Wnt-ß-catenin activation epigenetically reprograms Treg cells in inflammatory bowel disease and dysplastic progression.

The diversity of regulatory T (Treg) cells in health and in disease remains unclear. Individuals with colorectal cancer harbor a subpopulation of RORγt+ Treg cells with elevated expression of ß-catenin and pro-inflammatory properties. Here we show progressive expansion of RORγt+ Treg cells in individuals with inflammatory bowel disease during inflammation and early dysplasia. Activating Wnt-ß-catenin signaling in human and murine Treg cells was sufficient to recapitulate the disease-associated increase in the frequency of RORγt+ Treg cells coexpressing multiple pro-inflammatory cytokines. Binding of the ß-catenin interacting partner, TCF-1, to DNA overlapped with Foxp3 binding at enhancer sites of pro-inflammatory pathway genes. Sustained Wnt-ß-catenin activation induced newly accessible chromatin sites in these genes and upregulated their expression. These findings indicate that TCF-1 and Foxp3 together limit the expression of pro-inflammatory genes in Treg cells. Activation of ß-catenin signaling interferes with this function and promotes the disease-associated RORγt+ Treg phenotype.

A Genomic Link From Heart Failure to Atrial Fibrillation Risk: FOG2 Modulates a TBX5/GATA4-Dependent Atrial Gene Regulatory Network.

BACKGROUND: The relationship between heart failure (HF) and atrial fibrillation (AF) is clear, with up to half of patients with HF progressing to AF. The pathophysiological basis of AF in the context of HF is presumed to result from atrial remodeling. Upregulation of the transcription factor FOG2 (friend of GATA2; encoded by ZFPM2) is observed in human ventricles during HF and causes HF in mice. METHODS: FOG2 expression was assessed in human atria. The effect of adult-specific FOG2 overexpression in the mouse heart was evaluated by whole animal electrophysiology, in vivo organ electrophysiology, cellular electrophysiology, calcium flux, mouse genetic interactions, gene expression, and genomic function, including a novel approach for defining functional transcription factor interactions based on overlapping effects on enhancer noncoding transcription. RESULTS: FOG2 is significantly upregulated in the human atria during HF. Adult cardiomyocyte-specific FOG2 overexpression in mice caused primary spontaneous AF before the development of HF or atrial remodeling. FOG2 overexpression generated arrhythmia substrate and trigger in cardiomyocytes, including calcium cycling defects. We found that FOG2 repressed atrial gene expression promoted by TBX5. FOG2 bound a subset of GATA4 and TBX5 co-bound genomic locations, defining a shared atrial gene regulatory network. FOG2 repressed TBX5-dependent transcription from a subset of co-bound enhancers, including a conserved enhancer at the Atp2a2 locus. Atrial rhythm abnormalities in mice caused by Tbx5 haploinsufficiency were rescued by Zfpm2 haploinsufficiency. CONCLUSIONS: Transcriptional changes in the atria observed in human HF directly antagonize the atrial rhythm gene regulatory network, providing a genomic link between HF and AF risk independent of atrial remodeling.

Microbial signals drive pre-leukaemic myeloproliferation in a Tet2-deficient host.

Somatic mutations in tet methylcytosine dioxygenase 2 (TET2), which encodes an epigenetic modifier enzyme, drive the development of haematopoietic malignancies1-7. In both humans and mice, TET2 deficiency leads to increased self-renewal of haematopoietic stem cells with a net developmental bias towards the myeloid lineage1,4,8,9. However, pre-leukaemic myeloproliferation (PMP) occurs in only a fraction of Tet2-/- mice8,9 and humans with TET2 mutations1,3,5-7, suggesting that extrinsic non-cell-autonomous factors are required for disease onset. Here we show that bacterial translocation and increased interleukin-6 production, resulting from dysfunction of the small-intestinal barrier, are critical for the development of PMP in mice that lack Tet2 expression in haematopoietic cells. Furthermore, in symptom-free Tet2-/- mice, PMP can be induced by disrupting intestinal barrier integrity, or in response to systemic bacterial stimuli such as the toll-like receptor 2 agonist. PMP was reversed by antibiotic treatment and failed to develop in germ-free Tet2-/- mice, which illustrates the importance of microbial signals in the development of this condition. Our findings demonstrate the requirement for microbial-dependent inflammation in the development of PMP and provide a mechanistic basis for the variation in PMP penetrance observed in Tet2-/- mice. This study will prompt new lines of investigation that may profoundly affect the prevention and management of haematopoietic malignancies.

