key: cord-0722549-9e9r14n3
authors: Mwase, Chimwemwe; Phung, Thien-Khoi N.; O’Sullivan, Michael J.; Mitchel, Jennifer A.; De Marzio, Margherita; Kılıç, Ayşe; Weiss, Scott T.; Fredberg, Jeffrey J.; Park, Jin-Ah
title: Mechanical Compression of Human Airway Epithelial Cells Induces Release of Extracellular Vesicles Containing Tenascin C
date: 2022-01-13
journal: Cells
DOI: 10.3390/cells11020256
sha: 5452f99915df87bdabc234fbea2ee6d1ee6bb584
doc_id: 722549
cord_uid: 9e9r14n3

Aberrant remodeling of the asthmatic airway is not well understood but is thought to be attributable in part to mechanical compression of airway epithelial cells. Here, we examine compression-induced expression and secretion of the extracellular matrix protein tenascin C (TNC) from well-differentiated primary human bronchial epithelial (HBE) cells grown in an air–liquid interface culture. We measured TNC mRNA expression using RT-qPCR and secreted TNC protein using Western blotting and ELISA. To determine intracellular signaling pathways, we used specific inhibitors for either ERK or TGF-β receptor, and to assess the release of extracellular vesicles (EVs) we used a commercially available kit and Western blotting. At baseline, secreted TNC protein was significantly higher in asthmatic compared to non-asthmatic cells. In response to mechanical compression, both TNC mRNA expression and secreted TNC protein was significantly increased in both non-asthmatic and asthmatic cells. TNC production depended on both the ERK and TGF-β receptor pathways. Moreover, mechanically compressed HBE cells released EVs that contain TNC. These data reveal a novel mechanism by which mechanical compression, as is caused by bronchospasm, is sufficient to induce the production of ECM protein in the airway and potentially contribute to airway remodeling.

A hallmark of asthma is aberrant airway remodeling. While the origin of airway remodeling is not well understood, a growing body of evidence suggests that airway epithelial cells are a causal factor [1] [2] [3] [4] . In particular, during asthma exacerbations, airway narrowing causes mechanical compression of airway epithelial cells, which then produce pathologic mediators thereby contributing to airway remodeling [5] [6] [7] . Mechanical compression applied to well-differentiated human bronchial epithelial (HBE) cells activates multiple signaling cascades, including epidermal growth factor receptor (EGFR), protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and transforming growth factor-β (TGF-β) receptor, all of which are linked to a variety of pathophysiologic features of airway remodeling and asthma [8] [9] [10] [11] [12] [13] [14] [15] [16] . Furthermore, our recent RNA sequencing analysis revealed that mechanical compression of non-asthmatic HBE cells induces transcriptional changes that recapitulate an asthmatic phenotype, including the upregulation of type 2 inflammatory genes, epithelial repair genes, and extracellular matrix (ECM) remodeling

To mimic bronchospasm during an asthma exacerbation, well-differentiated primary HBE cells in ALI culture were subjected to apical-to-basal mechanical compression with a magnitude of 30 cm H 2 O pressure for 3 h, as previously described [6, 12, 47, 50] . Sham control cells received 0 cm H 2 O pressure.

To determine the cellular signaling pathways by which mechanical compression induced TNC expression and secretion, we used a MEK inhibitor (U0126; 10 µM, Tocris, Bristol, UK) and a TGF-β receptor I inhibitor (SB431542; 10 µM, Tocris). Either inhibitor was added to the basolateral conditioned media of HBE cells at 1 h prior to the application of mechanical compression. As a vehicle control for the inhibitors, we used 0.1% DMSO. Cells, basolateral conditioned media, and apical washes were collected at 3 h and 24 h after the initiation of compression. We harvested and stored 1.5 mL of the basolateral conditioned media for further analyses. To harvest the apical washes, we added 0.25 mL of culture media to the apical surface, incubated for 10 min at 34 • C in a 5% CO 2 incubator and then collected the apical washes for further analyses.

