Phosphatidylinositol 3‐kinase‐δ controls endoplasmic reticulum membrane fluidity and permeability in fungus‐induced allergic inflammation in mice

Background and Purpose Phosphatidylinositol 3‐kinase (PI3K), especially PI3K‐δ, and endoplasmic reticulum (ER) stress play important roles in refractory asthma induced by the fungus Aspergillus fumigatus through mechanisms that are not well understood. Here we have investigated these mechanisms, using BEAS‐2B human bronchial epithelial cells and a mouse model of A. fumigatus‐induced allergic lung inflammation. Experimental Approach A selective PI3K‐δ inhibitor, IC87114, and an ER folding chaperone, 4‐phenylbutyric acid (4‐PBA), were applied to a model of A. fumigatus‐induced asthma in female C57BL/6 mice. The therapeutic potential of IC87114 and 4‐PBA was assessed in relevant primary cell, tissue, and disease models, using immunohistochemistry, western blotting and assessment of ER redox state and membrane fluidity. Key Results Treatment with IC87114 or 4‐PBA alleviated pulmonary inflammation and airway remodelling and reduced ER stress and inflammation‐associated intra‐ER hyperoxidation, disrupting protein disulfide isomerase (PDI) chaperone activity. IC87114 and 4‐PBA also reversed changes in ER membrane fluidity and permeability and the resultant mitochondrial hyperactivation (i.e., Ca2+ accumulation) under hyperoxidation, thereby restoring the physiological state of the ER and mitochondria. These compounds also abolished mitochondria‐associated ER membrane (MAM) formation caused by the physical contact between these subcellular organelles. Conclusion and Implications PI3K‐δ and ER stress mediate A. fumigatus‐induced allergic lung inflammation by altering the ER redox state, PDI chaperone function, and ER membrane fluidity and permeability and by amplifying ER signalling to mitochondria through MAM formation. Thus, therapeutic strategies that target the PI3K‐δ–ER stress axis could be an effective treatment for allergic asthma caused by fungi.

PI3K-δ-ER stress axis could be an effective treatment for allergic asthma caused by fungi.

| INTRODUCTION
Fungi are increasingly recognised as the main cause of allergic asthma, one of the most common respiratory diseases that can lead to chronic airway inflammation, reversible airway obstruction, increased mucus production, and non-specific airway hyper-responsiveness (AHR; Leong & Huston, 2001). The fungus Aspergillus fumigatus is one of the most frequent stimuli inducing inflammation-related airway AHR and remodelling (Hoselton, Samarasinghe, Seydel, & Schuh, 2010;Schuh & Hoselton, 2013). Furthermore, corticosteroids are not effective in the treatment of severe asthma associated with fungal sensitisation (Denning et al., 2009) and the alleviation of A. fumigatus-induced allergic lung inflammation, including eosinophil-dominant inflammatory cell infiltration into the lungs, AHR, and elevation of TH2-dominant proinflammatory cytokines (Felton, Lucas, Rossi, & Dransfield, 2014;Lee et al., 2016). This suggests that A. fumigatus-induced lung inflammation represents a unique endotype of steroid-resistant, eosinophildominant, allergic lung inflammation (Felton et al., 2014;Lee et al., 2016). Exposure to the fungus activates Toll-like receptor 4 signalling and consequently, phosphoinositide 3-kinase (PI3K)/Akt, and downstream NF-κB pathways (Laird et al., 2009), leading to immune responses including the trafficking and activation of neutrophils and eosinophils (Kang et al., 2012;Puri et al., 2004). Thus, inhibition of PI3K-δ is a potential therapeutic strategy for refractory asthma.
PI3K activation is associated with high protein synthesis and folding load and affects the redox balance of protein folding organelles.
Hyper-loading of the endoplasmic reticulum (ER) also alters protein folding. To maintain its functional integrity, the ER must constantly balance the capacity of its protein chaperones with the load of newly synthesised unfolded proteins in the cell and disrupting this balance leads to the accumulation of unfolded or misfolded proteins in the ER (Hotamisligil, 2010). This process, known as ER stress, is associated with changes to the fluidity and permeability of the ER membrane (Kaplan, Racay, Lehotsky, & Mezesova, 1995;Pamplona, 2008) that result in the propagation of signals to nearby mitochondria (Malhotra & Kaufman, 2011;Marchi, Patergnani, & Pinton, 2014). The sites of physical contact between the ER and mitochondria-known as mitochondria-associated endoplasmic reticulum membranes (MAMs)-are determinants of cell survival and death induced by inflammation signalling through the transfer of Ca 2+ , ROS, and other metabolites (Friedman et al., 2011;Rowland & Voeltz, 2012). However, the relevance of this interaction between organelles to inflammation-related cellular dysfunction and metabolic homeostasis is not known.
In this study, we show that inhibiting PI3K-δ suppresses fungusinduced cytokine release and airway refractory asthma triggered by changes in ER membrane fluidity and permeability and abnormal MAM formation.

