Differences between cell profiles of different patients
We explored the immune PBMC from individuals with rheumatoid arthritis (RA, Group A), rheumatoid arthritis combined with typical interstitial pneumonia (RA + UIP, Group B), and rheumatoid arthritis combined with nonspecific interstitial pneumonia (RA + NSIP, Group C) through single-cell sequencing. Our findings revealed 14 distinct cell clusters (Figure.1A). By identifying marker genes, we categorized cells into 10 subgroups (T cell, NK cell, Neutrophils, Red blood cell, B cell, Monocyte, Macrophage, Dendritic cell, Platelet, Hematopoietic stem cell) (Figure.1B). UMAP visualization across groups highlighted T cell, NK cell, Neutrophils, Red blood cell, and B cell as the top five cell types. In comparison to Group A, Groups B and C showed increased T cell populations, while Group C displayed an increase in NK cells. Neutrophils and Red blood cells predominated in Group A, and B cells were most abundant in Group B (Figure.1C, G), warranting further investigation. Cell annotation marker genes, including CD8A, TRGC2, LTB, LEF1, PTGDS, GNLY, S100A9, LYZ, HBA1, HBB, IGHM, BANK1, IGKC, IGHA1, GZMK, KLRB1, IKZF2, C15orf53, LST1, ALF1, S100A8, MMP9, TUBA1B, STMN1, HLA-DRA, CST3, PPBP, PF4, TCF4, IRF8, showcased relative expression in cell groups and types through violin plots, scatter plots, and heat maps (Figure.1D-F). In pursuit of delving into evolutionary patterns of blood cells in RA and RA + UIP, RA + NSIP, we performed functional enrichment and subgroup analysis onto T cells, NK cells, and B cells.
RA-relevant T cell functional enrichment and subgroup analysis
In T cell category, a comparison within Group A and Group B unveiled noteworthy changes in gene expression for Group A (Figure.2A). GO analysis enrichment highlighted the top 10 significant enrichments. Biological processes (BPs) were primarily linked to activities such as cytoplasmic translation, B cell activation, and the processing and presentation of peptide antigens. Cellular components (CCs) were tied to the cytosolic ribosome, ribosomal subunit, and ribosome. Molecular functions (MFs) were engaged in the structural constituents of the ribosome, antigen binding, and peptide antigen binding (Figure.2A). The KEGG pathway analysis indicated that both Group A and Group B were notably associated with pathways such as Antigen processing and presentation, Viral myocarditis, Th17 cell differentiation, Inflammatory bowel disease, and Rheumatoid arthritis (Figure.2A). Figure.2B illustrated the comparison amid Group A and Group C. GO enrichment revealed terms associated with lymphocyte-mediated immunity, leukocyte-mediated immunity, and the immune response-regulating cell surface receptor signaling pathway. Concerning CCs, enrichment was observed in areas external to the plasma membrane, the MHC class II protein complex, and the MHC protein complex. MFs were related to MHC protein complex binding, antigen binding, and peptide antigen binding. KEGG pathway analysis indicated associations with Viral myocarditis, Inflammatory bowel disease, Th17 cell differentiation, Rheumatoid arthritis, and the IL-17 signaling pathway for both Group A and Group C. Likewise, in the comparison between Group B and Group C, GO enrichment included terms related to antigen processing and presentation of peptide antigen, antigen processing and presentation of exogenous peptide antigen via MHC class II, and peptide antigen assembly with MHC protein complex. Cellular components were associated with the cytosolic ribosome, ribosome, and ribosomal subunit, while molecular functions involved the structural constituent of ribosome, MHC class II protein complex binding, and antigen binding. KEGG enrichment was mainly associated with Th17 cell differentiation, Antigen processing and presentation, Viral myocarditis, Tuberculosis, and Rheumatoid arthritis (Figure.2C). In summary, DEGs were found to enrich pathways such as Rheumatoid arthritis, IL-17 signaling pathway, Th17 cell differentiation, and antigen processing and presentation of peptide antigen.
Conducting pseudo-temporal analysis revealed that with the increase in pseudo-temporal sequences, T cells showed a trend of initially slight decrease followed by an increase (Figure.2D). Differentiation trend of T cells followed the sequence of CD4 + T cell, Treg T cell, CD69 + T cell, and CD8 + T cell (Figure.2E). Genes such as EIF2AK2, NCOA2, PIAS1, PORA, RUNX1, SETBP1, SP100, and TCF12 displayed low expression during initial stages of differentiation. Their expression gradually increased with the elevation in pseudo-temporal sequences. Conversely, BTF3 presented high expression during early stages of differentiation but experienced a decline as pseudo-temporal sequences advanced. MEF2C showed relatively constant expressions throughout the differentiation process (Figure.2F).