Decentralized clinical trials for medications to reduce the risk of dementia: Consensus report and guidance.

INTRODUCTION: Recent growth in the functionality and use of technology has prompted an increased interest in the potential for remote or decentralized clinical trials in dementia. There are many potential benefits associated with decentralized medication trials, but we currently lack specific recommendations for their delivery in the dementia field. METHODS: A modified Delphi method engaged an expert panel to develop recommendations for the conduct of decentralized medication trials in dementia prevention. A working group of researchers and clinicians with expertise in dementia trials further refined the recommendations. RESULTS: Overall, the recommendations support the delivery of decentralized trials in dementia prevention provided adequate safety checks and balances are included. A total of 40 recommendations are presented, spanning aspects of decentralized clinical trials, including safety, dispensing, outcome assessment, and data collection. DISCUSSION: These recommendations provide an accessible, pragmatic guide for the design and conduct of remote medication trials for dementia prevention. HIGHLIGHTS: Clinical trials of medication have begun adopting decentralized approaches. Researchers in the field lack guidance on what would be appropriate circumstances and frameworks for what would be appropriate circumstances and frameworks for the use of decentralized trial methods in dementia prevention. The present report provides consensus-based expert recommendations for decentralized clinical trials for dementia prevention.

Computational Models of Claudin Assembly in Tight Junctions and Strand Properties.

Claudins are one of the major components of tight junctions (TJs) that polymerize within the cell membrane and form interactions between cells. Some claudins seal the paracellular space, limiting paracellular flux, while others form selectively permeable ion channels that control the paracellular permeability of small ions. Claudin strands are known to be dynamic and reshape within TJs to accommodate large-scale movements and rearrangements of epithelial tissues. Here, we summarize the recent computational and modeling studies on claudin assembly into tetrameric ion channels and their polymerization into µm long strands within the membrane. Computational studies ranging from all-atom molecular dynamics, coarse-grained simulations, and hybrid-resolution simulations elucidate the molecular nature of claudin assembly and function and provide a framework that describes the lateral flexibility of claudin strands.

OrganoID: A versatile deep learning platform for tracking and analysis of single-organoid dynamics.

Organoids have immense potential as ex vivo disease models for drug discovery and personalized drug screening. Dynamic changes in individual organoid morphology, number, and size can indicate important drug responses. However, these metrics are difficult and labor-intensive to obtain for high-throughput image datasets. Here, we present OrganoID, a robust image analysis platform that automatically recognizes, labels, and tracks single organoids, pixel-by-pixel, in brightfield and phase-contrast microscopy experiments. The platform was trained on images of pancreatic cancer organoids and validated on separate images of pancreatic, lung, colon, and adenoid cystic carcinoma organoids, which showed excellent agreement with manual measurements of organoid count (95%) and size (97%) without any parameter adjustments. Single-organoid tracking accuracy remained above 89% over a four-day time-lapse microscopy study. Automated single-organoid morphology analysis of a chemotherapy dose-response experiment identified strong dose effect sizes on organoid circularity, solidity, and eccentricity. OrganoID enables straightforward, detailed, and accurate image analysis to accelerate the use of organoids in high-throughput, data-intensive biomedical applications.

Direct Measurement of Ice-Ablator Interface Motion for Instability Mitigation in Indirect Drive ICF Implosions.

In indirect drive inertial confinement fusion (ICF) implosions hydrodynamic instability growth at the imploding capsule ablator-DT fuel interface can reduce fuel compressibility and inject ablator into the hot spot hence reducing hot spot pressure and temperature. As a mitigation strategy, a gentle acceleration of this interface is predicted by simulations and theory to significantly reduce this instability growth in the early stage of the implosion. We have performed high-contrast, time-resolved x-ray refraction enhanced radiography (RER) to accurately measure the level of acceleration as a function of the initial laser drive time history for indirect-drive implosions on the National Ignition Facility. We demonstrate a transition from no acceleration to 20±1.8 µm ns^{-2} acceleration by tweaking the drive that should reduce the initial instabilities by an order of magnitude at high modes.

