Airway epithelial integrin β 4 suppresses allergic inﬂammation by decreasing CCL17 production

Airway epithelial cells (AECs) play a key role in asthma susceptibility and severity. Integrin β 4 (ITGB4) is a structural adhesion molecule that is down-regulated in the airway epithelium of asthma patients. Although a few studies hint toward the role of ITGB4 in asthmatic inflammation pathogenesis, their specific resultant effects remain unexplored. In the present study, we determined the role of ITGB4 of AECs in the regulation of Th2 response and identified the underpinning molecular mechanisms. We found that ITGB4 deficiency led to exaggerated lung inflammation and AHR with higher production of CCL17 in house dust mite (HDM)-treated mice. ITGB4 regulated CCL17 production in AECs through EGFR, ERK and NF- κ B pathways. EFGR-antagonist treatment or the neutralization of CCL17 both inhibited exaggerated pathological marks in HDM-challenged ITGB4-deficient mice. Together, these results demonstrated the involvement of ITGB4 deficiency in the development of Th2 responses of allergic asthma by down-regulation of EGFR and CCL17 pathway in AECs.


Introduction
Airway epithelial cells (AECs) form a barrier to environment hazardous stimuli by tight intercellular junctions and adhesion on basal membrane. Integrins play important roles in the adhesion, tissue repair and homeostasis of these cells [1]. Among these molecules, integrin β4 (ITGB4) has been shown to regulate the adhesion of AECs on basal membrane through hemidesmosomal structure that is a specialized adhesion microstructure attached to the extracellular matrix [2]. ITGB4 has a unique long cytoplasmic domain subunit that could recruit a range of signaling molecules. As a result, ITGB4 has been proven to play a key role in the activation of several intracellular signaling pathways including phosphatidylinositol 3-kinase (PI3K) [3], extracellular signal-regulated kinases (ERK) 1/2 [4] and NF-κB [5], indicating that this integrin potentially contributes to the instigation of subsequent immune response and inflammation. Notably, ITGB4 is implicated in the pathogenesis of allergic asthma, and its level is significantly decreased by specific variation sites in AECs of asthma patients [6,7].
Allergic asthma is a chronic airway disorder that is characterized with epithelial desquamation, airway inflammation and airway hyperresponsiveness (AHR) to non-specific spasmogen. Although CD4 + T-helper 2 cells (Th2 cells) are widely recognized to orchestrate the development of the disease by expressing Th2 cytokines (e.g. interleukin (IL)-4, IL-5 and IL-13), there is compelling evidence that airway epithelium plays a vital role in the induction of aberrant immune responses underlying the pathogenesis

Induction of allergic asthma and administration of anti-CCL17 neutralizing antibody and EGFR antagonist
For the induction of allergic asthma, mice were intranasally (i.n.) exposed to 100 μg HDM on days 0, 7, 14 and 21 as previous described [25]. Nonsensitized mice were i.n. treated with phosphate-buffered saline (PBS). On day 24, AHR was assessed, and then inflammatory infiltrates and histological changes in the lung were quantified as described previously [26]. Some of ITGB4 −/− mice were intraperitoneally (i.p.) treated with 150 μg anti-mouse CCL17 monoclonal antibody (mAb, clone 110904, R&D Systems) or isotype control mAb (MAB006; R&D Systems, U.S.A.) in 200 μl PBS, twice a week for 3 weeks as previously described [27]. Some of ITGB4 −/− mice were given intraperitoneal (i.p.) injections of EGFR inhibitor AG1478 (20 mg/kg, Calbiochem, San Diego, CA) that was dissolved in CMC-Na (0.5%) or control vehicle three times weekly over the course of HDM treatment period [28].

BALF collection and cell counting
BALF was collected and processed as previously described [30]. In brief, after mice killed by exsanguination following an overdose of sodium pentobarbital, the lung was lavaged with 0.5 ml ice-cold PBS with 0.1 mM EDTA twice. Red blood cells were removed by using hypotonic red blood cell lysis buffer and BALF was then centrifuged to collect cellular infiltrate. The total number of cells was quantified using a hemocytometer and the cells were plated on a glass slide. Differential leukocyte counts were determined based on morphological criteria by light microscopy (×100) on May-Grunwald and Giemsa-stained slides.

