Dysregulation of PI3K and Hippo signaling pathways synergistically induces chronic pancreatitis via CTGF upregulation

The role of PI3K and Hippo signaling in chronic pancreatitis (CP) pathogenesis is unclear. Therefore, we assessed the involvement of these pathways in CP by examining the PI3K and Hippo signaling components PTEN and SAV1, respectively. We observed significant decreases in pancreatic PTEN and SAV1 levels in 2 murine CP models: repeated cerulein injection and pancreatic ductal ligation. Additionally, pancreas-specific deletion of Pten and Sav1 (DKO) induced CP in mice. Pancreatic connective tissue growth factor (CTGF) was markedly upregulated in both CP models and DKO mice, and pancreatic CCAAT/enhancer-binding protein-α (CEBPA) expression was downregulated in the CP models. Interestingly, in pancreatic acinar cells (PACs), CEBPA knockdown reduced PTEN and SAV1 and increased CTGF levels in vitro. Furthermore, CEBPA knockdown in PACs induced acinar-to-ductal metaplasia and activation of cocultured macrophages and pancreatic stellate cells. These results were mitigated by CTGF inhibition. CP in DKO mice was also ameliorated by Ctgf gene deletion, and cerulein-induced CP was alleviated by antibody-mediated CTGF neutralization. Finally, we observed significantly decreased PTEN, SAV1, and CEBPA and increased CTGF levels in human CP tissues compared with nonpancreatitis tissues. Taken together, our results indicate that dysregulation of PI3K and Hippo signaling induces CP via CTGF upregulation.     

DUOX2 variants associate with preclinical disturbances in microbiota-immune homeostasis and increased inflammatory bowel disease risk

A primordial gut-epithelial innate defense response is the release of hydrogen peroxide by dual NADPH oxidase (DUOX). In inflammatory bowel disease (IBD), a condition characterized by an imbalanced gut microbiota-immune homeostasis, DUOX2 isoenzyme is the highest induced gene. Performing multiomic analyses using 2872 human participants of a wellness program, we detected a substantial burden of rare protein-altering DUOX2 gene variants of unknown physiologic significance. We identified a significant association between these rare loss-of-function variants and increased plasma levels of interleukin-17C, which is induced also in mucosal biopsies of patients with IBD. DUOX2-deficient mice replicated increased IL-17C induction in the intestine, with outlier high Il17c expression linked to the mucosal expansion of specific Proteobacteria pathobionts. Integrated microbiota/host gene expression analyses in patients with IBD corroborated IL-17C as a marker for epithelial activation by gram-negative bacteria. Finally, the impact of DUOX2 variants on IL-17C induction provided a rationale for variant stratification in case control studies that substantiated DUOX2 as an IBD risk gene. Thus, our study identifies an association of deleterious DUOX2 variants with a preclinical hallmark of disturbed microbiota-immune homeostasis that appears to precede the manifestation of IBD.     

Toll-like receptor 4–mediated lymphocyte influx induces neonatal necrotizing enterocolitis

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1^–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3⁺CD4⁺IL-17⁺ and reduced tolerogenic Foxp3⁺ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.     

Micropeptide ASAP encoded by LINC00467 promotes colorectal cancer progression by directly modulating ATP synthase activity

Emerging evidence has shown that open reading frames inside long noncoding RNAs (lncRNAs) could encode micropeptides. However, their roles in cellular energy metabolism and tumor progression remain largely unknown. Here, we identified a 94 amino acid–length micropeptide encoded by lncRNA LINC00467 in colorectal cancer. We also characterized its conservation across higher mammals, localization to mitochondria, and the concerted local functions. This peptide enhanced the ATP synthase construction by interacting with the subunits α and γ (ATP5A and ATP5C), increased ATP synthase activity and mitochondrial oxygen consumption rate, and thereby promoted colorectal cancer cell proliferation. Hence, this micropeptide was termed ATP synthase–associated peptide (ASAP). Furthermore, loss of ASAP suppressed patient-derived xenograft growth with attenuated ATP synthase activity and mitochondrial ATP production. Clinically, high expression of ASAP and LINC00467 predicted poor prognosis of colorectal cancer patients. Taken together, our findings revealed a colorectal cancer–associated micropeptide as a vital player in mitochondrial metabolism and provided a therapeutic target for colorectal cancer.     