ZO-1 Regulates Intercalated Disc Composition and Atrioventricular Node Conduction.

RATIONALE: ZO-1 (Zona occludens 1), encoded by the tight junction protein 1 (TJP1) gene, is a regulator of paracellular permeability in epithelia and endothelia. ZO-1 interacts with the actin cytoskeleton, gap, and adherens junction proteins and localizes to intercalated discs in cardiomyocytes. However, the contribution of ZO-1 to cardiac physiology remains poorly defined. OBJECTIVE: We aim to determine the role of ZO-1 in cardiac function. METHODS AND RESULTS: Inducible cardiomyocyte-specific Tjp1 deletion mice (Tjp1fl/fl; Myh6Cre/Esr1*) were generated by crossing the Tjp1 floxed mice and Myh6Cre/Esr1* transgenic mice. Tamoxifen-induced loss of ZO-1 led to atrioventricular (AV) block without changes in heart rate, as measured by ECG and ex vivo optical mapping. Mice with tamoxifen-induced conduction system-specific deletion of Tjp1 (Tjp1fl/fl; Hcn4CreERt2) developed AV block while tamoxifen-induced conduction system deletion of Tjp1 distal to the AV node (Tjp1fl/fl; Kcne1CreERt2) did not demonstrate conduction defects. Western blot and immunostaining analyses of AV nodes showed that ZO-1 loss decreased Cx (connexin) 40 expression and intercalated disc localization. Consistent with the mouse model study, immunohistochemical staining showed that ZO-1 is abundantly expressed in the human AV node and colocalizes with Cx40. Ventricular conduction was not altered despite decreased localization of ZO-1 and Cx43 at the ventricular intercalated disc and modestly decreased left ventricular ejection fraction, suggesting ZO-1 is differentially required for AV node and ventricular conduction. CONCLUSIONS: ZO-1 is a key protein responsible for maintaining appropriate AV node conduction through maintaining gap junction protein localization.

Histopathology of Colectomy Specimens Predicts Endoscopic Pouch Phenotype in Patients with Ulcerative Colitis.

BACKGROUND: The endoscopic appearance in patients with "pouchitis" after ileal pouch-anal anastomosis (IPAA) for ulcerative colitis (UC) can be quite heterogenous. Patients with an endoscopic phenotype resembling Crohn's disease (CD) are at high risk of pouch loss. AIMS: We aimed to assess how the histopathology of colectomy specimens predicts endoscopic pouch phenotypes in UC. METHODS: We retrospectively assessed pouchoscopies from patients with UC who underwent IPAA and classified pouch findings into 7 main phenotypes: (1) normal, (2) afferent limb involvement, (3) inlet involvement, (4) diffuse, (5) focal inflammation of the pouch body, (6) cuffitis, and (7) pouch with fistulas noted ≥ 6 months from ileostomy takedown. We assessed the clinical and pathological data including deep, focal inflammation, granulomas, and terminal ileal involvement in the colectomy specimens. Logistic regression analysis was performed to identify contributing factors to each phenotype. RESULTS: This study included 1,203 pouchoscopies from 382 patients with UC. On multivariable analysis, deep inflammation was significantly associated with pouch fistulas (Odds ratio 3.27; 95% confidence interval 1.65-6.47; P = 0.0007). Of the 75 patients with deep inflammation, only two patients (2.7%) were diagnosed with CD based on pathology review. Terminal ileal involvement significantly increased the risk of afferent limb involvement (Odds ratio 2.96; 95% confidence interval 1.04-8.47; P = 0.04). There were no significant associations between other microscopic features and phenotypes. CONCLUSIONS: We identify histologic features of colectomy specimens in UC that predict subsequent pouch phenotypes. Particularly, deep inflammation in the resected colon was significantly associated with pouch fistulas, a pouch phenotype with poor prognosis.

Transcriptional factors in calcium mishandling and atrial fibrillation development.