| Animals
All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Chonbuk National University (CBNU2017-0039). All the animal studies complied with the principle of replacement, refinement, or reduction (the 3Rs). The animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010) and with the recommendations made by the What is already known • PI3K-δ and endoplasmic reticulum stress are involved in airway hyper-responsiveness and refractory asthma.

What this study adds
• PI3K-δ mediates changes in ER membrane fluidity and permeability in A.fumigatus-induced allergic lung inflammation.
• Excessive ER-mitochondrial coupling is an essential mechanism for refractory asthma associated with ER stress.
What is the clinical significance • PI3K-δ and ER stress, along with ER-mitochondria interactions, underlie the development of refractory asthma. and 4 days after intranasal challenge, the mice received 20 μg of A. fumigatus antigen dissolved in normal saline via the intratracheal route (Hogaboam et al., 2000). Non-sensitised control mice were given normal saline alone via the same routes at the same time points and received the same number of conidia. Bronchoalveolar lavage (BAL) was performed in mice 48 hr after the last challenge with A. fumigatus ( Figure S1).
A block randomisation technique was used to randomise the animals into groups of equal sample sizes at all time points. The optimum sample size and number of animals were determined by a power anal-

| Subcellular fractionation
Subcellular extractions (cytosol, nuclear, and ER) were performed as previously described (Kim et al., 2008). Briefly, lung tissue was resuspended in iso-osmotic buffer (0.32-M sucrose, 1-mM MgCl 2 , 10-mM Tris-HCl [pH 7.4]) and lysed by 20 passes with a Dounce homogeniser. The homogenate was centrifuged at 1,000× g for 10 min at 4 C to obtain the nuclear fraction (pellet). The supernatant was then centrifuged at 13,000× g for 30 min at 4 C, and the second supernatant was centrifuged at 100,000× g for 1 hr at 4 C using an SW32.1 rotor in an L8-80M ultracentrifuge (Beckman-Coulter) to obtain the cytosolic (supernatant) and ER (pellet) fractions. The fractions were stored at −80 C until use.
The samples were then washed five times with 10-mM Tris, pH 7.5 containing 0.1-M NaCl and 1% Triton X-100, and subsequently eluted in 50 μl of sample buffer. For SDS-PAGE, equal amounts of protein were separated on a 10% SDS gel and transferred to a PVDF membrane using a mini-transfer tank (Bio-Rad, Hercules, CA, USA). The membrane was probed with primary antibodies; after incubation with secondary antibody, protein bands were detected with a chemiluminescence system. Protein expression levels were quantified and normalised based on the band intensity by using ImageJ 1.48v (http:// imagej.nih.gov/ij, RRID:SCR_003070)..

| Detection of cytokines in BALF
Cytokine levels in the BALF supernatant were quantified by enzyme immunoassay using commercial kits (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer's protocol.