Total RNA was isolated from HBE cells (n = 6 donors with no history of lung disease) using the RNeasy Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. Bulk RNA sequencing analysis was performed using a NovaSeq instrument (Illumina, San Diego, CA, USA) by The Bauer Core Facility at Harvard University. Reads were mapped to the GRCh38 reference genome using Salmon [53] . Gene expression (pseudo counts from Salmon) was normalized using tximport (v 1.18.0) [54] and DESeq2 (v 1.30.1) [55] . Genes with low counts (less than or equal 10 counts in at least 70 of 72 samples) were filtered out, and differential expression analyses were performed with FDR < 0.05.

Using 1 ug of total RNA isolated as described above, we prepared 20 ng of cDNA using MultiScribe reverse transcriptase (Applied Biosystems, Forster City, CA, USA). The cDNA was used to perform real-time RT-qPCR using 2× SYBR Green master mix (Life Technologies, Grand Island, NY, USA) and the primers listed below ( Table 1 ). The foldchange for TNC normalized to GAPDH was calculated using the delta-delta Ct method [56] . Table 1 . Primer sequences used in RT-qPCR [42] .

Primer Sequences We quantified the amount of TNC in the basolateral conditioned media and apical washes using an enzyme-linked immunosorbent assay (ELISA) kit (ab213831; Abcam, Cambridge, UK). The validation of the specificity of the ELISA kit was performed using a recombinant human TNC protein (CC065; Sigma Aldrich).

As described previously [13, 16] , using Western blot analysis we detected cellular protein and secreted protein into basolateral conditioned media. Primary antibodies against TNC (33352; Cell Signaling Technology, Danvers, MA, USA), GAPDH (5174S; Cell Signaling Technology), and transferrin (PA3-913; Thermo Fisher Scientific, Waltham, MA, USA) were used. GAPDH and transferrin were detected as loading controls for cellular protein and secreted protein, respectively.

After the initiation of mechanical compression, basolateral conditioned media were collected at 24 h. Basolateral conditioned media were centrifuged at 1200× g for 20 min at 4 • C to remove cell debris and to collect cell-free supernatant. To isolate the EV fraction from cell-free supernatant, we used the commercially available kit, MagCapture Exosome Isolation Kit PS (293-77601; Fujifilm WAKO Pure Chemical Corporation, Tokyo, Japan). Following the instruction, we incubated cell-free supernatant for 6 h at 4 • C. Then, using a DynaMag TM 2 (12321D; Invitrogen, Waltham, MA, USA), we separated the magnetic bead-bound fraction as an EV fraction and the remaining supernatant as a non-EV fraction. The bead-bound EVs were boiled with 2× Laemmli sample buffer (1610737EDU; BIO-RAD, Hercules, CA, USA) containing 1 M dithiothreitol at 100 • C for 6 min and used for Western blot analysis. To validate EV fractions by Western blot analysis, we detected known EV markers [13, 44, 50, [57] [58] [59] , including CD9 (D801A; Cell Signaling Technology) and tissue factor (AF2339; R&D Systems, Minneapolis, MN, USA).

For each condition, the results are expressed as mean ± SEM, and statistical analysis was performed using GraphPad Prism 9 software (GraphPad Software Inc., San Diego, CA, USA). In all experiments, a two-way ANOVA with Bonferroni post-hoc test was utilized to analyze the differences between groups. A p-value less than 0.05 was considered statistically significant.

Using RNA sequence analysis, we previously identified that mechanical compression induces TNC expression in non-asthmatic HBE cells [42] . Here, we compared TNC mRNA expression in HBE cells cultured from non-asthmatic and asthmatic donors at baseline and in response to mechanical compression (Figure 1 ). At baseline, TNC mRNA expression was marginally higher in asthmatic cells (1.5-fold, p = 0.042) compared to non-asthmatic cells only at the 3 h time point, but not at the 24 h time point ( Figure S1 ). In the non-asthmatic cells, TNC mRNA expression was significantly increased by 3.9-fold (p < 0.001) at 3 h and 2.9-fold (p < 0.01) at 24 h after compression compared to their time-matched controls ( Figure 1A ). Conversely, in the asthmatic cells, TNC mRNA expression was significantly increased by 2.1-fold (p < 0.01) at 3 h and 1.4-fold at 24 h after compression ( Figure 1B ). At either 3 or 24 h, the extent of the increased TNC mRNA expression in response to compression did not differ between non-asthmatic and asthmatic cells.

