ITGB4 deficiency induces mucus hypersecretion by upregulating MUC5AC in RSV-infected airway epithelial cells

Respiratory syncytial virus (RSV) infection is the main cause of bronchiolitis in children. Excessive mucus secretion is one of the primary symbols in RSV related lower respiratory tract infections (RSV-related LRTI), which is closely associated with the occurrence and development of asthma in later life. Integrin β4 (ITGB4) is down-regulated in the airway epithelial cells (AECs) of asthma patients which plays a critical role in the pathogenesis of asthma. However, whether ITGB4 is involved in the pathological processes of RSV infection remains unclear. In this study, we found that decreased expression of ITGB4 was negatively correlated with the level of MUC5AC in childhood AECs following RSV infection. Moreover, ITGB4 deficiency led to mucus hypersecretion and MUC5AC overexpression in the small airway of RSV-infected mice. MUC5AC expression was upregulated by ITGB4 in HBE cells through EGFR, ERK and c-Jun pathways. EGFR inhibitors treatment inhibited mucus hypersecretion and MUC5AC overexpression in ITGB4-deficient mice after RSV infection. Together, these results demonstrated that epithelial ITGB4 deficiency induces mucus hypersecretion by upregulating the expression of MUC5AC through EGFR/ERK/c-Jun pathway, which further associated with RSV-related LRTI.


Introduction
Respiratory syncytial virus (RSV) is one of the most common respiratory pathogens causing lower respiratory tract infection among neonates and infants. RSV infection results about 33 million acute lower respiratory tract infections (LRTI) cases worldwide each year. Not only that, RSV-related LRTI would induce subsequent severe respiratory diseases and around 55,000-199,000 deaths in children younger than the age of 5 years old, which poses a heavy world medicine burden [1].
Airway epithelial cells (AECs), specifically ciliated AECs, is the main target for RSV infection. Of particular concern is that mucus hypersecretion in AECs is one of most important pathological events in severe LRTI, which is always associated with increased risk of subsequently wheezing and later asthma in childhood [2,3]. Mucus is important for the mucociliary clearance system, which is the first line against exogenous stimuli and pathogens [4]. However, mucus hypersecretion in pathological state would aggravate inflammatory response, which further led to airway hyperresponsiveness (AHR), airflow obstruction and weakened lung function [5]. Thus, exploring the intrinsic mechanism that causes Ivyspring International Publisher abnormal mucus secretion in RSV-infected AECs will contribute to prevent and reduce the clinically occurrence of severe LRTI.
Mucins, the main components of mucus, have many different types. In addition to forming a protective barrier against pathogens, mucins also have other unconventional functions, such as anti-inflammatory action and receptors for bacterium [6]. MUC5AC and MUC5B are the principal secretory mucins in the airway and MUC5AC is the primary gene expressed by airway epithelial goblet cells. Generally, MUC5AC is mainly expressed at relatively low level. However, the expression of MUC5AC increased significantly in inflammatory microenvironment which was usually caused by various external stimuli including RSV infection [6,7]. Studies have found that MUC5AC overexpression induced the hypersecretion of airway mucus in RSV-infected AECs [8,9]. However, the mechanisms underlying MUC5AC overexpression in RSV-infected AECs remain largely unclear.
Integrin family is a type of adhesion molecules that are constitutively expressed in AECs. Among these molecules, integrin β4 (ITGB4) deserves a special attention for its unique intracellular long subunit. ITGB4 has been proven to act a critical role in the activation of phosphatidylinositol 3-kinase (PI3K), extracellular signal-regulated kinases (ERK1/2), nuclear factor kappa-B (NF-κB) and other intracellular signaling pathways [10][11][12]. Of note, expression of ITGB4 decreased significantly by specific variation sites in the AECs of asthma patients [13]. Moreover, ITGB4 deficiency induces exaggerated lung inflammation and enhanced AHR in House Dust Mite (HDM) treated mice [14].
Although the association between ITGB4 and asthma has been verified, little is known about the contribution of ITGB4 to mucus hypersecretion after RSV infection in childhood asthma. Thus, we further though to unravel the role of ITGB4 in the regulation of mucus production following RSV infection in AECs. First, RSV-infected model was constructed both in vitro and in vivo to investigate mucus production in ITGB4 deficient AECs following RSV infection. Then, throat swab samples from children's cases of RSV induced bronchiolitis and control cases were collected to verify the correlation between ITGB4 and MUC5AC in RSV-infected airway epithelia.
Finally, corresponding intervention strategies were performed to identify the underpinning molecular mechanisms of MUC5AC overexpression in ITGB4 deficient AECs after RSV infection.

