Transient receptor potential ankyrin 1 promotes the expression of interferon-stimulated antiviral genes in human A549 lung epithelial cells

Abstract

Transient receptor potential ankyrin 1 (TRPA1) is an ion channel known for its chemosensory function in neurons, causing pain and neurogenic inflammation. TRPA1 is activated by many noxious compounds including some inflammatory mediators. We and others have shown that TRPA1 is also expressed in epithelial cells, but its function in the epithelial barrier remains unclear. Here, we discovered in RNA-seq studies that inhibition of TRPA1 reduced the expression of a large number of antiviral and inflammatory genes under the influence of the key antiviral cytokine interferon beta in human A549 lung epithelial cells. In the gene ontology analysis, the terms most strongly affected by TRPA1 antagonists included many associated with antiviral defense such as “defense response to virus” and “antiviral innate immune response.” To validate the RNA-seq results, selected antiviral genes such as myxovirus resistance protein 1 were further studied and found to be upregulated by TRPA1 by using reverse transcription quantitative polymerase chain reaction and western blotting, pharmacological TRPA1 inhibitors, TRPA1-targeting small interfering RNA, and ex vivo lung tissue cultures from TRPA1-deficient mice. Mechanistically, TRPA1 inhibitors partially reduced interferon beta-induced Ca²⁺ influx, phosphorylation of the transcription factor signal transducer and activator 1, and the interferon-sensitive response element-dependent transcription. These data suggest that TRPA1 mediates cellular signaling and biologically relevant changes in gene expression induced by type I interferons. The results offer TRPA1 as a novel treatment target for inflammatory conditions characterized by enhanced type I interferon activity such as hyperinflammatory states associated with viral infections and some autoimmune diseases, but TRPA1 inhibition may also influence interferon-induced antiviral immunity.

Significance Statement

We found that the transient receptor potential ankyrin 1 channel can promote gene expression changes induced by type I interferons in human lung epithelial cells. Inhibiting transient receptor potential ankyrin 1 could decrease interferon-induced inflammation but could also influence the antiviral state.

1. Introduction

Type I interferons IFN beta and IFN alfa are important innate immune system mediators that mediate the rapid antiviral immune defense and shape the subsequent inflammatory responses. They are produced by nearly all cells upon stimulation of the pattern recognition system. They bind the same, widely expressed receptor to initiate canonical signaling which ultimately induces the expression of interferon-stimulated genes (ISGs). The products of these genes function as effectors in antiviral immunity and modulate immune responses.1, 2, 3, 4

Transient receptor potential ankyrin 1 (TRPA1) is a nonselective cation channel especially permeable to Ca²⁺.^5,6 In sensory neurons TRPA1 is activated by a plethora of noxious chemical compounds, for instance acrolein in cigarette smoke⁷ and allyl isothiocyanate⁸ found in mustard oil—causing pain, itch, and cough.⁵ In addition, TRPA1 is also activated by endogenous compounds produced in inflammation, including reactive oxygen and nitrogen species.9, 10, 11, 12 It also mediates inflammation in many disease models. For example, TRPA1 deficiency is protective in carrageenan-induced paw edema¹³ as well as in gout^14,15 and asthma16, 17, 18, 19, 20 models in the mouse. These proinflammatory effects have mostly been attributed to neurogenic inflammation—a mechanism where TRPA1-dependent Ca²⁺ influx mediates the release of proinflammatory neuropeptides from sensory nerve endings, reviewed by Landini et al.²¹

TRPA1 expression is not restricted to sensory neurons; it is also expressed in many other cell types at varying expression levels.^5,22 In these non-neuronal cells, the consequences of TRPA1 function are not fully defined, but it seems to mediate inflammatory signals. For example, in the airways, TRPA1 is expressed in epithelial cells, fibroblasts, and smooth muscle cells and promotes the release of the chemokine interleukin 8 (IL-8).^23,24 In addition, we and others have shown that TRPA1 expression and activity is enhanced in response to IL-1β, and TRPA1 mediates some of the proinflammatory effects of IL-1β.^25,26 The inflammatory factor bradykinin also activates TRPA1 indirectly through phospholipase C signaling.^27,28

Canonical type I IFN signaling leads to phosphorylation of signal transducer and activator 1 and 2 (STAT1, STAT2) proteins which then assemble with IFN regulatory factor 9. The formed trimer, ISG factor-3, binds to interferon-sensitive response elements (ISRE) in the promoters in the ISGs to induce their transcription.¹ Wang et al²⁹ have shown that intracellular Ca²⁺ can augment this signaling through the Ca²⁺-dependent kinases, calmodulin kinase II, and the downstream proline-rich tyrosine kinase 2, which further enhances Janus kinase activity to promote STAT1 tyrosine phosphorylation and other responses induced by IFN alfa.²⁹ TRPA1 has been reported to activate both of these Ca²⁺-dependent kinases.30, 31, 32, 33

As airway epithelial cells are repeatedly exposed to pathogenic viruses being important in the generation of the antiviral innate immune responses, we investigated the potential role of TRPA1 in type I IFN responses in A549 lung epithelial cells which express the TRPA1 channel.^24,34 We report here, for the first time, that type I IFNs activate TRPA1 in these cells, and that the antiviral gene expression responses are partially mediated by TRPA1. This finding opens the door for further studies to find out whether type I IFN-mediated inflammation could be controlled by drugs targeting TRPA1.

