Mitochondrial Transfer Triggered by TSLP Promotes Epithelial-Mesenchymal Transition in Human Lung Epithelial Cells Exposed to House Dust Mite

Mitochondrial Transfer Triggered by TSLP Promotes Epithelial-Mesenchymal Transition in Human Lung Epithelial Cells Exposed to House Dust Mite | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitochondrial Transfer Triggered by TSLP Promotes Epithelial-Mesenchymal Transition in Human Lung Epithelial Cells Exposed to House Dust Mite Chung-Yu Yeh, Mei-Lan Tsai, Hsin-Ying Clair Chiou, Wei-Ting Liao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8162140/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Airway remodeling is a key pathological feature of asthma, closely linked to lung function decline and disease severity. Persistent inflammation, EMT, and mitochondrial dysfunction all contribute to this process. Although HDM strongly induces epithelial alarmins such as TSLP, how cytokine priming cooperates with subsequent allergen exposure to alter mitochondrial function and promote EMT remains unclear. Methods This study investigated the effects of sequential TSLP and HDM exposure in A549 lung epithelial cells. Cell motility and EMT markers were assessed using wound-healing assays and Western Blot. Mitochondrial membrane potential and oxidative stress were examined by JC-1 staining and ROS quantification. To determine how TSLP priming shapes subsequent HDM responses, we analyzed intercellular mitochondrial transfer, and further assessed EMT phenotypes in recipient cells acquiring mitochondria from TSLP-treated donors. Results Sequential TSLP priming followed by HDM exposure markedly enhanced epithelial migration and accelerated EMT. This was accompanied by a collapse of mitochondrial membrane potential, impaired biogenesis, increased mtDNA release, and robust mitophagy activation. Notably, TSLP priming potentiated intercellular mitochondrial transfer, and recipient cells that acquired TSLP-conditioned mitochondria displayed amplified migratory and EMT behavior. These findings identify mitochondrial transfer as an additional layer by which epithelial cytokine priming amplifies allergen-driven remodeling. Conclusions Collectively, these results indicate that TSLP primes epithelial cells by reprogramming mitochondrial dynamics and promoting intercellular mitochondrial transfer, thereby heightening their sensitivity to environmental stimuli. Targeting TSLP signaling and mitochondrial regulation may provide novel therapeutic opportunities to mitigate airway remodeling in chronic respiratory diseases. airway remodeling TSLP epithelial-mesenchymal transition mitochondrial transfer mitophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Asthma is a diverse condition marked by persistent airway inflammation, heightened airway responsiveness, and reversible airflow obstruction. Over time, airway remodeling becomes the underlying pathological factor contributing to the progression of fixed airflow limitation in individuals with asthma. [ 1 ]. During chronic inflammation, exaggerated repair responses contribute to airway remodeling, characterized by goblet cell hyperplasia, thickening of the epithelial basement membrane, neovascularization, and the development of subepithelial fibrotic lesions [ 2 ]. It’s essential to recognize that airway remodeling is not solely driven by chronic inflammation. Research indicates that this process can commence early in preschool-aged children who present with wheezing symptoms. [ 3 , 4 ]. At present, no asthma medications have been clinically confirmed to stop the remodeling process. [ 1 ]. As the first line of defense, airway epithelial cells are essential for initiating the innate immune response and contributing to the remodeling process. Growing evidence indicates that increased epithelial-mesenchymal transition (EMT) plays a significant role in airway remodeling in asthma. [ 5 ] For example, a study using a murine model sensitized to house dust mites (HDM) demonstrated that changes in EMT are major contributors to airway remodeling. This process involves airway epithelial cells losing their usual characteristics, marked by the downregulation of E-cadherin and the upregulation of mesenchymal markers like vimentin, α-SMA, and type I pro-collagen, along with the nuclear translocation of Snail1, a key EMT inducer. [ 6 ]. An epithelium-derived cytokine, thymic stromal lymphopoietin (TSLP) represents a master switch at the interface between environmental allergens and pulmonary allergic immunologic responses [ 7 ]. The airway epithelium is a key component of the innate immune system and the initiator of airway remodeling in asthma. Previous studies have shown that expressions of TSLP and TARC/CCL17 were correlated with airway obstruction in asthma [ 8 ]. TSLP-induced cellular senescence was required for airway remodeling, and inhibited cellular senescence blocks airway remodeling and relieves airway resistance [ 9 ]. In airway smooth muscle model, TSLP could induce proinflammatory mediators such as IL-6, IL-8 production and cell migration [ 10 ]. Cai et al. showed that TSLP alone could decrease the protein level of E-cadherin while increased mesenchymal proteins such as vimentin, collagen I and fibronectin levels in a concentration-dependent manner and promoted TGF-β1-induced EMT in human bronchial epithelial cells [ 11 ]. In addition, TSLP-transgenic mice developed spontaneous inflammation with infiltration of goblet cell hyperplasia, leukocytes, and subepithelial fibrosis in the lung [ 12 ]. In the chronic HDM-induced asthma model, neutralization of TSLP with an anti-TSLP mAb reversed airway inflammation, prevented structural alterations, and decreased AHR to methacholine [ 13 ]. It suggested that TSLP play a pivotal role in the initiation and persistence of airway inflammation and remodeling. Additionally, HDM, well-known allergens that induce asthma and contribute to airway remodeling along with alarmin production, present major concerns.[ 14 ]. In the contemporary environment, air pollution has been recognized as a significant factor in the exacerbation of asthma. Notably, diesel exhaust particles have been identified as potent inducers of alarmin production in epithelial cells, thereby contributing to airway inflammation and remodeling. [ 15 ]. This study aims to investigate the effects of sequential exposure to TSLP and HDM on epithelial-mesenchymal transition (EMT) and its associated signaling pathways. By elucidating critical exposure combinations that modulate EMT, this research seeks to advance the understanding of airway remodeling mechanisms and contribute to the development of targeted therapeutic strategies for asthma management. Materials and Methods Cell culture The human lung carcinoma cell line A549 (ATCC) was maintained in MEM supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% non-essential amino acids, and 1% antibiotic-antimycotic (Thermo Fisher Scientific Inc., Waltham, USA). After centrifugation, the cells were resuspended in fresh medium and seeded into 6-well plates at a density of 3 x 10 5 cells per well, followed by incubation for 24 h prior to experimentation. Wound healing assay Cells were seeded into 6-well plates and treated with IL-25 (1 or 10 ng/mL) or HDM (1 μg/mL) for 24 h, followed by a reversed stimulation in which cells were exposed to the alternate factor (HDM or IL-25) for an additional 24 h. To assess cell signaling, cells were pretreated for 1 h with either the antioxidant N-acetylcysteine (NAC), the mitochondrial ROS scavenger MitoTEMPO, the mitophagy inhibitor Mdivi-1, or the autophagy inhibitor chloroquine (CQ) (Merck Millipore) before alarmin stimulation. A wound was generated using 1000 μL pipette tips, followed by two washes with PBS to remove detached cells and debris, after which cells were maintained in complete medium for 24 h. Cell migration was examined under a microscope, and the wound closure was quantified as a percentage of the initial wound area using ImageJ software, calculated as [(wound area at indicated time point)/(wound area at 0 h)] × 100% and expressed as GAP %. Measurement of ΔψM and transmission electron microscopy Mitochondrial membrane potential was evaluated using JC-1 dye (Invitrogen, Waltham, USA) in combination with LSR II flow cytometry (Becton Dickinson, San Jose, CA) and confocal microscopy (LSM 700, Carl Zeiss Microscopy, Göttingen, Germany). JC-1 accumulates in mitochondria in a potential-dependent manner, exhibiting a fluorescence emission shift from green (525 nm) to red (590 nm). A reduction in the red/green fluorescence intensity ratio indicates mitochondrial depolarization. For ultrastructural analysis, harvested cells were examined using transmission electron microscopy (JEM-2000EXII, JEOL Ltd., Tokyo, Japan) to assess mitochondrial morphology and mitophagy. Quantitative real-time Reverse transcription polymerase chain reaction To examine mitochondrial involvement, gene expression of mitochondrial copy number and respiratory chain complexes I–V was analyzed, including NADH dehydrogenase subunit 1 (MT-ND1, complex I), succinate dehydrogenase complex subunit A flavoprotein (SDHA, complex II), mitochondrial cytochrome b (MT-CYTB, complex III), cytochrome c oxidase I (COXI, complex IV), ATP synthase H+ transporting mitochondrial Fo complex subunit F6 (MT-ATP6, complex V), as well as the mitochondrial proton carrier uncoupling protein 2 (UCP2). Following TSLP treatment for various time points, total RNA (2 μg) was isolated and reverse transcribed using SuperScript II with an anchored oligo-dT primer according to the manufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The synthesized cDNA was subsequently subjected to quantitative reverse transcription PCR (qRT–PCR). Primer sequences and qRT–PCR procedures were carried out as previously described in our study. [16]. Preparation of cDNA (mtDNA) and relative quantification of mtDNA fragments Total mitochondrial DNA (mtDNA) was extracted using a mitochondrial DNA isolation kit (BioVision, Milpitas, USA) according to the manufacturer’s protocol. Two primer sets targeting mitochondrial ribosomal 16S rRNA, mtDNA-79 and mtDNA-230, were applied as previously reported [16]. The 79-bp fragment (mtDNA-79) represents mitophagy-related mtDNA generated through enzymatic cleavage during apoptosis, whereas the 230-bp fragment (mtDNA-230) reflects mtDNA released by non-apoptotic cell death processes (e.g., necrosis) or by active secretion [17]. Quantitative real-time PCR was used to measure these mtDNA fragments, and results were normalized to the expression of the housekeeping gene ACTB. Western blotting At harvest, cells were lysed in equal volumes of ice-cold lysis buffer (Abcam, Cambridge, MA) supplemented with protease inhibitors (Merck Millipore). Equal protein amounts from the lysates were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in TBS containing 5% non-fat dry milk and 0.1% Tween-20, followed by incubation with primary antibodies against N-cadherin, E-cadherin (Genetex, Hsinchu, Taiwan), LC3, SQSTM1 (Abcam), PINK, SMAD2, or GAPDH (Cell Signaling Technology, Danvers, MA). Immunoreactive bands were detected using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence (ECL) system (Merck Millipore), and images were acquired with a ChemiDoc-It 810 Imager (Ultra-Violet Products, Upland, CA, USA). All procedures were performed according to the manufacturers’ instructions. Mitochondrial transfer assay Mitochondrial transfer was evaluated following a previously described protocol [17]. Donor A549 cells were labeled with 200 nM MitoTracker Deep Red FM (#M22426; Thermo Fisher Scientific) and CellTrace Violet (#C34557; Thermo Fisher Scientific) according to the manufacturer’s instructions. These labeled donor cells were then co-cultured with unlabeled recipient A549 cells, and mitochondrial transfer was assessed after 3 h by detecting MitoTracker signal in recipient cells lacking CellTrace labeling. All assays were conducted using control A549 cells. To define cell boundaries (regions of interest, ROI), cells were counterstained with phalloidin 488. Mitochondrial transfer was quantified as sum intensity per ROI (SI/ROI). For each condition, six wells were analyzed, with four images captured per well. In each image, 25 CellTrace-negative cells were measured for MitoTracker intensity and ROI, yielding a total of 600 cells per condition (6 wells × 4 images per well × 25 cells per image). Statistical analysis Data are expressed as mean ± standard deviation (SD). Differences between experimental and control groups in each independent experiment were analyzed using the Mann-Whitney U test. Densitometric values from western blot assays were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA) by measuring the optical density of individual bands. Statistical analyses were conducted using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA, USA). A p-value < 0.05 was considered statistically significant. Results Pre-treatment with TSLP prior to house dust mite exposure enhanced cell migration and altered the expression of EMT markers. To investigate the effect of the stimulation order of TSLP and HDM on cell migration, cells were first treated with either TSLP (10 ng/mL) or HDM (1 µg/mL) for 24 hours. This was followed by a reversed stimulation, in which cells were exposed to the alternate factor (HDM or TSLP) for an additional 24 hours. Cell migration was assessed using wound healing assays (Fig. 1 A and B). The results showed that TSLP at both 1 ng/mL and 10 ng/mL significantly enhanced cell migration. Moreover, treatment with TSLP followed by HDM further increased cell migration compared to TSLP alone. The group treated with TSLP at 10 ng/mL followed by HDM also displayed a pronounced increase in migration compared to HDM alone. While HDM alone significantly promoted migration, no additional effect was observed when HDM was applied first, followed by TSLP, compared to HDM alone. The influence of stimulation order on EMT-related protein expression was also examined. A549 cells were initially treated with either TSLP (1 or 10 ng/mL) or HDM (1 µg/mL) for 24 hours, followed by reversed stimulation with the alternate factor for another 24 hours. Protein expression was analyzed by Western blot. As shown in Fig. 1 C, TSLP significantly increased the mesenchymal marker N-Cadherin and the transcription factor SLUG while reducing the epithelial marker E-Cadherin. These effects were further enhanced when cells were treated with TSLP followed by HDM. Although HDM alone also significantly modulated EMT protein expression, applying HDM first followed by TSLP did not produce additional changes compared to HDM treatment alone. TSLP promoted EMT changes through oxidative stress. Oxidative stress, the physiological damage caused by reactive oxygen species (ROS), has been shown to affect smooth muscle contraction, induce airway hyperresponsiveness, and increase mucus secretion and epithelial shedding in respiratory cells. Excessive ROS production in asthma can lead to oxidative stress, contributing to pulmonary fibrosis associated with TGF-β [ 18 ]. To investigate the effect of the stimulation order of TSLP and HDM on ROS production, cells were first treated with either TSLP (10 ng/mL) or HDM (1 µg/mL) for 3 hours, followed by reversed stimulation with the alternate factor (HDM or TSLP) for an additional 3 hours. ROS generation was assessed using DHE and DCFH-DA staining. DHE is commonly used to detect intracellular superoxide (Fig. 2 A), whereas DCFH-DA reacts with H2O2, ONOO−, lipid hydroperoxides, and O2−, serving as a general indicator of total ROS (Fig. 2 B). The results demonstrated that stimulation with TSLP (10 ng/mL) or HDM significantly increased both superoxide and total intracellular ROS. Moreover, treatment with TSLP followed by HDM further enhanced ROS production compared to TSLP alone. We further examined the effects of ROS scavengers, N-acetylcysteine (NAC) and MitoTEMPO, on EMT-related proteins and cell migration. TSLP-induced decreases in E-cadherin and increases in N-cadherin and SMAD2 were reversed by ROS scavengers (Fig. 2 C). Additionally, TSLP-induced cell migration was significantly suppressed by both MitoTEMPO and NAC (Fig. 2 D). Pre-treatment with TSLP before house dust mite exposure induced mitochondrial dysfunction. Mitochondrial ROS production is a key indicator of mitochondrial dysfunction and cellular stress. To assess whether TSLP treatment followed by HDM exposure affects mitochondrial function and induces mitophagy, we performed mitochondrial membrane potential (MMP) analysis. A decrease in the red/green fluorescence intensity ratio indicated that TSLP promoted mitochondrial depolarization, which was further enhanced by subsequent HDM exposure (Fig. 3 A). We also evaluated mitochondrial DNA (mtDNA) release following TSLP treatment or TSLP treatment followed by HDM. Quantitative real-time PCR revealed that TSLP increased cytosolic mtDNA levels in A549 cells, and TSLP followed by HDM caused a further significant increase compared to TSLP alone (Fig. 3 B). Mitochondrial biogenesis was assessed by measuring mitochondrial copy number and the expression of respiratory chain genes. As shown in Fig. 3 C, stimulation with TSLP or HDM for 6 hours did not significantly alter mitochondrial copy numbers. The expression of ND1 (complex I), SDHA (complex II), CYTB (complex III), COX1 (complex IV), ATP6 (complex V), and uncoupling protein 2 (UCP2) was measured by qRT-PCR. TSLP followed by HDM significantly upregulated these genes compared to TSLP alone. Western blot analysis further showed that TSLP increased the levels of SQSTM1 and LC3, with this effect being enhanced by HDM treatment (Fig. 4 A). To examine the role of mitophagy in TSLP- and HDM-induced cell migration, cells were pretreated with the mitophagy inhibitor Mdivi-1. As shown in Fig. 4 C, TSLP-induced cell migration was suppressed by Mdivi-1, and similar suppression was observed for TSLP followed by HDM-induced migration. Collectively, these results indicate that TSLP followed by HDM alters mitochondrial function and promotes mitophagy in A549 cells. TSLP promoted mitochondrial transfer, leading to increased cell migration and modulation of EMT marker expression. Over the past few decades, studies have demonstrated that cells can transfer mitochondria to neighboring cells through intercellular mitochondrial transfer, a process observed in various tissues and involved in both normal physiology and disease development. While many studies emphasize that mitochondrial transfer supports the metabolism of recipient cells, recent evidence indicates it also helps maintain mitochondrial health in donor cells [ 19 ]. We next investigated how stimulation with TSLP alone or TSLP followed by HDM influences mitochondrial transfer. As shown in Fig. 5 A, mitochondria were observed moving to adjacent cells via tunneling nanotubes (TNT). Figure 5 B shows that stimulation with either TSLP or HDM alone significantly increased mitochondrial transfer to recipient cells. Notably, TSLP-induced mitochondrial transfer was further enhanced when followed by HDM, whereas no such increase was observed when