Effects of CPX on airway inflammation
All mice increased in weight over the course of the experiment, and the degree of weight changes were not significantly different among groups (Fig. 1B). PM group did not induce significant AHR, however, PM/OVA group showed significant AHR compared to CON group. CPX or dexamethasone ameliorated AHR compared to PM/OVA group (Fig. 1C). PM/OVA group showed significant increase of total cell, macrophage, and eosinophil in BALF. CPX or dexamethasone significantly reduced levels of total cell and eosinophil in BALF (Fig. 1D).
Effects of CPX on fibrosis
PM and PM/OVA group showed significant fibrotic changes around airway lumen and vessels in MT-staining pathologic findings. CPX or dexamethasone ameliorated these fibrotic changes (Fig. 2A). Quantitative analysis also showed that PM and OVA induced significant increase of fibrotic changes, and CPX or dexamethasone improved them (Fig. 2B). The mRNA expression of collagen, fibrotic marker, also increased in PM and OVA treated group, and decreased in CPX or dexamethasone treated group (Fig. 2C-D).
Effects of CPX on inflammatory markers
The level of TGF-β in lung homogenates were significantly increased in PM/OVA group, and it was significantly decreased in CPX or dexamethasone treated group (Fig. 3A). The level of IL-1β significantly increased in PM group and OVA/PMA group. That in CPX or dexamethasone treated group were decreased compared to that in PM group (Fig. 3B). The level of IL-6 was significantly increased in PM group. That in CPX treated group was decreased compared to PM group (Fig. 3C). The levels of TNF-α and IL-13 were not significantly changed (Fig. 3D-E).
CPX significantly suppressed target molecule, chitinase-1
In IF study, various cells including bronchial epithelial cells and interstitial cells expressed chitinase-1. The areas expressing chitinase-1 (green color on FITC and merge slides) were more obvious and larger in PM and PM/OVA group compared to control group. They were ameliorated in PM/OVA/CPX and PM/OVA/Dex group (Fig. 4A). The level of chitinase-1 in lung homogenates were significantly increased in PM and PM/OVA group compared to control group, and it was significantly decreased in CPX or dexamethasone treated group (Fig. 4B).
Differential Gene Expression Between Groups
The expression of a genes between the groups was shown as the scatter plots (Supplementary data 1). In PM group, genes associated skeletal or cardiac muscle (Nppa, Nppb, Bmp10, and Mb) were down regulated (Fig. 5A–gene cluster 5, 7, 9, and 11; and Supplementary data 1A). The PM also down regulated Bpifa1, which plays a role in the innate immune responses of the upper airways. In PM/OVA group, Cxcl3 which plays a role in inflammation and as a chemoattractant for neutrophils, Cxcl5 which recruit and activate leukocytes, and Ccl8 which displays chemotactic activity for monocytes, lymphocytes, basophils and eosinophils) were highly up regulated, and muscle related genes, Nppa, Nppb, Bmp10, and Mb were also down regulated comparing with control group (Fig. 5A–gene cluster 5, 6, 7, 10, and 11; and Supplementary data 1B). Most of highly variable genes in PM group, also up or down regulated in the PM/OVA group (gene cluster 5, 7, and 11), and genes in the cluster 11 more prominently up regulated in PM/OVA group than PM group. However, comparing with PM group, Rnase2a which encodes eosinophil-derived neurotoxin, Ccl7 and Ccl24 which attracts monocytes and eosinophils were highly up regulated. Irf7 which plays a role in the innate immune response against DNA and RNA viruses, Ly6i which associated with T cell physiology, oncogenesis and immunological regulation were down regulated in PM/OVA group (Fig. 5A–gene cluster 2, 3, 6, and 10; and Supplementary data 1C).
In PM/OVA/CPX group, chemokine genes (Cxcl3, Cxcl5, Cxcl10, Ccl2, and Ccl4) down regulated in PM/OVA/CPX than PM/OVA. However, inflammation and allergy associated genes (Chil3 and Chil4), eosinophil-associated gene (Ear7), and B lymphocyte-associated gene (Jchain) were prominently up regulated in PM/OVA/CPX group than PM/OVA group (Fig. 5A–gene cluster 8).
