Cellular and Molecular Gastroenterology and Hepatology最新文献

Background & aims: The liver has remarkable regenerative and detoxification capacities, which require the Nrf2 and NF-κB transcription factors. Although their individual functions in hepatocytes are well characterized, knowledge about their crosstalk in the adult liver is limited.

Methods: We performed AAV8-Cre inducible, hepatocyte-specific knockout of Nrf2, the NF-κB subunit p65, or both genes to determine the individual and combined roles of these transcription factors in the intact liver of male adult mice and after acute CCl₄ injury. Mice were characterized using histological and immunohistochemical stainings, serum and liver bile acid analysis, flow cytometry, and RNA sequencing. To distinguish between cell-autonomous and non-cell-autonomous mechanisms, we generated and analyzed knockout and knock-down AML12 liver cells. Clodronate liposome-mediated macrophage depletion was used to determine the role of these immune cells in hepatocyte proliferation after CCl₄ injection.

Results: Loss of p65 alone or p65 in combination with Nrf2 caused spontaneous liver inflammation and necrosis. Gene expression profiling identified individual and common target genes of both transcription factors, including genes involved in the control of cell proliferation. Consistent with the expression of these genes, hepatocyte proliferation was reduced by Nrf2 deficiency under homeostatic conditions and after CCl₄ injury, which was rescued by additional loss of p65. The increased hepatocyte proliferation in the double-knockout mice was non-cell-autonomous and correlated with macrophage accumulation in the liver. Depletion of macrophages in these mice suppressed hepatocyte proliferation after CCl₄ treatment.

Conclusions: These results reveal a crosstalk between Nrf2 and p65 in the control of hepatocyte proliferation and point to a key role of macrophages in this effect.

Background and aims: Necrotizing enterocolitis (NEC) is a life-threatening condition in premature infants, marked by acute intestinal necrosis. NEC develops in part after activation of the lipopolysaccharide receptor toll-like receptor 4 (TLR4) by intestinal microbes in the intestinal epithelium. Previous authors have shown an increased risk of NEC in human infants after cytomegalovirus (CMV) infection, which can affect mitochondrial function. We now seek to explore the impact and the mechanisms of CMV infection on NEC severity and its relationship with TLR4 signaling and mitochondria function.

Methods: NEC was induced in newborn mice with and without CMV infection. RNA sequencing and gene set enrichment analysis were performed to identify effects on inflammatory and metabolic pathways. The role of TLR4 signaling and mitochondrial function were investigated in wild-type and Tlr4-deficient mice. The adenosine receptor agonist 5'-N-ethylcarboxamido adenosine was tested for its ability to reduce CMV-induced effects on NEC severity.

Results: CMV infection significantly increased NEC severity in wild-type mice. Mechanistically, CMV infection triggered proinflammatory pathways, disrupted cellular metabolism, and upregulated Tlr4 expression, leading to mitochondrial dysfunction and nuclear factor-kB translocation. These effects were notably absent in Tlr4-deficient mice. 5'-N-ethylcarboxamido adenosine treatment reversed CMV-induced NEC severity by reducing mitochondrial dysfunction and TLR4-driven nuclear factor-kB activation.

Conclusions: CMV infection worsens NEC severity in mice by amplifying TLR4 signaling, inflammation, and mitochondrial dysfunction. Targeting CMV and its influence on TLR4 may offer novel therapeutic approaches for NEC.

Background & aims: Clostridioides difficile infection (CDI) causes colitis and diarrhea. C. difficile bacterium produces toxins A and B, which cause intestinal inflammation. A metabolomics analysis discovered fecal metabolites with anti-inflammatory effects in CDI. We aimed to identify an anti-CDI metabolite that can inhibit CDI-mediated colitis and prevent recurrence.

Methods: Fresh human colonic tissues and primary human cells were used to determine metabolite effects. Humanized C. difficile-infected HuCD34-NCG mice and antibiotics-treated human gut microbiota-treated (ABX + HGM) hamsters were used to simulate the human environment.

