P16 I NK 4a Deletion Ameliorates Damage of Intestinal Epithelial Barrier and Microbial Dysbiosis in a Stress-Induced Premature Senescence Model of Bmi-1 Deficiency.

Jiawen Zhou,Qing Li,Haiyun Chen,Guoping Zuo,Zi’An Miao,Jingyu Zhao,Chenxing Hou,Min Cui,Qiuyi Wang,Rong Wang,Ziyue Qin,Dengshun Miao,Jianliang Jin,Chunfeng Xie

doi:10.3389/fcell.2021.671564

Abstract

This study aimed to determine whether Bmi-1 deficiency leads to intestinal epithelial barrier destruction and microbiota dysfunction, which members of the microbial community alter barrier function with age, and whether p16INK4a deletion could reverse the damage of intestinal epithelial barrier and microbial dysbiosis. Intestines from Bmi-1–deficient (Bmi-1–/–), Bmi-1 and p16INK4a double-knockout (Bmi-1–/–p16INK4a–/–), and wild-type mice were observed for aging and inflammation. Duolink Proximity Ligation Assay, immunoprecipitation, and construction of p16INK4a overexpressed adenovirus and the overexpressed plasmids of full-length, mutant, or truncated fragments for occludin were used for analyzing the interaction between p16INK4a and occludin. High-throughput sequencing of V4 region amplicon of 16S ribosomal RNA was conducted using intestinal microbiota. We found Bmi-1 deficiency destructed barrier structure, barrier function, and tight junction (TJ) in intestinal epithelium; decreased the TJ proteins; increased tumor necrosis factor α (TNF-α)–dependent barrier permeability; and up-regulated proinflammatory level of macrophages induced by intestinal microbial dysbiosis. The transplantation of fecal microbiota from wild-type mice ameliorated TJ in intestinal epithelium of Bmi-1–/– and Bmi-1–/–p16INK4a–/– mice. Harmful bacteria including Desulfovibrio, Helicobacter, and Oscillibacter were at a higher level in Bmi-1–/– mice. More harmful bacteria Desulfovibrio entered the epithelium and promoted macrophages-secreted TNF-α and caused TNF-α–dependent barrier permeability and aging. Accumulated p16INK4a combined with occludin at the 1st–160th residue in cytoplasm of intestinal epithelium cells from Bmi-1–/– mice, which blocked formation of TJ and the repair of intestinal epithelium barrier. P16INK4a deletion could maintain barrier function and microbiota balance in Bmi-1–/– mice through strengthening formation of TJ and decreasing macrophages-secreted TNF-α induced by Desulfovibrio entering the intestinal epithelium. Thus, Bmi-1 maintained intestinal TJ, epithelial barrier function, and microbiota balance through preventing senescence characterized by p16INK4a accumulation. The clearance of p16INK4a-positive cells in aging intestinal epithelium would be a new method for maintaining barrier function and microbiota balance. The residues 1–160 of occludin could be a novel therapeutic target for identifying small molecular antagonistic peptides to prevent the combination of p16INK4a with occludin for protecting TJ.

Highlights

Mammalian intestine is the critical site of digestion, absorption and assimilation, and a highly immune-active ecosystem that harbors and preserves a large abundance of commensal microorganisms
These results demonstrated that p16 deletion ameliorated the damage of barrier structure and dysfunction in intestinal epithelium caused by Bmi-1 deficiency
Desulfovibrio treatment promoted the secretion of tumor necrosis factor α (TNF-α) (Figures 10A,B), and the expressions of nuclear factor κB (NF-κB)-p65, p-p65 (Ser536), IκBα, and p-IκBα (Ser32) in WT, Bmi-1−/−, and Bmi-1−/−p16−/− BMDMs compared with the same genotype BMDMs without Desulfovibrio treatment (Figures 10C,D). These results demonstrated that Desulfovibrio promoted the TNF-α secretion and activated the NF-κB signaling in BMDMs, and p16 deletion ameliorated the effect of Desulfovibrio on promoting macrophages-secreted TNF-α in Bmi-1−/− mice

Summary

Introduction

Mammalian intestine is the critical site of digestion, absorption and assimilation, and a highly immune-active ecosystem that harbors and preserves a large abundance of commensal microorganisms. With agingdependent decrease in the intestinal barrier function, microbial productions including proinflammatory factors enter the bloodstream, triggering systemic inflammation (Thevaranjan et al, 2017). Age-related microbial imbalance and intestinal barrier dysfunction have been associated with various senescence-associated intestinal and systemic diseases including inflammatory bowel diseases, celiac disease, type 1 diabetes, obesity, and Alzheimer disease (Lee, 2015). Previous study shows that tumor necrosis factor α (TNF-α) is the main proinflammatory cytokine that causes the destruction of epithelial tight junction (TJ), increases intestinal epithelial permeability, and aggravates senescence-associated systemic inflammation; which members of the microbial community alter barrier function with age have not been yet identified (Thevaranjan et al, 2017). Whether accumulated p16 plays a critical role in damaging intestinal epithelial barrier and microbial homeostasis in inflammaging process is unclear

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