Endoplasmic Reticulum Stress and Unfolded Protein Response Sensor ERN1 Regulates Organic Dust Induction of Lung Inflammation

ABSTRACT

Inhalation of organic dust increases the risk for respiratory symptoms and respiratory diseases, with chronic inflammation playing a major role in their development. Previously, we reported that organic dust induction of inflammatory mediators in bronchial epithelial cells is mediated through increase of intracellular reactive oxygen species (ROS) and activation of NFκB and Stat3. Oxidative stress caused by increased ROS has been linked to the activation of endoplasmic reticulum (ER) stress and unfolded protein response (UPR). UPR modulates immune responses and plays key roles in the development of acute and chronic diseases. Herein, we hypothesized that organic dust‐induced ER stress‐UPR regulates airway epithelial cell inflammatory responses. We found that poultry organic dust extract (referred to as dust extract) increased the expression of ER stress/UPR sensor ERN1 in Beas2B bronchial epithelial cells. Dust extract was also found to increase ERN1 protein levels in mouse lungs with ERN1 immunostaining detected predominantly in the bronchial epithelium. Additionally, dust extract increased Ser724 ERN1 phosphorylation in the mouse bronchial epithelium indicating activation. Chemical inhibition and mRNA knockdown studies revealed that TLR2/TLR4‐Myd88‐ROS‐NFκB/Stat3 pathway mediates ERN1 induction. ERN1 chemical inhibitors, KIRA6 and APY29, and ERN1 mRNA knockdown reduced the induction of IL6, CXCL8, and pro IL1β. KIRA6 inhibited dust extract stimulation of NFκB‐p65, Stat3, Jun and MAPK 8/9 phosphorylation. Our studies have shown that ER stress and ERN1 are new players in the control of organic dust induced lung inflammation. Cross‐regulation between members of cell signaling cascade, TLR2‐TLR4/MyD88/ROS/ERN1/NFκB/Stat3 may fine tune immune and inflammatory responses elicited by organic dust.

Endoplasmic Reticulum (ER) Stress‐Unfolded Protein Response (UPR) sensor ERN1 regulates organic dust induction of lung inflammation. Treatment of bronchial epithelial cells and mouse lungs with organic dust extract increased ERN1 expression and activation. ERN1 induction was mediated by a signaling pathway involving TLRs 2 and 4, MyD88, ROS,NFkB and Stat3. In turn, ERN1 was found to contribute to the induction of lung inflammation by regulating the levels of proinflammatory cytokines via NFkB, Stat3, and Jun activation. Cross‐regulation between ERN1 and NFkB/Stat3 suggests a potential feedback loop that may fine‐tune organic dust induced lung immune and inflammatory responses.

1. Introduction

Agriculture, which encompasses crop and livestock production, forestry, and fisheries, plays a major role in the United States economy [1]. Roughly 2.4 million workers were employed in the United States agricultural industry, generating a total income of $ 182 billion in 2022 [2, 3]. Industrial‐scale livestock production, also known as concentrated animal feeding operations (CAFO) has been practiced for poultry since the 1950s and was later adopted for swine and cattle farming [4]. Because of the large number of animals in confined spaces, CAFOs can generate high levels of aerosolized dust, contaminating the indoor environment. Exposure to aerosolized dust increases workers' risk of developing respiratory symptoms and respiratory diseases [5, 6, 7]. Respiratory diseases such as asthma, chronic bronchitis, chronic obstructive pulmonary disease (COPD), allergic alveolitis, and organic dust toxic syndrome (ODTS) are common among agricultural workers [5, 8]. Respiratory symptoms and chronic bronchitis were found to be more prevalent in poultry farm workers than in other agricultural workers [8, 9, 10].

Poultry organic dust is a complex mixture of feathers, dander, feed, feces, bedding material, microorganisms, and microbial products such as lipopolysaccharide, peptidoglycan, and others [5, 11]. Additionally, noxious gases produced during the decomposition of waste materials, such as methane, ammonia, and hydrogen sulfide, are found associated with the dust particles [5]. Recent studies have identified chicken feces as a major contaminant of poultry organic dust, with its content ranging from 60% to 95% [12]. We recently reported that bacterial extracellular vesicles [13] and chicken trypsin [14] serve as proinflammatory constituents of poultry organic dust. Aqueous extracts of different organic dusts were found to induce the secretion and expression of cytokines such as interleukin (IL) 6, CXC motif chemokine ligand (CXCL) 8, and tumor necrosis factor alpha (TNF) in vitro in airway epithelial, macrophage, and monocytic cell lines [15, 16, 17, 18, 19]. Intranasal administration of organic dust extracts [20, 21], organic dust‐derived bacterial EVs [13], and purified trypsin from organic dust [14] were found to increase the lung levels of CXCL1, IL6, and TNF in mice. Enhanced cytokine levels were found to be associated with increases in bronchoalveolar lavage fluid (BALF) inflammatory cell counts [13, 20]. Exposure to the swine barn environment induced bronchial responsiveness, fever, and malaise in naïve human subjects in as little as 2 to 5 h after the exposure [22, 23]. These symptoms were found to be associated with increased counts of BALF neutrophils, eosinophils, and lymphocytes [24]. Among the inflammatory cells, neutrophil counts increased by greater than 50‐fold, whereas alveolar macrophage and lymphocyte counts increased by 2–3 fold [25].

