Cellular and Molecular Gastroenterology and Hepatology最新文献

Background & Aims

Abdominal pain is a major symptom of diseases that are associated with microbial dysbiosis, including irritable bowel syndrome and inflammatory bowel disease. Germ-free mice are more prone to abdominal pain than conventionally housed mice, and reconstitution of the microbiota in germ-free mice reduces abdominal pain sensitivity. However, the mechanisms underlying microbial modulation of pain remain elusive. We hypothesized that disruption of the intestinal microbiota modulates the excitability of peripheral nociceptive neurons.

Methods

In vivo and in vitro assays of visceral sensation were performed on mice treated with the nonabsorbable antibiotic vancomycin (50 μg/mL in drinking water) for 7 days and water-treated control mice. Bacterial dysbiosis was verified by 16s rRNA analysis of stool microbial composition.

Results

Treatment of mice with vancomycin led to an increased sensitivity to colonic distension in vivo and in vitro and hyperexcitability of dorsal root ganglion (DRG) neurons in vitro, compared with controls. Interestingly, hyperexcitability of DRG neurons was not restricted to those that innervated the gut, suggesting a widespread effect of gut dysbiosis on peripheral pain circuits. Consistent with this, mice treated with vancomycin were more sensitive than control mice to thermal stimuli applied to hind paws. Incubation of DRG neurons from naive mice in serum from vancomycin-treated mice increased DRG neuron excitability, suggesting that microbial dysbiosis alters circulating mediators that influence nociception. The cysteine protease inhibitor E64 (30 nmol/L) and the protease-activated receptor 2 (PAR-2) antagonist GB-83 (10 μmol/L) each blocked the increase in DRG neuron excitability in response to serum from vancomycin-treated mice, as did the knockout of PAR-2 in NaV1.8-expressing neurons. Stool supernatant, but not colonic supernatant, from mice treated with vancomycin increased DRG neuron excitability via cysteine protease activation of PAR-2.

Conclusions

Together, these data suggest that gut microbial dysbiosis alters pain sensitivity and identify cysteine proteases as a potential mediator of this effect.

Background

A peculiar feature of the hepatitis E virus (HEV) is its reliance on the exosomal route for viral release. Genomic replication is mediated via the viral polyprotein pORF1, yet little is known about its subcellular localization.

Methods

Subcellular localization of pORF1 and its subdomains, generated and cloned based on a structural prediciton of the viral replicase, was analyzed via confocal laser scanning microscopy. Exosomes released from cells were isolated via ultracentrifugation and analyzed by isopycnic density gradient centrifugation. This was followed by fluorimetry or Western blot analyses or reverse transcriptase–polymerase chain reaction to analyze separated particles in more detail.

Results

We found pORF1 to be accumulating within the endosomal system, most dominantly to multivesicular bodies (MVBs). Expression of the polyprotein’s 7 subdomains revealed that the papain-like cysteine-protease (PCP) is the only domain localizing like the full-length protein. A PCP-deficient pORF1 mutant lost its association to MVBs. Strikingly, both pORF1 and PCP can be released via exosomes. Similarly, genomic RNA still is released via exosomes in the absence of pORF2/3.

Conclusions

Taken together, we found that pORF1 localizes to MVBs in a PCP-dependent manner, which is followed by exosomal release. This reveals new aspects of HEV life cycle, because replication and release could be coupled at the endosomal interface. In addition, this may mediate capsid-independent spread or may facilitate the spread of viral infection, because genomes entering the cell during de novo infection readily encounter exosomally transferred pORF1.

Alcohol-associated hepatitis (AH) is an acute-on-chronic liver injury that occurs in patients with chronic alcohol-associated liver disease (ALD). Patients with severe AH have high short-term mortality and lack effective pharmacologic therapies. Inflammation is believed to be one of the key factors promoting AH progression and has been actively investigated as therapeutic targets over the last several decades, but no effective inflammatory targets have been identified so far. In this review, we discuss how inflammatory cells and the inflammatory mediators produced by these cells contribute to the development and progression of AH, with focus on neutrophils and macrophages. The crosstalk between inflammatory cells and liver nonparenchymal cells in the pathogenesis of AH is elaborated. We also deliberate the application of recent cutting-edge technologies in characterizing liver inflammation in AH. Finally, the potential therapeutic targets of inflammatory mediators for AH are briefly summarized.

