Endoplasmic reticulum stress mediates house dust mite-induced airway epithelial apoptosis and fibrosis

A human bronchial epithelial cell line (HBE) was kindly provided by Dr. Albert van der Vliet-University of Vermont, and cultured as described previously [22, 23] and primary human nasal epithelial cells were cultured as described previously [24]. Human cell lines were exposed to either PBS or 25 μg/ml of HDM (Greer, Lenoir, NC). All protocols that utilize primary human nasal epithelial cells were approved by the University of Vermont Institutional Review Board. Cells were transfected with plasmids or siRNA as described [25, 26]. Caspase-3 activities were measured using Caspase-Glo 3 (Promega, Madison, WI) reagents, according to the manufacturer’s protocol (Promega, Madison, WI). Results were expressed in Relative Luminescence Units (RLU), after subtraction of background luminescence values. Cell death was measured by MTT assay [25]. All results were obtained from 3 independent experiments conducted in triplicate.

For all experiments, 8 to 12 wk old WT BALB/c mice (Jackson Laboratories) were used, as approved by the Institutional Animal Care and Use Committee. Mice (n = 10/group) were anesthetized with isofluorane and exposed to 50 μg of the allergen, HDM (GREER-containing 35 endotoxin units/mg) extract, resuspended in PBS, via intranasal administration on day 0 and boosted again on day 7. Mice were then administered 50 μg of HDM consecutively on days 14–18, and euthanized 48 h post final exposure. The control group was given 50 μl of sterile PBS alone at all time points. Alternatively, mice were sensitized via oropharyngeal administration of 100 μg of low endotoxin Ovalbumin (Grade V, Sigma Aldrich) in PBS with 0.1 μg of LPS on days 0 and 7, challenged using 6 doses of aerosolized 1% OVA in PBS for 30 min on days 14–19, and euthanized on day 21. This protocol was adapted from a previously described method of airway sensitization and challenge [27].

Mice (n = 10/group) were anesthetized with isofluorane and administered 10 mg/kg of scrambled small interfering (si) RNA or siRNA for ERp57 (Thermo Scientific-L45187) and ATF6α (ORIGENE-SR418766) oropharyngeally on days −1, 6, and 13, and again on days 16 and 19X. Simultaneously, mice were exposed to 50 μg of HDM resuspended in PBS, or PBS alone via intranasal administration on days 1 and 7. Mice were then administered 50 μg of HDM consecutively, on days 14–18 and euthanized 72 h following the final HDM exposure. On day 16, when siRNA administration coincided with HDM exposure, mice received siRNA 6 h prior to intranasal administration of HDM.

Mice (n = 10/group) were anesthetized with an intraperitoneal injection of pentobarbital sodium (90 mg/kg), tracheotomized using an 18 gauge cannula, then mechanically ventilated at 200 breaths/min using a FlexiVent™ computer controlled small animal ventilator (SCIREQ). While on the ventilator mice also received the paralytic, pancuronium bromide. The parameters Newtonian resistance (Rn), tissue damping (G), and elastance (H) were calculated as previously described [28, 29]. Airway responsiveness is represented as the average of the 3 peak measurements for each animal, obtained at incremental methacholine doses.

Bronchoalveolar lavage (BAL) from mice (n = 10/group) was collected. Total and differential cell counts were performed as previously described [20]. Briefly, cells were isolated by centrifugation and total cell counts were enumerated using the Advia 120 automated hematology analyzer system. Differential cell counts were obtained via cytospins using Hema3 stain reagents (Fisher Scientific). Differentials were performed on a minimum of 300 cells per animal.

Following dissection, right lung lobes were flash frozen for protein analysis. Lungs were pulverized, and lysed in buffer containing 137 mM Tris∙HCl (pH 8.0), 130 mM NaCl, and 1% NP-40. Proteins from cell lysates were prepared in the same buffer. Insoluble proteins were pelleted via centrifugation, and following protein quantitation of the supernatant, samples were resuspended in loading buffer with dithiothrietol (DTT), and resolved by SDS-PAGE. Proteins were transferred to PVDF and membranes were probed using a standard immunoblotting protocol using the following primary antibodies: P-IRE, IRE, GRP78, ATF6⁵⁰ and CHOP (Abcam), ERp57, GRP94 (Stressgen), Poly (ADP-ribose) polymerase (PARP) (BD Pharmingen) and β-actin (Sigma).