Our published studies have shown that poultry organic dust extract increases inflammatory mediators' levels in bronchial epithelial cells via elevated levels of reactive oxygen species (ROS) and activation of protein kinase C (PKC), nuclear factor kappa‐light‐chain‐enhancer of activated B cells (NFκB) and signal transducer and activator of transcription 3 (Stat3) [21, 26, 27]. Increased ROS levels were found to be produced by nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase (NOX) and xanthine oxidase enzymes [27]. Elevated ROS generation is known to play important roles in the development of lung diseases such as COPD, lung fibrosis, and pulmonary hypertension [28, 29]. Oxidative stress caused by excess levels of ROS is known to elicit endoplasmic reticulum (ER) stress and unfolded protein response (UPR), which are implicated in the development of pulmonary disorders [30]. The ER plays a central role in the biosynthesis, protein folding, and posttranslational modifications of secretory and membrane proteins [31]. Impaired protein glycosylation and protein disulfide bond formation, protein over‐expression, and mutated proteins entering the secretory pathway elicit ER stress, leading to the activation of UPR [32]. Heat shock protein family A (Hsp70) member 5 (HSPA5) is a central regulator of ER stress, functioning as a major chaperone controlling the activation of the three transmembrane gatekeepers of ER stress, endoplasmic reticulum to nucleus signaling 1 (ERN1), activating transcription factor 6 (ATF6), and eukaryotic translation initiation factor 2 alpha kinase 3 (EIF2AK3) through a binding‐release mechanism [32]. UPR signaling is known to modulate innate and adaptive immune responses at various levels [33].

There is no information on the role of ER stress‐UPR in the modulation of lung inflammation induced by organic dust. Our published studies have demonstrated that enhanced ROS generation mediates organic dust induction of inflammatory mediators in bronchial epithelial cells [27]. As elevated ROS levels and the ensuing oxidative stress are implicated in the activation of ER stress [30], we investigated if ER stress‐UPR modulates organic dust induction of inflammatory mediators in bronchial epithelial cells. Our findings demonstrated that the ER stress‐UPR sensor ERN1 is an important component of the cell signaling cascade modulating organic dust‐induced bronchial epithelial cell inflammatory response.

2. Materials and Methods

2.1. Chemicals and Reagents

VAS2870 (HY12804, MedChemExpress, NJ, USA) was used as a pan‐NOX inhibitor. Toll‐like receptor 2 (TLR2) inhibitor CU CPT 22 (4884, Tocris, Bristol, UK), TLR4 inhibitor resatorvid (HY‐11109, MedChemExpress, NJ, USA), NFκB inhibitor BAY 11–7082 (Sc‐200,615, Santa Cruz Biotechnology, Dallas, TX, USA) and Stat3 inhibitor Stattic (2798, Tocris, Bristol, UK) were used to inhibit the signaling pathways. KIRA6 (HY‐19708, MedChemExpress, NJ, USA) and APY29 (HY‐17537, MedChemExpress, NJ, USA) were used as ERN1 inhibitors.

2.2. Dust Extract Preparation

Dust settled on vertical surfaces such as ventilator fan blades and windowsills was collected during the summer season and provided by a local poultry farm. Dust was extracted with Dulbecco's phosphate buffered saline (D‐PBS, 21‐031‐CV, Corning, NY, USA) without calcium and magnesium at a ratio of 1:10 (w/v) as described previously [26] or with modification which included mixing dust suspension overnight at 4°C with a rotating mixer. Dust suspension was centrifuged twice at 800× g for 10 min at 4°C, and the supernatant was further centrifuged at 10,000× g for 10 min at 4°C. The supernatant was filtered with a 0.22 μm syringe filter and the filtrate was stored at −70°C until further use. The concentration of dust extract was arbitrarily considered as 100%. Protein concentration of dust extract was determined by the Bradford Assay.

2.3. Cell Culture and Treatment

Beas2B bronchial epithelial cells (ATCC CRL‐9609, Manassas, VA, USA) were grown on plastic culture dishes coated with fibronectin, bovine type 1 collagen and bovine serum albumin and maintained in LHC 9 (12680013, Thermo Fisher Scientific, Waltham, MA, USA) or BronchiaLife (LS‐1047, Lifeline Cell Technology, Oceanside, CA, USA) medium containing penicillin (100 U/mL), streptomycin (100 μg/mL), gentamicin (30 μg/mL) and amphotericin B (0.25 μg/mL). Cells when 80% confluent were maintained in serum‐free RPMI 1640 medium (10‐040‐CV, Corning, NY, USA) containing antibiotics and amphotericin overnight and subjected to treatments in the same medium.

2.4. Cell Viability

Cell viability was assessed by either trypan blue staining using Bio‐Rad TC10 automated cell counter or MTS assay (G3580, CellTiter 96 Aqueous One Solution Cell Proliferation Assay, Promega, Madison, WI, USA).

2.5. Animals

Animal experiments had previously been approved by our IACUC. Female C57BL6 mice (8–10 weeks, 18–20 g weight) (The Jackson Laboratory, Bar Harbor, ME, USA) were maintained under standard housing conditions with a 12:12 h light–dark cycle and fed water and standard mouse diet. Mice were acclimatized for at least 1 week before treatments. Mice were anesthetized by IP injection of ketamine/xylazine (100 mg/kg/8.5 mg/kg). After deep anesthesia was confirmed by lack of response to toe pinch, 50 μL DPBS (endotoxin‐free) or 50 μL 20% dust extract was administered via intranasal instillation once daily (Monday–Friday) for 3 weeks. On the last day of treatment, mice were euthanized 3 h after administration of dust extract and lungs were fixated in situ under anesthesia by transcardial perfusion of PBS containing 4% paraformaldehyde. In separate experiments, lungs were fixated by inflating with ExCell fixative under a constant hydrostatic pressure of 20 cm. Lungs were also collected for western blot analysis.

2.6. Immunohistochemical Staining

Lung sections were subjected to antigen retrieval by incubating the rehydrated slides in sodium citrate buffer (10 mM sodium citrate, pH 6, 0.05% tween) for 5 min at 95°C. Post antigen retrieval, the lung sections were immunostained with anti‐ ERN1 antibody (3294, IRE1alpha (14C10), Cell Signaling Technology, Danvers, MA, USA), anti‐phospho Ser724 ERN1 antibody (NBP3‐12124, Novus Biologicals, Centennial, CO, USA) and non‐immune rabbit IgG antibody using VECTASTAIN Elite ABC kit (PK‐6101, Vector Laboratories, Newark, CA, USA) and ImmPACT 3, 3′‐diaminobenzidine (DAB) substrate (SK‐4105, Vector Laboratories, Newark, CA, USA) according to the kit instructions. Immunohistochemical images were captured using an Olympus BX41 microscope and the images were blinded for analysis.