Throat swab sample collection
All participants provided written informed consent, approved by the No.2020KT-52 Central South University Research Ethics Committee. Children cases of RSV infection and healthy controls were recruited at Hunan Provincial People's Hospital between September 2020 and February 2021. Eligible patients (RSV patients' group) were aged under 18 and diagnosed with bronchiolitis and RSV-RNA positive. Patients who had pneumonia, acute heart failure within a month were excluded. Healthy controls were aged under 18 without respiratory disease. The demographic and clinical characteristics are shown in Table S2. Throat swab samples were collected for extracting RNAs and qPCR analysis by using BIOG RNA Swab Kit (BAIDAI, China).

Animals and challenge protocols
This animal studies were approved by the Xiangya Animal Protection and Use Committee of Central South University (No. 2020sydw0305). ITGB4 conditionally knocked out mice (ITGB4 -/mice) was constructed according to our previous work [15]. The AECs-specific ITGB4 knock out was controlled by doxycycline (Dox) induction. After birth, Dox was placed in drinking water (1mg/ml) for mother. Infant mice are breastfed and injected with Dox (i.p. 25mg/kg) on day 7 and 14 after birth. ITGB4 fl/fl male littermates lacking either CCSP-rtTA or TetO-Cre were served as controls (ITGB4 +/+ mice) which were treated with Dox synchronously. For the induction of RSV infection, mice were intranasally (i.n) stimulated with 1×10 6 pfu RSV-A2 in 20 ul on day 15 after birth, and the control group received an equal volume of PBS [16]. Lung tissues were collected on day 17, 19, and 21 after birth (mice killed by exsanguination following an overdose of sodium pentobarbital). All the animal work was taken place in Department of Laboratory Animals, Central South University.

RNA extraction, RT-PCR and qPCR
Total RNA was extracted by Trizol and quantified on a Varioskan microplate reader (Thermo Scientific, USA). PCR and qPCR were conducted according to the Kit protocol (PrimeScript RT Master Mix Kit, Takara, Japan) [15]. Primer sequences were shown in Table S1.

Immunoprecipitation
Immunoprecipitation was performed according to our previous publications [19]. Briefly, protein from HBE cells was extracted with weak RIPA lysis buffer. An appropriate dilution of anti-ITGB4 antibody (Ab197772, Abcam, USA) or anti-EGFR antibody (sc-373746, Santa Cruz, USA) were added to the centrifuge tube which conjugate with suspension of Protein G. Mix the antibody-bead mixture at 4°C for 4 h using tube rotator. Then, 50μg of cell lysates were added into the mixtures and the lysate-bead/antibody conjugate mixtures were incubated overnight at 4°C. Mixtures were further resuspended in 5xSDS loading buffer. Samples were boiled for 5 minutes and analyzed by Western blot. Immunocomplexes were stained with anti-ITGB4 (ab197772, Abcam, USA) as well as EGFR (sc-373746, Santa Cruz, USA).

Immunocytochemical staining
HBE cells slides were fixed with 4% paraformaldehyde and incubated with PBS (containing 0.1% Triton X-100) for 10 minutes. After blocking with 1% BSA, cells were incubated with antibodies for MUC5AC (sc-21701, Santa Cruz, USA) or c-Jun (sc-74543, Santa Cruz) in a humidified box and incubate overnight at 4°C. Then, cells were incubated with DAPI for DNA staining (Solarbio, China). Results were visualized using a fluorescence microscope (Carl Zeiss MicroImaging GmbH, Göttingen, Germany).

