1. Transcriptomic profiling unveils immune dysregulation in IMQ-induced lupus model via single-cell RNA sequencing. Utilizing scRNA-seq, we have provided an unprecedented comprehensive characterization of the cellular landscape in the spleens of a lupus animal model induced by IMQ. After rigorous quality control, a total of 33,091 splenic cells were available for downstream analysis, encompassing 21,321 cells from three IMQ-induced lupus model mice and 11,770 cells from two control mice. Based on classical gene expression markers, the cells were categorized into eight distinct subgroups, including CD4/CD8 T cells (CD3d), B cells (CD79a), dendritic cells (DCs, Flt3), plasma cells (Jchain), neutrophils (S100a8/Mki67), macrophages (C1qa), and monocytes (Ace/Vcan) (Fig. 1A and C). Each cell type displayed unique gene expression profiles (Fig. 1B and C). Notably, the proportions of plasma cells, macrophages, and neutrophils in the splenic tissue of lupus model mice (3.14%, 8.37%, 58.68%, respectively) were significantly higher than those in the controls (0.96%, 2.84%, 7.99%, respectively) (Fig. 1B), implicating these cell types in the pathogenesis of lupus. In comparison to the control splenic cells, there was a decrease in the proportion of T cells and B cells in the model mice. Collectively, prioritized DEGs were plotted for each cell type, demonstrating extensive transcriptomic alterations between the lupus model mice and the controls (Fig. 1E).
2. Elucidating the splenic B cell heterogeneity and pathway enrichment in an IMQ-induced lupus model. Following the separation and re-clustering of B cells from the combined pool, we identified seven B cell subtypes within the spleen based on expression markers Stmn1, Mki67, Xbp1, Jchain, Il7r, Vpreb3, Cr2, Plac8, Ms4a1, and Ighd. These subtypes included Follicular_B_cells, Follicular_B_cycling, MZ_B_cells, pre-pro_B cells, pre-pro_B_cycling, Plasma, and Plasma_cycling cells (Fig. 2A, B). The composition of B cells within the splenic tissue exhibited distinct differences between the lupus model and the control groups. Novel populations of pre-pro_B cells, pre-pro_B_cycling, and Plasma_cycling cells, absent in the control, were identified in the model group (Fig. 2C, D), highlighting their potential importance in the IMQ-induced lupus mouse model. We observed an elevated proportion of Follicular_B_cells, Plasma, and Follicular_B_cycling cells in the model group, whereas MZ_B_cells were more prevalent in the control group (Fig. 2C, D). Subsequent KEGG pathway and GO enrichment analyses revealed that pathways such as "B cell receptor signaling," "response to alkaloid," and "rRNA processing" were predominantly upregulated in Plasma cells. In contrast, pathways like "retrograde protein transport, ER to cytosol" and "endoplasmic reticulum to Golgi vesicle-mediated transport" were chiefly enriched in Plasma cells of the model group as compared to the controls (Fig. 2E). Plasma_cycling cells in the model group were primarily enriched for "adaptive immune response," "protein autoubiquitination," and "mRNA splicing, via spliceosome" pathways. Follicular_B_cells and MZ_B_cells in the model group were predominantly associated with "immune system process" pathways. To further explore the origins and development of Follicular_B_cells in the model group, RNA velocity analysis was performed, suggesting that lupus model group Follicular_B_cells may derive from MZ_B_cells (Fig. 2F).
3. Identification of T Cell Subtypes and Immune Pathways in the Spleen Reveals Key Insights into Lupus Pathogenesis in Mice. Based on the expression of Stmn1, Mki67, Foxp3, Ikzf2, Lag3, Pdcd1, Id2, Cxcr6, Cxcr3, Gzmk, Ccl5, Cd44, Ccr7, and Sell, we identified nine T cell subtypes in the spleen, including CD8 TN cells, CD8 TCM cells, CD4 TEM cells, CD8 TEM cells, CD4 TN cells, T cycling cells, Treg cells, Tfh cells, and CD4 TRM cells (Fig. 3A, B). In the model group, the proportions of CD4_TEM, CD8_TEM, T_cycling, Tfh, and CD4_TRM cells were higher, whereas those of CD8_TN, CD8_TCM, and CD4_TN cells were relatively lower (Fig. 3C, D). GO and KEGG analyses revealed that pathways related to "adaptive immune response" and "immune system process" were highly enriched in CD4 TEM cells in the model group, indicating that immune system activation is involved in the pathogenesis of lupus in mice. The most enriched pathways for CD4 TN, CD4 TRM, CD8 TCM, CD8 TN, and T cycling cells were those related to "translation" (Fig. 3E and Extended Data Fig. 1A-C, E). Trajectory analysis suggested that T cycling cells may differentiate into CD4 TEM cells, and CD4 TN cells may differentiate into Tfh cells (Fig. 3F).
