Levels of serum CC16 in mice before and after HDM inhalation
Using ELISA assay, serum CC16 concentrations of the control and HDM groups were detected on Days 0, 7, 21, and 35, respectively. As summarized in Tab.1, baseline levels of serum CC16 on Day 0 prior to HDM inhalation did not differ between the two groups. Although the HDM group had slightly higher serum CC16 levels on Day 7 (one week after initial HDM exposure) than those before HDM sensitization (baseline), the differences were not statistically significant. Compared to baseline, a sharply elevation of serum CC16 levels was observed on Day 21 post-HDM challenge in asthmatic mice. Thereafter, serum levels of CC16 in HDM group on Day 35 were significantly declined to a very low extent, which were even less than the control.
CC16 alleviates airway inflammation and airway epithelial injury in HDM-induced asthmatic mice
To explore the possible protective role of CC16 in allergic airway inflammation and airway epithelial injury, we established a HDM-induced murine asthmatic model and treated mice with or without ranged doses of recombinant CC16 (5ug/g/mouse or 10ug/g/mouse) prior to HDM challenge (Fig.1a). BALF was collected 24h after the mice were sacrificed, and then total and differential inflammatory cells were counted. As shown in Fig.1b, the number of total cells was significantly elevated in HDM-induced asthmatic group compared with that from the saline-challenged group, mainly represented in alveolar macrophages (AM), eosinophils (Eos), lymphocytes (Lym), and neutrophils (Neu). BALF inflammatory cell counts from HDM-challenged mice that received low or high dose of CC16 (5μg/g, 10μg/g) were both reduced compared with those from HDM-challenged control. Of note, administration with high dose of CC16 (10μg/g) exhibited a markedly reduction in the infiltration of BALF inflammatory cells in contrast to low dose of CC16 (10μg/g). Similarly, compared with the control, the HDM-induced asthmatic group showed a significant elevation in the production of Th2-associated inflammatory cytokines including IL-4, IL-5,IL-13 and HDM-specific IgE (sIgE) via ELISA assay, which were inhibited by CC16 treatment in a dose-dependent manner (Fig. 1c,d,e,f).
By histopathological analysis of lung tissues stained with hematoxylin-eosin, our findings showed that HDM challenge resulted in extensive airway wall thickening, mucosal metaplasia as well as the recruitment and infiltration of inflammatory cells in peribronchial and perivascular areas. Further PAS staining exhibited collagen deposition, goblet cell hyperplasia and mucus hypersecretion that constituted airway damage. Intriguingly, asthmatic mice with administration of CC16 had less inflammatory cell infiltration especially eosinophils together with mild histological damage of airway epithelial tissues. Moreover, a greater pathological alleviation in 10ug/g CC16-treated asthmatic group had been discovered than that in 5ug/g CC16-treated group (Fig.1g). All these results indicated that CC16 treatment dose-dependently suppressed HDM-induced airway injury and inflammation.
CC16 treatment prevents airway epithelia against apoptosis exposed to HDM in mice.
HDM allergen can induce airway epithelium dysfunction and promote epithelial cells apoptosis that have been considered to be strongly associated with airway injury[19].To further explore the antiapoptotic impact of CC16 on airway epithelium, we performed TUNEL experiment to evaluate the level of apoptosis in airway tissues under HDM exposure. The quantitation of TUNEL-positive cells was based on the average from five randomly chosen areas per sample. In normal lung, few airway epithelia expressed TUNEL-positive cells. In contrast, HDM challenge significantly augmented the number of TUNEL-positive cells in airway epithelium in asthmatic mice (Fig.2a), suggesting that HDM allergen caused severe airway damage. Upon pretreatment with CC16 during HDM challenge, especially high-dose CC16 treatment, the ratio of TUNEL-labeled airway epithelial cells was significantly decreased compared with that in HDM alone group (Fig. 2b). Importantly, the extent of TUNEL positivity was parallel to pathological changes of airway epithelium. Thus, epithelial cells apoptosis reflected the degree of airway injury and inflammation.
