Bronchial epithelial DNA methyltransferase 3b dampens pulmonary immune responses during Pseudomonas aeruginosa infection

Abstract

DNA methyltransferase (Dnmt)3b mediates de novo DNA methylation and modulation of Dnmt3b in respiratory epithelial cells has been shown to affect the expression of multiple genes. Respiratory epithelial cells provide a first line of defense against pulmonary pathogens and play a crucial role in the immune response during pneumonia caused by Pseudomonas (P.) aeruginosa, a gram-negative bacterium that expresses flagellin as an important virulence factor. We here sought to determine the role of Dntm3b in respiratory epithelial cells in immune responses elicited by P. aeruginosa. DNMT3B expression was reduced in human bronchial epithelial (BEAS-2B) cells as well as in primary human and mouse bronchial epithelial cells grown in air liquid interface upon exposure to P. aeruginosa (PAK). Dnmt3b deficient human bronchial epithelial (BEAS-2B) cells produced more CXCL1, CXCL8 and CCL20 than control cells when stimulated with PAK, flagellin-deficient PAK (PAKflic) or flagellin. Dnmt3b deficiency reduced DNA methylation at exon 1 of CXCL1 and enhanced NF-ĸB p65 binding to the CXCL1 promoter. Mice with bronchial epithelial Dntm3b deficiency showed increased Cxcl1 mRNA expression in bronchial epithelium and CXCL1 protein release in the airways during pneumonia caused by PAK, which was associated with enhanced neutrophil recruitment and accelerated bacterial clearance; bronchial epithelial Dnmt3b deficiency did not modify responses during pneumonia caused by PAKflic or Klebsiella pneumoniae (an un-flagellated gram-negative bacterium). Dnmt3b deficiency in type II alveolar epithelial cells did not affect mouse pulmonary defense against PAK infection. These results suggest that bronchial epithelial Dnmt3b impairs host defense during Pseudomonas induced pneumonia, at least in part, by dampening mucosal responses to flagellin.

An epigenetic and transcriptomic signature of immune tolerance in human monocytes through multi-omics integration

“…Next, we quantified 5-methylcytosine (5-mC) levels in monocytes obtained from a subset of CAP patients ( n =21), captured during the acute disease stage, as well as control participants ( n =16) by methylated DNA immunoprecipitation (MeDIP) [ 41 ]. The characteristics of CAP patients and controls are tabulated in Additional file 3 : Table S2.…”

Induction of Acute or Disseminating Bacterial Pneumonia in Mice and Sampling of Infected Organs for Studying the Host Response to Bacterial Pneumonia

“…Lung inflammation is determined by pathology analysis and by measurement of myeloperoxidase and elastase levels in lung homogenates using ELISA. In BALF, leukocyte influx is determined using flow cytometry (Qin et al, 2020 and2021a). The epithelial cell responses are evaluated by measuring mRNA levels of inflammatory genes (relative to a housekeeping gene) in lung brushes using RT-qPCR analysis (Qin et al, 2020(Qin et al, , 2021a(Qin et al, and 2021b.…”

“…by measuring bacterial burden, inflammatory cytokine production, and pathological changes in both lung and distal organs. Procedure A is also applicable to study lung immune responses or inflammation induced by intranasal inoculation of Methicillin-resistant Staphylococcus aureus (Meijer et al, 2021), or bacterial compounds, such as flagellin (Qin et al, 2021b) and lipopolysaccharide (Otto et al, 2020). according to formula: final volume (mL)= 10 × OD620/0.5.…”

The Role of Host Cell DNA Methylation in the Immune Response to Bacterial Infection

“…DNA methylation enzymes are important for regulating host immune responses against this bacterium infection, as indicated by the association between genetic variants of DNMT3B and P. aeruginosa infection in children ( 168 ). We recently identified a role for DNMT3B in bronchial epithelial cells during P. aeruginosa pneumonia ( 169 ). DNMT3B deficient human bronchial epithelial cells produced more CXCL1 and related chemokines than control cells when stimulated with P. aeruginosa .…”

“…These in vitro findings were corroborated by studies in mice with bronchial epithelial Dntm3b deficiency infected with viable P. aeruginosa via the airways, which showed increased Cxcl1 expression in bronchial epithelium and CXCL1 protein release together with enhanced neutrophil recruitment and accelerated bacterial clearance. Additional studies using purified flagellin (an important virulent factor expressed by Pseudomonas ) and a flagellin-deficient P. aeruginosa strain demonstrated that bronchial epithelial DNMT3b impaired host defense during Pseudomonas induced pneumonia at least in part by diminishing mucosal responses to flagellin ( 169 ). In separate investigations we showed that the DNA methylation eraser TET2 maintains epithelium barrier function during acute P. aeruginosa infection in mice ( 204 ).…”