of putative molecular targets for therapeutic intervention in colorectal cancer.

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection.

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease.IMPORTANCE Acute viral infections are thought to be cleared by the host with few lasting consequences. However, there may be much broader and long-lasting effects of viruses on immune homeostasis. Infection with reovirus, a common, nonpathogenic virus, triggers inflammation against innocuous food antigens, implicating this virus in the development of celiac disease, an autoimmune intestinal disorder triggered by exposure to dietary gluten. Using two reovirus strains that differ in the capacity to abrogate oral tolerance, we found that strain-specific differences in the capacity to replicate in the intestine inversely correlate with the capacity to induce apoptotic death of intestinal epithelial cells, providing a host-mediated process to restrict intestinal infection. This work contributes new knowledge about virus-host interactions in the intestine and establishes a foundation for future studies to define mechanisms by which viruses break oral tolerance in celiac disease.

Selenoproteins and oxidative stress-induced inflammatory tumorigenesis in the gut.

Selenium is an essential micronutrient that is incorporated into at least 25 selenoproteins encoded by the human genome, many of which serve antioxidant functions. Because patients with inflammatory bowel disease (IBD) demonstrate nutritional deficiencies and are at increased risk for colon cancer due to heightened inflammation and oxidative stress, selenoprotein dysfunction may contribute to disease progression. Over the years, numerous studies have analyzed the effects of selenoprotein loss and shown that they are important mediators of intestinal inflammation and carcinogenesis. In particular, recent work has focused on the role of selenoprotein P (SEPP1), a major selenium transport protein which also has endogenous antioxidant function. These experiments determined SEPP1 loss altered immune and epithelial cellular function in a murine model of colitis-associated carcinoma. Here, we discuss the current knowledge of SEPP1 and selenoprotein function in the setting of IBD, colitis, and inflammatory tumorigenesis.

BVES regulates c-Myc stability via PP2A and suppresses colitis-induced tumourigenesis.

OBJECTIVE: Blood vessel epicardial substance (BVES) is a tight junction-associated protein that regulates epithelial-mesenchymal states and is underexpressed in epithelial malignancy. However, the functional impact of BVES loss on tumourigenesis is unknown. Here we define the in vivo role of BVES in colitis-associated cancer (CAC), its cellular function and its relevance to patients with IBD. DESIGN: We determined BVES promoter methylation status using an Infinium HumanMethylation450 array screen of patients with UC with and without CAC. We also measured BVES mRNA levels in a tissue microarray consisting of normal colons and CAC samples. Bves-/- and wild-type mice (controls) were administered azoxymethane (AOM) and dextran sodium sulfate (DSS) to induce tumour formation. Last, we used a yeast two-hybrid screen to identify BVES interactors and performed mechanistic studies in multiple cell lines to define how BVES reduces c-Myc levels. RESULTS: BVES mRNA was reduced in tumours from patients with CAC via promoter hypermethylation. Importantly, BVES promoter hypermethylation was concurrently present in distant non-malignant-appearing mucosa. As seen in human patients, Bves was underexpressed in experimental inflammatory carcinogenesis, and Bves-/- mice had increased tumour multiplicity and degree of dysplasia after AOM/DSS administration. Molecular analysis of Bves-/- tumours revealed Wnt activation and increased c-Myc levels. Mechanistically, we identified a new signalling pathway whereby BVES interacts with PR61α, a protein phosphatase 2A regulatory subunit, to mediate c-Myc destruction. CONCLUSION: Loss of BVES promotes inflammatory tumourigenesis through dysregulation of Wnt signalling and the oncogene c-Myc. BVES promoter methylation status may serve as a CAC biomarker.

BVES Regulates Intestinal Stem Cell Programs and Intestinal Crypt Viability after Radiation.

Blood vessel epicardial substance (BVES/Popdc1) is a junctional-associated transmembrane protein that is underexpressed in a number of malignancies and regulates epithelial-to-mesenchymal transition. We previously identified a role for BVES in regulation of the Wnt pathway, a modulator of intestinal stem cell programs, but its role in small intestinal (SI) biology remains unexplored. We hypothesized that BVES influences intestinal stem cell programs and is critical to SI homeostasis after radiation injury. At baseline, Bves(-/-) mice demonstrated increased crypt height, as well as elevated proliferation and expression of the stem cell marker Lgr5 compared to wild-type (WT) mice. Intercross with Lgr5-EGFP reporter mice confirmed expansion of the stem cell compartment in Bves(-/-) mice. To examine stem cell function after BVES deletion, we used ex vivo 3D-enteroid cultures. Bves(-/-) enteroids demonstrated increased stemness compared to WT, when examining parameters such as plating efficiency, stem spheroid formation, and retention of peripheral cystic structures. Furthermore, we observed increased proliferation, expression of crypt-base columnar "CBC" and "+4" stem cell markers, amplified Wnt signaling, and responsiveness to Wnt activation in the Bves(-/-) enteroids. Bves expression was downregulated after radiation in WT mice. Moreover, after radiation, Bves(-/-) mice demonstrated significantly greater SI crypt viability, proliferation, and amplified Wnt signaling in comparison to WT mice. Bves(-/-) mice also demonstrated elevations in Lgr5 and Ascl2 expression, and putative damage-responsive stem cell populations marked by Bmi1 and TERT. Therefore, BVES is a key regulator of intestinal stem cell programs and mucosal homeostasis. Stem Cells 2016;34:1626-1636.