B cell functional enrichment and subgroup analysis
Within B cell group, a comparison from Group A and Group B illustrated the existence of genes that showed increased and decreased expression in Group A (Figure.3A). GO enrichment functional analysis emphasized the top 10 significantly enriched terms. BPs primarily involved activities such as cytoplasmic translation, myeloid cell differentiation, and erythrocyte homeostasis. CCs were predominantly associated with the cell-substrate junction, focal adhesion, and secretory granule lumen. MFs mainly included cadherin binding, actin binding, and structural constituent of ribosome (Figure.3A). Results from KEGG pathway analysis indicated predominant associations of Group A and Group B with pathways including Chemokine signaling, Shigellosis, Pathogenic Escherichia coli infection, Salmonella infection, and Regulation of actin cytoskeleton (Figure.3A). In the comparison between Group A and Group C (Figure.3B), GO enrichment primarily encompasses myeloid cell differentiation, positive regulation of cellular catabolic processes, and the T cell receptor signaling pathway. CCs were primarily linked to focal adhesion, cell-substrate junction, and secretory granule membrane. MFs predominantly involved GTPase regulator activity, nucleoside-triphosphatase regulator activity, and protein serine/threonine kinase activity. In a parallel analysis of Group A and Group C, KEGG pathways association encompassed Chemokine signaling, Hepatitis B, Proteoglycans in cancer, Th17 cell differentiation, and Th1 and Th2 cell differentiation (Figure.3B). When comparing Group B with Group C, GO enrichment includes biological processes like positive regulation of leukocyte activation, positive regulation of cell activation, and positive regulation of lymphocyte activation. CCs showed association with cytosolic ribosome, cytosolic small ribosomal subunit, and secretory granule lumen. MFs primarily participated in structural constituent of ribosome, antigen binding, and RAGE receptor binding. Further KEGG analysis (Figure.3C) indicated that DEGs were enriched in pathways such as Viral myocarditis, Th1 and Th2 cell differentiation, Rheumatoid arthritis, Th17 cell differentiation, and Antigen processing and presentation.
Temporal analysis revealed that with the increase in pseudotime sequences, the overall trend of B cells in Group B shows a decrease, while Group A and Group C show an increasing trend, with Group A showing the most pronounced increase (Figure.3D). B cells differentiate into B1 B cells in the early stage and later differentiate into B2 B cells (Figure.3E). Genes such as BACH2, CDCA7L, DDX5, POU2F2, SF1, TRIM22, and ZFP36L1 posed lower expression in the early stages of differentiation, increasing in relative expression as the pseudotime sequence progresses. On the other hand, HMGB2 and PREB unveiled higher expression in the early stages, decreasing in relative expression with the increase in pseudotime sequences. Relative expression of ID3 showed an initial increase followed by a decrease in trend (Figure.3F).
NK cell functional enrichment and subgroup analysis
In NK cell group, comparison between Group A and Group B revealed both upregulated and downregulated genes in Group A (Figure.4A). GO enrichment functional analysis highlighted the top 10 enrichments, with BPs primarily involving cytolysis, leukocyte-mediated cytotoxicity, and natural killer cell-mediated immunity. CCs were mainly associated with cytosolic ribosome, coated vesicle membrane, and MHC protein complex, while MFs were primarily involved in MHC protein complex binding, structural constituent of ribosome, and catalytic activity acting on DNA (Figure.4A). KEGG pathway analysis indicated that Groups A and B were mainly associated with Antigen processing and presentation, Viral myocarditis, Th1 and Th2 cell differentiation, Th17 cell differentiation, and Inflammatory bowel disease pathways (Figure.4A). Comparison between Groups A and C was shown in Figure.4B. GO enrichment included BPs related to positive regulation of DNA-templated transcription elongation, antigen processing and presentation of exogenous antigen, and protein targeting. CCs were associated with ficolin-1-rich granule lumen, ficolin-1-rich granule, and mitochondrial protein-containing complex. Pathway analysis indicated associations with Tuberculosis, Huntington disease, Diabetic cardiomyopathy, Antigen processing and presentation, and Viral myocarditis pathways (Figure.4B). Similarly, comparison between Groups B and C revealed GO enrichments with BPs involving cytoplasmic translation, lymphocyte proliferation, and leukocyte proliferation. CCs were associated with cytosolic ribosome, ribosomal subunit, and cytosolic large ribosomal subunit. MFs primarily involved structural constituent of ribosome, proton-transporting ATP synthase activity (rotational mechanism), and MHC protein binding. Top five enriched KEGG pathways were Alzheimer's disease, Amyotrophic lateral sclerosis, Th17 cell differentiation, Ribosome, and Th1 and Th2 cell differentiation (Figure.4C).
