Soluble advanced glycosylation receptor is a potential target for the treatment of neutrophilic asthma

Neutrophilic asthma (NA) was a subtype of asthma. Soluble advanced glycosylation receptor (sRAGE) was considered to be associated with the neutrophilic airway. However, the role of sRAGE in NA still limited. A NA mouse model was established and the levels of sRAGE in the bronchoalveolar lavage uid (BALF) were measured by ELISA. Hematoxylin-eosin (HE) and Masson trichrome staining were used to identifying airway remodeling. Adeno-associated virus 9 (AAV9) overexpressed sRAGE and inhibitors for HMGB1, RAGE, and PI3K were used to intervene NA mouse model via tail-vein injection and intraperitoneally injection. Expressions of airway remodeling, EMT, and signaling markers were detected using qRT-PCR or western blotting. The levels of IL-17 and IL-6 in BALF were measured by ELISA. HMGB1 was applied to induce EMT of human bronchial epithelial cells (16HBE), then E-cadherin and vimentin expressions were examined after sRAGE, RAGE inhibitor, and PI3K inhibitor administration.


Background
Asthma is a common chronic airway in ammatory disease affecting more than 300 million people in the world [1]. Neutrophilic asthma (NA) is a subtype of asthma occurring in 15-25% of the asthma cases [2]. Some patients with NA exhibited a poor response to treatment and may have irreversible air ow obstruction [3]. A long-term cohort study has shown that children with asthma reported a 10-32-fold increased risk of developing adult COPD which was also characterized by airway neutrophilic in ammation [4]. Airway remodeling is a common pathological process of asthma and COPD and it is also an important cause of air ow limitation. Studies con rmed that children with asthma have existed airway remodeling in early childhood. Therefore, better understanding the mechanism of airway remodeling in NA may pave the way for targeted treatment in the key asthma subtype and bene t to COPD management in the future.
Soluble advanced glycosylation receptor (sRAGE) as a predictive indicator of airway neutrophil in ammation and a biomarker for COPD, which was decreased in the blood and bronchoalveolar lavage uid (BALF) of patients with COPD [5]. Moreover, it was negatively correlated with COPD grade and lung function [6,7]. We hypothesized that sRAGE may be involved in the pathogenesis of NA and the common pathological process of NA and COPD.
To study the role of sRAGE in NA, we established a mouse model with NA to explore sRAGE expression in BALF and the function of sRAGE on airway remodeling by an adeno-associated virus 9 (AAV9) overexpressed sRAGE administration. We proposed that sRAGE overexpression may yield bene ts in the treatment of neutrophil airway in ammation and airway remodeling for NA.

Animals
Female C57BL/6 mice aged 6-8 weeks with weight 18-20 g were purchased from Guangxi Medical University Animal Center. Mice were housed under speci c pathogen-free conditions in separate cages with a relatively stable temperature (20-24 °C) and humidity (55 ± 10%) at 12-h light/dark cycles and given food and water freely. All animal experiments were approved by The Ethics Committee of The First Sigma-Aldrich; Merck KGaA) in a total volume of 50 µl PBS on days 1, 7 and 14. The OVA + LPS mixture was instilled along the posterior oropharyngeal wall, and the mixed solution was inhaled into the airway, followed by a challenge with 1% OVA aerosol for 1 h from day 21 for 7 consecutive days. In the NA + sRAGE group and NA + sRAGE control group, mice were administered with AAV9-sRAGE (0.8E + 12 v.g./mL × 100 µL, Shanghai GeneChemCo., Ltd, China) and AAV9-control (0.8E + 12 v.g./mL × 100 µL, Shanghai GeneChemCo., Ltd, China) via tail-vein injection 1 week before sensitization and challenge, respectively. HMGB1 inhibitor, RAGE inhibitor, and PI3K inhibitor were intraperitoneally injected on days 21,23,25, and 27 at 30 min before each OVA challenge. Mice in the PBS group received the equivalent amount of PBS treatment instead of OVA + LPS for sensitization and challenge. The NC group received no treatment.