Healthy cardiac conduction relies on the coordinated electrical activity of distinct populations of cardiomyocytes. Disruption of cell-cell conduction results in cardiac arrhythmias, a leading cause of morbidity and mortality worldwide. Recent genetic studies have highlighted a major heritable component and identified numerous loci associated with risk of atrial fibrillation, including transcription factor genes, particularly those important in cardiac development, microRNAs, and long noncoding RNAs. Identification of such genetic factors has prompted the search to understand the mechanisms that underlie the genetic component of AF. Recent studies have found several mechanisms by which genetic alterations can result in AF formation via disruption of calcium handling. Loss of developmental transcription factors in adult cardiomyocytes can result in disruption of SR calcium ATPase, sodium calcium exchanger, calcium channels, among other ion channels, which underlie action potential abnormalities and triggered activity that can contribute to AF. This review aims to summarize the complex network of transcription factors and their roles in calcium handling.

Histologic changes caused by injection of a novel submucosal lifting agent for endoscopic resection in GI lesions.

BACKGROUND AND AIMS: ORISE Gel is a recently introduced, U.S. Food and Drug Administration-approved submucosal lifting agent used in endoscopic resection of GI lesions. Histologically evident gel deposits in resected specimens may pose a potential diagnostic pitfall. To aid in recognition of this procedure-related artifact, we report the largest histologic series of ORISE Gel in endoscopic and surgical resection specimens to date. METHODS: Fifty-eight EMR/endoscopic submucosal dissection (ESD) specimens with ORISE Gel injection and 5 interval surgical resection specimens with previous ORISE Gel injection were included. Patient demographics and endoscopy reports were obtained. Histologic slides from all cases were reviewed. Histochemical stains were performed on select cases. RESULTS: Fifty-one EMR and 7 ESD specimens were identified. In 51 of 58 (88%) endoscopic resection specimens, amorphous, pale blue-gray, finely granular material was evident in the submucosa, as well as focally within the mucosa in 4 cases. Most cases showed homogeneous near-complete filling of the submucosa with this material, whereas a few demonstrated areas of condensation and retraction. Mucicarmine and periodic acid-Schiff stains were negative for mucin. Interval surgical resection specimens revealed extensive deposition of dense, eosinophilic material with associated multinucleated giant cells in the submucosa in all cases, with transmural extension in 3 cases. CONCLUSION: ORISE Gel injection during endoscopic resection of GI lesions results in deposition of amorphous, blue-gray material seen in histologic sections, whereas interval surgical resection specimens demonstrate dense, eosinophilic material with an associated giant cell reaction. Awareness of these artifacts will help avoid misinterpretation of their presence as pathologic findings.

The cerebral cavernous malformation disease causing gene KRIT1 participates in intestinal epithelial barrier maintenance and regulation.

Epithelial barrier maintenance and regulation requires an intact perijunctional actomyosin ring underneath the cell-cell junctions. By searching for known factors affecting the actin cytoskeleton, we identified Krev interaction trapped protein 1 (KRIT1) as a major regulator for epithelial barrier function through multiple mechanisms. KRIT1 is expressed in both small intestinal and colonic epithelium, and KRIT1 knockdown in differentiated Caco-2 intestinal epithelium decreases epithelial barrier function and increases cation selectivity. KRIT1 knockdown abolished Rho-associated protein kinase-induced and myosin II motor inhibitor-induced barrier loss by limiting both small and large molecule permeability but did not affect myosin light chain kinase-induced increases in epithelial barrier function. These data suggest that KRIT1 participates in Rho-associated protein kinase- and myosin II motor-dependent (but not myosin light chain kinase-dependent) epithelial barrier regulation. KRIT1 knockdown exacerbated low-dose TNF-induced barrier loss, along with increased cleaved caspase-3 production. Both events are blocked by pan-caspase inhibition, indicating that KRIT1 regulates TNF-induced barrier loss through limiting epithelial apoptosis. These data indicate that KRIT1 controls epithelial barrier maintenance and regulation through multiple pathways, suggesting that KRIT1 mutation in cerebral cavernous malformation disease may alter epithelial function and affect human health.-Wang, Y., Li, Y., Zou, J., Polster, S. P., Lightle, R., Moore, T., Dimaano, M., He, T.-C., Weber, C. R., Awad, I. A., Shen, L. The cerebral cavernous malformation disease causing gene KRIT1 participates in intestinal epithelial barrier maintenance and regulation.