HDM preceded TSLP. Mitochondrial transfer occurs through three primary mechanisms: (1) formation of temporary intercellular structures such as TNTs, (2) export of mitochondria within extracellular vesicles, and (3) release of mitochondria as free organelles [ 19 ]. To determine whether blocking TSLP-induced mitochondrial transfer affects cell migration, we used the TNT inhibitor AraC, the extracellular vesicle inhibitor Y-27632, and the exosome inhibitor GW4869. As shown in Fig. 5 C–F, all three inhibitors reduced TSLP-induced mitochondrial transfer, suggesting that TSLP-mediated transfer occurs via TNT formation and vesicle-mediated transport. We further evaluated the functional impact on recipient cells. Untreated cells were co-cultured with either untreated or TSLP-treated donor cells for 24 hours, followed by staining with MitoTracker and CellTrace. Only MitoTracker-positive recipient cells were isolated and replated overnight. Wound healing assays and analysis of EMT-related markers revealed that recipient cells acquiring mitochondria from TSLP-treated donors exhibited increased migration compared to controls. These cells also showed reduced E-cadherin expression and elevated N-cadherin and SMAD2 levels (Fig. 6 A–C). Together, these results indicate that TSLP priming induces mitochondrial dysfunction and transfer, which alters the behavior of neighboring cells, promotes migration, and contributes to tissue remodeling. Discussion TSLP is a cytokine mainly produced by epithelial cells, keratinocytes, and stromal cells. Structurally similar to IL-7 and part of the IL-2 family, it functions as an alarmin that is rapidly upregulated in response to stress or injury to barrier tissues such as the skin, lungs, and gut, especially following exposure to environmental triggers like allergens, pollutants, or pathogens [ 20 ]. Initially linked to allergic diseases such as atopic dermatitis, asthma, and eosinophilic esophagitis, TSLP has also been implicated in chronic inflammatory conditions (e.g., COPD, celiac disease), autoimmune disorders (e.g., psoriasis, rheumatoid arthritis), and various cancers [ 21 ]. In asthma, TSLP activates dendritic cells, which in turn prime naïve T cells to differentiate into Th2 cells. These Th2 cells secrete cytokines such as IL-4, IL-5, and IL-13, leading to eosinophilic inflammation, airway hyper responsiveness, and mucus overproduction, thereby contributing to sustained allergic inflammation [ 22 ]. A study found that elevated baseline pulmonary TSLP levels are associated with greater asthma severity in a subset of children. These findings suggest that TSLP plays a crucial role in the development of pediatric asthma [ 23 ]. Recent studies have shown that TSLP stimulation of human nasal epithelial cells reduced E-cadherin expression, increased FSP1, vimentin, and collagen I/III levels, and induced a fibroblast-like morphology [ 24 ]. TSLP also drives asthmatic airway remodeling via p38 MAPK-STAT3 activation, with epithelial-fibroblast interactions further exacerbating the remodeling process [ 25 ]. This indicates that the alarmin TSLP plays a role not only in inflammatory responses but also in promoting airway remodeling. Airway epithelial cells serve as the first line of defense against environmental stimuli by rapidly secreting alarmin cytokines. CM Weng et al. demonstrated that exposure of primary epithelial cells to diesel exhaust particles significantly elevated IL-25, IL-33, and TSLP levels via activation of the aryl hydrocarbon receptor.[ 15 ]. Previous studies have shown that stimulation of human bronchial epithelial cells with either Der p1 or PM2.5 alone increases the production of IL-25, IL-33, and TSLP, with a more pronounced effect observed when both stimuli are applied together [ 14 ]. Both pollutants and allergens like house dust mites (HDM) can stimulate TSLP production, promote airway remodeling, and worsen asthma symptoms. TSLP signals via an IL-17RA/TSLPR heterodimer, recruiting JAK1 and JAK2 in a cell type–dependent manner. These kinases can activate STATs, NF-κB, and MAPK through PI3K. The effects vary by cell type, including upregulation of IL-6 and IL-8, increased cell proliferation, and, in dendritic cells, migration and maturation toward a Th2 phenotype [ 26 ]. Previous studies have demonstrated that HDM allergens, such as Der p 5, bind to TLR2 on airway epithelial cells, activating NF-κB through IκBα degradation. This triggers nuclear translocation of RelA (canonical pathway) and RelB (non-canonical pathway), promoting the production of proinflammatory cytokines, including IL-8 and IL-33[ 26 , 27 ]. Additionally, Der p 1 directly activates ERK1/2 and p38 MAPK in airway smooth muscle (ASM) cells via an IgE-independent mechanism, where ERK1/2 contributes to ASM hyperresponsiveness and p38 MAPK regulates ERK1/2 activity [ 28 ]. These findings suggest that TSLP and HDM share overlapping signaling pathways. This study aimed to determine whether TSLP exposure prior to allergen stimulation is more effective in promoting airway remodeling and asthma exacerbation compared to the reverse sequence. Our results showed that TSLP pretreatment followed by HDM exposure enhanced cell migration and induced more pronounced changes in EMT-related markers than the opposite order. These observations indicate that TSLP may prime the airway epithelium for EMT, thereby amplifying the remodeling response to subsequent allergen exposure. Mitochondrial dysfunction contributes to the development of chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD), and is characterized by increased mitochondrial ROS, reduced mitochondrial membrane potential, elevated calcium influx, mitochondrial damage and DNA release, as well as impaired mitophagy [ 29 , 30 ]. Our previous study demonstrated that TSLP modulates mitochondrial ROS-induced mitophagy through histone modifications in human monocytes [ 16 ]. A previous study found that cigarette smoke induced small airway remodeling by enhancing EMT and PINK1-Parkin–mediated mitophagy [ 31 ]. In this study, intracellular ROS and superoxide levels were assessed using DCFH-DA and DHE. We found that TSLP treatment increased the production of intracellular ROS and superoxide, and this effect was further enhanced when TSLP was applied prior to HDM stimulation in A549 cells. It has also been reported that within mitochondria, NAC is converted into hydrogen sulfide and various sulfane sulfur compounds, which serve as more effective oxidant scavengers [ 32 ]. MitoTEMPO is a newly developed antioxidant specifically targeting mitochondria [ 33 ]. In our study, pretreatment of A549 cells with MitoTEMPO or NAC before TSLP stimulation suppressed cell migration, decreased the expression of mitophagy markers, N-cadherin, and SMAD2, while increasing E-cadherin expression. In our previous work, we also observed that TSLP stimulation induces hyperactivity of mitochondrial complexes I and II/III, accompanied by a reduction in mitochondrial membrane potential [ 16 ]. In this study, we further demonstrated that the effect was amplified when TSLP was administered prior to HDM stimulation in A549 cells. Notably, while UCP-2 decreases ROS production by uncoupling mitochondrial respiration, elevated ROS levels can in turn induce cellular adaptations that upregulate UCP expression [ 34 ]. Mdivi-1, a cell-permeable quinazolone compound, effectively inhibits mitophagy. Pretreating cells with Mdivi-1 prior to TSLP stimulation or sequential TSLP and HDM exposure reduced cell migration. These findings suggest that suppressing ROS and mitophagy can mitigate TSLP-induced EMT, indicating that TSLP may promote EMT progression through ROS-mediated mitophagy activation. Mitochondrial transfer, the process by which mitochondria are exchanged between cells, has recently gained significant attention for its potential therapeutic role in mitochondrial diseases, age-related conditions, and various other health disorders [ 35 ]. Previous studies have shown that mitochondrial dysfunction plays a critical role in driving airway remodeling by enhancing oxidative stress, epithelial damage, and EMT-related changes [ 36 ]. This study is the first to demonstrate that TSLP can induce mitochondrial transfer and mediate intercellular transport of mitochondria among human lung epithelial cells. Similarly, TSLP pretreatment combined with HDM exposure resulted in increased mitochondrial transfer and more pronounced changes. Mitochondrial transfer can occur through multiple mechanisms, including exosome-mediated pathways and TNTs [ 37 ]. Notably, TSLP-induced mitochondrial transfer may involve both exosome- and TNT-dependent pathways. To investigate the role and mechanism of TSLP-induced mitochondrial transfer in cell migration, we used the vesicle inhibitor Y-27632, the exosome inhibitor GW4869, and the TNT inhibitor AraC. Results showed that all three inhibitors suppressed TSLP-induced cell migration, indicating that mitochondrial transfer triggered by TSLP contributes to this process. We further assessed the functions of untreated cells that received mitochondria from TSLP-treated cells. These recipient cells displayed enhanced migratory capacity and altered EMT-related marker expression. Previous studies have shown that platelets recruited to injury sites transport mitochondria to the plasma membrane and release them as extracellular vesicles (EVMs) and free mitochondria during degranulation [ 38 ]. These mitochondria are subsequently taken up by subendothelial mesenchymal stem cells, which in turn secrete pro-angiogenic factors to promote wound healing within the