RNA extraction, RT-PCR and quantitative RT-PCR
Total RNA was prepared using TRIzol reagent (Invitrogen) and phenolchloroform extraction from whole-lung tissues of mice and quantified on a SmartSpecTM Plus spectrophotometer (Bio-rad, U.S.A.) [35]. cDNA was synthesized by RT-PCR using oligo d(T) primer (Invitrogen) on a T100 thermal cycler (Bio-Rad). Quantitative PCR (qPCR) was performed on a CFX96 Touch™ Deep Well Real-Time PCR Detection System (Bio-rad, U.S.A.) using TaqMan Gene Expression Master Mix (Applied Biosystems) with thermal cycling conditions. Primer sequences were described in Supplementary Table S2. Resulting mRNA levels were normalized to GAPDH and expressed as a fold-change relative to control samples.

Western blot
Fifty micrograms of cell protein was separated by 10% SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Levels of ITGB4, CCL17, EGFR, phosphorylated EGFR (p-EGFR), ERK, phosphorylated ERK (p-ERK) and NF-κB p65 were determined respectively with anti-mouse antibodies against these proteins by Western blot as previously reported [37]. GAPDH and Lamin B1 were used as controls as indicated.

Statistical analysis
All data were analyzed using GraphPad Prism Software (Version 6; San Diego, CA) and presented as median with range. Two-way ANOVA followed by Fisher post hoc test was used to identify differences in lung function. Chemokines' expression in sputum samples was analyzed by Mann-Whitney U Test. One-way ANOVA followed by Dunnett's post hoc test was used for all other comparisons. Differences were considered statistically significant for *P<0.05, **P<0.01 and ***P<0.001.

Deficiency of ITGB4 in AECs aggravates the development of HDM-induced allergic asthma
AECs specific ITGB4 conditionally knockout mice were treated with doxycycline to deplete this integrin as previously described ( Figure 1A) [24]. ITGB4 knockout efficiency was verified by immunofluorescence (Supplementary Figure  Figure 1. AHR and allergic disease of the lung is markedly exaggerated in the absence of airway epithelial ITGB4 (A) Mice were sensitized to HDM on days 0, 7, 14 or 21. Some mice were treated with 1% Dox in drinking water to specifically delete ITGB4 in airway epithelial cells. Control non-sensitized mice received PBS. (B) AHR was represented as airway resistance in response to methacholine. Data represent the median with range of six mice per group. **P<0.01 by two-way ANOVA followed by Fisher post hoc test. (C) Lung histology was assessed (n=8), bars: 50 μm. Values represented as median with range. **P<0.01 using one-way ANOVA followed by Dunnett's post hoc test. (D) BALF inflammatory cell was counted (n=10). Values represented as median with range. **P<0.01, ***P<0.001 compared with controls using one-way ANOVA followed by Dunnett's post hoc test. S1). Mice were exposed HDM to induce allergic asthma. HDM exposure resulted in significantly increased airway reactivity in lung of ITGB4 +/+ mice, as compared with PBS exposed control. Following HDM exposure, ITGB4 −/− mice exhibited significantly higher AHR than that of ITGB4 +/+ mice ( Figure 1B). HDM exposed ITGB4 −/− mice displayed significantly elevated histopathological scores due to exacerbated inflammation in airway, vascular and parenchyma, as compared with either PBS exposed ITGB4 −/− mice or HDM exposed ITGB4 +/+ mice ( Figure 1C). In line with aforementioned findings, the levels of BALF inflammatory infiltrates of HDM exposed ITGB4 −/− mice also augmented significantly when compared with those of HDM-exposed ITGB4 +/+ mice, and increased inflammatory cells in BALF were mainly lymphocytes, eosinophils and neutrophils ( Figure 1D). These observations reveal that ITGB4 deletion exacerbates the development of allergic asthma. (B and C) Two days after the final challenge, the levels of IFN-γ, IL-4, IL-13 and IL-17A protein in BALF (n=8) and their transcripts in lung (n=6) were examined by ELISA and qPCR, respectively. Values represented as median with range. **P<0.01 using one-way ANOVA followed by Dunnett's post hoc test.

