Single-cell Transcriptome Study On Cutaneous Lesions Of Lupus Erythematosus

Lupus erythematosus（LE）is a severe and heterogeneous autoimmune disease characterized by the immune cells,especially T and B cells lacking of tolerance of self-antigens,the formation of autoreactive antibodies and the deposition of the complex which eventually leads to damage to one or more tissues or organs such as the skin,kidneys,joints,and nervous system.Clinically,LE is divided into two categories,one is only manifested as damage to skin tissue and does not involve systemic damage,which is called CLE,and the other is manifested as damage to multiple tissues and organs throughout the body such as kidneys,blood,joints,and is called SLE.DLE,which is featured by erythema,telangiectasia,follicle plugging and scarring,is the most common type of CLE.More and more research evidences show that IRF5 mutation,IFN regulatory factors,p DCs,Th1 cells and B cell abnormalities may be related to the development of LE.Recent studies on the mechanism of skin damage in lupus erythematosus have shown that abnormal circulating inflammatory cells and a large number of Ig G autoantibodies are closely related to SLE skin damage,but there is little evidence that serum autoantibodies and circulating inflammatory cells are involved in the pathogenesis of CLE,especially DLE.Thus,the pathogenesis and molecular/cellular mechanisms of DLE and SLE are still far from clear.scRNA-seq,which has been widely used since its introduction in2009,provides an unbiased way to define cell types based on the transcriptome of single cells in healthy or disease.sc RNA-seq can be used to define skin cell composition and discover new or rare cell populations.At present,sc RNA-seq studies have described the heterogeneity of PBMC in SLE patients.However,there is no report on the cutaneous heterogeneity of DLE and SLE.Objective:（1）To detect the cell composition and their functions between DLE and SLE skin lesions,and compare them with healthy controls through sc RNA-seq.Methods:（1）We collected the skin tissues of 23 lupus erythematosus patients and age-sex-matched HCs,separated their epidermis and dermis,and made a single-cell suspension which was then sequenced on the 10×Genomics Chromium system.（2）The sequencing data was first compared with the GRCh38genome by Cell Ranger,and the downstream data analysis was performed with the Seurat package,including quality control,data homogenization and standardization,finding variable genes,principal component analysis,data dimensionality reduction,and calculating DEGs,further defining cell types and comparing the proportion of each cell type in HC,DLE and SLE.（3）The cluster Profiler package was adopted for GO functional analysis,and the DEGs were calculated for the expanded cell subgroups in skin stromal cells,and the enrichment analysis of GO biological processes was performed based on the differentially expressed genes of the expanded skin stromal cells.For the immune cell population,the DEGs of the immune cells were calculated in groups of HC,DLE and SLE,and then the GO enrichment analysis was performed.The results of the GO analysis were visualized using the ggplot2 package.（4）Monocle2 was used to infer the pseudotime differentiation trajectory between some expanded skin stromal cell populations.The genes calculated by the Find Allmarkers function in the Seurat package were filtered by pct.1>0.5 and pct.2<0.5,and then used for speculating pseudotime trajectory.（5）We compared the immune cell subsets identified in this study,such as T cell subclusters and B cell subclusters with previously reported PBMC sc RNA-seq data of patients with SLE.The similarity analysis were performed on the corresponding immune cell subclusters based on their DEGs.（6）Cell Phone DB was used to analyze the cell communication between various cell types in skin tissues of HC,DLE and SLE.Each cell type was randomly arranged 1000 times,and the average expression level of each ligand-receptor pair was calculated,and the P value was further represented to screen out meaningful pair of ligand-reptors.