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Summary The 30+ unique ligands of the TGFβ family signal by forming complexes using different combinations of type I and type II receptors. Therapeutically, the extracellular domain of a single receptor fused to an Fc molecule can effectively neutralize subsets of ligands. Increased ligand specificity can be accomplished by using the extracellular domains of both the type I and type II receptor to mimic the naturally occurring signaling complex. Here, we report the structure of one “type II-type I-Fc” fusion, ActRIIB-Alk4-Fc, in complex with two TGFβ family ligands, ActA, and GDF11, providing a snapshot of this therapeutic platform. The study reveals that extensive contacts are formed by both receptors, replicating the ternary signaling complex, despite the inherent low affinity of Alk4. Our study shows that low-affinity type I interactions support altered ligand specificity and can be visualized at the molecular level using this platform., Graphical abstract, Highlights • Structure of type I and type II Fc receptor trap bound to TGF- β ligand • Similar ligand-binding interactions observed for low-affinity type I receptors • Structure of Alk4 reveals differences relative to other type I TGF-β receptors • Structures are consistent with the conformational selection model, Biochemistry; Molecular biology; Structural biology

ObjectiveThe clinical presentation and course of Crohn's disease (CD) is highly variable. We sought to better understand the cellular and molecular mechanisms that guide this heterogeneity, and characterise the cellular processes associated with disease phenotypes.DesignWe examined both gene expression and gene regulation (chromatin accessibility) in non-inflamed colon tissue from a cohort of adult patients with CD and control patients. To support the generality of our findings, we analysed previously published expression data from a large cohort of treatment-naïve paediatric CD and control ileum.ResultsWe found that adult patients with CD clearly segregated into two classes based on colon tissue gene expression—one that largely resembled the normal colon and one where certain genes showed expression patterns normally specific to the ileum. These classes were supported by changes in gene regulatory profiles observed at the level of chromatin accessibility, reflective of a fundamental shift in underlying molecular phenotypes. Furthermore, gene expression from the ilea of a treatment-naïve cohort of paediatric patients with CD could be similarly subdivided into colon-like and ileum-like classes. Finally, expression patterns within these CD subclasses highlight large-scale differences in the immune response and aspects of cellular metabolism, and were associated with multiple clinical phenotypes describing disease behaviour, including rectal disease and need for colectomy.ConclusionsOur results strongly suggest that these molecular signatures define two clinically relevant forms of CD irrespective of tissue sampling location, patient age or treatment status.

Intestinal macrophages are uniquely programmed to tolerate exposure to bacteria without mounting potent inflammatory responses. The cytokine IL-10 maintains the macrophage anti-inflammatory response such that loss of IL-10 results in chronic intestinal inflammation. To investigate how IL-10-deficiency alters intestinal macrophage programming and bacterial tolerance, we studied changes in chromatin accessibility in response to bacteria in macrophages from two distinct niches, the intestine and bone-marrow, from both wild-type and IL-10-deficient mice. In both bone-marrow-derived and intestinal macrophages, we identified chromatin accessibility changes associated with bacterial exposure and IL-10-deficiency. Surprisingly, IL-10-deficient intestinal macrophages adopted chromatin and gene expression patterns characteristic of an inflammatory response, even in the absence of bacteria. Further, if IL-10 protein was added to cells that had previously been IL-10-deficient, it could not revert the chromatin landscape to a normal state. Our results demonstrate that IL-10 deficiency results in stable chromatin alterations in macrophages, even in the absence of bacteria. This supports a model where IL-10-deficiency leads to chromatin alterations that contribute to a loss of intestinal macrophage tolerance to bacteria, which is a primary initiating event in chronic intestinal inflammation.

Article first published online 6 July 2015. Supplemental Digital Content is Available in the Text., Background: There is a dire need for reliable prognostic markers that can guide effective therapeutic intervention in Crohn's disease (CD). We examined whether different phenotypes in CD can be classified based on colonic microRNA (miRNA) expression and whether miRNAs have prognostic utility for CD. Methods: High-throughput sequencing of small and total RNA isolated from colon tissue from patients with CD and controls without Inflammatory Bowel Disease (non-IBD) was performed. To identify miRNAs associated with specific phenotypes of CD, patients were stratified according to disease behavior (nonstricturing, nonpenetrating; stricturing; penetrating), and miRNA profiles in each subset were compared with those of the non-IBD group. Validation assays were performed using quantitative reverse transcription polymerase chain reaction. These miRNAs were further evaluated by quantitative reverse transcriptase polymerase chain reaction on formalin-fixed, paraffin-embedded tissue (index biopsies) of patients with nonpenetrating CD at the time of diagnosis that either retained the nonpenetrating phenotype or progressed to penetrating/fistulizing CD. Results: We found a suite of miRNAs, including miR-31-5p, miR-215, miR-223-3p, miR-196b-5p, and miR-203 that stratify patients with CD according to disease behavior independent of the effect of inflammation. Furthermore, we also demonstrated that expression levels of miR-215 in index biopsies of patients with CD might predict the likelihood of progression to penetrating/fistulizing CD. Finally, using a novel statistical simulation approach applied to colonic RNA-sequencing data for patients with CD and non-IBD controls, we identified miR-31-5p and miR-203 as candidate master regulators of gene expression profiles associated with CD. Conclusions: miRNAs may serve as clinically useful prognostic markers guiding initial therapy and identifying patients who would benefit most from effective intervention.

