HDM-induced Atg5 Mediates TSLP Expressing to disrupt epithelial barrier

Background: Asthma is a complex and heterogeneous disease. Autophagy, process of self-protection in cells, is an intracellular process when cells are being attacked by certain stress. Our team focused upon the disruption of airways epithelial barrier in asthma, and we interested in whether autophagy played a key role in asthma. Methods: 400U HDM was used to treat HBECs and established asthmatic mice model. Western blotting, RT-PCR and immunofluorescence were mainly used to detect autophagy process in vivo and in vitro. One way ANOVA and Mann Whitney test were used for statistic. Results: After treated with HDM, expression of LC3ab increased in vivo and in vitro. Using Rapamycin, 3-MA and Chloroquine to treat HBECs, then we surprisingly found that HDM disrupts epithelial barrier through incomplete autophagy. To find out the connection between asthma and autophagy, we chose known autophagy related genes to determine the association between autophagy and disruption of airway epithelial barrier. Atg5 and atg12 were chosen because these two genes varied upon the time dependent manner. Knocked down the expression of atg5 or atg12 by siRNA, the expression of TSLP, which can induce the disruption of airway epithelial barrier, remarkably reduced. Conclusions: These results demonstrated that HDM induced inflammatory in airway epithelium through autophagy, and then knocked down autophagy related genes alleviated the inflammatory in HBECs. through upregulating Atg5 and Atg12


Background
Asthma is a complex chronic inflammatory heterogeneous lung disease [1]. Chronic inflammation may finally lead to hyperresponsiveness to common allergens, microorganisms, oxidants, pollutants, and It is also associated with immune system activation, airway hyperresponsiveness (AHR), epithelial cell activation, airway epithelial barrier disruption, mucus overproduction and airway remodeling [2,3]. Under normal circumstances, airway epithelial cells connected by tight junctions secrete mucus, airway surface lining fluid, host defense peptides, and antioxidants and express innate immune pattern recognition receptors to respond to inhaled foreign substances and pathogens [4]. Airway epithelium has been thought to function mainly as the first defensive barrier to protect lung from allergens. The integrity of airway epithelial barrier depends on apical tight junctions composed of zonula occludens 1-3, occludin, and claudin 1-5, and on adherens junctions, which consist of E-cadherin, β-catenin, and junctional adhesion molecule, that keep bronchial epithelial cells together and maintain their apicobasal polarity. When asthma patients were exposed to allergens, such as House Dust Mites (HDM), airway epithelial cells activated and released cytokines, such as IL-33, IL-25 and TSLP, induced Th2 inflammation that play a critical role in asthma [3].
Autophagy, connect to a large number of human diseases, is a process that intracellular components and dysfunctional organelles are delivered to the lysosome for degradation and recycling, and it is essential for cell survival, bioenergetic homeostasis, organism development, and cell death regulation [5]. Recent studies showed that dysfunction in autophagy have been associated with environmental pollutant and allergen-induced oxidative stress, mitochondrial dysfunction, secretion of multiple inflammatory proteins, and subsequently development of asthma [6]. During the past two decades, some evidences have revealed that autophagy upregulation or activation may help to reduce levels of toxic protein species and apoptosis, thereby alleviate disease [7][8][9]. On the other hands, researchers also found that inhibition of autophagy can enhance the action of anti-cancer drugs by modulating the apoptotic response (Regulation of Apoptosis by Autophagy to Enhance Cancer Therapy.). Whether autophagy plays a positive or negative role in certain diseases is still under argument.
Our team focus on the airway epithelial barrier disruption induced by HDM. We hypothesized autophagy process plays critical roles in HDM-induced asthma, and in this study we evaluated that different stage of autophagic process played distinct roles in HDM-induced asthma.

Animals and experimental protocol
Specific Pathogen Free (SPF) C57BL/6 mice (female, 6 weeks old) were purchased from Southern Medical University (Guangzhou, China). The mice were housed in the SPF environment (room temperature 24°C, humidity range 40-70%, 12 h light/dark cycle).
Providing sterilized water and food. All experiments were conducted under the guidelines outlined by the committee of Southern Medical University on the Use and Care of Laboratory Animals. Standard guidelines for laboratory animal care followed the Guide for the Care and Use of Laboratory Animals. HDM was purchased from ALK-Abello A/S (Denmark). C57BL/6 mice were randomly assigned into each of following 2 groups: (1) control group, phosphate-buffered saline (PBS; Gibco, Life Technology); (2) HDM group, treated intranasally with HDM (400 U/mouse each irritation, dissolved in sterilized PBS). The protocol of establishing the mouse model of HDM-induced asthma was as follows: The mice were injected 100 µl HDM solution intraperitoneally once a week in total two weeks. Three days after the last injection, mice were exposed to 25 ul of all treatments twice (50ul in total) for each irritation under inhaled isoflurane-anesthesia two times every week, for a total of 8 weeks.