vascular microenvironment [ 39 ]. Additionally, EVMs can mediate intercellular communication by transferring proteins, nucleic acids, microRNAs, lipids, and metabolites to nearby or distant target cells [ 40 ]. This suggests that exosome-mediated transfer induced by TSLP may also deliver additional mediators that influence recipient cell functions. Further research is required to confirm this possibility. Mitochondrial transfer has been applied in various animal models and holds promise as a therapeutic strategy for multiple human diseases, including neurodegenerative disorders, central nervous system injuries, and cardiovascular diseases[ 35 ]. In this study, we demonstrated that TSLP primes epithelial cells by regulating mitochondrial dynamics and promoting mitochondrial transfer, thereby enhancing their responsiveness to environmental stimuli. Thus, targeting TSLP signaling and mitochondrial transfer pathways may provide new strategies to prevent airway remodeling. Conclusion This study revealed that TSLP pretreatment sensitizes airway epithelial cells to subsequent allergen exposure, leading to enhanced cell migration and more pronounced EMT alterations. TSLP stimulation triggered oxidative stress, mitochondrial dysfunction, and mitochondrial transfer, all of which were further intensified by subsequent HDM exposure. Furthermore, TSLP primes epithelial cells through the regulation of mitochondrial dynamics and the promotion of intercellular mitochondrial transfer, thereby heightening their reactivity to environmental stimuli. Collectively, these results indicate that targeting TSLP signaling and mitochondrial transfer mechanisms could represent a potential therapeutic approach to mitigate airway remodeling in asthma and other chronic respiratory diseases. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding The study was supported by grants from the Ministry of Science and Technology of the Republic of China (grant numbers MOST 113-2314-B-037-094-MY3), and the Research Center for Precision Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan and by Kaohsiung Medical University Research Center Grant (grant numbers KMU-TC114A01) Funding The study was supported by grants from the National Science and Technology Council of the Republic of China (grant numbers MOST 113-2314-B-037-094-MY3), and the Research Center for Precision Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan and by Kaohsiung Medical University Research Center Grant (grant numbers KMU-TC114A01) Author Contribution Conceptualization: Chang-Yu Yeh, Wei-Ting Liao, and Chih-Hsing Hung; Methodology: Chang-Yu Yeh and Chih-Hsing Hung; Formal analysis: Mei-Lan Tsai and Hsin-Ying Clair Chiou; Investigation: Wei-Ting Liao; Data curation: Chang-Yu Yeh and Mei-Lan Tsai; Writing-original draft preparation: Chang-Yu Yeh; Writing-reviewing and editing: Wei-Ting Liao and Chih-Hsing Hung. 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09:21:09","extension":"html","order_by":34,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":126036,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8162140/v1/ebbe27b78fecc1871aaf5519.html"},{"id":97416521,"identity":"f44b10a8-5aab-4783-86f6-52ef37a0d807","added_by":"auto","created_at":"2025-12-04 07:07:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1426703,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP stimulation prior to house dust mite (HDM) exposure enhanced cell migration and altered epithelial-mesenchymal transition (EMT) markers expression. \u003c/strong\u003eA549 cells were treated as follows: control (‒, white bar), TSLP alone (1 or 10 ng/mL, gray bar), HDM alone (1 μg/mL, black bar), TSLP for 24 h followed by HDM for 24 h (diagonal white bar), or HDM for 24 h followed by TSLP for 24 h (diagonal black bar). (A) Wound healing assay was performed and quantified 24 h after scratching. (B) Transwell migration assay with quantitative analysis. (C) Western blot showing EMT-related protein expression with quantification. Data shown as mean ± SEM, N = 6. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8162140/v1/2e5eabec52d1c488efa3e4b8.png"},{"id":97416520,"identity":"bc725423-63ee-4137-8e03-83e9bd61005a","added_by":"auto","created_at":"2025-12-04 07:07:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1232707,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP enhanced EMT changes via oxidative stress. \u003c/strong\u003eA549 cells were treated with control (‒, white bar), TSLP alone (gray bar), HDM alone (1 μg/mL, black bar), TSLP for 3 h then HDM for 3 h (diagonal white bar), or HDM followed by TSLP (diagonal black bar). The intracellular superoxide (A) and intracellular total H2O2 (B) were measured by ELISA reader. A549 cells were pretreated with N-acetylcysteine (NAC) or MitoTEMPO 1 hours before TSLP treatment. After another 48 hours of combined treatment. (C) The Western blot shows the expressions of EMT-related proteins. (D) Wound healing assay assessed migration 24 h after scratching. Data shown as mean ± SEM, N = 6. Wound closure presented as GAP%. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8162140/v1/7f08bda61cf2e3a5122e5574.png"},{"id":97666587,"identity":"7fd7a811-d814-4ee2-ab91-1112d42afb3d","added_by":"auto","created_at":"2025-12-08 09:21:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1118446,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP stimulation prior to HDM exposure induced mitochondrial dysfunction. \u003c/strong\u003eA549 cells received the following treatments: control (‒, white bar), IL-25 (10 ng/mL, gray bar), HDM (1 μg/mL, black bar), TSLP for 24 h followed by HDM for 24 h (diagonal white bar), or HDM followed by IL-25 (diagonal black bar). (A) The mitochondrial membrane potential was observed by confocal microscope using the JC-1 staining method. (B) A549 cells were treated with the following conditions: untreated control (‒), TSLP (10 ng/mL) alone, HDM (1 μg/mL) alone, TSLP for 4 hours followed by HDM stimulation for another 4 hours, or HDM for 4 hours followed by TSLP stimulation for another 4 hours. The released mtDNA was analyzed by qRT-PCR. (C) A549 cells were treated with the following conditions: untreated control (‒), TSLP (10 ng/mL) alone, HDM (1 μg/mL) alone, TSLP for 3 hour followed by HDM stimulation for another 3 hours, or HDM for 3 hour followed by TSLP stimulation for another 3 hours. The expression of mitochondrial complex genes was analyzed by qRT-PCR. Data presented as mean ± SEM. N = 6. *: p\u0026lt;0.05; **: p\u0026lt;0.01; *: p\u0026lt;0.001.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8162140/v1/6b3a1a7c1e55b2c9dde1c476.png"},{"id":97416522,"identity":"28ed2d0d-9306-493a-8147-82c1d4b0bf54","added_by":"auto","created_at":"2025-12-04 07:07:39","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":940385,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP amplified EMT changes triggered by HDM through mitophagy. \u003c/strong\u003eA549 cells were treated with control (‒, white bar), TSLP alone (gray bar), HDM alone (1 μg/mL, black bar), TSLP for 24 h then HDM for 24 h (diagonal white bar), or HDM followed by TSLP (diagonal black bar). (A) Western blot analysis was performed to evaluate the expression of mitophagy-related proteins. (B) Quantification of PINK1, SQSTM1, and LC3 expression levels is shown in the right panel. (C) For functional analysis, cells were pretreated with Mdivi-1 (1 μM) for 1 h prior to TSLP exposure, then stimulated with HDM as above. Wound healing assays were conducted, and wound closure was assessed 24 h post- ounding. (D) The corresponding quantification of wound healing is displayed in the right panel. Data are presented as mean ± SEM (N = 6). Wound area was quantified and expressed as GAP %. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8162140/v1/be7bde32fbe9002fb6385134.png"},{"id":97416533,"identity":"5923a42a-2ed7-4cab-9eea-1c15cf5f70c0","added_by":"auto","created_at":"2025-12-04 07:07:39","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1577339,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTSLP pretreatment followed by HDM stimulation enhanced mitochondrial transfer.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA549 cells were treated with control (‒, white bar), TSLP alone (gray bar), HDM alone (1 μg/mL, black bar), TSLP for 12 h then HDM for 12 h (diagonal white bar), or HDM followed by TSLP (diagonal black bar). After treatment, the cells were co-cultured with untreated cells at a 1:1 ratio for 3 hours. The quantitative results of mitochondrial transfer are presented in panel (B). A549 cells received a 1-hour pretreatment with Y-27632, GW4869, or AraC prior to a 24-hour IL-33 stimulation. Wounds were then created for a wound healing assay, and coverage was assessed 20 hours post-wounding (C). The right panel presents the corresponding quantitative results (D-F). Data are presented as mean ± SEM, with N = 4. Wound area is expressed as GAP %. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8162140/v1/3d7ee2dbc23bd234c4bc27cf.png"},{"id":97666145,"identity":"668581ba-429b-449d-9033-3a5f8de36ed8","added_by":"auto","created_at":"2025-12-08 09:20:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":551367,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMitochondria transferred from TSLP-treated cells altered the function of recipient cells. \u003c/strong\u003eMitoTracker-positive cells were isolated and replated overnight. Wounds were created for the wound healing assay, and wound closure was measured 20 hours after scratching (C). Quantitative data are shown in the right panel (D). Western blot analysis displays the expression of EMT-related proteins (E). Data are presented as mean ± SEM, with N = 3. Wound area is expressed as GAP %. *p \u0026lt; 0.05; **p \u0026lt; 0.01; ***p \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8162140/v1/f56f7cf36f304736e7b1eecf.png"},{"id":98628643,"identity":"b55ada3c-ff34-4c40-8429-f390ef6415b4","added_by":"auto","created_at":"2025-12-19 17:11:56","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10651324,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8162140/v1/0f64f9a4-9dba-424f-b3b5-29c1a055b669.