While mechanical compression induced TNC mRNA expression in HBE cells, we did not detect increased cellular TNC protein at any time points we examined (at 24, 48, and 72 h post-compression) ( Figure S2 ). To compare the amount of TNC secretion from HBE cells between non-asthmatic and asthmatic cells from four donors each, we measured the level of TNC protein by ELISA in the basolateral conditioned media collected at 24 h after compression ( Figure 1C ). Similar to our previous report [42] , TNC was constitutively secreted to the basolateral side of the well-differentiated HBE cells in ALI culture. At baseline, constitutive TNC secretion was significantly higher in asthmatic HBE cells than in non-asthmatic HBE cells (asthma: 22.3 vs. non-asthma: 5.8 ng/well, p < 0.05). In both non-asthmatic and asthmatic HBE cells, mechanical compression induced TNC secretion. While constitutive TNC secretion was higher in asthmatic cells, the amount of compressioninduced TNC secretion did not differ between non-asthmatic and asthmatic cells (nonasthma: 56 vs. asthma: 60 ng/well). . TNC mRNA expression was normalized to GAPDH. The amount/well of TNC secreted in the basolateral conditioned media was measured by ELISA (mean ± SEM, 4 non-asthma and 4 asthma donors) (C). ** p < 0.01, *** p < 0.001, **** p < 0.0001, significantly different from time-matched no pressure control, # p < 0.05 significantly different between non-asthma and asthma, analyzed by two-way ANOVA with Bonferroni's post-hoc test. Each symbol represents each of the four donors. The open symbols represent control condition, and the closed symbols represent compressed condition in both non-asthmatic (blue symbols; A and C) and asthmatic HBE cells (red symbols; B and C).

While mechanical compression induced TNC mRNA expression in HBE cells, we did not detect increased cellular TNC protein at any time points we examined (at 24, 48, and 72 h post-compression) ( Figure S2 ). To compare the amount of TNC secretion from HBE cells between non-asthmatic and asthmatic cells from four donors each, we measured the level of TNC protein by ELISA in the basolateral conditioned media collected at 24 h after compression ( Figure 1C ). Similar to our previous report [42] , TNC was constitutively secreted to the basolateral side of the well-differentiated HBE cells in ALI culture. At baseline, constitutive TNC secretion was significantly higher in asthmatic HBE cells than in non-asthmatic HBE cells (asthma: 22.3 vs. non-asthma: 5.8 ng/well, p < 0.05). In both nonasthmatic and asthmatic HBE cells, mechanical compression induced TNC secretion. While constitutive TNC secretion was higher in asthmatic cells, the amount of compression-induced TNC secretion did not differ between non-asthmatic and asthmatic cells (non-asthma: 56 vs. asthma: 60 ng/well). . TNC mRNA expression was normalized to GAPDH. The amount/well of TNC secreted in the basolateral conditioned media was measured by ELISA (mean ± SEM, 4 non-asthma and 4 asthma donors) (C). ** p < 0.01, *** p < 0.001, **** p < 0.0001, significantly different from time-matched no pressure control, # p < 0.05 significantly different between non-asthma and asthma, analyzed by two-way ANOVA with Bonferroni's post-hoc test. Each symbol represents each of the four donors. The open symbols represent control condition, and the closed symbols represent compressed condition in both non-asthmatic (blue symbols; A,C) and asthmatic HBE cells (red symbols; B,C).

