The Mechanism Of PM2.5 Exacerbates Th17/Treg Imbalance In COPD Mice Through AhR Pathway

Part 1:Exploring the impact of PM2.5 on Th17 cell differentiation based on transcriptomics and bioinformatics.Background and Objective:Fine particulate matter（PM2.5）is one of the main components of air pollution.Because the diameter of PM2.5 is small and usually accesses easily inhaled into the terminal airways and alveoli,and even penetrate into the alveolar space and ultimately into the blood circulation,causing airway inflammation,systemic inflammation,and occurrence of multiple chronic conditions.T helper（Th）17 cells are a class of effector T cells of that secrete Interleukin（IL）-17.IL-17 exerts pro-inflammatory effects in multiple diseases,which exacerbates the inflammatory responses and the patient’s condition.Various studies in vitro and in vivo have shown that PM2.5 could influence Th17 and Treg differentiation.After in vitro and in vivo experiments,bioinformatics has become the new means of biomedical research.This study aimed to evaluate the mechanism of PM2.5’s effect on Treg and Th17 differentiation.and provide new ideas for the influence of PM2.5 on Treg/Th17imbalance in multiple diseases.Method:Using keywords“PM2.5”and“T cell”,transcriptomes data and RNA sequencing（RNA-seq）data on the effects of PM2.5 on T cells in the NCBI Gene Expression Omnibus（GEO）database were searched.We searched transcriptome sequencing GSE124660 data sets and downloaded Original counts matrix papers.Network Analyst 3.0 was employed for difference analysis.Differential analysis was performed with the DEseq2 package.Screened conditions are|log₂FC|>1（FC,fold change）and P-value<0.05.GO,KEGG,and Wiki Pathway database metabolic pathways enrichment analyses were performedusing the R package,‘cluster Profiler’.Enrichment analysis in transcriptomic data was performed using gene set enrichment analysis（GSEA）.Protein-protein interaction（PPI）network of the aggregated DEGs was constructed using STRING database.Finally,a PPI network was visualized using Cytoscape software.We identified hub genes PPI network through Cyto Hubba plugin.Results:The study obtained RNA-seq datasets GSE124660.Gene differential expression analysis of data was performed with the DEseq2 package.A total of 919significant differences genes were obtained,contain 227 up-regulated genes and 524down-regulated genes.Function enrichment analysis showed that Go functions mainly concentrated on protein secretion,granulocyte chemotaxis,cell chemotaxis,leukocyte chemotaxis,positive regulation of cytokine production,regulation of peptide secretion,receptor complex,N-acetyllactosaminide beta-1,3-N-acetylglucosaminyltransferase activity,cytokine receptor binding,acetylglucosaminyltransferase activity,chemokine activity,acetylgalactosaminyltransferase activity and galactosyltransferase activity.KEGG pathway enrichment analysis demonstrated that differential genes were mainly enriched in cytokine-cytokine receptor interaction,hematopoietic cell lineage,glycosphingolipid biosynthesis lacto and neolacto series,cell adhesion molecules,chemokine signaling pathway,ECM-receptor interaction,MAPK signaling pathway,NF-kappa B signaling pathway,and PI3K-Akt signaling pathway.Wiki Pathway databases pathway analysis revealed differential genes were mainly enriched in Aryl Hydrocarbon Receptor,inflammatory Response Pathway,IL-6 signaling Pathway,T-Cell Receptor Co-stimulatory Signaling,and chemokine signaling pathway.GSEA analysis suggested that Th0 cells exposure to PM2.5,various signaling pathways are activated in Th0 cell,including protein processing in endoplasmic reticulum,IL-17signaling pathway,cytokine-cytokine receptor interaction,JAK-STAT