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mitochondrial Transfer Triggered by TSLP Promotes Epithelial-Mesenchymal Transition in Human Lung Epithelial Cells Exposed to House Dust Mite","fulltext":[{"header":"Background","content":"\u003cp\u003eAsthma is a diverse condition marked by persistent airway inflammation, heightened airway responsiveness, and reversible airflow obstruction. Over time, airway remodeling becomes the underlying pathological factor contributing to the progression of fixed airflow limitation in individuals with asthma. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. During chronic inflammation, exaggerated repair responses contribute to airway remodeling, characterized by goblet cell hyperplasia, thickening of the epithelial basement membrane, neovascularization, and the development of subepithelial fibrotic lesions [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. It\u0026rsquo;s essential to recognize that airway remodeling is not solely driven by chronic inflammation. Research indicates that this process can commence early in preschool-aged children who present with wheezing symptoms. [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. At present, no asthma medications have been clinically confirmed to stop the remodeling process. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAs the first line of defense, airway epithelial cells are essential for initiating the innate immune response and contributing to the remodeling process. Growing evidence indicates that increased epithelial-mesenchymal transition (EMT) plays a significant role in airway remodeling in asthma. [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e] For example, a study using a murine model sensitized to house dust mites (HDM) demonstrated that changes in EMT are major contributors to airway remodeling. This process involves airway epithelial cells losing their usual characteristics, marked by the downregulation of E-cadherin and the upregulation of mesenchymal markers like vimentin, α-SMA, and type I pro-collagen, along with the nuclear translocation of Snail1, a key EMT inducer. [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eAn epithelium-derived cytokine, thymic stromal lymphopoietin (TSLP) represents a master switch at the interface between environmental allergens and pulmonary allergic immunologic responses [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. The airway epithelium is a key component of the innate immune system and the initiator of airway remodeling in asthma. Previous studies have shown that expressions of TSLP and TARC/CCL17 were correlated with airway obstruction in asthma [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. TSLP-induced cellular senescence was required for airway remodeling, and inhibited cellular senescence blocks airway remodeling and relieves airway resistance [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. In airway smooth muscle model, TSLP could induce proinflammatory mediators such as IL-6, IL-8 production and cell migration [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Cai et al. showed that TSLP alone could decrease the protein level of E-cadherin while increased mesenchymal proteins such as vimentin, collagen I and fibronectin levels in a concentration-dependent manner and promoted TGF-β1-induced EMT in human bronchial epithelial cells [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In addition, TSLP-transgenic mice developed spontaneous inflammation with infiltration of goblet cell hyperplasia, leukocytes, and subepithelial fibrosis in the lung [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. In the chronic HDM-induced asthma model, neutralization of TSLP with an anti-TSLP mAb reversed airway inflammation, prevented structural alterations, and decreased AHR to methacholine [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. It suggested that TSLP play a pivotal role in the initiation and persistence of airway inflammation and remodeling.\u003c/p\u003e\u003cp\u003eAdditionally, HDM, well-known allergens that induce asthma and contribute to airway remodeling along with alarmin production, present major concerns.[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In the contemporary environment, air pollution has been recognized as a significant factor in the exacerbation of asthma. Notably, diesel exhaust particles have been identified as potent inducers of alarmin production in epithelial cells, thereby contributing to airway inflammation and remodeling. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. This study aims to investigate the effects of sequential exposure to TSLP and HDM on epithelial-mesenchymal transition (EMT) and its associated signaling pathways. By elucidating critical exposure combinations that modulate EMT, this research seeks to advance the understanding of airway remodeling mechanisms and contribute to the development of targeted therapeutic strategies for asthma management.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eCell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe human lung carcinoma cell line A549 (ATCC) was maintained in MEM supplemented with 10% fetal bovine serum, 1% sodium pyruvate, 1% non-essential amino acids, and 1% antibiotic-antimycotic (Thermo Fisher Scientific Inc., Waltham, USA). After centrifugation, the cells were resuspended in fresh medium and seeded into 6-well plates at a density of 3 x 10\u003csup\u003e5\u003c/sup\u003e cells per well, followed by incubation for 24 h prior to experimentation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWound healing assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCells were seeded into 6-well plates and treated with IL-25 (1 or 10 ng/mL) or HDM (1 \u0026mu;g/mL) for 24 h, followed by a reversed stimulation in which cells were exposed to the alternate factor (HDM or IL-25) for an additional 24 h. To assess cell signaling, cells were pretreated for 1 h with either the antioxidant N-acetylcysteine (NAC), the mitochondrial ROS scavenger MitoTEMPO, the mitophagy inhibitor Mdivi-1, or the autophagy inhibitor chloroquine (CQ) (Merck Millipore) before alarmin stimulation. A wound was generated using 1000 \u0026mu;L pipette tips, followed by two washes with PBS to remove detached cells and debris, after which cells were maintained in complete medium for 24 h. Cell migration was examined under a microscope, and the wound closure was quantified as a percentage of the initial wound area using ImageJ software, calculated as [(wound area at indicated time point)/(wound area at 0 h)] \u0026times; 100% and expressed as GAP %.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMeasurement of \u0026Delta;\u0026psi;M and transmission electron microscopy\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial membrane potential was evaluated using JC-1 dye (Invitrogen, Waltham, USA) in combination with LSR II flow cytometry (Becton Dickinson, San Jose, CA) and confocal microscopy (LSM 700, Carl Zeiss Microscopy, G\u0026ouml;ttingen, Germany). JC-1 accumulates in mitochondria in a potential-dependent manner, exhibiting a fluorescence emission shift from green (525 nm) to red (590 nm). A reduction in the red/green fluorescence intensity ratio indicates mitochondrial depolarization. For ultrastructural analysis, harvested cells were examined using transmission electron microscopy (JEM-2000EXII, JEOL Ltd., Tokyo, Japan) to assess mitochondrial morphology and mitophagy.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eQuantitative real-time Reverse transcription polymerase chain reaction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo examine mitochondrial involvement, gene expression of mitochondrial copy number and respiratory chain complexes I\u0026ndash;V was analyzed, including NADH dehydrogenase subunit 1 (MT-ND1, complex I), succinate dehydrogenase complex subunit A flavoprotein (SDHA, complex II), mitochondrial cytochrome b (MT-CYTB, complex III), cytochrome c oxidase I (COXI, complex IV), ATP synthase H+ transporting mitochondrial Fo complex subunit F6 (MT-ATP6, complex V), as well as the mitochondrial proton carrier uncoupling protein 2 (UCP2). Following TSLP treatment for various time points, total RNA (2 \u0026mu;g) was isolated and reverse transcribed using SuperScript II with an anchored oligo-dT primer according to the manufacturer\u0026rsquo;s protocol (Invitrogen, Carlsbad, CA, USA). The synthesized cDNA was subsequently subjected to quantitative reverse transcription PCR (qRT\u0026ndash;PCR). Primer sequences and qRT\u0026ndash;PCR procedures were carried out as previously described in our study. [16].