Competition between the Brain and Testes under Selenium-Compromised Conditions: Insight into Sex Differences in Selenium Metabolism and Risk of Neurodevelopmental Disease.

Selenium (Se) is essential for both brain development and male fertility. Male mice lacking two key genes involved in Se metabolism (Scly(-/-)Sepp1(-/-) mice), selenoprotein P (Sepp1) and Sec lyase (Scly), develop severe neurological dysfunction, neurodegeneration, and audiogenic seizures that manifest beginning in early adulthood. We demonstrate that prepubescent castration of Scly(-/-)Sepp1(-/-) mice prevents behavioral deficits, attenuates neurodegeneration, rescues maturation of GABAergic inhibition, and increases brain selenoprotein levels. Moreover, castration also yields similar neuroprotective benefits to Sepp1(-/-) and wild-type mice challenged with Se-deficient diets. Our data show that, under Se-compromised conditions, the brain and testes compete for Se utilization, with concomitant effects on neurodevelopment and neurodegeneration. SIGNIFICANCE STATEMENT: Selenium is an essential trace element that promotes male fertility and brain function. Herein, we report that prepubescent castration provides neuroprotection by increasing selenium-dependent antioxidant activity in the brain, revealing a competition between the brain and testes for selenium utilization. These findings provide novel insight into the interaction of sex and oxidative stress upon the developing brain and have potentially significant implications for the prevention of neurodevelopmental disorders characterized by aberrant excitatory/inhibitory balance, such as schizophrenia and epilepsy.

The transcriptional corepressor MTGR1 regulates intestinal secretory lineage allocation.

Notch signaling largely determines intestinal epithelial cell fate. High Notch activity drives progenitors toward absorptive enterocytes by repressing secretory differentiation programs, whereas low Notch permits secretory cell assignment. Myeloid translocation gene-related 1 (MTGR1) is a transcriptional corepressor in the myeloid translocation gene/Eight-Twenty-One family. Given that Mtgr1(-/-) mice have a dramatic reduction of intestinal epithelial secretory cells, we hypothesized that MTGR1 is a key repressor of Notch signaling. In support of this, transcriptome analysis of laser capture microdissected Mtgr1(-/-) intestinal crypts revealed Notch activation, and secretory markers Mucin2, Chromogranin A, and Growth factor-independent 1 (Gfi1) were down-regulated in Mtgr1(-/-) whole intestines and Mtgr1(-/-) enteroids. We demonstrate that MTGR1 is in a complex with Suppressor of Hairless Homolog, a key Notch effector, and represses Notch-induced Hairy/Enhancer of Split 1 activity. Moreover, pharmacologic Notch inhibition using a γ-secretase inhibitor (GSI) rescued the hyperproliferative baseline phenotype in the Mtgr1(-/-) intestine and increased production of goblet and enteroendocrine lineages in Mtgr1(-/-) mice. GSI increased Paneth cell production in wild-type mice but failed to do so in Mtgr1(-/-) mice. We determined that MTGR1 can interact with GFI1, a transcriptional corepressor required for Paneth cell differentiation, and repress GFI1 targets. Overall, the data suggest that MTGR1, a transcriptional corepressor well characterized in hematopoiesis, plays a critical role in intestinal lineage allocation.

Baseline neurological evaluations in a hyperbaric trial of post-concussive syndrome.

Standard neurologic examinations may not detect abnormalities in U.S. military service members with persistent post-concussive symptoms following mild traumatic brain injury. The Brain Injury and Mechanisms of Action of Hyperbaric Oxygen for Persistent Post-Concussive Symptoms after Mild Traumatic Brain Injury Study (BIMA) enrolled 71 participants September 2012-May 2014. Participants received: comprehensive neurological and oculomotor exam; balance testing (Berg Balance Scale-BBS; Romberg Test-RT, Sharpened Romberg Test-SRT); olfactory function (Brief Smell Identification Test-BSIT). Two trained neurologists conducted the examinations at a central facility in Colorado Springs. Median age was 32 years (range 21-53), 99% male, 82% Caucasian, 49% PTSD, 28% most recent qualifying injury three months to one year prior to enrollment, 32% blast injuries only, and 73% multiple injuries. Some participants presented with abnormal facial sensation (15%), abnormal tandem gait (13%), and tremor (11%). 54% had abnormal near point of convergence (abnormal range 13-80 cm). 86% scored ≥ 55 on the BBS, with no participant scoring ⟨ 50. 49% scored ⟨ 30 seconds on the best trial of the SRT. RT was abnormal in 10%. 15% of participants scored ≤ 9 (out of 12) on BSIT, about twice what is expected in a normal population. The neurological examination found abnormalities across a range of testing, with convergence insufficiency and SRT having the most sensitivity. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01611194; https://clinicaltrials.gov/show/NCT01611194.

