LRRC8A drives NADPH oxidase-mediated mitochondrial dysfunction and inflammation in allergic rhinitis

Abstract

Objectives

Allergic rhinitis (AR) is a complex disorder with variable pathogenesis. Increasing evidence suggests that the LRRC8A is involved in maintaining cellular stability, regulating immune cell activation and function, and playing significant roles in inflammation. However, the involvement of LRRC8A in AR inflammation and its underlying mechanisms remain unclear.

Methods

LRRC8A expression in AR patients, confirmed by qRT-PCR and Western blotting, was analyzed to investigate its relationship with the clinical characteristics of AR patients. In vitro, IL-13 stimulated HNEpCs to establish a Th2 inflammation model, with subsequent LRRC8A knockout or overexpression. NOX1/NOX4 inhibitor (GKT137831) and chloride channel inhibitor (DCPIB) were utilized to investigate AR development mechanisms during LRRC8A overexpression. An OVA-induced AR model with nasal mucosa LRRC8A knockdown confirmed LRRC8A's regulatory role in AR inflammation.

Results

LRRC8A mRNA and protein levels were significantly elevated in AR patients, positively correlating with NADPH oxidase subunits and Th2 inflammatory markers. In vitro, IL-13 stimulation of HNEpCs resulted in upregulation of LRRC8A and increased expression of NOX1, NOX4, and p22^phox, along with mitochondrial dysfunction and NF-κB pathway activation. The knockout of LRRC8A reversed these effects. In nasal mucosal epithelial cells, DCPIB and GKT137831 completely blocked mitochondrial dysfunction caused by the overexpression of LRRC8A, which led to up-regulation of NOX1, NOX4, and p22^phox. In vivo, knocking down LRRC8A reduced eosinophil infiltration, downregulated the expression of NOX1, NOX4, p22^phox IL-4, IL-5, and IL-13, and decreased NF-κB pathway activation.

Conclusion

LRRC8A drives the upregulation of NOX1, NOX4, and p22^phox, leading to ROS overproduction and mitochondrial dysfunction. It also activates NF-κB, ultimately leading to nasal mucosal epithelial inflammation. LRRC8A may be a potential target for the treatment of AR.

Introduction

Allergic rhinitis (AR) is caused by an IgE-mediated response to specific inhalant allergens. Although it is not a life-threatening disease, it has a serious impact on quality of life, affecting about 10–40% of the global population [1]. There are currently many therapeutic agents for AR, including glucocorticoids, antihistamines, and anticholinergics. However, for some patients, standard treatments fail to achieve adequate symptom control [2].Therefore, it is necessary to discover new safe and effective targets for the treatment of AR.

Oxidative stress is the result of overproduction of ROS and damage to the antioxidant system, promotes the inflammatory response in AR [3]. In addition, some researchers have suggested that the degree of oxidative stress is related to the severity of AR [4]. Mitochondria and NADPH oxidase are the main producers of ROS in inflammatory diseases. They were initially found in phagocytes and later found to be present in different epithelial and inflammatory cells as well [5]. Studies have shown that subunits NOX1 and NOX4 of NADPH oxidase are expressed in the nasal mucosal epithelium and are differentially expressed between AR patients and non-AR patients [6]. This suggests that NOX1 and NOX4 may be associated with the pathogenesis of AR. Moreover, the active subunit p22^phox has been demonstrated to mediate airway hyperresponsiveness in models of asthma [7]. Multiple lines of evidence suggest that NOX4 may be localized to mitochondria due to the presence of mitochondrial targeting signals [8]. This suggests that high expression of NOX4 may lead to mitochondrial dysfunction. Despite advancements in our understanding of the role of ROS in cellular function, the effects of NADPH oxidases in patients with AR remain poorly understood.

The volume-regulated anion channel (VRAC) was first identified in human lymphocytes and epithelial cells [9]. VRAC mainly mediates chloride ion efflux. The Cl- channel is also usually represented by VRAC [10]. Inhibition of VRAC also has anti-inflammatory effects in vivo, and DCPIB, an inhibitor of VRAC, reduces microglia inflammation and ROS production in cardiomyocytes [11, 12]. LRRC8A was identified as a key protein component of VRAC. The LRRC8 family consists of five members—LRRC8A/B/C/D/E—with LRRC8A being the only essential subunit, and at least one other subunit is required to form VRAC [13, 14]. LRRC8A has been extensively studied and is implicated in the activation of NLRP3 inflammasome, T cells and increased oxidative stress [15,16,17]. Recently, a correlation has been identified between LRRC8A and NADPH oxidase. Studies have demonstrated co-localization of LRRC8A with Nox1 and p22^phox subunits in vascular smooth muscle cells [18]. Knockdown of LRRC8A reduces ROS production by decreasing NOX2 and NOX4 expression and inhibiting p47^phox and p67^phox translocations [19]. LRRC8A channels support TNFα-induced superoxide production by Nox1, a process required for TNF receptor endocytosis [20]. These findings suggest that LRRC8A might regulate the expression of NADPH oxidase. However, it remains unclear whether LRRC8A influences disease progression by regulating NADPH oxidase/ROS production in AR.

In this study, we investigated the role and mechanism of LRRC8A in AR. We observed increased LRRC8A expression in AR and IL-13-stimulated cells. Knockdown of LRRC8A reduced the production of key disease markers associated with AR by inhibiting NADPH oxidase expression and antioxidant enzyme (Mn-SOD). Our findings suggest that LRRC8A plays a role in the development of AR patients and may represent a potential therapeutic target.

Materials and methods

In vitro experiments

Cell culture and stimulation

Human Nasal Mucosal Epithelial Cells (HNEpCs) were purchased from BeiNa Culture Collection in China (Catalog No: BNCC356247). The cells were cultured in Eagle's Minimum Essential Medium (EMEM) supplemented with 10% fetal bovine serum, 100 μg/mL streptomycin, and 100 U/mL penicillin. All cell experiments were conducted using third-generation cells. All in vitro experiments were performed at least three times.