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePreparation of cDNA (mtDNA) and relative quantification of mtDNA fragments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTotal mitochondrial DNA (mtDNA) was extracted using a mitochondrial DNA isolation kit (BioVision, Milpitas, USA) according to the manufacturer\u0026rsquo;s protocol. Two primer sets targeting mitochondrial ribosomal 16S rRNA, mtDNA-79 and mtDNA-230, were applied as previously reported [16]. The 79-bp fragment (mtDNA-79) represents mitophagy-related mtDNA generated through enzymatic cleavage during apoptosis, whereas the 230-bp fragment (mtDNA-230) reflects mtDNA released by non-apoptotic cell death processes (e.g., necrosis) or by active secretion [17]. Quantitative real-time PCR was used to measure these mtDNA fragments, and results were normalized to the expression of the housekeeping gene ACTB.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eWestern blotting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt harvest, cells were lysed in equal volumes of ice-cold lysis buffer (Abcam, Cambridge, MA) supplemented with protease inhibitors (Merck Millipore). Equal protein amounts from the lysates were resolved by SDS-PAGE and transferred onto PVDF membranes. Membranes were blocked in TBS containing 5% non-fat dry milk and 0.1% Tween-20, followed by incubation with primary antibodies against N-cadherin, E-cadherin (Genetex, Hsinchu, Taiwan), LC3, SQSTM1 (Abcam), PINK, SMAD2, or GAPDH (Cell Signaling Technology, Danvers, MA). Immunoreactive bands were detected using horseradish peroxidase-conjugated secondary antibodies and an enhanced chemiluminescence (ECL) system (Merck Millipore), and images were acquired with a ChemiDoc-It 810 Imager (Ultra-Violet Products, Upland, CA, USA). All procedures were performed according to the manufacturers\u0026rsquo; instructions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMitochondrial transfer assay\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMitochondrial transfer was evaluated following a previously described protocol [17]. Donor A549 cells were labeled with 200 nM MitoTracker Deep Red FM (#M22426; Thermo Fisher Scientific) and CellTrace Violet (#C34557; Thermo Fisher Scientific) according to the manufacturer\u0026rsquo;s instructions. These labeled donor cells were then co-cultured with unlabeled recipient A549 cells, and mitochondrial transfer was assessed after 3 h by detecting MitoTracker signal in recipient cells lacking CellTrace labeling. All assays were conducted using control A549 cells. To define cell boundaries (regions of interest, ROI), cells were counterstained with phalloidin 488. Mitochondrial transfer was quantified as sum intensity per ROI (SI/ROI). For each condition, six wells were analyzed, with four images captured per well. In each image, 25 CellTrace-negative cells were measured for MitoTracker intensity and ROI, yielding a total of 600 cells per condition (6 wells \u0026times; 4 images per well \u0026times; 25 cells per image).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData are expressed as mean \u0026plusmn; standard deviation (SD). Differences between experimental and control groups in each independent experiment were analyzed using the Mann-Whitney U test. Densitometric values from western blot assays were quantified with ImageJ software (National Institutes of Health, Bethesda, MD, USA) by measuring the optical density of individual bands. Statistical analyses were conducted using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, CA, USA). A p-value \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003ePre-treatment with TSLP prior to house dust mite exposure enhanced cell migration and altered the expression of EMT markers.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the effect of the stimulation order of TSLP and HDM on cell migration, cells were first treated with either TSLP (10 ng/mL) or HDM (1 \u0026micro;g/mL) for 24 hours. This was followed by a reversed stimulation, in which cells were exposed to the alternate factor (HDM or TSLP) for an additional 24 hours. Cell migration was assessed using wound healing assays (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and B). The results showed that TSLP at both 1 ng/mL and 10 ng/mL significantly enhanced cell migration. Moreover, treatment with TSLP followed by HDM further increased cell migration compared to TSLP alone. The group treated with TSLP at 10 ng/mL followed by HDM also displayed a pronounced increase in migration compared to HDM alone. While HDM alone significantly promoted migration, no additional effect was observed when HDM was applied first, followed by TSLP, compared to HDM alone. The influence of stimulation order on EMT-related protein expression was also examined. A549 cells were initially treated with either TSLP (1 or 10 ng/mL) or HDM (1 \u0026micro;g/mL) for 24 hours, followed by reversed stimulation with the alternate factor for another 24 hours. Protein expression was analyzed by Western blot. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC, TSLP significantly increased the mesenchymal marker N-Cadherin and the transcription factor SLUG while reducing the epithelial marker E-Cadherin. These effects were further enhanced when cells were treated with TSLP followed by HDM. Although HDM alone also significantly modulated EMT protein expression, applying HDM first followed by TSLP did not produce additional changes compared to HDM treatment alone.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTSLP promoted EMT changes through oxidative stress.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOxidative stress, the physiological damage caused by reactive oxygen species (ROS), has been shown to affect smooth muscle contraction, induce airway hyperresponsiveness, and increase mucus secretion and epithelial shedding in respiratory cells. Excessive ROS production in asthma can lead to oxidative stress, contributing to pulmonary fibrosis associated with TGF-β [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. To investigate the effect of the stimulation order of TSLP and HDM on ROS production, cells were first treated with either TSLP (10 ng/mL) or HDM (1 \u0026micro;g/mL) for 3 hours, followed by reversed stimulation with the alternate factor (HDM or TSLP) for an additional 3 hours. ROS generation was assessed using DHE and DCFH-DA staining. DHE is commonly used to detect intracellular superoxide (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), whereas DCFH-DA reacts with H2O2, ONOO\u0026minus;, lipid hydroperoxides, and O2\u0026minus;, serving as a general indicator of total ROS (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). The results demonstrated that stimulation with TSLP (10 ng/mL) or HDM significantly increased both superoxide and total intracellular ROS. Moreover, treatment with TSLP followed by HDM further enhanced ROS production compared to TSLP alone. We further examined the effects of ROS scavengers, N-acetylcysteine (NAC) and MitoTEMPO, on EMT-related proteins and cell migration. TSLP-induced decreases in E-cadherin and increases in N-cadherin and SMAD2 were reversed by ROS scavengers (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Additionally, TSLP-induced cell migration was significantly suppressed by both MitoTEMPO and NAC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003ePre-treatment with TSLP before house dust mite exposure induced mitochondrial dysfunction.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eMitochondrial ROS production is a key indicator of mitochondrial dysfunction and cellular stress. To assess whether TSLP treatment followed by HDM exposure affects mitochondrial function and induces mitophagy, we performed mitochondrial membrane potential (MMP) analysis. A decrease in the red/green fluorescence intensity ratio indicated that TSLP promoted mitochondrial depolarization, which was further enhanced by subsequent HDM exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). We also evaluated mitochondrial DNA (mtDNA) release following TSLP treatment or TSLP treatment followed by HDM. Quantitative real-time PCR revealed that TSLP increased cytosolic mtDNA levels in A549 cells, and TSLP followed by HDM caused a further significant increase compared to TSLP alone (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Mitochondrial biogenesis was assessed by measuring mitochondrial copy number and the expression of respiratory chain genes. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC, stimulation with TSLP or HDM for 6 hours did not significantly alter mitochondrial copy numbers. The expression of ND1 (complex I), SDHA (complex II), CYTB (complex III), COX1 (complex IV), ATP6 (complex V), and uncoupling protein 2 (UCP2) was measured by qRT-PCR. TSLP followed by HDM significantly upregulated these genes compared to TSLP alone. Western blot analysis further showed that TSLP increased the levels of SQSTM1 and LC3, with this effect being enhanced by HDM treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). To examine the role of mitophagy in TSLP- and HDM-induced cell migration, cells were pretreated with the mitophagy inhibitor Mdivi-1. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, TSLP-induced cell migration was suppressed by Mdivi-1, and similar suppression was observed for TSLP followed by HDM-induced migration. Collectively, these results indicate that TSLP followed by HDM alters mitochondrial function and promotes mitophagy in A549 cells.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTSLP promoted mitochondrial transfer, leading to increased cell migration and modulation of EMT marker expression.