Baseline EEG abnormalities in mild traumatic brain injury from the BIMA study.

The Brain Injury and Mechanisms of Action of HBO2 for Persistent Post-Concussive Symptoms after Mild Traumatic Brain Injury (BIMA), sponsored by the Department of Defense, is a randomized, double-blind, sham-controlled trial of hyperbaric oxygen (HBO2) in service members with persistent post-concussive symptoms following mild TBI, undergoing comprehensive assessments. The clinical EEG was assessed by neurologists for slow wave activity, ictal/interictal epileptiform abnormalities, and background periodic discharges. There is scant literature about EEG findings in this population, so we report baseline clinical EEG results and explore associations with other evaluations, including demographics, medication, neurological assessments, and clinical MRI outcomes. Seventy-one participants were enrolled: median age 32 years, 99% male, 49% comorbid PTSD, 28% with mTBI in the previous year, 32% blast injuries only, and 73% multiple injuries. All participants reported medication use (mean medications = 8, SD = 5). Slowing was present in 39%: generalized 37%, localized 8%, both 6%. No other abnormalities were identified. Slowing was not significantly associated with demographics, medication or neurological evaluation. Participants without EEG abnormalities paradoxically had significantly higher number of white matter hyperintensities as identified on MRI (p = 0.003). EEG slowing is present in more than one-third of participants in this study without evidence of associations with demographics, medications or neurological findings. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01611194; https://clinicaltrials.gov/show/NCT01611194.

ERBB4 is over-expressed in human colon cancer and enhances cellular transformation.

The ERBB4 receptor tyrosine kinase promotes colonocyte survival. Herein, we tested whether ERBB4's antiapoptotic signaling promotes transformation and colorectal tumorigenesis. ERBB4 alterations in a The Cancer Genome Atlas colorectal cancer (CRC) data set stratified survival, and in a combined Moffitt Cancer Center and Vanderbilt Medical Center CRC expression data set, ERBB4 message levels were increased at all tumor stages. Similarly, western blot and immunohistochemistry on additional CRC tissue banks showed elevated ERBB4 protein in tumors. ERBB4 was highly expressed in aggressive, dedifferentiated CRC cell lines, and its knockdown in LIM2405 cells reduced anchorage-independent colony formation. In nude mouse xenograft studies, ERBB4 alone was insufficient to induce tumor establishment of non-transformed mouse colonocytes, but its over-expression in cells harboring Apc(min) and v-Ha-Ras caused a doubling of tumor size. ERBB4-expressing xenografts displayed increased activation of survival pathways, including epidermal growth factor receptor and Akt phosphorylation and COX-2 expression, and decreased apoptotic signals. Finally, ERBB4 deletion from mouse intestinal epithelium impaired stem cell replication and in vitro enteroid establishment. In summary, we report that ERBB4 is over-expressed in human CRC, and in experimental systems enhances the survival and growth of cells driven by Ras and/or WNT signaling. Chronic ERBB4 over-expression in the context of, for example, inflammation may contribute to colorectal carcinogenesis. Tumors with high receptor levels are likely to have enhanced cell survival signaling through epidermal growth factor receptor, PI3K and COX-2. These results suggest ERBB4 as a novel therapeutic target in a subset of CRC.

Transcriptional corepressor MTG16 regulates small intestinal crypt proliferation and crypt regeneration after radiation-induced injury.