To determine intracellular signaling pathways by which TNC is induced by mechanical compression, we first used bulk RNA sequencing analysis to identify mechanical compression-responsive genes that are regulated by either ERK or TGF-β receptor signaling pathways. We assessed differentially expressed (DE) genes at 3 and 24 h post-compression in the presence or absence of either inhibitor for MEK (U0126) or TGF-β receptor (SB431542). We identified TNC as one of the compression-responsive DE genes that was downregulated in the presence of either ERK or TGF-β inhibition. At 3 h, treatment with vehicle, U0126, or SB431542 did not affect the expression level of TNC. Contrarily, only pretreatment with U0126 (p < 0.0001) significantly decreased the mechanical compression-induced TNC expression (Figure 2A ). At 24 h, we observed that the baseline expression of TNC was decreased only by treatment with U0126 (p < 0.0001) but not with vehicle or SB431542. However, pretreatment with either U0126 (p < 0.0001) or SB431542 (p < 0.0001) blocked the mechanical compression-induced TNC expression ( Figure 2B ). vehicle, U0126, or SB431542 did not affect the expression level of TNC. Contrarily, only pretreatment with U0126 (p < 0.0001) significantly decreased the mechanical compressioninduced TNC expression (Figure 2A ). At 24 h, we observed that the baseline expression of TNC was decreased only by treatment with U0126 (p < 0.0001) but not with vehicle or SB431542. However, pretreatment with either U0126 (p < 0.0001) or SB431542 (p < 0.0001) blocked the mechanical compression-induced TNC expression ( Figure 2B ). To validate the data from the RNA sequencing analysis, we measured TNC expression by RT-qPCR in an independent experiment. Here, in three non-asthmatic donor HBE cells, we determined TNC mRNA expression at 24 h post-compression in the presence or absence of either U0126 or SB431542. At baseline, TNC mRNA expression was not significantly different in the cells between pretreated with the vehicle and either U0126 or SB431542 ( Figure 2C ). In response to mechanical compression, TNC mRNA expression To validate the data from the RNA sequencing analysis, we measured TNC expression by RT-qPCR in an independent experiment. Here, in three non-asthmatic donor HBE cells, we determined TNC mRNA expression at 24 h post-compression in the presence or absence of either U0126 or SB431542. At baseline, TNC mRNA expression was not significantly different in the cells between pretreated with the vehicle and either U0126 or SB431542 ( Figure 2C ). In response to mechanical compression, TNC mRNA expression was increased by 2.3-fold (p < 0.05) in the presence of vehicle control, while it was significantly attenuated by 0.97-fold (p < 0.01) and 0.93-fold (p < 0.01) in the presence of U0126 and SB431542, respectively ( Figure 2C ). These data demonstrate that pretreatment with either U0126 or SB431542 significantly attenuated the mechanical compression-induced TNC mRNA expression ( Figure 2C ).

In ALI culture, HBE cells are well-differentiated and polarized. In the polarized cells, intracellular signaling pathways may have a preferential effect on the protein trafficking to the basolateral side versus the apical side. Thus, here we measured both basolateral and apical secretions of TNC. First, we detected basolateral secretion of TNC by both Western blot analysis and ELISA ( Figure 3A,B) . The levels of secreted TNC detected by Western blot analysis and measured by ELISA were comparable. Similar to TNC mRNA expression, either inhibitor did not affect the constitutive secretion of basolateral TNC. At baseline, the amount of basolateral TNC was 10 ng/well. In response to mechanical compression, basolateral TNC was increased to 62 ng/well ( Figure 3B ). In the presence of either inhibitor, compression-induced basolateral TNC was significantly attenuated to 9 ng/well (U0126) and 12 ng/well (SB431542) ( Figure 3B ). Next, we examined the apical Cells 2022, 11, 256 7 of 13 secretion of TNC by ELISA because we validated the specificity of the ELISA assay using basolateral conditioned media. Similar to basolateral secretion, either inhibitor did not affect the constitutive secretion of apical TNC. At baseline, the amount of apical TNC was 3 ng/well. In response to mechanical compression, apical TNC was increased to 17 ng/well ( Figure 3C ). In the presence of either inhibitor, compression-induced apical TNC was significantly attenuated to 5 ng/well (U0126) and 11 ng/well (SB431542) ( Figure 3C ). These data indicate that TNC is secreted both apically and basolaterally, but the basolateral secretion of TNC is predominant and could be the major source of extracellular TNC. blot analysis and ELISA (Figures 3A,B) . The levels of secreted TNC detected by Western blot analysis and measured by ELISA were comparable. Similar to TNC mRNA expression, either inhibitor did not affect the constitutive secretion of basolateral TNC. At baseline, the amount of basolateral TNC was 10 ng/well. In response to mechanical compression, basolateral TNC was increased to 62 ng/well ( Figure 3B ). In the presence of either inhibitor, compression-induced basolateral TNC was significantly attenuated to 9 ng/well (U0126) and 12 ng/well (SB431542) ( Figure 3B ). Next, we examined the apical secretion of TNC by ELISA because we validated the specificity of the ELISA assay using basolateral conditioned media. Similar to basolateral secretion, either inhibitor did not affect the constitutive secretion of apical TNC. At baseline, the amount of apical TNC was 3 ng/well. In response to mechanical compression, apical TNC was increased to 17 ng/well ( Figure 3C ). In the presence of either inhibitor, compression-induced apical TNC was significantly attenuated to 5 ng/well (U0126) and 11 ng/well (SB431542) ( Figure 3C ). These data indicate that TNC is secreted both apically and basolaterally, but the basolateral secretion of TNC is predominant and could be the major source of extracellular TNC. Representative Western blot of three independent experiments shows the detection of basolateral secretion of TNC. Transferrin was detected as a loading control (A). Secreted TNC were detected by ELISA in the basolateral conditioned media (B) and in the apical washes (C) (mean ± SEM, 3 non-asthma donors). **** p < 0.0001, significantly different from vehicle control; $$$$ p < 0.0001, significantly different from vehicle with pressure, analyzed by two-way ANOVA with Bonferroni's post-hoc test. Each symbol represents each of the three donors. The open symbols represent control condition, and the closed symbols represent compressed condition in non-asthmatic HBE cells (blue symbols; B,C).