In order to determine the antiapoptotic regulatory mechanism of CC16, the protein levels of Bcl-2, Bax and cleaved caspase-3 of airway tissues were evaluated by Western blot among different groups (Fig.2c). Compared to control group, the expression of antiapoptotic protein Bcl-2 was significantly decreased in asthmatic mice under HDM conditions companied with the upregulation of proapoptotic protein Bax and cleaved caspase-3 expression, indicative of HDM allergen-induced propensity to epithelial apoptosis. However, treatment with CC16 exhibited a dose-dependent improvement of Bcl-2/Bax protein ratio and downregulated cleaved caspase-3 expression in contrast to HDM alone (Fig.2d). These data displayed that CC16 administration might protect against airway epithelial cell injury and apoptosis through inhibiting the activation of mitochondrial apoptotic pathway.
The cytoprotective effect of CC16 on HDM-induced injury and inflammatory cytokines production in BEAS-2B cells
HDM-induced airway injury and inflammatory response are closely related to airway epithelial cell dysfunction, which aggravate the pathogenesis of asthma [20]. To elucidate the protective effect of CC16 in airway epithelia under HDM conditions, we performed experiments on HDM-stimulated normal human airway epithelial BEAS-2B cells. CC16 was applied to pretreat BEAS-2B cells with various concentrations ranged from 5ng/ml to 200ng/ml for 24h. As a result, CC16 showed little cytotoxicity in BEAS-2B cells at concentrations less than 200ng/ml (Fig.3a). Subsequently, BEAS-2B cells were incubated with CC16 (100ng/ml and 200ng/ml, respectively) for 24h and then exposed to 300ng/ml HDM for 48h. PBS was used as a negative control. The proliferation of HDM-stimulated BEAS-2B cells was further assessed by CCK-8 assay. As displayed in Fig.3b, HDM exposure significantly restrained cell viability in comparison with the untreated control cells, while the growth rate of BEAS-2B cells was significantly attenuated by CC16 treatment dose-dependently. Since HDM stimulation contributes to the production of epithelial-derived proinflammatory mediators via its protease activity, BEAS-2B cells were additionally coincubated with HDM (300ng/ml) and cysteine protease activity inhibitor E64 (20μM) (Sigma-Aldrich, Stockholm, Sweden). Our results showed increased mRNA levels of epithelial-derived cytokines IL-25, IL-33 and TSLP in HDM-treated BEAS-2B cells compared to control group, whereas they were significantly blocked in the E64-treated group. Noticeably, dose-dependently decreases of IL-25, IL-33 and TSLP expression were also observed in CC16 groups compared to HDM alone group, although the degree of suppression was less than that of E64-treated cells (Fig.3c).
We also investigated whether CC16 treatment could ameliorate HDM-evoked apoptosis in BEAS-2B cells. The flow cytometry results showed a dramatic elevation in the proportion of apoptotic cells upon HDM stimulation, while CC16 pretreatment reversed HDM-induced BEAS-2B cells apoptosis by 26% and 50%, respectively, according to different doses of CC16 (Fig.3d). The percentage of cleaved caspase-3 positive cells was also significantly increased in HDM group compared to the control, and declined to varying degrees with different doses of CC16 treatment (Fig.3e). These data indicate that CC16 exerts an anti-inflammatory and antiapoptotic influence on HDM-induced BEAS-2B cells which is similar to airway tissues described above.
CC16 suppresses HDM-induced overexpression of HMGB1 in vivo and in vitro.