Myeloid translocation genes (MTGs) are transcriptional corepressors implicated in development, malignancy, differentiation, and stem cell function. While MTG16 loss renders mice sensitive to chemical colitis, the role of MTG16 in the small intestine is unknown. Histological examination revealed that Mtg16(-/-) mice have increased enterocyte proliferation and goblet cell deficiency. After exposure to radiation, Mtg16(-/-) mice exhibited increased crypt viability and decreased apoptosis compared with wild-type (WT) mice. Flow cytometric and immunofluorescence analysis of intestinal epithelial cells for phospho-histone H2A.X also indicated decreased DNA damage and apoptosis in Mtg16(-/-) intestines. To determine if Mtg16 deletion affected epithelial cells in a cell-autonomous fashion, intestinal crypts were isolated from Mtg16(-/-) mice. Mtg16(-/-) and WT intestinal crypts showed similar enterosphere forming efficiencies when cultured in the presence of EGF, Noggin, and R-spondin. However, when Mtg16(-/-) crypts were cultured in the presence of Wnt3a, they demonstrated higher enterosphere forming efficiencies and delayed progression to mature enteroids. Mtg16(-/-) intestinal crypts isolated from irradiated mice exhibited increased survival compared with WT intestinal crypts. Interestingly, Mtg16 expression was reduced in a stem cell-enriched population at the time of crypt regeneration. This is consistent with MTG16 negatively regulating regeneration in vivo. Taken together, our data demonstrate that MTG16 loss promotes radioresistance and impacts intestinal stem cell function, possibly due to shifting cellular response away from DNA damage-induced apoptosis and towards DNA repair after injury.

STAT6 deficiency ameliorates severity of oxazolone colitis by decreasing expression of claudin-2 and Th2-inducing cytokines.

Patients suffering from ulcerative colitis (UC) exhibit chronic colonic inflammation caused by a dysregulated mucosal immune response and epithelial barrier disruption. Th2 cytokines, including IL-13, have been implicated in the pathogenesis of UC. IL-13 induces phosphorylation of STAT6, and we previously demonstrated increased epithelial p-STAT6 in children with UC. In this study, we investigated the role of STAT6 in oxazolone colitis, a murine model of UC, by inducing colitis in STAT6-deficient (STAT6(-/-)) and wild type (WT) mice. We observed increased epithelial cell, T cell, macrophage, and NKT cell STAT6 phosphorylation, as well as increased p-STAT6(+) IL-13-producing NKT cells, in colitic WT mice. Colitis was attenuated in STAT6(-/-) mice, with improvements in weight, colon length, and histopathology. There was decreased induction of the pore-forming tight junction protein claudin-2 in STAT6(-/-) mice. Similarly, short hairpin RNA STAT6 knockdown reduced claudin-2 induction and transepithelial resistance decrease in IL-13-treated human T84 cells. Tissue expression of IL-13, IFN-γ, IL-17, and IL-10 mRNA was similarly induced in WT and STAT6(-/-) colitic mice; however, we observed increased mRNA expression for the Th2-inducing cytokines IL-33 and thymic stromal lymphopoietin in WT mice with colitis, which was abrogated in STAT6(-/-) mice. Mesenteric lymph node cells from STAT6(-/-) mice with colitis exhibited reduced secretion of IL-4, IL-5, IL-13, and IFN-γ. IL-33 augmented mesenteric lymph node cell secretion of IL-5, IL-13, IL-6, and IFN-γ. These data implicate STAT6 in the pathogenesis of colitis in vivo with important roles in altering epithelial barrier function and regulating Th2-inducing cytokine production.

MTG16 contributes to colonic epithelial integrity in experimental colitis.

OBJECTIVE: The myeloid translocation genes (MTGs) are transcriptional corepressors with both Mtg8(-/-) and Mtgr1(-/-) mice showing developmental and/or differentiation defects in the intestine. We sought to determine the role of MTG16 in intestinal integrity. METHODS: Baseline and stress induced colonic phenotypes were examined in Mtg16(-/-) mice. To unmask phenotypes, we treated Mtg16(-/-) mice with dextran sodium sulphate (DSS) or infected them with Citrobacter rodentium and the colons were examined for ulceration and for changes in proliferation, apoptosis and inflammation. RESULTS: Mtg16(-/-) mice have altered immune subsets, suggesting priming towards Th1 responses. Mtg16(-/-) mice developed increased weight loss, diarrhoea, mortality and histological colitis and there were increased innate (Gr1(+), F4/80(+), CD11c(+) and MHCII(+); CD11c(+)) and Th1 adaptive (CD4) immune cells in Mtg16(-/-) colons after DSS treatment. Additionally, there was increased apoptosis and a compensatory increased proliferation in Mtg16(-/-) colons. Compared with wild-type mice, Mtg16(-/-) mice exhibited increased colonic CD4;IFN-γ cells in vehicle-treated and DSS-treated mice. Adoptive transfer of wild-type marrow into Mtg16(-/-) recipients did not rescue the Mtg16(-/-) injury phenotype. Isolated colonic epithelial cells from DSS-treated Mtg16(-/-) mice exhibited increased KC (Cxcl1) mRNA expression when compared with wild-type mice. Mtg16(-/-) mice infected with C rodentium had more severe colitis and greater bacterial colonisation. Last, MTG16 mRNA levels were reduced in human ulcerative colitis versus normal colon tissues. CONCLUSIONS: These observations indicate that MTG16 is critical for colonocyte survival and regeneration in response to intestinal injury and provide evidence that this transcriptional corepressor regulates inflammatory recruitment in response to injury.