signaling pathway,pentose and glucuronate interconversions,and gycosphingolipid biosynthesis lacto and neolacto series.MCC algorithm showed that the top 15 hub genes were Itgam,Cxcl10,Il2,Csf2,Cd40,Vegfa,Irf7,Usp18,Rtp4,Sdc1,Plcg1,Ccl4,Oasl2,Parp14,Ccl3.Conclusion:PM2.5 induced CD4⁺T cells and differentiation into Th17 cells,and its detailed mechanism might be correlated with activation of the IL-17,Jak/Stat and AhR signaling pathway.key genes involved in the process are Itgam、Cxcl10、Il2、Cd40、Usp18、Parp14.The study investigated molecular mechanisms of PM2.5induced CD4⁺T cells and differentiation into Th17 cells and provide new ideas and targets for the prevention and treatment of air pollution-related diseases.Part 2: Effect and mechanism of AhR pathway in PM2.5-aggravated Th17/Treg immune imbalance in mouse models of chronic obstructive pulmonary diseaseObjective: Chronic obstructive pulmonary disease（COPD）is a chronic inflammatory disease.The immune imbalance plays a key role in the pathogenesis of COPD.PM2.5 is closely associated with Th17/Treg imbalance.Detailed mechanisms are unclear.Several studies showed that AhR pathway plays a critical regulatory role in the regulation of Treg/Th17 balance.Whether PM2.5 through AhR signaling pathway the Th17/Treg balance is exacerbated in COPD remains unknown.This study aimed to explore the mechanisms of AhR signaling pathway exacerbating Treg/Th17 immune imbalance of COPD mice.Thus,this study provides new therapeutic targets and strategies for COPD treatment.Method: 40 BALB/c mice（8 weeks old）were randomized into four groups.COPD,control,COPD+PM2.5,COPD+CH（CH223191）,COPD+PM2.5+CH groups.A COPD mouse model was established by cigarette smoke（CS）exposure for 90 days.COPD+CH and COPD+PM2.5+CH mice groups were injected with CH（10 mg/kg body wt,intraperitoneally）once every other day for 3 weeks.We observed the general conditions of the mice after modeling,the mice underwent tracheal intubation and general anesthesia.Mice were sacrificed by cervical dislocation.The lung tissues of mice were fixed in 4% paraformaldehyde.The histopathology of the lung was assessed by performing HE staining.The lymphocytes were detached under sterile conditions.Flow cytometry analysis of the proportion of Treg and Th17 cells in CD4+T cells.We calculated ratios of Th17/Treg frequencies.Lung tissue RNA was extracted.Real-time quantitative PCR（RT-q PCR）was used to determine the mRNA levels of AhR,and CYP1A1 of Lung tissue.AhR and CYP1A1 protein expression was measured using Western blot analysis.Serum IL-17 A and IL-10 levels and bronchoalveolar lavage fluid（BALF）were detected by enzyme-linked immunosorbent assay（ELISA）.Results: 1.lung function assessment and pathological changes of the lung: Forced expiratory volume at one second（FEV1）/forced vital capacity（FVC）ratio,peak expiratory flow（PEF）,and mean mid-expiratory flow（MMEF）,50% tidal volume expiratory flow（EF50）,and mean linear interceptin（MLI）in COPD mice significantly decreased compared with the healthy control group.All results met the characteristics of COPD,the model is successful.2.Detection of Th17 and Treg cell subsets: Th17%（4.86±0.27）% in COPD group was significantly higher than that in healthy control group（1.57±0.21）%（P<0.01）,and Treg%（6.27±0.42）% was significantly lower than that in healthy control group（7.87±0.27）%（P<0.01）.Th17/Treg ratio in COPD group（4.55±0.40）was significantly higher than that in healthy control group（0.22±0.05）（P < 0.01）,suggesting Th17/Treg cell imbalance in COPD mice.After PM2.5 exposure,Th17%（5.84±0.24）% of COPD+PM2.5 group was further increased（P<0.01）,Treg% was further decreased（1.03±0.43）%（P<0.01）,and Th17/Treg ratio was further