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GEO dataset integration and immune landscape of CD

We constructed a combined dataset covering 279 CD samples and 224 control samples from mucosa after the removal of batch effects (Fig.2A,B). A broadly uncoordinated immune response is an indispensable hallmark of CD. With the aim of revealing the immune landscape, we scored the immune cell infiltration of CD patients and controls via the ssGSEA method. As illustrated in Fig.2C, the infiltration of 20 immune cells in the CD group and control group was significantly different, among which only the scores of T helper 17 (Th17) cells were lower in CD tissues than in control tissues. We then performed a correlation analysis of distinct immune cells, as shown in Fig.2D. Interestingly, Th17 cells, CD56bright natural killer (NK) cells, CD56dim NK cells and monocytes showed inverse correlations with almost all other immune cells, whereas the other immune cells were generally positively correlated with one another, which deserves special attention.

GEO dataset combination and immune landscape of CD. (A) PCA between datasets before removal of batch effects. (B) PCA between integrated datasets after removal of batch effects. (C) Infiltration levels of 28 immune cell subtypes in CD samples and controls. The blue bars represent controls, and the red bars represent CD samples. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. (D) Pearson correlation analysis of distinct immune cells. The purple squares represent positive correlations, and the orange squares represent inverse correlations. GEO Gene Expression Omnibus, CD Crohns disease, PCA principal component analysis.

A total of 1265 DEGs, consisting of 592 upregulated and 673 downregulated genes, were identified through differential expression analysis (Fig.3A). A list of possible PRGs was produced from previous research (Supplementary file 1: Table S1). Subsequently, we intersected the 1265 DEGs with 930 PRGs via a Venn diagram; thus, 130 DE-PRGs were identified (Fig.3B), which were further grouped in a heatmap (Fig.3C). The overall expression of these DE-PRGs in the CD group and control group is shown in Supplementary file 3: Fig. S1. We could conclude that the vast majority of DE-PRGs were expressed at higher levels in CD tissues than in control tissues.

Identification of DE-PRGs. (A) Volcano map of the DEGs with the cutoff threshold set at |log2 (fold change)|>1 and adj. p<0.05. The blue dots represent downregulated DEGs, the red dots represent upregulated DEGs, and the gray dots represent genes with no significant difference. (B) Venn diagram of DEGs and PRGs. Pink circle represents DEGs, blue circle represents PRGs, and their overlapping area represents DE-PRGs. (C) Clustered heatmap of the top 40 DE-PRGs. Each row represents one of the top 40 DE-PRGs, and each column represents one sample, either CD or normal. DE-PRGs differentially expressed PANoptosis-related genes, DEGs differentially expressed genes, PRGs PANoptosis-related genes, CD Crohns disease.

We then examined the latent functions and signaling pathways of the DE-PRGs. GO analysis revealed that these DE-PRGs were predominantly involved in regulation of apoptotic signaling pathway, leukocyte cellcell adhesion, regulation of inflammatory response (biological process); membrane raft, membrane microdomain, focal adhesion (cellular component); ubiquitin-like protein ligase binding, ubiquitin protein ligase binding, and phosphatase binding (molecular function) (Supplementary file 4: Fig. S2A). Additionally, DE-PRGs were notably enriched in apoptosis, proteoglycans in cancer, NOD-like receptor signaling pathway, among others, according to the KEGG results (Supplementary file 4: Fig. S2B). Moreover, a PPI network analysis of the DE-PRGs was performed and a complex network of the DE-PRGs was constructed (Supplementary file 5: Fig. S3).