Temporal analysis revealed that with the increase in pseudotime sequences, NK cells in Groups A, B, and C exhibit a trend of initial increase followed by a decrease (Figure.4D). In the early stages, NK cell-1 has a higher proportion, while in the later stages, NK cell-2 became more predominant (Figure.4E). HLA-DR85 showed relatively low expression in the early stages of differentiation, and with the increase in pseudotime sequences, its relative expression increases. On the other hand, HDACC9 and KLF2 represent a trend of initial increase followed by a decrease in relative expression over the pseudotime.
RA group's potential for a heightened inflammatory expression in contrast to UIP group
Single-cell map of patients revealed T cells as the predominant cells, constituting over 50% of all cells (Figure.1G). Significant differential genes in T cells between different groups were enriched in signaling pathways closely associated with RA, such as the IL-17 signaling pathway, Th17 cell differentiation, and antigen processing and presentation of peptide antigen (Figure.2B-C). To delve deeper into the cells most closely related to RA and their potential mechanisms within patient PBMCs, we isolated T cells for further analysis. Our results indicated that T cells differentiate into four cell subgroups in pseudotime analysis: CD4 + T cells, Treg T cells, CD69 + T cells, and CD8 + T cells (Figure.2E). Proportions from high to low were CD8 + T cells, CD4 + T cells, Treg T cells, and CD69 + T cells (Figure.5A). Comparison between Groups A and B revealed significant upregulation of 52 genes, including CTSW, and significant downregulation of 65 genes, including IGHA1, in the CD8 + T cell subgroup. GO enrichment functional analysis highlighted the top 10 enrichments, with BPs mainly involving antigen receptor-mediated signaling pathway, immune response-activating cell surface receptor signaling pathway, and immune response-activating signal transduction. CCs were mainly associated with cytolytic granule, endocytic vesicle, and antigen binding, while MFs primarily involved RAGE receptor binding, immunoglobulin receptor binding, and calcium-dependent protein serine and threonine kinase activity (Figure.5B). KEGG pathway analysis indicated that CD8 + T cells in Groups A and B were primarily associated with pathways such as Th1 and Th2 cell differentiation, Antigen processing and presentation, and Th17 cell differentiation (Figure.5B). Comparison between Groups A and B for CD4 + T cells was illustrated in Figure.5C, where 53 genes were significantly upregulated, and 242 genes were significantly downregulated in Group A. Enrichment of significant differential genes revealed that BPs mainly involved leukocyte proliferation, T cell differentiation, and B cell receptor signaling pathway. CCs were primarily associated with cytosolic ribosome, ribosomal subunit, and ribosome, while MFs mainly included structural constituent of ribosome, protein serine/threonine kinase activity, and protein serine kinase activity. KEGG pathway analysis showed associations with pathways such as Ribosome, Growth hormone synthesis, secretion and action, and T cell receptor signaling pathway (Figure.5C). In the comparison of Treg T cells, 25 genes are significantly upregulated, and 43 genes are significantly downregulated in Group A. GO enrichment involved BPs related to defense response to bacterium, defense response to virus, and defense response to symbiont. CCs were associated with cytosolic ribosome, cytosolic small ribosomal subunit, and ribosome, while MFs involve Antigen processing and presentation, IL-17 signaling pathway, and Th17 cell differentiation. KEGG enrichment is mainly associated with pathways such as Ribosome, Influenza A, and IL-17 signaling pathway (Figure.5D). Similarly, in the comparison of CD69 + T cells in Groups A and B, 117 genes, including CTSW, are significantly upregulated, and 170 genes, including FKBP5, were significantly downregulated in Group A. GO enrichment involved BPs related to positive regulation of cell activation, positive regulation of lymphocyte activation, and adaptive immune response according to somatic recombination of immune receptors built from immunoglobulin superfamily domains. CCs were associated with endocytic vesicle lumen, cytosolic ribosome, and cytosolic small ribosomal subunit, while MFs involve MHC class II protein complex binding, MHC protein complex binding, and haptoglobin binding. KEGG enrichment is mainly associated with pathways such as Antigen processing and presentation, Th1 and Th2 cell differentiation, Th17 cell differentiation, and Rheumatoid arthritis (Figure.5E). In summary, Groups A and B showcased differential genes, such as CTSW, which are enriched in pathways including Antigen processing and presentation, Th1 and Th2 cell differentiation, Th17 cell differentiation, Rheumatoid arthritis, and IL-17 signaling pathway.