Lung homogenates were resuspended in loading buffer without the reducing agent dithiothrietol (DTT). A separate set of samples were resuspended in loading buffer with DTT to reduce the disulfide bonds. The samples were resolved by SDS-PAGE and subjected to western blot analysis.

Following euthanization, left lobes were fixed with 4% paraformaldehyde, stored at 4°C overnight for fixation of the tissue, mounted in paraffin, and 5 μm sections were affixed to glass microscope slides for histopathology as previously described [30]. Sections were prepared for immunofluorescence by deparaffinizing with xylene and rehydrating through a series of ethanols [30]. For antigen retrieval, slides were heated for 20 min in 95°C citrate buffer (pH 6.0) with 0.05% TWEEN-20 then rinsed in distilled water. Sections were then blocked for 1 h in 1% bovine serum albumin (BSA) in PBS, followed by incubation with primary antibody for ERp57 (Stressgen), and Caspase-3 (Cell Signal) at 1:500, overnight at 4°C. Slides were then washed 3x5min in PBS, incubated with Alexafluor 647 at 1:1000 in 1% BSA, and counterstained with DAPI in PBS at 1:4000 for nuclear localization. Sections were imaged using a Zeiss 510-META confocal laser scanning microscope.

Collagen content was measured via the Sircol assay (n = 10/group) (Biocolor Ltd, UK). Briefly, lung lobes were diced and placed in 500 μl of 10 mg/mL pepsin in 0.5 M acetic acid for 3 h at 37°C, or until lungs were completely digested. The digest was spun at 10,000 g for 10 min at room temperature. Fifty microliters of the supernatant was mixed vigorously with 500 μL of sircol dye solution for 30 min and then spun again at 10,000 g for 10 min. Excess dye was decanted off, and the resulting pellet was dissolved in 500 μL of an alkaline solution, 200 μL of which was pipetted in duplicates into a 96 well plate and measured at 540 nm. To evaluate regional changes in alpha-smooth muscle actin (αSMA), fixed sections were prepared for immunostaining by deparaffinizing with xylene and rehydrating through a series of ethanols. For antigen retrieval, slides were heated for 20 min in 95°C citrate buffer (pH 6.0), then rinsed in distilled water. Sections were then blocked for 1 h in blocking serum as per manufacturer’s instructions (Vectastain Alkaline Phosphatase Universal, Vector). Slides were then washed in TBS with 0.1% TWEEN-20 3×5 min, followed by incubation with primary antibody for αSMA (Sigma) overnight at 4°C. Sections were washed again and incubated with a biotinylated universal secondary antibody (Vectastain Alkaline Phosphatase Universal, Vector) for 30 min at room temperature. Slides were washed and incubated with the Vectastain ABC-AP reagent (prepared as per manufacturer’s instructions) for 30 min at room temperature. Sections were then incubated with Vector Red Alkaline Phosphatase Substrate Kit I (Vector) for 10 min at room temperature, rinsed with tap water, and counterstained with Mayer’s Hemotoxylin.

All assays were performed in triplicates. Data were analyzed by one-way analysis of variance (ANOVA) using the Tukey’s test to adjust for multiple comparisons or student’s t test where appropriate. Histopathological scores were analyzed using the Kruskal-Wallis test and Dunn's multiple comparison post hoc tests. Data from multiple experiments were averaged and expressed as mean values ± SEM.

HDM is a complex allergen known to activate multiple receptors and their consequent downstream pathways [5]. In the current study, we hypothesized that these events would result in increased ER stress in epithelial cells. To address this hypothesis, primary human nasal epithelial (PHNE) cells from two non-asthmatic subjects and a human bronchial epithelial (HBE) cell line were challenged with either HDM or PBS as a control. The optimal dose of 25 μg/ml-HDM was selected based on our prior analysis in the laboratory, which showed induction of inflammatory as well as robust ER stress responses in lung epithelial cells (data not shown). Seventy-two hours following repeated challenge, both subjects exhibited increases in phosphorylation of IRE1 (P-IRE), albeit to a greater extent in cells from subject 2, as well as increases in ER chaperone GRP78 (Bip), GRP94, and ERp57. ER stress transducer-ATF6α and downstream transcriptional effector CHOP were also increased after HDM exposure (Figure 1A). With the exception of P-IRE, HBE cells responded in a similar manner with slight differences in kinetics between members of the ER stress responders (Figure 1A). As previously shown, physiological processes demanding a high rate of protein synthesis and secretion may lead to unresolved ER stress resulting in apoptosis [14]. Accordingly, allergen exposure resulted in significant activation of caspase-3 at varying levels in both PHNE and HBE cells (Figure 1B).