To determine the mechanism of extracellular TNC secretion from compressed HBE cells, we isolated the EV fraction from basolateral conditioned media. We used Western blot analysis to assess the three fractions obtained during the isolation process: (1) the whole conditioned media, (2) non-EV fraction, and (3) EV fraction. In the whole conditioned media from compressed cells, we detected TNC and the following three proteins: YKL-40, a marker for the non-EV fraction, and CD9 and tissue factor, the markers for the EV fraction [13, 44, 50, [57] [58] [59] (Figure 4 ). In the EV fraction, we detected CD9 and tissue factor. In the non-EV fraction, we detected YKL-40. While TNC protein was detected predominantly in the non-EV fraction, it was also prominently detected in the EV fraction suggesting TNC is secreted by multiple mechanisms. tioned media from compressed cells, we detected TNC and the following three proteins: YKL-40, a marker for the non-EV fraction, and CD9 and tissue factor, the markers for the EV fraction [13, 44, 50, [57] [58] [59] (Figure 4 ). In the EV fraction, we detected CD9 and tissue factor. In the non-EV fraction, we detected YKL-40. While TNC protein was detected predominantly in the non-EV fraction, it was also prominently detected in the EV fraction suggesting TNC is secreted by multiple mechanisms. 

This study examines the role of mechanical compression that is associated with bronchospasm in the modulation of the extracellular environment during the development of asthma. We focused on TNC, an ECM glycoprotein that is highly expressed in asthmatic airways and differentially overexpressed in compressed HBE cells [29] [30] [31] [32] [33] [34] 42] . The mechanical compression that we applied to cells was comparable to the magnitude that is associated with maximal bronchospasm during asthma exacerbations [5, 60] . In both nonasthmatic and asthmatic HBE cells, our data demonstrate that mechanical compression increased TNC mRNA expression and protein secretion. Constitutive TNC secretion detected at baseline was greater in asthmatic cells compared to non-asthmatic cells, but stimulated TNC secretion in response to mechanical compression was substantially greater than at baseline but not different between asthmatic and non-asthmatic cells. Our data further indicate that TNC mRNA expression and protein secretion depended on the activation of ERK or TGF-β receptors. Furthermore, our data reveal that mechanically compressed HBE cells released EVs that contain TNC.

