Functional Classification Of Dendritic Cells And The Potential Role In Atherosclerosis: Based On Single-Cell Technologies

As the most potent professional antigen-presenting cells,dendritic cells（DCs）can capture,process and present antigens to T cells.In this way,DCs provide an important link between the innate and adaptive systems and play crucial roles in immune system.DCs can be divided into different subsets based on different phenotypic markers and show great phenotypic heterogeneity in vivo.It was reported that DCs of the same subset participate in different immune responses under different physiological and pathological conditions,and show different functions through a series of functional-related molecules,which suggest functional heterogeneity of DCs.However,functional classification of DCs is still not clear for their rareness in tissues and limitation of detection.DC heterogeity based on important functional markers may lay the foundation for deep exploring the roles of DCs in different physiological conditions and inflammatory diseases.Using flow cytometry,single-cell qPCR（sc-qPCR）and bioinformatic analysis,a method was established to identify DC classification based on not only phenotypic but also functional markers.Firstly,48 target genes were selected,including classic phenotypic markers and important functional markers of DCs（i.e.,maturation,migration,adhesion,antigen recognition,lipid uptake and T-cell stimulation）.The primers of the target genes were designed by Primer Premier 3.0,and the specificity and linearity of these primers were validated.Then,liver was selected as the representative of non-lymphoid tissue and spleen was chosen as the representative of lymphoid tissue.Using a FACS Aria II machine,single CD11c⁺MHCII⁺DCs from the liver or spleen of C57BL/6 mice were sorted.The expression profiles of 48 target genes of the sorted single DCs were obtained by Biomark high-throughput gene analysis system.Futhermore,bioinformatic analysis,including hierarchical clustering,principal component analysis,phenotypic classification and functional classification,was performed with the R software.At last,this method was applied to analyze the liver and spleen DCs of the wild type（WT）and atherosclerotic mice.Immunofluorescence was used to confirm the expression of CCR7,a key migration-related molecule.Under physiological condition,DCs in mouse lymphoid tissue and non-lymphoid tissue showed that:1)In the liver DCs,phenotypic genes,i.e.BST2 and XCR1 were increased;scavenger receptor MSR1was enriched,revealing greater lipid uptake ability;chemokine receptors CCR1,CCR2 and CCR4 were increased,indicating enhanced migration properties.2)In the spleen DCs,phenotypic gene SIRPA was enriched;costimulatory molecule CD80 was upregulated,suggesting a greater maturation state;Toll-like receptor（TLR）adaptor molecule MYD88 was increased,revealing an increased ability to activate T cells;Toll-like receptor TLR9 was elevated,which indicated increased pathogen recognition.3)Immunofluorescence results showed CCR7,a key migration-related molecule,was slightly upregulated in liver DCs.These results indicate that the mouse spleen DCs and liver DCs showed obvious phenotypic and functional heterogeneity under physiological condition.Migration and maturation are the core functions that discriminate non-lymphoid tissue and lymphoid tissue DCs.CCR1,CCR2,CCR4 and CCR7,as the important genes indicating migration heterogeneity,and CD80,as the key gene suggesting maturation state,are expected to become important targets for exploring functional heterogeneity of DCs.DCs in the atherosclerotic mouse model showed that:1)compared with the WT mice,phenotypic genes ITGAM and DC-SIGN were enriched in the ApoE^-/-liver DCs;costimulatory molecule CD40 was upregulated,suggesting a greater maturation state;scavenger receptor OLR1 was elevated,revealing enhanced lipid uptake;chemokine receptors CCR2 and CX3CR1 were increased,and CCR7 was down-regulated;TLR adaptor MYD88 was upregulated,which indicated increased T-cell stimulation;adhesion molecule CDH1 was down-regulated,suggesting decreased adhesion property.2)In ApoE^-/-spleen DCs,expression of LY6C and BST2 was increased;chemokine receptor CCR1 was upregulated,suggesting increased migration;Toll-like receptor genes TLR7 and TLR9 were enriched,which indicated increased antigen recognition;costimulatory molecule CD80was down-regulated,suggesting a less maturation state.3)Immunofluorescence results showed CCR7 was upregulated in ApoE^-/-spleen and aortic DCs,and ApoE^-/-liver DCs exhibited a down-regulation of CCR7,which was consistent with the previous qPCR analysis.These results indicate that compared with the WT mice,the spleen DCs and liver DCs from the atherosclerotic mice showed obvious phenotypic and functional heterogeneity.Migration and maturation are the core heterogeneity functions between WT and ApoE^-/-mice.CCR1,CCR2,CCR7 and CX3CR1,are the important genes indicating migration heterogeneity of DCs in the ApoE^-/-mice,while CD40 and CD80,are the key genes suggesting the maturation state of DCs in the ApoE^-/-mice.Decreased expression of CCR7 in ApoE^-/-liver DCs may indicate impaired migration property under sustained hyperlipidemia conditions,which remain to be explained.In our approaches,functional heterogeneity was brought into DCs classification,which was established to classify DCs based on important functional markers.By a combination of flow cytometry,sc-qPCR and bioinformatic analysis,the 6 important functions of DCs under physiological and pathological conditions were explored.Our study showed that migration and maturation are the core functions that discriminate DCs in non-lymphoid tissue and lymphoid tissue,and in the WT and atherosclerotic mice.Important function-related genes were suggested.Our study provided new insights into understanding the molecular mechanism of immune responses during physiology and pathology process,and provided new target molecules for study and therapy based on DCs.