Cellular and Molecular Gastroenterology and Hepatology最新文献

Background & aims

Humans with WNT2B deficiency have severe intestinal disease, including significant inflammatory injury, highlighting a critical role for WNT2B. We sought to understand how WNT2B contributes to intestinal homeostasis.

Methods

We investigated the intestinal health of Wnt2b knock out (KO) mice. We assessed the baseline histology and health of the small intestine and colon, and the impact of inflammatory challenge using dextran sodium sulfate (DSS). We also evaluated human intestinal tissue.

Results

Mice with WNT2B deficiency had normal baseline histology but enhanced susceptibility to DSS colitis because of an increased early injury response. Although intestinal stem cells markers were decreased, epithelial proliferation was similar to control subjects. Wnt2b KO mice showed an enhanced inflammatory signature after DSS treatment. Wnt2b KO colon and human WNT2B-deficient organoids had increased levels of CXCR4 and IL6, and biopsy tissue from humans showed increased neutrophils.

Conclusions

WNT2B is important for regulation of inflammation in the intestine. Absence of WNT2B leads to increased expression of inflammatory cytokines and increased susceptibility to gastrointestinal inflammation, particularly in the colon.

Background & Aims

Dysregulated colonic epithelial cell (CEC) proliferation is a critical feature in the development of colorectal cancer. We show that NF-κB-inducing kinase (NIK) attenuates colorectal cancer through coordinating CEC regeneration/differentiation via noncanonical NF-κB signaling that is unique from canonical NF-kB signaling.

Methods

Initial studies evaluated crypt morphology/functionality, organoid generation, transcriptome profiles, and the microbiome. Inflammation and inflammation-induced tumorigenesis were initiated in whole-body NIK knockout mice (Nik^-/-) and conditional-knockout mice following administration of azoxymethane and dextran sulfate sodium.

Results

Human transcriptomic data revealed dysregulated noncanonical NF-kB signaling. In vitro studies evaluating Nik^-/- crypts and organoids derived from mature, nondividing CECs, and colonic stem cells exhibited increased accumulation and stunted growth, respectively. Transcriptomic analysis of Nik^-/- cells revealed gene expression signatures associated with altered differentiation-regeneration. When assessed in vivo, Nik^-/- mice exhibited more severe colitis with dextran sulfate sodium administration and an altered microbiome characterized by increased colitogenic microbiota. In the inflammation-induced tumorigenesis model, we observed both increased tumor burdens and inflammation in mice where NIK is knocked out in CECs (Nik^ΔCEC). Interestingly, this was not recapitulated when NIK was conditionally knocked out in myeloid cells (Nik^ΔMYE). Surprisingly, conditional knockout of the canonical pathway in myeloid cells (RelA^ΔMYE) revealed decreased tumor burden and inflammation and no significant changes when conditionally knocked out in CECs (RelA^ΔCEC).

Conclusions

Dysregulated noncanonical NF-κB signaling is associated with the development of colorectal cancer in a tissue-dependent manner and defines a critical role for NIK in regulating gastrointestinal inflammation and regeneration associated with colorectal cancer.

Background & Aims

The immune tolerance induced by hepatitis B virus (HBV) is a major challenge for achieving effective viral clearance, and the mechanisms involved are not well-understood. One potential factor involved in modulating immune responses is mesencephalic astrocyte-derived neurotrophic factor (MANF), which has been reported to be increased in patients with chronic hepatitis B. In this study, our objective is to examine the role of MANF in regulating immune responses to HBV.

Methods

We utilized a commonly used HBV-harboring mouse model, where mice were hydrodynamically injected with the pAAV/HBV1.2 plasmid. We assessed the HBV load by measuring the levels of various markers including hepatitis B surface antigen, hepatitis B envelope antigen, hepatitis B core antigen, HBV DNA, and HBV RNA.

Results

Our study revealed that following HBV infection, both myeloid cells and hepatocytes exhibited increased expression of MANF. Moreover, we observed that mice with myeloid-specific MANF knockout (Manf^Mye-/-) displayed reduced HBV load and improved HBV-specific T cell responses. The decreased HBV-induced tolerance in Manf^Mye-/- mice was associated with reduced accumulation of myeloid-derived suppressor cells (MDSCs) in the liver. Restoring MDSC levels in Manf^Mye-/- mice through MDSC adoptive transfer reinstated HBV-induced tolerance. Mechanistically, we found that MANF promoted MDSC expansion by activating the IL-6/STAT3 pathway. Importantly, our study demonstrated the effectiveness of a combination therapy involving an hepatitis B surface antigen vaccine and nanoparticle-encapsulated MANF siRNA in effectively clearing HBV in HBV-carrier mice.

Conclusion

The current study reveals that MANF plays a previously unrecognized regulatory role in liver tolerance by expanding MDSCs in the liver through IL-6/STAT3 signaling, leading to MDSC-mediated CD8⁺ T cell exhaustion.

