NOX1 is essential for TNFα-induced intestinal epithelial ROS secretion and inhibits M cell signatures.

OBJECTIVE: Loss-of-function mutations in genes generating reactive oxygen species (ROS), such as NOX1, are associated with IBD. Mechanisms whereby loss of ROS drive IBD are incompletely defined. DESIGN: ROS measurements and single-cell transcriptomics were performed on colonoids stratified by NOX1 genotype and TNFα stimulation. Clustering of epithelial cells from human UC (inflamed and uninflamed) scRNASeq was performed. Validation of M cell induction was performed by immunohistochemistry using UEA1 (ulex europaeus agglutin-1 lectin) and in vivo with DSS injury. RESULTS: TNFα induces ROS production more in NOX1-WT versus NOX1-deficient murine colonoids under a range of Wnt-mediated and Notch-mediated conditions. scRNASeq from inflamed and uninflamed human colitis versus TNFα stimulated, in vitro colonoids defines substantially shared, induced transcription factors; NOX1-deficient colonoids express substantially lower levels of STAT3 (signal transducer and activator of transcription 3), CEBPD (CCAAT enhancer-binding protein delta), DNMT1 (DNA methyltransferase) and HIF1A (hypoxia-inducible factor) baseline. Subclustering unexpectedly showed marked TNFα-mediated induction of M cells (sentinel cells overlying lymphoid aggregates) in NOX1-deficient colonoids. M cell induction by UEA1 staining is rescued with H2O2 and paraquat, defining extra- and intracellular ROS roles in maintenance of LGR5+ stem cells. DSS injury demonstrated GP2 (glycoprotein-2), basal lymphoplasmacytosis and UEA1 induction in NOX1-deficiency. Principal components analyses of M cell genes and decreased DNMT1 RNA velocity correlate with UC inflammation. CONCLUSIONS: NOX1 deficiency plus TNFα stimulation contribute to colitis through dysregulation of the stem cell niche and altered cell differentiation, enhancing basal lymphoplasmacytosis. Our findings prioritise ROS modulation for future therapies.

Single-cell transcriptomics reveals conserved cell identities and fibrogenic phenotypes in zebrafish and human liver.

The mechanisms underlying liver fibrosis are multifaceted and remain elusive with no approved antifibrotic treatments available. The adult zebrafish has been an underutilized tool to study liver fibrosis. We aimed to characterize the single-cell transcriptome of the adult zebrafish liver to determine its utility as a model for studying liver fibrosis. We used single-cell RNA sequencing (scRNA-seq) of adult zebrafish liver to study the molecular and cellular dynamics at a single-cell level. We performed a comparative analysis to scRNA-seq of human liver with a focus on hepatic stellate cells (HSCs), the driver cells in liver fibrosis. scRNA-seq reveals transcriptionally unique populations of hepatic cell types that comprise the zebrafish liver. Joint clustering with human liver scRNA-seq data demonstrates high conservation of transcriptional profiles and human marker genes in zebrafish. Human and zebrafish HSCs show conservation of transcriptional profiles, and we uncover collectin subfamily member 11 (colec11) as a novel, conserved marker for zebrafish HSCs. To demonstrate the power of scRNA-seq to study liver fibrosis using zebrafish, we performed scRNA-seq on our zebrafish model of a pediatric liver disease with mutation in mannose phosphate isomerase (MPI) and characteristic early liver fibrosis. We found fibrosis signaling pathways and upstream regulators conserved across MPI-depleted zebrafish and human HSCs. CellPhoneDB analysis of zebrafish transcriptome identified neuropilin 1 as a potential driver of liver fibrosis. Conclusion: This study establishes the first scRNA-seq atlas of the adult zebrafish liver, highlights the high degree of similarity to human liver, and strengthens its value as a model to study liver fibrosis.

A Role for CXCR3 Ligands as Biomarkers of Post-Operative Crohn's Disease Recurrence.