Background & Aims

Metabolic dysfunction-associated steatotic liver disease (MASLD) has become the most prevalent chronic liver disorder, with no approved treatment. Our previous work demonstrated the efficacy of a pan-ErbB inhibitor, Canertinib, in reducing steatosis and fibrosis in a murine fast-food diet (FFD) model of MASLD. The current study explores the effects of hepatocyte-specific ErbB1 (ie, epidermal growth factor receptor [EGFR]) deletion in the FFD model.

Methods

EGFR^flox/flox mice, treated with AAV8-TBG-CRE to delete EGFR specifically in hepatocytes (EGFR-KO), were fed either a chow-diet or FFD for 2 or 5 months.

Results

Hepatocyte-specific EGFR deletion reduced serum triglyceride levels but did not prevent steatosis. Surprisingly, hepatic fibrosis was increased in EGFR-KO mice in the long-term study, which correlated with activation of transforming growth factor-β/fibrosis signaling pathways. Further, nuclear levels of some of the major MASLD regulating transcription factors (SREBP1, PPARγ, PPARα, and HNF4α) were altered in FFD-fed EGFR-KO mice. Transcriptomic analysis revealed significant alteration of lipid metabolism pathways in EGFR-KO mice with changes in several relevant genes, including downregulation of fatty-acid synthase and induction of lipolysis gene, Pnpla2, without impacting overall steatosis. Interestingly, EGFR downstream signaling mediators, including AKT, remain activated in EGFR-KO mice, which correlated with increased activity pattern of other receptor tyrosine kinases, including ErbB3/MET, in transcriptomic analysis. Lastly, Canertinib treatment in EGFR-KO mice, which inhibits all ErbB receptors, successfully reduced steatosis, suggesting the compensatory roles of other ErbB receptors in supporting MASLD without EGFR.

Conclusions

Hepatocyte-specific EGFR-KO did not impact steatosis, but enhanced fibrosis in the FFD model of MASLD. Gene networks associated with lipid metabolism were greatly altered in EGFR-KO, but phenotypic effects might be compensated by alternate signaling pathways.

Background & Aims

Necrotizing enterocolitis (NEC) is a life-threatening disease affecting mostly the ileum of preemies. Intestinal epithelial cell (IEC) apoptosis contributes to NEC pathogenesis. However, how scattered crypt IEC apoptosis leads to NEC with excessive villus epithelial necrosis remains unclear.

Methods

A novel triple-transgenic mouse model, namely, 3xTg-iAP^cIEC (inducible apoptosis phenotype in crypt-IEC), was developed to induce IEC-specific overexpression of Fasl transgene using doxycycline (Dox)-inducible tetO-rtTA system and villin-cre technology. The 3-days-old neonatal 3xTg-iAP^cIEC mice and their littermate controls were subcutaneously (s.c.) challenged with a single dose of Dox. Intestinal tissues were processed at different time points to examine scattered crypt IEC apoptosis-mediated NEC development. Gene knockout technology, antibody-mediated cell depletion, and antibiotic-facilitated Gram-positive bacteria depletion were used to study mechanisms.

Results

Treatment of 3xTg-iAP^cIEC mouse pups with Dox induces scattered crypt IEC apoptosis followed by crypt inflammation and excessive villous necrosis resembling NEC. This progression correlated with elevated Ifng, Rip3, CD8⁺ T cells, and Gram-positive bacteria in the ileum. Mechanistically, IFN-γ and RIP3-activated signals mediate the effect of scattered crypt IEC apoptosis on the induction of intestinal crypt inflammation and villous necrosis. Meanwhile, pathophysiological events of CD8⁺ T cell infiltration and dysbiosis with Gram-positive bacteria primarily contribute to excessive villous inflammation and necrosis. Notably, blocking any of these events protects against NEC development in 3xTg-iAP^cIEC mouse pups, underlining their central roles in NEC pathogenesis.

Conclusions

Scattered crypt IEC apoptosis induces NEC in mouse pups via IFN-γ, RIP3, CD8⁺ T cells, and Gram-positive bacteria-mediated comprehensive pathophysiological events. Our findings may advance knowledge in the prevention and treatment of NEC.