Allergens such as HDM can lead to the upregulation of HMGB1 protein by injuried airway epithelial cells[21]. Many studies have confirmed that HMGB1 plays an essential role in allergic airway inflammation as a signal for DNA repair and cell death. In view of CC16-mediated protective effect on HDM-challenged airway epithelial cells damage, we hypothesized that regulation of HMGB1 expression might be involved in the underlying molecular mechanism of CC16 treatment. In the current study, the expression of HMGB1 was investigated in vitro and in vivo. In the HDM-induced asthma model, immunochemistry findings showed that HMGB1 was mainly expressed in airway epithelium and some peripherally infiltrative lymphocytes of lung tissues, suggesting that airway epithelial cell was an important source of HMGB1 production. In particular, HMGB1 expression was distinctly detected in the nuclei and cytoplasm of airway epithelia in HDM-challenged asthmatic group, whereas HMGB1 was only weekly or modestly stained in airway epithelia nuclei in control group (Fig.4a). These results illustrated that HDM could induce HMGB1 to be translocated from the nucleus to the cytoplasm. Furthermore, the elevated expression especially cytoplasmic HMGB1 in HDM-induced mice was partially diminished by CC16 administration, with better improvement observed in high-dose CC16-treated group. In addition, HMGB1 protein levels in BALF and lung tissue were evaluated by ELISA assay and Western blot respectively. As shown in Fig.4b, c and d, HMGB1 expression was significantly increased in both the BALF and lungs of asthmatic mice in contrast to those in control group, indicating that extracellular release of HMGB1 was also actively promoted after HDM exposure except for nucleocytoplasmic translocation. As expected, these changes of HMGB1 expression were reversed by pretreatment with CC16 in a dose-dependent manner. To focus on the modulatory function of CC16 on HMGB1 expression in airway epithelial cells, immunofluorescence assay was conducted to detect the cellular localization of HMGB1 protein following the in vitro HDM-mediated damage. Likewise, HDM-challenged BEAS-2B cells showed significantly increased cytoplasmic and extracellular expression of HMGB1 whilst a little faint HMGB1 immunofluorescence staining was detected in the nuclei of PBS-treated control cells. In contrast, HDM+CC16 group showed a markedly reduction of HMGB1 staining as compared with HDM group, suggesting that HDM-stimulated HMGB1 upregulation was dramatically abolished by CC16 especially extracellular HMGB1 release, as displayed in Fig.4e. Moreover, the cells treated with high-dose CC16 showed less HMGB1 expression than CC16 low-dose group. Altogether, based on these data, it was referred that CC6 could suppress HDM-induced HMGB1 activation in airway epithelial cells.
HMGB1 contributes to HDM-induced airway epithelia damage through TLR4/NF-κB signaling pathway
It is well accepted that TLR4, a crucial PRR interacted with HMGB1, is generally expressed by airway epithelial cells in response to inhaled HDM allergen and is required for the subsequent activation of NF-κB that modulates apoptosis and inflammatory cytokine genes. Since HDM allergen led to upregulation of HMGB1 expression and the latter was negatively regulated by CC16, we next explored the role of HMGB1 as well as its potential signal pathway involved in HDM-mediated airway epithelial cell injury and apoptosis. To determine the in vivo effect of HMGB1 on HDM-induced allergic airway injury, HMGB1 expression was intervened via intratracheally administration of Ad-sh-HMGB1 or Ad-GEP vector before the establishment of asthma model. As shown in Fig.5a, relative mRNA level of HMGB1 was markedly downregulated in airway tissues of the mice receiving Ad-sh-HMGB1 relative to the Ad-GFP-treated mice under both PBS and HDM conditions. Histological analysis revealed that there was a noticeable augmentation in inflammatory cells infiltration and airway wall thicken in Ad-GEP-treated mice with HDM inhalation compared to those receiving Ad-GEP alone. However, in contrast with that in the HDM+Ad-GEP group, administration of Ad-sh-HMGB1 into the lungs of mice exhibited relatively less severe damage of airway tissues under HDM conditions (Fig.5b). Similarly, HDM remarkably promoted caspase-3 activity in the lungs of Ad-GEP-treated group, which was partially decreased by shRNA-mediated HMGB1 knockdown (Fig.5c).
In the in vitro experiment, following transfected with si-NC and si-HMGB1 respectively, BEAS-2B cells were incubated in the presence or absence of HDM stimulation for 12h. As expected, HDM stimulation indeed suppressed the growth rate of BEAS-2B cells in contrast to that of PBS-treated control cells via CCK-8 assay. Simultaneously, transfection with si-HMGB1 effectively attenuated the decrease of cell viability caused by HDM (Fig.6a). Accordingly, for flow-cytometry assay, it was found that silencing HMGB1 remarkably antagonized the facilitative effect of HDM on cell apoptosis (Fig.6b). There was no significant difference between PBS+si-NC group and PBS+si-HMGB1 group. These findings suggested that HMGB1 was essential during the process of HDM-induced cell apoptosis.
