Cellular and Molecular Gastroenterology and Hepatology最新文献

Background & aims: Stress granules (SGs) represent membrane-free cytoplasmic structures rapidly aggregating during cellular stress responses arguably useful as markers of molecular inflammation. To provide an automated, reproducible, and unbiased analytic workflow, we used the open-source software CellProfiler to quantify SGs in distinct cell types in inflammatory bowel disease.

Methods: The EpiCellProfiler (ECP) and PropiCellProfiler (PCP) pipelines enable segmentation within intestinal epithelial cells and lamina propria cells, respectively. The SG marker Ras GTPase-activating protein-binding protein 1 (G3BP1) was quantified for fluorescence intensity, granule size, and morphology on tissue sections of patients with ulcerative colitis (UC) and Crohn's disease (CD) in deep remission.

Results: Both pipelines detected elevated G3BP1 fluorescence intensities in inactive UC and CD. Additionally, SGs spot counts and spot sizes were increased in CD and UC compared with controls. The distribution of G3BP1 was homogenous in intestinal epithelial cells, without SG typical aggregations. In UC, PCP analysis revealed nuclear morphology alterations in terms of size, regularity, and compactness.

Conclusions: Herein, we provide a powerful, reproducible, versatile and open-source software tool to quantify remnant molecular inflammation in patients with CD and UC, enabling research to openly share, reproduce and compare results within the field of quantitative image analysis. Our pipeline separates and distinguishes between epithelial and lamina propria events and provides insights into the spatial distribution and dynamics of SGs, revealing their homogeneous distribution and persistent accumulation in patients with CD and UC, notably in such without clinical, endoscopic, biochemical and histological disease activity. The sensitivity of the pipelines allows detection of subtle morphologic alterations that warrant further investigation, as does the usage of G3BP1 as an inflammatory bowel disease stress marker.

Background & aims: Hepatic ischemia-reperfusion injury (HIRI) is one of the common complications of liver transplantation. Rhomboid 5 homolog 2 (Rhbdf2) plays a crucial role in apoptosis, inflammation, and liver injury, but its role and regulatory mechanism in HIRI remain unclear. The aim of this study was to investigate the role of Rhbdf2 in HIRI and elucidate its molecular mechanism.

Methods: Rhbdf2 expression levels were detected in pre-ischemia-reperfusion (Pre) and post-ischemia-reperfusion (Post) livers. Western blot analysis, flow cytometry, quantitative real-time polymerase chain reaction, and immunofluorescence staining were used to investigate the effects of Rhbdf2 on hepatic ischemia-reperfusion (HI/R). The potential molecular mechanisms of the effects of Rhbdf2 on HI/R were investigated by combining RNA sequencing and mass spectrometry analysis, as well as co-immunoprecipitation and in vitro ubiquitination assays.

Results: The level of Rhbdf2 protein was significantly increased in HI/R. Overexpression of Rhbdf2 in mice exacerbated HI/R-induced liver injury, apoptosis, and the inflammatory response, whereas knockdown of Rhbdf2 produced the opposite results. Mechanistically, overexpression of Rhbdf2 promoted the phosphorylation of mitogen-activated protein kinase kinase kinase 7 (MAP3K7, also known as Tak1), thereby activating the JNK/p38 signaling pathway and ultimately exacerbating HIRI. Mass spectrometry analysis, co-immunoprecipitation, and in vitro ubiquitination assays revealed that the E3 ubiquitin ligase constitutive photomorphogenic 1 (Cop1) interacts with Rhbdf2 and mediates its degradation through K48-linked ubiquitination, thereby inhibiting the TAK1- JNK/p38 axis and reducing HIRI.

Conclusions: This study revealed that Rhbdf2 exacerbates HIRI by activating the TAK1- JNK/p38 axis, whereas Cop1-mediated Rhbdf2 ubiquitination and degradation can significantly inhibit this process. These findings provide potential therapeutic targets and insights for the clinical treatment of HIRI.

Background & aims: Duodenal adenomas have malignant potential, yet the drivers of duodenal tumorigenesis remain unclear. Duodenal adenomas robustly develop in villin- Toll-like receptor 4 (TLR4) mice, a transgenic mouse model of increased innate immune signaling in the intestinal epithelium. Here, we sought to test the contributions of the microbiota and bile acids to duodenal adenoma development.

Methods: Duodenal tissue was analyzed for proliferation rate and histology in villin-TLR4 vs wild-type mice. Mice were rederived into germ-free conditions and administered a diet containing the bile acid sequestering resin cholestyramine or treated with the NADPH oxidase inhibitor apocynin. Chemokine expression and myeloid cell recruitment were measured. Findings from mouse studies were corroborated by RNA sequencing and tissue microarray analyses of human duodenal adenomas.