| Lipid peroxidation assay
Lipid peroxidation was assessed with a lipid hydroperoxide assay kit (Cayman Chemicals, Ann Arbor, MI, USA). Lung microsomes (1 mg) were homogenised in 1 ml of ice-cold 2% SDS buffer. Sample homogenates and MDA standards were incubated with SDS and 0.8% thiobarbituric acid (20% acetic acid [pH 3.5]) in the presence of 0.8% butylated hydroxytoluene at 95 C for 1 hr. After cooling on ice and centrifuging at 1,000× g for 15 min, peroxidation levels in the supernatant were assessed by measuring the absorbance at 532 nm using a spectrophotometer.

| GSH/GSSG ratio assay
Oxidative stress in the lung was evaluated using a GSH assay kit (Cayman Chemicals) according to the manufacturer's instructions.

| OxyBlot assay
The oxidative protein carbonylation assay was performed on lung tissue using an OxyBlot Protein Detection Kit (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. The carbonyl groups in the protein side chains were derivatised to dinitrophenylhydrazine (DNP)-hydrazone by reaction with 2,4-DNP.
The proteins were then separated by 10% SDS-PAGE and transferred to a PVDF membrane. After incubation with an anti-DNP antibody, the protein band was detected with a chemiluminescence system. 2.14 | Membrane fluidity

| Measurement of intracellular Ca 2+ ([Ca 2+ ] i )
[Ca 2+ ] i was measured as previously described (Kim et al., 2008). The 229 nM was assumed for the binding of Ca 2+ to Fura-2/AM. R max and R min were determined for each experimental group following the consecutive addition of 30-μM Triton (R max ) and 50-mM EGTA (R min ).

| Data and statistical analysis
The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). The data are presented as the means ± SD of the measurements made with 10 mice in each group per experiment. The statistical analysis was performed using

| PI3K-δ is involved in airway inflammation, ER redox imbalance, and associated ROS accumulation in A. fumigatus-induced allergic lung inflammation
Previous studies have suggested that PI3K-δ and ER stress are involved in airway inflammation and remodelling (Kim et al., 2018).
To confirm the pathological role of PI3K-δ and ER stress, we examined the effects of the PI3K-δ inhibitor IC87114 and the chemical chaperone 4-PBA that regulates ER stress in an A. fumigatus-induced mouse model of airway inflammation. Histological analysis revealed greater infiltration of various types of inflammatory cells into the bronchioles in A. fumigatus-exposed mice than in the control group ( Figure 1a). As expected, A. fumigatus-exposed mice treated with IC87114 or 4-PBA showed a marked reduction in inflammatory cell infiltration. In particular, the eosinophil fraction was significantly increased in the BALF of A. fumigatus-exposed mice, an effect that was mitigated by IC87114 or 4-PBA administration (Figure 1b). Airway responses to methacholine were measured 1 hr before and 0, 4, and 8 hr after aspiration. Prior to aspiration, A. fumigatus-exposed mice showed AHR, as shown by increased enhanced pause (Penh) values in response to methacholine. In contrast, IC87114 or 4-PBA treatment reduced Penh values in A. fumigatus-exposed mice ( Figure 1c). PI3K activity and phosphorylation of the downstream target Akt were also inhibited by the administration of IC87114 or 4-PBA ( Figure S2A, B) whereas PAS staining and immunolabelling of mucin 5AC-a marker of mucus secretion-showed a decreased intensity ( Figure S3). These results indicate that submucosal oedema and mucus hyper-secretion are reduced by inhibiting PI3K-δ and ER stress.
Given that oxidative stress is a hallmark of allergic lung inflammation (Lee et al., 2016), we analysed membrane lipid peroxidation, protein oxidation, and GSH redox status (GSH/GSSG balance) in our model of A. fumigatus-induced asthma. The increase in protein oxidation observed in A. fumigatus-challenged mice was reduced by treatment with IC87114 or 4-PBA ( Figure 1d). As protein oxidation may be linked to ER-associated ROS (Tu & Weissman, 2004), we assessed intra-ER lipid peroxidation status with the 4-HNE and MDA assays and by measuring H 2 O 2 production and the GSH/GSSG ratio in mice exposed to A. fumigatus, with and without IC87114 or 4-PBA treatment. The 4-HNE and MDA levels in the ER fraction were reduced in the presence of IC87114 or 4-PBA (Figure 1e), which also blocked the increase in intra-ER H 2 O 2 level in A. fumigatus-exposed mice ( Figure 1f). The balance between GSH and GSSG in the ER reflects ER protein oxidation status (Appenzeller-Herzog, 2011); we found here that the GSH:GSSG ratio was decreased in A. fumigatus-challenged mice and restored by IC87114 and 4-PBA treatment (Figure 1g).