Western blot analysis was performed to determine the alterations of the signaling proteins including TLR4, NF-κB and phosphorylated(p)-NF-κB under the mimic asthmatic condition. As shown in fig.6c and d, HDM exposure significantly led to overexpression of HMGB1 that was associated with the upregulation of TLR4/p-NF-κB axis and corresponding changes of apoptosis-related markers compared with control cells. At the same time, si-HMGB1 transfection obviously blocked the increased expression of HMGB1 in BEAS-2B cells induced by HDM, concomitant with the downregulation of TLR4 and p-NF-κB expression. The difference between PBS+si-NC and PBS+si-HMGB1 groups was slight significant. Besides, compared with PBS+si-NC group, a higher level of caspase-3 cleavage was observed by flow cytometry in HDM-treated cells with si-NC transfection, yet decreased by si-HMGB1 transfection (Fig.6e). Overall, these data demonstrated that HMGB1-mediated airway epithelial cell apoptosis was correlated with TLR4/NF-κB signaling activation in the in vitro model of HDM-induced asthma.
HMGB1 signaling is involved in CC16-mediated cytoprotection in airway epithelial cells exposed to HDM.
As mentioned above, CC16 could negatively regulate the expression of HMGB1 which imperatively contributed to HDM-mediated airway epithelia damage. To further ascertain whether CC16 exerted protective influence in a HMGB1-dependent manner, the HMGB1-overexpressing mice were generated by the intratracheal injection of Ad-HMGB1 vector prior to HDM exposure. As shown in Fig.7a, Ad-HMGB1-injected mice had significantly higher mRNA HMGB1 levels in the lungs than GFP-injected mice. Then the HMGB1-modified mice were treated with CC16 (10g/g) administration or the negative vehicle in response to HDM. Our results demonstrated that CC16 treatment could effectively alleviate airway epithelium edema and mucus production in the mice receiving Ad-GFP under HDM conditions, whereas HDM-induced inflammatory responses and airway damage did not have a great improvement in Ad-HMGB1-injected mice after CC16 administration(Fig.7b). Caspase-3 activity assay also confirmed that HMGB1 overexpression abated the protective effect of CC16 on HDM-induced apoptosis in mice (Fig.7c). Additionally, BEAS-2B cells were transfected with the recombinant pcDNA3.1-HMGB1 plasmid to elevate HMGB1 expression. The mechanisms underlying the association between HMGB1-mediated signaling and CC16 were then excavated by Western blot. Consistently, flow-cytometry analysis showed that the increased apoptosis index of BEAS-2B cells exposed to HDM (300ng/ml) was abrogated by 200ng/ml CC16 treatment. HMGB1 overexpression abolished the antiapoptotic effect of CC16 on HDM-induced BEAS-2B cells, as proved by the enhancement of cellular apoptosis index (Fig.8a). The immunoblotting results further displayed that HDM stimulation significantly enhanced protein expression of HMGB1, TLR4 as well as p-NF-kB compared with the pcDNA3.1-vector control group. In contrast to the HDM+pcDNA3.1-vector group, overexpression of HMGB1 rendered an enhancement of HMGB1-mediated TLR4/NF-κB signaling activity caused by HDM. Meanwhile, CC16 treatment markedly reversed the increased activation of HMGB1 signaling in the cells exposed to HDM, suggesting that CC16 acted as a key agent of HMGB1-mediated signaling molecules leading to HDM-evoked inflammation and damage. More importantly, it was found that rescued HMGB1 level by transfection with recombinant plasmid blunted CC16-mediated inhibition on HMGB1/TLR4/NF-κB signaling. The downstream apoptosis regulators of HMGB1-TLR4/NF-κB axis were also detected by immunoblotting analysis. As expected, the ratio of Bcl-2/Bax in HDM-induced group was dramatically lower than that in the control, while cleaved caspase-3 expression level was higher. Accordingly, the alterations in the expression of Bcl-2, Bax, and cleaved caspase-3 upon HDM exposure were abolished by CC16 treatment. However, the antiapoptotic ability of CC16 was subsequently compromised by HMGB1 overexpression, in keeping with the augmentation of HMGB1-mediated TLR4/NF-κB signaling activity (Fig.8b and c). Flow cytometry of cleaved caspase-3 detection also demonstrated that the adverse effect of CC16 on HDM-induced caspase-3 activation was overturned after the overexpression of HMGB1 (Fig.8d).
Collectively, our findings indicate that CC16 alleviates HDM-activated airway epithelial injury and apoptosis via inhibition of HMGB1 expression, while HMGB1-mediated signaling proteins such as TLR4 and NF-κB are potentially modulators involved in the airway protection of CC16.