Silencing LRRC8A: HNEpCs were seeded at a density of 3.5 × 10⁵ cells/mL in 6-well plates. After 24 h of culture, the cells in the 6-well plates were randomly divided into three groups: NC group, where cells were transfected with NCsiRNA for 36 h followed by medium replacement according to the Lipofectamine™ 3000 transfection reagent protocol(#L3000015, Invitrogen); IL-13 + NC group, where cells were transfected with NCsiRNA for 36 h, followed by stimulation with 50 ng/mL IL-13 for 12 h; IL-13 + siLRRC8A group, where cells were transfected with siLRRC8A for 36 h, followed by stimulation with 50 ng/mL IL-13 for 12 h. The siRNAs used in this study were obtained from genepharma, China Sequences are as follows: siLRRC8A: forward: 5′-GCCUUAAGCUGUGGUACAATT-3′, reverse: 5′-UUGUACCACAGCUUAAGGCTT-3′.

LRRC8A overexpression and inhibition: HNEpCs were plated at a density of 4.0 × 10⁵ cells/mL in 6-well plates. After 24 h, the cells were randomly divided into four groups: pcDNA-NC group, in which cells were transfected with pcDNA3.1-NC vector for 24 h and then replaced with medium for another 24 h; pcDNA-LRRC8A group, where cells were transfected with the overexpression plasmid pcDNA3.1 (+)-LRRC8A and similarly cultured; pcDNA-LRRC8A + GKT137831 group and pcDNA-LRRC8A + DCPIB group, where cells were pre-treated with specific NOX1/NOX4 inhibitor GKT137831 (10 μmol/L, #S7171, Selleck) and specific VRAC inhibitor DCPIB (20 μmol/L, #1540,Tocris) respectively for 1 h before transfection. HNEpCs were transfected with the pcDNA3.1-LRRC8A for 24 h, followed by 24 h with fresh medium. Cells were harvested for subsequent experiments. Overexpression plasmids were constructed by GenePharma, China.

ROS assay

ROS levels in HNEpCs were measured using the oxidative stress-sensitive fluorescent probe DCFH-DA (10 μM, #S0033S, Beyotime). To assess mitochondrial ROS, cells were treated with MitoSOX Red reagent (5 μM, #M36008, Invitrogen). Briefly, cell slides were removed and washed twice with PBS. Cells were then incubated in the dark at 37 °C with DCFH-DA and MitoSOX Red dyes for 30 min, followed by three washes with PBS, and immediate observation under a fluorescence microscope.

Mitochondrial membrane potential and intramitochondrial NOX4

To detect changes in the mitochondrial membrane potential of stimulated cells, cell slides were incubated in the dark at 37 °C with TMRM (1:1000, #I34361, Invitrogen) for 30 min. To assess NOX4 expression within mitochondria, initially, cell slides were incubated with MitoTracker Red (500 nM, #M7512, Invitrogen) in the dark at 37 °C for 30 min. Cells were then fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and washed three times with PBS. After blocking with 10% goat serum at room temperature for 1 h, anti-NOX4 antibody (1:50, #14347-1-AP, Proteintech) was added, and the slides were incubated overnight at 4 °C. Slides were then brought to room temperature, incubated with a fluorescent secondary antibody (1:500, #35552, Invitrogen) in the dark at room temperature for 1 h, washed with PBS, stained with DAPI for 10 min. Finally, co-localization was observed using a confocal microscope.

Measurement of mitochondrial copy number (mtDNA)

After treatment, total DNA was extracted from HNEpCs using a genomic DNA extraction kit according to the manufacturer's instructions (#DP304-02, Tiangen Biotechnology). DNA concentration was determined using a microvolume spectrophotometer, and sample DNA concentrations were adjusted to ensure uniformity. Following this, samples with equalized concentrations were further processed by adding Human Mitochondrial DNA (mtDNA) Monitoring Primer (#7246, Takara) and TB Green^® Premix Ex Taq™ (#RR420Q, Takara), and quantitative amplification was performed using the Mastercycler ep realplex system.

Intracellular Cl⁻ fluorescence assay

To measure the intracellular chloride content, MQAE, a chloride-sensitive fluorescent probe, was used (5 Mm, #HY-D0090, Medchemexpress). Following the manufacturer's protocol, stimulated HNEpCs on slides were washed three times with Krebs-HEPES buffer, then incubated with diluted MQAE working solution in the dark at 37 °C for 1 h. The fluorescence intensity of MQAE was observed using a fluorescence microscope.

Human and in vivo experiments

Human sample collection

This study was approved by the Ethics Review Committee of Shandong Provincial ENT Hospital. Informed consent was obtained from all participants. We collected nasal mucosal tissues from 48 patients undergoing nasal septum or turbinate surgery. The diagnostic criteria for AR included two or more of the following symptoms: frequent sneezing, nasal itching, watery rhinorrhea, and nasal congestion, with symptoms lasting or accumulating for more than one hour; nasal endoscopy showing pale and edematous nasal mucosa; and positive specific IgE test or positive skin prick test [21]. Exclusion criteria included ongoing immunotherapy and other systemic allergic diseases. Thirty patients meeting the criteria were included in the AR group, with the remaining 18 non-AR patients serving as the control group.

Establishment and treatment of AR mouse model

All animal experiments were conducted in accordance with the regulations for the management of experimental animals and were approved by the Animal Ethics Committee of Shandong Provincial ENT Hospital. Fifty-four female BALB/c mice were purchased from Apex Experimental Animal Breeding, China. Before modeling, adaptive breeding was conducted for one week under the following environmental conditions: isolated cage breeding, room temperature at 22.5 ± 2.5 °C, relative humidity 50 ± 10%, ventilation 10–15 times/h, airflow speed < 0.18 m/s, pressure difference 2–5 mmH₂O, cleanliness level of 100,000, colony count < 12.2 per plate, ammonia concentration < 14 mg/L, noise level < 60 dB, and illumination 150–300 lx. AAV5-siLRRC8A used in this study was sourced from GenePharma, China, with the sequence: 5′-GCCTTCATGTTGCACCTCATC-3′. Mice were anesthetized with isoflurane and then administered 20 μL of either AAV5-siLRRC8A or AAV5-NC (containing NCsiRNA) intranasally, repeated after 10 min for a total of two times.