4. Identifying dendritic and neutrophil cell subtypes in the spleen reveals immune system activation in an IMQ-induced lupus model. Based on the expression of CD209a, Itgax, Clec10a, Cd300a, Ccl5, Ccr7, Clec9a, Xcr1, Tcf4, and Siglech, we identified five subtypes of dendritic cells (DCs) within the spleen, including CD209a_cDC2, Itgax_cDC2, Ccr7_cDC1, Clec9a_cDC1, and pDC cells (Fig. 4A, B). Based on the expression of Ccl6, Csf3r, Retnlg, Mmp8, Camp, Ltf, Elane, and Mpo, four groups of Neutrophil cells were identified in the spleen, including G1_Neu, G2_Neu, G3_Neu, and G4_Neu (Fig. 5A, B). We observed a significant increase in the expression of CD209a_cDC2 in the model group (Fig. 4C, D), with G2_Neu levels significantly elevated and G4_Neu expression reduced in the model group (Fig. 5C, D). The results of GO and KEGG analyses indicated that "immune system process" pathways were highly enriched in CD209a_cDC2 cells of the model group, suggesting the involvement of immune system activation in the IMQ-induced lupus model. Trajectory analysis showed that CD209a_cDC2 might differentiate into Itgax_cDC2 (Fig. 4F), and G1_Neu might differentiate into G2_Neu, G3_Neu, and G4_Neu (Fig. 5F).
5. Enhanced Immune Cell Interactions and Regulatory Network Analysis in an IMQ-Induced Lupus Mouse Model. Understanding the networks of intercellular communication will aid in elucidating the cellular mechanisms of disease pathogenesis. We utilized CellChat to explore the presumed interactions among major cell types in both model groups and controls. The results indicated altered activities of pathways including BAFF, IL1, C-X-C motif chemokine ligand (CXCL), BTLA, IL6, TNF, and VISFATIN in the model group (Fig. 6A and Extended Data Fig. 2). Within the model group, the ANGPTL signaling pathway was enriched from CD4 T cells to DCs, macrophages, monocytes, and neutrophils. The BAFF signaling pathway was enriched from macrophages to B cells and plasma cells. The GALECTIN signaling pathway was enriched from CD4 T cells to monocytes and plasma cells. The IL1 signaling pathway was enriched from macrophages to CD4 T cells. However, the activities of ANNEXIN, BMP, and CXCL pathways were reduced in the model group compared to controls (Fig. 6A and Extended Data Fig. 2). To further investigate the pathways involved in immune system activation, we explored the interactions between different immune cells. In the model group, interactions among macrophages, DCs, and monocytes were notably enhanced (Fig. 6B). Interactions among CD8 T cells, B cells, plasma cells, CD4 T cells, and neutrophils were weaker, yet the interactions of neutrophils and CD4 T cells with macrophages, DCs, and monocytes were intensified. The interactions of CD8 T cells, B cells, and plasma cells with other cells showed no significant change (Fig. 6B). These results suggest an enhancement in innate immune functions and signal transmission among CD4 T cells in the IMQ-induced lupus mouse model. To identify transcription factors (TFs) that regulate differential gene expression across different cell populations, we employed single-cell regulatory network inference and clustering analysis. This analysis revealed an upregulation of Foxp3, Lef1, Eomes, and Tcf7 in T cells, and increased expression of Cebpa, Ehf, Nr1h3, Tcf7l2, Mafb, Spic, and Hoxb5 in macrophage cells. In neutrophils, Cebpb, Foxg1, Klf5, Cebpe, Cebpd, and Pbx1 were significantly enriched (Fig. 6C).