In patients with asthma, abundant TNC expression in the airway is detected in the subepithelial basement membrane and significantly increased after allergen challenge [29] [30] [31] [32] . While the source of this extracellular TNC protein in the airway basement membrane has not previously been elucidated, our data suggest that airway epithelial cells are a major source of this elevated extracellular TNC. Here, we validated that well-differentiated bronchial epithelial cells are a source of extracellular TNC [42] and discovered that TNC is constitutively secreted from HBE cells even in the absence of stimulation ( Figures  1C and 3 ). At baseline, the constitutive secretion of TNC from asthmatic HBE cells was significantly higher than that from non-asthmatic HBE cells ( Figure 1C ). Similar to the previously reported RNA sequencing analysis [8] , we detected higher TNC mRNA Representative Western blots of four independent experiments show that CD9, tissue factor, YKL-40, and TNC were detected in the whole conditioned media with a greater abundance in conditioned media from compressed cells. YKL-40 was detected in the non-EV fraction, while CD9 and tissue factor were detected in the EV fraction. TNC was detected in both the non-EV and the EV fractions.

This study examines the role of mechanical compression that is associated with bronchospasm in the modulation of the extracellular environment during the development of asthma. We focused on TNC, an ECM glycoprotein that is highly expressed in asthmatic airways and differentially overexpressed in compressed HBE cells [29] [30] [31] [32] [33] [34] 42] . The mechanical compression that we applied to cells was comparable to the magnitude that is associated with maximal bronchospasm during asthma exacerbations [5, 60] . In both non-asthmatic and asthmatic HBE cells, our data demonstrate that mechanical compression increased TNC mRNA expression and protein secretion. Constitutive TNC secretion detected at baseline was greater in asthmatic cells compared to non-asthmatic cells, but stimulated TNC secretion in response to mechanical compression was substantially greater than at baseline but not different between asthmatic and non-asthmatic cells. Our data further indicate that TNC mRNA expression and protein secretion depended on the activation of ERK or TGF-β receptors. Furthermore, our data reveal that mechanically compressed HBE cells released EVs that contain TNC.

In patients with asthma, abundant TNC expression in the airway is detected in the subepithelial basement membrane and significantly increased after allergen challenge [29] [30] [31] [32] . While the source of this extracellular TNC protein in the airway basement membrane has not previously been elucidated, our data suggest that airway epithelial cells are a major source of this elevated extracellular TNC. Here, we validated that well-differentiated bronchial epithelial cells are a source of extracellular TNC [42] and discovered that TNC is constitutively secreted from HBE cells even in the absence of stimulation ( Figures 1C and 3 ). At baseline, the constitutive secretion of TNC from asthmatic HBE cells was significantly higher than that from non-asthmatic HBE cells ( Figure 1C ). Similar to the previously reported RNA sequencing analysis [8] , we detected higher TNC mRNA expression in asthmatic cells at one of the two time points ( Figure S1 ). However, due to the small sample size and the high variability among asthmatic cells, this has to be further examined using asthmatic cells that can be associated with the status and history of the disease. In both non-asthmatic and asthmatic HBE cells, mechanical compression caused increased mRNA expression and secreted protein of TNC ( Figure 1A-C) . However, there was no significant difference in the levels of stimulated TNC production between non-asthmatic and asthmatic HBE cells. These findings indicate that, regardless of the disease state and in the absence of inflammatory cells, bronchospasm alone can induce TNC mRNA expression and extracellular TNC production from airway epithelial cells. These findings highlight the important role of bronchospasm in airway physiology beyond asthma because bronchospasm is also experienced in individuals with obesity, with other chronic lung diseases including COPD, or after exposure to environmental pollutants such as particulate matter or ozone [61] [62] [63] [64] .