To screen the hub DE-PRGs, we first capitalized on three algorithms, LASSO, SVM and RF, and discovered 20, 34 and 33 potential hub DE-PRGs, respectively (Fig.4AE). Afterward, 10 hub DE-PRGs were identified through the intersection of the machine learning results, namely CD44, cell death inducing DFFA like effector c (CIDEC), N-myc downstream regulated 1 (NDRG1), nuclear mitotic apparatus protein 1 (NUMA1), proliferation and apoptosis adaptor protein 15 (PEA15), recombination activating 1 (RAG1), S100 calcium binding protein A8 (S100A8), S100 calcium binding protein A9 (S100A9), TIMP metallopeptidase inhibitor 1 (TIMP1) and X-box binding protein 1 (XBP1) (Fig.4F). Next, we probed their interactions, as shown in Fig.4G. Most hub DE-PRGs, such as CD44, PEA15, S100A8, S100A9, TIMP1 and XBP1, were closely interrelated. Moreover, NDRG1, NUMA1 and RAG1 generally presented antagonistic effects on the other hub DE-PRGs. Finally, the diagnostic value of each hub DE-PRG in predicting CD was calculated based on our combined dataset (Fig.4H). All 10 hub DE-PRGs exhibited outstanding predictive performance with area under the curve (AUC) values greater than 0.740. Notably, the AUC reached as high as 0.871 when the 10 hub DE-PRGs were combined (Fig.4H). In addition, we conducted external validation on the GSE102133 and GSE207022 datasets, respectively. The results were satisfactory, with high AUC values (Supplementary file 6: Fig. S4).

Identification of the hub DE-PRGs. (A) Cross-validations of adjusted parameter selection in the LASSO model. Each curve corresponds to one gene. (B) LASSO coefficient analysis. Vertical dashed lines are plotted at the best lambda. (C) SVM algorithm for hub gene selection. (D) Relationship between the number of random forest trees and error rates. (E) Ranking of the relative importance of genes. (F) Venn diagram showing the 10 hub DE-PRGs identified by LASSO, SVM and RF. Pink circle represents potential hub DE-PRGs identified by RF, blue circle represents potential hub DE-PRGs identified by SVM, green circle represents potential hub DE-PRGs identified by LASSO, and their overlapping area represents the final hub DE-PRGs. (G) Chord diagram showing the correlations between the hub DE-PRGs. Red represents positive correlations between different genes and green represents negative correlations between different genes. (H) ROC curves of the hub DE-PRGs in CD diagnosis. DE-PRGs differentially expressed PANoptosis-related genes, LASSO least absolute shrinkage and selection operator, RF random forest, SVM support vector machine, ROC receiver operating characteristic, AUC area under the curve, CD Crohns disease.

Spearman correlation analysis was carried out to determine the interactions between the hub DE-PRGs and immune cells (Fig.5). CD44, PEA15, S100A8, S100A9, TIMP1 and XBP1 demonstrated noteworthy positive correlations with the infiltration of an abundance of immune cells, except for certain immune cells, such as monocytes and CD56bright NK cells. In contrast, NDRG1, NUMA1, and RAG1 were negatively associated with most types of immune cells, excluding a few immune cells such as monocytes. In addition, the CIDEC fell somewhere between these two extremes.

Spearman correlation analysis of hub DE-PRGs with immune cells. The correlations between CD44 (A), CIDEC (B), NDRG1 (C), NUMA1 (D), PEA15 (E), RAG1 (F), S100A8 (G), S100A9 (H), TIMP1 (I) and XBP1 (J) gene expressions with immune cells, respectively. The size of the dots represents the strength of gene correlation with immune cells; the larger the dot, the stronger the correlation. The color of the dots represents the p-value; the greener the color, the lower the p-value. p<0.05 was considered statistically significant. DE-PRGs differentially expressed PANoptosis-related genes.

The top 30 crucial genes related to CD were extracted from the GeneCards database, and their expression levels were compared between CD samples and normal samples (Fig.6A). We could easily conclude that a majority of the CD-related genes (26 out of 30) were differentially expressed, especially COL1A1, CTLA4, IL10 and NOD2. Pearson correlation analysis was subsequently conducted to scrutinize the relationships between these CD-related genes and the hub DE-PRGs (Fig.6B). Notably, CTLA4, one of the most differentially expressed CD-related genes, was significantly associated with each hub DE-PRG. COL1A1, IL10 and NOD2 also presented varying levels of correlation with the hub DE-PRGs. Nevertheless, there were no significant correlations between the hub DE-PRGs and some CD-related genes, including CYBB, IL10RA, RET and VCP.

