In this study, we generated the functional network using STRING combined with KEGG. Additionally, we also analyzed the 93 genes using the network dataset generated by Cui and colleagues
. Cui’s dataset, including 1,634 genes, is comprehensive for analyzing signaling pathways in cancers. However, only 15 genes overlap with our 93 genes. The signaling network could not be generated using these 15 genes. It may be due to our dataset is related with proinflammatory effects and Cui’s dataset is focused on cancer signaling.
Under asthmatic conditions, ECP would be released by activated eosinophils and damage bronchial epithelial cells. At the same time, ECPsp is cleaved by human SP and SPP, and the resulting ECPsp triggers overexpression of TGF-α and EGFR. It suggested that the short signal peptide of ECP might function not only in protein secretion but also to regulate gene expression
. Similarly, the signal peptide may provide other functions besides protein targeting. Localization of the signal peptide of the envelope glycoprotein (Env) of Jaagsiekte sheep retrovirus to the nucleus regulates viral gene expression by mediating a beneficial post-translational step in the replication cycle
. In addition, interaction of calmodulin with the signal peptide fragments of preprolactin and HIV-1 gp160 processed by SPP triggers Ca2+/calmodulin-dependent cellular signaling
Our study provides evidence that ECPsp does indeed regulate gene expression in cells and that the upregulated genes participate in inflammation-related processes. Under inflammatory conditions, eosinophils might release cytokines to stimulate cell growth and activate immune cells; they might release chemokines to attract macrophages to the bronchia to eliminate the pathogens and damaged cell debris after secretion of ECP. The present microarray profiles show the upregulation of four chemokines, namely CCL5, interferon-induced protein of 10 kDa (IP-10/CXCL10), interferon-inducible T-cell alpha chemoattractant (I-TAC/CXCL11), and CXCL16. Also called RANTES, the chemotactic cytokine CCL5 is the most potent of these four chemokines, as it is able to both recruit eosinophils to tissues (via binding to CCR3) and attract macrophages (via CCR5), Th1 cells, and basophils to the site of inflammation
[30–33]. Moreover, CXCL10 binds CXCR3 and enrolls Th1 cells to resist intracellular pathogen infection, such as viruses
. In addition, CXCL10 is an important contributor to airway hyperactivity
. Another ligand of CXCR3, CXCL11, attracts Th1 cells, NK cells, and eosinophils to inflammatory sites
. The cytokine CXCL16 interacts with CXCR6 on Th1 and CD8 effector T cells and plays a crucial role in recruiting these cells to sites of inflammation
. Apart from the molecules mentioned above, we observed IFN-β and many interferon-related molecules in the dataset generated by DNA microarray analysis. Interferons have several functions in common, many of which involve anti-viral and anti-tumor activities. By interacting with their specific receptors, interferons can activate signaling pathways transmitted by STAT proteins. The STATs (STAT1–STAT6) are a family of transcriptional factors that regulate the expression of particular immunoregulatory genes. They can be activated by type I (IFN-α and IFN-β), type II (IFN-γ), and type III (IFN-λ) interferons
[38, 39]. For example, type-I IFNs can induce gene expression via either the ISRE (interferon-sensitive response element) or GAS (gamma interferon activation site) transcription elements through the actions of the STAT1/STAT2 heterodimer. In contrast, type II IFNs can transactivate a gene only if it contains the GAS element by means of the STAT1 homodimer
In our DNA microarray dataset, the IFNB1 (IFN-β) gene is upregulated 80%. It belongs to the type I IFNs which can activate the JAK/STAT1 and STAT2 pathways to regulate the expression of genes encoding chemokines that act downstream in this pathway. We thus suggested that ECPsp may regulate chemokine production by this pathway. Transcriptomic analyses indicate that the upregulation of JAK-STAT1 signaling plays a crucial role in protecting against microbial infection
[40, 41] by enhancing the expression of genes encoding chemokines (CXCL1, CXCL2, CCL2, and CCL5), a cytokine (IL-6), and components of JAK-STAT1 signaling (IFN regulatory factor 7 [IRF7], IRF9, and STAT1)
. Similarly, our investigation showed that genes including IRF7, IFN-induced protein with tetratricopeptides (IFIT1, IFIT2, IFIT3, and IFIT5), myxovirus resistance 1 and 2 (MX1 and MX2), and 2′,5′-oligoadenylate synthetases (OAS1, OAS2, OAS3, and OASL) are upregulated by ECPsp. In all of these instances, transactivation may depend on JAK/STAT signaling. Therefore, we hypothesized that under inflammatory conditions, eosinophils release ECP to damage pathogens and that ECPsp simultaneously induces JAK/STAT signaling to protect host cells from microbial infection and trigger chemokine production that might recruit immune cells, such as macrophages, to inflammatory sites.