2.7. siRNA Transfection

Cells were transfected with non‐targeting control (SIC001, Millipore Sigma, Burlington, MA, USA), ERN1 (SASI_Hs01_00194923), Stat3 (SASI_Hs01_00121206), and MyD88 (SASI_Hs01_00111539) siRNAs (Millipore Sigma, Burlington, MA, USA) using Lipofectamine 3000 transfection reagent (L3000015, Invitrogen, Waltham, MA, USA) according to the manufacturer's protocol. After transfection, cells were grown for 48–63 h and subjected to treatments.

2.8. RNA Isolation and Quantitative Real Time RT‐PCR

Total RNA from cells was isolated using TRI‐Reagent (TR118, Molecular Research Center, Cincinnati, OH, USA) and RNA purity checked by analyzing A260/A280 and A260/A230 ratios. For the determination of mRNA levels, total RNA was treated with DNase (AM1907, Turbo DNA‐free kit, Invitrogen, Waltham, MA, USA) to remove genomic DNA contamination and cDNA was synthesized using iScript Reverse Transcription kit (1,708,841, Bio‐Rad, Hercules, CA, USA). Levels of mRNAs were determined by TaqMan assays (Bio‐Rad, Hercules, CA, USA) using Bio‐Rad CFX96 real time PCR detection system and normalized to actin mRNA. The normalized gene expression data (^ΔΔCt) relative to control sample, arbitrarily assigned as 1, was obtained using CFX Manager Software. TaqMan gene expression assay IDs for the measurement of mRNA levels are listed in Table 1.

TABLE 1.

TaqMan Assay IDs.

Genes	Assay IDs
ERN1	qHsaCIP0030524 (Bio‐Rad, Hercules, CA, USA)
EIF2AK3	Hs00984005_m1 (Thermo Fisher, Waltham, MA, USA)
ATF6	Hs00232586_m1 (Thermo Fisher, Waltham, MA, USA)
Actin	qHsaCEP0036280 (Bio‐Rad, Hercules, CA, USA)

2.9. Western Immunoblotting

Equal amounts of proteins (20–30 μg) were separated by SDS‐PAGE in 10% or 4%–12% Bis‐Tris gels (QP3520, SMOBio, Paramount, CA, USA) alongside protein markers using MOPS‐SDS running buffer. Separated proteins were transferred to Hybond‐PVDF membranes (0.2 μm) (88,520, Thermo Scientific, Waltham, MA, USA) by electroblotting. Membranes were reacted sequentially with a primary antibody and a secondary antibody linked to horseradish peroxidase (HRP) and protein bands were visualized by the enhanced chemiluminescence (ECL) method using the ChemiDoc MP imaging system (Bio‐Rad, Hercules, CA, USA). Protein bands were quantified and normalized to actin using Image Lab software version 6.0.1 (Bio‐Rad, Hercules, CA, USA). Levels in control samples were arbitrarily assigned as 1, and levels in treated samples are shown relative to the control sample. Antibodies used and their dilutions are listed in Table 2.

TABLE 2.

List of primary and secondary antibodies.

Primary antibody	Dilutions	Catalog number and company
β‐Actin antibody (C‐4)	1:1000	sc‐47778, Santa Cruz Biotechnology, Dallas, TX, USA
IRE1alpha (ERN1) (14C10)	1:1000	3294, Cell Signaling Technology, Danvers, MA, USA
ATF6	1:1000	24169‐1‐AP, Proteintech, Rosemont, IL, USA
PERK (EIF2AK3) (D11A8)	1:1000	5683, Cell Signaling Technology, Danvers, MA, USA
Anti‐IRE‐1α (pSer724)	1:1000	NBP3‐12124, Novus Biologicals, Centennial, CO, USA
MyD88 (D80F5)	1:1000	4283, Cell Signaling Technology, Danvers, MA, USA
BiP (HSPA5) (C50B12)	1:1000	3177, Cell Signaling Technology, Danvers, MA, USA
XBP1	1:1000	PA5‐27650, Invitrogen, Waltham, MA, USA
IL1β (D3U3E)	1:1000	12703, Cell Signaling Technology, Danvers, MA, USA
ICAM1 (G‐5)	1:1000	sc‐8439, Santa Cruz Biotechnology, Dallas, TX, USA
Phospho‐SAPK/JNK (MAPK 8/9) (Thr183/Tyr185) (81E11)	1:1000	4668, Cell Signaling Technology, Danvers, MA, USA
SAPK/JNK (MAPK 8/9)	1:1000	9252, Cell Signaling Technology, Danvers, MA, USA
Phospho‐NFκB p65 (Ser536) (93H1)	1:500	3033, Cell Signaling Technology, Danvers, MA, USA
NFκB p65 (C‐20)	1:1000	sc‐372, Santa Cruz Biotechnology, Dallas, TX, USA
Phospho‐Stat3 (Tyr705) (D3A7) XP	1:500	9145, Cell Signaling Technology, Danvers, MA, USA
Stat3 (79D7)	1:1000	4904, Cell Signaling Technology, Danvers, MA, USA
Phospho‐c‐Jun (pJun) (Ser73) (D47G9) XP (R) rabbit mAb	1:1000	3270, Cell signaling Technology, Danvers, MA, USA
c‐Jun (60A8) (Jun) rabbit mAb	1:1000	9165, Cell signaling Technology, Danvers, MA, USA

Secondary antibody		Catalog number and company
Anti‐mouse IgG, HRP‐linked antibody	1:5000	7076, Cell Signaling Technology, Danvers, MA, USA
Anti‐rabbit IgG, HRP‐linked antibody	1:5000	7074, Cell Signaling Technology, Danvers, MA, USA
Anti‐rabbit IgG, AP‐linked antibody	1:5000	7054, Cell Signaling Technology, Danvers, MA, USA
Anti‐mouse IgG, AP‐linked antibody	1:5000	7056, Cell Signaling Technology, Danvers, MA, USA

2.10. Enzyme‐Linked Immunosorbent Assay (ELISA)

The levels of IL6 and CXCL8 in cell culture medium were measured by ELISA (DY206 and DY208, R and D Systems, Minneapolis, MN, USA) according to the manufacturer's protocol.