During ER stress, activation of ATF6α is known to specifically up regulate PDIs, chaperones, as well as CEBP homologous protein CHOP, and consequently, these events are known to lead to apoptosis [13, 31]. To address the contribution of ATF6α activation, chaperone induction, and downstream activation of apoptosis, HBE cells were transfected with either scrambled small interfering (si) RNA (Si-scr) or siRNA for ATF6α. Twenty-four hours following transfection, cells were stimulated with 25 μg of HDM or PBS and harvested at 48 and 72 h after exposure. Knockdown of ATF6α in HBE cells resulted in decreased activation of the 50 kD fragment of ATF6α, CHOP, and ERp57 in whole cell lysates, indicating a requirement for ATF6 in HDM-driven expression of CHOP and ERp57 (Figure 1C). Following HDM administration, active caspase-3 was increased and cell survival was decreased in scrambled siRNA transfected HBE cells. Knockdown of ATF6α in HBE cells showed significant decrease in caspase-3 activity and an increase in cell survival (Figure 1D and E) in cell treated with HDM. These results indicate that allergen (HDM) exposure can induce ER stress, and in turn, lead to apoptosis in human lung epithelial cells.

To elicit allergic airways disease we challenged mice with a ubiquitous allergen-HDM, or Ovalbumin and compared the responses with mice treated with LPS (a model of acute lung injury and inflammation). Mice were initially sensitized and challenged via intranasal administration of HDM, LPS or low endotoxin OVA with 0.1 μg of LPS (as an adjuvant) and were euthanized on day 21 [27] (Figure 2A). Results in Figure 2B and Additional file 1: Figure S1 demonstrates activation of ER stress in response to HDM or OVA/LPS in the whole lung, as evidenced by increases in phosphorylation of IRE1, as well as increased expression of GRP78, GRP94 and ERp57. ATF6α and CHOP were also increased after HDM exposure as compared to controls. In contrast to our observation in the HDM model, ATF6α did not appear to increase after OVA/LPS, but we observed a slight elevation in GRP94 and CHOP expression (Figure 2B and Additional file 1: Figure S1). Analysis of inflammatory cells showed a significant increase in eosinophils and lymphocytes in both models as compared to controls (Table 1). Macrophages were decreased in HDM challenged mice as compared to PBS controls, while in LPS and OVA/LPS challenged mice there was a significant increases in macrophages (Table 1). Immunofluorescence of HDM-instilled lungs indicated increased ERp57 as well as active caspase-3 predominantly in the bronchiolar epithelium, as compared to controls (Figure 2C). Collectively these results demonstrate that HDM is a potent activator of ER stress in murine models of allergic airway disease, in comparison to Ovalbumin. Based on these results we continued our evaluations of the role of ER stress in allergic airways disease using the HDM model.