2. Methods

2.1. Cell culture

Human A549 lung epithelial cells (American Type Culture Collection) were cultured in Ham’s F–12 K (Kaighn’s modification) medium with 10% heat-inactivated FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B (all from Gibco/Life Technologies) at 37 ^◦C in 5% CO₂ atmosphere. Cells wereseeded on 24-well plates and grown for 48 hours before the experiments were started. In the experiments, the cells were cultured with the following compounds or their combinations as indicated: IFN alfa-2a, IFN beta (both from R&D Systems Europe Ltd) and the TRPA1 antagonists HC-030031 and A-967079 (both from Sigma Aldrich).

2.2. RNA sequencing and data analysis

Total RNA was extracted using GenElute Mammalian Total RNA Miniprep kit, and columns were treated with DNAse I, using On-Column DNAse I Digestion Set (Sigma Aldrich). RNA concentration and integrity were confirmed with the 2100 Bioanalyzer (Agilent Technologies). RNA-seq was carried out by Novogene using Illumina sequencing. Quality control was performed using Novogene’s in-house perl scripts. Reads were aligned to the reference genome (GRCh38/hg38) using Hisat2 v2.0.5 by Kim et al.³⁵ featureCounts v1.5.0-p3 (Liao et al³⁶) was used to count the reads mapped to each gene. Differential expression was determined using the DESeq2 R package.³⁷ Further analysis was performed with biologically relevant genes which were determined as up- or downregulated by IFN beta with a fold change (FC) minimum of ±5 and false discovery rate-corrected P value of <.05 unless indicated otherwise. Functional analysis was performed against the gene ontology (GO) database^38,39 using the DAVID (database for annotation visualization and integrated discovery) tool.⁴⁰ Heatmaps were generated with Heatmapper.⁴¹

2.3. Reverse transcription quantitative polymerase chain reaction

Total RNA was extracted from A549 cells using GenElute Mammalian Total RNA Miniprep kit (Sigma Aldrich) at indicated time points and reverse transcribed to cDNA using TaqMan Reverse Transcription Reagents (Applied Biosystems). Polymerase chain reaction (PCR) was carried out by using the Applied Biosystems 7500 Real-Time PCR instrument and Taqman Universal PCR Master Mix reagent. The following TaqMan Gene Expression assays were used: 2’-5’-oligoadenylate synthetase 2 (OAS2, Hs00942643_m1), myxovirus resistance protein 1 (MX1, Hs00895608_m1), DExD/H-box helicase 58 (DDX58, Hs01061436_m1), TRPA1 (Hs00175798_m1), and ribosomal protein L30 (RPL30, Hs00265497_m1). These were obtained from Life Technologies (Life Technologies Europe BV). In data analysis, mRNA expression levels were first normalized against RPL30 mRNA levels, and the ΔΔCt method was used in the calculations.

In mouse lung tissue culture experiments, RNA extraction and reverse transcription were performed as described under “Mouse lung tissue culture” subheading. Mx1 (Mm00487796_m1; Life Technologies) TaqMan Gene Expression assay was used, normalized against Gapdh levels, and the ΔΔCt method was used in the calculations. The in-house primers and probes for Gapdh (for sequences and concentrations, refer to Mäki-Opas et al.⁴²) were purchased from Metabion.

2.4. Protein extraction and western blot

After indicated incubation times, cell culture medium was removed and adherent cells were lysed in ice-cold extraction buffer (10 mM Tris base, pH 7.4, 5 mM EDTA, 50 mM NaCl, 1% Triton X-100, 1x Halt Protease Inhibitor Cocktail [Thermo Scientific], 1 mM Na₃VO₄, 5 mM NaF, and 10 μM N-octyl-β-D-glucopyranoside). Samples were incubated on ice for 15 minutes and centrifuged for 10 minutes 12 000 rpm. Protein concentration of the supernatant was measured using the Coomassie blue method.⁴³ Sample buffer (6.25 mM Tris-HCl, 10% glycerol, 2% SDS, and 0.025% 2-mercapto-ethanol) was mixed with a ratio of 1:4, and samples were boiled for 10 minutes. Approximately 20 μg of protein from each sample was loaded on each well on 8% (for detection of STAT1) or 10% (other proteins) polyacrylamide gels and transferred to iBlot nitrocellulose membranes (Invitrogen). The membranes were blocked with TBS/T [20 mM Tris base (pH 7.6), 150 mM NaCl, 0.1 % Tween-20] containing 5% nonfat milk (in the case of actin) or 5% BSA (other proteins) for 1 hour at room temperature. Thereafter, the membranes were incubated overnight at +4 °C with primary antibodies, and for 1 hour at room temperature with the secondary antibody. Proteins were visualized using SuperSignal West Pico PLUS or Dura Extended Duration chemiluminescent substrates (Pierce). The chemiluminescent signal was quantified with ImageQuant TL 7.0 Image Analysis Software (GE Healthcare Bio-Sciences AB). The following primary antibodies diluted 1:1000 in 5% BSA were used: OAS2 (E2G4K) Rabbit mAb #24344, MX1 (D3W7I) Rabbit mAb #37849, Rig-I (D33H10) Rabbit mAb #4200, Phospho-Stat1 (Tyr701) (D4A7) Rabbit mAb #7649, and Stat1 antibody #9172 (all from Cell Signaling Technology, CST). Sc-1616R (Santa Cruz) in 5% nonfat milk was used as the primary antibody against actin. Goat anti-rabbit horseradish peroxidase-linked IgG antibody CST#7074 diluted 1:2000 in 5% nonfat milk (in the case of actin) or 5% BSA (other proteins) was used as the secondary antibody. In the case of STAT1, phosphatase inhibitors (5 mM Na₃VO₄ and 5 mM NaF) were added to all blocking and antibody solutions.