BACKGROUND AND AIMS: Crohn's disease [CD] recurrence following ileocolic resection [ICR] is common. We sought to identify blood-based biomarkers associated with CD recurrence. METHODS: CD patients undergoing ICR were recruited across six centres. Serum samples were obtained at post-operative colonoscopy. A multiplex immunoassay was used to analyse 92 inflammation-related proteins [Olink Proteomics]. Bayesian analysis was used to identify proteins associated with increasing Rutgeerts score. Identified proteins were used in receiver operating characteristic [ROC] analysis to examine the ability to identify CD recurrence [Rutgeerts score ≥i2]. Existing single cell data were interrogated to further elucidate the role of the identified proteins. RESULTS: Data from 276 colonoscopies in 213 patients were available. Median time from surgery to first and second colonoscopy was 7 (interquartile range [IQR] 6-9) and 19 [IQR 16-23] months, respectively. Disease recurrence was evident at 60 [30%] first and 36 [49%] second colonoscopies. Of 14 proteins significantly associated with Rutgeerts score, the strongest signal was seen for CXCL9 and MMP1. Among patients on anti-tumour necrosis factor drugs, CXCL9 and CXCL11 were most strongly associated with Rutgeerts score. Both are CXCR3 ligands. Incorporation of identified proteins into ROC analysis improved the ability to identify disease recurrence as compared to C-reactive protein alone: area under the curve [AUC] 0.75 (95% confidence interval [CI]: 0.66-0.82] vs 0.64 [95% CI 0.56-0.72], p = 0.012. Single cell transcriptomic data provide evidence that innate immune cells are the primary source of the identified proteins. CONCLUSIONS: CXCR3 ligands are associated with CD recurrence following ICR. Incorporation of novel blood-based candidate biomarkers may aid in identification of CD recurrence.

From single-target to cellular niche targeting in Crohn's disease: intercepting bad communications.

The mainstay of moderate to severe Crohn's disease (CD), anti-TNF treatment, shows no clinical benefit in ∼40% of patients, likely due to incomplete cellular targeting and delayed treatment institution. While single-target therapeutics have been highly effective for some CD patients, substantial limitations with respect to safety, efficacy, and long-term, complete remission remain. Deconvolution of the cellular and molecular circuitry of tissue lesions underscores the importance of combinatorial strategies targeting cellular niches. This review aims to evaluate current therapeutic approaches used to manage CD, and highlight recent advances to our cellular, genetic, and molecular understanding of mechanisms driving pathogenic niche activation in CD. We propose new frameworks outlining that combinatorial therapies, along with serial tissue sampling and studies guided by genetics and genomics, can advance on current treatment approaches and will inform newer strategies upon which we can move towards precision therapeutics in IBD.

A myeloid-stromal niche and gp130 rescue in NOD2-driven Crohn's disease.

Crohn's disease is a chronic inflammatory intestinal disease that is frequently accompanied by aberrant healing and stricturing complications. Crosstalk between activated myeloid and stromal cells is critical in the pathogenicity of Crohn's disease1,2, and increases in intravasating monocytes are correlated with a lack of response to anti-TNF treatment3. The risk alleles with the highest effect on Crohn's disease are loss-of-function mutations in NOD24,5, which increase the risk of stricturing6. However, the mechanisms that underlie pathogenicity driven by NOD2 mutations and the pathways that might rescue a lack of response to anti-TNF treatment remain largely uncharacterized. Here we use direct ex vivo analyses of patients who carry risk alleles of NOD2 to show that loss of NOD2 leads to dysregulated homeostasis of activated fibroblasts and macrophages. CD14+ peripheral blood mononuclear cells from carriers of NOD2 risk alleles produce cells that express high levels of collagen, and elevation of conserved signatures is observed in nod2-deficient zebrafish models of intestinal injury. The enrichment of STAT3 regulation and gp130 ligands in activated fibroblasts and macrophages suggested that gp130 blockade might rescue the activated program in NOD2-deficient cells. We show that post-treatment induction of the STAT3 pathway is correlated with a lack of response to anti-TNF treatment in patients, and demonstrate in vivo in zebrafish the amelioration of the activated myeloid-stromal niche using the specific gp130 inhibitor bazedoxifene. Our results provide insights into NOD2-driven fibrosis in Crohn's disease, and suggest that gp130 blockade may benefit some patients with Crohn's disease-potentially as a complement to anti-TNF therapy.

Inflamed Ulcerative Colitis Regions Associated With MRGPRX2-Mediated Mast Cell Degranulation and Cell Activation Modules, Defining a New Therapeutic Target.