Results: High-throughput screening and fecal metabolomics analysis identified anti-inflammatory metabolites. Compared with other tested metabolites, citrulline preserved the mucosal integrity of toxin-exposed fresh human colonic tissues with reduced macrophage inflammatory protein 1 alpha (MIP-1a) and increased interleukin-10 (IL-10) expression. Oral citrulline treatment alleviated cecal inflammation in hamsters infected with C. difficile ribotype 027. This was accomplished by the augmented expression of cecal IL-10 and the diminished level of cecal MIP-1a. Citrulline and vancomycin synergistically prevented recurrence in the infected ABX + HGM hamsters. In C57BL/6J mice infected with C. difficile VPI10463, citrulline ameliorated colitis by reducing colonic Ccl3 mRNA expression. In immunologically humanized HuCD34-NCG mice infected with toxin B-expressing C. difficile ribotype 017, citrulline ameliorated colitis with increased human IL-10 expression in colonic macrophages. Citrulline suppressed MIP-1a secretion and GSK3a/b dephosphorylation in the toxin A-exposed human colonic epithelial cells and promoted IL-10 expression in toxin B-exposed human macrophages and heat shock protein 27 phosphorylation.

Conclusion: Citrulline exerts anti-inflammatory effects in the intestines against C. difficile toxins and inhibits CDI recurrence in mice and hamsters.

Background & aims: Congestion alters the microenvironment of the liver sinusoid along the portal-central axis. We studied spatial changes in immune cells in the sinusoid that contribute to congestive fibrosis and portal hypertension (PHTN).

Methods: To visualize the distribution of immune cells in congestive hepatopathy (CH), we performed imaging mass cytometry (IMC) on liver tissue from patients with CH, Fontan-associated liver disease (FALD), and controls. We performed partial ligation of the inferior vena cava (pIVCL) to simulate CH in mice and isolated primary liver cells for single-cell RNA-sequencing (scRNA-seq) to study zonation of liver sinusoidal endothelial cells (LSECs). After pIVCL, mice were treated with intraperitoneal injections of AMG487, an inhibitor of the CXCL9 receptor, or a neutralizing antibody to CXCL9.

Results: Peri-central macrophages are enriched in CH and FALD. Given the role of CXCL9 in macrophage patterning in the liver, we performed RNA in situ hybridization (RNAish) in CH and determined that CXCL9 was highly expressed in LSECs in FALD, suggesting that LSECs recruit macrophages in CH. After pIVCL, treatment with AMG487 or an antibody to CXCL9 attenuated portal pressures, fibrosis, and intra-hepatic macrophages. To study changes in LSECs that promote macrophage chemotaxis, we performed scRNA-seq after pIVCL and sham procedures. Analysis revealed 3 LSEC subpopulations according to sinusoidal location. RNAish identified peri-central LSECs as the predominant source of CXCL9 in FALD. In vitro analyses revealed that β-catenin and hypoxia inducible factor-1 α regulate CXCL9 transcription in peri-central LSECs.

Conclusions: CXCL9 derived from peri-central LSECs enriches intra-hepatic macrophages in CH and FALD, contributing to congestive fibrosis and PHTN. Strategies to target LSEC-derived CXCL9 may prevent the progression of CH and FALD.

Background & aims: Following liver damage, ductular reaction often coincides with liver fibrosis. Proliferation of hepatic progenitor cells is observed in ductular reaction, whereas activated hepatic stellate cells (HSCs) are the main drivers of liver fibrosis. These observations may suggest a functional interaction between these 2 cell types. Here, we report on an in vitro co-culture system to examine these interactions and validate their co-expression in human liver explants.

Methods: In a 3D organoid co-culture system, we combined freshly isolated quiescent mouse HSCs and fluorescently labeled progenitor cells (undifferentiated intrahepatic cholangiocyte organoids), permitting real-time observation of cell morphology and behavior. After 7 days, cells were sorted based on the fluorescent label and analyzed for changes in gene expression.

Results: In the 3D co-culture system, the proliferation of progenitor cells is enhanced, and HSCs are activated, recapitulating the cellular events observed in the patient liver. Both effects in 3D co-culture require close contact between the 2 different cell types. HSC activation during 3D co-culture differs from quiescent (3D mono-cultured) HSCs and activated HSCs on plastic (2D mono-culture). Upregulation of a cluster of genes containing Aldh1a2, Cthrc1, and several genes related to frizzled binding/Wnt signaling were exclusively observed in 3D co-cultured HSCs. The localized co-expression of specific genes was confirmed by spatial transcriptomics in human liver explants.