Two weeks later, an AR mouse model was induced using ovalbumin (OVA). On days 0, 7, and 14, the AR group mice received intraperitoneal injections of 25 μg of OVA mixed with 2 mg of aluminum hydroxide and PBS, totaling 250 μL. From days 21 to 30, mice were challenged daily with 20 μL of 3% OVA solution intranasally. The control group received identical steps and volumes of PBS. Mice were euthanized 24 h after the final challenge to collect protein samples, while other samples were taken 3 h after the last stimulus. The 54 mice were randomly divided into four groups: NC (stimulated with PBS only), OVA (stimulated with OVA to induce AR), OVA + AAV5-NC (OVA stimulated and treated intranasally with NCsiRNA), and OVA + AAV5-siLRRC8A (OVA stimulated and treated intranasally with siLRRC8A).

Extraction of mouse nasal mucosal cells and ROS measurement

Mice were euthanized via decapitation after being anesthetized with 10% chloral hydrate. Under aseptic conditions in a biosafety cabinet, nasal mucosal tissues were dissected under a microscope and washed with DPBS containing antibiotics. The tissues were then minced and digested in Dispase II enzyme solution (10 mg/mL, #D4693, Sigma) at 37 °C for 2 h. After digestion, the suspension was centrifuged to discard the supernatant and the pellet was further digested with 0.25% trypsin for 10 min. The digestion was terminated. The cells were resuspended in DMEM, passed through a 70 μm filter, and the cell suspension was incubated with DCFH-DA and MitoSOX Red at 37 °C in the dark for 30 min. After washing with PBS, cell slides were prepared using a Cytopin centrifugal cytospinner at 800 rpm for 5 min. The slides were then immediately examined under a fluorescence microscope.

Mn-SOD activity

Collected cells or tissues were placed in ice-cold PBS. Cells were lysed using an ultrasonic homogenizer in ice-cold PBS, while tissues were homogenized using a tissue grinder. After treatment, the samples were lysed on ice for 20 min with shaking once during the process. The supernatant was collected, and CuZn/Mn-SOD activity was measured using an activity assay kit as per the manufacturer’s instructions (#S0103, Beyotime). The optical density (OD) at 450 nm was read using a multifunctional enzyme reader to calculate the Mn-SOD activity.

Primary culture of mouse splenocytes

Following euthanasia, the spleens were dissected out in a laminar flow hood, washed with PBS containing antibiotics, and mechanically dissociated through a 70 μm mesh filter until no visible tissue fragments remained. Red blood cells were lysed for 5 min using RBC lysis buffer (#00-4333-57, Invitrogen). After centrifugation, the cell pellet was resuspended in RPMI 1640 medium supplemented with 200 ng/mL of OVA and plated at a density of 2 × 10⁷ cells/mL in 6-well plates. After 72 h, the culture medium was collected for subsequent experiments.

ELISA

The levels of OVA-specific IgE in mouse serum (#3010, Chondrex) and IL-5 (#BMS610, Invitrogen), IL-4, and IL-13 (#EMC003.96, #EMC124.96, Neobioscience) in spleen cell supernatants were measured using corresponding ELISA kits.

Preparation of RNA and quantitative real-time PCR, qRT-PCR

Total RNA was extracted from human and mouse nasal mucosal tissues using the TRIzol method. mRNA was reverse transcribed into cDNA using the PrimeScript RT Master Mix (#RR036Q, Takara). Quantitative PCR was conducted using the Mastercycler ep realplex system and TB Green^® Premix Ex Taq™ (#RR420Q, Takara). GAPDH was used as the reference gene, and reactions were performed in duplicate to minimize error. The relative gene expression levels were calculated using the 2^−ΔΔCt method. Primer sequences are listed in Supplementary Material 1.

Immunohistochemistry

Nasal mucosal tissues were fixed in 4% paraformaldehyde, embedded in paraffin, and cut into 4 μm thick sections, which were then heated to 65 °C. Sections were deparaffinized with sodium hyaluronate and a graded series of alcohols, followed by antigen retrieval buffer (#ab93678, Abcam) and cooling to room temperature. After washing with PBS, sections were incubated in 3% H₂O₂ in the dark for 15 min to block endogenous peroxidase activity. After blocking with 10% goat serum for 1 h, sections were incubated overnight at 4 °C with primary antibodies against NOX1(1:400, #PA5-103220, Invitrogen), NOX4 (1:300, #14347-1-AP, Proteintech), and LRRC8A (1:400, #PA5-72870, Invitrogen). Following warming to room temperature, sections were washed with PBS, incubated with HRP-conjugated secondary antibodies against rabbit and mouse for 1 h, and then washed again. DAB was used for color development (#GK801030, Genetech), hematoxylin for nuclear staining, followed by dehydration in reverse order and mounting with neutral resin for observation.

H&E staining

Mouse heads were fixed in 4% paraformaldehyde for 24 h and decalcified with EDTA (#E1170, Solarbio) for 10 days. After embedding in paraffin, sections were cut to a thickness of 4 μm and heated to 65 °C. The sections underwent deparaffinization with sodium hyaluronate and graded alcohols and were then rehydrated. Hematoxylin and eosin (H&E) staining was performed to assess eosinophil infiltration in the nasal mucosa, observed under an optical microscope.

Western blotting

Collected cells or tissues were lysed in RIPA buffer containing protease and phosphatase inhibitors. The lysates were collected and protein concentration was determined using a BCA assay kit. Protein samples were mixed with SDS-PAGE loading buffer and boiled in a 100 °C metal bath for 10 min. Twenty micrograms of protein per well were loaded and separated on an 8–10% SDS-PAGE gel. The proteins were then transferred to a PVDF membrane and blocked with 5% non-fat milk for 1.5 h. The membranes were incubated overnight at 4 °C with primary antibodies against NOX1 (1:1000, #17772-1-AP, proteintech), NOX4 (1:4000), p22^phox (1:1000, #sc-271968, Santa Cruz Biotechnology), LRRC8A (1:2000, #ab254389, Abcam), NF-κB p65, and phospho-NF-κB p65 (1:1000, #8242, #3033, Cell Signaling Technology). After incubation with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h, the proteins were detected using enhanced chemiluminescence.

Statistical analysis

This experiment presents data from our study in the format of mean ± SEM unless otherwise specified. Differences between two groups were evaluated using independent samples t-test. One-way analysis of variance (ANOVA) was conducted for comparisons among multiple groups, while the Kruskal–Wallis Test was used for non-normally distributed data. Welch’s ANOVA accounted for unequal sample sizes and variances. Relative protein expression from Western blot analysis was quantified using ImageJ software, normalizing to β-actin levels for each sample. Immunohistochemical analysis was performed by quantifying the integrated optical density (IOD) per area using ImageJ software to evaluate LRRC8A and other marker expression levels. Statistical analyses were performed using GraphPad Prism software, and a significance level of P < 0.05 was considered statistically significant.