Cerebral Cavernous Malformation Proteins in Barrier Maintenance and Regulation.

Cerebral cavernous malformation (CCM) is a disease characterized by mulberry shaped clusters of dilated microvessels, primarily in the central nervous system. Such lesions can cause seizures, headaches, and stroke from brain bleeding. Loss-of-function germline and somatic mutations of a group of genes, called CCM genes, have been attributed to disease pathogenesis. In this review, we discuss the impact of CCM gene encoded proteins on cellular signaling, barrier function of endothelium and epithelium, and their contribution to CCM and potentially other diseases.

Computational Modeling of Claudin Structure and Function.

Tight junctions form a barrier to control passive transport of ions and small molecules across epithelia and endothelia. In addition to forming a barrier, some of claudins control transport properties of tight junctions by forming charge- and size-selective ion channels. It has been suggested claudin monomers can form or incorporate into tight junction strands to form channels. Resolving the crystallographic structure of several claudins in recent years has provided an opportunity to examine structural basis of claudins in tight junctions. Computational and theoretical modeling relying on atomic description of the pore have contributed significantly to our understanding of claudin pores and paracellular transport. In this paper, we review recent computational and mathematical modeling of claudin barrier function. We focus on dynamic modeling of global epithelial barrier function as a function of claudin pores and molecular dynamics studies of claudins leading to a functional model of claudin channels.

Safety and Efficacy of Combination Treatment With Calcineurin Inhibitors and Vedolizumab in Patients With Refractory Inflammatory Bowel Disease.

BACKGROUND & AIMS: Little is known about the efficacy and safety of induction therapy with calcineurin inhibitors in combination with vedolizumab for patients with Crohn's disease (CD) or ulcerative colitis (UC). We analyzed the outcomes of patients receiving vedolizumab along with calcineurin inhibitors. METHODS: We collected data on patients with CD (n = 9) or UC (n = 11) who began treatment with vedolizumab from May 20, 2014, through March 30, 2015, and received calcineurin inhibitors (tacrolimus or cyclosporin) during the first 12 months of vedolizumab therapy. Clinical activity scores and inflammatory markers were measured at baseline and at weeks 14, 30, and 52 of vedolizumab treatment. Clinical remission was defined as a Harvey-Bradshaw index score ≤4 or short clinical colitis activity index score ≤2; steroid-free clinical remission was defined as clinical remission without corticosteroids. RESULTS: By week 14 of treatment, 44% of the patients with CD and 55% of the patients with UC achieved steroid-free clinical remission; after 52 weeks of treatment, 33% of the patients with CD and 45% of the patients with UC were in steroid-free clinical remission. Seven patients received salvage therapy with a calcineurin inhibitor after primary nonresponse to vedolizumab-1 of the 2 patients with UC and 2 of 5 patients with CD stopped taking the calcineurin inhibitors and achieved steroid-free remission at week 52. In total, 16 patients (59%) received 52 weeks of treatment with vedolizumab. Three serious adverse events were associated with calcineurin inhibitors. CONCLUSIONS: Combination therapy of vedolizumab with either cyclosporin or tacrolimus is effective and safe at inducing and maintaining clinical remission in patients with CD and UC with up to 52 weeks of follow-up evaluation. Larger studies of the ability of calcineurin inhibitors to induce remission in patients on vedolizumab are warranted.

Patients With Ulcerative Colitis and Primary Sclerosing Cholangitis Frequently Have Subclinical Inflammation in the Proximal Colon.

