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Active or Autoclaved Akkermansia muciniphila Relieves TNF-α-Induced Inflammation in Intestinal Epithelial Cells Through Distinct Pathways.

ABSTRACT: Intestinal inflammation is a major threat to the health and growth of young animals such as piglets. As a next-generation probiotics, limited studies have shown that Akkermansia muciniphila could alleviate inflammation of intestinal epithelial cells (IECs). In this study, a TNF-α-induced inflammatory model of IPEC-J2 cells, the intestinal porcine enterocytes, was built to evaluate the effects of active or inactive A. muciniphila on the inflammation of IECs. The viability of IPEC-J2 cells was the highest when treated with active (10⁸ copies/mL) or inactive (10⁹ copies/mL) A. muciniphila for 7.5 h (P < 0.01). Treated with 20 ng/mL of TNF-α and followed by a treatment of A. muciniphila, the mRNA level of proinflammatory cytokines (IL-8, IL-1β, IL-6 and TNF-α) was remarkably reduced (P < 0.05) along with the increased mRNA level of tight junction proteins (ZO-1 and Occludin, P < 0.05). Flow cytometry analysis showed that active or inactive A. muciniphila significantly suppressed the rate of the early and total apoptotic of the inflammatory IPEC-J2 cells (P < 0.05). According to results of transcriptome sequencing, active and inactive A. muciniphila may decline cell apoptosis by down-regulating the expression of key genes in calcium signaling pathway, or up-regulating the expression of key genes in cell cycle signaling pathway. And the bacterium may alleviate the inflammation of IECs by down-regulating the expression of PI3K upstream receptor genes. Our results indicate that A. muciniphila may be a promising NGP targeting intestinal inflammation.

SUBMITTER: Luo Y

PROVIDER: S-EPMC8716699 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe intestinal epithelial barrier is the interface for interaction between gut microbiota and host metabolic systems. Akkermansia muciniphila (A. muciniphila) is a key player in the colonic microbiota that resides in the mucus layer, whose abundance is selectively decreased in the faecal microbiota of inflammatory bowel disease (IBD) patients. This study aims to investigate the regulatory mechanism among A. muciniphila, a transcription factor cAMP-responsive element-binding protein H (CREBH), and microRNA-143/145 (miR-143/145) in intestinal inflammatory stress, gut barrier integrity and epithelial regeneration.MethodsA novel mouse model with increased colonization of A muciniphila in the intestine of CREBH knockout mice, an epithelial wound healing assay and several molecular biological techniques were applied in this study. Results were analysed using a homoscedastic 2-tailed t-test.ResultsIncreased colonization of A. muciniphila in mouse gut enhanced expression of intestinal CREBH, which was associated with the mitigation of intestinal endoplasmic reticulum (ER) stress, gut barrier leakage and blood endotoxemia induced by dextran sulfate sodium (DSS). Genetic depletion of CREBH (CREBH-KO) significantly inhibited the expression of tight junction proteins that are associated with gut barrier integrity, including Claudin5 and Claudin8, but upregulated Claudin2, a tight junction protein that enhances gut permeability, resulting in intestinal hyperpermeability and inflammation. Upregulation of CREBH by A. muciniphila further coupled with miR-143/145 promoted intestinal epithelial cell (IEC) regeneration and wound repair via insulin-like growth factor (IGF) and IGFBP5 signalling. Moreover, the gene expressing an outer membrane protein of A. muciniphila, Amuc_1100, was cloned into a mammalian cell-expression vector and successfully expressed in porcine and human IECs. Expression of Amuc_1100 in IECs could recapitulate the health beneficial effect of A. muciniphila on the gut by activating CREBH, inhibiting ER stress and enhancing the expression of genes involved in gut barrier integrity and IEC's regeneration.ConclusionsThis study uncovers a novel mechanism that links A. muciniphila and its membrane protein with host CREBH, IGF signalling and miRNAs in mitigating intestinal inflammatory stress-gut barrier permeability and promoting intestinal wound healing. This novel finding may lend support to the development of therapeutic approaches for IBD by manipulating the interaction between host genes, gut bacteria and its bioactive components.

Project description:BACKGROUND: The human gastrointestinal tract contains a complex community of microbes, fulfilling important health-promoting functions. However, this vast complexity of species hampers the assignment of responsible organisms to these functions. Recently, Akkermansia muciniphila, a new species from the deeply branched phylum Verrucomicrobia, was isolated from the human intestinal tract based on its capacity to efficiently use mucus as a carbon and nitrogen source. This anaerobic resident is associated with the protective mucus lining of the intestines. METHODOLOGY/PRINCIPAL FINDINGS: In order to uncover the functional potential of A. muciniphila, its genome was sequenced and annotated. It was found to contain numerous candidate mucinase-encoding genes, but lacking genes encoding canonical mucus-binding domains. Numerous phage-associated sequences found throughout the genome indicate that viruses have played an important part in the evolution of this species. Furthermore, we mined 37 GI tract metagenomes for the presence, and genetic diversity of Akkermansia sequences. Out of 37, eleven contained 16S ribosomal RNA gene sequences that are >95% identical to that of A. muciniphila. In addition, these libraries were found to contain large amounts of Akkermansia DNA based on average nucleotide identity scores, which indicated in one subject co-colonization by different Akkermansia phylotypes. An additional 12 libraries also contained Akkermansia sequences, making a total of ?16 Mbp of new Akkermansia pangenomic DNA. The relative abundance of Akkermansia DNA varied between <0.01% to nearly 4% of the assembled metagenomic reads. Finally, by testing a large collection of full length 16S sequences, we find at least eight different representative species in the genus Akkermansia. CONCLUSIONS/SIGNIFICANCE: These large repositories allow us to further mine for genetic heterogeneity and species diversity in the genus Akkermansia, providing novel insight towards the functionality of this abundant inhabitant of the human intestinal tract.

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Active or Autoclaved Akkermansia muciniphila Relieves TNF-α-Induced Inflammation in Intestinal Epithelial Cells Through Distinct Pathways.

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