There are many different animal models available for studying the pathogenesis of human inflammatory bowel diseases (IBD), each with its own advantages and disadvantages. We describe here an experimental colitis model that is initiated by adoptive transfer of syngeneic splenic CD4+CD45RBhigh T cells into T and B cell deficient recipient mice. The CD4+CD45RBhigh T cell population that largely consists of naive effector cells is capable of inducing chronic intestinal inflammation, closely resembling key aspects of human IBD. This method can be manipulated to study aspects of disease onset and progression. Additionally it can be used to study the function of innate, adaptive, and regulatory immune cell populations, and the role of environmental exposures, i.e., the microbiota, in intestinal inflammation. In this article we illustrate the methodology for inducing colitis with a step-by-step protocol. This includes a video demonstration of key technical aspects required to successfully develop this murine model of experimental colitis for research purposes.

Crohn's disease (CD) is a chronic inflammatory disease of the gastrointestinal tract, characterized by an inappropriate immune response to the enteric microbiota. Chromatin based assays have successfully identified cell type and disease-specific regions of DNA accessibility, and have been linked to differential transcription of target genes, but this approach has yet to be explored in CD. In this study we performed formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) to identify nucleosome-depleted regions associated with regulatory elements, and RNA-seq to quantify expression, to characterize disease-relevant changes in chromatin and establish links to putative target genes.Mucosal Biopsies for 21 CD and 12 non-IBD individuals were taken from macroscopically uninflamed portions of the ascending colon at time of surgery. We performed FAIRE-seq and RNA-seq on each tissue sample. Reads were aligned to personalized genomes constructed with genotypes assayed on the Illumina Immunochip platform. A genome-wide scan for differential regions and genes was performed using 2 sided t-tests on normalized read counts in 300bp regions (FAIRE-seq) and RPKM (RNA-seq).RNA-seq data analysis across all samples identified 2 distinct classes of patients associated with epithelial cell lipid transport (class I, n = 10, CD; n = 1, non-IBD) and maintenance of cellular and luminal pH (class II, n = 11, CD, n = 11, non-IBD). To unravel potential gene regulatory mechanisms we analyzed chromatin profiles using FAIRE-seq and histone annotations using tissue specific ChIP-seq data sets. Class I patients showed increased accessibility at distal sites marked for enhancer activity in small intestine, and reduced accessibility at sites marked as enhancers in colon, suggesting 2 distinct regulatory programs driving the expression of differential genes. This pattern was reversed in class II samples. In an analysis restricting to class II, which was characterized by normal colon enhancer activity, we found regulatory regions specific to CD and non-IBD, respectively, as well as 51 and 507 genes upregulated in the respective cohorts. Top ontologies of genes upregulated in CD included host response to bacteria and immune response. Differential regulatory genes were enriched near TSSs of differentially expressed genes, implying putative causal effect. In an integrative analysis, we identified 72 regulatory regions that associate with both disease status and expression of nearby genes. A retrospective analysis of patients in class I and II revealed that the majority of patients (10/11) in class I required post-operative anti-TNF therapy for disease recurrence compared to only 1/11 CD patients in the class II group.We used chromatin status as a novel marker of disease in Crohn's. We identified a subset of CD patients that are characterized at the chromatin level in the colon by enrichment of enhancer marks specific to small intestine, and loss of enhancer activity specific to colon. Among individuals with enhancer activity indicative of normal colon, we integrated data from well-described transcriptional markers with novel chromatin variables to identify molecular associations and interactions that affect disease predisposition. Despite small patient numbers we were able to use chromatin profiles to identify patients that required post-operative management with anti-TNF agents.

