Suppression of combined granulocyte inflammation, airway structural remodeling, and AHR by CpG-ODN plus BUD in chronic CS-exposed asthmatic mice.
Histological data showed that lung specimens from OVA/CS mice had substantial peribronchial and perivascular connective tissues (e-Fig. 3A), multiple airway goblet cells containing mucus (e-Fig. 3B,D), and peribronchial collagen deposition (e-Fig. 3C,E). A combined granulocyte (neutrophil and eosinophil) inflammatory phenotype was confirmed as indicated by elevated Gr-1 (neutrophil-specific marker; Fig. 1B,E) and ECP (eosinophil-specific marker, Fig. 1A,D) immunohistochemical signals in the lungs, as well as marked expression of eotaxin 1 in BALF (Fig. 1C), which facilitates the recruitment of eosinophils and neutrophils[25]. Predominance of airway inflammation associated with neutrophils mixed with eosinophils was reduced; airway remodeling factors such as goblet hyperplasia and collagen accumulation were also diminished in the airway of CpG-ODN or BUD treated mice (e-Fig. 3) compared with CS-exposure asthmatic mice. Meanwhile, treatment with combined CpG-ODN and BUD caused almost no mucus hypersecretion alteration, negligible cell infiltration and airway wall thickness alteration, with suppression of AHR upon methacholine administration in animals with CS-exposure asthma (Fig. 1, e-Fig. 3).
Alteration of Th2/Th17 polarization and reduction of pro-inflammatory cytokines by CpG-ODN and BUD in CS-associated asthmatic mice.
Th2 markers (IL-5 and IL-13) were induced, while the Th1 marker IFN-γ was reduced after OVA + CS co-exposure in the mouse model (p < 0.01; e-Fig. 4C,D,E and p < 0.01; e-Fig. 4B). Pro-inflammatory cytokines (IL-8 and TNF-α) and TGF-β1, and serum anti-OVA IgE increased also (all p < 0.01, e-Fig. 4A,F,G,H,I). The above-mentioned values changed substantially after treatment with CpG-ODN (e-Fig. 4). BUD also somewhat attenuated CS associated increase in pro-inflammatory cytokines and serum anti-OVA IgE. However, we also noted that CpG-ODN had additive beneficial effects with BUD treatment on Th1/Th2 homeostasis modulation, pro-inflammatory cytokines, TGF-β1, and anti-OVA IgE, in the co-administration group (e-Fig. 4), which showed that CpG-ODN potentiated the effects of the corticosteroid.
Th17 cells exert their effects by producing multiple inflammatory cytokines such as IL-17A, which is known to enhance the chemotaxis of neutrophils in bronchial epithelial cells and airway smooth muscle cells[26]. More evidences have claimed Th17-associated neutrophilic airway inflammation in the mouse is GC insensitive[27]. As expected, Th17 cells in CS-exposure asthmatic mouse models were markedly elevated compared with the vehicle control group according to flow cytometry data (Fig. 2A,B). Moreover, significantly elevated serum, lung, and BALF IL-17A protein and mRNA amounts were found in the CS, OVA, and OVA/CS groups compared with the vehicle control group (Fig. 2C-G). Both CpG-ODN and BUD decreased the percentage of Th17 positive cells, and IL-17 mRNA and protein levels compared with untreated CS-related asthmatic mice (Fig. 2A-G). Meanwhile, joint treatment with CpG-ODN and BUD remarkably reduced Th17 cells, IL-17 mRNA and protein levels compared with the monotherapy groups (Fig. 2A-G).
Taken together, these data indicated that CS-exposure associated asthma induced a Th17/Th2-type response, and CpG-ODN and BUD synergistically decreased the exacerbated Th17- and Th2-associated cytokine amounts, enhancing the biosynthesis of IFN-γ, a Th1-associated cytokine.
HDAC2 activity and expression recovery upon treatment with CpG-ODN and BUD in CS-exposure asthmatic mice
CS reduces steroid responsiveness by modifying histone acetyltransferase, an essential epigenetic enzyme that mediates steroid anti-inflammatory action[28, 29]. Furthermore, HDAC2 activity and levels are substantially decreased by oxidative/nitrative stress, causing inflammation to be insensitive to the anti-inflammatory effects of GCs[30]. In this study, we assessed the levels of secreted HDAC2 in lung tissue samples by immunohistochemistry, ELISA and Western blotting. As shown in Fig. 3, OVA challenge and CS exposure both remarkably decreased HDAC2 mRNA and protein amounts (Fig. 3A-E). We also investigated the effects of CpG-ODN and BUD on CS-induced changes in HDAC2 mRNA and protein expression levels to verify whether CpG-ODN affect HDAC2 expression. Interestingly, it was found that after treatment with CpG-ODN or BUD only, HDAC2 gene expression levels were reversed compared with the untreated group, and this was more apparent after co-administration of CpG-ODN and BUD (Fig. 3A,B,C,E), although a statistically insignificant increase in HDAC2 protein expression levels was observed in mice administered CpG-ODN plus BUD (P = 0.06,Fig. 3D).