Expression levels of the top 30 CD-related genes and relationships between them and hub DE-PRGs. (A) Boxplot of the top 30 crucial genes in relation to CD. The blue bars represent controls, and the red bars represent CD samples. (B) Pearson correlation analysis between the top 30 CD-related genes and the 10 hub DE-PRGs. *p<0.05; **p<0.01; ***p<0.001. CD Crohns disease, DE-PRGs differentially expressed PANoptosis-related genes.

Subsequently, a genemiRNA interaction network of the 10 hub DE-PRGs consisting of 226 nodes and 338 edges was constructed (Supplementary file 7: Fig. S5 and Supplementary file 8: Table S3). Apparently, miR-124-3p, miR-34a-5p and miR-27a-3p were most strongly associated with the hub DE-PRGs in CD. After that, we generated a geneTF regulatory network of the 10 hub DE-PRGs (Supplementary file 9: Fig. S6). The 10 hub DE-PRGs were regulated by 35 total TFs. Among them, FOXC1 was found to regulate as many as 7 hub DE-PRGs and S100A8 was regulated by 13 miRNAs (Supplementary file 10: Table S4). In addition, we looked for available drugs that act on the hub DE-PRGs, and a host of drugs were involved (Supplementary file 11: Fig. S7 and Supplementary file 12: Table S5). Specifically, a total of 19 drugs interacted with XBP1, 8 of which inhibited it.

To distinguish different PANoptosis patterns in CD patients, we adopted the NMF method for unsupervised clustering on the basis of the 10 hub DE-PRGs. At k=2, the most stable and optimal PANclusters were identified (Fig.7A). There were 101 and 178 CD samples in PANcluster A and PANcluster B, respectively. The geometrical distance between the two clusters is shown in Fig.7B, validating their gene expression heterogeneity. Thereafter, a boxplot and a heatmap were generated to compare the expression levels of the hub DE-PRGs between PANcluster A and PANcluster B (Fig.7C,D). Specifically, PANcluster A was distinguished by the considerably high expression levels of CIDEC, NDRG1, NUMA1 and RAG1, while the other hub DE-PRGs, that is, CD44, PEA15, S100A8, S100A9, TIMP1 and XBP1, were expressed at higher levels in PANcluster B.

Recognition of PANclusters in CD. (A) Unsupervised clustering matrix generated using NMF method when k=2. (B) PCA plot showing the distribution of PANcluster A and PANcluster B. The red dots represent PANcluster A and the blue dots represent PANcluster B. (C) Boxplot of the expression levels of the hub DE-PRGs in PANcluster A and PANcluster B. The red bars represent PANcluster A, and the blue bars represent PANcluster B. (D) Heatmap of the expression levels of the hub DE-PRGs in PANcluster A and PANcluster B. Each row represents one hub DE-PRG, and each column represents one CD sample. PANclusters PANoptosis patterns, CD Crohns disease, NMF nonnegative matrix factorization, PCA principal component analysis, DE-PRGs differentially expressed PANoptosis-related genes.

GSVA was performed with the aim of shedding light on the functional diversity patterns of the recognized PANclusters. With regard to Hallmark pathways, increased activity of p53 pathway, androgen response and hypoxia were detected in PANcluster A, whereas mTORC1 signaling, inflammatory response, TNF- signaling via NF-B, IL-6/JAK/STAT3 signaling and epithelial mesenchymal transition were increased in PANcluster B (Supplementary file 13: Fig. S8A). In addition, results from the KEGG analysis suggested that PANcluster A had hypoactive ECMreceptor interaction and endocytosis but expressed high levels of genes associated with cytokinecytokine receptor interaction and numerous signaling pathways, including toll-like receptor signaling pathway and NOD-like receptor signaling pathway (Supplementary file 13: Fig. S8B). Concerning the Reactome-based pathways, PANcluster A showed an increase in the cell cycle pathway, while most pathways, such as cytokine signaling in immune system and extracellular matrix-related pathways, were significantly enriched in PANcluster B (Supplementary file 13: Fig. S8C).