2.5. Reporter gene assay

A549 cells (2 × 10⁴) were seeded on 96-well plates in 100 μL of medium containing no antibiotics. After 24 hours, cells were transfected with 60 ng/well of vectors containing a Firefly luciferase reporter under an ISRE-dependent promoter or a control promoter (“empty”); and Renilla luciferase under a constitutive promoter (BPS-60613; BPS Bioscience) as an internal control for 24 hours. Lipofectamine 3000 Transfection Reagent (ThermoFisher) was used with 0.3 μL of Lipofectamine 3000 and P3000 Reagent on each well. Thereafter, transfection medium was replaced with media containing indicated compounds. After 7 hours 30 minutes, culture media was removed, wells were rinsed with ice-cold PBS and lysed in 50 μL of lysis buffer provided in Luc-Pair Duo-Luciferase HS Assay Kit (Genecopoeia), and dual luciferase assay was performed according to manufacturer’s instructions. Luminescence was read using Victor3 multilabel counter (PerkinElmer). Firefly luciferase activity of each sample was normalized to the Renilla activity of the respective sample. Values obtained from samples transfected with ISRE-dependent vector were thereafter normalized to the mean of empty-transfected ones of the respective treatment.

2.6. Downregulating TRPA1 by small interfering RNA

A549 (3 × 10⁴) cells were seeded on 24-well plates in 500 μL of medium containing no antibiotics. After 20 hours, cells were transfected using TRPA1 TriFECTa DsiRNA Kit (hs.Ri.TRPA1.13.; Integrated DNA Technologies) and 1 μL/well of Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to manufacturer’s instructions. Transfection efficiency at 24 hours was >90% as determined by TYE 563 DS transfection control supplied with the kit. TRPA1 DsiRNA 2 (hs.Ri.TRPA1.13.2) was used, the sequences of which (reported by the manufacturer) were as follows: hs.Ri.TRPA1.13.2-SEQ1-rGrGrA rGrCrA rArUrU rGrCrU rGrUrU rUrArC rUrUrC rUrAT T and hs.Ri.TRPA1.13.2-SEQ2-rArArU rArGrA rArGrU rArArA rCrArG rCrArA rUrUrG rCrUrC rCrArC. Negative control DsiRNA (DS NC1) was used as a negative control. Medium was replaced with fresh medium without antibiotics 5 hours 30 minutes and 48 hours after transfection. Cell culture treatments were conducted as indicated 72 hours after transfection. TRPA1 knockdown was confirmed by reverse transcription (RT)-quantitative PCR (qPCR).

2.7. Intracellular Ca²⁺ measurements

TRPA1-dependent changes in intracellular Ca²⁺ levels were determined using fluo-3-acetoxymethyl ester (Fluo 3-AM, Sigma Aldrich), a fluorescent indicator of free Ca²⁺. A549 cells were seeded (4 × 10⁴ cells/well in 100 μL) on 96-well black Nunc Optical-Bottom Plates with Polymer Base (Thermo Scientific). After 48 hours, confluent cultures were loaded with 4 μM Fluo 3-AM and 0.08% Pluronic F-127 in Hanks’ balanced salt solution containing Ca²⁺ and Mg²⁺, without phenol red (Gibco), 1 mg/mL bovine serum albumin, 2.5 mM probenecid, and 25 mM HEPES pH 7.45 (all from Sigma Aldrich) in the volume of 100 μL/well for 30 minutes at room temperature. As a negative control, cells were sham loaded with a similar medium without Fluo 3-AM. The cells were then washed twice with 100 μL of Hanks’ balanced salt solution and preincubated for 30 minutes at room temperature with the TRPA1 antagonist HC-030031 or A-967079 (both 100 μM, Sigma Aldrich) or the vehicle (DMSO), in the volume of 75 μL. Measurements were carried out using the Victor Nivo plate reader (PerkinElmer) at 37 ^◦C, and the plate was prewarmed for 5 minutes before starting the measurements. Excitation/emission wavelengths of 480/530 nm (bandwidth of 30 nm) were read every 2.5 seconds for 1 second as an indicator of free intracellular Ca²⁺. Background fluorescence was measured for 15 seconds before IFN beta was administered using a dispenser in the volume of 25 μL to yield a total volume of 100 μL in the well and an IFN beta concentration of 10 ng/mL, after which fluorescence was measured for 5 minutes 30 seconds. The change in fluorescence was normalized to the mean background fluorescence of the respective well.

2.8. Animals and mouse lung tissue culture

Wild type (WT) and TRPA1-deficient (knockout, KO) male B6; 129P-Trpa1(tm1-Kykw)/J mice (Charles River Laboratories) at about the age of 12 months were used in lung tissue experiments. The mice were housed under standard conditions (12–12 hour light-dark cycle, temperature 22 ± 1 °C, and humidity 50%–60%) with food and water provided ad libitum. The experiments were conducted in compliance with legislation for the protection of animals used for scientific purposes (Directive 2010/63/EU) and under Tampere University license EKS-2018 (issued 12 April 2018).