Measurement of airway hyper-responsiveness (AHR)
Airway responses to aerosolized methacholine were measured using a lung function test instrument for the mouse (FinePointe Resistance and Compliance; Data Sciences International; Harvard Bioscience, Inc.). Mice were anesthetized with 1% pentobarbital sodium (50 mg/kg body weight) by intraperitoneal injection, and the trachea was cannulated with a needle, followed by mechanical ventilation. Airway resistance (R; cmH 2 O.s/ml) was measured after aerosolization of 10 µl PBS and administration of increasing doses of aerosolized methacholine (3.125, 6.25, 12.5, 25 and 50 mg/ml in 10 µl; Sigma-Aldrich; Merck KGaA) sequentially. The results are presented as fold-increase of R (cmH 2 O.s/ml) above the baseline and were calculated as follows: [R(response) -R(baseline)]/R(baseline) [9].

Cell classi cation of BALF
Mice were sacri ced 24 h after the nal aerosolization. The cervical dislocation was used for euthanasia and death was con rmed by the onset of rigor mortis, according to The National Institutes of Health Guide for the Care and Use of Laboratory Animals. The trachea was exposed, and a 22-gauge needle was used for endotracheal intubation. The lungs were subjected to bronchoalveolar lavage twice with 0.5 ml PBS (recovery rate ≥ 80%) and the total volume of BALF was 0.8 ml. Total and differential cell counts from BALF were determined by staining with Diff-Quick (Beijing Solarbio Science & Technology Co., Ltd.) for 1 min at room temperature. BALF was centrifuged at 160 x g for 10 min at 4˚C and the supernatants were stored at -20˚C for further experiments.

Histopathological analysis
Lungs were xed in 4% paraformaldehyde solution for 24 h at room temperature and subjected to gradient alcohol dehydration and para n-embedding, which were cut into 5-7-µm thick sections and subsequently stained with hematoxylin-eosin (HE) and Masson's trichrome. The sections were subsequently stained with hematoxylin at room temperature for 2-3 min and then with eosin at room temperature for 30-60 sec. The score of peribronchiolar in ammation was determined according to the in ltration of in ammatory cells as follows: 0, normal; 1, few in ammatory cells; 2, a layer of in ammatory cells; 3, two to four layers of in ammatory cells; and 4, more than four layers of in ammatory cells. An Olympus CX31 light microscope (Olympus Corporation) was used to evaluate the general in ammation and the airway morphology.

Statistical analysis
Data were analyzed by GraphPad Prism (San Diego, CA, USA) and expressed as mean ± SD. The statistical signi cance was determined by unpaired Student's t-test or ANOVA test for multiple comparisons. For non-normally distributed data, the signi cance was performed using a non-parametric one-way ANOVA with a post hoc Kruskal-Wallis multiple comparisons test. A p-value of less than 0.05 was considered to be statistically signi cant.

NA mouse model was established
A mouse model with NA was induced by OVA plus LPS sensitization followed by seven sequential daily OVA challenges (Fig. 1a). sRAGE and inhibitors were administered to NA mice as illustrated in Fig. 1b and 1c. As shown in HE staining, the control group manifested a normal bronchial lumen and alveolar structure, while NA mice exhibited a disordered structure with epithelial cell shedding, thicken bronchial wall, and basement membrane with multi-layer in ammatory cell in ltration around (Fig. 2a). Compared with the NC group and PBS group, airway resistance increased signi cantly in the NA group after methacholine challenge at doses of 12.5, 25, and 50 mg/mL (P < 0.0001) (Fig. 2b). The in ammatory cells in BALF signi cantly elevated (including total cells, neutrophils, and lymphocytes) in NA mice when compared with the levels in control mice and the PBS group (Fig. 2c).