Results

Correlation analysis of LRRC8A mRNA with NADPH oxidase subunits and clinical features

The clinical characteristics of the subjects included in this study are presented in Table 1. Compared to the control group, patients with AR showed significant differences in blood total IgE, blood Phadiatop test, and percentage of blood eosinophils. To investigate whether LRRC8A is involved in the pathogenesis of AR, we performed qRT-PCR to verify the mRNA expression in collected samples. We found that mRNA levels of LRRC8A, NOX1, NOX4, p22^phox, IL-4, IL-5, and IL-13 were significantly higher in AR patients compared to controls (Fig. 1A–G).

Table 1 Basic clinical characteristics of subjects

Fig. 1

LRRC8A mRNA expression correlates with NADPH oxidase subunits and clinical characteristics, with LRRC8A, NOX1, NOX4, and p22^phox upregulated in the nasal mucosa of allergic rhinitis patients compared to controls. A–G qRT-PCR was used to detect the mRNA expression levels of LRRC8A, NOX1, NOX4, p22^phox, IL-4, IL-5, and IL-13 in human nasal mucosa (Con n = 18, AR n = 30). H–O Correlation analysis was conducted between LRRC8A mRNA and the mRNA levels of NOX1, NOX4, p22^phox, IL-4, IL-5, IL-13(Con n = 18, AR n = 30), serum total IgE(Con n = 15, AR n = 26), and serum Phadiatop Test-specific IgE (Con n = 15, AR n = 24). P Protein expression of LRRC8A, NOX1, NOX4, and p22^phox in human nasal mucosal homogenates as detected by Western blotting (Con n = 5, AR n = 5). Q Immunohistochemical analysis showing the expression of LRRC8A, NOX1, and NOX4 in the epithelial layer(scale bar: 20 μm). The circled regions highlight the areas where these proteins are expressed (Con n = 8, AR n = 8). Data are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Correlation analysis revealed that the expression of LRRC8A mRNA was positively correlated with Th2 inflammatory factors IL-4 (r = 0.472, P < 0.001), IL-5 (r = 0.562, P < 0.0001), IL-13 (r = 0.635, P < 0.0001) mRNA, NADPH oxidase subunits NOX1 (r = 0.301, P < 0.05), NOX4 (r = 0.739, P < 0.0001, p22^phox (r = 0.710, P < 0.0001) mRNA, and serum total IgE (r = 0.479, P < 0.01) as well as Phadiatop test-specific IgE (r = 0.376, P < 0.05). The correlation analysis revealed varying levels of association, with r values ranging from 0.301 (weak) to 0.739 (strong), indicating different strengths of correlation between LRRC8A and the inflammatory markers. Notably, IL-5, IL-13, NOX4, and p22^phox exhibited strong correlations with LRRC8A (Fig. 1H–O).

Upregulation of LRRC8A, NOX1, NOX4, p22^phox in nasal mucosal tissue of AR patients

These results suggest that LRRC8A may be involved in the pathogenesis of AR. We further validated the protein levels of these molecules in collected human nasal mucosal tissue via Western blotting and analyzed their spatial localization using immunohistochemistry. Our data, for the first time, showed increased protein expression of LRRC8A, NOX1, NOX4, p22^phox in AR patients compared to controls (Fig. 1P). Moreover, immunohistochemical analysis revealed the expression of LRRC8A, NOX1, NOX4 in the epithelial layer of the nasal mucosa (Fig. 1Q), indicating that LRRC8A, NOX1, and NOX4 may affect epithelial function and contribute to the development of epithelial inflammation in AR patients.

LRRC8A regulates NADPH oxidase subunit expression and ROS production

Correlation analysis showed that the expression of LRRC8A is positively correlated with IL-13 expression, suggesting LRRC8A may be involved in the regulation of epithelial inflammation caused by IL-13. Thus, we used IL-13 to stimulate HNEpCs to induce epithelial cell inflammation. The results showed that with increasing doses of IL-13, the expression of LRRC8A increased at 50 ng/mL (Fig. 2A, E). Subsequently, we knocked out LRRC8A and monitored the production of NADPH oxidase subunits and ROS. First, we confirmed that siLRRC8A had effective knockdown efficiency (Fig. 2B, F, G). we explored whether an increase in LRRC8A following IL-13 stimulation would activate the VRAC channel. The results showed that after IL-13 stimulation, the chloride-sensitive fluorescent probe (MQAE) showed a decrease in intracellular Cl⁻ and an increase in intracellular chloride ions in LRRC8A knockout cells (Fig. 2C, M). Fluorescence results from DCFH-DA indicated that ROS levels increased after IL-13 stimulation, but significantly decreased upon LRRC8A knockdown (Fig. 2D, N). Studies have shown that LRRC8A plays an important role in various physiological activities by activating the VRAC channel [15, 22]. Therefore, Further, we observed the effects of LRRC8A knockdown on NADPH subunits. The results indicated that IL-13 stimulation led to an increase in the production of NADPH oxidase subunits NOX1, NOX4, and p22^phox. However, knocking out LRRC8A could inhibit the protein expression of NOX1, NOX4, and p22^phox (Fig. 2H–L).