Immediately after euthanization, both lungs of the mice were taken into Ham’s F12-K (Kaighn’s modification) medium containing 100 U/mL penicillin, 100 μg/mL streptomycin, and 250 ng/mL amphotericin B. Samples were immediately processed at room temperature in a sterile environment. Lungs were first rinsed in PBS and cut to small pieces (1–3 mm in diameter, weighing 5–10 mg). The pieces were then transferred on 24-wells containing 1 mL of medium supplemented 10% heat-inactivated FBS, antibiotics (see above), and the indicated treatments. Lung tissue was then cultured at 37 °C in 5% CO₂ atmosphere for 24 hours. Thereafter, tissue pieces were rinsed in PBS and collected in 2 mL Precellys CKMix tubes (Bertin Technologies) containing 600 μL of RNEasy lysis buffer (Qiagen) supplemented with 1% 2-mercaptoethanol (Sigma Aldrich) and were disrupted using the Precellys 24 homogenizer (6000g for 15 seconds, 2 times). To ensure complete homogenization, samples were centrifuged through QiaShredder (Qiagen) columns. Thereafter, 500 μL of the sample was used to extract total RNA using the RNEasy Mini Kit (Qiagen) with DNAse I on-column treatment according to manufacturer’s instructions. Total RNA was reverse transcribed to cDNA using Maxima Reverse Transcription Reagents (ThermoFisher) according to manufacturer’s instructions and RT-qPCR was performed as described above.

2.9. Statistics

Statistical analysis was performed using the Graphpad Prism 9 software (GraphPad). Student’s t test, one-way ANOVA (with or without repeated measures) and repeated measures two-way ANOVA (mixed effects model), was used in the analysis, followed by multiple comparison post-tests as indicated. Next generation sequencing (NGS) data analysis and statistical testing are described under the subheading “RNA sequencing and data analysis.”

3. Results

3.1. TRPA1 mediates the expression of an array of ISGs in A549 human lung epithelial cells

We tested the hypothesis that TRPA1 has a mediator role in IFN-beta-induced changes in gene expression and took a NGS-based approach to investigate changes in the transcriptome. A549 cells were cultured with IFN beta (10 ng/mL), with or without the TRPA1 antagonists HC-030031 or A-967079 (both 100 μM) for 24 hours, and RNA was sequenced.

IFN beta treatment altered the expression of 532 genes by a FC of at least ±5. Of these genes, 456 were upregulated and 76 downregulated (Supplemental Table 1). Of the 456 upregulated genes, 133 (29.2%) were downregulated and of the 76 downregulated genes, 2 genes were upregulated by both TRPA1 antagonists (by FC of at least +1.3). Of the genes downregulated by IFN beta, no genes were further downregulated by the 2 TRPA1 antagonists; and of the upregulated ones, 2 genes were further upregulated by the 2 TRPA1 antagonists (Supplemental Table 2).

The 133 genes whose upregulation was inhibited by both TRPA1 antagonists were further subjected to GO analysis. Twenty-nine statistically significant GO terms were yielded (Table 1, Supplemental Table 3). The terms were related particularly to antiviral immunity, inflammation and immune response (Table 1). The most highly statistically significant term “defense response to virus” included 30 genes downregulated by both TRPA1 antagonists including genes classically involved in antiviral innate immunity, such as MX1. In addition, terms such as “Innate immune response,” “Response to virus,” and “Inflammatory response” were also affected, including for example the proinflammatory cytokine IL-6. Gene expression data of the genes included in selected GO terms are visualized as heatmaps in Fig. 1.

Table 1.

GO terms regulated by TRPA1 antagonists in A549 cells

GO analysis (using the DAVID tool) was performed for the 133 genes whose expression was enhanced by IFN beta (10 ng/mL, 24 hours) by FC of 5 or more, and downregulated by both TRPA1 antagonists HC-030031 and A-967079 (both 100 μM) by FC of 1.3 or more in A549 cells (n = 4 independent experiments). The affected genes are listed in the middle column. Six statistically most significant GO terms are shown (all 29 terms are shown in Supplemental Table 3).

Term	Genes	FDR
Defense response to virus	IFITM3, RTP4, IFIT5, DDX60L, SAMHD1, IFI44L, IFIT3, IFIT2, IFIH1, IFI16, CASP1, GBP1, TRIM22, GBP3, ZBP1, GBP5, APOBEC3G, DTX3L, RSAD2, MX2, MX1, ISG20, BST2, IL6, ZNFX1, OAS2, IRF1, OAS3, APOBEC3A, TLR3	8.00E-27
Innate immune response	C1S, SAMD9, IFIT5, UBE2L6, SAMHD1, IFIH1, C4B, C4A, IFI16, DHX58, TRIM25, APOL1, SLC15A3, GBP1, TRIM22, GBP3, GBP5, APOBEC3G, DTX3L, RSAD2, MX2, MX1, HLA-B, NMI, ISG20, BST2, ZNFX1, OAS2, OAS3, SERPING1, TRIM38, APOBEC3A, TLR3	2.05E-20
Negative regulation of viral genome replication	IFITM3, APOBEC3G, RSAD2, MX1, IFIT5, IFIH1, BST2, ISG20, ZNFX1, IFI16, OAS2, OAS3, CCL5, APOBEC3A	2.16E-16
Response to virus	IFITM3, RSAD2, MX2, MX1, NMI, IFIT3, IFIT2, IFIH1, BST2, ISG20, OAS2, OAS3, CCL5, DHX58, TRIM22	1.18E-12
Antiviral innate immune response	IFIH1, ZBP1, CXCL10, MX1, DHX58, TRIM25, USP18, IFIT3, IFIT2	3.15E-07
Inflammatory response	GBP5, PTGIR, PLA2G4C, TNFRSF1B, C4B, CXCL10, C4A, CXCL11, IL6, IFI16, CCL5, TXNIP, LGALS9, APOL3, TLR3, IDO1	6.81E-06

FDR = P value corrected for false discovery rate.