sRAGE level decreased in NA mouse
Compared with the control group, the sRAGE level was signi cantly decreased in NA mouse BALF determined by ELISA (Fig. 2d). Given it was negatively correlated with lung function in COPD [6,7], we evaluated the relation of sRAGE and airway resistance in NA. It showed that there was an opposite trend between sRAGE levels in BALF and airway resistance, however, with no statistical differences (Fig. 2e). A study revealed that sRAGE may be a predictor of neutrophilic airway in ammation of asthma and COPD [5]. Our data veri ed that sRAGE was negatively related to BALF neutrophils, especially in the NA group with r=-0.9217 and P = 0.0078 by Spearman correlation analysis (Fig. 2f). 3.3 Overexpressed sRAGE decreased neutrophilic airway in ammation in NA mice AAV-9 was used to overexpress sRAGE and then the transfection e ciency was assessed by the green uorescent protein (GFP) expression. Compared to the NC and NA groups, the NA + sRAGE group exhibited a distinct expression of GFP (Fig. 3a). Further, the sRAGE administration inhibited the number of neutrophils in BALF cytology smears (Fig. 3b). More importantly, sRAGE sharply reduced the in ux of total cells and neutrophils in NA mouse BALF (Fig. 3c). Compared with the airway showing increased in ammatory cell in ltration and thick basement membrane in NA mice, sRAGE overexpression signi cantly inhibited the airway in ammation and the thicknesses of the basement membrane (Fig. 3d). sRAGE intervention signi cantly inhibited the in ammation score in NA mice (P < 0.05) (Fig. 3e). Interleukin (IL) -17 and IL-6 were reported as the in ammatory mediator of NA, in this study, sRAGE decreased IL-17 and IL-6 levels in BALF (Fig. 3f). BALF. n = 6 in each group. Results were depicted as means ± SD. * P < 0.05 and **** P < 0.0001 using oneway ANOVA for analysis.

sRAGE reduced the airway remodeling of NA mice
Masson trichrome staining was widely used to identify collagen deposits which were proved to be the typical phenotype of airway remodeling. Compared to the NC group, NA mice exhibited more blue-stained collagen deposits around the bronchus and blood vessels (Fig. 4a). sRAGE administration highly decreased the collagen deposition, whereas the NA + sRAGE control group showed no reduction. Airway remodeling markers were detected by RT-qPCR (Fig. 4b) and Western Blotting (Fig. 4c). The result revealed that TGF-β1 mRNA was increased in NA mice lung (P < 0.05), while VEGF, MMP-9, and α-SMA expression showed no elevation. Consistently, TGF-β1 protein expression was increased in the NA group with VEGF, MMP-9, and α-SMA protein expression no change. Overexpression of sRAGE signi cantly decreased the mRNA and protein expression of TGF-β1 (P < 0.05), suggesting sRAGE may inhibit airway remodeling in the NA mouse model. group. Results were depicted as means ± SD. ns: no signi cant difference. * P < 0.05, ** P < 0.01. One-way ANOVA was performed for analysis.

Effects of sRAGE on EMT and signaling pathway in NA mice
EMT is one of the mechanisms of airway remodeling. The expression of E-cadherin and vimentin were evaluated using RT-qPCR (Fig. 5a) and Western Blotting (Fig. 5b). Compared with the control mice, Ecadherin expression was decreased, while vimentin level was increased in the NA group. sRAGE administration markedly upregulated the level of E-cadherin and reduced the vimentin expression, indicating sRAGE regulated the EMT of NA mouse.
As reported that RAGE and PI3K participated in the EMT process of human airway epithelial cells. sRAGE as a decoy receptor competitively bound the ligands of RAGE (HMGB1, S100, etc) to inhibit the in ammatory effect. In this study, RAGE, PI3K, and HMGB1 expressions were elevated in the lung of NA mice measured by RT-qPCR, while the sRAGE administration inhibited the expressions (Fig. 5c, d). To determine whether sRAGE modulated EMT via RAGE/PI3K, we assessed E-cadherin and vimentin expressions after HMGB1 inhibitor, RAGE inhibitor, and PI3K inhibitor intervention (Fig. 5e, f). It showed that the mRNA and protein levels of E-cadherin were improved in the NA + HMGB1 inhibitor group, NA + RAGE inhibitor group, and NA + PI3K inhibitor group, whereas the administration of the inhibitor could not affect the vimentin expression. The data suggesting that RAGE/PI3K may partly participate in the EMT process of NA. no signi cant difference. n = 6 in each group. Results were depicted as means ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. One-way ANOVA was performed for analysis.

The effect of sRAGE on EMT in 16HBE cells
HMGB1 was reported to induce EMT of airway epithelial cells. We assessed the function of sRAGE on HMGB1-induced EMT in 16HBE cells. The results showed that the migration ability of 16HBE cells was enhanced in the HMGB1 group (P < 0.0001), while the sRAGE administration inhibited this effect (P < 0.001) (Fig. 6a, b). The mRNA expression of E-cadherin decreased and vimentin elevated in HMGB1 group (1000 ng/mL) (P < 0.05) (Fig. 6c). Compared to the HMGB1 group, E-cadherin mRNA was signi cantly elevated (P < 0.0001), while the vimentin level was decreased in the HMGB1 + sRAGE group (P < 0.05) (Fig. 6d). Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. One-way ANOVA was performed for analysis.