Background & Aims

Heterozygous SPINK1 mutations are strong risk factors for chronic pancreatitis in humans, yet heterozygous disruption of mouse Spink1 yielded no pancreatic phenotype. To resolve this contradiction, we used CRISPR/Cas9-mediated genome editing to generate heterozygous Spink1-deleted mice (Spink1-KO^het) in the C57BL/6N strain and studied the effect of this allele in trypsin-independent and trypsin-dependent pancreatitis models.

Methods

We investigated severity of acute pancreatitis and progression to chronic pancreatitis in Spink1-KO^het mice after transient (10 injections) and prolonged (2 × 8 injections) cerulein hyperstimulation. We crossed Spink1-KO^het mice with T7D23A and T7D22N,K24R mice that carry strongly autoactivating trypsinogen mutants and exhibit spontaneous chronic pancreatitis.

Results

Prolonged but not transient cerulein stimulation resulted in increased intrapancreatic trypsin activity and more severe acute pancreatitis in Spink1-KO^het mice relative to the C57BL/6N control strain. After the acute episode, Spink1-KO^het mice developed progressive disease with chronic pancreatitis-like features, whereas C57BL/6N mice recovered rapidly. Trypsinogen mutant mice carrying the Spink1-KO^het allele exhibited strikingly more severe chronic pancreatitis than the respective parent strains.

Conclusions

Heterozygous Spink1 deficiency caused more severe acute pancreatitis after prolonged cerulein stimulation and promoted chronic pancreatitis after the cerulein-induced acute episode, and in two strains of trypsinogen mutant mice with spontaneous disease. In contrast, acute pancreatitis induced with limited cerulein hyperstimulation was unaffected by heterozygous Spink1 deletion, in agreement with recent observations that trypsin activity does not mediate pathologic responses in this model. Taken together, the findings strongly support the notion that loss-of-function SPINK1 mutations in humans increase chronic pancreatitis risk in a trypsin-dependent manner.

Background & Aims

Colorectal cancer (CRC) is the third most common cancer in the world. Gut microbiota has recently been implicated in the development of CRC. Actinomyces odontolyticus is one of the most abundant bacteria in the gut of patients with very early stages of CRC. A odontolyticus is an anaerobic bacterium existing principally in the oral cavity, similar to Fusobacterium nucleatum, which is known as a colon carcinogenic bacterium. Here we newly determined the biological functions of A odontolyticus on colonic oncogenesis.

Methods

We examined the induction of intracellular signaling by A odontolyticus in human colonic epithelial cells (CECs). DNA damage levels in CECs were confirmed using the human induced pluripotent stem cell-derived gut organoid model and mouse colon tissues in vivo.

Results

A odontolyticus secretes membrane vesicles (MVs), which induce nuclear factor kappa B signaling and also produce excessive reactive oxygen species (ROS) in colon epithelial cells. We found that A odontolyticus secretes lipoteichoic acid-rich MVs, promoting inflammatory signaling via TLR2. Simultaneously, those MVs are internalized into the colon epithelial cells, co-localize with the mitochondria, and cause mitochondrial dysfunction, resulting in excessive ROS production and DNA damage. Induction of excessive DNA damage in colonic cells by A odontolyticus-derived MVs was confirmed in the gut organoid model and also in mouse colon tissues.

Conclusions

A odontolyticus secretes MVs, which cause chronic inflammation and ROS production in colonic epithelial cells, leading to the initiation of CRC.

Background & Aims

Inflammatory bowel disease is associated with carcinogenesis, which limits the prognosis of the patients. The local expression of proteinases and proteinase-activated receptor 1 (PAR₁) increases in inflammatory bowel disease. The present study investigated the therapeutic effects of PAR₁ antagonism on colitis-associated carcinogenesis.

Methods

A colitis-associated carcinogenesis model was prepared in mice by treatment with azoxymethane (AOM) and dextran sulfate sodium (DSS). PAR₁ antagonist E5555 was administered in long- and short-term protocol, starting on the day of AOM injection and 1 week after completing AOM/DSS treatment, respectively. The fecal samples were collected for metagenome analysis of gut microbiota. The intestinal myofibroblasts of the Crohn’s disease patients were used to elucidate underlying cellular mechanisms. Caco-2 cells were used to investigate a possible source of PAR₁ agonist proteinases.

Results

AOM/DSS model showed weight loss, diarrhea, tumor development, inflammation, fibrosis, and increased production of inflammatory cytokines. The β-diversity, but not α-diversity, of microbiota significantly differed between AOM/DSS and control mice. E5555 alleviated these pathological changes and altered the microbiota β-diversity in AOM/DSS mice. The thrombin expression was up-regulated in tumor and non-tumor areas, whereas PAR₁ mRNA expression was higher in tumor areas compared with non-tumor areas. E5555 inhibited thrombin-triggered elevation of cytosolic Ca²⁺ concentration and ERK1/2 phosphorylation, as well as IL6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation in intestinal myofibroblasts. Caco-2 cell-conditioned medium contained immunoreactive thrombin, which cleaved the recombinant protein containing the extracellular domain of PAR₁ at the thrombin cleavage site.

Conclusions

PAR₁ antagonism is proposed to be a novel therapeutic strategy for treatment of inflammatory bowel disease and its associated carcinogenesis.

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Background & Aims

Methods

Results

Conclusions