Cell culture and treatment
The human bronchial epithelial cell line, HBE-135 (ATCC, USA) was raised in Kerotinocyte Medium (Sciencell) and placed in a humidified incubator at 37°C with an atmosphere of 5%

Pulmonary histologic examination
Left lungs were gently infused with 4% neutral formalin to fully inflate all lobes (inflation was judged visually) and immersed in formalin for at least 48h, then fixed, paraffin-embedded, cut in 5μm sections, and stained with hematoxylin and eosin (H&E) for blinded histopathologic assessment. For immunohistochemistry of LC3a/b, lung sections (5μm) were prepared with a Leica microtome 2030 (Leica Microsystems Nussloch GmbH, Nussloch, Germany), and then submerged in citrate buffer (pH 6.0) for antigen retrieval. Samples were treated with PBS contained 5%BSA for 10 min at room temperature to block endogenous peroxidases, and then incubated overnight at 4°C in recommended dilutions of anti-LC3ab (Cell Signaling Technology). After washing with PBS three times, slices were incubated with a secondary antibody for 30 min at room temperature. Signals were visualized with a DAB peroxidase substrate kit (ZhongShanJinQiao, Beijing).

Evaluation of epithelial barrier function
Epithelial barrier integrity was assessed by measuring TEER and FITC-Dx flux across the monolayers of cultured epithelial cells. Briefly, confluent monolayers of HBECs, polarized at an air-liquid interface, were cultured in 12-well Transwell inserts (Corning Costar). TEER was measured using a Millicell ERS-2 Epithelial Volt-Ohm meter with an STX01 electrode (Millipore Corp, Billerica, MA, USA). Then, the apical medium (luminal side) was replaced with 200 μL of phenol red-free RPMI 1640 containing 0.5 mg/ml FITC-Dx (Sigma, USA), and the basal medium (non-luminal side) was replaced with 800 μL of phenol red-free RPMI 1640 without FITC-Dx, and the cells were incubated at 37°C for 90 min. Samples were analyzed by fluorimetry (excitation 492nm; emission 530nm). Epithelial permeability was expressed as the percent leakage of FITC-Dx from apical to basolateral compartments.

Western blotting analysis
Cells were lysed in the RIPA lysis buffer (KeyGEN Biotech) containing PMSF (KeyGEN Biotech) at 4°C, 15min. After ultrasonicated 10s, the samples were centrifuged at 12000 rpm for 10min, and the supernatants were collected and boiled with standard SDS sample buffer.

Quantitative real-time PCR (RT-qPCR)
Total RNA was extracted from the treated cells by using Cell Total RNA Isolation Kit (Foregene, China). RNA samples were then reverse transcribed into first-strand cDNA using the PrimeScriptTM RT reagent kit (Takara). SYBR Green (Yeasen, China) was used to perform (reverse). The amplification protocol was set as follows: denaturation at 95°C for 10 min; 40 or 50 cycles denaturation at 95°C for 15 s; followed by 1 min of annealing/extension at 60°C.
The levels of mRNA were normalized to those of GAPDH mRNA (the internal control; Δ Ct method) and were calculated and displayed as 2−ΔCt values.

Adenovirus mRFP-GFP-LC3 infected
HBECs were seeded in confocal plates at a density of 5x103 cells per well, cultured in a

Statistical analysis
Statistical analysis was performed using the GraphPad Prism 5 software package. Data are means ± SD and assessed for significance by one way ANOVA accompanied by Bonferonni's post hoc test or by Mann-Whitney U test as specified on figure legends. Values of P < 0.05 were considered significant.

The expression of LC3ab could be induced by treatment of HDM both in vivo and in vitro.
Autophagy is a common intracellular process induced by varied stimulation, so we Bronchial Epithelial Cells (HBECs) after treated with HDM in indicated concentration for 24h.
As we thought, the expression of LC3ab upregulated in HBECs (Fig2 A). These data suggested that HDM induced autophagy in both mice and HBECs.

The autophagy process was induced by treating with HDM in HBECs.
According to our team researched before, HDM disrupted airway epithelial barrier in HBECs [10]. As autophagy was induced by starvation, virus infection and other stress, we hypothesized that airway epithelial barrier disruption associated with autophagy. To confirm that the association between asthma and autophagy in HBECs, we used three reagents respectively to verify our hypothesis. Rapamycin (Rap) induces autophagy through suppressed mTOR pathway. 3-MA, suppresses autophagy through PI3K pathway.