While TNC mRNA expression and protein secretion are associated with multiple pathologic conditions [17, 18, [36] [37] [38] [39] [40] [41] , underlying regulatory mechanisms in HBE cells are yet to be fully elucidated. Among multiple pathologic factors, TGF-β is a potent inducer of TNC expression in various cell types, including fibroblasts [26, 38, 65] . The TGF-β receptor transduces signals through canonical and non-canonical pathways [66] . The canonical TGF-β pathway is primarily mediated through the SMAD-dependent pathway, while the non-canonical pathway is mediated through several SMAD-independent pathways, including ROCK, JNK, and ERK [66] . In fibroblasts, both SMAD and non-SMAD pathways are involved in the TGF-β-induced TNC expression [38, 67] . In HBE cells, TGF-β secretion is increased by mechanical compression and plays an integral role in goblet cell hyperplasia, fibrosis, and subepithelial collagen deposition [9, 14, 68] . Thus, we hypothesized that in HBE cells TGF-β signaling pathway could regulate mechanical compression-induced TNC production. Pharmacological inhibitors of either ERK or TGF-β receptor I activity significantly attenuated mechanical compression-induced TNC mRNA expression (Figure 2) , suggesting that the transcriptional regulation of epithelial TNC expression depends on ERK or the TGF-β receptor. In addition, inhibition of ERK or TGF-β receptor subsequently attenuated mechanical compression-induced secretion of TNC both basolaterally and apically ( Figure 3 ). While we did not further determine if the attenuated secretion was the result of attenuated TNC mRNA expression or an attenuated secretory mechanism, our data clearly indicate that TNC secretion from bronchial epithelial cells depends on either ERK or the TGF-β receptor. Another limitation is that we did not compare apical secretion between non-asthmatic HBE cells and asthmatic HBE cells. Because the apical and basal surfaces of the epithelium interact with different types of immune and resident cells, the function and preference of the secreted TNC to either compartment should be a future direction to investigate. Despite this limitation, our data show that the amount of basal secretion is significantly greater than that of an apical secretion at either baseline or in response to mechanical compression.

EVs play a key role in cell-to-cell communication by transporting various cellular cargo from the donor cells to the recipient cells [44, 45, 59, 69, 70] . Thus, EVs were explored as a mechanism of exporting various biological components from the cells to the extracellular environment [43] . Extracellular TNC was detected in EVs released from various cancer cell types [43, 71] Clinically, EVs containing TNC were detected in the plasma of patients with cancer [43, 72, 73] . In patients with COVID-19, EVs isolated from plasma contain abundant TNC compared with that from healthy controls [74] . This study further revealed that these circulating EVs can trigger proinflammatory signals in the cells of a distant organ [74] . In addition to the observations of TNC contained in EVs, EVs are suggested as a required mechanism for the extracellular deposition of TNC by tumor cells and fibroblasts [43, 75, 76] . In a study using mouse embryonic fibroblasts, the abrogation of EV release correlates with impaired TNC deposition and leads to intracellular accumulation of TNC [75] . In the same study, the delivery of TNC-containing EVs into TNC knockout mice by intravenous injection resulted in the accumulation of TNC in different organs including the liver and lungs, suggesting that EVs contribute significantly to extracellular TNC deposition. Additionally, in the study using bronchial epithelial cell line, BEAS-2B cells, TNC secretion is equally dependent on the non-EV mediated and EV-mediated pathways [41] .

Here we examined extracellular TNC secretion from HBE cells in response to mechanical compression. Our previous studies demonstrate that mechanically compressed HBE cells release EVs that contain tissue factor protein [13, 50] . The tissue factor was exclusively contained in the EV fraction, and here we used tissue factor as a marker of the EV fraction. In this study, we identified that TNC secreted from compressed HBE cells was contained in both non-EV and EV fractions (Figure 4 ). This suggests that extracellular TNC secreted from airway epithelial cells depends on both non-EV mediated and EV-mediated pathways in the lung. While abundant TNC is detected in the subepithelial basement membrane, the EV-mediated secretion of TNC potentiates the role of extracellular TNC not only in the lung but also outside of the lung. For example, in the patients with refractory neutrophilic asthma, serum TNC level is significantly higher than that in either the subjects with non-refractory asthma or healthy individuals [34] suggesting that TNC may contribute to persistent asthma symptoms or inflammation in patients with uncontrolled and severe asthma. The cellular cargo contained in EVs can remain stable for a prolonged period of time and be transported to cells in distant organs [44, 45, 59, 69, 70] . This is the first study to show that a non-inflammatory cue can induce the release of EVs that contain an ECM protein, TNC. To identify additional proteins contained in epithelial cell-derived EVs that may play critical roles in ECM deposition in asthma, proteomic analysis on the EVs is required.