To clarify the disparities in the immune system among the PANclusters, we compared their immune microenvironments, as shown in Fig.8A. Remarkably, the enrichment scores of 26 immune cells were much greater in PANcluster B than in PANcluster A. Consequently, CD56bright NK cells and monocytes were the only two exceptions with higher infiltration degrees in PANcluster A, the explanations behind which demand further investigation. In addition, differential gene analysis revealed 533 DEGs, including 171 upregulated and 362 downregulated genes (Fig.8B). To learn more about the biological functions and processes linked to these DEGs, GO and KEGG analyses were performed. The 533 DEGs were markedly enriched in the following terms: positive regulation of cell adhesion, leukocyte cellcell adhesion, and extracellular matrix organization (biological process); collagen-containing extracellular matrix, secretory granule membrane, and basement membrane (cellular component); and extracellular matrix structural constituent, glycosaminoglycan binding, and integrin binding (molecular function) (Fig.8C,D). Moreover, the 533 DEGs were principally involved in many pathways, such as cell adhesion molecules, ECMreceptor interaction and PI3K-Akt signaling pathway (Fig.8E).

Characterization of different PANclusters. (A) Infiltration levels of 28 immune cell subtypes in PANclusters A and B. The red bars represent PANcluster A, and the blue bars represent PANcluster B. (B) Volcano map of DEGs between PANclusters A and B. The blue dots represent downregulated DEGs, the red dots represent upregulated DEGs, and the gray dots represent genes with no significant difference. (C,D) Enriched items in GO analysis based on the DEGs between PANclusters A and B. (E) Enriched items in KEGG analysis based on the DEGs between PANclusters A and B. Node color indicates gene expression level; quadrilateral color indicates z-score. PANclusters PANoptosis patterns, DEGs differentially expressed genes, BP biological process, CC cellular component, MF molecular function, GO Gene Ontology, KEGG Kyoto Encyclopedia of Genes and Genomes.

CD and control samples were acquired from 10 patients who were diagnosed with CD, and their demographic and clinical information is presented in Table 1. qRT-PCR was subsequently conducted to determine the relative expression levels of the 10 hub DE-PRGs (Fig.9A). As expected, the levels of CD44, PEA15, S100A8, S100A9, TIMP1 and XBP1 increased in CD samples compared with those in control samples; while the opposite trend was observed for NDRG1. Moreover, there was no significant difference in the mRNA expression levels of CIDEC, NUMA1 or RAG1. Furthermore, we established classic TNBS and DSS mouse models of CD and collected colon tissues to analyze the expression levels of the hub DE-PRGs in murine colon tissues from the TNBS, DSS and control groups (Fig.9B,C). Generally, the results of the TNBS model were in line with expectations. Specifically, in TNBS-induced colitis, Cd44, Numa1, S100a8, S100a9, Timp1 and Xbp1 were more highly expressed, while Cidec and Rag1 were less expressed. In addition, the levels of Ndrg1 and Pea15a did not significantly differ between the TNBS group and the control group. Consistent with previous work, in the DSS mouse model, the expression levels of Cd44, S100a8, S100a9 and Timp1 were greater in the mice with colitis; while the expression level of Ndrg1 was lower in the mice with colitis. In addition, no significant difference in the expression levels of Cidec, Pea15a or Xbp1 was detected. Unexpectedly, the expression levels of Numa1 and Rag1 in the DSS group were different from those in the CD and TNBS colitis groups.

qRT-PCR validation of the hub DE-PRGs in CD patients (A), TNBS-induced colitis model (B) and DSS-induced colitis model (C). The blue dots represent the normal/control tissues, and the red dots represent the diseased tissues. qRT-PCR quantitative real-time PCR, DE-PRGs differentially expressed PANoptosis-related genes, CD Crohns disease, TNBS 2,4,6-trinitrobenzene sulfonic acid, DSS dextran sodium sulfate, GAPDH glyceraldehyde-3-phosphate dehydrogenase.

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