BACKGROUND & AIMS:Tumor necrosis factor (TNF) is a key driver of intestinal epithelial inflammation. The baculoviral inhibitor of apoptosis protein repeat-containing 3 (BIRC3) gene encodes the cellular inhibitor of apoptosis protein 2 (cIAP2), a known regulator of TNF signaling. Although genetic variants in components of the TNF signaling pathway have been reported, no human BIRC3 variants have been previously identified. METHODS:We screened exomes obtained from Crohn's disease (CD) patients from multiple centers for BIRC3 variants. We used cellular, mouse organoids, induced pluripotent stem cells-derived intestinal organoids, knock-in and knockout mouse models, and knockout zebrafish, as well as transcriptome analysis of various samples to determine pathogenicity of BIRC3 variants. RESULTS:Rare and damaging BIRC3 variants were identified in 14 patients from 10 unrelated families with CD diagnosed between infancy and adulthood. Functional studies showed that BIRC3 deficiency caused impaired receptor-interacting protein kinase 1 (RIPK1) ubiquitylation, leading to RIPK1 autophosphorylation resulting in increased epithelial cell death. The p.H312Y cIAP2 variant identified in both our index and in another independent patient was mislocalizaed, and a knock-in mouse model of this BIRC3 variant (cIAP2H312Y/+) had exacerbation of chemically induced colitis, whereas ciap1-/+ zebrafish developed spontaneous colitis. Transcriptome analysis of mice organoids and zebrafish showed that BIRC3 deficiency led to inappropriate sustained activation of TNF-responsiveness genes in the absence of stimuli. Small molecule pharmacologic inhibition of RIPK1 or caspases attenuated intestinal inflammation in BIRC3-deficient intestinal organoids and cIAP2H312Y/+ mice. CONCLUSIONS:We establish BIRC3 deficiency as a cause of monogenic CD in both pediatric- and adult-onset patients and identify RIPK1 as a therapeutic target.

BACKGROUND:Siblings of individuals with Crohn's disease (CD) are at increased risk for developing CD but the underlying mechanisms remain unclear. OBJECTIVE:We hypothesised that childhood (vs adult) exposure to a sibling with CD drives microbial perturbations, increasing CD susceptibility. DESIGN:We used the prospective Genetic Environmental Microbial (GEM) Project and the nationwide South Korean database to assess the association between childhood exposure to affected siblings and CD onset. The association between childhood exposure and gut microbiome was evaluated in the GEM cohort. A T-cell transfer model of colitis in germ-free mice was conducted to investigate the effects of stool from childhood-exposed versus adult-exposed siblings. Finally, an integrative risk model combining faecal calprotectin (FCP) and microbial enterotypes was trained and internally validated within the GEM cohort. RESULTS:In the GEM cohort, childhood exposure to an affected sibling was associated with a fourfold higher risk of developing CD compared with adult exposure (adjusted HR (aHR) (95% CI) 4.00 (1.83 to 8.75); p=5.3×10-4), which was validated in the South Korean dataset (aHR (95%CI) 2.54 (1.70 to 3.81; p=6.0×10-6)). Childhood exposure was associated with reduced abundances of Lachnospira, Roseburia and Colidextribacter, reductions that mediation analysis identified as partially mediating CD risk. In vivo, transplantation of childhood exposure-associated microbiota into germ-free recipient mice exacerbated colitis and elevated mucosal interleukin-22 expression, supporting a potential mechanistic role. Our model identified siblings with elevated FCP and Blautia-enriched or Prevotella-enriched enterotypes as highest risk, with a 10-year cumulative incidence of 22.5%. CONCLUSION:Childhood exposure to siblings with CD is an independent risk factor of CD onset, potentially mediated by early-life microbial perturbation.

We describe biochemically and functionally validated primary bile acid malabsorption caused by novel biallelic SLC10A2 variants in a child initially diagnosed with Crohn's disease. Pediatric IBD cohort reanalysis identified PBAM-compatible genotypes, supporting selective testing when clinical features are suggestive.

Inflammatory bowel disease (IBD) causes chronic suffering from gastrointestinal inflammation and dysfunction that can progress to colon cancer1,2. The prevalence of the disease is increasing, and there is an urgent need to better understand its pathogenic mechanisms to improve treatment. We show that GPR15-a G-protein-coupled receptor expressed in immune cells and described previously as an entry co-factor for human and simian immunodeficiency viruses3-is a marker and homing receptor for a subset of intramucosal GPR15-guided regulatory CD8+ T lymphocytes (CD8+ TIGR cells). Deleterious GPR15 gene variants in humans cause defective homing of CD8+ TIGR cells and are associated with severe early-onset IBD. Moreover, CD8+ TIGR cells are reduced in the intestinal mucosa of individuals with sporadic IBD. In mice, GPR15 deficiency impairs colonic homing of CD8+ TIGR cells, leading to accumulation of inflammatory macrophages and increased susceptibility to colitis. CD8+ TIGR cells potently kill macrophages activated by intestinal damage or disease using Fas ligand and TNF-related weak inducer of apoptosis (TWEAK). The identification of CD8+ TIGR cells yields new insights into organ-specific immune regulation and potential therapeutics for IBD.