BACKGROUND & AIMS: Recent literature has implicated a key role for mast cells in murine models of colonic inflammation, but their role in human ulcerative colitis (UC) is not well established. A major advance has been the identification of mrgprb2 (human orthologue, MRGPX2) as mediating IgE-independent mast cell activation. We sought to define mechanisms of mast cell activation and MRGPRX2 in human UC. METHODS: Colon tissues were collected from patients with UC for bulk RNA sequencing and lamina propria cells were isolated for MRGPRX2 activation studies and single-cell RNA sequencing. Genetic association of all protein-altering G-protein coupled receptor single-nucleotide polymorphism was performed in an Ashkenazi Jewish UC case-control cohort. Variants of MRGPRX2 were transfected into Chinese hamster ovary (CHO) and human mast cell (HMC) 1.1 cells to detect genotype-dependent effects on ß-arrestin recruitment, IP-1 accumulation, and phosphorylated extracellular signal-regulated kinase. RESULTS: Mast cell-specific mediators and adrenomedullin (proteolytic precursor of PAMP-12, an MRGPRX2 agonist) are up-regulated in inflamed compared to uninflamed UC. MRGPRX2 stimulation induces carboxypeptidase secretion from inflamed UC. Of all protein-altering GPCR alleles, a unique variant of MRGPRX2, Asn62Ser, was most associated with and was bioinformatically predicted to alter arrestin recruitment. We validated that the UC protective serine allele enhances ß-arrestin recruitment, decreases IP-1, and increases phosphorylated extracellular signal-regulated kinase with MRGPRX2 agonists. Single-cell RNA sequencing defines that adrenomedullin is expressed by activated fibroblasts and epithelial cells and that interferon gamma is a key upstream regulator of mast cell gene expression. CONCLUSION: Inflamed UC regions are distinguished by MRGPRX2-mediated activation of mast cells, with decreased activation observed with a UC-protective genetic variant. These results define cell modules of UC activation and a new therapeutic target.

Machine learning identifies novel blood protein predictors of penetrating and stricturing complications in newly diagnosed paediatric Crohn's disease.

BACKGROUND: There is a need for improved risk stratification in Crohn's disease. AIM: To identify novel blood protein biomarkers associated with future Crohn's disease complications METHODS: We performed a case-cohort study utilising a paediatric inception cohort, the Risk Stratification and Identification of Immunogenetic and Microbial Markers of Rapid Disease Progression in Children with Crohn's disease (RISK) study. All patients had inflammatory disease (B1) at baseline. Outcomes were development of stricturing (B2) or penetrating (B3) complications. We assayed 92 inflammation-related proteins in baseline plasma using a proximity extension assay (Olink Proteomics). An ensemble machine learning technique, random survival forests (RSF), selected variables predicting B2 and B3 complications. Selected analytes were compared to clinical variables and serology only models. We examined selected proteins in a single-cell sequencing cohort to analyse differential cell expression in blood and ileum. RESULTS: We included 265 patients with mean age 11.6 years (standard deviation [SD] 3.2). Seventy-three and 34 patients, respectively, had B2 and B3 complications within mean 1123 (SD 477) days for B2 and 1251 (442) for B3. A model with 5 protein markers predicted B3 complications with an area under the curve (AUC) of 0.79 (95% confidence interval [CI] 0.76-0.82) compared to 0.69 (95% CI 0.66-0.72) for serologies and 0.74 (95% CI 0.71-0.77) for clinical variables. A model with 4 protein markers predicted B2 complications with an AUC of 0.68 (95% CI 0.65-0.71) compared to 0.62 (95% CI 0.59-0.65) for serologies and 0.52 (95% CI 0.50-0.55) for clinical variables. B2 analytes were highly expressed in ileal stromal cells while B3 analytes were prominent in peripheral blood and ileal T cells. CONCLUSIONS: We identified novel blood proteomic markers, distinct for B2 and B3, associated with progression of paediatric Crohn's disease.

Glycemic Index of Wheat and Rice are Similar When Consumed as Part of a North Indian Mixed Meal.

INTRODUCTION: Wheat is preferable over rice due to its lower glycemic index (GI). It is not known if the same is true when these staples are a part of mixed meals, hence we compared the Glycemic responses of wheat/rice containing mixed meals. MATERIALS AND METHODS: Glycemic responses of 2 mixed meals were compared with reference meal (glucose) where each was designed to provide a total of 50 g of available carbohydrate (AvCHO), in 10 healthy adult volunteers as per recent recommendations. Test meal 1 comprised of a pulse preparation (green gram dal), a vegetable (ladies' finger), and 2 wheat chapattis. In test meal 2 these wheat chapattis were replaced by cooked rice supplying an equal amount of AvCHO. After an overnight fast of 10- 14 h, capillary blood glucose estimations were done subsequent to eating each test meal or glucose. GI of test meals was calculated by comparing their area under curve (AUCs) with AUC for glucose. GI of test meals were compared using unpaired t test. RESULTS: The study sample comprised of 7 males and 3 females with mean age 30.9 ± 5.1y. The GI of test meal 1 (85.5 ± 11.8%) and test meal 2 (83.6 ± 11.4%) was not significantly different (P = 0.7095). CONCLUSION: The present study found no differences in glycemic index of wheat chapatti and rice based mixed meals with equivalent AvCHO content of the staple.

