Cellular and Molecular Gastroenterology and Hepatology最新文献

Background and aims: Gastrointestinal motility persists when peripheral cholinergic signaling is blocked genetically or pharmacologically, and a recent study suggests nitric oxide drives propagating neurogenic contractions.

Methods: To determine the neuronal substrates that underlie these contractions, we measured contractile-associated movements together with calcium responses of cholinergic or nitrergic myenteric neurons in un-paralyzed ex vivo preparations of whole mouse colon. We chose to look at these two subpopulations because they encompass nearly all myenteric neurons.

Results: Many but not all cholinergic neurons of the middle colon exhibited contractile-associated calcium responses with distinct features. By contrast, a large population of nitrergic neurons of the middle colon shut their activity off just before contraction onset, whereas another population of nitrergic neurons initiated a response just after contraction onset. When contractions were evoked by a variety of stimuli to the proximal and distal colon, the same neuronal subtypes exhibited the same activity patterns during the contraction. However, stimulation of proximal colon produced a transient, stimulation-locked response before the ensuing contraction in a subpopulation of cholinergic neurons and in nearly all nitrergic neurons, suggesting that distinct neuronal activity patterns underlie specific stimuli. Finally, although blockade of nitric oxide failed to arrest the generation or propagation of neurogenic contractions, chemogenetic elimination of nitrergic activity impaired their propagation to middle and distal colon.

Conclusions: Genetic approaches were used to study the activity patterns of enteric neurons underlying spontaneous and evoked neurogenic contractions in un-paralyzed colon. These approaches can be combined with a variety of other approaches to identify the neuronal subtypes and subclasses that coordinate colonic motility.

Background & aims: The incidence of graft fibrosis is elevated after pediatric liver transplantation (pLT) and is influenced by cold ischemic time (CIT). Myosin light chain 9 (MYL9), a member of the myosin family, could act on hepatic stellate cells (HSCs) and induce a transition to active phase. We hypothesized that cold ischemic injury could stimulate MYL9 expression and lead to graft fibrosis.

Methods: We tested the hypothesis by analyzing multi-omics data from human protocol liver biopsy samples 2 years after LT, performing rat LT with different CIT and conducting in vitro studies in HSC cell lines with MYL9 knockdown and overexpression.

Results: Clinically, CIT is an independent risk factor for graft fibrosis after pLT. Omics analysis identified MYL9 as a prominent contributor in graft fibrosis. MYL9 is strongly correlated with liver fibrosis grade and the progression of fibrosis. The study of rat LT model demonstrated MYL9 expression increases with the prolongation of CIT, and its role is specific to transplant setting. Mechanistically, in vitro experiments with HSCs exposed to hypoxia/reoxygenation revealed a substantial decrease in HSCs activation after MYL9 knockdown. Conversely, overexpression of MYL9 significantly enhanced the activation of HSCs. Subsequent transcriptome sequencing of HSCs with MYL9 knockdown unveiled that MYL9 primarily functions through the TLR4/MYD88/NF-κB signaling pathway. Liver graft fibrosis was ameliorated when toll like receptor 4 signaling was inhibited in rats.

Conclusions: Our findings demonstrate that prolonged CIT up-regulates the expression of MYL9 in liver graft after LT. MYL9 activates HSCs and promotes fibrosis through a TLR4/MYD88/NF-κB signaling dependent manner.

Background & aims: Hepatocellular carcinoma (HCC) frequently undergoes regional chromosomal amplification, resulting in elevated gene expression levels. We aimed to elucidate the role of these poorly understood genetic changes by using CRISPR activation (CRISPRa) screening in mouse livers to identify which genes within these amplified loci are cancer driver genes.

Methods: We used data from The Cancer Genome Atlas to identify that frequently copy number-amplified and up-regulated genes all reside on human chromosomes 1q and 8q. We generated CRISPRa screening transposons that contain oncogenic Myc to drive tumor formation. We conducted CRISPRa screens in vivo in the liver to identify tumor driver genes. We extensively validated the findings in separate mice and performed RNA sequencing analysis to explore mechanisms driving tumorigenesis.