Fig. 2

LRRC8A regulates the expression of NADPH oxidase subunits and ROS production. A, E Western blotting analysis of LRRC8A protein expression in HNEpCs after stimulation with IL-13 for 12 h. B, F, G Validation of siLRRC8A knockdown efficiency at the mRNA and protein levels. C, M Measurement of Cl⁻ expression in HNEPCs using MQAE (scale bar = 50 μm). D, N After being stimulated with IL-13, HNEPCs transfected with siLRRC8A and NCsirna were measured for ROS expression using DCFH-DA (scale bar = 50 μm). H–L Western blotting analysis of LRRC8A, NOX1, NOX4, and p22^phox protein expression in HNEPCs. * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Knocking out LRRC8A improves mitochondrial oxidative stress and function in HNEpCs

Studies have indicated that NOX4 is expressed within mitochondria and involved in the production of mitochondrial ROS (mtROS); knocking out NOX4 can improve mitochondrial function [23, 24]. Therefore, we first examined whether NOX4 is expressed in HNEpCs mitochondria. The results showed that after IL-13 stimulation, the co-localization of NOX4 and mitochondrial marker MitoTracker Red increased, but decreased after LRRC8A knockdown (Fig. 3A, B). To further verify the functional state of mitochondria, we examined mtROS, mitochondrial membrane potential (TMRM), and mitochondrial DNA (mtDNA) copy number. The results showed that mtROS expression increased after IL-13 stimulation (Fig. 3C, D), and the fluorescence intensity of TMRM weakened (Fig. 3E, F), indicating that IL-13 caused mitochondrial damage. However, knocking out LRRC8A in HNEpCs cells reduced mtROS fluorescence intensity and increased TMRM fluorescence intensity, revealing that LRRC8A knockdown could inhibit mitochondrial damage caused by IL-13. Since mtDNA encodes various proteins, changes in mtDNA copy number significantly affect mitochondrial function. Thus, we examined changes in mtDNA copy number after IL-13 stimulation. The results showed that mtDNA copy number decreased after IL-13 stimulation, but increased after LRRC8A knockdown (Fig. 3G). The occurrence of oxidative stress is the result of an imbalance in ROS production. Therefore, we measured the activity of a major mitochondrial antioxidant enzyme, Mn-SOD. The results showed that Mn-SOD activity decreased after IL-13 stimulation, indicating a disruption in mitochondrial oxidative balance. However, when LRRC8A was knocked out, Mn-SOD activity increased, suggesting that knocking out LRRC8A could improve Mn-SOD enzyme activity (Fig. 3H).

Fig. 3

Knockout of LRRC8A improves mitochondrial oxidative stress and function in HNEpCs, and activates the NF-κB pathway. A Representative fluorescence co-localization images of NOX4 and Mito Tracker Red in HNEpCs transfected with siLRRC8A and NCsirna before IL-13 stimulation (scale bar = 10 μm). B Fluorescence intensity curves of NOX4 (Green) and Mito Tracker Red (Red) co-localization images in treated HNEpCs. C, D Mitosox staining of HNEPCs post-IL-13 stimulation reveals mitochondrial ROS expression (scale bar = 20 μm). E, F Changes in mitochondrial membrane potential were assessed using TMRM staining (scale bar = 50 μm). G Detection of mitochondrial DNA copy number levels using a mitochondrial mtDNA copy number assay kit. H Measurement of Mn-SOD enzyme activity expression levels. I–K Before IL-13 stimulation, HNEpCs were treated with siLRRC8A or NCsirna, followed by Western blotting to assess NF-κB p-65 and P-p65 protein expression. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

LRRC8A regulates the activation of the NF-κB pathway

The NF-κB pathway plays a crucial role in the development of allergic rhinitis, with mitochondrial ROS and NADPH oxidase known to activate the NF-κB inflammatory pathway [25, 26]. Therefore, we investigated changes in the NF-κB pathway activation-associated proteins p65 and phosphorylated p65. Results showed that NF-κB pathway activation increased after IL-13 stimulation, as evidenced by elevated levels of phosphorylated p65 while the total p65 remained unchanged. However, upon LRRC8A knockdown, the expression of the NF-κB-related inflammatory protein p65 was significantly downregulated (Fig. 3I–K).

LRRC8A mediates mitochondrial damage in HNEpCs by activating VRAC channels and promoting NOX1/NOX4 expression

To verify whether LRRC8A-mediated ROS production and mitochondrial damage in HNEpCs are regulated through the activation of the LRRC8A-dependent VRAC channel affecting NOX1/NOX4 protein expression, we first overexpressed LRRC8A in HNEpCs via plasmid transfection. The results indicated that cells transfected with pcDNA-LRRC8A showed a significant increase in LRRC8A expression compared to the pcDNA-NC control (Fig. 4A, B). Subsequently, in cells overexpressing LRRC8A, we added GKT137831 and DCPIB one hour prior. The results showed that overexpression of LRRC8A increased NOX1 and NOX4 protein levels, the addition of GKT137831 and DCPIB could inhibit the expression of NOX1 and NOX4 (Fig. 4C–E). Since NOX1 and NOX4 are major sources of ROS, we further measured the ROS and mtROS levels. The fluorescence intensity of ROS and mtROS staining increased in HNEpCs overexpressing LRRC8A compared to controls, and the addition of GKT137831 and DCPIB inhibited these changes (Fig. 4F–I).

Fig. 4

LRRC8A Mediates Mitochondrial Damage in HNEpCs by Activating VRAC channels and Promoting NOX1/NOX4 Expression. A, B LRRC8A protein expression levels in HNEpCs overexpressing LRRC8A were analyzed. C–E Expression levels of NOX1 and NOX4 in HNEpCs overexpressing LRRC8A, treated with GKT137831 (pcDNA-LRRC8A + GKT) and DCPIB (pcDNA-LRRC8A + DCPIB), were determined. F, H ROS expression levels were quantified using DCFH-DA fluorescence assay in HNEpCs overexpressing LRRC8A (scale bar = 50 μm). G, I Mitochondrial ROS expression was observed by Mitosox staining in HNEpCs overexpressing LRRC8A (scale bar = 20 μm). J Mn-SOD enzyme activity levels determined. K, L Changes in mitochondrial membrane potential assessed using TMRM staining (scale bar = 50 μm). M mtDNA copy number levels detected using a mtDNA copy number assay kit. *P < 0.05, **P < 0.01, ***P < 0.001****, P < 0.0001

To further understand the oxidative stress state within mitochondria, we measured the activity of the antioxidant enzyme Mn-SOD post-treatment. The results demonstrated that overexpression of LRRC8A reduced Mn-SOD enzyme activity compared to the control group. Interestingly, the addition of the DCPIB and GKT137831 increased Mn-SOD activity (Fig. 4J). We then continued to monitor whether inhibiting the activation of the LRRC8A-dependent VRAC channel and the expression of NOX1/NOX4 could improve mitochondrial function. The results confirmed that overexpression of LRRC8A reduced mitochondrial membrane potential and mtDNA copy number compared to the control group. The addition of GKT137831 and DCPIB improved mitochondrial membrane potential and restored mtDNA copy numbers (Fig. 4K–M). These results indicate that LRRC8A activates the VRAC channel regulating NOX1/NOX4 expression, inducing ROS production, and inhibiting the antioxidant enzyme Mn-SOD activity, thereby mediating mitochondrial damage and contributing to nasal mucosal epithelial cell inflammation and damage.