Statistical analysis
Pearson' correlation was used to access the association between the expression of ITGB4 and MUC5AC, which was done with SPSS. 19. Other data were analyzed with GraphPad Prism Software (Version 7; San Diego, CA) and presented as mean ± SEM. Statistical comparisons were made by one-way ANOVA followed by Dunnett's post hoc test. Differences were considered statistically significant for *p < 0.05, **p < 0.01 and ***p < 0.001.

ITGB4 deficiency in AECs induced mucus hypersecretion in RSV-infected mice
To explore the influence of ITGB4 deficiency on the mucus secretion in airway epithelia following RSV infection, RSV-infected mice animal was constructed ( Figure 1A). PAS staining showed that, on day 2 post RSV infection, a small amount of mucus was detected in bronchial lumen of both ITGB4 -/mice and ITGB4 +/+ mice. On day 4 and day 6 post infection, goblet cells in the bronchial lumen of ITGB4 -/mice were significantly proliferated and mucus secretion was greatly increased, whereas ITGB4 +/+ mice showed no obvious goblet cell proliferation and mucus secretion ( Figure 1B). Meanwhile, during days 4 to 6 post RSV infection, the expression of MUC5AC in ITGB4 -/mice were significantly higher than that in ITGB4 +/+ mice ( Figure 1C, Figure S1).

Involvement of ITGB4 deficiency on the overexpression of MUC5AC in RSV patients
Both clinic and animal studies have shown that mucus hypersecretion from airway epithelia is one of most important pathological events in severe LRTI following RSV infection. To further determine the impact of ITGB4 on mucus hypersecretion, we examined the levels of MUC5AC through throat swab samples from RSV patients and healthy controls. Compared with healthy controls, expression of ITGB4 decreased significantly in RSV patients, (Figure 2A), while levels of MUC5AC were higher in RSV patients compared with healthy controls ( Figure 2B). Of note, expression of ITGB4 was negatively associated with the level of MUC5AC in RSV patients ( Figure 2C). This correlation strongly indicated that ITGB4 deficiency in airway epithelia was involved in mucus hypersecretion and MUC5AC overexpression following RSV infection.

Level of MUC5AC was induced significantly in ITGB4 deficient HBE cells after RSV infection
To further verify the effect of mucus hypersecretion caused by ITGB4 deficiency, HBE cells were transfected with ITGB4 siRNA, respectively. The silence efficiency of ITGB4 was verified by western blot ( Figure 3A). After RSV infection, the mRNA expression of MUC5AC enhanced significantly in ITGB4 silenced HBE cells. Besides, comparing with control HBE cells, the protein expression of MUC5AC in ITGB4 silenced HBE cells also increased obviously following RSV infection ( Figure 3B, C, D).

. The interaction between ITGB4 and EGFR and the phosphorylation of EGFR in ITGB4 silenced HBE cells after RSV infection. A-B. Immunoprecipitation of RSV-infected HBE cells for interaction between ITGB4 and EGFR. C. ITGB4 deficiency enhanced EGFR phosphorylation in RSV-infected HBE cells, as compared with that in control HBE cells.
Values are represented as mean ± SEM of at least three independent experiments. ** p<0.05; *** p<0.01.

ITGB4 deficiency increased EGFR phosphorylation in RSV-infected HBE cells
Previous literatures have showed that ITGB4 reduces the binding of ligand to EGFR and the mobility of EGFR in the plasma membrane through physical interaction with EGFR [20][21][22]. In this study, we further determined whether ITGB4 regulates EGFR phosphorylation in RSV-infected HBE cells. Assay with immunoprecipitation revealed a direct interaction between ITGB4 and EGFR in RSV-infected HBE cells (Fig 4A, B). Basal level of EGFR phosphorylation in RSV-infected HBE cells was higher than that in control HBE cells without outside stimuli. Moreover, after RSV infection, ITGB4 silence further enhanced EGFR phosphorylation in HBE cells compared with that in control HBE cells (Fig 4C). Either at baseline or after RSV infection, there is no significant difference of total EGFR between ITGB4 silenced HBE cells and control HBE cells. The above results indicated that ITGB4 was involved in the activation of EGFR during RSV infection.