| PI3K-δ mediates changes in ER membrane fluidity and permeability in A. fumigatus-induced allergic lung inflammation
ER membrane properties such as fluidity and permeability can reflect ER stress status (Castuma & Brenner, 1983;Kaplan et al., 1995;Yang, Sheng, Sun, & Lee, 2011). In mice exposed to A. fumigatus and exhibiting an ER stress response (Figure 2a), we examined whether A. fumigatus-induced allergic lung inflammation alters ER membrane fluidity, which could lead to the disruption of calcium homeostasis and increase in ER stress. In A. fumigatus-exposed lung tissue and lung epithelial cells, ER membrane fluidity was decreased relative to the control, shown by the decrease in PDA excimer-to-monomer ratio (Figure 2b,c). Administration of IC87114 or 4-PBA reversed this effect. These results were validated by using DPH polarisation anisotropy-which is based on the elimination of temperaturedependent DPH polarisation (Stott et al., 2008)-to measure the membrane fluidity of purified ER fractions following treatment with IC87114 or 4-PBA (Figure 2d, (Figure 2f,g). By adding ionomycin at different times after thapsigargin, we determined that the kinetics of Ca 2+ release were markedly accelerated in A. fumigatus-exposed cells; both IC87114 and 4-PBA delayed Ca 2+ release relative to the control group. In addition, we detected higher mitochondrial Ca 2+ uptake after stimulating ER Ca 2+ release with ATP in A. fumigatus-challenged cells compared with the control group (Figure 2h). In the former, mitochondrial Ca 2+ intake was reduced by IC87114 or 4-PBA, while adding carbonyl cyanide mchlorophenyl hydrazone increased cytosolic Ca 2+ content compared with the control (Figure 2i), an effect that was reversed by treatment with IC87114 or 4-PBA.