Results: We targeted genes that frequently undergo amplification in human HCC using an in vivo CRISPRa screening system in mice, which induced extensive liver tumorigenesis. Human chromosome 1q genes Zbtb7b, Vps72, Gba1, and Mrpl9 emerged as drivers of liver tumorigenesis. In human HCC there is a trend in correlation between levels of MRPL9, VPS72, or GBA1 and poor survival. In validation assays, activation of Vps72, Gba1, or Mrpl9 resulted in extensive liver tumorigenesis and decreased survival in mice. RNA sequencing revealed different mechanisms driving HCC, with Mrpl9 activation altering genes functionally related to mitochondrial function, Vps72 levels altering phospholipid metabolism, and Gba1 activation enhancing endosomal-lysosomal activity, all leading to promotion of cellular proliferation. Analysis of human tumor tissues with high levels of MRPL9, VPS72, or GBA1 revealed congruent results, indicating conserved mechanisms driving HCC.

Conclusions: This study reveals chromosome 1q genes Vps72, Gba1, and Mrpl9 as drivers of HCC. Future efforts to prevent or treat HCC can focus on these new driver genes.

Background & aims: Coronavirus disease (COVID-19), caused by severe acquired respiratory syndrome-Coronavirus-2 (SARS-CoV-2), triggered a global pandemic with severe medical and socioeconomic consequences. Although fatality rates are higher among the elderly and those with underlying comorbidities, host factors that promote susceptibility to SARS-CoV-2 infection and severe disease are poorly understood. Although individuals with certain autoimmune/inflammatory disorders show increased susceptibility to viral infections, there is incomplete knowledge of SARS-CoV-2 susceptibility in these diseases. The aim of our study was to investigate whether the autoimmunity risk gene, PTPN2, which also confers elevated risk to develop inflammatory bowel disease, affects susceptibility to SARS-CoV-2 viral uptake.

Methods: Using samples from PTPN2 genotyped patients with inflammatory bowel disease, PTPN2-deficient mice, and human intestinal and lung epithelial cell lines, we investigated how PTPN2 affects expression of the SARS-CoV-2 receptor angiotensin converting enzyme 2 (ACE2), and uptake of virus-like particles expressing the SARS-CoV2 spike protein and live SARS-CoV-2 virus.

Results: We report that the autoimmune PTPN2 loss-of-function risk variant rs1893217 promotes expression of the SARS-CoV-2 receptor, ACE2, and increases cellular entry of SARS-CoV-2 spike protein and live virus. Elevated ACE2 expression and viral entry were mediated by increased Janus kinase-signal transducers and activators of transcription signaling and were reversed by the Janus kinase inhibitor, tofacitinib.

Conclusion: Collectively, our findings uncover a novel risk biomarker for increased expression of the SARS-CoV-2 receptor and viral entry and identify a clinically approved therapeutic agent to mitigate this risk.

Background & aims: The interactions between human milk and the regulation of innate immune homeostasis in newborns, and their impact on intestinal health, are not fully understood. This study aimed to explore the role of peptides in human milk extracellular vesicles (EVs) in this process.

Methods: A comprehensive screening of peptides within human milk EVs was performed, leading to the identification of a beta-casein-derived peptide (CASB_135-150). The effects of CASB_135-150 on intestinal injury were evaluated in a rat necrotizing enterocolitis (NEC) model. Immunofluorescence analysis was used to determine its distribution, and its impact on NF-κB signaling and inflammation was studied in bone marrow-derived macrophages (BMDMs) and intestinal macrophages. Protein-protein interaction (PPI) analysis, single-cell RNA-seq (scRNA-seq), and co-immunoprecipitation (co-IP) experiments were conducted to explore the mechanism underlying CASB_135-150 function.

Results: CASB_135-150 significantly mitigated intestinal injury in the rat NEC model. Immunofluorescence analysis revealed that CASB_135-150 could target intestinal macrophages and rapidly inhibited NF-κB signaling and reduced inflammation. ScRNA-seq analyses indicated a strong association between FHL2 and NEC development, and co-IP confirmed the interaction between CASB_135-150 and FHL2. CASB_135-150 disrupted the FHL2/TRAF6 complex, reducing TRAF6 protein levels. Mutation of key amino acids in CASB_135-150 disrupted its interaction with FHL2 and abolished its ability to inhibit NF-κB signaling, which also prevented its protective effect in vivo. RNA-seq of intestinal tissue further highlighted the impact of CASB_135-150 on the NF-κB signaling pathway.

