We focused on elucidating (1) the expression of Mas receptor in OVA-induced acute allergic asthma and (2) whether activating Mas receptor could attenuate inflammatory cells, especially macrophage infiltration in vivo and in vitro. Our results demonstrated that Mas receptor mainly functioned in bronchial epithelium and activating Mas receptor attenuates allergic airway inflammation, especially CCL2 dependent macrophages recruitment via JNK pathways.
In acute allergic airway inflammation, we found that after consecutive OVA challenge for seven days, the expression of Mas receptor decreased significantly in lung tissue. The decrease probably presented in the whole pathological process of asthma, because in chronic asthma mice model, after OVA challenge from days 21 to 46, decreased expression of Mas receptor in lung tissue was also detected through western blot and immunohistochemical [17, 18]. Generally, decreased expression of Mas receptor was involved in the occurrence and development of many diseases. In the hippocampus of diabetic rats, the decreased expression of Mas receptor was involved in Alzheimer's disease like functional and pathological changes in streptozotocin-induced diabetes [30]. In balloon-injured rat aorta, the decrease of Mas receptor expression could be observed in the injured aorta, while valsartan could improve the expression of Mas receptor and reduce the neointimal hyperplasia of aorta [31]. Besides, chronic hypoxia-induced pulmonary hypertension also presented decreased expression of Mas receptor in the right ventricle of rats, which contributed to right ventricular remodeling and fibrosis [32]. Most of the studies mentioned above were mainly chronic diseases, and were related to tissue regeneration and remodeling. For acute inflammatory diseases, there were few studies discussing about changes of Mas receptor expression. In fact, different expression patterns of Mas receptor might exist in different stages of the disease. For example, the expression level of Mas receptor was significantly increased in acute phase and decreased in post-acute phase during the process of peripheral nerve injury, reflecting the transformation from anti-inflammatory to pro-inflammatory [33]. Compared with chronic OVA stimulation, our study found that even a short-term OVA stimulation could significantly reduce the expression of Mas receptor, which suggested that the decrease of Mas receptor might involve in the initiation and development of asthma in the early stage. Hence, it was of great significance to study the role of Mas receptor in acute allergic airway inflammation. Considering that Ang-(1–7) exerted a short duration of biological effect in vivo for rapid degradation by ACE which was rich in lung [34], we employed AVE0991, a synthetic non-peptide agonist of Mas receptor, in subsequent experiments to further evaluate the role of Mas receptor in acute allergic airway inflammation. Different from studies in MG Rodrigue-Machado [17, 18], we further performed the immunofluorescence double staining to co-localize Mas receptor with EpCAM, which confirmed that Mas receptor mainly acted on airway epithelial cells. It suggested that the method of intratracheal infusion AVE0991 to activate Mas receptor was credible.
Indeed, MG Rodrigue-Machado’s research of AVE0991 focused more on airway and vascular remodeling in chronic asthma [18]. Nevertheless, we found that pretreatment of AVE0991 activating Mas receptor before OVA challenge significantly attenuated acute airway inflammation and decreased macrophage infiltration. In our study, increase in IL-4 induced by OVA was blocked by AVE0991. IL-4 was important cytokine for alternative activation of infiltrated macrophages and enhancement of allergic inflammation [35]. IL-10, a potent anti-inflammatory cytokine [36], exhibited an opposite result to IL-4. Our data argued that AVE0991 could serve as an important tool for treatment of acute allergic airway inflammation. As for signaling pathways, the Ang-(1–7)/Mas receptor axis reportedly inhibited phosphorylation of JNK, ERK1/2, and p38 in different physiological conditions [37, 38, 39]. To elucidate the mechanisms by which Mas receptor mediated their effects on acute allergic airway inflammation, we investigated MAPK signaling pathways that were involved in asthma, including JNK, ERK1/2, and p38. In vivo studies revealed that activating Mas receptor inhibited the phosphorylation of JNK, ERK1/2, and p38 induced by OVA. In chronic asthma, MG Rodrigue-Machado discussed more about the protective effect of AVE0991 on pathology and changes in expression of renin-angiotensin axis related receptors and angiotensin peptides, rather than signaling pathways [18]. Researches based on Ang-(1–7) focused more on the role of ERK pathway in eosinophil infiltration and airway remodeling [14, 15, 16, 17]. In fact, all three subfamilies of MAPKs were supposedly involved in the pathogenesis of asthma. The phosphorylation states of all three MAPK members were up-regulated in asthma animal models, which identified them as potential novel targets for asthma treatment [40]. Various small-molecular inhibitors of MAPK tested in animal models of asthma hold potential for treatment of severe corticosteroid resistant asthma and warrant further clinical investigation. The ERK1/2 signaling pathway reportedly played a more important role in the process of airway remodeling rather than acute allergic airway inflammation, and p-ERK1/2 inhibitor effectively inhibited airway remodeling in chronic asthma [41]. Kim et al. reported that inhibition of p38 MAPK might attenuate allergen-induced airway inflammation and vascular leakage through modulation of VEGF expression in mice [42]. JNK pathway regulated various physiological conditions including inflammatory responses, expression of proteins and so on [43]. An only JNK inhibitor, SP600125, showed significant reduction of eosinophil, lymphocyte, neutrophil, and macrophage counts in BALF as well as decreased expression of pro-inflammatory cytokines in an OVA-induced murine acute asthma model [44].