3.9
The signaling pathway involved in EMT of 16HBE cells RAGE and PI3K mRNA expressions improved in 16HBE cells treated with HMGB1 (P < 0.01) (Fig. 7a), while the sRAGE administration inhibited the improvement. RAGE inhibitor and PI3K inhibitor were used to intervene in the HMGB1-induced 16HBE cells. It showed that E-cadherin mRNA was increased, while vimentin mRNA was declined in the HMGB1 + RAGE inhibitor group and HMGB1 + PI3K inhibitor group (Fig. 7b). Western Blotting demonstrated that HMGB1 down-regulated the protein expression of Ecadherin (P < 0.001) and up-regulated vimentin expression (P < 0.05) in 16HBE cells (Fig. 7c). However, the E-cadherin level was elevated (P < 0.01) with vimentin protein decreasing (P < 0.01) in HMGB1 + RAGE inhibitor group and HMGB1 + PI3K inhibitor group, suggesting that RAGE/PI3K may be involved in EMT of 16HBE cells. were detected by Western Blotting. n = 6 in each group. Data are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. One-way ANOVA or Mann-Whitney test was performed for analysis.

Discussion
NA was rst described in 1993 in a patient with sudden-onset fatal asthma [11]. With the deepening of research, it was gradually discovered that NA existed not only in the adult with severe asthma but also in children with mild and remission asthma. In recent years, the relationship between childhood asthma and COPD in adulthood has attracted much attention. Airway neutrophilic in ammation in asthma was considered to be related to airway remodeling and air ow limitation, suggesting NA and COPD may have a potential connection. sRAGE has been well understood in COPD and also be discussed in some studies with asthma. Here, we tried to explore the function of sRAGE in NA.
In the present study, a NA mouse model was established based on the work by Wilson et al [8] and our previous study (published in Chinese). NA mice exhibited increased AHR, an elevated number of neutrophils in BALF, thicken bronchial wall, and accumulated in ltration of in ammatory cells around the bronchial wall. Consistent with the sRAGE expression in patients with COPD and asthmatic patients with airway neutrophil in ammation [5], sRAGE was reduced in NA mouse BALF. Moreover, we found that the lower the sRAGE level in the BALF, the higher the airway resistance after the 50 mg/mL acetylcholine atomization excitation. Furthermore, the expression of sRAGE was signi cantly negatively correlated with the proportion of neutrophils in BALF. Studies have shown that serum sRAGE levels were inversely related to the severity of asthma in children [12], and asthmatic patients with higher airway neutrophils exhibited lower plasma sRAGE levels [13]. The research reported that sRAGE as a predictive indicator of airway neutrophils for it was related to airway neutrophilic in ammation both in patients with asthma and COPD [14]. Similar to the existing studies, the sRAGE level in NA mouse BALF was decreased and the reduced sRAGE was negatively correlated with the airway neutrophil in ammation, suggesting the loss of sRAGE may be involved in the development of NA.
Further, we transfected the NA mouse model with an AAV9-overexpressed sRAGE by tail vein injection. AAV is a common vector for gene manipulation because of its low immunogenicity, high transfection e ciency, and long-term stable gene expression. AAV9 is widely used in the research of respiratory for it is one of the most e cient serotypes to infect lung epithelium cells. Studies have reported that sRAGE reduced the neutrophil in ltration of lung tissue in LPS-induced acute lung injury mouse model [15] and the airway neutrophil in ammation in asthmatic mice [16]. Consistent with the existing research, overexpressed sRAGE not only signi cantly reduced the in ltration of in ammatory cells and in ammation score of lung tissue in NA mice but also sharply inhibited the in ammatory cells and neutrophils in BALF, which implying that sRAGE de ciency may participate in airway neutrophilic in ammation of NA mice. As reported, administration with mesenchymal stem cells overexpressing sRAGE reduced the expression of Th17 cells and IL-17 in the spleen of mice with rheumatoid arthritis [17]. Similarly, we found that IL-17 and IL-6 levels signi cantly decreased in BALF of AAV9-sRAGE overexpressed NA mice. Studies proposed that both IL-17 and IL-6 were involved in airway remodeling, especially the relationship between IL-17 and airway remodeling has been con rmed in asthma and COPD [18,19], suggesting that sRAGE may be involved in airway remodeling of NA mice. Airway remodeling is a common pathological process of asthma and COPD, which leads to progressive and irreversible damage to lung function [20,21]. We explored the effect of overexpression of sRAGE on airway remodeling in the NA mouse model. The results showed that sRAGE administration signi cantly inhibited airway collagen deposition and TGF-β1 expression in NA mice. TGF-β1 can be secreted by neutrophils [22], and it was involved in the airway remodeling of asthma by mediating the differentiation, proliferation, survival, and apoptosis of airway structural cells [23]. It was also an important inducer of EMT which was one of the mechanisms of airway remodeling [24]. More importantly, TGF-β1 was considered to be a key molecule connecting children asthma and COPD [25]. Limited animal models suggested that there was a potential relationship between sRAGE, TGF-β1, and tissue remodeling [26]. We con rmed that sRAGE intervention declined the TGF-β1 expression, suggesting that sRAGE may play a role in the airway remodeling through TGF-β1 in NA.
EMT was one of the mechanisms of airway remodeling for it resulted in epithelial barrier destruction, subepithelial brosis, smooth muscle proliferation, and airway in ammation [27], it was also considered to be involved in airway remodeling of asthma and COPD [28][29][30]. In this study, epithelial marker Ecadherin expression was lost and interstitial marker vimentin was enriched in NA mice. However, AAV9-sRAGE restored the expressions of E-cadherin and Vimentin, implying that sRAGE overexpression may reduce airway EMT in NA mice. A study has revealed that sRAGE alleviated the Th17 cell response and airway neutrophilic in ammation in NA mice through competing with a RAGE ligand -HMGB1 [16]. RAGE was highly expressed in the lung tissues of asthmatic mice and was involved in the mucus hypersecretion and airway remodeling of asthma [31]. Moreover, RAGE inhibitor reduced airway in ammation and airway remodeling in asthmatic mice [32]. RAGE induced the activation of PI3K in human peripheral blood neutrophils while antagonizing RAGE/PI3K inhibited the adhesion activation of neutrophils [33]. In the present study, HMGB1, RAGE, and PI3K expressions were increased in NA mice, whereas the levels were inhibited by sRAGE administration. Besides, we observed the HMGB1-induced EMT in 16HBE cells in vitro, while this process was suppressed by sRAGE intervention. Furthermore, rescue assay showed that HMGB1 induced the EMT via RAGE and PI3K, suggesting sRAGE may regulate the EMT caused by HMGB1 through RAGE/PI3K in 16HBE cells.