| PI3K-δ is involved in the communication between ER and mitochondria in A. fumigatus-induced allergic lung inflammation
As ER-associated redox imbalance is amplified by juxtaposed mitochondria, the main source of cellular ROS (Yoboue, Sitia, & Simmen, 2018), we first confirmed the ER-mitochondria association under conditions of A. fumigatus-induced ROS disturbance. In the lungs of A. fumigatus-challenged mice, the ER membrane showed an aberrant morphology with an increased thickness and decreased perimeter, while the juxtaposed mitochondria appeared swollen, and the distance between the ER and mitochondria was smaller than normal; F I G U R E 2 IC87114 and 4-PBA restore ER membrane fluidity and calcium permeability in Af-induced allergic lung inflammation. (a) Lung tissue samples were stained for GRP78 or CHOP. Scale bar 10 μm. (b-e) Lung tissue was obtained from A. fumigatus-challenged and salinetreated (Af) mice and A. fumigatus-challenged mice treated with 1 mg kg −1 IC87114 (Af + IC) or 80 mg kg −1 4-PBA (Af + 4-PBA) (b, c); and BEAS-2B cells were treated with 0.1 mg ml −1 IC87114 or 5-mM 4-PBA with or without 100 μg ml −1 A. fumigatus for 24 hr prior to purification of ER fractions (d, e). ER membrane fluidity in lung tissues (b, c) and cells (d, e) were measured as the excimer-to-monomer ratio (b, d) or by the inverse correlation of DPH polarisation anisotropy, whereby decreased DPH polarisation indicates increased membrane fluidity (c, e). (f, g) Cells were treated with 0.1 mg ml −1 IC87114 or 5-mM 4-PBA with or without 100 μg ml −1 A. fumigatus for 24 hr before 1-μM thapsigargin was added for 1 min; 10-μM ionomycin was added either concomitantly with thapsigargin or at 1-min intervals from 1 to 4 min. Ionomycin-releasable Ca 2+ (expressed as a percentage of the initial peak [Ca 2+ ] i ) is plotted as a function of time after thapsigargin addition. (h) [Ca 2+ ] m dynamics were measured following 100-μM ATP stimulation. (i) Cytosolic Ca 2+ was measured with 4-μM Fura2-AM after 1-μM CCCP treatment. Data are expressed as the mean ± SD (n = 10). # P < .05, significantly different from saline; *P < .05, significantly different from A. fumigatus alone; ANOVA. CCCP, carbonyl cyanide m-chlorophenyl hydrazine; i, intracellular; m, mitochondria these phenotypes were reduced by treatment with IC87114 or 4-PBA (Figure 3a-c). The interaction between the mitochondrial outer membrane protein, voltage-dependent anion channel (VDAC)1, and the ER membrane-associated channel protein IP 3 R1 in the asthmatic condition was abolished in the presence of IC87114 or 4-PBA, as determined with the proximity ligation assay (Figure 3d,e). We also analysed functional parameters of mitochondria including ROS levels, ATP, and cytochrome c oxidase (COX) I and COX III activity in A. fumigatus-challenged mice (Figure 3f-i) and found that the exposure to A. fumigatus increased mitochondrial ROS and decreased ATP levels, while inhibiting COX I and COX III activities. These effects were reversed by IC87114 or 4-PBA administration.
3.4 | IC87114 and 4-PBA attenuates the formation and activity of the NLRP3 inflammasome, induced by exposure to A. fumigatus, in airway inflammation The kinase IRE1α is the main ER stress sensor, and its RNAse activity is linked to ER-mitochondria communication through contact between the organelles ( Carreras-Sureda et al., 2019). Upon the activation of IRE1α, the binding proteins GRP78 and PDIA6 dissociate from it. As expected, PDIA6 dissociated from IRE1α, yielding p-IRE1α in the A. fumigatus-challenged mice, but it was stably bound to IRE1α in the presence of IC87114 or 4-PBA (Figure 4a). In the A. fumigatuschallenged mice, the activation of IRE1α increased the mitochondrial enrichment of thioredoxin interacting protein (TXNIP; Figure 4b), which shuttles to mitochondria and binds thioredoxin-2, thereby increasing the concentration of mitochondrial ROS (Lerner et al., 2012). The mitochondrial expression of TXNIP was significantly decreased by the administration of IC87114 or 4-PBA. The interaction between TXNIP and NLRP3-the main mechanism for inflammasome formation and the resultant NF-κB activation during allergic lung inflammation-was clearly observed in the A. fumigatus-challenged condition, whereas administration of IC87114 or 4-PBA suppressed the association between TXNIP and NLRP3 (Figure 4c).
Allergic lung inflammation evokes a clear pro-inflammatory shift in cytokine expression through the formation of the inflammasome (Chen & Nunez, 2010;Rincon & Irvin, 2012). We confirmed the association of NLRP3, ASC, and caspase-1 in A. fumigatus-exposed mice permeable to both ROS and calcium leaked from the ER through VDAC1, which is linked to mitochondrial metabolic activity and ultimately required for ROS production (Colombini, 2004). As expected, VDAC1 was highly expressed in A. fumigatus-induced asthma, and its expression was controlled by treatment with IC87114 or 4-PBA ( Figure S6A). In cells with VDAC1 knockdown, the associations of ASC (apoptosis-associated speck-like protein containing a CARD) and activated caspase-1 with NLRP3 were markedly blocked ( Figure S6B), indicating that in the inflamed condition, a substantial proportion of the inflammasome is associated with MAMs. Furthermore, NLRP3 checks and modulates mitochondrial ROS and the subsequently linked NF-κB activity and that association is negatively controlled by treatment with either a PI3K-δ inhibitor or a chemical chaperone.