Knockdown of LRRC8A inhibits inflammation in the AR mouse model

The AR model in mice was established using OVA (Fig. 5A). Initially, we confirmed the expression of LRRC8A in the nasal mucosal tissue of the mouse model using Western blotting. The results showed increased LRRC8A expression in the OVA and AAV5-NC groups compared to the control group, while in the AAV5-siLRRC8A group, LRRC8A expression was reduced, indicating that AAV5-siLRRC8A effectively inhibited LRRC8A expression in vivo (Fig. 5B, C). After the final stimulation, we recorded the number of sneezes and nose scratches within 10 min. The results showed a significant increase in sneezing and scratching in the OVA and AAV5-NC groups compared to controls. However, these behaviors were suppressed in the AAV-siLRRC8A group (Fig. 5D, E). Further, we used qRT-PCR and ELISA to demonstrate that in vivo knockdown of LRRC8A significantly reduced the expression of Th2 inflammatory cytokines and OVA-specific IgE (Fig. 5F–L). Additionally, H&E staining of nasal mucosa showed that, compared to the control group, the number of eosinophils was significantly increased in the OVA and AAV5-NC groups. However, knockdown of LRRC8A inhibited this change (Fig. 5M, N).

Fig. 5

Suppression of inflammation in the AR mouse model by knocking down LRRC8A. A Schematic diagram of AR models. B, C Western blotting was used to detect LRRC8A protein expression in different groups of AR mouse models. D, E Comparison of sneezing and nasal rubbing frequencies within a 10-min period among different groups of mice. F–H qRT-PCR analysis of mRNA levels of Th2 cytokines IL-4, IL-5, and IL-13 in mouse nasal mucosa tissues. I–L ELISA was used to detect levels of OVA-specific IgE in serum and IL-4, IL-5, and IL-13 in supernatants of splenocytes in different groups. M, N HE staining of mouse nasal mucosa sections to monitor eosinophil infiltration (scale bar = 20 μm), with arrows indicating typical eosinophils. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

In the AR mouse model, we also monitored whether knocking down LRRC8A could suppress the expression of NADPH oxidase. Using Western blotting, we verified the expression of NADPH oxidase subunits. We found that OVA stimulation upregulated the expression of NOX1, NOX4, and p22^phox, while knockdown of LRRC8A decreased the protein expression levels of these subunits (Fig. 6A–D). We further used DCFH-DA and MitoSOX Red staining to observe the expression of ROS and mtROS in the nasal mucosal cell suspension of mice. Results showed that the fluorescence intensity of ROS and mtROS was weak in the control group but enhanced after OVA stimulation. However, after treatment with AAV5-siLRRC8A, the fluorescence intensity of ROS and mtROS in the nasal mucosal cell suspension was significantly weaker than in the OVA-treated group, indicating that knocking down LRRC8A could reduce the expression of ROS and mtROS in the nasal mucosa of AR mice (Fig. 6E, F). To confirm whether LRRC8A affects the mitochondrial redox balance in vivo, we further measured the activity of the major mitochondrial antioxidant enzyme Mn-SOD. Results indicated that Mn-SOD activity was reduced in the OVA-stimulated AR mouse model, but knockdown of LRRC8A promoted the recovery of Mn-SOD activity (Fig. 6G). The NF-κB pathway, a major pathway for inflammation activation, where ROS has been shown to be involved in the activation of the NF-κB pathway, participating in the inflammatory response of the airways [27]. Therefore, we measured the expression of NF-κB pathway-related proteins in vivo. Results showed that in the OVA-stimulated AR mouse model, NF-κB p65 expression remained unchanged, but NF-κB P-p65 expression was upregulated. However, after transfection with AAV5-siLRRC8A, the expression of NF-κB P-p65 was significantly reduced (Fig. 6H–I). These results suggest that knocking down LRRC8A can reduce ROS production by NOX1/NOX4 and inhibit the activity of the antioxidant enzyme Mn-SOD, thereby improving mitochondrial damage, inhibiting the NF-κB pathway, and ameliorating upper airway inflammation in AR mice.

Fig. 6

Knockdown of LRRC8A suppresses NOX1, NOX4, and p22^phox-mediated mitochondrial dysfunction and NF-κB pathway activation in the AR mouse model. A–D Protein expression levels of NOX1, NOX4, and p22^phox were assessed by Western blotting analysis. E, F ROS and mtROS levels were measured using DCFH-DA and Mitosox fluorescent probes, respectively. (scale bar: 50 μm). G Mn-SOD enzyme activity levels were measured among different groups. H, I Protein expression levels of NF-κB pathway-related proteins, including p65 and P-p65, were determined by Western blotting analysis in the AR mouse. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Discussion

In this study, we report for the first time the association between the VRAC-associated protein LRRC8A and AR, and elucidate its role and potential mechanisms in AR. We observed that LRRC8A expression is upregulated in patients with AR and in the OVA-induced AR mouse model, and it is expressed in nasal mucosal epithelial cells. In vitro, we used IL-13 as an inflammatory stimulus to observe the role of LRRC8A in epithelial inflammation. We found that IL-13 treated HNEpCs showed increased LRRC8A expression. Subsequently, we knocked down and overexpressed LRRC8A in HNEpCs using siRNA and plasmids respectively, to explore its role and potential mechanisms in IL-13 induced epithelial inflammation. We discovered that inhibiting LRRC8A expression significantly reduced the production of IL-13 induced ROS and the activation of the VRAC channel. By adding specific inhibitors of NOX1/NOX4 and the VRAC channel, we confirmed that inhibiting the VRAC-associated protein LRRC8A targets NOX1/NOX4, inhibits mtROS production, and enhances the activity of the antioxidant enzyme Mn-SOD, improving mitochondrial function and inhibiting NF-κB pathway activation. In vivo models further validated these findings, demonstrating that inhibiting LRRC8A could suppress oxidative stress and mitochondrial damage in the body, reduce the expression of Th2 inflammatory cytokines and eosinophil infiltration. This study is the first to demonstrate the role of LRRC8A in AR, particularly in modulating Th2 cytokines (IL-4, IL-5, IL-13). Our findings suggest that LRRC8A may regulate these cytokines through NADPH oxidase activity, highlighting its potential as a mediator in AR by linking oxidative stress with immune response regulation.