ITGB4 deficiency enhanced the activation of EGFR/ERK/c-Jun pathway in RSV-infected HBE cells
To further explore the role of EGFR mediated signaling pathways in ITGB4 deficiency-induced MUC5AC overexpression, we examined the downstream pathway of EGFR activation in ITGB4 silenced HBE cells after RSV infection. The expression of ERK1/2, phosphorylated ERK1/2 (p-ERK1/2), c-Jun and phosphorylated c-Jun (p-c-Jun) were detected in ITGB4 silenced HBE cells after RSV infection using multiple inhibitors, including AG1478, U0126 and SP600125. Western blot analysis showed that treatment with AG1478, U0126 and SP600125 significantly inhibited the level of p-EGFR, p-ERK1/2 and p-c-Jun in ITGB4 silenced HBE cells after RSV infection, respectively ( Figure 5A, B, C).
As c-Jun is an important binding site on MUC5AC promoter, we further verify the possible involvement of c-Jun in the overexpression of MUC5AC. The nuclear c-Jun subunit was detected upon stimulation with AG1478 or U0126. Immunofluorescence results demonstrated the nuclear translocation of c-Jun in ITGB4 silenced HBE cells following RSV infection ( Figure 6A). As expected, treatments with AG1478, U0126 reduced the nuclear translocation of c-Jun in ITGB4 silenced HBE cells after RSV infection ( Figure 6B).

Blockade of EGFR/ERK1/2/c-Jun pathway inhibited MUC5AC overproduction in ITGB4 silenced HBE cells after RSV infection
To further verify the involvement of EGFR-ERK1/2-c-Jun pathway in the regulation of MUC5AC expression, we detected the level of MUC5AC in ITGB4 silenced HBE cells after RSV infection using AG1478, U0126, SP600125, respectively. Administration of AG1478, U0126 and SP600125 significantly blocked the phosphorylation of EGFR, ERK, c-Jun in ITGB4 silenced HBE cells ( Figure  5A, B, C). More importantly, these inhibitor treatments blocked MUC5AC overexpression in ITGB4 silenced HBE cells after RSV infection ( Figure  7A, B). These findings verified that ITGB4 deficiency upregulate MUC5AC expression through activation of EGFR/ERK/c-Jun pathway in RSV-infected HBE cells.

Inhibition of EGFR phosphorylation reduced mucus hypersecretion and MUC5AC overexpression in ITGB4 -/-mice after RSV infection
As the key role for EGFR is indicated in the pathogenesis of mucus secretion, we block EGFR phosphorylation with AG1478 in ITGB4 -/mice after RSV infection. On day 6 post RSV infection, treatment with AG1478 significantly decreased both mucus hypersecretion in airway lumen and MUC5AC overexpression in airway epithelia ( Figure 8A, B). These results verified that EGFR signaling pathway underpins ITGB4-regulated MUC5AC expression in AECs which further contributes to mucus hypersecretion after RSV infection.

