RAGE-mediated functional DNA methylated modification contributes to cigarette smoke-induced airway inflammation in mice

Abstract Our previous study indicated knockout of receptor for advanced glycation end-products (RAGE) significantly attenuated cigarette smoke (CS)-induced airway inflammation in mice. In the present study, we aim to further detect the mediatory effects of RAGE in DNA methylated modification in CS-induced airway inflammation. Lung tissues from the CS-exposed mouse model of airway inflammation were collected for profiling of DNA methylation by liquid hybridization capture-based bisulfite sequencing, which were used for conjoint analysis with our previous data of gene expression by cDNA microarray to identify functional methylated genes, as well as hub genes selected by protein–protein interaction (PPI) network analysis, and functional enrichment analyses were then performed. After RAGE knockout, 90 genes were identified by intersection of the differentially methylated genes and differentially expressed genes. According to the reversed effects of methylation in promoters on gene transcription, 14 genes with functional methylated modification were further identified, among which chemokine (C–X–C motif) ligand 1 (CXCL1), Toll-like receptor 6 (TLR6) and oncostatin M (OSM) with hypomethylation in promoters, were selected as the hub genes by PPI network analysis. Moreover, functional enrichment analyses showed the 14 functional methylated genes, including the 3 hub genes, were mainly enriched in immune-inflammatory responses, especially mitogen-activated protein kinase, tumor necrosis factor, TLRs, interleukin (IL)-6 and IL-17 pathways. The present study suggests that RAGE mediates functional DNA methylated modification in a cluster of 14 targeted genes, particularly hypomethylation in promoters of CXCL1, TLR6 and OSM, which might significantly contribute to CS-induced airway inflammation via a network of signaling pathways.


Introduction
Chronic obstructive pulmonary disease (COPD) is characterized by persistent respiratory symptoms and airflow limitation that are associated with persistent airway inflammation induced by cigarette smoke (CS), a major causative factor for COPD [1].
Receptor for advanced glycation end-products (RAGE), a membrane protein from the immunoglobulin superfamily, has been implicated in the pathogenesis of COPD [2].
Overexpression of RAGE contributed to airway inflammation in CS-associated COPD [3]. Our previous study further indicated knockout (KO) of RAGE gene significantly attenuated CS-induced airway inflammation in a mouse model [4]. However, the mechanisms regarding the effects of RAGE on airway inflammation in COPD remain not clear. Recently, some evidences implied DNA methylation, an epigenetic regulation, played important roles in RAGE-mediated inflammatory responses in various diseases [5][6][7], although the role in DNA methylated modification mediated by RAGE in airway inflammation in COPD was not reported.
In consequence, we did this study, using the established mouse model of CS-induced airway inflammation, to explore the underlying mechanisms of RAGE-mediated DNA methylated modification in airway inflammation in COPD.

Animal model
The animal model of CS-induced airway inflammation has been established in our former study [4]. C57BL/6 mice (7-9 week old, 20-22 g weight) were used to generate RAGE KO mice through CRISPR/Cas9 gene targeting technology by bioray biotechnology (Shanghai, China). The four experimental groups (n=3 mice per group) were included in the present study, as follows: i) wild-type (WT) group, ii) CS+WT group, iii) CS+KO group, iv) WT+KO. All mice were specific pathogen-free and kept on a 12-h light/12-h dark cycle, at a room temperature of 22±2℃, with free access to food and water. WT and RAGE KO mice were exposed to mainstream CS or room air for 2 h twice daily, 6 days per week for consecutive 4 weeks. After 4-week CS exposure, the mice were anesthetized intraperitoneally with pentobarbital sodium and sacrificed by femoral artery transection. The animal study was approved by the Panel on Laboratory Animal Care of West China Hospital of Sichuan University and took place at the Experimental Animal Center of West China Hospital of Sichuan University.

DNA extraction
The total DNA of lung tissues was extracted and purified by DNeasy Blood Tissue Kit (Qiagen), according to the manufacturer's instructions. Purified DNA was then quantified by NanoDrop 2000 Spectrophotometer (Thermo) and agarose gel electrophoresis. Only DNA samples with A260/280 ratio between 1.8 and 2.0 were used for further experiments.