| DISCUSSION
The results of this study demonstrate that inhibiting PI3K-δ or ER stress can mitigate airway inflammation and remodelling in an A. fumigatus-induced mouse model of severe asthma characterised by ER stress, changes in ER membrane fluidity and permeability, close contact between the ER and mitochondria, and amplified Ca 2+ and ROS-induced signalling. In airway remodelling, the PI3K-δ-ER stress axis provides a physical link between ER and mitochondria and mitochondrial ROS accumulation, which leads to NF-κB activation.
In The A. fumigatus-induced ER stress resulted in ER membrane peroxidation both in vivo and in vitro, which decreased ER membrane fluidity, increased its permeability, and altered the capacity of ER vesicles to sequester Ca 2+ ions ( Figure 2). Previous reports indicate that ER membrane-associated oxidative stress induces crosslinking via formation of disulfide bridges from two intermolecular thiol (─SH) groups in proteins to lipid membrane rafts as well as formation of adducts with MDA, a typical lipid peroxidation product. This can lead to peroxidative changes in polyunsaturated fatty acid content and membrane dysfunction caused by increased tissue permeability through the loss of membrane fluidity, which is implicated in lung inflammation (Chen & Yu, 1994). Our study also showed that ER membrane peroxidation decreased ER membrane fluidity in lung tissue and pulmonary epithelial cells, which could underlie the observed imbalance in ER redox state and Ca 2+ sequestration. Thus, activation of the PI3K-δ-ER stress signalling axis is associated with altered ER membrane fluidity and permeability and may serve as the pathological basis for fungus-induced airway remodelling.
The functional alteration of ER membranes affects the physical state of ER and mitochondria leading to MAM formation. Crosscommunication between these two organelles might be due to the close physical proximity of the two organelles (Figure 3a,e). In the presence of severe inflammation and damage manifested by alteration of ER membrane fluidity and permeability, the MAM, a platform for crosstalk between the ER and mitochondria, might be easily formed, promoting increased production of ROS and release of mitochondrial DAMPs into the cytosol (Thoudam, Jeon, Ha, & Lee, 2016). In the process of inflammasome formation, NLRP3 binds to the signalling adaptor TXNIP, whose translation is controlled by the activated IRE1α ( Figure 4), an ER stress signalling protein, that also controls the opening of the mitochondrial Ca 2+ intake pore VDAC. Mitochondrial electron transfer chain coupling and uncoupling, a key signalling pathway for mitochondrial ROS, is greatly affected by mitochondrial Ca 2+ , an important regulator of mitochondrial depolarisation linked to inflammation (Giorgi et al., 2012;Tait & Green, 2012). VDAC expression was increased at the mitochondrial membrane-enriched sites of the ER in the A. fumigatus-induced recruitment of inflammatory cells and NF-κB signalling ( Figure S6A). Silencing VDAC with siRNA emphasised the critical role of mitochondrial Ca 2+ in NLRP3, caspase-1, and mitochondrial damage in the active state of the PI3K-δ-ER stress axis ( Figure S6B). Consistent with our results, mitochondrial signalling pathways that communicate with the other organelles has been demonstrated in asthma-associated inflammations (Arruda et al., 2014;Thoudam et al., 2016) and is thought to contribute to Ca 2+ and lipid transfer and ROS amplification in the inflammatory NF-κB signalling (Csordas & Hajnoczky, 2009;Gorlach, Bertram, Hudecova, & Krizanova, 2015). In this study, the PI3K-ER stress axis has been strongly suggested to be a NF-κB signalling pathway for the amplification of inflammation, where ER-mitochondria connection-based mitochondrial characteristics were functionally changed through ER membrane fluidity and alteration in the permeability.
In conclusion, our results suggest that PI3K-δ and ER stress along with ER-mitochondria interactions are involved in the mechanism behind refractory asthma and inflammation, which is challenging to study. Further studies should consider testing components of this mechanism as therapeutic targets against pulmonary diseases involving inflammation and immunity.

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

RIGOUR
This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, Immunoblotting and Immunochemistry and Animal Experimentation, and as recommended by funding agencies, publishers and other organizations engaged with supporting research.