Discussion
Airway mucus is the first biological hydrogel barrier of airway to defend against outside insults. Mucin, as a critical component of mucus, plays an important role in innate immunity which goes beyond the scope of mucociliary. MUC5AC and MUC5B are the principal gel secretory mucins which are important hallmarks of airway pathological state. MUC5B mainly exerts a role in ensuring normal mucus clearance, whereas MUC5AC is the main up-regulated mucin which was involved in the development of AHR, mucous metaplasia, and airway mucus plugging. Moreover, overexpression of MUC5AC is also accompanied with the inducement of airway inflammation [7,23,24]. It is well known that the expression of MUC5AC in AECs increased obviously in allergic asthma patients [25]. Animal models further demonstrated that MUC5AC deficiency enhanced mucociliary transport and antibacterial defense in AECs [26]. Meanwhile, mucus plugs were significantly reduced in MUC5ACdeficient mice after allergen stimulation [27]. In addition, MUC5AC overexpression in AECs could also imply airway goblet cell metaplasia, which is a key feature of airway remodeling in asthma [28,29].
Notably, RSV infection is the major trigger for airway immune response and mucus production, which further led to airway obstruction, AHR and airway inflammation. A large body of evidence from epidemiologic studies have shown that RSV infection in infancy and early childhood was associated with elevated susceptibility of later asthma [30]. Early RSV infection may have sustained effects on subsequent local lung immune responses [31]. Moreover, RSV infection and asthma may share a common host genetic background [30,32]. Intriguingly, there is an obvious heterogeneity in the severity of RSV infection, which may be related to the susceptibility of subsequent asthma. However, the intrinsic mechanism underlying the heterogeneity of RSV infection is far from unclear.
Our previous work found that ITGB4 deficiency in AECs was involved in the development of Th2 responses of allergic asthma though ITGB4-mediated signaling events [13,33]. In this present study, we further pointed out that the decreased expression of ITGB4 was negatively correlated with the level of MUC5AC in throat swab of children with RSV infection. Moreover, ITGB4 deficiency enhances mucus hypersecretion and MUC5AC overexpression following RSV infection in mice model. Furthermore, our findings identify the underpinning molecular mechanisms of mucus hypersecretion in ITGB4 deficient AECs after RSV infection and highlight the potential of targeting this pathway for the treatment of mucus hypersecretion. Studies have reported that EGFR pathway is necessary for mucus production in AECs [34,35]. The binding of EGF and EGFR in epithelial cells will be triggered after subjection to external stimuli. Activation of EGFR induced receptor dimerization and intracellular domain phosphorylation, which can further activate downstream regulators, including c-Jun N-terminal kinase (JNK), AKT, NF-κB. Of note, the expression of MUC5AC is associated with the activation of c-Jun, AKT and NF-κB pathway [36]. Besides, EGFR tyrosine kinase inhibition has also been shown to block the expression of MUC5AC. It has confirmed that EGFR phosphorylation was regulated by ITGB4 in gastric cancer, breast cancer and liver cancer [37]. In this study, we further demonstrated that ITGB4 deficiency in AECs induces overproduction of MUC5AC through EGFR activation, and use of EGFR phosphorylation inhibitors could restore expression of MUC5AC significantly [20][21][22]. EGFR is a kind of receptor tyrosine kinase with intrinsic tyrosine kinase activity, which can induce phosphorylation of multiple intracellular substrates, of which ERK1/2 is the most widely known downstream pathway [38]. ERK1/2 is a central component in the MAPK decades. After activated by phosphorylated EGFR, ERK1/2 enters the nucleus and acts on transcription factors such as NF-κB and AP-1 [39,40]. AP-1, as a type of transcription factor, is composed by subunit c-Jun and subunit c-Fos, which is involved in basal transcription of MUC5AC [41]. Our results also proved that ITGB4 and EGER have a direct interaction in human AECs, which is in line with previous findings [11,22]. Intriguingly, our data also found that ITGB4 deficiency in AECs increased the phosphorylation of EGFR slightly even without RSV infection. However, the level of MUC5AC was enhanced in ITGB4 deficient AECs only under RSV infection. This phenomenon suggests that the expression of MUC5AC was regulated mainly under specific stimulus, such as RSV. Moreover, apart from the ITGB4-EGFR pathways, other components pathways may also participate in the regulation of MUC5AC in AECs [42][43][44]. Not only that, Lakshmanan et al. have discovered that MUC5AC interacts directly with ITGB4 in genetically engineered mouse adenocarcinoma tissues [45]. However, such direct interaction between ITGB4 and MUC5AC was not observed in our primary AECs which may due to the distinct cellular functions and regulation mechanism of AECs.
In summary, this study found that decreased expression of ITGB4 in AECs was involved in mucus hypersecretion and MUC5AC overexpression through EGFR, ERK and c-Jun pathway following RSV infection. This could serve to better understand the mechanism of ITGB4 deficiency on mucus hypersecretion in RSV-infected AECs. Complete elucidation of the underlying mechanism might provide novel therapeutic targets to reduce mucus overproduction in RSV-related LRTI.