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eOver the past few decades, studies have demonstrated that cells can transfer mitochondria to neighboring cells through intercellular mitochondrial transfer, a process observed in various tissues and involved in both normal physiology and disease development. While many studies emphasize that mitochondrial transfer supports the metabolism of recipient cells, recent evidence indicates it also helps maintain mitochondrial health in donor cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We next investigated how stimulation with TSLP alone or TSLP followed by HDM influences mitochondrial transfer. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, mitochondria were observed moving to adjacent cells via tunneling nanotubes (TNT). Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB shows that stimulation with either TSLP or HDM alone significantly increased mitochondrial transfer to recipient cells. Notably, TSLP-induced mitochondrial transfer was further enhanced when followed by HDM, whereas no such increase was observed when HDM preceded TSLP. Mitochondrial transfer occurs through three primary mechanisms: (1) formation of temporary intercellular structures such as TNTs, (2) export of mitochondria within extracellular vesicles, and (3) release of mitochondria as free organelles [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. To determine whether blocking TSLP-induced mitochondrial transfer affects cell migration, we used the TNT inhibitor AraC, the extracellular vesicle inhibitor Y-27632, and the exosome inhibitor GW4869. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC\u0026ndash;F, all three inhibitors reduced TSLP-induced mitochondrial transfer, suggesting that TSLP-mediated transfer occurs via TNT formation and vesicle-mediated transport. We further evaluated the functional impact on recipient cells. Untreated cells were co-cultured with either untreated or TSLP-treated donor cells for 24 hours, followed by staining with MitoTracker and CellTrace. Only MitoTracker-positive recipient cells were isolated and replated overnight. Wound healing assays and analysis of EMT-related markers revealed that recipient cells acquiring mitochondria from TSLP-treated donors exhibited increased migration compared to controls. These cells also showed reduced E-cadherin expression and elevated N-cadherin and SMAD2 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA\u0026ndash;C). Together, these results indicate that TSLP priming induces mitochondrial dysfunction and transfer, which alters the behavior of neighboring cells, promotes migration, and contributes to tissue remodeling.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eTSLP is a cytokine mainly produced by epithelial cells, keratinocytes, and stromal cells. Structurally similar to IL-7 and part of the IL-2 family, it functions as an alarmin that is rapidly upregulated in response to stress or injury to barrier tissues such as the skin, lungs, and gut, especially following exposure to environmental triggers like allergens, pollutants, or pathogens [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Initially linked to allergic diseases such as atopic dermatitis, asthma, and eosinophilic esophagitis, TSLP has also been implicated in chronic inflammatory conditions (e.g., COPD, celiac disease), autoimmune disorders (e.g., psoriasis, rheumatoid arthritis), and various cancers [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In asthma, TSLP activates dendritic cells, which in turn prime na\u0026iuml;ve T cells to differentiate into Th2 cells. These Th2 cells secrete cytokines such as IL-4, IL-5, and IL-13, leading to eosinophilic inflammation, airway hyper responsiveness, and mucus overproduction, thereby contributing to sustained allergic inflammation [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. A study found that elevated baseline pulmonary TSLP levels are associated with greater asthma severity in a subset of children. These findings suggest that TSLP plays a crucial role in the development of pediatric asthma [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Recent studies have shown that TSLP stimulation of human nasal epithelial cells reduced E-cadherin expression, increased FSP1, vimentin, and collagen I/III levels, and induced a fibroblast-like morphology [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. TSLP also drives asthmatic airway remodeling via p38 MAPK-STAT3 activation, with epithelial-fibroblast interactions further exacerbating the remodeling process [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This indicates that the alarmin TSLP plays a role not only in inflammatory responses but also in promoting airway remodeling.\u003c/p\u003e\u003cp\u003eAirway epithelial cells serve as the first line of defense against environmental stimuli by rapidly secreting alarmin cytokines. CM Weng et al. demonstrated that exposure of primary epithelial cells to diesel exhaust particles significantly elevated IL-25, IL-33, and TSLP levels via activation of the aryl hydrocarbon receptor.[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Previous studies have shown that stimulation of human bronchial epithelial cells with either Der p1 or PM2.5 alone increases the production of IL-25, IL-33, and TSLP, with a more pronounced effect observed when both stimuli are applied together [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Both pollutants and allergens like house dust mites (HDM) can stimulate TSLP production, promote airway remodeling, and worsen asthma symptoms. TSLP signals via an IL-17RA/TSLPR heterodimer, recruiting JAK1 and JAK2 in a cell type\u0026ndash;dependent manner. These kinases can activate STATs, NF-κB, and MAPK through PI3K. The effects vary by cell type, including upregulation of IL-6 and IL-8, increased cell proliferation, and, in dendritic cells, migration and maturation toward a Th2 phenotype [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have demonstrated that HDM allergens, such as Der p 5, bind to TLR2 on airway epithelial cells, activating NF-κB through IκBα degradation. This triggers nuclear translocation of RelA (canonical pathway) and RelB (non-canonical pathway), promoting the production of proinflammatory cytokines, including IL-8 and IL-33[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Additionally, Der p 1 directly activates ERK1/2 and p38 MAPK in airway smooth muscle (ASM) cells via an IgE-independent mechanism, where ERK1/2 contributes to ASM hyperresponsiveness and p38 MAPK regulates ERK1/2 activity [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. These findings suggest that TSLP and HDM share overlapping signaling pathways. This study aimed to determine whether TSLP exposure prior to allergen stimulation is more effective in promoting airway remodeling and asthma exacerbation compared to the reverse sequence. Our results showed that TSLP pretreatment followed by HDM exposure enhanced cell migration and induced more pronounced changes in EMT-related markers than the opposite order. These observations indicate that TSLP may prime the airway epithelium for EMT, thereby amplifying the remodeling response to subsequent allergen exposure.\u003c/p\u003e\u003cp\u003eMitochondrial dysfunction contributes to the development of chronic respiratory diseases, including chronic obstructive pulmonary disease (COPD), and is characterized by increased mitochondrial ROS, reduced mitochondrial membrane potential, elevated calcium influx, mitochondrial damage and DNA release, as well as impaired mitophagy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Our previous study demonstrated that TSLP modulates mitochondrial ROS-induced mitophagy through histone modifications in human monocytes [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. A previous study found that cigarette smoke induced small airway remodeling by enhancing EMT and PINK1-Parkin\u0026ndash;mediated mitophagy [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In this study, intracellular ROS and superoxide levels were assessed using DCFH-DA and DHE. We found that TSLP treatment increased the production of intracellular ROS and superoxide, and this effect was further enhanced when TSLP was applied prior to HDM stimulation in A549 cells. It has also been reported that within mitochondria, NAC is converted into hydrogen sulfide and various sulfane sulfur compounds, which serve as more effective oxidant scavengers [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. MitoTEMPO is a newly developed antioxidant specifically targeting mitochondria [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. In our study, pretreatment of A549 cells with MitoTEMPO or NAC before TSLP stimulation suppressed cell migration, decreased the expression of mitophagy markers, N-cadherin, and SMAD2, while increasing E-cadherin expression. In our previous work, we also observed that TSLP stimulation induces hyperactivity of mitochondrial complexes I and II/III, accompanied by a reduction in mitochondrial membrane potential [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. In this study, we further demonstrated that the effect was amplified when TSLP was administered prior to HDM stimulation in A549 cells. Notably, while UCP-2 decreases ROS production by uncoupling mitochondrial respiration, elevated ROS levels can in turn induce cellular adaptations that upregulate UCP expression [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Mdivi-1, a cell-permeable quinazolone compound, effectively inhibits mitophagy. Pretreating cells with Mdivi-1 prior to TSLP stimulation or sequential TSLP and HDM exposure reduced cell migration. These findings suggest that suppressing ROS and mitophagy can mitigate TSLP-induced EMT, indicating that TSLP may promote EMT progression through ROS-mediated mitophagy activation.