RA group's potential for a heightened inflammatory presentation compared to NSIP group
When comparing Groups A and C, within the CD8 + T cell subpopulation, 19 genes including CTSW were significantly upregulated in Group A, while 37 genes including GZMK were significantly downregulated. GO enrichment analysis revealed the top 10 significantly enriched terms, with BPs mainly involving B cell activation, mononuclear cell differentiation, and lymphocyte differentiation. CCs were primarily associated with the external side of the plasma membrane, cytosolic ribosome, and cytosolic small ribosomal subunit. MFs included antioxidant activity, haptoglobin binding, and chemokine binding (Figure.6A). KEGG pathway analysis indicated that CD8 + T cells from Groups A and C were primarily associated with pathways such as Antigen processing and presentation and Th1 and Th2 cell differentiation (Figure.6A). In the comparison between Groups A and C for CD4 + T cells, as shown in Figure.6B, there were 22 significantly upregulated genes and 97 significantly downregulated genes in Group A. Enrichment analysis of the DEGs revealed that BPs were mainly associated with the antigen receptor-mediated signaling pathway, regulation of antigen receptor-mediated signaling pathway, and leukocyte cell-cell adhesion. CCs were primarily related to the cytosolic ribosome, cytosolic small ribosomal subunit, and haptoglobin-hemoglobin complex, while MFs included haptoglobin binding, protein serine kinase activity, and oxygen carrier activity. KEGG pathway analysis indicated that CD4 + T cells from Groups A and C were primarily associated with pathways such as the TNF signaling pathway and Th17 cell differentiation (Figure.6B). In the comparison of Treg T cells, 19 genes were significantly upregulated, and 25 genes were significantly downregulated in Group A. GO enrichment analysis showed that BPs were mainly involved in cytoplasmic translation, carbon dioxide transport, and oxygen transport. CCs were mainly associated with the cytosolic ribosome, ribosomal subunit, and cytosolic small ribosomal subunit, while MFs included structural constituent of ribosome, haptoglobin binding, and oxygen carrier activity. KEGG enrichment analysis revealed associations with Th1 and Th2 cell differentiation, IL-17 signaling pathway, Th17 cell differentiation, and TNF signaling pathway (Figure.6C). Similarly, in the comparison of CD69 + T cells between Groups A and C, 56 genes, including CTSW, were significantly upregulated, while 47 genes, including MT-ND1, were significantly downregulated in Group A. GO enrichment analysis revealed that BPs were mainly associated with cytoplasmic translation, gamma-delta T cell activation, and leukocyte-mediated immunity. CCs were primarily related to the cytosolic small ribosomal subunit, ribosome, and small ribosomal subunit, while MFs included structural constituent of ribosome, MHC protein complex binding, and haptoglobin binding. KEGG enrichment analysis indicated associations with Antigen processing and presentation, Rheumatoid arthritis, Th1 and Th2 cell differentiation, Th17 cell differentiation, and TNF signaling pathway (Figure.6D). In other words, there are DEGs, such as CTSW, which are enriched in pathways such as Rheumatoid arthritis, IL-17 signaling pathway, Th1 and Th2 cell differentiation, Th17 cell differentiation, and TNF signaling pathway.