Conclusions: Our study identifies CASB_135-150, a novel peptide in human milk EVs, that rapidly regulates macrophage inflammatory responses and protects against NEC-induced intestinal injury. These findings provide new insights into the role of human milk in modulating the infant immune system and intestinal health.

The onset of colorectal cancer (CRC) in patients younger than 50 continues to rapidly increase. This study highlights the epidemiologic changes, risk factors, clinical characteristics, and molecular profiles prevalent in early onset CRC patients, and identifies key areas for future research. It has been noted that only a small fraction of early onset CRC cases is attributed to known hereditary mutations and fit the canonical pathway of late-onset colorectal cancer development. To highlight this, we review the genetic and epigenetic modifications specific to early onset CRC. We also discuss the synergetic effect of single-nucleotide polymorphisms and environmental factors on the early onset of CRC. Additionally, we discuss the potential of noninvasive biomarker assays to enhance early detection, screening, diagnosis, and prognostic outcome predictions.

Background & aims: The immunological mechanisms underpinning the pathogenesis of alcoholic-associated liver disease (ALD) remain incompletely elucidated. This study aims to explore the transcriptomic profiles of hepatic immune cells in ALD compared with healthy individuals and those with metabolic dysfunction-associated steatotic liver disease (MASLD).

Methods: We utilized single-cell RNA sequencing to analyze liver samples from healthy subjects and patients with MASLD and ALD, focusing on the immune cell landscapes within the liver. Key alterations in immune cell subsets were further validated using liver biopsy samples from additional patient cohorts.

Results: We observed a significant accumulation of CD4⁺ T cells in livers of patients with ALD, surpassing the prevalence of CD8⁺ T cells, in contrast to patients with MASLD and healthy counterparts, whereas natural killer (NK) cells and γδT cells exhibited reduced intrahepatic infiltration. In-depth transcriptional and developmental trajectory analyses unveiled that a distinct CD4⁺ subset characterized by granzyme K (GZMK) expression, displaying a tissue-resident signature and terminal effector state, prominently enriched among CD4⁺ T cells infiltrating the livers of patients with ALD. Subsequent examination of an independent ALD patient cohort corroborated the substantial enrichment of GZMK⁺CD4⁺ T lymphocytes, primarily within liver fibrotic zones, suggesting their potential involvement in disease progression. Additionally, we noted shifts in myeloid populations, with expanded APOE⁺ macrophage and FCGR3B⁺ monocyte subsets in ALD samples relative to MASLD and healthy tissues.

Conclusions: In summary, this study unravels the intricate cellular diversity within hepatic immune cell populations, highlighting the pivotal immune pathogenic role of the GZMK⁺CD4⁺ T lymphocyte subset in ALD pathogenesis.

Background & aims: Cystic fibrosis (CF) is an autosomal recessive genetic disorder, affecting multiple organ systems. CF intestinal disease develops early, manifesting as intestinal bacterial overgrowth/dysbiosis, neutrophilic inflammation, and obstruction. As unresolvable infection and inflammation reflect host immune deficiency, we sought to determine if the CF-affected immune system plays any significant role in CF intestinal disease pathogenesis.

Methods: CF and sibling wild-type (WT) mice underwent reciprocal bone marrow transplantation. After immune reconstitution, their mortality, intestinal transit, fecal inflammatory markers, and mucosal immune cell composition were assessed. Moreover, reciprocal neutrophil transfusion was conducted to determine if neutrophil function affects intestinal movement. Furthermore, expression of induced nitric oxide synthase (iNOS) and production of nitric oxide (NO) in CF and WT neutrophils were compared. Lastly, specific iNOS inhibitor 1400W was tested to prevent CF intestinal obstruction.

Results: Immune restoration in CF mice reversed the intestinal neutrophilic inflammation, improved the intestinal dysmotility, and rescued the mice from mortality. Transfusion of WT neutrophils into CF mice ameliorated the retarded bowel movement. CF neutrophils expressed significantly more iNOS and produced significantly more NO. Pharmaceutical blocking of iNOS significantly improved intestinal transit and survival of CF mice.

Conclusions: CF immune defect plays a critical role in CF intestinal disease development. Activation of iNOS in inflammatory cells produces excessive NO, slows the bowel movement, and facilitates intestinal paralysis and obstruction in CF. Thus, normalization of the CF immune system may offer a novel therapy to treat CF intestinal disease.