\u003c/p\u003e\u003cp\u003eMitochondrial transfer, the process by which mitochondria are exchanged between cells, has recently gained significant attention for its potential therapeutic role in mitochondrial diseases, age-related conditions, and various other health disorders [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Previous studies have shown that mitochondrial dysfunction plays a critical role in driving airway remodeling by enhancing oxidative stress, epithelial damage, and EMT-related changes [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. This study is the first to demonstrate that TSLP can induce mitochondrial transfer and mediate intercellular transport of mitochondria among human lung epithelial cells. Similarly, TSLP pretreatment combined with HDM exposure resulted in increased mitochondrial transfer and more pronounced changes. Mitochondrial transfer can occur through multiple mechanisms, including exosome-mediated pathways and TNTs [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Notably, TSLP-induced mitochondrial transfer may involve both exosome- and TNT-dependent pathways. To investigate the role and mechanism of TSLP-induced mitochondrial transfer in cell migration, we used the vesicle inhibitor Y-27632, the exosome inhibitor GW4869, and the TNT inhibitor AraC. Results showed that all three inhibitors suppressed TSLP-induced cell migration, indicating that mitochondrial transfer triggered by TSLP contributes to this process. We further assessed the functions of untreated cells that received mitochondria from TSLP-treated cells. These recipient cells displayed enhanced migratory capacity and altered EMT-related marker expression. Previous studies have shown that platelets recruited to injury sites transport mitochondria to the plasma membrane and release them as extracellular vesicles (EVMs) and free mitochondria during degranulation [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. These mitochondria are subsequently taken up by subendothelial mesenchymal stem cells, which in turn secrete pro-angiogenic factors to promote wound healing within the vascular microenvironment [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Additionally, EVMs can mediate intercellular communication by transferring proteins, nucleic acids, microRNAs, lipids, and metabolites to nearby or distant target cells [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. This suggests that exosome-mediated transfer induced by TSLP may also deliver additional mediators that influence recipient cell functions. Further research is required to confirm this possibility. Mitochondrial transfer has been applied in various animal models and holds promise as a therapeutic strategy for multiple human diseases, including neurodegenerative disorders, central nervous system injuries, and cardiovascular diseases[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In this study, we demonstrated that TSLP primes epithelial cells by regulating mitochondrial dynamics and promoting mitochondrial transfer, thereby enhancing their responsiveness to environmental stimuli. Thus, targeting TSLP signaling and mitochondrial transfer pathways may provide new strategies to prevent airway remodeling.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study revealed that TSLP pretreatment sensitizes airway epithelial cells to subsequent allergen exposure, leading to enhanced cell migration and more pronounced EMT alterations. TSLP stimulation triggered oxidative stress, mitochondrial dysfunction, and mitochondrial transfer, all of which were further intensified by subsequent HDM exposure. Furthermore, TSLP primes epithelial cells through the regulation of mitochondrial dynamics and the promotion of intercellular mitochondrial transfer, thereby heightening their reactivity to environmental stimuli. Collectively, these results indicate that targeting TSLP signaling and mitochondrial transfer mechanisms could represent a potential therapeutic approach to mitigate airway remodeling in asthma and other chronic respiratory diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003ch2\u003eCompeting Interests\u003c/h2\u003e\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe study was supported by grants from the Ministry of Science and Technology of the Republic of China (grant numbers MOST 113-2314-B-037-094-MY3), and the Research Center for Precision Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan and by Kaohsiung Medical University Research Center Grant (grant numbers KMU-TC114A01)\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThe study was supported by grants from the National Science and Technology Council of the Republic of China (grant numbers MOST 113-2314-B-037-094-MY3), and the Research Center for Precision Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan from The Featured Areas Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education (MOE) in Taiwan and by Kaohsiung Medical University Research Center Grant (grant numbers KMU-TC114A01)\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization: Chang-Yu Yeh, Wei-Ting Liao, and Chih-Hsing Hung; Methodology: Chang-Yu Yeh and Chih-Hsing Hung; Formal analysis: Mei-Lan Tsai and Hsin-Ying Clair Chiou; Investigation: Wei-Ting Liao; Data curation: Chang-Yu Yeh and Mei-Lan Tsai; Writing-original draft preparation: Chang-Yu Yeh; Writing-reviewing and editing: Wei-Ting Liao and Chih-Hsing Hung. All authors reviewed the manuscript. Wei-Ting Liao and Chih-Hsing Hung contributed equally.\u003c/p\u003e\u003ch2\u003eAcknowledgement\u003c/h2\u003e\u003cp\u003eThe authors gratefully acknowledge the Center for Research Resources and Development at Kaohsiung Medical University for their support with flow cytometry using the FACSMelody and LSRII systems, as well as assistance with confocal image analysis using the LSM 700.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eHuang Y, Qiu C: \u003cstrong\u003eResearch advances in airway remodeling in asthma: a narrative review.\u003c/strong\u003e \u003cem\u003eAnn Transl Med \u003c/em\u003e2022, \u003cstrong\u003e10:\u003c/strong\u003e1023.\u003c/li\u003e\n\u003cli\u003eSingla A, Reuter S, Taube C, Peters M, Peters K: \u003cstrong\u003eThe molecular mechanisms of remodeling in asthma, COPD and IPF with a special emphasis on the complex role of Wnt5A.\u003c/strong\u003e \u003cem\u003eInflammation Research \u003c/em\u003e2023, 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\u003cstrong\u003e19:\u003c/strong\u003e104.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"airway remodeling, TSLP, epithelial-mesenchymal transition, mitochondrial transfer, mitophagy","lastPublishedDoi":"10.21203/rs.3.rs-8162140/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8162140/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eAirway remodeling is a key pathological feature of asthma, closely linked to lung function decline and disease severity. Persistent inflammation, EMT, and mitochondrial dysfunction all contribute to this process. Although HDM strongly induces epithelial alarmins such as TSLP, how cytokine priming cooperates with subsequent allergen exposure to alter mitochondrial function and promote EMT remains unclear.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eThis study investigated the effects of sequential TSLP and HDM exposure in A549 lung epithelial cells. Cell motility and EMT markers were assessed using wound-healing assays and Western Blot. Mitochondrial membrane potential and oxidative stress were examined by JC-1 staining and ROS quantification. To determine how TSLP priming shapes subsequent HDM responses, we analyzed intercellular mitochondrial transfer, and further assessed EMT phenotypes in recipient cells acquiring mitochondria from TSLP-treated donors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eSequential TSLP priming followed by HDM exposure markedly enhanced epithelial migration and accelerated EMT. This was accompanied by a collapse of mitochondrial membrane potential, impaired biogenesis, increased mtDNA release, and robust mitophagy activation. Notably, TSLP priming potentiated intercellular mitochondrial transfer, and recipient cells that acquired TSLP-conditioned mitochondria displayed amplified migratory and EMT behavior. These findings identify mitochondrial transfer as an additional layer by which epithelial cytokine priming amplifies allergen-driven remodeling.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eCollectively, these results indicate that TSLP primes epithelial cells by reprogramming mitochondrial dynamics and promoting intercellular mitochondrial transfer, thereby heightening their sensitivity to environmental stimuli. Targeting TSLP signaling and mitochondrial regulation may provide novel therapeutic opportunities to mitigate airway remodeling in chronic respiratory diseases.\u003c/p\u003e","manuscriptTitle":"Mitochondrial Transfer Triggered by TSLP Promotes Epithelial-Mesenchymal Transition in Human Lung Epithelial Cells Exposed to House Dust Mite","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-04 07:07:34","doi":"10.21203/rs.3.rs-8162140/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"20a03381-e83b-4a0e-a35c-b8dfdc3ccbe3","owner":[],"postedDate":"December 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-12-19T13:23:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-04 07:07:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8162140","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8162140","identity":"rs-8162140","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}