Fig. 1.

Genes included in selected GO terms regulated by TRPA1 antagonists in A549 cells. A heatmap showing genes included in the GO terms: defense response to virus (A), reponse to virus (B), and inflammatory response (C) whose expression was enhanced by IFN beta (10 ng/mL, 24 hours) by an FC of 5 or more compared with control (ctrl), and downregulated by both HC-030031 (HC; 100 μM) and A-967079 (A96; 100 μM) by FC of 1.3 or more. Expression levels are DESeq2 normalized and row scaled, with red denoting higher and white lower expression. The heatmaps were created with Heatmapper.⁴¹

In the further studies, we chose to analyze the effects the TRPA1 antagonists on the expression of ISGs by using RT-qPCR and western blot. Three well-established ISGs were selected as examples: the antiviral GTPase MX1/MXA, and the viral RNA sensors OAS2 and DDX58/retinoic-acid-inducible gene I (RIG-I).

A549 cells were cultured with IFN beta in the presence or absence of the TRPA1 antagonists. To confirm that the effect was not restricted to IFN beta, the experiment was replicated using another type I interferon, IFN alfa (subtype 2a). RT-qPCR and western blot data indicated that the expression of the selected ISGs, MX1, OAS2, and DDX58, was highly enhanced by both IFN beta and IFN alfa, and that both of the 2 TRPA1 antagonists inhibited their expression (Figs. 2 and 3). In the case of HC-030031, 30 μM concentration was required for a statistically significant effect, and 100 μM increased the inhibition further. On the other hand, already 10 μM concentration of A-967079 produced a statistically significant downregulation (Fig. 2).

Fig. 2.

The effect of TRPA1 antagonists on the mRNA expression of the IFN-stimulated antiviral genes MX1, OAS2, and DDX58. A549 cells were cultured with IFN beta or IFN alfa-2a (both 10 ng/mL) in the presence or absence of the TRPA1 antagonists HC-030031 or A-967079 at indicated concentrations for 24 hours. Thereafter, total RNA was extracted, and RT-qPCR of MX1, OAS2, and DDX58 was carried out against RPL30 as an endogenous control gene. Individual values and mean ± SD are presented, n = 5–6. Control was set as 1, and the other values are given in relation to that value. Statistical analysis was performed using one-way ANOVA with Holm-Šídák post-test, and comparisons were carried out against the IFN-treated condition (second bar from the left). ∗, ∗∗, ∗∗∗, and ∗∗∗∗ denote P < .05, <.01, <.001, or <.0001, respectively.

Fig. 3.

The effect of the TRPA1 antagonists on the protein expression of the IFN-stimulated antiviral genes MX1, OAS2. and DDX58. A549 cells were cultured with IFN beta (10 ng/mL; A, C) or IFN alfa-2a (10 ng/mL; B, D) for 24 hours with or without the TRPA1 antagonists HC-030031 (HC; 100 μM) or A-967079 (A96; 100 μM), total protein was extracted, and indicated proteins were detected by western blotting. In panels A and B, representative blots of the indicated proteins (with 2 biological replicates of each treatment) are shown following IFN beta (A) and IFN alfa-2a (B) treatment, respectively. Actin was detected as a loading control. Densitometric analysis is shown in panels C and D. Individual values and mean ± SD are presented, n = 5–6. IFN-treated conditions (second bar from the left) were set as 1, and the other values are given in relation to and statistically tested against that value. Statistical analysis was performed using one-way ANOVA with Holm-Šídák post-test. ∗, ∗∗, ∗∗∗, and ∗∗∗∗ denote P < .05, <.01, <.001, or <.0001, respectively.

3.2. Genetic TRPA1 inhibition downregulates MX1 expression induced by IFN beta

To confirm our pharmacological data, A549 cells were transfected with TRPA1 targeting small interfering RNA (siRNA), nontargeting siRNA (negative control; Neg) or were not transfected. Using TRPA1 siRNA, TRPA1 was downregulated by 84%. In cells transfected with TRPA1 siRNA, IFN-beta-induced MX1 upregulation was lower than in nontransfected and Neg siRNA transfected cells (which were similar), indicating that this effect was TRPA1-dependent (Fig. 4, A and B).

Fig. 4.