Since the discovery of VRAC, its physiological functions have been a focus of scientific interest [28]. However, it was not until the LRRC8 protein family was identified as the molecular structure of the VRAC channel that this mystery was resolved [13, 29]. The relationship between the VRAC-associated protein LRRC8A with inflammation and oxidative stress has been of particular interest. Studies have found that the VRAC-associated protein LRRC8A is a key regulator of the NLRP3 inflammasome and innate immunity [15]. Extensive research indicates that activation of VRAC is necessary for ROS production, and inhibition of VRAC can also prevent the accumulation of ROS within cells [12, 18, 30]. NADPH oxidase, as a major producer of ROS, can cause excessive production of ROS in epithelial cells, leading to dysfunction of airway epithelia [31,32,33,34]. Previous studies have shown that increased oxidative stress correlates with elevated LRRC8A levels, suggesting a common pathway in various inflammatory conditions [18, 19]. Therefore, we continue to focus on the relationship between LRRC8A and NADPH oxidase in AR.

In this experiment, we used IL-13 to stimulate epithelial cells in vitro not only because IL-13 can simulate a Th2-type inflammation model in vitro, but also because ROS are considered intermediate signaling molecules downstream of IL-13 [35]. This study is the first to conduct a correlation analysis between LRRC8A and NOX1, NOX4, p22^phox, as well as IL-4, IL-5, IL-13, total serum IgE, and inhalant-specific IgE in patients with AR. The results indicate a strong correlation between LRRC8A and NADPH oxidase, as well as Th2-type inflammation. Due to cultural factors in our region, fewer female participants were willing to undergo nasal tissue biopsy, which may have led to a sampling bias. This could have introduced bias in our study, as studies have shown that sex hormones can modulate immune responses [36], and sex-related biological factors might influence LRRC8A’s role in AR. However, our subsequent in vitro and in vivo experiments validated similar findings as in the human samples, suggesting that sex differences may not significantly affect LRRC8A's role in AR. Subsequently, we continued to verify the possibility of LRRC8A targeting NOX1/NOX4 to regulate AR through both in vitro and in vivo experiments. Our findings reveal that IL-13 can activate the VRAC channel and produce ROS in HNEpCs cells, and knocking down LRRC8A can inhibit the activation of the VRAC channel and reduce ROS production. This suggests that IL-13-induced activation of the LRRC8A-dependent VRAC channel promotes ROS production. Studies have shown that NOX1 and NOX4 expression is higher in AR patients than in controls, consistent with literature reports [6]. p22^phox, as the most important active subunit of NADPH oxidase, maintains the stability of NOX proteins and promotes the assembly of the NADPH oxidase complex. A reduction in p22^phox leads to decreased activity of NOX1-4 [37]. Therefore, through both in vitro and in vivo experiments, we continue to verify the possibility of LRRC8A targeting NOX1, NOX4, and p22^phox to regulate ROS production. Results indicate that knocking down LRRC8A can inhibit the protein expression of NOX1, NOX4, and p22^phox, and the reduction of these proteins is accompanied by an improvement in ROS and mitochondrial function, potentially through regulation of the redox balance within mitochondria. Existing research primarily focuses on the harmful effects of ROS in AR progression, with limited attention to its sources in AR patients [4]. This study identifies excessive ROS production in AR as likely driven by the LRRC8A-mediated expression of NADPH oxidase subunits (NOX1, NOX4, p22^phox), providing new insights into ROS sources in AR. These findings bridge the gap between Th2-type immune responses and oxidative stress, echoing previous studies on the role of NADPH oxidase in immune responses [31,32,33].

Mitochondrial oxidative stress causes mitochondrial dysfunction, not only damaging the integrity of the mitochondrial membrane and leading to a reduction in mitochondrial membrane potential and mtDNA copy number but also releasing mtROS into the cytoplasm, exacerbating oxidative stress [38]. The intracellular localization of NOX4—whether in the endoplasmic reticulum, mitochondria, or the nucleus—has been a long-debated topic; now, most scholars believe NOX4 can be localized in all these structures [39]. The role of NOX4 in mitochondria, particularly its interaction with LRRC8A, remains underexplored in previous studies. This study is the first to show that LRRC8A inhibits NOX4’s mitochondrial localization in HNEpCs, broadening our understanding of LRRC8A’s biological role beyond its function as an ion channel. Additionally, in vascular smooth muscle cells, zinc overload stimulates NOX1 to produce ROS, which in turn stimulates the production of mtROS [25]. Excessive NOX4 within mitochondria promotes mitochondrial damage, and an increase in mtROS further leads to mitochondrial dysfunction, creating a vicious cycle [40]. We continue to explore whether LRRC8A regulates the expression of NOX1/NOX4, leading to the production of mtROS and mitochondrial damage. To ensure the specificity of LRRC8A's effects, we overexpressed LRRC8A in HNEpCs using a plasmid and added specific inhibitors for NOX1/NOX4 and DCPIB. Our study shows that overexpression of LRRC8A leads to increased protein expression of NOX1 and NOX4, and adding NOX1/NOX4 and VRAC channel inhibitors can inhibit the expression of these proteins. These findings suggest that LRRC8A may influence mitochondrial function by regulating the NOX pathway, aligning with evidence that NADPH oxidase overexpression leads to mitochondrial dysfunction [40]. While previous studies have noted a connection between oxidative stress and VRAC channel activity, the regulatory mechanism remains unclear [22] Our study provides the first experimental evidence that in AR, LRRC8A may regulate NOX1/NOX4 expression via the VRAC pathway, controlling mtROS production and impairing mitochondrial membrane potential. These findings are crucial as they establish a direct link between LRRC8A, mitochondrial dysfunction, and oxidative stress, providing novel insights into the pathophysiology of AR and potential therapeutic targets.