Infants and children with primary and secondary immune deficiency often present with gastrointestinal symptoms, including diarrhea, abdominal pain, failure to thrive, intestinal infection, malabsorption, and intestinal inflammation. In addition, certain immune deficiencies predispose patients to gastrointestinal and hepatic malignancy. This chapter reviews the development of the mucosal immune system, the pathogenesis of the immune response and intestinal inflammation, and the gastrointestinal manifestations of immune deficiency. In addition, monogenic defects that result in very early onset inflammatory bowel disease are discussed in detail.

Crohn’s disease is an inflammatory bowel disease (IBD) commonly treated through anti-TNF blockade. However, most patients still relapse and inevitably progress. Comprehensive single-cell RNA-sequencing (scRNA-seq) atlases have largely sampled patients with established treatment-refractory IBD, limiting our understanding of which cell types, subsets, and states at diagnosis anticipate disease severity and response to treatment. Here, through combining clinical, flow cytometry, histology, and scRNA-seq methods, we profile diagnostic human biopsies from the terminal ileum of treatment-naïve pediatric patients with Crohn’s disease (pediCD; n=14), matched repeat biopsies (pediCD-treated; n=8) and from non-inflamed pediatric controls with functional gastrointestinal disorders (FGID; n=13). To resolve and annotate epithelial, stromal, and immune cell states among the 201,883 baseline single-cell transcriptomes, we develop a principled and unbiased tiered clustering approach, ARBOL. Through flow cytometry and scRNA-seq, we observe that treatment-naïve pediCD and FGID have similar broad cell type composition. However, through high-resolution scRNA-seq analysis and microscopy, we identify significant differences in cell subsets and states that arise during pediCD relative to FGID. By closely linking our scRNA-seq analysis with clinical meta-data, we resolve a vector of T cell, innate lymphocyte, myeloid, and epithelial cell states in treatment-naïve pediCD (pediCD-TIME) samples which can distinguish patients along the trajectory of disease severity and anti-TNF response. By using ARBOL with integration, we position repeat on-treatment biopsies from our patients between treatment-naïve pediCD and on-treatment adult CD. We identify that anti-TNF treatment pushes the pediatric cellular ecosystem towards an adult, more treatment-refractory state. Our study jointly leverages a treatment-naïve cohort, high-resolution principled scRNA-seq data analysis, and clinical outcomes to understand which baseline cell states may predict Crohn’s disease trajectory.

Inflammatory bowel disease (IBD) burden is rising globally, yet only subsets of patients benefit from available therapies, underscoring the need for more precise molecular and cellular stratification. In the PREDICT study, we enrolled treatment-naïve pediatric patients with IBD, alongside disorders of gut-brain interaction (DGBI) controls and healthy donors, and profiled their intestinal and blood-derived T cells using single-cell RNA sequencing (scRNA-seq). Across 107 participants, we identify a unique population of cytotoxic CD4+ T cells (CD4 CTL) enriched in the inflamed gut of patients with Crohn's disease (CD) and ulcerative colitis. CD4 CTLs are clonally expanded and express cytotoxic effector molecules and IFNG, consistent with antigen-driven activation. Cell-cell interaction analyses implicate macrophage-derived IL-27 as the top candidate for CD4 CTL differentiation, and IL-27 blockade in a mouse model limits CD4 CTL formation. Notably, elevated CD4 CTL frequencies in gut and peripheral blood at diagnosis are associated with subsequent poor outcome of anti-TNF therapy in pediatric CD. Findings in our identification cohort are validated in an independent cohort and through reanalysis of published datasets. Importantly, we designed a simple flow cytometry panel to isolate blood CD4+ CXCR6+ CD27- T cells, which displayed a CD4 CTL transcriptional phenotype. Together, our results link CD4 CTLs to anti-TNF nonresponse and support their potential as an early, blood-accessible biomarker for treatment stratification in pediatric CD.

