Fig 1.
Identification and analysis of key cells.
All analyses were performed on the basis of the GSE122960 dataset, and all samples in this dataset were included simultaneously. Before the analysis, all samples underwent unified quality control, standardization, and integration to ensure the comparability of the results. (a) Screening for highly variable genes. The red dots represent the top 2000 highly variable genes, while the black dots indicate the genes with smaller coefficients of variation. The top 10 highly variable genes are labeled in the figure. (b) Principal component (PC) plot based on the Jackstraw function permutation test algorithm. (c) Scree plot generated by the ElbowPlot function. (d) UMAP diagram for cell cluster classification. (e) Bubble plot of the expression of marker genes in different cells. (f) Cell annotation diagrams for different cell types. Twelve cell types were annotated for different cell clusters, with different colors representing different cell types.
Fig 2.
Cell communication of key cell and pseudotime analysis.
(a) The number of interactions in the communication network of the control group. (b) The number of interactions in the communication network of the SSc disease group. (c) The intensity of communication interaction in the communication network of the control group. (d) Interaction intensity of communication networks in the SSc disease group. The thickness of the connection lines represents the intensity of communication. (e–f) Ligand–receptor interaction point diagram. From left to right are the ligand–receptor interaction point maps of the control and SSc disease groups, respectively. The colors of the dots from red to blue represent the intensity of communication from strong to weak, and the size of the dots represents the P value. The more significant the P value, the larger the dots. (g) Distribution of different subgroups of macrophages; a total of 14 subgroups were obtained from clusters 0–13. (h) Differences in the proportion of cells are found between different subgroups of macrophages in the disease and control groups, with red indicating the proportion of the SSc disease group and blue indicating the proportion of the control group. (i) The differentiation trajectory of macrophages, from left to right, consists of the cell differentiation time, cell state, and cell-type trajectories. The different colors represent the different states of cell differentiation.
Fig 3.
hdWGCNA identifies the module genes of macrophages.
(a) Main candidate feature genes for different modules. The top 25 genes with the highest pairwise correlation between genes and module feature genes in each module. (b) The differences in candidate feature gene expression levels between the SSc-ILD group and the control group. The size of the circle represents the number of genes in the module, and the horizontal axis represents the different multiple log2FC values.
Fig 4.
Identification of differentially expressed and candidate genes.
(a) The volcano map of DEGs. The horizontal axis represents the log2FoldChange, and the vertical axis represents −log10 (Padj), with each point representing a gene. The horizontal reference line represents Padj = 0.05, and the vertical reference line represents log2FoldChange = ±0.5. Divided by reference lines, the red dots represent the upregulated differentially expressed genes, while the blue dots represent the downregulated differentially expressed genes. The genes labeled in the figure are the top 10 upregulated genes and top 10 downregulated genes sorted by log2FC. (b) Heat map of DEGs. The top is the density distribution heatmap of differentially expressed genes, and the bottom is the heatmap of differentially expressed gene expression levels, showing the gene expression and clustering of the top 50 upregulated genes and top 50 downregulated genes sorted by log2FC. The color bars represent the relative changes in gene expression levels, from blue (low expression) to red (high expression). (c) Identification of candidate genes. The green circle represents DEGs; the blue circle represents the key module genes (Mes) obtained from WGCNA, and the overlapping area in the middle represents the genes that are present in both gene sets simultaneously. (d) GO and KEGG enrichment analyses. The size of the dots represents the number of enriched genes, with larger dots indicating more enriched genes. The color of the dot represents P value, with redder dots indicating more significant enrichment result. (e) DO enrichment results. The size of the dots represents the number of enriched genes, with larger dots indicating more enriched genes.
Fig 5.
(a) Protein interaction network. The nodes represent the candidate genes, and the color of the nodes from red to yellow represents the degree of the node, which is the number of interactions between each gene and other genes. The redder the color, the more interactions the gene has and the higher the metric value. The size of the node also represents its degree. The larger the size, the more frequent the interaction between the gene and the larger the degree. (b) The important modules selected by MCODE and the hub genes obtained from MCC algorithm. (c) The expression of hub genes in the training set. The box plot indicates the differential expression of key genes in the training set, with red representing the SSc group and blue representing the normal group. P < 0.05 is considered statistically significant. (d) The expression of hub genes in the validation set.
Fig 6.
Chromosomal localization, functional similarity analysis, and subcellular localization of key genes.
(a) Subcellular localization. The radar map localization points are the subcellular localization distribution probabilities predicted for each key gene, with blue representing ARG2, yellow representing ELF3, and orange representing NKX2−1. (b) GeneMANIA analysis of key genes. (c) GSEA enrichment analysis results of the key gene ARG2. The result chart is divided into three parts from top to bottom: the first part is the enrichment score (ES), with the horizontal axis representing the tested gene set sorted by correlation coefficient and the vertical axis representing the corresponding running ES. The peak of the line chart represents the ES of the enriched pathway, and the genes before the peak are the core genes located in the enriched pathway of the tested gene set. The second part is “hit,” which marks the genes located under the test gene set with lines. The vertical lines are concentrated at the front or back of the gene sorting list, indicating that the gene set pathway is upregulated or downregulated. If the vertical lines are evenly distributed in the gene sorting list, then the gene set pathway has no significant changes in the two compared data. The third part is the rank value distribution map of all genes, and the Signal2Niose algorithm is used by default. (d) GSEA enrichment analysis results of the key gene ELF3. (e) GSEA enrichment analysis results of the key gene NKX2−1.
Fig 7.
Analysis of immune cell infiltration.
(a) Differential analysis of immune cells between the SSc-ILD group and the control group. The box plot indicates the differential expression of key genes in the validation set, with red representing the SSc group and blue representing the normal group. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001. (b) Correlation analysis between differential immune cells. The labeled value represents the intercellular correlation coefficient cor, where cor > 0 is red and cor < 0 is blue. * indicates P < 0.05; ** indicates P < 0.01. (c) Correlation analysis between differential immune cells and key genes. The asterisk indicates the significant P value of gene cell correlation, where cor > 0 is red and cor < 0 is blue. * indicates P < 0.05; ** indicates P < 0.01.
Fig 8.
Interaction network identified between key genes, TFs, and miRNAs.
TF–mRNA–miRNA regulatory network. Yellow represents the key genes; green represents the predicted TFs; and blue represents the predicted miRNAs.
Table 1.
Binding energies of the molecular docking.
Fig 9.
Drug prediction and molecular docking.
(a) The drug prediction network diagram of ARG2 and ELF3. Red squares represent genes, and blue squares represent related drugs. (b) The molecular docking results of ARG2, ELF3, and NKX2−1 with drugs. The left part is the overall graph, and the right part is the locally magnified graph.
Fig 10.
Analysis of the functional characteristics of macrophage subsets.
(a) Cluster 3 difference analysis. Blue represents the genes downregulated in cluster 3, and red represents the genes upregulated in cluster 3. (b) Biochemical processes in GO enrichment analysis of cluster 3. (c) Molecular function in GO enrichment analysis of cluster 3. (d) Cellular component in GO enrichment analysis of cluster 3. (e) Enrichment results of KEGG in cluster 3. (f) Cluster 5 difference analysis. Blue represents the genes downregulated in cluster 5, and red represents the genes upregulated in cluster 5. (g) Biochemical processes in GO enrichment analysis of cluster 5. (h) Molecular function in GO enrichment analysis of cluster 5. (i) Cellular component in GO enrichment analysis of cluster 5. (j) Enrichment results of KEGG in cluster 5.