Moreover, based on studies reporting that patients with severe asthma have diminished GC sensitivity of peripheral blood monocytes (PBMCs) in comparison with non-severe asthma cases, in association with decreased HDAC2 activity that parallels the impaired GC sensitivity[31], we tested HDAC2 activity with a HDAC2 activity assay kit. As expected, similar to HDAC2 expression, HDAC2 activity in OVA + CS challenged mice was obviously suppressed and markedly recovered after administration of CpG-ODN or BUD, with significant differences between the OVA/CS and CpG-ODN/BUD groups, reflecting HDAC2 protein expression changes (Fig. 3F) .
These data suggested that HDAC2 was impaired, both at the expression and activity levels, in chronic asthmatic murine models. Meanwhile, CpG-ODN restored responsiveness to GC therapy by restoring HDAC2 expression and enhancing HDAC2 activity. When combined with BUD, CpG-ODN more substantially recovered HDAC2 activity and expression than either CpG-ODN or BUD alone.
Decreased RORγt expression and Th17 response under CS and OVA challenge after CpG-ODN and BUD treatment
HDAC2 is important in Th-17 cell differentiation from naive CD4+ T cells, and RORγt involvement attracts increasing attention[32, 33]. The catalytic activity of HDAC2 is important in inhibiting RORγt’s transcriptional activity, and SUMOylated RORγt recruits HDAC2 to the IL-17 promoter for gene downregulation[34]. To explore the mechanism by which CpG-ODN treatment regulates the IL-17A cytokine due to HDAC2 up-regulation, we next examined the amounts of RORγt, an important biomarker of HDAC2-mediated Th17 response under CS-induced asthmatic conditions, by immunohistochemistry, ELISA and Western blotting. The results exhibited a distinct increasing trend in RORγt mRNA and protein expression levels in CS-exposed asthmatic mice in comparison with the vehicle control group (Fig. 4). Meanwhile, upon joint administration of CpG-ODN and BUD, the animals showed significantly decreased RORγt mRNA and protein amounts (Fig. 4), indicating that CpG-ODN in combination with BUD suppressed RORγt to a certain extent, thereby inhibiting IL-17A expression in Th17 cells.
CpG-ODN and BUD synergistically regulate interplay of HDAC2, RORγt and IL-17A, orchestrating inflammatory reactions in HBE cells.
Airway epithelial cells play a primordial role on body defense against allergens, viruses, and environmental pollutants, which are involved in asthma pathogenesis. Meanwhile, IL-17A is found in airway epithelial cells[35]. To further confirm whether CpG-ODN inhibit RORγt-mediated Th17 response via HDAC2, we next performed in vitro cultures of HBE cells exposed to OVA and/or CSE, and administered CpG-ODN and/or BUD. We performed ELISA, qRT-PCR, Western blotting, immunofluorescence and flow cytometry to assess the levels of cytokines, HDAC2 and RORγt in all groups.
Consistent with animal data, it is found that CSE-exposed or OVA-challenged HBE cells had elevated IL-5, IL-13 (Th2 cytokine) and IL-17A (Th17 cytokine) levels compared with the vehicle control group. The abovementioned cytokines were markedly increased in HBE cells after co-exposure to CSE and OVA (all p < 0.01, Fig. 5A-F). Moreover, OVA-stimulation only, CSE-exposure only, or both in HBE cells significantly reduced HDAC2 amounts, with remarkable RORγt and IL-17A amount increases at both the gene and protein levels, suggesting specific associations of HDAC2 and RORγt with the IL-17 promoter in HBE cells (all p < 0.05,Fig. 5G-H, Fig. 6). Since HDAC2 is the main HDAC contributing to GC effects, whether CpG-ODN influence the interplay of HDAC2, RORγt and IL-17A in HBE cells was examined. Thus, OVA and CSE-exposed HBE cells were treated with CpG-ODN and BUD. Interestingly, after administration of CpG-ODN or BUD, HDAC2 protein levels showed an increasing trend. Notably, the increasing trend of HDAC2 expression changes was more meaningfully after joint treatment with CpG-ODN and BUD. However, contrary to HDAC2 results, RORγt and IL-17A amounts were decreased in mice administered CpG-ODN and notably reduced after co-administration of BUD and CpG-ODN (all p < 0.05, Fig. 5G-H, Fig. 6).
Collectively, these data provided convincing evidence of an interplay between HDAC2 and RORγt in OVA-induced and CSE-exposed airway epithelial cells, substantially affecting allergic airway inflammation. Moreover, CpG-ODN could partly affect this interplay, specifically by improving HDAC2 expression and inhibiting RORγt expression simultaneously.