2.11. Statistical Analyses

All experiments were performed at least three times independently and the data are shown as mean ± SE. Statistical significance between two groups was analyzed by two‐tailed unpaired t‐test and that between multiple groups by one‐way or two‐way analysis of variance (ANOVA) followed by Dunnett's, Sidak's or Tukey's post hoc test, using GraphPad Prism 9.4.0 (GraphPad Software Inc., Boston, MA, USA).

3. Results

3.1. Organic Dust Extract Increases ER Stress Sensors HSPA5 and ERN1 but Not EIF2AK3 and ATF6 Levels

To determine if organic dust modulates ER stress‐UPR in Beas2B cells, we first studied the effects of varying concentrations (0.25%, 1% and 5%) of dust extract on the levels of ER stress sensor proteins HSPA5, ERN1, EIF2AK3, and ATF6 after incubation for 9 h. Our exploratory studies (data not shown) indicated that the stimulatory effects of dust extract on HSPA5 and ERN1 protein and mRNA levels increased with increasing concentrations of dust extract, leveling at 1%. On the other hand, increasing concentrations of dust extract did not affect EIF2AK3 protein and mRNA levels but reduced ATF6 protein and mRNA levels. Based on these data, we chose to study the effects of 1% dust extract in subsequent experiments. Treatment with 1% dust extract for 3, 9, and 24 h increased HSPA5 and ERN1 protein levels in a time‐dependent manner, whereas EIF2AK3 protein levels were unchanged and ATF6 protein levels decreased at 24 h (Figure 1A,E). Increases in ERN1 and HSPA5 protein levels were associated with increases in their mRNA levels (Figure 1F,G). Although ATF6 protein levels decreased with treatment time, ATF6 mRNA levels did not change (Figure 1E,I). Treatment with 1% dust extract did not affect Beas2B cell viability (Figure S1). Treatment of mice once daily (Monday–Friday) with dust extract (50 μL of 20% dust extract/mouse) for 3 weeks increased lung ERN1 protein levels (Figure 2A,B) and lung ERN1 immunostaining predominantly in the bronchial epithelium (Figure 2C–F).

FIGURE 1.

Time‐course effects of dust extract on the expression of ER stress‐UPR sensors in Beas2B bronchial epithelial cells. Cells were treated with 1% dust extract for 3, 9, and 24 h and protein and mRNA levels of ER stress‐UPR sensors were determined. (A) Representative western blots are shown. (B–E) HSPA5, ERN1, EIF2AK3 and ATF6 protein levels were determined by western blotting and normalized to Actin levels. (F–I) HSPA5, ERN1, EIF2AK3, and ATF6 mRNA levels were determined by real‐time qRT‐PCR and normalized to Actin mRNA levels. Data shown are mean ± SE (n = 3–4). Statistical significance was analyzed by one‐way ANOVA followed by Dunnett's post hoc test. *p < 0.05, **p < 0.01. C, control; DE, dust extract.

FIGURE 2.

Effects of dust extract on ERN1 protein levels in mouse lungs. (A, B) Fifty microliters of DPBS or 20% dust extract were instilled intranasally once daily (Monday to Friday) into female C57BL6 mice for 3 weeks. Lung ERN1 protein levels were determined by western blotting and normalized to Actin levels. Data shown are mean ± SE (n = 5–6). Statistical significance was analyzed by two‐tailed unpaired t‐test. **p < 0.01. C, control; DE, dust extract. (C–F) Lung sections from mice (n = 5) were immunostained with ERN1 (C, E) and non‐immune rabbit IgG antibodies (D, F) and developed with DAB substrate. Immunohistochemical images were blinded. Representative images are shown at 10 and 40× magnifications (Scale bar = 500 μm).

Treatment of Beas2B cells with dust extract appeared to increase ERN1 serine 724 phosphorylation in a time‐dependent manner; however, the increase was not statistically significant (Figure 3A,B). Immunostaining for ERN1 Ser 724 phosphorylation in lungs from mice treated repeatedly with dust extract for 3 weeks showed increased staining predominantly in the bronchial epithelium (Figure 3E–H). X‐box binding protein 1 (XBP‐1) is a transcription factor that is spliced by ERN1 in response to ER stress–UPR and regulates the expression of genes involved in the mitigation of cellular stress such as chaperons, folding enzymes, and others [34]. Although dust extract treatment increased HSPA5 and ERN1 protein levels and activated ERN1, there was no increase in the spliced form of XBP1 protein (Figure 3C,D). Cells treated with increasing concentrations of dust extract showed no XBP1 splicing, while DTT (1 mM) (a positive control for ER stress) treated cells showed increased XBP‐1 splicing (Figure S2).

FIGURE 3.