T cell protein tyrosine phosphatase protects intestinal barrier function by restricting epithelial tight junction remodeling

Genome-wide association studies revealed that loss-of-function mutations in protein tyrosine phosphatase non-receptor type 2 (PTPN2) increase the risk of developing chronic immune diseases, such as inflammatory bowel disease (IBD) and celiac disease. These conditions are associated with increased intestinal permeability as an early etiological event. The aim of this study was to examine the consequences of deficient activity of the PTPN2 gene product, T cell protein tyrosine phosphatase (TCPTP), on intestinal barrier function and tight junction organization in vivo and in vitro. Here, we demonstrate that TCPTP protected against intestinal barrier dysfunction induced by the inflammatory cytokine IFN-γ by 2 mechanisms: it maintained localization of zonula occludens 1 and occludin at apical tight junctions and restricted both expression and insertion of the cation pore-forming transmembrane protein, claudin-2, at tight junctions through upregulation of the inhibitory cysteine protease, matriptase. We also confirmed that the loss-of-function PTPN2 rs1893217 SNP was associated with increased intestinal claudin-2 expression in patients with IBD. Moreover, elevated claudin-2 levels and paracellular electrolyte flux in TCPTP-deficient intestinal epithelial cells were normalized by recombinant matriptase. Our findings uncover distinct and critical roles for epithelial TCPTP in preserving intestinal barrier integrity, thereby proposing a mechanism by which PTPN2 mutations contribute to IBD.     

Dysregulation of the NRG1/ERBB pathway causes a developmental disorder with gastrointestinal dysmotility in humans

Hirschsprung disease (HSCR) is the most frequent developmental anomaly of the enteric nervous system, with an incidence of 1 in 5000 live births. Chronic intestinal pseudo-obstruction (CIPO) is less frequent and classified as neurogenic or myogenic. Isolated HSCR has an oligogenic inheritance with RET as the major disease-causing gene, while CIPO is genetically heterogeneous, caused by mutations in smooth muscle–specific genes. Here, we describe a series of patients with developmental disorders including gastrointestinal dysmotility, and investigate the underlying molecular bases. Trio-exome sequencing led to the identification of biallelic variants in ERBB3 and ERBB2 in 8 individuals variably associating HSCR, CIPO, peripheral neuropathy, and arthrogryposis. Thorough gut histology revealed aganglionosis, hypoganglionosis, and intestinal smooth muscle abnormalities. The cell type–specific ErbB3 and ErbB2 function was further analyzed in mouse single-cell RNA sequencing data and in a conditional ErbB3-deficient mouse model, revealing a primary role for ERBB3 in enteric progenitors. The consequences of the identified variants were evaluated using quantitative real-time PCR (RT-qPCR) on patient-derived fibroblasts or immunoblot assays on Neuro-2a cells overexpressing WT or mutant proteins, revealing either decreased expression or altered phosphorylation of the mutant receptors. Our results demonstrate that dysregulation of ERBB3 or ERBB2 leads to a broad spectrum of developmental anomalies, including intestinal dysmotility.     

Mitochondrial dysfunction in inflammatory bowel disease alters intestinal epithelial metabolism of hepatic acylcarnitines

As the interface between the gut microbiota and the mucosal immune system, there has been great interest in the maintenance of colonic epithelial integrity through mitochondrial oxidation of butyrate, a short-chain fatty acid produced by the gut microbiota. Herein, we showed that the intestinal epithelium could also oxidize long-chain fatty acids, and that luminally delivered acylcarnitines in bile could be consumed via apical absorption by the intestinal epithelium, resulting in mitochondrial oxidation. Finally, intestinal inflammation led to mitochondrial dysfunction in the apical domain of the surface epithelium that may reduce the consumption of fatty acids, contributing to higher concentrations of fecal acylcarnitines in murine Citrobacter rodentium–induced colitis and human inflammatory bowel disease. These results emphasized the importance of both the gut microbiota and the liver in the delivery of energy substrates for mitochondrial metabolism by the intestinal epithelium.     

Protein tyrosine phosphatase nonreceptor type 2 controls colorectal cancer development

Protein tyrosine phosphatase nonreceptor type 2 (PTPN2) recently emerged as a promising cancer immunotherapy target. We set out to investigate the functional role of PTPN2 in the pathogenesis of human colorectal carcinoma (CRC), as its role in immune-silent solid tumors is poorly understood. We demonstrate that in human CRC, increased PTPN2 expression and activity correlated with disease progression and decreased immune responses in tumor tissues. In particular, stage II and III tumors displayed enhanced PTPN2 protein expression in tumor-infiltrating T cells, and increased PTPN2 levels negatively correlated with expression of PD-1, CTLA4, STAT1, and granzyme A. In vivo, T cell– and DC-specific PTPN2 deletion reduced tumor burden in several CRC models by promoting CD44⁺ effector/memory T cells, as well as CD8⁺ T cell infiltration and cytotoxicity in the tumor. In direct relevance to CRC treatment, T cell–specific PTPN2 deletion potentiated anti–PD-1 efficacy and induced antitumor memory formation upon tumor rechallenge in vivo. Our data suggest a role for PTPN2 in suppressing antitumor immunity and promoting tumor development in patients with CRC. Our in vivo results identify PTPN2 as a key player in controlling the immunogenicity of CRC, with the strong potential to be exploited for cancer immunotherapy.