The p110δ subunit of class IA PI3K modulates signaling in innate immune cells. We previously demonstrated that mice harboring a kinase-dead p110δ subunit (p110δKD) develop spontaneous colitis. Macrophages contributed to the Th1/Th17 cytokine bias in p110δKD mice through increased IL-12 and IL-23 expression. In this study, we show that the enteric microbiota is required for colitis development in germfree p110δKD mice. Colonic tissue and macrophages from p110δKD mice produce significantly less IL-10 compared with wild-type mice. p110δKD APCs cocultured with naive CD4+ Ag-specific T cells also produce significantly less IL-10 and induce more IFN-γ– and IL-17A–producing CD4+ T cells compared with wild-type APCs. Illustrating the importance of APC–T cell interactions in colitis pathogenesis in vivo, Rag1−/−/p110δKD mice develop mild colonic inflammation and produced more colonic IL-12p40 compared with Rag1−/− mice. However, CD4+CD45RBhigh/low T cell Rag1−/−/p110δKD recipient mice develop severe colitis with increased percentages of IFN-γ– and IL-17A–producing lamina propria CD3+CD4+ T cells compared with Rag1−/− recipient mice. Intestinal tissue samples from patients with Crohn’s disease reveal significantly lower expression of PIK3CD compared with intestinal samples from non–inflammatory bowel disease control subjects (p < 0.05). PIK3CD expression inversely correlates with the ratio of IL12B:IL10 expression. In conclusion, the PI3K subunit p110δ controls homeostatic APC–T cell interactions by altering the balance between IL-10 and IL-12/23. Defects in p110δ expression and/or function may underlie the pathogenesis of human inflammatory bowel disease and lead to new therapeutic strategies.

Because of emerging resistance to existing drugs, new chemical classes of antimalarial drugs are urgently needed. We have rationally designed a library of compounds that were predicted to accumulate in the digestive vacuole and then decrystallize hemozoin by breaking the iron carboxylate bond in hemozoin. We report the synthesis of 16 naphthothiazolium salts with amine-bearing side chains and their activities against the erythrocytic stage of Plasmodium falciparum in vitro. KSWI-855, the compound with the highest efficacy against the asexual stages of P. falciparum in vitro, also had in vitro activity against P. falciparum gametocytes and in vivo activity against P. berghei in a murine malaria model.

Introduction: Genome-wide association studies suggest that there is dysregulation of distinct biological circuits involved in the maintenance of intestinal homeostasis in patients with inflammatory bowel diseases (IBD). Broadly, we seek to understand the relationship between patient genotype and disease-specific, tissue-dependent gene expression to elucidate critical pathways at play in health and disease, identify novel, clinically-applicable biomarkers, guide therapeutic discovery, and move towards personalized care for patients with IBD. Methods: Using the most-recent GWAS data for IBD, identifying 163 risk loci, a custom NanoString probeset of 683 IBD-associated genes and 15 housekeeping genes was designed; 25% of these genes were not assayed by conventional microarrays. Patients and controls were recruited at five IBD centers nationwide as part of the Sinai-Helmsley Alliance for Research Excellence (SHARE) consortium. To date, we have collected 352 specimens from 172 unique patients (Crohn's disease, n=142, ulcerative colitis, n=28, indeterminate colitis, n=2). Total mRNA was isolated from 2-3 mucosal biopsies obtained at colonoscopy or taken from surgical specimens and used for downstream processing. A custom bioanalytic pipeline was designed for quality control and normalization to permit differential expression, data-driven clustering, and genotype-based expression quantitative trait loci (eQTL) analysis. Results: To serve as a basis for analysis in patients, we defined the expression patterns of these selected genes in healthy controls in multiple anatomic locations, including terminal ileum, ascending and descending colon, observing distinct expression patterns between ileum and colon and high correlation between controls. In the second phase of this project, we compared the expression patterns between diseased tissue versus healthy controls, inflamed versus uninflamed tissues from the same patient, and ulcerative colitis versus Crohn's colitis. Preliminary analysis demonstrates a strong differential gene expression pattern with 120 upregulated genes and 8 down-regulated genes in the inflamed tissue as compared to the uninflamed tissue. Pathway analysis suggests differences in activity between inflamed and uninflamed tissues in pathways related to cell adhesion, immune system signaling, and biosynthetic processing. With targeted examination for eQTLs for four disease-related SNPs (NOD2, ATG16L1, IRGM, MST1), we have identified genotype-dependent, fold-change gene expression differences between paired inflamed and uninflamed specimens. Conclusions: Selected profiling of IBD-related genes in disease-relevant tissues by NanoString technology and in patients with known genotypes can robustly detect novel patterns of gene epistasis, identify eQTLs, and highlight dominant pathways perturbed in disease.