Group 3 innate lymphoid cells (ILC3s) are key sensors of the intestinal environment, integrating dietary and microbial cues to maintain intestinal immunity. We found that intestinal ILC3s were reduced in overweight and obese humans and in high-fat diet (HFD)-fed mice. ILC3 loss occurred independently of caloric excess, weight gain, or glucose intolerance. Instead, impairment arose within hours of HFD consumption and was initiated by microbiota-driven intestinal barrier permeability and concomitant activation of inflammatory mononuclear phagocytes (MNPs). This response to inflammation impaired fatty acid oxidation in lipid-loaded ILC3s, resulting in mitochondrial damage and cell death. Intestinal ILC3 cell death was rescued by removal of excess fats from the diet. ILC3s from individuals with obesity also exhibited impaired fatty acid oxidation. Together, our findings define a malleable mechanism whereby dietary fats and microbial cues drive ILC3 maladaptation and death, with consequences for intestinal homeostasis.

Objectives:Monogenic causes of congenital diarrheas and enteropathies (CoDE) and very early onset inflammatory bowel disease (VEOIBD) are mostly recessive and therefore more prevalent in populations with increased consanguinity rates. To assess the genetic basis of these disorders in a likely high-prevalence population, we established a multi-center cohort of patients across the Middle East. Methods:Patients were enrolled across four centers in the Middle East. Clinical data, including self-reported consanguinity, were collected. Trio whole exome sequencing (WES) was performed, and genomic data were processed through a standardized variant identification pipeline. Patients who inherited a rare variant consistent with the disease in a gene known to be causative for CoDE or VEOIBD were classified as having a monogenic etiology. Self-reported consanguinity and homozygosity mapping were analyzed and correlated with monogenic diagnoses. Results:WES from trios of 27 patients (13 CoDE and 14 VEOIBD) were analyzed. Higher frequency of consanguinity was self-reported by the CoDE families (11/13, 84.6%) and confirmed by homozygosity mapping than in the VEOIBD group (4/14, 28.6%). A monogenic cause of disease was identified in 10 of 13 CoDE patients (76.9%) in the following genes: EPCAM, SPINT2, DGAT1, MYO5B, SPINT2, and STXBP2. A single VEOIBD patient (7.1%) had an identifiable monogenic cause of disease in SKIV2L. Conclusions:Within this limited cohort, the diagnostic yield for known genes associated with CoDE was high, consistent with the primarily monogenic etiology and high consanguinity. For VEOIBD, the diagnostic yield was lower and similar to that reported in North America.

BACKGROUND:Interleukin-10 (IL-10) is an essential regulator of intestinal immune homeostasis. Neutralising autoantibodies against IL-10 (anti-IL-10) have been identified in children and also adult patients with IBD. Positivity for anti-IL-10 autoantibodies was associated with carriage of HLA-DRB1*01:03 allele. OBJECTIVE:To determine the prevalence of anti-IL-10 in paediatric IBD and assess the associated clinical phenotype. DESIGN:We conducted a cross-sectional multicentre study across paediatric IBD cohorts from four countries. Anti-IL-10 antibodies were investigated in serum and plasma from paediatric patients with IBD (mean age of IBD onset 11.2±3.8 years). IL-10-neutralisation capacity was confirmed by functional IL-10 reporter assay, competitive ELISA and cytokine release assay. Clinical data were analysed to evaluate disease phenotype and treatment outcomes, with comparison with matched controls. HLA-DRB1*01:03 analysis was performed. RESULTS:Anti-IL-10 positivity was identified in 26/1045 paediatric patients with IBD (2.5%) (Crohn's disease n=6, UC n=19, IBD unclassified n=1; IBD diagnosis age of 13±3 years). Anti-IL-10 autoantibodies were of the IgG class and amplified pro-inflammatory cytokine responses in vitro. Anti-IL-10-positive patients exhibited more severe disease compared with matched controls, including an increased prevalence of difficult-to-treat disease (23% vs 6%, p=0.03), higher rate of acute severe UC (26% vs 6%; p=0.038) and higher rates of colectomy (27% vs 6%, p=0.01). Eighty percent (16/20) of anti-IL-10-positive patients with available HLA data carried the HLA-DRB1*01:03 allele, compared with 1.5% in the anti-IL-10-negative group. CONCLUSION:Anti-IL-10 autoantibodies are present in a subgroup of paediatric patients with IBD and are associated with difficult-to-treat disease.