increased（5.29±1.67）（P<0.01）.It is suggested that PM2.5 can further aggravate the immune imbalance of COPD mice.After CH intervention,Th17% and Th17/Treg ratio [（2.05±0.17）%,（0.34±0.04）%],[（3.01±0.26）%,（1.50 ± 1.10）] in COPD+CH group and COPD+PM2.5+CH group were significantly lower than those in COPD group and COPD+PM2.5 group（both P < 0.01）;Treg%（3.58±0.30）% in COPD+CH group was significantly higher than that in COPD +CH group（P < 0.01）.3.IL-17 A and IL-10 in serum and BALF: The levels of IL-17 A in serum and BALF in COPD group [（77.19±6.28）,（29.04±3.32）] were significantly higher than those in healthy control group [（49.94±2.62）pg/ml,（14.90±3.88）pg/ml]（P<0.01）.The level of IL-10 [（572.30±26.20）pg/ml,（210.60±19.36）pg/ml] was significantly lower than that of healthy control group [（648.10±41.66）pg/ml,（268.90±10.22）pg/ml]（P<0.01）.IL-17A/IL-10 in serum and BALF ratio [（0.19±0.04）,（0.14±0.01）] compared with healthy controls [（0.08±0.01）,（0.06±0.01）] in increased significantly;After exposure to PM2.5,The IL-17 A content [（100.30±7.18）pg/ml,（36.20±3.97）pg/ml] and the ratio of IL-17A/IL-10 [（0.44±0.13）,（0.21±0.02）] in serum and BALF of mice in COPD+PM2.5 group were further increased（P<0.01）.Content of IL-10 [（226.30±46.18）pg/ml,（171.10±13.38）pg/ml] further reduced（P<0.01）;After CH intervention,IL-17 A content in serum and BALF of mice in COPD+CH group and COPD+PM2.5+CH group [（65.06±8.00）,（17.54±3.46）],[（86.67±6.81）,（33.22 + 2.70）] and the ratio of IL-17 A / IL-10 [（0.13 ± 0.03）,（0.26±0.09）],[（0.07±0.01）,（0.16±0.01）] are decreased compared with respective control groups（P<0.05）,but the content of IL-10[（510.80±15.13）,（379.70±18.89）],[（235.90±34.13）,（211.40±16.13）] was significantly higher than respective control groups（P<0.05）.4.mRNA expression of AhR and CYP1A1: The mRNA expression of AhR and CYP1A1 in lung tissue of COPD mice [（1.90±0.08）,（2.49±0.33）] was significantly increased compared with that of healthy control mice（1.00±0.00）（P<0.01）.The mRNA expressions of AhR and CYP1A1 in PM2.5 group were significantly increased（2.43±0.26）and（4.64±0.25）compared with healthy control group and COPD group（P<0.05）.After CH intervention,mRNA expressions of AhR and CYP1A1 in COPD+CH group and COPD+PM2.5+CH group [（1.46±0.31）,（1.58±0.26）],[（1.79±0.23）,（2.63±0.34）] were significantly decreased compared with the control group（P<0.05）.5.Protein expression of AhR and CYP1A1: Relative protein expression levels of AhR and CYP1A1 in COPD mice [（0.64±0.01）,（0.78±0.02）] were significantly higher than those in healthy control mice [（0.46±0.04）,（0.43±0.01）]（P<0.01）.The protein relative expression levels of AhR and CYP1A1 in PM2.5 group [（0.84±0.03）,（1.08±0.02）] were further increased compared with that in COPD group（P<0.01）.After CH intervention,the relative expression levels of AhR and CYP1A1 protein in COPD+CH group and COPD+PM2.5+CH group [（0.48±0.01）,（0.37±0.01）,（0.65±0.03）,and（0.76±0.03）] were significantly decreased compared with control group（P<0.01）,respectively.6.Correlation analysis: AhR mRNA and protein in each group were positively correlated with Th17% and Th17/Treg ratio（P<0.05）,and negatively correlated with Treg%.CYP1A1 mRNA and protein were positively correlated with Th17% and Th17/Treg ratio（P<0.05）,and negatively correlated with Treg%（P<0.05）.Conclusion: Cigarette smoke exposure alone can establish a mouse model of COPD,and exposure to PM2.5 exacerbates the Th17/Treg immune imbalance in COPD mice.The AhR pathway plays an important role in PM2.5-induced immune imbalance,and the AhR inhibitor CH223191 can partially improve Th17/Treg of COPD mice caused by PM2.5.These findings provide new insights into the pathogenesis and potential therapeutic targets for COPD.