Effects of dust extract on ERN1 phosphorylation (Ser724) and XBP‐1 splicing in Beas2B bronchial epithelial cells and mouse lungs. (A, B) Cells were treated with 1% dust extract (DE) for 5, 10, 30, 60, and 120 min, and the levels of phosphorylated and total ERN1 were detected by western blotting. Levels of phospho ERN1 were normalized to total ERN1. Data shown are mean ± SE (n = 3). A representative western blot is shown (A). (C, D) Cells were treated with 1% dust extract for 3, 9, and 24 h. Unspliced (XBP1u) and spliced (XBP1s) protein levels were determined by western blotting and normalized to Actin levels. A representative western blot is shown (C). Data shown are mean ± SE (n = 4). Statistical significance for (B, D) was analyzed by one‐way ANOVA followed by Dunnett's post hoc test. C, control; DE, dust extract. (E–H) Lung sections from mice (n = 4) repeatedly treated with dust extract, as indicated in Figure 2 legend, were immunostained with phosphorylated ERN1 (pIRE) and non‐immune rabbit IgG antibodies and developed with DAB substrate. The immunohistochemical images were blinded. Representative images are shown at 10 and 40× magnifications (Scale bar = 500 μm).

3.2. TLR2/TLR4‐MyD88‐ROS‐NFκB/Stat3 Mediate Organic Dust Extract Induction of ERN1

Our studies showed that dust extract increased ERN1 protein levels by increasing ERN1 mRNA expression in bronchial epithelial cells. To understand the molecular mechanisms regulating increase of ERN1 expression, we investigated the roles of TLR activators, ROS and NFκB and Stat3 as poultry dust contains both gram‐positive and gram‐negative bacteria and their byproducts [13] and dust extract is known to induce inflammatory mediators via increased intracellular ROS levels produced by NOX and xanthine oxidase enzymes and activation of NFκB and Stat3 [27]. Additionally, published studies have shown that NFκB and Stat3 are activated during ER stress and there is crosstalk between UPR and NFκB signaling pathways [35, 36, 37]. The involvement of TLR2 and TLR4 were investigated by studying the effects of chemical inhibitors and MyD88 knockdown on the induction of ERN1. TLR2 inhibitor CU CPT 22 and TLR4 inhibitor resatorvid as well as MyD88 knockdown inhibited the increase of ERN1 indicating the importance of TLR signaling in the induction (Figure 4A–G). VAS2870, a pan‐NOX inhibitor, reduced ERN1 induction indicating the involvement of NOX‐derived ROS (Figure 5A,B). Treatment of Beas2B cells with NFκB inhibitor BAY 11–7082 or Stat3 inhibitor stattic inhibited the induction of ERN1 protein levels by dust extract (Figure 6A–D). Knockdown of Stat3 by siRNA transfection similarly inhibited ERN1 induction by dust extract (Figure 6E–G). These data demonstrated that NFκB and Stat3 activation controls ERN1 induction.

FIGURE 4.

Effects of TLR chemical inhibitors and MyD88 knockdown on dust extract induction of ERN1 protein levels in Beas2B bronchial epithelial cells. (A–D) Cells were treated with medium alone, 10 μM CU CPT 22, or 10 μM resatorvid for 1 h prior to incubation with 1% dust extract for 24 h. (E–G) Cells were transfected with 10 nM non‐targeting control siRNA or MyD88 siRNA and treated with 1% dust extract for 24 h. ERN1 and MyD88 protein levels were determined by western blotting and normalized to Actin levels. Representative western blots are shown (A, C, E). Data shown are mean ± SE (n = 4 for chemical inhibitors and n = 3 for MyD88 siRNA transfection). Statistical significance was analyzed by two‐way ANOVA followed by Sidak's post hoc test for analysis between specific groups (F) and Tukey's post hoc test for analysis between multiple groups (B, D, G). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C, control; DE, dust extract.

FIGURE 5.

Effect of NOX chemical inhibitor VAS2870 on dust extract induction of ERN1 protein levels in Beas2B bronchial epithelial cells. Cells were treated with medium alone or 5 μM VAS2870 for 1 h prior to incubation with 1% dust extract for 24 h. ERN1 protein levels were determined by western blotting and both ERN1 minor upper band and major lower band were quantified and normalized to Actin levels. A representative western blot is shown with noncontiguous lanes demarcated by broken lines (A). Data shown are mean ± SE (n = 4) and statistical significance was analyzed by two‐way ANOVA followed by Tukey's post hoc test (B). **p < 0.01. C, control; DE, dust extract.

FIGURE 6.

Effects of NFκB and Stat3 chemical inhibitors and Stat3 knockdown on dust extract induction of ERN1 protein levels in Beas2B bronchial epithelial cells. (A–D) Cells were first treated for 1 h with medium alone, 5 μM BAY 11–7082 (BAY), or 5 μM Stattic prior to incubation with 1% dust extract for 24 h. (E–G). Cells were transfected with 33 nM non‐targeting control siRNA or Stat3 siRNA and treated with 1% dust extract for 24 h. ERN1 and Stat3 protein levels were determined by western blotting and normalized to Actin levels. In (A, C), both ERN1 minor upper band and major lower band were quantified and normalized to Actin. Representative western blots are shown with noncontiguous lanes demarcated by broken lines (A, C). Data shown are mean ± SE (B: n = 6; D: n = 4; F: n = 4–5; G: n = 5). Statistical significance was analyzed by two‐way ANOVA followed by Sidak's post hoc test for analysis between specific groups (F) and Tukey's post hoc test for analysis between multiple groups (B, D, G). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C, control; DE, dust extract.

