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 were collected 24 h 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 those from the saline challenged group, which mainly represented elevation 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 (5 µg/g, 10 µg/g) were both reduced compared with those from HDM-challenged control. Of note, administration with high dose of CC16 (10 µg/g) exhibited a markedly reduction in the infiltration of BALF inflammatory cells in contrast to low dose of CC16 (10 µg/g). Similarly, compared with control group, 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 constitute 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.To further explore the anti-apoptosis impact of CC16 on airway epithelium, we performed TUNEL immunofluorescence to evaluate the level of apoptosis in airway tissues under HDM exposure. DAPI was used as staining marker of nucleus. In normal lung, few airway epithelia expressed TUNEL-positive cells. In contrast, HDM challenge significantly augmented the number of TUNEL-positive cells around 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 staining cells were significantly reduced by 29% in lung tissue 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. We also investigated whether CC16 treatment could ameliorate HDM-evoked apoptosis in BEAS-2B cells.
In order to determine the anti-apoptosis regulatory mechanism of CC16, the balance between Bcl-2 and Bax proteins were evaluated by western blot. Compared to control group, the expression of antiapoptotic protein Bcl-2 was significantly decreased under HDM conditions companied with the upregulation of proapoptotic protein Bax expression, indicative of HDM allergen-induced propensity to epithelial apoptosis. However, treatment with CC16 exhibited an improvement of Bcl-2/Bax protein ratio in contrast to HDM alone. These data displayed that CC16 may prevent against airway epithelial cell injury and apoptosis through a Bcl-2/Bax-dependent way.
The cytoprotective effect of CC16 on HDM-induced injury and inflammatory cytokines production in BEAS-2B cells
HDM-induced airway injury and inflammatory response is closely related to airway epithelial cell dysfunction, which aggravates the pathogenesis of asthma. 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 5 ng/ml to 200 ng/ml for 24 h. As a result, CC16 showed little cytotoxicity in BEAS-2B cells at concentrations less than 200 ng/ml (Fig. 3a). Subsequently, BEAS-2B cells were incubated with CC16 (100 ng/ml and 200 ng/ml, respectively) for 24 h and then exposed to 300 ng/ml HDM for 48 h. 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. Additionally, for real-time RT-PCR, the mRNA expression of epithelial-derived pro-inflammatory mediators including IL-25, IL-33 and TSLP were upregulated in BEAS-2B cells upon HDM stimulation compared to control group, whereas it was noticeably overturned following treatment with CC16 (Fig. 3c,d,e).
We also investigated whether CC16 treatment could ameliorate HDM-evoked apoptosis in BEAS-2B cells. By flow cytometry, we found that the ratio of apoptotic cells were dramatically increased upon HDM stimulation, while CC16 pretreatment reversed HDM-induced BEAS-2B cells apoptosis by 13% and 17%, respectively, according to different dose of CC16 (Fig. 3f).These data indicate that CC16 may exert an anti-inflammatory and anti-apoptosis 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. 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 may 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 the lung tissue, suggesting that the 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, the protein level of HMGB1 in BALF was evaluated by ELISA assay. As shown in Fig. 4b and c, HMGB1 expression was significantly increased in BALF from the mice in asthmatic group 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 a significantly increased cytoplasmic and extracellular expression of HMGB1 whilst a few 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 200 ng/ml CC16 showed lower HMGB1 than those in 100 ng/ml CC16 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. Given that 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. In our study, after transfected with si-NC and si-HMGB1 respectively, BEAS-2B cells were incubated in the presence or absence of HDM stimulation for 12 h. As expected, HDM stimulation indeed suppressed the growth rate of BEAS-2B cells in contrast to PBS-treated control cells via CCK-8 assay. Simultaneously, transfection with si-HMGB1 effectively attenuated the decrease of cell viability caused by HDM (Fig. 5a). Accordingly, for flow-cytometry assay, it was found that silencing HMGB1 remarkably antagonized the facilitative effect of HDM on cell apoptosis (Fig. 5b). 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. 5(c), HDM exposure significantly promoted 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 down-regulation of TLR4 and p-NF-κB expression. The difference between PBS + si-NC and PBS + si-HMGB1 groups was slight significant. 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, BEAS-2B cells were transfected with the recombinant pcDNA3.1-HMGB1 plasmid to elevate HMGB1 expression. Next, the mechanisms underlying the association between HMGB1-mediated signaling and CC16 were excavated by the western blot. Consistently, flow-cytometry analysis showed that the increased apoptosis percentages of BEAS-2B cells under HDM condition were abrogated by CC16 treatment. HMGB1 overexpression abolished the anti-apoptosis effect of CC16 on HDM-induced BEAS-2B cells, as proved by the enhancement of cellular apoptosis index(Fig. 6a). 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 (Fig. 6b). 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(Fig. 6c,d,e,f). In addition, Bax and Bcl-2, the downstream apoptosis regulators of HMGB1-TLR4/NF-κB axis were also detected by immunoblotting analysis(Fig. 6g,h). As expected, the expression level of epithelial protein Bax stimulated by HDM was dramatically higher than in control cells, while Bcl-2 expression level was lower. Accordingly, the alterations in the expression of Bax and Bcl-2 upon HDM exposure were abolished by CC16 treatment. Of note, we further detected that the anti-apoptosis ability of CC16 was subsequently compromised by HMGB1 overexpression, in keeping with the augmentation of HMGB1-mediated TLR4/NF-κB signaling activity.
Collectively, our findings indicated that CC16 alleviated HDM-activated airway epithelial injury and apoptosis via inhibition of HMGB1 expression, while HMGB1-mediated signaling proteins such as TLR4 and NF-κB were potentially modulators involved in the airway protection of CC16.