AECs-specific ITGB4 deficiency increases the numbers of Th2 and Th17 cells and the levels of their cytokines
To further characterize the impact of ITGB4 defect on the activation of T cells, we examined the infiltration of Th1, Th2 and Th17 cells by flow cytometry and the levels of their cytokines in lung by ELISA and qPCR, respectively. The levels of CD4 + IL-4 + T cells, CD4 + IL-13 + T cells and CD4 + IL-17 + T cells increased significantly in the lung of both ITGB4 +/+ and ITGB4 −/− groups following HDM exposure as compared with respective PBS control groups, and the levels of CD4 + IFN-γ + T cells did not display significant changes (Figure 2A and Supplementary Figure S2). Of note, the infiltration of Th2 and Th17 cells were significantly greater in HDM exposed ITGB4 −/− animals than that of ITGB4 +/+ animals. Likewise, the protein and transcript levels of IL-4, IL-5, IL-13 and IL-17A were significantly elevated as demonstrated in the BALF and lung of HDM exposed ITGB4 −/− group, when compared with those of HDM exposed ITGB4 +/+ group ( Figure 2B,C). HDM exposure resulted in significantly increased levels of these cytokines in BALF and lung of ITGB4 +/+ mice, as compared with those PBS exposed control. These data provide evidence that ITGB4 deficiency engages in the activity of Th2 and Th17 cells but not Th1 cells.

Involvement of ITGB4 deficiency on the increased production of CCL17
Both clinic and animal studies have shown that Th2 cells orchestrate the development of allergic asthma [9]. To determine how ITGB4 contributes the activation of Th2 cells, we examined the levels of CCL3, CCL5, CCL17 and CCL22 that could bind to CCR4, a Th2 chemotactic receptor [38][39][40]. Significant higher levels of CCL17 protein and transcript were found in the BALF and lung of HDM exposed ITGB4 −/mice, as compared with those of HDM exposed ITGB4 +/+ mice ( Figure 3A,B). Similar results could also be observed in HDM stimulated AECs ( Figure 3C). Sputum samples from patients with allergic asthma and healthy subjects were also collected to determine the secretion of CCL17. By using ELISA, levels of CCL17 were found significantly higher in the sputum of patients with allergic asthma, as compared with that of healthy controls ( Figure 3D). Interestingly, there was no significant difference in the transcript levels of CCL3, CCL5 and CCL22 in HDM exposed ITGB4 −/− mice, as compared with those of HDM exposed ITGB4 +/+ mice (Supplementary Figure S3A). Also, similar results could also be found in HDM stimulated  Figure S3, B and C). Taken together, these findings indicate that ITGB4 in AECs critically regulates the expression of CCL17 and is associated with the pathogenesis of allergic asthma.

ITGB4 inhibits the EGFR phosphorylation of AECs
ITGB4 has been shown to regulate EGFR phosphorylation in liver cancer cells, breast cancer cells and gastric cancer cells, which is an important signaling mechanism for ERK activation [41][42][43]. To determine whether ITGB4 regulates EGFR phosphorylation in our model, we treated isolated AECs from ITGB4 +/+ and ITGB4 −/− mice with recombinant EGF to investigate the levels of EGFR phosphorylation. Basal level of EGFR phosphorylation in ITGB4 −/− AECs was higher than that in ITGB4 +/+ AECs in the absence of EGF ( Figure 4A). However, EGF significantly enhanced EGFR phosphorylation in ITGB4 −/− AECs, as compared with that in ITGB4 +/+ AECs. Furthermore, assays with immunoprecipitation ( Figure 4B) in cultured AECs and immunofluorescence in AECs and lung tissue ( Figure 4C,D) revealed a direct binding between ITGB4 and EGFR. These results demonstrate the involvement of ITGB4 deficiency on the increased production of CCL17 through the regulation of EGFR phosphorylation.