3.3. ERN1 Mediates Organic Dust Extract Induction of Pro IL1β, IL6, and CXCL8 Levels

Apart from its primary role as an UPR sensor, ERN1 has been implicated as a signal transducer regulating immune and inflammatory responses [38, 39, 40]. Because organic dust is a strong inducer of lung inflammatory responses, we investigated the role of ERN1 in the induction of inflammatory mediators levels. Firstly, we studied the effects of chemical inhibitors targeting the kinase and RNase activities of ERN1 on the induction of inflammatory mediators by dust extract. Among the chemical inhibitors, we selected APY29 and KIRA6, which are ATP‐competitive type I and II ERN1 kinase inhibitors, respectively. APY29 inhibits ERN1 autophosphorylation and enhances its RNase function [41] whereas KIRA6 prevents ERN1 oligomerization, resulting in the attenuation of RNase activity [42]. KIRA6 was reported to directly bind to the ERN1 kinase domain by FRET assay and display greater selectivity to inhibit ERN1 than other chemical inhibitors [41, 43]. Initial dose–response experiments showed that APY29 and KIRA6 at 1 μΜ markedly inhibited dust extract induction of inflammatory mediators (data not shown) leading us to select this concentration for subsequent studies. APY29 and KIRA6 potently reduced the induction of pro IL1β, IL6, and CXCL8 protein levels by dust extract (Figure 7A–E). Intercellular adhesion molecule 1 (ICAM1) was significantly inhibited by APY29 but not by KIRA6 (Figure 7A,C). Effects on Beas2B cell viability by MTS assay showed that KIRA6 alone and in combination with dust extract did not significantly affect cell viability, whereas APY29 reduced cell viability by 20%–25% (Figure S3). To corroborate the inhibitory effects of chemical inhibitors, the effects of ERN1 knockdown on dust extract induction of inflammatory mediators in Beas2B cells were determined. In agreement with the results of the effects of chemical inhibitors, ERN1 knockdown caused significant decreases in the induction of pro IL1β and CXCL8 but not ICAM1 protein levels, whereas reduction in IL6 levels was not statistically significant (Figure 7F–K).

FIGURE 7.

Effects of ERN1 chemical inhibitors and ERN1 knockdown on dust extract induction of inflammatory mediator protein levels in Beas2B bronchial epithelial cells. (A–E). Cells were first treated for 1 h with medium alone, 1 μM KIRA6, or 1 μM APY29 prior to incubation with 1% dust extract for 3 h. (F–K). Cells were transfected with 10 nM non‐targeting control siRNA or ERN1 siRNA and treated with 1% dust extract for 24 h. Cellular pro IL1β, ICAM1, and ERN1 protein levels and secreted IL6 and CXCL8 levels were determined by western blotting and ELISA, respectively. Pro IL1β, ICAM1, and ERN1 protein levels were normalized to Actin levels. Representative western blots are shown (A, F). Data shown are mean ± SE (n = 4–8 for chemical inhibitor experiments and n = 4–5 for siRNA transfection experiments). Statistical significance was analyzed by two‐way ANOVA followed by Sidak's post hoc test for analysis between specific groups (G) and Tukey's post hoc test for analysis between multiple groups (B–E, H–K). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. C, control; DE, dust extract.

3.4. ERN1 Chemical Inhibitor Targets NFκB, Stat3, Jun and MAPK 8/9 to Reduce Induction of Inflammatory Mediators

ERN1 chemical inhibitors KIRA6 and APY29 and ERN1 knockdown potently inhibited the induction of pro IL1β, IL6 and CXCL8 protein levels. To understand mechanisms by which ERN1 mediates induction of inflammatory mediators, we determined the effects of KIRA6 on dust extract stimulation of NFκB‐p65 (Ser526), Stat3 (Tyr705), Jun (Ser73) and mitogen‐activated protein kinase (MAPK) 8/9 (Thr183/Tyr185) phosphorylation. We have found previously that NFκB‐p65 and Stat3 activation controls induction of inflammatory mediator levels [21, 26] and that NFκB and AP1 binding sites control increase of CXCL8 promoter activity [26] by dust extract. We found that KIRA6 reduced phosphorylation of NFκB‐p65 (Ser 526) (Figure 8A,B), Stat3 (Tyr705) (Figure 8C,D), Jun (Ser73) (Figure 8E,F) and MAPK 8/9 (Thr183/Tyr185) (Figure 8G,H) in dust extract treated cells. These data collectively indicated that ERN1 regulates activation of the transcription factors important for dust extract induction of inflammatory mediators.

FIGURE 8.

Effects of ERN1 chemical inhibitor KIRA6 on dust extract induced NFκB‐p65, Stat3, Jun and MAPK 8/9 phosphorylation in Beas2B bronchial epithelial cells. Cells were first treated for 1 h with medium alone or 1 μM KIRA6. After treatment, cells were incubated with medium alone or 1% dust extract for 10 min to analyze NFκB‐p65 phosphorylation, 1 h to analyze Stat3 and Jun phosphorylation, and 20 min to analyze MAPK 8/9 phosphorylation. Phosphorylated and total levels of NFκB‐p65, Stat3, Jun and MAPK 8/9 were determined by western blotting and the levels of phosphorylated proteins were normalized to total protein levels. Representative western blots are shown (A, C, E and G). Data shown are mean ± SE (B and D: N = 5; F and H: N = 6). Statistical significance was analyzed by two‐way ANOVA followed by Tukey's post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001. C, control; DE, dust extract.

4. Discussion

Organic dusts are complex mixtures of feed, bedding material, feathers, feces, microbes and microbial byproducts [5, 11]. Organic dust inhalation increases the risk for respiratory symptoms and respiratory diseases in agricultural workers [5, 8, 10]. We have found previously that exposure of airway epithelial cells and mouse lungs to poultry organic dust extracts [18, 21], poultry organic dust derived bacterial extracellular vesicles (EVs) [13], and purified trypsin from poultry organic dust [14] induces the expression of IL6, CXCL8, TNF, pro IL1β and ICAM1. To date, we have tested dust samples from three different poultry farms located in East Texas [18, 27] and Louisiana (data not shown) and found that they elicit similar inflammatory responses in Beas2B cells and mouse lungs. We have found that elevated ROS generation is a key mediator of induction of inflammatory cytokines and ICAM1 by organic dust and organic dust derived constituents, bacterial EVs and trypsin [19, 27]. NOX and xanthine oxidase enzymes were found to contribute to increased ROS production in dust extract treated bronchial epithelial cells [27]. Additionally, our studies have shown that dust extract and serine protease purified from poultry organic dust extract increased staining for mitochondrial ROS in Beas2B airway epithelial cells [14, 27].

