Dynamic Expression Of MicroRNAs In The M2b Polarized Macrophages Associated With Systemic Lupus Erythematosus

Macrophage polarization has been found contributing to the initiation andperpetuation of systemic lupus erythematosus (SLE). Increasing studies revealed thatthe M2b macrophages have a role in causing SLE disease directly. Our previous studyhas also demonstrated that M2b polarized macrophages induced by activatedlymphocyte-derived DNA (ALD-DNA) play a crucial role in the initiation and progressof SLE disease. Accumulating evidences suggested that microRNAs (miRNAs) mightserve as critical regulators to control macrophage polarization. However, the regulationof miRNAs in the macrophage M2b polarization of SLE is not very clear. Besides, thedynamic patterns of miRNAs in the M2b polarized macrophages are still elusive.In our study, to investigate the regulation of miRNAs in the macrophage M2bpolarization of SLE, murine BMDMs were treated with ALD-DNA for different timeperiods (0,6, and36h). Consistent with our previous studies, the macrophages treatedwith ALD-DNA displayed the M2b phenotype in vitro. Further analysis of themacrophage polarization at different time points showed that the BMDMs has done ofM2b polarization6h after stimulation, whereas the expression of the M2b marker geneswere declined after36h, indicating that ALD-DNA induced macrophage polarizationstatus changes over time.To investigate miRNA expression patterns associated with macrophage M2bpolarization dynamically, miRNA microarray of murine BMDMs following stimulationwith ALD-DNA for6h and36h was performed. Quantitative real-time PCR wasutilized for validation of miRNA expression changes. Cluster analysis of the microarraydata was conducted with the Mfuzz package and unsupervised hierarchical clusteringmethod. Ingenuity Pathway Analysis (IPA) and Gene Ontology (GO) analysis were performed to investigate the functional relationships between differentially expressedmiRNAs.Compared to untreated macrophages, expression of over11%of the1111miRNAsappeared differentially expressed in ALD-DNA induced macrophage M2b polarization,suggesting that the majority of miRNAs were not affecting the macrophage M2bpolarization. Cluster analysis revealed several patterns of miRNAs expression duringmacrophage polarization, revealing that miRNA expression levels have a time coursereproducible dynamic changes. Analysis of the structure of the network showed the predictedfunctions of the differentially regulated miRNAs at6h time-point were significantly associated withinflammatory response and inflammatory disease, it supports the conclusion from the GOanalysis that6h-altered miRNAs were involved in the signaling cascade of macrophagepolarization. However, the differentially regulated miRNAs identified at36h time-point weresignificantly related to cell proliferation by biological network analysis, this was consistent withthe conclusion from the GO analysis of the36h-altered miRNAs. In conclusion, therewas great difference between the bio-functions of the two time-points of the ALD-DNAexperiment, indicating that the differentially regulated miRNAs in6h with ALD-DNAstimulation were more specific for M2b polarization.To further define the miRNAs that may contribute to macrophage M2b polarization,we compared the miRNA microarray data sets of the ALD-DNA experiment to those ofthe UnALD-DNA experiment. In the6h ALD-DNA versus6h UnALD-DNA,50miRNAs were differentially regulated,28miRNAs were differentially regulated in the36h ALD-DNA versus36h UnALD-DNA, these indicated that the difference betweenthe miRNA expression profile of the ALD-DNA and UnALD-DNA experimentdecreased with time. The most impacted biological functions for the differentiallyregulated miRNAs in the6h ALD-DNA versus6h UnALD-DNA were inflammatorydisease and inflammatory response. This was in accordance with our finding of themiRNAs that specific to6h-ALD-DNA datasets. However, the biological functions suchas cancer, cell proliferation and growth were predicted to significantly associated withthe differentially regulated miRNAs in the36h ALD-DNA versus36h UnALD-DNA. It supports the conclusion of the miRNAs that specific to36h-ALD-DNA datasets. Theseresults also indicated that the differentially regulated miRNAs in6h with ALD-DNAstimulation were more specific for M2b polarization.In this study, we have for the first time described the dynamic miRNA expressionpatterns and pathway analysis in ALD-DNA induced macrophage M2b polarization,these data provide us not only a better understanding of miRNA mediate macrophagepolarization but also the future therapeutic potential of targeting miRNAs in SLEpatients.