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. 2024 Jan;12(1):e1162.
doi: 10.1002/iid3.1162.

The functional role of CST1 and CCL26 in asthma development

Angela Hoyer  1   2 , Sandip Chakraborty  1   2 , Ingrid Lilienthal  3 , Jon R Konradsen  1   2 , Shintaro Katayama  4   5   6 , Cilla Söderhäll  1   2
Affiliations

The functional role of CST1 and CCL26 in asthma development

Angela Hoyer et al. Immun Inflamm Dis. 2024 Jan.

Abstract

Background: Asthma is the most common chronic disease in children with an increasing prevalence. Its development is caused by genetic and environmental factors and allergic sensitization is a known trigger. Dog allergens affect up to 30% of all children and dog dander-sensitized children show increased expression of cystatin-1 (CST1) and eotaxin-3 (CCL26) in nasal epithelium. The aim of our study was to investigate the functional mechanism of CST1 and CCL26 in the alveolar basal epithelial cell line A549.

Methods: A549 cells were transfected with individual overexpression vectors for CST1 and CCL26 and RNA sequencing was performed to examine the transcriptomics. edgeR was used to identify differentially expressed genes (= DEG, |log2 FC | ≥ 2, FDR < 0.01). The protein expression levels of A549 cells overexpressing CST1 and CCL26 were analyzed using the Target 96 inflammation panel from OLINK (antibody-mediated proximity extension-based assay; OLINK Proteomics). Differentially expressed proteins were considered with a |log2 FC| ≥ 1, p < .05.

Results: The overexpression of CST1 resulted in a total of 27 DEG (1 upregulated and 26 downregulated) and the overexpression of CCL26 in a total of 137 DEG (0 upregulated and 137 downregulated). The gene ontology enrichment analysis showed a significant downregulation of type I and III interferon signaling pathway genes as well as interferon-stimulated genes. At the protein level, overexpression of CST1 induced a significantly increased expression of CCL3, whereas CCL26 overexpression led to increased expression of HGF, and a decrease of CXCL11, CCL20, CCL3 and CXCL10.

Conclusion: Our results indicate that an overexpression of CST1 and CCL26 cause a downregulation of interferon related genes and inflammatory proteins. It might cause a higher disease susceptibility, mainly for allergic asthma, as CCL26 is an agonist for CCR-3-carrying cells, such as eosinophils and Th2 lymphocytes, mostly active in allergic asthma.

Keywords: A549; CCL26; CST1; OLINK; RNA sequencing; allergic asthma.

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Conflict of interest statement

The authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Flow chart of the study. A549 cells were separately transfected with the expression plasmids containing human CST1 and CCL26 as well as the empty vector pCMV6‐entry as control, obtained from Origene Technologies Inc. In total, 12 samples were included (4 biological replicates of CST1, 4 biological replicates of CCL26, 4 biological replicates of pCMV6‐Entry). On the one hand, RNA was extracted with the Qiagen RNeasy Micro kit 48 h after transfection and on the other hand, the cell lysates were collected with RIPA lysis buffer. Transcriptomic analysis was conducted via RNA sequencing and proteomic analysis via the Target 96 inflammation panel from OLINK. The transcriptomic analysis was conducted in biological quadruplicates and the proteomic analysis in biological duplicates. The results identified a downregulation of type I and III interferon genes as well as differentially expression of inflammatory proteins. Created with BioRender.
Figure 2
Figure 2
Volcano plot of the differentially expressed genes after the overexpression of (A) CST1 and (B) CCL26, separately. CST1 and CCL26 are in the upper right corner of the respective volcano plot. Gray: nonsignificantly expressed genes, blue: significantly downregulated genes, red: significantly upregulated genes with a |log2FC | ≥ 2. FDR, false discovery rate.
Figure 3
Figure 3
Gene ontology enrichment analysis for the downregulated genes due to (A) CST1 overexpression (n downregulated = 26) and (B) CCL26 overexpression (ndownregulated = 137). The x‐axis represents the GeneRatio, number of gene count indicated by the dot size, adjusted p‐value indicated by color (red = high significant, blue = low significant); the y‐axis represents the gene ontology biological processes. Network analysis of the relationship between the enriched pathways for the downregulated genes due to (C) CST1 overexpression (ndownregulated = 26) and (D) CCL26 overexpression (ndownregulated = 137). Two nodes are connected if they share 20% (default) or more genes. Darker nodes = more significantly enriched gene sets, bigger nodes = larger gene sets, thicker edges = more overlapped genes.
Figure 4
Figure 4
Confirmation of the RNAseq results via rt‐qPCR for the CST1 samples.
Figure 5
Figure 5
Confirmation of the RNAseq results via rt‐qPCR for the CCL26 samples.
Figure 6
Figure 6
Venn diagram of differentially expressed downregulated protein coding genes after either CST1 or CCL26 overexpression.
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