HDM induced autophagy through Atg5 & Atg12 in HBECs.
Our results mentioned above demonstrated that HDM induced autophagy in HBECs in initial stage, we hypothesized that block autophagy initially may be useful for alleviating the epithelial barrier disruption. To clarify how HDM induced autophagy to disrupted epithelial barrier, we used HDM to stimulate HBECs for indicated times, we found that autophagy was induced from 12 to 24h (Fig4 A). Autophagy related genes (ATGs) associated with both autophagy and human disease pathogenesis, especially neurodegenerative, inflammatory disorders and cancer [14], so we screened the autophagy related genes, including atg5, atg7, atg10, atg16L1 and atg12, for finding out the most important gene in autophagic process in HBECs. Under the treatment with HDM for indicated times, the expressions of Atg5 and Atg12 upregulated most remarkable in HBECs from 12 to 24h(Fig4 B), these two proteins were also a known complex in the autophagy process [15], on the other hand, the levels of ATGs mRNA were not change under the treatment (Fig4 C). These results suggested that HDM induced autophagy in HBECs through Atg5/Atg12, and the two proteins expression upregulated in 12h.

TSLP plays critical role in HDM-induced allergic inflammation.
And the immunochemistry was used to detected the alarmins, including TSLP, IL-25 and IL-33 which are the alarmins that is released in the lung mainly by epithelial cells [17,18], for our next investigation. Our team had confirmed that the airway epithelial barrier was disrupted by HDM in vivo and in vitro, and the TSLP played essential role in the epithelial barrier disruption, and the expression of TSLP also upregulated in 12h under the treatment with HDM in HBECs [10,16].

The expression of TSLP was blocked after silencing Atg5.
We hypothesized that the increased expression of TSLP was associated with Atg5 and Atg12 upregulating. Then we interfered protein levels of ATG5 and of ATG12 respectively (Fig6 A), for further investigating the association between Atg5/Atg12 and TSLP in HBECs under HDM-treated, western blotting was used to assess the protein expression of TSLP while Atg5

Discussion
Autophagy is a conserved process that substrates are endocytosed by the double membrane vesicles and are transported to the lysosomes for degradation [5,19]. The process plays an important role in many diseases, such as Crohn disease, a type of inflammatory bowel disease.
A recent research had demonstrated that ATG16L1, one of the initial genes of autophagy, affects the respond of autophagy in Salmonella typhimurium, which means host cells and autophagy response to stimulation in the pathogenesis of Crohn disease [20]. Another research found that the autophagy receptor p62 are able to mediate the anti-inflammation in LPS-induced macrophages, it has revealed a novel mechanism of macrophage inflammation [21]. As to asthma, plenty of evidence showed that autophagy plays an important role in it, such as environmental particulate matter (PM) induced autophagy in bronchial epithelial cells, while airway inflammation reduced after blocked autophagy in asthmatic mice [22]. More and more evidence showed that autophagy is associated with asthma, but the mechanisms of autophagy affecting airway epithelial inflammation in allergic asthma are not really understood. We attempted to make clear the underlying mechanisms of the function of autophagy in allergic asthma.
In this study, we have demonstrated that autophagy plays different roles in its different stage of process in HDM-induced asthma. Our team have investigated the disruption of airway epithelial barrier for years, and we found that the 400U HDM destroyed the barrier by inducing TSLP expression, then redistributed E-cadherin and β-catenin [10,16]. Blocking the autophagy process induced by HDM at the early stage, like knocked down the autophagy related gene 5(Atg5), we surprisingly found the expression of TSLP, which damaged the airway epithelial barrier, was downregulated in HBECs. On the other hand, complete autophagy process and blocked autophagic flux both disrupted epithelial barrier in our study. These data suggested initial autophagy process is a matter of TSLP expressing resulting in epithelial barrier disruption.
Interestingly, our research has found that the expression of P62 in HBECs wasn't affected by HDM. Only the CQ, a reagent could induce P62 accumulation due to inhibit the autophagolysosome formation, increased the expression of P62 in vitro (Fig2 C), it was a pity that we couldn't explorer further reasons of this phenomenon as our research limited.
There are more and more evidences showed that autophagy highly associated with inflammation in asthma [9,[23][24][25], but to our knowledge, researchers studied autophagy, considered it plays either good or bad role in certain disease [26][27][28][29]. Hence, our findings demonstrated that HDM induced Atg5 to mediate TSLP expressing resulted in disruption of airway epithelial barrier.

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Consent for publication
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