Enrichment of DEGs in T cell subpopulations between UIP and NSIP groups into inflammation-related pathways
In the comparison between Groups B and C, 37 genes were significantly upregulated, and 16 genes were significantly downregulated in the CD8 + T cell subgroup. GO enrichment analysis highlighted the top 10 significantly enriched terms, with BPs mainly involving antigen receptor-mediated signaling pathway, immune response-regulating cell surface receptor signaling pathway, and immune response-activating cell surface receptor signaling pathway. CCs were associated with cytosolic small ribosomal subunit, endocytic vesicle lumen, and blood microparticle, while MFs included haptoglobin binding, MHC protein complex binding, and oxygen carrier activity (Figure.7A). KEGG pathway analysis revealed that CD8 + T cell subsets in Groups B and C were primarily associated with Antigen processing and presentation, Th1 and Th2 cell differentiation, and IL-17 signaling pathway (Figure.7A). In the comparison of CD4 + T cells between Groups B and C, there were 34 upregulated and 76 downregulated DEGs in Group B (Figure.7B). Enrichment analysis of the significantly different genes indicated that BPs were mainly involved in B cell receptor signaling pathway, antigen receptor-mediated signaling pathway, and regulation of B cell activation. CCs were mainly associated with IgA immunoglobulin complex, blood microparticle, and cytosolic small ribosomal subunit. MFs were primarily involved in immunoglobulin receptor binding, haptoglobin binding, and oxygen carrier activity. KEGG pathway analysis suggests that CD4 + T cells in Groups B and C were mainly associated with Coronavirus disease - COVID − 19, Measles, and IL-17 signaling pathway (Figure.7B). In the comparison of Treg T cells, Group B presented 34 significantly upregulated genes and 12 significantly downregulated genes when compared to Group C. Enrichment analysis of these DEGs revealed that BPs were primarily associated with response to virus, defense response to virus, and defense response to symbiont. CCs were mainly related to cytosolic small ribosomal subunit, cytosolic ribosome, and small ribosomal subunit, while MFs were mainly involved in haptoglobin binding, oxygen carrier activity, and antioxidant activity. KEGG pathway enrichment indicates significant associations with Coronavirus disease - COVID-19, Ribosome, and IL-17 signaling pathway (Figure.7C). Similarly, in the comparison of CD69 + T cells between Groups B and C, there were 66 significantly upregulated genes and 48 significantly downregulated genes in Group B. Enrichment analysis showed that BPs were primarily involved in immune response-regulating cell surface receptor signaling pathway, immune response-activating cell surface receptor signaling pathway, and immune response-activating signal transduction. CCs were mainly associated with cytosolic small ribosomal subunit, cytosolic ribosome, and endocytic vesicle lumen, while MFs were mainly related to haptoglobin binding, oxygen carrier activity, and lamin binding. KEGG pathway enrichment suggested significant associations with Chemokine signaling pathway, Cytokine-cytokine receptor interaction, and Rheumatoid arthritis (Figure.7D). This implied that there were significant DEGs in Groups B and C, and they were enriched in pathways related to Rheumatoid arthritis, IL-17 signaling pathway, and TNF signaling pathway.
Cellular communication unveiling the pivotal role of T cells
Our results of T cell subset analysis indicated a crucial role for T cells in the inflammatory response. In order to gain a deeper understanding of the interactions between various cell types, this study utilized CellChat to pairwise compare the quantity and strength of interactions between different cell groups (Figure.8–10). Then the results turned out that, compared to the AB group, there was strong interaction between T cells, B cells, and NK cells, making them the major cell types expressing ligands (Figure.8A). These interactions were critical for the inflammatory environment, with T cells acting as a hub in this context. Further exploration of ligand-receptor pairs involved in crosstalk between cell groups (Figure.8B) revealed that strong communication mainly occurs between T cells, NK cells, and B cells. In the A group, increased signals in strong communication include ligands such as HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and receptors like CD8A, CD8B. Decreased signals in strong communication include ligands like MIF, CLEC2C, CD99, and receptors like KLRB1, CD74, CD44, CXCR4. To uncover the impact of signaling pathways on intercellular communication, the strength of signaling pathways in the AB group was calculated (Figure.8C). Compared to other signaling pathways, MHC-1 was identified as the most prominent signaling pathway, with higher intensity in the B group, although the difference is not statistically significant. In addition, an increase in signals such as IL2 and IL16 was observed in group A. In comparison to group AC, strong interactions were found within T cells, but the intercellular interaction strength was slightly lower (Figure.9A). Increased signals in strong communication mainly occurred between ligands like MIF, CLEC2B, CLEC2C, CLEC2D, and receptors like CD74, CD44, CXCR4, KLRB1. Decreased signals in strong communication mainly occurred between ligands like HLA-A, HLA-B, HLA-C, HLA-E, and receptors like CD8A, CD8B (Figure.9B). Compared to other signaling pathways, MHC-1 was identified as the most significant signaling pathway, with IL2 signaling intensity significantly higher in group A than in group C (Figure.9C). In group BC, there were more ligand-receptor pairs in Monocytes, B cells, Neutrophils, and Dendritic cells, but the intercellular interaction strength was slightly lower (Figure.10A). Increased signals in strong communication in the B group mainly occurred between ligands like MIF, CD99, CLEC2D, and receptors like KLRB1, CD74, CD44, CXCR4. Decreased signals in strong communication included ligands like HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, and receptors like CD8A, CD8B (Figure.10B). MHC-1 was identified as the most significant signaling pathway, but there was no significant difference between groups B and C. Signaling intensity of IL16 pathway in group C was significantly higher than that in group B (Figure.10C). These results indicated that T cells play a central role in cellular interactions, mediating the activity of B cells and NK cells, among others.