Conclusions
In summary, we found that sRAGE expression was remarkably reduced in the NA mouse model. AAV9-sRAGE administration signi cantly inhibited the airway neutrophilic in ammation, airway remodeling, the expression of TGF-β1, and EMT markers in the NA mouse model. Moreover, sRAGE reversed the EMT in 16HBE cells induced by HMGB1 in vitro via RAGE/PI3K. This suggests sRAGE overexpression may yield bene ts in the treatment of neutrophilic airway in ammation and airway remodeling of NA. The authors declare that they have no competing interests.    Results were depicted as means ± SD. *P < 0.05 and ****P < 0.0001 using one-way ANOVA for analysis. (c) TGF-β1, VEGF, MMP-9, and α-SMA protein were detected by Western Blotting. n=6 in each group. Results were depicted as means ± SD. ns: no signi cant difference. *P <0.05, **P <0.01. One-way ANOVA was performed for analysis. signi cant difference. n=6 in each group. Results were depicted as means ± SD. *P <0.05, **P <0.01, ***P <0.001, ****P <0.0001. One-way ANOVA was performed for analysis.  The signaling pathway involved in EMT of 16HBE cells. (a) The mRNA expression of RAGE and PI3K was examined by RT-qPCR. (b) E-cadherin and Vimentin expressions were detected using RT-qPCR after RAGE inhibitor and PI3K inhibitor intervention. (c) The protein levels of E-cadherin and Vimentin were detected by Western Blotting. n=6 in each group. Data are presented as mean ± SD. *P <0.05, **P <0.01, ***P <0.001. One-way ANOVA or Mann-Whitney test was performed for analysis.