The state of redox balance within mitochondria influences their function. SOD is a group of endogenous antioxidant enzymes, including cytoplasmic Cu/Zn-SOD, Mn-SOD located in the mitochondrial matrix and inner membrane, and extracellular SOD. Our discussion extends the current understanding by highlighting the unique genetic and functional aspects of Mn-SOD, which exhibits distinct homology compared to Cu/Zn-SOD and extracellular SOD, implying its specialized transcriptional pathway and unique role in mitochondrial maintenance [41]. Notably, previous studies have suggested that an increase in Mn-SOD enhances the activity of NADPH oxidase, while overexpression of Mn-SOD weakens its activity [42]. The mitochondrial inner membrane is a site of free radical production, and mtDNA, located near the mitochondrial inner membrane, is more susceptible to oxidative damage and degradation than nuclear DNA [43]. The D-loop region of mtDNA is sensitive to oxidative stress and prone to mutations and other damages, affecting mtDNA replication and transcription, thereby causing a decline in mtDNA copy number or altering mtDNA gene expression [44]. Our findings advance this knowledge by revealing that inhibiting NOX1/NOX4 mitigates Mn-SOD activity loss and mtDNA copy reduction induced by LRRC8A overexpression, suggesting a protective effect of VRAC inhibition in maintaining mitochondrial homeostasis. Our study also provides new insights into the role of LRRC8A in allergic inflammation by detailing its interaction with the NF-κB signaling pathway, a key player in immune response modulation. Recent studies have shown that the occurrence of allergic inflammation is accompanied by the activation of NF-κB [45]. Additionally, NF-κB is a redox-sensitive transcription factor, where ROS can enhance [46] or inhibit [47] its activation, depending on the inflammatory environment. mtROS are considered secondary messengers of NF-κB [48]. Our results, while differing slightly from those in chronic granulomatous disease research [49], align closely with findings in allergic disease models that underscore ROS’s active role in inflammation [50]. This study is among the first to show that LRRC8A inhibition reduces NF-κB activation and phosphorylated p65 levels, both in vitro and in vivo, offering a potential pathway by which LRRC8A influences allergic responses. Given that the activation response of NF-κB depends on the stimulus, timing, and cell type [51], the variability of NF-κB responses seems to be highly sensitive to differences in experimental models and protocols. Furthermore, this study does not clarify whether LRRC8A-induced NF-κB activation is due to an increase in ROS production by NADPH oxidase or through mitochondrial oxidative stress mediated by the production of mtROS. This requires further investigation.

Our in vivo findings strongly support the hypothesis that LRRC8A modulates AR pathophysiology by influencing Th2 cytokine secretion and eosinophilic infiltration in the nasal mucosa. We utilized AAV5, a vector with high affinity for the respiratory system, to knock down LRRC8A in the nasal mucosa of mice through local nasal administration. We found that knocking down LRRC8A significantly improved the classic symptoms and pathological changes associated with AR, including reducing the expression of Th2 inflammatory cytokines and the infiltration of eosinophils in the nasal mucosa. This study investigates LRRC8A’s role in allergic reactions, specifically its involvement in regulating NADPH oxidase activity and Th2-type cytokine secretion. Our findings align with previous reports identifying LRRC8A as a "gatekeeper" for cell volume control, promoting T cell activation [16] Another typical pathological manifestation of AR is the epithelial-mesenchymal transition (EMT) of the nasal mucosa, which affects the normal function of epithelial cells. This study does not elaborate on whether LRRC8A influences the epithelial-mesenchymal transition of the nasal mucosa. However, according to literature reports, knocking down LRRC8A can attenuate TGF-β1-induced EMT in HEK293 cells by controlling GSH and ROS level [22]. We speculate that LRRC8A could delay the epithelial-mesenchymal transition of the nasal mucosa by affecting the expression of its associated proteins. Finally, although our findings show that LRRC8A knockdown reduces inflammation, we acknowledge potential off-target effects and compensatory mechanisms. We used validated siRNA sequences with high specificity and confirmed target suppression, but further investigation into compensatory pathways, such as other ion channels, is needed. Future studies will include rescue experiments and transcriptomic analyses to clarify these mechanisms.

Conclusion

Our findings indicate that LRRC8A is a crucial mediator in the pathophysiological development of AR. Further studies show that activation of the VRAC pathway by LRRC8A can regulate the expression of NOX1/NOX4, promote ROS-induced oxidative stress, induce mtROS, and inhibit the activity of Mn-SOD, thereby causing an imbalance between oxidation and antioxidation within mitochondria. It leads to mitochondrial dysfunction. Additionally, LRRC8A can activate the NF-κB pathway, ultimately promoting inflammatory responses in the nasal mucosa epithelium (Fig. 7). Our research could provide new insights for the treatment of AR, and drugs targeting LRRC8A may represent innovative therapeutic options for future AR treatment. While this study offers valuable insights, several limitations should be noted. First, we initially used DCPIB, a VRAC inhibitor, to suggest that LRRC8A regulates NOX1 and NOX4 through VRAC activation; however, additional pathways may contribute, such as calcium influx and ROS feedback loops, potentially enhancing NOX1 and NOX4 expression. Secondly, as the study primarily relied on IL-13-stimulated models to assess LRRC8A's role, it remains unclear whether other Th2 cytokines, such as IL-4 or IL-5, would yield similar results. This limitation is notable as different cytokines may exhibit synergistic or antagonistic effects on cellular responses, with potential co-stimulation influencing LRRC8A’s expression and function in diverse ways. Future studies should examine these aspects to better understand LRRC8A's full role in AR inflammation. Lastly, targeting LRRC8A with small molecule inhibitors or gene therapies could suppress NOX1 and NOX4, reduce oxidative stress, and complement existing treatments to enhance efficacy and lower dosages. Genomic approaches may also explore LRRC8A as a biomarker across diverse populations to support personalized allergy treatment.

Fig. 7

LRRC8A-mediated mechanism of nasal mucosal epithelial cell inflammation: LRRC8A activation by allergen inhalation increases Th2 cytokine secretion, leading to VRAC-associated LRRC8A upregulation. This induces up-regulation of NOX1, NOX4 and p22^phox, forming NOX1/NOX4 and p22^phox complexes, causing overproduction of ROS, impairing Mn-SOD activity, TMRM, and mtDNA copy number, leading to mitochondrial dysfunction in epithelial cells. At the same time, the NF-κB pathway was activated, which ultimately promoted nasal mucosal epithelial inflammation