Single-Cell Analysis of Crohn's Disease Lesions Identifies a Pathogenic Cellular Module Associated with Resistance to Anti-TNF Therapy.

Clinical benefits of cytokine blockade in ileal Crohn's disease (iCD) are limited to a subset of patients. Here, we applied single-cell technologies to iCD lesions to address whether cellular heterogeneity contributes to treatment resistance. We found that a subset of patients expressed a unique cellular module in inflamed tissues that consisted of IgG plasma cells, inflammatory mononuclear phagocytes, activated T cells, and stromal cells, which we named the GIMATS module. Analysis of ligand-receptor interaction pairs identified a distinct network connectivity that likely drives the GIMATS module. Strikingly, the GIMATS module was also present in a subset of patients in four independent iCD cohorts (n = 441), and its presence at diagnosis correlated with failure to achieve durable corticosteroid-free remission upon anti-TNF therapy. These results emphasize the limitations of current diagnostic assays and the potential for single-cell mapping tools to identify novel biomarkers of treatment response and tailored therapeutic opportunities.

Zebrafish modeling of intestinal injury, bacterial exposures and medications defines epithelial in vivo responses relevant to human inflammatory bowel disease.

Genome-wide association studies have identified over 200 genomic loci associated with inflammatory bowel disease (IBD). High-effect risk alleles define key roles for genes involved in bacterial response and innate defense. More high-throughput in vivo systems are required to rapidly evaluate therapeutic agents. We visualize, in zebrafish, the effects on epithelial barrier function and intestinal autophagy of one-course and repetitive injury. Repetitive injury induces increased mortality, impaired recovery of intestinal barrier function, failure to contain bacteria within the intestine and impaired autophagy. Prostaglandin E2 (PGE2) administration protected against injury by enhancing epithelial barrier function and limiting systemic infection. Effects of IBD therapeutic agents were defined: mesalamine showed protective features during injury, whereas 6-mercaptopurine displayed marked induction of autophagy during recovery. Given the highly conserved nature of innate defense in zebrafish, it represents an ideal model system with which to test established and new IBD therapies targeted to the epithelial barrier.This article has an associated First Person interview with the first author of the paper.

MPI depletion enhances O-GlcNAcylation of p53 and suppresses the Warburg effect.

Rapid cellular proliferation in early development and cancer depends on glucose metabolism to fuel macromolecule biosynthesis. Metabolic enzymes are presumed regulators of this glycolysis-driven metabolic program, known as the Warburg effect; however, few have been identified. We uncover a previously unappreciated role for Mannose phosphate isomerase (MPI) as a metabolic enzyme required to maintain Warburg metabolism in zebrafish embryos and in both primary and malignant mammalian cells. The functional consequences of MPI loss are striking: glycolysis is blocked and cells die. These phenotypes are caused by induction of p53 and accumulation of the glycolytic intermediate fructose 6-phosphate, leading to engagement of the hexosamine biosynthetic pathway (HBP), increased O-GlcNAcylation, and p53 stabilization. Inhibiting the HBP through genetic and chemical methods reverses p53 stabilization and rescues the Mpi-deficient phenotype. This work provides mechanistic evidence by which MPI loss induces p53, and identifies MPI as a novel regulator of p53 and Warburg metabolism.

trappc11 is required for protein glycosylation in zebrafish and humans.

Activation of the unfolded protein response (UPR) can be either adaptive or pathological. We term the pathological UPR that causes fatty liver disease a "stressed UPR." Here we investigate the mechanism of stressed UPR activation in zebrafish bearing a mutation in thetrappc11gene, which encodes a component of the transport protein particle (TRAPP) complex.trappc11mutants are characterized by secretory pathway defects, reflecting disruption of the TRAPP complex. In addition, we uncover a defect in protein glycosylation intrappc11mutants that is associated with reduced levels of lipid-linked oligosaccharides (LLOs) and compensatory up-regulation of genes in the terpenoid biosynthetic pathway that produces the LLO anchor dolichol. Treating wild-type larvae with terpenoid or LLO synthesis inhibitors phenocopies the stressed UPR seen intrappc11mutants and is synthetically lethal withtrappc11mutation. We propose that reduced LLO level causing hypoglycosylation is a mechanism of stressed UPR induction intrappc11mutants. Of importance, in human cells, depletion of TRAPPC11, but not other TRAPP components, causes protein hypoglycosylation, and lipid droplets accumulate in fibroblasts from patients with theTRAPPC11mutation. These data point to a previously unanticipated and conserved role for TRAPPC11 in LLO biosynthesis and protein glycosylation in addition to its established function in vesicle trafficking.