Oxidative stress due to elevated ROS generation is known to impair disulfide bond formation, protein glycosylation as well as ATP production, leading to the accumulation of misfolded and unfolded proteins in the ER and subsequent activation of ER stress and UPR signaling pathways [44]. Accumulation of misfolded proteins in the ER further increase ROS production by the actions of protein disulfide isomerases (PDI) to rectify protein misfolding exacerbating ER stress [45, 46]. Additionally, increased ER stress can result in elevated calcium transport to the mitochondrial outer membrane enhancing ROS generation [47]. Lungs are exposed to environmental toxins such as cigarette smoke, ambient air pollution and microbial pathogens that lead to oxidative stress triggering activation of ER stress and UPR [45, 48, 49]. Inability to restore cellular homeostasis due to persistent ER stress can lead to cell death and development of diseases [44]. ER stress and UPR have been implicated in the development of lung fibrosis, asthma, and lung cancer [30, 49]. UPR activation has been reported to be commonly associated with sporadic idiopathic interstitial pneumonia and idiopathic pulmonary fibrosis [49, 50]. Although organic dust exposure is associated with the development of respiratory symptoms and respiratory diseases with inflammation playing a key role, it is not known whether dust exposure results in ER stress and UPR activation in the lung and whether UPR activation modulates lung inflammation.

Beas2B cells have been used extensively as a bronchial epithelial cell model to investigate the effects of environmental agents and toxic chemicals [51, 52, 53] as well as organic dust extracts [13, 14, 15, 16, 17]. Our studies of the effects of poultry organic dust extract [18, 21, 26, 27] and organic dust derived bacterial EVs [13] and trypsin protease [14] on inflammatory responses showed similarities between Beas2B and NHBE cells and mouse lungs with variation in the time of response indicating that Beas2B cells are an appropriate cell model to understand the mechanisms of inflammatory responses. In our study, ERN1 immunostaining was predominantly increased in the bronchial epithelium of mouse lungs exposed to dust extract consistent with the inductive effects on ERN1 expression in Beas2B cells. We found that treatment of Beas2B cells with dust extract increased HSPA5 and ERN1 protein levels in a time‐ and concentration‐dependent manner whereas EIF2AK3 levels were unchanged and ATF6 levels were reduced. Increase in ERN1 protein level was associated with increase in its mRNA level indicating regulation at the transcriptional level. Although ATF6 protein levels were reduced, ATF6 mRNA levels remained unchanged suggesting regulation at the posttranslational level. Dust extract in addition to inducing ERN1 protein levels increased ERN1 Ser724 phosphorylation in Beas2B cells and in the bronchial epithelium of mouse lungs. Phosphorylation at Ser724 was reported to be important for the kinase/autophosphorylation status and RNase activities of ERN1 [54]. Although dust extract increased ERN1 Ser724 phosphorylation, the levels of spliced form of XBP1 (XBP1s) protein were not increased indicating a lack of role for XBP1s in the modulation of inflammatory mediators. ERN1 was previously found to act independently of XBP1 splicing for photoreceptor differentiation in drosophila [55] and glucagon regulation of glucose metabolism in the liver [56]. HSPA5, an ER‐localized HSP70 chaperone, is strongly induced under ER stress and functions to facilitate correct protein folding and protein oligomerization as well as binds to misfolded proteins to target them for degradation [57]. Our finding of increased HSPA5 levels in Beas2B cells treated with dust extract is consistent with enhanced ER stress. Mechanisms controlling HSPA5 induction by organic dust are yet to be studied.

Activation of UPR is regulated by the three ER stress transmembrane sensor proteins, ERN1, EIF2AK3, and ATF6. Under normal conditions, ER‐resident chaperone HSPA5 is bound to all the three UPR sensors and dissociates upon ER stress resulting in the activation of downstream signaling pathways aimed at restoring cellular homeostasis [32]. The three UPR signaling pathways display differential sensitivities to different forms of ER stress in terms of activation kinetics [58, 59]. Oxidative stress differentially activates UPR signaling pathways; whereas TNF induced oxidative stress activated ERN1, EIF2AK3, and ATF6 pathways, oxidative stress induced by H₂O₂ and arsenite selectively activated EIF2AK3‐mediated eIF2α phosphorylation [60, 61]. Dust extract treatment increased ERN1 mRNA and protein levels but had no effect on EIF2AK3 protein levels and decreased ATF6 protein levels. Hence our study focused on the dust extract regulation of ERN1 expression and its impact on the induction of inflammatory mediators. ERN1 is the most evolutionarily conserved protein among the three ER stress sensors and possess Ser/Thr kinase and endoribonuclease domains [62]. Increase of ERN1 protein levels by dust extract was reduced by TLR2 and TLR4 chemical inhibitors and by MyD88 knockdown indicating the involvement of TLR2/TLR4‐MyD88 pathway in the induction. Extracts of poultry organic dust contain bacterial products such as LPS, lipoteichoic acid, and peptidoglycan which may serve as TLR ligands to activate cell signaling pathways controlling induction of ERN1 gene expression [11, 26]. Additionally, bacterial EVs present in organic dust [13] could be a source for TLR ligands. The strong inhibitory effect of MyD88 knockdown further supports regulation of ERN1 expression by the TLR pathways. TLR2 and MyD88 have been reported to mediate the effects of swine farm organic dust extracts to induce inflammatory gene expression in mice [63].