MX1 induction by IFN beta is inhibited by genetic TRPA1 knockdown. (A, B) A549 cells were transfected with TRPA1 siRNA, nontargeting (negative control; Neg) siRNA or were not transfected. After 72 hours, the cells were cultured with IFN beta (10 ng/mL) with or without the TRPA1 antagonist A-967079 (100 μM) for 24 hours. Thereafter, total RNA was extracted, and RT-qPCR was performed. RPL30 was used as an endogenous control gene. In panel A is shown the level of MX1 expression relative to the respective controls of each transfection condition which were set as 1. In panel B, TRPA1 knockdown by siRNA relative to Neg siRNA (which was set as 1) is shown. N = 5–6 (A) and n = 6 (B). In panel C, a summary of Mx1 expression data from ex vivo experiments is presented. Lung tissue cultures from WT (n = 10) and TRPA1-deficient (KO; n = 7) mice were exposed to IFN beta (10 ng/mL) for 24 hours, tissue pieces were homogenized, total RNA was extracted, and Mx1 expression was measured by RT-qPCR. Gapdh was used as an endogenous control gene. The control of each mouse was set as 1, and the treated condition of the same mouse was given in relation to that value. In all figures, individual values and mean ± SD are presented. Statistical analysis was carried out using one-way ANOVA with Holm-Šídák post-test (A) or Student’s t test (B). In panel C, two-way repeated measures ANOVA (mixed effects model) with Holm-Šídák post-test was used and the interaction between the genotype and the treatment was statistically significant (P < .05). ∗∗ and ∗∗∗∗ denote <.01 and .0001, respectively.

We next took another genetic approach using lung tissue from WT and TRPA1-deficient (KO) mice to establish an ex vivo model. Lung tissue cultures of WT and TRPA1-deficient mice were treated with IFN beta, and Mx1 expression was measured. The data indicated that IFN beta enhanced Mx1 expression in both genotypes, but the response was clearly attenuated in TRPA1 KO mice, and the interaction between the genotype and the treatment was statistically significant when tested with two-way ANOVA (Fig. 4C).

3.3. TRPA1 partially mediates ISRE-dependent transcription induced by IFN beta

Based on the gene expression results, we hypothesized that TRPA1 could promote ISRE-dependent transcription induced by type I IFNs. To test the hypothesis, A549 cells were transfected with dual luciferase plasmids with Firefly luciferase under the control of an ISRE-dependent promoter or control promoter (“empty”); and in both cases, Renilla luciferase as a control reporter. IFN beta increased ISRE-dependent luciferase activity, and both TRPA1 antagonists (HC-030031 and A-967079) partially inhibited this effect to the same level. These data suggest that ISRE-dependent transcription induced by IFN beta was partially TRPA1 dependent (Fig. 5).

Fig. 5.

TRPA1 antagonists inhibit ISRE-dependent transcription induced by IFN beta. A549 cells were transfected for 24 hours with dual luciferase plasmids with Firefly luciferase as a reporter gene under the control of an ISRE-dependent promoter or control promoter (“empty”); and in both cases, Renilla luciferase under a constitutive promoter was used as an internal control. Thereafter, cells were treated with IFN beta (10 ng/mL), with or without the TRPA1 antagonists HC-030031 (HC; 100 μM) or A-967079 (A96; 100 μM), or vehicle (DMSO) for 7 hours 30 minutes. After protein extraction, Firefly and Renilla luciferase activity was read. Firefly activity in each sample was normalized to the respective Renilla activity, and ISRE-transfected activity was then normalized to the mean empty-transfected activity of the respective treatment. Control was set as 1, and the other values are given in relation to that value. Individual values and mean ± SD are presented, n = 6. Statistical analysis was performed using one-way ANOVA with Holm-Šídák post-test. Asterisks on bars indicate comparison against the condition treated with IFN beta, and asterisks on brackets indicate comparison between indicated conditions. ∗∗, ∗∗∗, and ∗∗∗∗ denote P < .01, <.001, and <.0001. Ns = not significant.

3.4. TRPA1 partially mediates IFN-beta-induced STAT1 tyrosine phosphorylation at the site 701

We hypothesized that the observed reduction in ISRE-dependent transcription by TRPA1 antagonists could be STAT1 dependent and examined STAT1 tyrosine phosphorylation at the site 701 (pSTAT1-Y701) as a marker of STAT1 activation. A549 cells were incubated with IFN beta or IFN alfa-2a for 15 minutes, in the presence or absence of HC-030031 or A-967079 and western blotting of the phosphorylated and total STAT1 was carried out. No STAT1 phosphorylation was detected in controls, and both IFNs induced a robust increase in pSTAT1-Y701. This increase was modestly (but in a statistically significant manner) inhibited by both TRPA1 antagonists, suggesting that TRPA1 did promote the tyrosine phosphorylation of STAT1 at the site 701 (Fig. 6).

Fig. 6.

TRPA1 antagonists inhibit IFN-beta-induced STAT1 tyrosine phosphorylation at the position 701. A549 cells were cultured for 15 minutes with 10 ng/mL of IFN beta (A, C) or IFN alfa-2a (B, D), with or without the TRPA1 antagonists HC-030031 (HC; 100 μM) or A-967079 (A96; 100 μM). Thereafter, total protein was extracted and western blot of tyrosine phosphorylated STAT1 (at the position 701; pSTAT1-Y701) and total STAT1 was performed. In panels A and B, representative blots (with 2 biological replicates of each treatment) are shown. In panels C and D, densitometric data of pSTAT1-Y701 normalized to the total STAT1 signal of the respective sample are presented as individual values and mean ± SD, n = 6. Statistical analysis was performed using one-way ANOVA with Holm-Šídák post-test against the IFN-treated condition (second bar from the left). ∗ and ∗∗∗∗ indicate P < .05 and P < .0001, respectively.