Conclusion: An in vitro 3D co-culture system provides evidence for direct interactions between HSCs and progenitor cells, which are sufficient to drive responses that are similar to those seen during ductular reaction and fibrosis. This model paves the way for further research into the cellular basis of liver pathology.

Helicobacter pylori (H. pylori) successfully and chronically colonizes the gastric mucosa of approximately 43% of the world's population. Infection with this organism is the strongest known risk factor for the development of gastric cancer, and disease development is dependent on several interactive components. One H. pylori determinant that augments cancer risk is the strain-specific cag type IV secretion system, which not only translocates a pro-inflammatory and oncogenic protein, CagA, into host cells but also DNA, peptidoglycan, and a lipopolysaccharide intermediate, heptose-1,7-bisphosphate. However, cognate interactions between certain microbial and host constituents can also attenuate pro-inflammatory responses, and H. pylori harbors multiple effectors that function differently than the respective counterparts in other mucosal pathogens. In this review, we discuss current data related to mechanisms utilized by H. pylori to evade the immune response, sustain its longevity in the host, and further disease progression, as well as implications for developing targeted, immune-based eradication strategies.

Background & aims: Loss of the tumor suppressor gene Apc in Lgr5+ intestinal stem cells results in aberrant Wnt signaling and colonic tumorigenesis. In the setting of injury, however, we and others have also shown that non-stem cells can also give rise to colonic tumors. The mechanism by which inflammation leads to cellular plasticity and cancer, however, remains largely unknown.

Methods: RNA expression analysis of Wnt, COX, and Akt signaling was assessed in patients with quiescent or active ulcerative colitis (UC) and patients with UC-associated neoplasia using available datasets. The role of COX signaling in colonic tumorigenesis was examined using epithelial and doublecortin-like kinase 1 (Dclk1)+ cell-specific conditional COX-1 knockout mice and pharmacologic treatment with different nonsteroidal anti-inflammatory drugs.

Results: In this study, we show that prostaglandins and phospho-Akt are key inflammatory mediators that promote stemness in Apc mutant Dclk1+ cells that give rise to colorectal cancer. Moreover, prostaglandin E₂ (PGE₂) and Akt are increased in colitis in both mice and humans, leading to inflammation-associated dysplasia upon activation of Wnt signaling. Importantly, inhibition of epithelial-derived COX-1 by aspirin or conditional knockout in Dclk1+ cells reduced PGE₂ levels and prevented the development of inflammation-associated colorectal cancer.

Conclusions: Our data shows that epithelial and Dclk1+ cell-derived COX-1 plays an important role in inflammation-associated tumorigenesis. Importantly, low-dose aspirin was effective in chemo-prevention through inhibition of COX-1 that reduced colitis-associated cancer.

Background & aims: Eosinophilic esophagitis (EoE) is a chronic esophageal inflammatory disorder characterized by eosinophil-rich mucosal inflammation and tissue remodeling. Prior research has revealed the upregulation of interferon (IFN) response signature genes (ISGs) in biopsy tissue from patients with EoE, but the specific cell types that contribute to this IFN response and the effect of interferons on the esophageal epithelium remain incompletely understood. Here, we use single-cell RNA sequencing (scRNA-seq) to examine the expression of IFN and ISGs during EoE and explore how IFN-α and IFN-γ treatments affect epithelial function.

Methods: Epithelial gene expression from patients with EoE was examined using scRNA-seq and a confirmatory bulk RNA-seq experiment of isolated epithelial cells. The functional impact of IFN-α and IFN-γ on epithelial cells was investigated using organoid models.

Results: Using scRNA-seq, the highest number of differentially regulated ISGs was found in the epithelial cells of patients with active EoE, and ISGs in transitional epithelial cells correlated significantly with eosinophil counts and endoscopic reference scores. IFN-γ and IFN-α treatments reduced organoid formation rate and size in a dose-dependent manner, with IFN-γ showing a more pronounced impact on measures of epithelial barrier formation and induction of caspase activity. We identify high IFNG expression in a cluster of majority CD8+ T cells with high expression of CD69 and FOS.

Conclusions: These findings reveal that interferon, especially IFN-γ, plays a central role in epithelial cell dysfunction, significantly affecting gene expression, cellular differentiation, and barrier integrity. Clarifying the contribution of varied cytokine signals in EoE may help explain the heterogeneity in patient presentation and therapeutic response.