BACKGROUND & AIMS: Patients with primary sclerosing cholangitis (PSC) and ulcerative colitis (UC) have a high risk of colonic neoplasia. Neoplasia frequently develops in the proximal colon in patients with PSC. Histologic inflammation is an independent risk factor for the development of neoplasia; we investigated whether patients with UC and PSC have more subclinical disease activity than patients with UC alone. METHODS: We performed a retrospective analysis of data from 143 patients (205 examinations) with ulcerative pancolitis who were in clinical remission and treated at a tertiary medical center from May 2011 through May 2016. Endoscopic and histologic activity were compared between patients with PSC (from 36 examinations) and without PSC (from 169 examinations). Disease activity was scored per colonic segment using a modified Mayo endoscopic subscore and histologic assessment. In each colonic segment, differences in disease activity and the degree of discordance between endoscopic and histologic inflammation among UC patients with and without PSC were compared. RESULTS: Patients with UC-PSC had significantly more subclinical endoscopic (odds ratio [OR], 4.21; 95% CI, 1.67-10.63) and histologic activity (OR, 5.13; 95% CI, 2.25-11.68) in the right colon, as well as greater degree of histologic than endoscopic inflammation in the proximal colon (OR, 3.14, 95% CI, 1.24-7.97), compared with patients without PSC. Patients with UC-PSC had significantly less histologic activity in the rectum on multivariate analysis (OR, 0.24; 95% CI, 0.08-0.72). CONCLUSIONS: Patients with UC and PSC who are in clinical remission are significantly more likely to have endoscopic and histologic inflammation in the right colon than patients with UC without PSC. Our findings provide insight into cause of colorectal cancer in UC patients with PSC.

Spectral biomarkers for chemoprevention of colonic neoplasia: a placebo-controlled double-blinded trial with aspirin.

OBJECTIVE: A major impediment to translating chemoprevention to clinical practice has been lack of intermediate biomarkers. We previously reported that rectal interrogation with low-coherence enhanced backscattering spectroscopy (LEBS) detected microarchitectural manifestations of field carcinogenesis. We now wanted to ascertain if reversion of two LEBS markers spectral slope (SPEC) and fractal dimension (FRAC) could serve as a marker for chemopreventive efficacy. DESIGN: We conducted a multicentre, prospective, randomised, double-blind placebo-controlled, clinical trial in subjects with a history of colonic neoplasia who manifested altered SPEC/FRAC in histologically normal colonic mucosa. Subjects (n=79) were randomised to 325 mg aspirin or placebo. The primary endpoint changed in FRAC and SPEC spectral markers after 3 months. Mucosal levels of prostaglandin E2 (PGE2) and UDP-glucuronosyltransferase (UGT)1A6 genotypes were planned secondary endpoints. RESULTS: At 3 months, the aspirin group manifested alterations in SPEC (48.9%, p=0.055) and FRAC (55.4%, p=0.200) with the direction towards non-neoplastic status. As a measure of aspirin's pharmacological efficacy, we assessed changes in rectal PGE2 levels and noted that it correlated with SPEC and FRAC alterations (R=-0.55, p=0.01 and R=0.57, p=0.009, respectively) whereas there was no significant correlation in placebo specimens. While UGT1A6 subgroup analysis did not achieve statistical significance, the changes in SPEC and FRAC to a less neoplastic direction occurred only in the variant consonant with epidemiological evidence of chemoprevention. CONCLUSIONS: We provide the first proof of concept, albeit somewhat underpowered, that spectral markers reversion mirrors antineoplastic efficacy providing a potential modality for titration of agent type/dose to optimise chemopreventive strategies in clinical practice. TRIAL NUMBER: NCT00468910.

The Thr300Ala variant in ATG16L1 is associated with improved survival in human colorectal cancer and enhanced production of type I interferon.