In PM/OVA/Dex group, Nppa, Chil4, Clca1, and Bpifa1 were highly up regulated, on the contrary, Cxcl10, Ccl4, Ccl2, and Cxcl5 were down regulated comparing with PM/OVA group (Fig. 5A–gene cluster 1, 3, 5, 10, and 11; and Supplementary data 1E). Especially, genes in cluster 1 were prominently up regulated than all other groups.
Gene Ontology Analysis Between Groups
The analysis for Gene Ontology (GO)-enrichment in the biological process category was shown in the heatmap plot and upset plots (Fig. 5B and Supplementary data 2). The result demonstrated that the enriched GOs in the PM and PM/OVA group were up regulated in activation of immune systems, such as immune cells migration and chemotaxis, cytokine or chemokine mediated inflammation, and antigen processing and presentation.
In both PM/OVA/CPX and PM/OVA/Dex group, granulocyte migration/chemotaxis, neutrophil migration/chemotaxis, acute-phase response, chemokine-medicated signaling pathway, and response to chemokine were significantly down regulated comparing with PM/OVA group. However, antigen processing and presentation (of peptide antigen), (positive) regulation of leukocyte mediated immunity, leukocyte mediated cytotoxicity, and regulation of innate immune response of PM/OVA/CPX group were not significantly different with those of PM/OVA group. On the contrary, these pathway activities of PM/OVA/Dex group were significantly down regulated comparing PM/OVA group, additionally, chemokine-mediated signaling pathway, (cellular) response to chemokine, response to interferon-gamma, (regulation of) adaptive immune response, (regulation of) leukocyte mediated immunity, lymphocyte mediated immunity, adaptive immune response based on somatic recombination of immune receptors built from immunoglobulin superfamily domains, and (defense) response to virus were more down regulated than PM/OVA/CPX group in the comparison with PM/OVA group. Common up or down regulated pathways in the top 10 significant GOs between PM/OVA vs. control group, PM/OVA/CPX vs. PM/OVA, and PM/OVA/Dex vs. PM/OVA were neutrophil migration, granulocyte chemotaxis, myeloid leukocyte migration, and leukocyte chemotaxis (Fig. 5C).
Muscle related GOs, including striated or cardiac muscle, were down regulated in all experimental groups comparing with control group (Fig. 5B). Although significant down regulation was found in PM/OVA/Dex comparing with control group, PM/OVA/Dex group tend to more upregulate muscle related GO activity than PM/OVA or PM/OVA/CPX group.
Asthma And Asthma Related Pathway Analysis With Kegg Database
The single exposure of PM affected the immediate reaction of asthma by upregulating of the antigen processing and presentation via MCHII and the FcεRI in the mast cell (Fig. 6A). In the comparison between PM/OVA and PM group, OVA affected both the immediate and late reaction of asthma by upregulating of the interleukin-4 (IL-4), FcεRI, eotaxin, and MBP in asthma pathway, and cytokine-cytokine receptor interaction pathway (Fig. 6A and Fig. 6C, respectively). The concurrent exposure of PM and OVA significantly increased entire asthma pathway and associated genes (Fig. 6A and Fig. 6D).
The CPX decreased the expression of Il-4, Il-9, FcεRI on immediate asthmatic reaction, and TNF-α on late asthmatic reaction. On the contrary, the CPX increased the expression of MHCII, CD40L, Il-13, eotaxin (Ccl11), Mbp, and Epo on late reaction (Fig. 6B), therefore, CPX did not significantly decrease the entire asthma pathway (Fig. 6C). The dexamethasone decreased the expression of MHCII, CD40L, Il-13, Il-9, FcεRI on immediate reaction, TNF-α, Il-5, and MBP on late reaction (Fig. 6B). In addition, dexamethasone significantly decreased entire asthma pathway, B cell receptor signaling pathway, and FcεRI signaling pathway, cytokine-cytokine receptor interaction, and JAK-STAT signaling pathway (Fig. 6C and Supplementary data 3). Especially, cytokine-cytokine receptor interaction and JAK-STAT signaling pathway are commonly decreased pathway between PM/OVA/CPX and PM/OVA/Dex.