（7）We adopted multi-color fluorescence immunohistochemistry to verify the increase of T cells,B cells and NK cells in cutanious of DLE and SLE,to verify the expressions of CCL20 in epidermal keratinocytes and CXCL1 in dermal fibroblasts,and to verify the increased expression of CCL19＿CCR7 in the fibroblasts or macrophage/DC cells of DLE and SLE.Results（1）The cell proportions analysis found that the ratio of T cells,B cells and NK cells in DLE and SLE skin lesions increased compared to HC,there are significantly more numbers of T cells,B cells and NK cells in dermis of DLE than HC and SLE.Multicolor fluorescence immunohistochemistry verified that the proportion of T,B and NK cells in skin lesions increased in DLE.（2）Sub-clustering analysis of keratinocyte identified 6 types of keratinocyte subpopulations,among which DLE and SLE-derived cells accounted for more than 75%of Diff kera-3,4,5,6 and CCL20⁺keratinocytes.Multicolor fluorescence immunohistochemistry verified the expansion of CCL20⁺keratinocytes in the epidermis of DLE and SLE.DEGs analysis showed that Diff kera-4 highly expressed interferon-stimulated genes,chemokines CXCL10 and CXCL11,and antigen processing and presentation-related molecules HLA-DRA,HLA-DRB;Diff kera-6 highly expressed CCL4,CCL5 and CXCR4,suggesting that these two groups of keratinocytes may play an important role in chemotaxis and activation of epidermal immune cells.Pseudotime trajectory analysis suggested a potential differentiation trajectory from Diff kera-2 to Diff kera-1,Diff kera-6,and finally towards terminally differentiated keratinocyte,or CCL20⁺kera and Diff kera-4.（3）Sub-clustering analysis of fibroblast defined 4 major fibroblast subpopulations,among which cells derived from SLE accounted for more than 75%of CXCL1⁺Fib,HLA⁺Fib1,pericyte 2 and Fib4 cells.The genes expression of CXCL1 and HLA-DRB1 in fibroblasts of DLE and SLE were higher than that of HC.Multiple fluorescence immunohistochemistry verified the increased expression of CXCL1 and HLA-DRB1 in fibroblasts of DLE and SLE.GO analysis showed that HLA⁺Fib1 and CXCL1⁺Fib were enriched in leukocyte adhesion and regulation of leukocyte activity pathways,while Pericyte2 and CXCL1⁺Fib separately enriched in the response to typeⅠinterferon and response to virus.The pseudotime trajectory analysis suggested the potential differentiation trajectories from Fib2 to HLA⁺Fib1,HLA⁺Fib2,and finally to part of Fib1 or CXCL1⁺Fib.（4）Epidermal T cell sub-clustering analysis divided epidermal T cells into 7 T cell subclusters,among which DLE and SLE cells account for more than 75%of epidermal T＿SC1 to T＿SC5,and dermal T cell subclustering analysis divided dermal T cells into 7 subclusters,DLE and SLE cells account for more than 75%of other dermal T cell subclusters except for dermal T＿SC2.DEGs analysis showed that epidermal T＿SC1and dermal T＿SC5 highly expressed the interferon-stimulated gene IFI6,IFI44,IFIH1 and DDX58,these two subclusters were highly similar to T-SC4 which were identified in PBMC of SLE patients by sc RNA-seq.Epidermal T＿SC3 and dermal T＿SC3 both highly express Treg-related genes（IKZF2,IL2RA and FOXP3）,and epidermal T＿SC2 and dermal T＿SC5 both highly expressed Th1 cell marker genes（IFNG and TBX21）and cytotoxic T cell-related genes（XCL2,XCL1 and GZMA）.GO pathway enrichment analysis showed that epidermal T cells of DLE and SLE were enriched in type I interferon pathway,while GO enrichment analysis of dermal T cells showed that regulating T cell activation and cell adhesion were simultaneously enriched in T cells of HC,DLE and SLE,dermal T cells of DLE enriched in response to the virus,while dermal T cells of SLE enriched in the type I interferon signaling pathway.