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. 2022 Apr 6:13:861459.
doi: 10.3389/fimmu.2022.861459. eCollection 2022.

Upregulated of ANXA3, SORL1, and Neutrophils May Be Key Factors in the Progressionof Ankylosing Spondylitis

Jie Jiang  1 , Xinli Zhan  1 , Haishun Qu  2 , Tuo Liang  1 , Hao Li  1 , Liyi Chen  1 , Shengsheng Huang  1 , Xuhua Sun  1 , Wenyong Jiang  1 , Jiarui Chen  1 , Tianyou Chen  1 , Yuanlin Yao  1 , Shaofeng Wu  1 , Jichong Zhu  1 , Chong Liu  1
Affiliations

Upregulated of ANXA3, SORL1, and Neutrophils May Be Key Factors in the Progressionof Ankylosing Spondylitis

Jie Jiang et al. Front Immunol. .

Abstract

Introduction: The specific pathogenesis of ankylosing spondylitis (AS) remains unclear, and our study aimed to investigate the possible pathogenesis of AS.

Materials and methods: Two datasets were downloaded from the GEO database to perform differentially expressed gene analysis, GO enrichment analysis, KEGG pathway analysis, DO enrichment analysis, GSEA analysis of differentially expressed genes, and construction of diagnostic genes using SVM and WGCNA along with Hypoxia-related genes. Also, drug sensitivity analysis was performed on diagnostic genes. To identify the differentially expressed immune genes in the AS and control groups, we analyzed the composition of immune cells between them. Then, we examined differentially expressed genes in three AS interspinous ligament specimens and three Degenerative lumbar spine specimens using high-throughput sequencing while the immune cells were examined using the neutrophil count data from routine blood tests of 1770 HLA-B27-positive samples and 7939 HLA-B27-negative samples. To assess the relationship between ANXA3 and SORL1 and disease activity, we took the neutrophil counts of the first 50 patients with above-average BASDAI scores and the last 50 patients with below-average BASDAI scores for statistical analysis. We used immunohistochemistry to verify the expression of ANXA3 and SORL1 in AS and in controls.

Results: ANXA3 and SORL1 were identified as new diagnostic genes for AS. These two genes showed a significant differential expression between AS and controls, along with showing a significant positive correlation with the neutrophil count. The results of high-throughput sequencing verified that these two gene deletions were indeed differentially expressed in AS versus controls. Data from a total of 9707 routine blood tests showed that the neutrophil count was significantly higher in AS patients than in controls (p < 0.001). Patients with AS with a high BASDAI score had a much higher neutrophil count than those with a low score, and the difference was statistically significant (p < 0.001). The results of immunohistochemistry showed that the expression of ANXA3 and SORL1 in AS was significantly higher than that in the control group.

Conclusion: Upregulated of ANXA3, SORL1, and neutrophils may be a key factor in the progression of Ankylosing spondylitis.

Keywords: Ankylosing spondylitis; BASDAI; high-throughput sequencing; hypoxia; immune cell correlation analysis; immune cell infiltration; immunohistochemical analysis.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Heat map and volcano plots of differentially expressed genes. (A) shows the heat map of differentially expressed genes, red indicates high expression genes and blue indicates low expression genes. (B) shows the volcano plots of differentially expressed genes, red is for high expression genes and green is for low expression genes.
Figure 2
Figure 2
GO and KEGG pathway enrichment analysis of differentially expressed genes. (A) shows the first 10 entries of GO enrichment analysis of differentially expressed genes. (B) shows the top 7 pathways of KEGG pathway.
Figure 3
Figure 3
SVM analysis, GSEA, DO analysis, veen plot and ROC curve. (A) shows the results of SVM analysis. (B) shows the results of intersection of GEO_grey module, hypoxia-related genes and SVM analysis. (C, D) show the results of GO and KEGG from GSEA enrichment analysis. (E) shows the results of DO enrichment analysis. (F, G) show the ROC diagnostic curves of 2 genes of the model. (F, G) show the ROC curves of 2 genes of the model.
Figure 4
Figure 4
Results of WGCNA analysis. (A-E) shows the results of cluster analysis. (G) shows the Person correlation with disease and normal groups. (F, H) show the scatter plot of correlation.
Figure 5
Figure 5
Drug sensitivity analysis. The graph shows the drug sensitivity analysis of ANXA3 and SORL1 with various drugs. If Cor > 0, it means the gene is positively correlated with drug sensitivity, the higher the gene expression, the stronger the sensitivity of this drug; conversely, Cor < 0 means the gene is negatively correlated with drug sensitivity, the higher the gene expression, the worse the sensitivity of this drug.
Figure 6
Figure 6
Immune cell composition analysis and differential expression analysis of immune cells. (A) shows the immune cell composition of all GEO downloaded samples, each sample consists of 22 immune cells with a total ratio of 100%. (B) shows the differences in immune cells in AS and control group, as seen in Neutrophils, NK cells activated, T cells gamma delta, T cells CD8 and B cells memory were significantly different (p < 0.05).
Figure 7
Figure 7
Correlation analysis of ANXA3, SORL1 and immune cells respectively. (A-D) shows the correlation analysis of ANXA3 and 4 immune cells. (E-I) shows the correlation analysis of SORL1 and 5 immune cells. If R > 0, it means higher gene expression and higher immune cell content; if R < 0, it means higher gene expression and lower immune cell content.
Figure 8
Figure 8
Immune correlation lollipop plot. (A) shows the immune cells with significant difference of ANXA3 in AS and control. (B) shows the immune cells with significant difference of SORL1 in AS and control.
Figure 9
Figure 9
Results of external validation sets are shown. (A, B) show heat and volcano maps of differentially expressed genes measured by high-throughput sequencing, confirming that ANXA3 and SORL1 are also differentially expressed genes in the interspinous ligament of AS. (C-F) show the variance analysis of the routine blood tests. (G) shows the difference in neutrophils between high BASDAI cases and low BASDAI cases. (H) shows the relationship between neutrophils and CRP.
Figure 10
Figure 10
Immunohistochemistry and positive rate statistics. (A1-D2) show the specific expression of ANXA3 with SORL1 in AS and controls. (E, F) shows the positive rate statistics of all immunohistochemical images of SORL1 in AS and control groups. “*” indicates p < 0.05, “***” indicates p < 0.001.
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