Liquid hybridization capture-based bisulfite sequencing (LHC-BS)
Genomic DNA (1μg per sample) was randomly fragmented into approximately 200-300bp by sonication. After purification, the DNA fragments were repaired in the

DNA methylation data analyses
The Fastp software (version 1.2.1) was used to process methylated raw data and remove the low quality reads, including i) contaminated sequences; ii) the Q value of 3' end is less than 20; iii) reads with less than 15bp; iv) reads with 40% base Q value less than 15; v) reads containing N bases greater than 5 (Q =-10*log10(p), P is the probability of error) [8]. The clean reads were aligned to the reference genome using Bismark software (version 0.19.0) with bowtie2 (version 2.3.4.2) to attain the methylated type, status, and proportion [9]. Differential methylated regions (DMRs) were subsequently analyzed using the R package methylKit (version 1.6.1) [10] and eDMR [11], with an adjusted P-value <0.05 and absolute differential methylation levels (absolute meth. diff >5%). Finally, related differential methylated genes (DMGs) were located and annotated in the DMRs by the ChIPseeker software. The data of gene expression by cDNA microarray have been obtained in our former study [4]. In the present study, differential expressed genes (DEGs) were identified by fold-change (FC), and only genes that at least 1.2-fold upregulated or downregulated were analyzed.

Candidate genes selection
To identify the candidate genes with methylated modification mediated by RAGE, a novel intersection model was performed ( Figure 1). Briefly, CS-associated (CS+WT vs WT) and RAGE-associated (CS+KO vs CS+WT plus WT+KO vs WT) DMGs intersected to get the overlapped DMGs, and so did CS-associated and RAGE-associated DEGs (overlapped DEGs). Then, the overlapped DMGs and DEGs intersected again to select the candidate genes.

Functional methylated genes and hub genes selection
The candidate genes with functional methylated modification, also called functional methylated genes, were identified according to the reversed effects of methylation in promoters on gene transcription. To further identify the hub genes that were most correlated with other genes, protein-protein interaction (PPI) network analysis among the functional methylated genes was performed using the online database SRTING [12] and displayed using Cytoscape [13].

Functional enrichment analyses
Functional enrichment analyses on the functional methylated genes, as well as the hub genes, using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases were performed with the clusterProfiler package using P-value < 0.05 was set as the threshold.

Data production
Based on the LHC-BS method, 15 Gbp raw sequence data were generated on average for each sample. More than 79% were mapped to at less one genomic position, covering ~77% of the target regions, with an average of 57 × sequencing depth per CpG and 12.5% duplication data, and the duplicated sequence reads were filtered for the subsequent analyses.

CS-induced airway inflammation
After RAGE KO, as reported in our previous study, CS-induced airway inflammation was significantly improved [4]. Meanwhile, 90 overlapping candidate genes were identified via the intersection of DMGs with DEGs (Table S1). It is well-known that DNA methylation occurs almost in CpG islands that are primarily located in the regions of promoter and are negatively correlated with gene expression [14]. As a result, 14 genes from the 90 candidate genes, with functional methylated modification in promoters were identified (Table 1)  which is positively correlated with pulmonary function decline [23,27]. Noticeably, as suggested in our study, CXCL1, TLR6 and OSM have been documented to be targeted genes of DNA methylated modification in other diseases, such as schizophrenia [28], type 1 diabetes [29], and Richter syndrome [30].
However, some limitations in this study should be considered. First, the sample size of mice in each group was relatively small, although the minimum requirement for biological repeat was reached. Second, the novel intersection model might be theoretically imperfect. Third, validation was thus needed in future studies.
In summary, the present study performed intersections of DMGs with DEGs in a CS-exposed mouse model and indicated RAGE could mediate functional methylated modification in multiple targeted genes, especially CXCL1, TLR6 and OSM, which might significantly contribute to airway inflammation in COPD.