In summary, our data provide a causal link between bronchospasm and extracellular TNC production from airway epithelial cells. We showed that asthmatic HBE cells constitutively secrete greater amounts of TNC compared to non-asthmatic HBE cells, suggesting that epithelial cell-derived TNC could be a major source of extracellular TNC deposited in the airway of patients with asthma. TNC secretion from HBE cells is substantially induced by mechanical compression in the absence of inflammatory cells, suggesting that bronchospasm alone can further augment the extracellular TNC production during asthma exacerbations. Importantly, the role of bronchospasm in TNC secretion may not be limited to patients with asthma because the cells from donors with no history of lung diseases also showed increased secretion of TNC by mechanical compression. These data indicate that TNC can be increasingly secreted from individuals who are prone to experience bronchospasm due to underlying conditions such as obesity or recurrent exposures to environmental pollutants. Finally, TNC contained in EVs that are released from compressed HBE cells could be transported to distal tissues. Our data reveal a novel mechanism by which bronchospasm alone is sufficient to induce the deposition of extracellular matrix protein in the airway, thereby contributing to remodeling.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/cells11020256/s1, Figure S1 : At baseline, TNC mRNA expression is marginally higher in asthmatic HBE cells at 3hr; Figure S2 : Compression does not increase the cellular expression of TNC; Table S1 : Donor information for primary HBE cells. 

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Biological properties of extracellular vesicles and their physiological functions

Extracellular vesicles as emerging intercellular communicasomes

In well-differentiated primary human bronchial epithelial cells, TGF-beta1 and TGF-beta2 induce expression of furin

Unjamming and cell shape in the asthmatic airway epithelium

IL-13 Augments Compressive Stress-Induced Tissue Factor Expression in Human Airway Epithelial Cells

Transcriptional profiling of mucociliary differentiation in human airway epithelial cells

Well-differentiated human airway epithelial cell cultures

Salmon provides fast and bias-aware quantification of transcript expression

Differential analyses for RNA-seq: Transcript-level estimates improve gene-level inferences

Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2

Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method

Minimal experimental requirements for definition of extracellular vesicles and their functions: A position statement from the International Society for Extracellular Vesicles

Immobilization and detection of platelet-derived extracellular vesicles on functionalized silicon substrate: Cytometric and spectrometric approach

Specificities of secretion and uptake of exosomes and other extracellular vesicles for cell-to-cell communication

Mechanotransduction through growth-factor shedding into the extracellular space

Effects of obesity on respiratory resistance

Obesity alters the topographical distribution of ventilation and the regional response to bronchoconstriction

Chronic obstructive pulmonary disease: Molecular and cellular mechanisms

Risk of bronchi obstruction among non-smokers-Review of environmental factors affecting bronchoconstriction

The regulation of tenascin expression by tissue microenvironments

Smad-dependent and Smad-independent pathways in TGF-beta family signalling

Tenascin-C upregulation by transforming growth factor-beta in human dermal fibroblasts involves Smad3, Sp1, and Ets1

Effect of bronchoconstriction on airway remodeling in asthma

Intercellular Communication between Airway Epithelial Cells Is Mediated by Exosome-Like Vesicles

Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles

Proteome profiling of exosomes derived from human primary and metastatic colorectal cancer cells reveal differential expression of key metastatic factors and signal transduction components

Tenascin-C: Form versus function

Extracellular matrix proteins and carcinoembryonic antigen-related cell adhesion molecules characterize pancreatic duct fluid exosomes in patients with pancreatic cancer

Exosomes from COVID-19 Patients Carry Tenascin-C and Fibrinogenbeta in Triggering Inflammatory Signals in Cells of Distant Organ

ECM deposition is driven by caveolin-1-dependent regulation of exosomal biogenesis and cargo sorting

Caveolin-1-containing extracellular vesicles transport adhesion proteins and promote malignancy in breast cancer cell lines

The authors thank Scott Randell and the Cystic Fibrosis Center Tissue Procurement and Cell Culture Core (The University of North Carolina, Chapel Hill) for providing primary human bronchial epithelial cells.

The authors declare no conflict of interest.