ITGB4 regulates the production of CCL17 through the activation of EGFR, ERK and NF-κB pathways
To determine the role of EGFR mediated signaling pathways in ITGB4-regulated CCL17 production, we examined the expression of CCL17 in isolated AECs and their culture supernatants using multiple inhibitors, including AG1478 for EGFR phosphorylation, U0126 for ERK1/2 phosphorylation and SC75741 for NF-κB p65 activation. Western blot analysis revealed that treatments with AG1478, U0126 or SC75741 significantly inhibited the levels of p-EGFR, p-ERK1/2 or nucleus NF-κB p65 in HDM stimulated ITGB4 −/− AECs, respectively ( Figure 5A-F). Importantly, these inhibitor treatments blocked heightened CCL17 production in HDM stimulated ITGB4 −/− AECs. ERK-activated pathways have been described to induce phosphorylation and translocation of the transcription factor NF-κB to the nucleus [43]. Meanwhile, NF-κB inhibitors have been reported to block CCL17 expression in the alveolar epithelial cell line [44]. To verify the possible involvement of NF-κB in the up-regulation of CCL17, we studied p65 subunit of the active NF-κB complex upon stimulation with AG1478, U0126 or SC75741. As anticipated, treatments with AG1478, U0126 and SC75741 reduced the levels of nucleus NF-κB p65 whose binding sites are present in the CCL17 promoter in HDM stimulated ITGB4 −/− AECs ( Figure 5G). Together, these findings indicate that blocking EGFR, ERK and NF-κB pathways suppresses augmented CCL17 production in ITGB4 −/− AECs following HDM exposure.

Blockade of EGFR phosphorylation inhibits CCL17 production and exaggerated AHR, airway inflammation and Th2 cells infiltration
To investigate the role of EGFR signaling pathway in ITGB4-regulated CCL17 production, we blocked EGFR phosphorylation with AG1478 in HDM exposed ITGB4 −/− mice. Administration of AG1478 inhibited the levels of p-EGFR in AECs, as compared to vehicle treatment ( Figure 6A). Furthermore, inhibition of EGFR phosphorylation by AG1478 led to significantly decreased levels of CCL17 in cultured ITGB4 −/-AECs and cell culture supernatants ( Figure 6B). Exaggerated AHR and histopathological scores were also significantly reduced by EGFR blockade in HDM exposed ITGB4 −/− mice, as compared with those of vehicle treatment ( Figure 6C,D). Flow cytometry revealed that AG1478 administration significantly reduced the levels of IL-4 + CD4 + T cells and IL-13 + CD4 + T cells but not IFN-γ + CD4 + T cells and IL-17 + CD4 + T cells in the lung of HDM treated ITGB4 −/− mice ( Figure 6E). Meanwhile, AG1478 administration significantly reduced the expression of IL-4, IL-5 and IL-13 in the BALF and lung of HDM exposed ITGB4 −/− mice ( Figure 6F,G). These data suggest that EGFR signaling pathway underpins ITGB4-regulated CCL17 production in AECs and thus contributes to the pathogenesis of Th2 inflammation.
Neutralization of CCL17 diminishes exaggerated AHR, airway inflammation and Th2 responses in HDM exposed ITGB4 −/− mice As a key role for CCL17 is indicated in the pathogenesis, we neutralized this chemokine with anti-CCL17 mAb in HDM exposed ITGB4 −/− mice. Treatment with anti-CCL17 mAb significantly and greatly decreased the levels the chemokine in lung, as compared with isotype Ab treatment ( Figure 7A). Neutralization of CCL17 significantly suppressed exaggerated AHR, airway inflammation in HDM exposed ITGB4 −/− mice ( Figure 7B,C). Of note, anti-CCL17 mAb treatment reduced the levels of CD4 + IL-4 + T cells and CD4 + IL-13 + T cells but not CD4 + IFN-γ + T cells and CD4 + IL-17 + T cells in the lung of HDM exposed ITGB4 −/− mice ( Figure 7D). Similarly, neutralizing CCL17 decreased the protein and transcript levels of IL-4, IL-5, and IL-13, but not those of IFN-γ and IL-17A in the BALF and lung of HDM exposed ITGB4 −/− mice, as compared to isotype Ab treatment ( Figure 7E,F).