To evaluate the role of ATF6α and ERp57 in HDM-induced allergic airways disease in vivo, mice were administered 10 mg/kg oropharyngeal scrambled siRNA (Si-scr) or siRNA targeting ERp57 and ATF6α. Subsequently, the mice were challenged via the intranasal administration with 50 μg of HDM consecutively on days 15–19 and euthanized 72 h after final challenge (Figure 3A). As expected, protein expression of ERp57 and ATF6α in the whole lung was decreased after repeated HDM exposure in mice that received siRNA for ERp57 and ATF6α (Si-ERp57 + ATF6) compared to siRNA control samples (Si-scr). We also observed decreases in CHOP and GRP78 in HDM challenged mice treated with Si-ERp57 + ATF6α as compared to Si-scr. The elevated expression of GRP94 was not altered following HDM challenge and treatment with Si-ERp57 + ATF6α relative to Si-scr HDM groups (Figure 3B and Additional file 1: Figure S2), demonstrating that not all ER stress responders were altered by knockdown of ERp57 and ATF6α. ER stress-induced ERp57 is known to form disulfide (−S-S-) bridges in proapoptotic Bak [16]. Results in Figure 3C and Additional file 1: Figure S2 demonstrate that siRNA-mediated knockdown of ERp57 and ATF6α resulted in significantly decreased -S-S- mediated oligomerization of Bak, manifested by decreases in the 75 and 50 kD forms of Bak. Furthermore, we also observed that caspase-3 activity and Poly (ADP-ribose) Polymerase (PARP), a target for caspase-3 cleavage during apoptosis, was significantly decreased in the whole lung lysates after ATF6α and ERp57 knockdown following HDM challenge compared to Si-scr controls (Figure 3D and E). This indicates that knockdown of ATF6α and ERp57 results in significant decrease in HDM-induced, ER stress-mediated apoptotic cascade in the lung.

As expected, animals in the Si-scr or Si-ERp57 + ATF6 groups that were not exposed to HDM exhibited primarily macrophages in BALF. However, mice that received Si-scr and were challenged with HDM showed a marked influx of cells into the airways, characterized by increases in eosinophils, lymphocytes, and to a lesser extent, neutrophils. In contrast, knockdown of ERp57 and ATF6α resulted in decreases in HDM-induced eosinophils and lymphocytes (Figure 4A, B, C, and D). As expected, we observed significant increases in IL-13 mRNA in HDM challenged mice as compared to PBS controls. Additionally, there was no significant increase in IFNγ mRNA (Additional file 1: Figure S3), indicating that instillation of double stranded siRNA did not alter HDM-induced responses from Th2 to Th1. To address the functional effects of siRNA-mediated knockdown of ERp57 and ATF6α in the airways, a forced oscillation technique was used to evaluate alterations in respiratory mechanics [28, 29] in response to HDM challenge. We did not observe any significant differences in central airway resistance (R_n) at either the lowest or highest dose of methacholine in HDM-challenged siRNA-treated groups of mice, but we did observe significant increases in R_n in mice that received Si-ERp57 + ATF6α as compared to Si-scr, after HDM challenge (Figure 5A). In the peripheral airways, tissue resistance/dampening (G) and elastance/stiffness (H) were significantly decreased in mice that received Si-ERp57 + ATF6α as compared to Si-scr, after HDM challenge (Figure 5B and C). Although our analysis revealed an increase in Muc5AC mRNA levels after HDM challenge, there were no significant differences in levels of Muc5AC mRNA or Periodic Acid Schiff (PAS) staining in HDM-challenged ERp57 + ATF6α-siRNA treated groups (data not shown).

To examine the role of ATF6α and ERp57 in airway remodeling, collagen deposition was evaluated following siRNA-administration and HDM exposure. Analysis of deposition of collagen by Masson’s Trichrome in mice receiving HDM and Si-scr exhibited significant increases over PBS controls, whereas animals that were treated with HDM and received si-ERp57 + ATF6α showed decreases in Masson’s trichrome staining (Figure 6A) as compared to HDM treated, Si-scr animals. Semi quantitative scoring for Masson’s trichrome staining by three independent scientists blinded to the identity of the samples revealed significant decreases in HDM challenged si-ERp57 + ATF6α treated mice (Figure 6B) over HDM Si-scr treated animals. Additionally, alpha-smooth muscle actin (αSMA) was increased in the peribronchiolar region of HDM challenged mice treated with Si-scr (Figure 6C) as compared to HDM challenged si-ERp57 + ATF6α treated mice. Western blot analysis of whole lung lysates also showed a significant increase in αSMA and Fibroblast Specific Protein (FSP-1) in HDM challenged mice treated with Si-scr (Figure 6D and Additional file 1: Figure S4). These increases were attenuated in mice challenged with HDM and treated with si-ERp57 + ATF6α (Figure 6D and Additional file 1: Figure S4). Furthermore, we also observed an increase in total collagen content in whole lung lysates from HDM challenged mice treated with Si-scr as compared to mice challenged with HDM and treated with si-ERp57 + ATF6α (Figure 6E). Collectively these results indicate that ER stress mediators act to control allergen induced airway fibrosis in the lung.