Increase of ERN1 levels by dust extract was inhibited by the pan NOX inhibitor VAS2870 indicating that NOX‐derived ROS may mediate the increase. NFκB inhibitor BAY 11‐7082 and Stat3 inhibitor stattic suppressed ERN1 increase indicating that NFκB and Stat3 activations are necessary for the increase. During chronic stress inhibition of Stat3 phosphorylation has been reported to decrease ERN1 phosphorylation [37]. In our study, we observed that Stat3 knockdown significantly reduced dust extract induction of ERN1 expression, confirming a positive role for Stat3. Both NFκB and Stat3 are redox‐sensitive transcription factors [64, 65, 66] and control dust extract induction of inflammatory mediators [21, 26, 27]. ER stress and UPR modulate immune and inflammatory responses via regulation of cytokine production at various levels [40] and ERN1 has been reported to act as a pattern recognition receptor (PRR), controlling inflammatory gene expression [67]. Similar to our results, TLR stimulation in macrophages has been found to induce inflammatory cytokines through activation of ERN1 [68, 69]. Additionally, a fungal pathogen Candida albicans was found to induce ERN1 dependent expression of cytokines via NOX generated ROS in myeloid cells through activation of MyD88 and the C‐type lectin receptor dectin‐1 [70]. Our studies have shown that NOX‐derived ROS mediate induction of inflammatory cytokines by dust extract via activation of transcription factors NFκB and Stat3 [27].

Our studies showed that dust extract treatment increased ERN1 protein levels by increasing ERN1 mRNA expression and increased ERN1 phosphorylation at Ser724. ERN1 signaling is a critical arm of UPR involved in the regulation of cytokine production [39, 71, 72]. ERN1 is a Ser/Thr kinase and an endoribonuclease (RNase) whose activities reside in its cytoplasmic domain [31]. ERN1 activation by dimerization/oligomerization and autophosphorylation leads to signal transduction events aimed at maintaining cellular homeostasis and cytokine regulation [31, 40]. Based on the important roles of ERN1 in the regulation of immune and inflammatory responses, we investigated its involvement in the organic dust induction of inflammatory mediators in airway epithelial cells. ERN1 chemical inhibitors KIRA6 and APY29 and ERN1 knockdown inhibited dust extract induction of pro IL1β, IL6 and CXCL8 protein levels in Beas2B cells underscoring ERN1's role in the organic dust induction of lung inflammatory responses.

Cell stress leading to UPR activation is known to regulate immune responses via modulation of cytokine production [40]. We found KIRA6 inhibited dust extract activation of NFκB‐p65, Stat3, Jun as well as MAPK 8/9 indicating that ERN1 controls induction of inflammatory mediators at the transcriptional level. ERN1 has been shown to activate NFκB via TRAF2 mediated increase of IκB kinase (IKK) and AP1 family members via ASK‐1 mediated activation of MAPK 8/9 and MAPK 14 [73]. In primary effusion lymphoma, Stat3 activation was reported to be medited by the ERN1‐XBP1 pathway [74]. Our finding of cross‐regulation between ERN1 and NFκB/Stat3 in the organic dust extract induction of inflammatory mediators might suggest a feedback loop regulation necessary for fine‐tuning of immune and inflammatory responses. Similar feedback loop regulation between TLR4 and NFκB/Stat3 has been reported [75, 76].

Collectively, our studies have shown that ERN1 signaling is an important pathway controlling organic dust induction of inflammatory mediators in bronchial epithelial cells. Although our studies have found that ERN1 regulates the induction of inflammatory mediators, its relative role in the overall induction of lung inflammatory mediators by organic dust is yet to be understood. Microorganisms and their byproducts present in organic dust may differentially modulate ERN1 and the other UPR sensors to regulate the production of inflammatory mediators. Additionally, interactions between different lung cell types such as epithelial cells, alveolar macrophages and other immune cells may shape the inflammatory outcome induced by organic dust. Future animal (mice) studies using pharmacological inhibitors targeting UPR sensors and mice lacking UPR sensors would be important to understand the role of ER stress‐UPR in organic dust induced lung inflammation at the physiological level. Additionally, studies using air‐liquid interface cultures of differentiated human bronchial epithelium can provide insights into mechanisms underlying ER stress‐UPR modulation of human airway inflammation induced by organic dust.

Our study focused on the investigation of cell and molecular mechanisms mediating organic dust induction of inflammatory mediators in bronchial epithelial cells and mice. The concentration of dust extract used in our studies may not correlate with dust exposure levels encountered by poultry farmworkers. Future studies in cell culture and animal (mice) models using dust particles or dust extracts to simulate exposure levels of poultry farm workers would be useful to understand the pathogenesis of lung diseases in these workers.

In summary, our studies have shown that organic dust extract enhanced ER stress‐UPR in bronchial epithelial cells. Organic dust extract increased the expression of HSPA5 and ERN1 but not EIF2AK3 and ATF6. ERN1 was found to serve a positive role in the modulation of organic dust induction of lung inflammatory mediators through activation of NFκB, Stat3 and Jun. A cell signaling cascade involving TLR2/TLR4, MyD88, ROS, ERN1, NFκB, Stat3 and Jun was found to mediate organic dust induction of inflammatory mediators. Cross‐regulation between members of the signaling cascade may fine tune inflammatory responses of bronchial epithelial cells to organic dust.

Author Contributions

Shilpa Kusampudi and Vijay Boggaram conceived the study and wrote the manuscript. Shilpa Kusampudi and Velmurugan Meganathan performed experiments and Shilpa Kusampudi, Velmurugan Meganathan and Vijay Boggaram analyzed the data.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

FIGURE S1. Effect of dust extract on the viability of Beas2B bronchial epithelial cells.

FIGURE S2. Effects of dust extract on XBP1 splicing in Beas2B bronchial epithelial cells.

FIGURE S3. Effects of KIRA6 and APY29 chemical inhibitors on the viability of Beas2B bronchial cells.

Kusampudi S., Meganathan V., and Boggaram V., “Endoplasmic Reticulum Stress and Unfolded Protein Response Sensor ERN1 Regulates Organic Dust Induction of Lung Inflammation,” FASEB BioAdvances 7, no. 7 (2025): e70031, 10.1096/fba.2025-00069.

Data Availability Statement

The data that support the findings of this study are available in the methods and/or [Link], [Link], [Link] of this article.