Discussion
Integrins are critical molecules for airway epithelial integrity and their expression is dramatically altered during chronic airway diseases such as allergic asthma [45][46][47]. However, the functional roles of these molecules in the regulation of aberrant immune responses and in the pathogenesis of disease are only beginning to be understood. We have previously reported that ITGB4 is down-regulated in AECs of asthma patients and shown that it negatively regulates thymic stromal lymphopoietin (TSLP) production and antigen presenting process by DCs [24]. In the present study, we observed a regulatory role of ITGB4 deficiency in modulating the activity of Th2 cells through promoting CCL17 production in ITGB4 −/− mice, indicating an unidentified role of this integrin in the mechanisms underpinning the development of allergic asthma. Furthermore, our findings establish a new link between ITGB4, EGFR and CCL17 in AECs, and highlight the potential of targeting this pathway for the treatment of allergic asthma.
AECs are known as the first cell barrier to inhaled pollutants and allergen and play a vital role in driving immune responses in respiratory disorders. Not only as the structural component that maintains epithelial architecture, integrins also play a key role in asthma pathogenesis. A recent study has shown that α5β1 integrin deficiency protects mice from cytokine-enhanced bronchoconstriction in a mouse model of asthma [45]. However, it is difficult to specifically identify the mechanism for this protection, since integrin has been reported to regulate a wide range of biological processes by targeting intracellular pathways including FAK, Src, Ras, RhoA, EGFR, ERK, p53, etc. [41,48,49]. In this Figure 6. Blockade of EGFR phosphorylation inhibits both CCL17 production and subsequent enhanced AHR, lung inflammation in HDM exposed ITGB4 −/− mice (A and B) AECs-specific ITGB4 conditional knock out mice were constructed and exposed HDM (HDM+) or PBS (HDM-) on days 0, 7, 14 or 21. Some mice received injection with either AG1478 or same volume of the control vehicle. Two days after the final challenge, AECs were isolated from these mice. The protein levels of EGFR and p-EGFR in AECs were detected by Western blot. The CCL17 level in the cell culture medium of AECs were detected by Western blot and ELISA. Values represented as median with range for five samples from one experiment and representative of three independent experiments. **P<0.01, ***P<0.001 using one-way ANOVA followed by Dunnett's post hoc test. (C) Two days after last HDM exposure, AHR was represented as airway resistance in response to methacholine. Data represent the median with range of six mice per group. **P<0.01 by two-way ANOVA followed by Fisher post hoc test. (D and E) Lung histology was assessed (n=8) and the levels of IFN-γ + CD4 + , IL-4 + CD4 + , IL-13 + CD4 + and IL-17A + CD4 + T cells in lung were determined (n=10), bars: 50 μm. (F and G) The levels of IFN-γ, IL-4, IL-13, and IL-17A protein in BALF (n=8) and their transcripts in lung (n=6) were examined by ELISA and qPCR, respectively. Values represented as median with range. *P<0.05, **P<0.01, ***P<0.001 using one-way ANOVA followed by Dunnett's post hoc test.
context, our observation that the specific disruption of ITGB4 in AECs protects animal from allergic asthma makes this problem more tractable and suggests a central role of AECs in the induction of allergic asthma. In our study, exposure to HDM in the airways of ITGB4 −/− mice led to exaggerated airway inflammation and heightened AHR (Figure 1), which is in line with our previous findings [29]. We have also shown that the effect of ITGB4 deficiency in AECs are not just limited on pathophysiological changes in airway but also on the activation of T helper cells as AECs-specific ITGB4 deficiency regulates the infiltration of Th2 and Th17 cells and the production of their cytokines ( Figure 2). This observation suggests that ITGB4 contributes to the disease progress through the regulation of Th2 and Th17 activation. The infiltration of T helper cells is exquisitely regulated by a wide range of chemokines [50,51]. While changes in levels of other CCR4-binding chemokines (e.g. CCL3, CCL5 and CCL22) did not show significant differences, the level of CCL17 was significantly elevated in the BALF and the lung of HDM exposed ITGB4 −/− mice. Notably, increased level of CCL17 was also detected in the sputum of allergic asthma patients (Figure 3). CCL17 plays a crucial role in the recruitment of Th2 cells in lung, as demonstrated by a mouse model of RSV infection and HDM stress [52]. AECs are the most important cellular source of CCL17 in the lung of asthma patients although macrophages and DCs have also been shown to produce CCL17 in the sputum [22,53]. Moreover, increased CCL17 expression from AECs has been verified to plays a key role in the allergic inflammatory