3.5. TRPA1 is activated by IFN beta in A549 cells

Motivated by the effects of the TRPA1 antagonists on the effects of type I IFNs, we tested the hypothesis that TRPA1 is activated in response to those cytokines. A549 lung epithelial cells (endogenously expressing TRPA1) were loaded with the fluorescent Ca²⁺ indicator Fluo 3-AM and exposed to IFN beta, in the presence or absence of the 2 TRPA1 antagonists HC-030031 and A-967079. Fluorescence was measured as an indicator of changes in intracellular Ca²⁺. IFN-beta-induced an increase in fluorescence within seconds, followed by a slower increase which seemed to plateau within the measurement time of 5 minutes 30 seconds. This increase was partially inhibited by the pretreatment with both TRPA1 antagonists, suggesting TRPA1 activation in response to IFN beta (Fig. 7).

Fig. 7.

TRPA1 is activated in response to IFN beta in A549 cells. A549 cells were loaded with the Ca²⁺-responsive fluorescent indicator Fluo 3-AM or were sham loaded for 30 minutes. Thereafter, cells were pretreated with the TRPA1 antagonists HC-030031 (HC; 100 μM) or A-967079 (A96; 100 μM) or with the vehicle (DMSO) for 30 minutes before starting the measurements. After measuring baseline fluorescence for 15 seconds, IFN beta (10 ng/mL) was dispensed and fluorescence was measured for 5 minutes 30 seconds. In both figures, data are presented as the relative change in fluorescence (ΔF) over the background fluorescence of the respective well (F0). The mean sham loaded value of each time point was subtracted from the values of the respective time point. In panel A, a time curve representing the mean fluorescence (±SEM) sampled from n = 18 wells at the indicated times is shown. In panel B, area under curve (AUC) data are presented as individual values and mean ± SD, n = 18. In panel B, statistical analysis was carried out using one-way ANOVA with Holm-Šídák post-test. ∗, ∗∗ denote P < .05 and <.01, respectively.

4. Discussion

The present data show that the TRPA1 ion channel mediates the effects of type I IFNs to promote the expression of antiviral genes in human A549 lung epithelial cells. TRPA1 seems to be activated in response to IFN beta in intracellular Ca²⁺ measurements and based on experiments with pharmacological or genetic inhibition of TRPA1 and MX1 as an example of antiviral genes, TRPA1 seemed to present its effect in an ISRE and pSTAT1-Y701 dependent manner.

The involvement of TRPA1 in the type I IFN-induced gene expression was studied by several methods. NGS-based RNA sequencing was used to explore the effects of TRPA1 on gene expression induced by IFN beta. To reduce the chance of off-target effects, we chose to use 2 TRPA1 antagonists (HC-030031 and A967079) belonging to different classes based on their chemical structure⁴⁴ and included only those genes which were affected by both of the antagonists. We found that TRPA1 antagonists inhibited the upregulation of a large fraction (133/456; 29.2%) of genes induced by IFN beta (by more than 5-fold) and that these genes were included in GO terms related to antiviral immunity and inflammation, such as the terms “defense response to virus,” “negative regulation of viral genome replication,” and “inflammatory response.” On the other hand, fewer (76) genes were downregulated by IFN beta and only in the case of 2 genes (2.6%) TRPA1 antagonists partly reversed this downregulation. These data suggest that TRPA1 is particularly involved in promoting the induction of ISGs rather than mediating the downregulation of genes by IFN beta.

Following the RNA sequencing analysis, IFN-stimulated genes OAS2, DDX58, and particularly MX1 were selected for the further studies, and RT-qPCR and western blot measurements first repeated the effects seen in the RNA-seq. These genes were chosen as examples due to their relevance in antiviral innate immunity. MX1 is a large GTPase protein with broad antiviral activity. It recognizes the structures of many different viruses and has been reported to carry out antiviral effector functions at multiple steps of the viral life cycle.⁴⁵ OAS proteins (including OAS2) induce the activation of the endonuclease RNAse L, which exerts antiviral effects by degrading viral RNA.⁴⁶ DDX58/RIG-I on the other hand recognizes dsRNA to induce the production of type I IFNs and other innate immunity responses.⁴⁷ TRPA1 antagonists inhibited the upregulation of these genes induced not only by IFN beta, but also by another type I interferon IFN alfa. Similar results to the pharmacological antagonists of TRPA1 were obtained by genetic inhibition using TRPA1 siRNA or TRPA1 deficient mice, supporting the TRPA1 dependency of these effects.

In addition to antiviral effects, type I IFNs possess immunomodulatory properties. For example, in viral lung infections, type I IFNs amplify the inflammatory response, recruiting and activating immune cells. Excessive type I IFN response may, however, drive overt proinflammatory responses and detrimental effects such as alveolar epithelial cell damage.^48,49 The inflammatory role of type I IFNs is highlighted by their pathogenetic role in some inflammatory rheumatoid diseases such as systemic lupus erythematosus.^50,51 Anifrolumab, a monoclonal antibody inhibiting the type I IFN receptor, has indeed proven efficacious in the treatment of systemic lupus erythematosus.⁵²

In our RNA sequencing data, the GO terms “innate immune response” and “inflammatory response” were also altered by TRPA1 antagonists. The terms involved several proinflammatory and/or immune cell activating factors, for example the cytokine IL-6 (consistent with our previous observations⁵³) and chemokines CCL5, CXCL10, and CXCL11. These data suggest that inhibition of TRPA1 attenuated the inflammatory response induced by type I IFNs in these cells. Inhibiting TRPA1 could therefore be a potential therapeutic strategy in treating conditions characterized by an excessive type I IFN signature, such as the previously mentioned rheumatological diseases or hyperinflammatory conditions associated with severe viral infections.