Results: Constitutive activation of epithelial TLR signaling in the duodenum led to adenomas with an intestinal phenotype. Non-adenomatous duodenal tissue showed increased expression of Cxcl1 and Cxcl2 by intestinal epithelial cells and recruitment of S100A8⁺ and myeloperoxidase⁺ myeloid cells. Re-deriving villin-TLR4 mice in germ-free conditions or feeding them a cholestyramine-supplemented diet prevented tumor initiation, epithelial expression of CXCR2 ligands, and myeloid cell recruitment. Apocynin supplementation slowed tumor progression without affecting chemokine expression or myeloid cell recruitment. In humans, duodenal adenomas had enriched neutrophil activation pathways, increased chemokine expression, and infiltration of S100A8⁺ and myeloperoxidase⁺ myeloid cells.

Conclusions: Bile acids and the microbiota are necessary for duodenal adenoma development and are potentially modifiable risk factors in humans at risk of duodenal adenomas. The recruitment of myeloid cells may promote tumor progression via the release of reactive oxygen species.

Background & aims: Fibrostenosis is a major complication of Crohn's disease (CD) characterized by intestinal remodeling and excessive extracellular matrix (ECM) deposition. A prominent feature is bowel wall muscularization, involving expansion of submucosal myoid cells and muscularis propria smooth muscle cell (SMC) hyperplasia. However, the cellular identity and molecular mechanisms underlying submucosal myoid cell hyperplasia remain poorly characterized.

Methods: Preoperative intestinal ultrasound from 117 patients with CD was retrospectively reviewed, and ileal tissues from 25 normal, 35 nonstenotic CD, and 44 stenotic CD cases were analyzed histologically. High-resolution spatial transcriptomics was applied to 1 nonfibrotic and 1 fibrostenotic ileal specimen with marked submucosal myoid cell expansion. Findings were validated using public single-cell RNA sequencing datasets (n = 158), immunofluorescence, primary pericyte cultures, and quantitative polymerase chain reaction.

Results: Submucosal myoid cells were predominantly identified as high ECM-producing myofibroblasts, possibly representing the dominant stromal population expanded in fibrotic submucosa. Spatial and pseudotemporal analyses demonstrated their origin from muscularis mucosae and submucosal vascular SMCs. Additionally, pericytes underwent significant expansion and transcriptional reprogramming toward a myofibroblast-like phenotype. Fibroblast sub-clustering revealed spatial heterogeneity, with FAP⁺ fibroblasts enriched specifically in fibrotic regions. Inflammatory monocytes colocalized with stromal cells, exhibiting robust predicted ligand-receptor interactions indicative of immune-stromal crosstalk.

Conclusions: This case-level, high-resolution spatial analysis delineates a spatially organized fibrotic niche within a CD stricture, composed of distinct stromal and immune populations. We define the identity and origins of profibrotic myofibroblasts and characterize pericyte-to-myofibroblast reprogramming, thereby highlighting specific cell subtypes as prime therapeutic targets for antifibrotic strategies.

Background & aims: Interleukin (IL)-1β is a key cytokine in hepatitis-related inflammation, but its role in metabolic dysfunction-associated steatotic liver disease (MASLD) or steatohepatitis (MASH) remains unclear. This study investigated IL-1β-mediated interactions in nonparenchymal liver cells to elucidate their contributions to pathological MASH progression.

Methods: We used the THP-1 monocyte-derived macrophage line and TMNK-1 liver sinusoidal endothelial cell (LSEC) line for in vitro assays. Endothelial cell-specific Il1r1-knockout (Il1r1^ΔEC) and systemic Cxcl10-knockout (Cxcl10^-/-) mice were subjected to a Western diet (WD) to establish a MASH model. An additional WD-fed cohort received the IL1R1 antagonist anakinra during the final 4 weeks. RNA sequencing data from liver tissues from patients with MASLD and spatial transcriptomic analyses focusing on nontumor regions of MASH-related hepatocellular carcinoma samples were evaluated.

Results: Liver levels of mature IL-1β were elevated in WD-fed mice compared with ND-fed mice. Il1r1 was highly expressed in LSECs, and Ccl2 and Cxcl10 expression were upregulated in LSECs under WD conditions. Palmitic acid inhibited autophagy in THP-1 macrophages, increasing IL-1β secretion. IL-1β enhanced CCL2 and CXCL10 expression in TMNK-1 LSECs via JNK activation. In Il1r1^ΔEC and Cxcl10^-/- mice, WD-induced inflammatory cell infiltration and fibrosis were attenuated, and anakinra produced similar effects. In human datasets, CCL2 and CXCL10 were upregulated in MASH livers and correlated with NAFLD activity scores. Spatial transcriptomics revealed a dominant periportal macrophage-to-LSEC IL1B-IL1R1 interaction that generates chemokine-enriched LSECs, forming inflammatory-fibrotic niches that facilitate immune cell recruitment.

Conclusions: Macrophage-derived IL-1β promotes hepatic inflammation and fibrosis through IL1R1-dependent chemokine induction in LSECs, highlighting IL1R1 signaling as a therapeutic target in MASH.