（5）Epidermal B cell were divided into 5 subclusters,and almost all the cells of epidermal B cell subclusters came from DLE and SLE,and dermal B cell were divided into 7 subclusters,and more than 75%of the cells in each dermal B cell subclusters came from DLE and SLE.DEGs analysis showed that epidermal B＿SC3 and dermal B＿SC4,B＿SC5highly expressed interferon-stimulated genes,and also highly expressed plasma cell-related genes（CD38,IGHG4,IGHG1）,and they were highly similar to the plasma cell population P-SC0 which was previously identified in PBMCs of SLE patients by sc RNA-seq.GO analysis showed the epidermal B cells of DLE and SLE enriched in type I interferon signaling pathway,the response to interferon gamma,regulation of innate immune cell response,immune response signaling pathway involved in neutrophil activation and leukocyte adhesion pathway,and epidermal DLE B cells enriched in the autophagy pathway.Unlike to epidermal B cells,dermal B cells in HC,DLE,and SLE enriched in the response signaling pathway to interferonγ,while only SLE dermal B cells enriched in type I Interferon signaling pathway.（6）Epidermal Macro/DCs cells were divided into 6 subclusters,among which more than 75%of the cells came from DLE and SLE in the other 5 epidermal Macro/DCs subclusters except epidermal M＿SC5.Dermal Macro/DCs were divided into 7 subclusters,except for dermal M＿SC3,M＿SC5 and M＿SC6,more than 75%of the cells in the other 4subclusters came from DLE and SLE.DEGs analysis showed that epidermal M＿SC1 and dermal M＿SC6 highly expressed interferon-stimulated genes,epidermal M＿SC2 and dermal M＿SC3 highly expressed p DC-related genes（JCHAIN and MZB1）.GO enrichment analysis showed that antigen processing and presentation,T cell activation and the immune response pathways involved in granulocyte activation were enriched in the epidermal Macro/DCs of HC,DLE and SLE,while only the epidermal Macro/DCs of SLE were enriched in type I interferon-related pathways.The dermal Macro/DCs of HC,DLE and SLE enriched in antigen processing and presentation and T cell activation,while dermal Macro/DCs of DLE and SLE also enriched in the production of type I interferon,in response to viruses.Dermal Macro/DCs of SLE enriched in autophagy pathway.（7）Sub-clustering analysis of epidermal and dermal NK cells separately generated 4 epidermal and 4 dermal NK subclusters.Except for epidermal N＿SC3 and dermal N＿SC3,more than 75%of the cells in the other NK cell subclusters came from DLE and SLE.Interferon-stimulated genes were highly expressed in dermal NK cells of DLE and SLE.Epidermal NK cells of DLE and SLE enriched in typeⅠinterferon signaling pathway,while only dermal NK cells of SLE enriched in typeⅠinterferon signaling pathways and regulation of inflammatory responses,dermal NK cells of DLE enriched in response to viruses and regulation of cytokine production.（8）Cell communication analysis revealed that the cell communication scores of epidermal keratinocytes and Macro/DCs cells of DLE and SLE were significantly higher than those of HC,and the score of those cells in DLE was higher than that of SLE.The communication scores of dermal fibroblasts,endothelial cells and Macro/DCs of DLE was also higher than that of SLE and HC.The average expression levels of CCL20＿CXCR3,TNFSF9＿TNFRSF9 and SPN＿ICAM1 in the epidermal keratinocytes and immune cells of DLE and SLE were significantly higher than those in HC,while the average expression level of CCL19＿CCR7,CCL19＿CCR2,CCL19＿CXCR3,CXCL12＿CXCR4,FAM3C＿CLEC2D,TNFSF13B＿CD40 in dermal fibroblasts and immune cells in DLE was higher than those in SLE and HC.Multicolor fluorescence immunohistochemistry verified that the expression of CCL19＿CCR7 in fibroblasts＿Macro/DCs was increased in DLE and SLE.Conclusions（1）The proportions of immune cells such as T cells,B cells and NK cells in skin lesions of DLE and SLE are higher than those in HC,and these immune cell proportions of DLE are higher than those of SLE.（2）CCL20⁺keratinocytes,ISG^hiCD4/CD8 T cells,ISG^hiplasma cells,p DC and NK cell subsets expand in cutaneous lesions of DLE and SLE.The genes expression of CXCL1 and HLA-DRB1 are increased in dermal fibroblasts of DLE and SLE.However,there are no significant differences between DLE and SLE.（3）The type I interferon signaling pathway is enriched in multiple epidermal and dermal immune cells of SLE and DLE,and targeting this signaling pathway may be a potential therapeutic target for LE.（4）The cell communications in epidermal cells of DLE and SLE are significantly higher than those of HC,among which the cell communications in DLE is higher than those in SLE.The cell communications of dermal cells in DLE are higher than those in SLE and HC.