OBJECTIVE: ATG16L1 is an autophagy gene known to control host immune responses to viruses and bacteria. Recently, a non-synonymous single-nucleotide polymorphism in ATG16L1 (Thr300Ala), previously identified as a risk factor in Crohn's disease (CD), was associated with more favourable clinical outcomes in thyroid cancer. Mechanisms underlying this observation have not been proposed, nor is it clear whether an association between Thr300Ala and clinical outcomes will be observed in other cancers. We hypothesised that Thr300Ala influences clinical outcome in human colorectal cancer (CRC) and controls innate antiviral pathways in colon cancer cells. DESIGN: We genotyped 460 patients with CRC and assessed for an association between ATG16L1 Thr300Ala and overall survival and clinical stage. Human CRC cell lines were targeted by homologous recombination to examine the functional consequence of loss of ATG16L1, or introduction of the Thr300Ala variant. RESULTS: We found an association between longer overall survival, reduced metastasis and the ATG16L1 Ala/Ala genotype. Tumour sections from ATG16L1 Ala/Ala patients expressed elevated type I interferons (IFN-I)-inducible, MxA, suggesting that differences in cytokine production may influence disease progression. When introduced into human CRC cells by homologous recombination, the Thr300Ala variant did not affect bulk autophagy, but increased basal production of type I IFN. Introduction of Thr300Ala resulted in increased sensitivity to the dsRNA mimic poly(I:C) through a mitochondrial antiviral signalling (MAVS)-dependent pathway. CONCLUSIONS: The CD-risk allele, Thr300Ala, in ATG16L1 is associated with improved overall survival in human CRC, generating a rationale to genotype ATG16L1 Thr300Ala in patients with CRC. We found that Thr300A alters production of MAVS-dependent type I IFN in CRC cells, providing a mechanism that may influence clinical outcomes.

Insights into the pathogenesis of ulcerative colitis from a murine model of stasis-induced dysbiosis, colonic metaplasia, and genetic susceptibility.

Gut dysbiosis, host genetics, and environmental triggers are implicated as causative factors in inflammatory bowel disease (IBD), yet mechanistic insights are lacking. Longitudinal analysis of ulcerative colitis (UC) patients following total colectomy with ileal anal anastomosis (IPAA) where >50% develop pouchitis offers a unique setting to examine cause vs. effect. To recapitulate human IPAA, we employed a mouse model of surgically created blind self-filling (SFL) and self-emptying (SEL) ileal loops using wild-type (WT), IL-10 knockout (KO) (IL-10), TLR4 KO (T4), and IL-10/T4 double KO mice. After 5 wk, loop histology, host gene/protein expression, and bacterial 16s rRNA profiles were examined. SFL exhibit fecal stasis due to directional motility oriented toward the loop end, whereas SEL remain empty. In WT mice, SFL, but not SEL, develop pouchlike microbial communities without accompanying active inflammation. However, in genetically susceptible IL-10-deficient mice, SFL, but not SEL, exhibit severe inflammation and mucosal transcriptomes resembling human pouchitis. The inflammation associated with IL-10 required TLR4, as animals lacking both pathways displayed little disease. Furthermore, germ-free IL-10 mice conventionalized with SFL, but not SEL, microbiota populations develop severe colitis. These data support essential roles of stasis-induced, colon-like microbiota, TLR4-mediated colonic metaplasia, and genetic susceptibility in the development of pouchitis and possibly UC. However, these factors by themselves are not sufficient. Similarities between this model and human UC/pouchitis provide opportunities for gaining insights into the mechanistic basis of IBD and for identification of targets for novel preventative and therapeutic interventions.

Dynamic migration of γδ intraepithelial lymphocytes requires occludin.

γδ intraepithelial lymphocytes (IELs) are located beneath or between adjacent intestinal epithelial cells and are thought to contribute to homeostasis and disease pathogenesis. Using in vivo microscopy to image jejunal mucosa of GFP γδ T-cell transgenic mice, we discovered that γδ IELs migrate actively within the intraepithelial compartment and into the lamina propria. As a result, each γδ IEL contacts multiple epithelial cells. Occludin is concentrated at sites of γδ IEL/epithelial interaction, where it forms a ring surrounding the γδ IEL. In vitro analyses showed that occludin is expressed by epithelial and γδ T cells and that occludin derived from both cell types contributes to these rings and to γδ IEL migration within epithelial monolayers. In vivo TNF administration, which results in epithelial occludin endocytosis, reduces γδ IEL migration. Further in vivo analyses demonstrated that occludin KO γδ T cells are defective in both initial accumulation and migration within the intraepithelial compartment. These data challenge the paradigm that γδ IELs are stationary in the intestinal epithelium and demonstrate that γδ IELs migrate dynamically to make extensive contacts with epithelial cells. The identification of occludin as an essential factor in γδ IEL migration provides insight into the molecular regulation of γδ IEL/epithelial interactions.