EP300 encodes p300, a histone acetyltransferase and essential regulator of chromatin accessibility and transcription. We describe a novel human mutation in EP300 causing very early onset IBD (VEOIBD)-like disease, and employ patient-derived organoids to characterize its effects on the intestinal epithelium and validate a precision therapy. A 20-month-old female presented with secretory diarrhea and hypovolemic shock, necessitating prolonged intensive care and parenteral nutrition use. Intestinal histology exhibited striking abnormalities, with severe chronic mucosal injury in the colon, and foveolar metaplasia, with abnormal mucosal architecture in the duodenum, implicating defects in epithelial cell differentiation and identity. Whole genome sequencing revealed a novel, heterozygous truncating variant in EP300, which in combination with hallmark syndromic features, confirmed Rubinstein-Taybi syndrome (RTS). Gastrointestinal inflammation or metaplasia have not previously been described in RTS, but two additional subsequently identified cases displayed similar features. We therefore hypothesized that partial loss of p300 function induces decreased chromatin acetylation, leading to impaired cellular differentiation and abnormal epithelial maturation. Analysis of patient duodenoids from the index EP300 variant showed severely impaired viability, abnormal morphology, and reduced growth. Western blot revealed reduced histone H3 acetylation, consistent with defective p300 activity. Transcriptional analysis showed aberrant upregulation of Pepsinogen and reduced Epcam, Alpi, and Neurog3, suggestive of abnormal ectopic lineage differentiation. We hypothesized that treatment with the histone deacetylase inhibitor (HDACi) valproic acid (VPA) would compensate for reduced EP300 function by favoring a state of histone acetylation. VPA-treated EP300 variant duodenoids exhibited increased viability and H3 acetylation and normalization of morphology. VPA also modulated cell differentiation with reduction in pepsinogen expression. We identified EP300 as a novel monogenic cause of VEOIBD/congenital enteropathy. p300 loss of function leads to significant epithelial disorganization, gastrointestinal metaplasia/dysplasia and altered cell identity in intestinal tissue and enteroids. In vitro application of VPA resulted in significant improvement in epithelial viability and a reduction in ectopic lineage differentiation, supporting its use as precision therapy for our EP300 patient. These results suggest that EP300 function is critical for defining intestinal epithelial differentiation programs and cellular identity. Our work supports a precision approach for epigenetic therapies in genetically defined GI disease and highlights the translational utility of organoids to guide individualized clinical care.

BACKGROUND:Pediatric Inflammatory Bowel Disease (IBD) is a chronic condition characterized by persistent intestinal inflammation in children. It often presents with distinct clinical phenotypes and is more frequently linked to rare monogenic variants affecting epithelial barrier function or mucosal immunity. Although over 100 genes are associated with monogenic IBD, their roles in the intestinal epithelium remain poorly defined. This study aimed to improve our understanding of epithelial dysfunction in early-onset IBD through molecular and cellular analyses to uncover patient-specific phenotypes and potential therapeutic targets. METHODS:We generated intestinal epithelial organoids (IEOs) from 94 pediatric IBD patients, including those with monogenic variants (BTK, TTC7A, IL10RA, LRBA, STXBP2, TRNT1, SKIV2L), along with 46 non-IBD controls. RNA sequencing was performed on 38 patient and 20 control lines, under both baseline conditions and after immunological stimulation, yielding a valuable dataset for studying epithelial responses in IBD. RESULTS:IEOs effectively initiated inflammation upon bacterial lysate stimulation, regardless of disease status, origin, or genotype. Inflammatory stimulation triggered upregulation of IBD-linked genes SERPINA1 and LIFR in IBD organoids, suggesting their role in epithelial innate immunity. However, network analysis showed no consistent transcriptional signatures across all IBD cases. Instead, specific genotypes (TTC7A, STXBP2, LRBA) revealed responses, with STXBP2 and LRBA showing shared upregulation of IL-1 and SLC30-mediated zinc trafficking pathways. CONCLUSIONS:These findings underscore the potential of IEOs as a valuable model for studying IBD and offer key insights that could guide the development of targeted therapies for both monogenic and non-monogenic forms of IBD.

Skeletal muscle regeneration is a multistep process involving the activation, proliferation, differentiation, and fusion of muscle stem cells, known as satellite cells. Fusion of satellite cell-derived myoblasts (SCMs) is indispensable for generating the multinucleated, contractile myofibers during muscle repair. However, the molecular and cellular mechanisms underlying SCM fusion during muscle regeneration remain incompletely understood. Here, we reveal a critical role for branched actin polymerization in SCM fusion during mouse skeletal muscle regeneration. Using conditional knockouts of the Arp2/3 complex and its actin nucleation-promoting factors N-WASP and WAVE, we demonstrate that branched actin polymerization is specifically required for SCM fusion but dispensable for satellite cell proliferation, differentiation, and migration. We show that the N-WASP and WAVE complexes have partially redundant functions in regulating SCM fusion and that branched actin polymerization is essential for generating invasive protrusions at fusogenic synapses in SCMs. Together, our study identifies branched-actin regulators as key components of the myoblast fusion machinery and establishes invasive protrusion formation as a critical mechanism enabling myoblast fusion during skeletal muscle regeneration.