In viral infections, TRPA1 antagonists could have beneficial or harmful effects depending on the course and type of the disease. For example, in the early phase of coronavirus disease 2019 (COVID-19) infection, an early and potent type I IFN response is protective.^54,55 On the other hand, a delayed and sustained systemic type I IFN response has been postulated to promote excessive inflammation.⁵⁵ Supporting this, the Janus kinase inhibitor baricitinib, which among its other effects also inhibits type I IFN responses, is associated with reduced mortality in severe COVID-19.⁵⁶ Therefore, inhibiting TRPA1 and consequently the effects of type I IFNs could be harmful in the early stage of COVID-19 but in the later, hyperinflammatory state, it is likely to be beneficial. We suggest that the incidence and severity of viral infections should be monitored when targeting TRPA1 for pharmacotherapy. Perhaps, activating TRPA1 by agonists could even be beneficial in the early stage of viral infections to potentially amplify the effects of type I IFNs, and inhibiting TRPA1 could be beneficial in later stages of severe viral infections characterized by a hyperinflammatory state.

Based on intracellular Ca²⁺ measurements, reporter gene experiments, and western blot data, we suggest that TRPA1 activation by type I IFNs promotes ISRE-dependent transcription, potentially via increased Y701 phosphorylation of STAT1.

This concept is supported by previous literature. It has been reported that IFN beta induces a rapid (in 10–20 seconds) increase in intracellular Ca²⁺ dependent on extracellular Ca²⁺ in RPMI-4788 cells.⁵⁷ Another study showed that intracellular Ca²⁺ augments type I IFN signaling in primary human macrophages and other cell types.²⁹ In the present study, IFN beta increased intracellular Ca²⁺ in A549 cells and TRPA1 antagonists partially reduced this effect, suggesting that TRPA1 is activated in response to IFN beta stimulation and contributes to the IFN-beta-induced calcium signaling. Furthermore, tyrosine phosphorylation of STAT1 is regulated by Ca²⁺-dependent kinases calmodulin kinase II and in its downstream, proline-rich tyrosine kinase 2.²⁹ By increasing Ca²⁺ influx, TRPA1 could activate these kinases which then might increase STAT1 phosphorylation and mediate the TRPA1-dependent potentiation of type I IFN-induced gene expression changes observed in the present study. Our data indicate that TRPA1 antagonists inhibited STAT1 phosphorylation (at the site 701), but the effect was rather modest. On the other hand, a modulatory effect rather than a large inhibition was expected as the increase in intracellular Ca²⁺ by IFN beta was only partially TRPA1 dependent.

Further studies are needed to determine the mechanism by which IFN beta activates TRPA1, but we propose that it is not a direct effect on the channel. TRPA1 is typically activated by electrophilic and other small chemical compounds and low temperature,⁵ but to our knowledge, not directly by macromolecules. It is known that an increase in intracellular Ca²⁺ alone is sufficient to activate TRPA1.⁸ A possible mechanism could therefore be that IFN beta activates first another mechanism which induces an increase in intracellular Ca²⁺ which results in TRPA1 activation. The primary mechanism of Ca²⁺ mobilization induced by IFN beta is poorly understood but seems be dependent on extracellular Ca²⁺.⁵⁷ Nevertheless, as TRPA1 antagonists inhibited the increase in intracellular Ca²⁺ induced by IFN beta only partially, it seems clear that other factors in addition to TRPA1 are also involved which remains to be studied.

Finally, some aspects should be considered when replicating and/or interpreting these results. A549 cells endogenously express TRPA1.^24,34 However, A549 cells are an immortalized cell line and TRPA1 expression is known to be altered in many cancer cells.³³ The antiviral mechanisms and type I IFN responses could potentially also be altered. This could affect the generalizability of these results from A549 cells to other cell types, and the results should be confirmed using primary cells in further studies. Secondly, the microenvironment likely plays an important role. In our previous studies, we have shown that TRPA1 expression is upregulated by (especially T helper 1 type) inflammatory cytokines.^24,58 The present study was carried out without inflammatory stimulation before IFN exposure, but despite this, TRPA1 was activated in response to type I IFNs and mediated biologically relevant gene expression changes. In an inflammatory microenvironment, TRPA1 expression and function would be expected to be upregulated as has been demonstrated previously.^24,25,58, 59, 60, 61 Therefore, under pre-existing inflammatory conditions, the role of TRPA1 in mediating type I IFN responses could be more pronounced. Finally, the calcium signal elicited by IFN beta in the present study could be an underestimation of the true biological response, as probenecid was present in the loading buffer. Probenecid has been shown to desensitize TRPA1 activation but importantly does not influence TRPA1 blocker responses.⁶²

5. Conclusions

Our results support a novel model in which the TRPA1 channel can promote the signaling and biologically relevant gene expression changes induced by type I IFNs. Therefore, TRPA1 could be a novel target for treating inflammatory conditions characterized by type I IFN activity such as excessive lung inflammation associated with viral infections and some autoimmune diseases—but TRPA1 inhibition could also carry adverse outcomes related to poorer IFN-induced antiviral immunity. This should be considered when targeting TRPA1 for pharmacotherapy.

Conflict of interest

The authors declare no conflicts of interest.