of allergic asthma patients by recruiting Th2 cells [54,55]. Although isolating primary AECs from human samples could be a better way to detect AECs' CCL17 expression, there is a certain risk during the collection of primary AECs and the number of collected primary AECs is also relatively small. Therefore, we extracted AECs from ITGB4 +/+ and ITGB4 −/− mice and then stimulated them with HDM to determine the CCL17 expression in AECs. The results demonstrated that elevated CCL17 was secreted from AECs after HDM exposure. The expression of CCL17 was also significantly enhanced in HDM stimulated ITGB4 −/− AECs compared with HDM stimulated ITGB4 +/+ AECs. In this regard, our study is important in understanding the pathogenesis of allergic asthma, by demonstrating that the AEC-derived CCL17 is critical for the activation of Th2 cells.
Although the production of CCL17 by AECs is known, it is still largely obscure how integrins contribute to the increased expression of CCL17 in allergen-induced asthma. With ITGB4 −/− mice, we were able to identify that the synchronization between ITGB4 and EGFR essentially orchestrates CCL17 production in AECs through the action of multiple downstream signaling pathways including EGFR, ERK1/2 and NF-κB (Figures 4 and 5). Studies in cancer research have shown that interaction between ITGB4 and EGFR phosphorylation has a significant impact on the progression and metastasis of hepatocellular carcinoma cells, mammary tumor cells and mammary tumor cells [41][42][43]. EGFR, a receptor tyrosine kinase, can induce phosphorylation of multiple intracellular transcriptional regulators including ERK1/2, and PI3K pathway [56,57]. Among these signaling regulators, EGFR is essential for the activation of ERK signaling that critically mediate the production of pro-inflammatory factors [58]. Our results are in line with the aforementioned findings, and have identified that a direct interaction between ITGB4 and EGFR in airway epithelial cells drives the development of Th2 cell-associated allergic responses. Interestingly, AG1478 treatment completely blocked the phosphorylation of EGFR and reduced CCL17 production in the AECs of HDM-exposed ITGB4 −/− mice to basal level. It also significantly inhibited the development of AHR, airway inflammation and the infiltration of Th2 cells ( Figure 6). Furthermore, neutralizing CCL17 also dramatically suppressed the manifestation of the disease in our model (Figure 7). Therefore, our data have shown that EGFR and CCL17 critically contribute to the disease, as demonstrated by its impact on Th2 cells infiltration, exaggerated AHR, airway inflammation in HDM-exposed ITGB4 −/− mice.
Interestingly, we have also found that ITGB4 defect leads to heightened infiltration of Th17 cells and higher production of IL-17A ( Figure 2). The importance of Th17 cells and their cytokines is implied not only in autoimmune diseases but also in allergic asthma [59,60]. Although EGFR antagonist or CCL17 mAb treatments significantly suppressed the migration of Th2 cells in HDM exposed ITGB4 −/− mice, they had no impact on Th17 cells infiltration. Furthermore, CCL17 mAb treatment did not affect IL-17A production. According to our previous work, this may be related to the increased production of TSLP in AECs [24]. Indeed, unaffected Th17 function is likely the reason why there exists residual AHR and airway inflammation after EGFR antagonist or anti-CCL17 mAb treatments in HDM-exposed ITGB4 −/− mice. These observations indicate the differential and profound roles of ITGB4 in the induction of allergic asthma and highlight the complex and heterogeneous nature of allergic asthma. Furthermore, the infiltration of Th1 cells and the production of IFN-γ were not affected after EGFR-inhibitor or anti-CCL17 mAb treatments in HDM exposed ITGB4 −/− mice, suggesting a specific role of ITGB4 in the pathophysiology of allergic asthma by the regulation of Th2 and Th17 responses. Recent researches show that ILC2 initiate and maintain the adaptive type immune response in the pathogenesis of allergic asthma [61]. Then, the pivotal role of integrin in AECs during the recruitment of ILC2 deserves our further attention and exploration.
In summary, our data provide an insight that ITGB4 defect in AECs leads to elevated Th2 responses and exaggerated AHR and airway inflammation. By focusing on the role of ITGB4, we have developed models that allow dissection of the mechanisms predisposing to the development of Th2 responses, allergic inflammation and AHR. Here we demonstrate the importance of integrated signaling events between ITGB4, EGFR, ERK1/2 and NF-κB pathways specifically in AECs for the induction of allergic disease which may be clinically relevant. Understanding the contribution of this molecular network within AECs to the pathogenesis of allergic asthma may provide new therapeutic approaches for the treatment of the disease.