Although multiple pathways and cytokines were involved in pathogenesis of acute allergic airway inflammation, one of critical pathways and its crucial downstream might be inhibited by Mas receptor activation and fail to induce inflammation. We detected mRNA levels of several known cytokines in lung samples, including IL-25, IL-33, TSLP [45], TNF-α, and chemokines (CCL2, CCL3, CCL4) [46, 47]. Our results demonstrated that an apparent increased expression of CCL2 in asthma mice could be sharply inhibited by AVE0991. CCL2 is closely associated with airway inflammation [48, 49], especially in recruiting macrophages [7]. Agache et al. observed a significant correlation between increased levels of CCL2 and asthma severity and fast LF decline [50]. Viral-induced exacerbation was accompanied by increased levels of CCL2. Although RSV-induced exacerbation was resistant to steroid treatment, inhibition of CCL2 function suppressed features of disease, including AHR and macrophage infiltration [51]. Previous studies demonstrated that either blocking the CCR2 or neutralizing CCL2 could effectively block the recruitment of inflammatory macrophages [52]. Though MG Rodrigue-Machado had mentioned that Ang-(1–7) prevented CCL2 increase induced by OVA, a further analysis of underlying mechanism was not performed [17]. In fact, there were evidences which suggested JNK pathway regulated the expression CCL2 under different processes. In the tumor microenvironment, increase of fatty acid oxidation promoted JNK activation, which enhanced the expression of CCL2 [53]. In non-alcoholic steatohepatitis, JNK activation promoted the release of CCL2 and thus attracted macrophages to injured liver [54]. In vitro model of neuroinflammation presented upregulation of CCL2, while JNK inhibitor dose-dependently inhibited LPS-induced CCL2 upregulation [55]. In acute asthma, it was reported that OVA activated TLR-2/JNK pathway signaling and increase CCL2 secretion in vivo, while no further validation of TLR2/JNK/CCL2 was performed [56]. Thus, the axis linking JNK and CCL2 in asthma remains unclear.
Since researches on Mas receptor activated by AVE0991 or Ang-(1–7) were mainly in vivo studies and mostly discussed about the effect of ERK pathway on eosinophil infiltration and airway remodeling [14, 15, 16, 17, 18], the crucial pathway and its downstream molecules remained unclear in acute allergic airway inflammation especially in macrophage recruitment and further validation of underlying mechanism in vitro were needed. According to our studies in vivo, activation of Mas receptor attenuated acute allergic airway inflammation probably through the mechanism that activating Mas receptor inhibited JNK/CCL2 pathway and reduced macrophage recruitment. Hence, we investigated it next at cellular level to explore the key molecule and pathway. The bronchial epithelium was the major source for CCL2 in allergic lung inflammation [57]. Based on the findings that the recruitment of macrophages was parallel to the up-regulation of CCL2 and that Mas receptor was co-localized with EpCAM, we explored the underlying mechanisms in 16HBE, a human bronchial epithelial cell line. Our work revealed that activating Mas receptor directly inhibited CCL2 secretion and THP-1 macrophages migration stimulated by LPS via decreasing phosphorylation of JNK. LPS-induced conditioned media promoted THP-1 macrophages migrations, while anti-CCL2 neutralizing antibodies blocked the migrations. The specific effect of JNK pathways on CCL2 was further proved by anisomycin, a JNK activator. Different from results of in vivo studies, AVE0991 treatment did not inhibit the phosphorylation of ERK induced by LPS in vitro. Previous studies had shown that ERK signaling pathway was mainly related to airway remodeling, especially the proliferation and migration of airway smooth muscle cells [58, 59]. Similarly, AVE0991 could not inhibit anisomycin-induced phosphorylation of p38, which was discussed more on differentiation of T cells as well as the proliferation and migration of airway smooth cells [60, 61]. However, the phosphorylation of JNK was coincident in vivo and in vitro. Indeed, airway epithelial JNK activation was found critical in initiation of allergic inflammation, especially house dust mite, fungi or OVA induced TLR2/JNK activation [56]. Rhinoviruses infection of airway epithelial cell frequently produced higher levels of inflammatory cytokines via TLR4/JNK pathway and exacerbated inflammation in asthma, causing worse airway obstruction and symptoms [62]. In clinical, systemic glucocorticoid (GC) inhibited phosphorylation of JNK in bronchial mucosal cells in GC responsiveness asthma, while the phenomenon was not observed in GC-resistant asthmatic subjects [63]. Thus, the JNK pathway could serve as a potential target for future therapy in asthma.