Investigation of the Pathomechanism of Chronic Cough Using an In Vitro Approach

Abstract

Previous ATP inhalation studies and clinical trials have demonstrated an anti-tussive effect of P2X3 antagonists, supporting the mechanism of purinergic P2X3 receptor activation leading to unexplained chronic cough (UCC). However, the role of TRP receptors in UCC is currently unclear. In this study, an interaction between P2X3 receptors on airway bronchial epithelial cells (BECs) and TRPA1 channels on upper airway nerves was explored in vitro as a potential mechanism for UCC (Figure 1A) [1-3]. It was hypothesised that irritants activating TRP channels can result in ATP release from airway nerves resulting in P2X3 receptor activation on adjacent airway epithelial cells [3-6]. To investigate the role of TRPA1 channel activation of P2X3 receptors, an indirect in vitro cell model was developed to demonstrate whether activation of TRPA1 channels expressed on neuronal cells result in ATP release that subsequently activates P2X3 receptors on adjacent BECs.

Dorsal root ganglion cells (DRGN, ND8/34 cell line, Sigma), known to express TRPA1 were used as a surrogate for airway neuronal cells [7]. Functional assays were performed on DRGNs to quantify TRPA1 activation using the TRPA1 specific agonist (JT010) by observing a change in fluorescence measured by [Ca²⁺]i of 10% or greater from baseline under confocal microscopy. The DRGNs cultured on poly-Lysine coated plates and loaded with FLUO4, were preincubated in the presence or absence of the TRPA1 antagonist HC-030031 (EMD Milipore) and then treated with a TRPA1-specific agonist (JT010 100 nM, Tocris) (n = 3 experiments). Culture media from the DRGNs cell wells were collected after stimulation and assayed for ATP. ATP release in response to JT010-induced TRPA1 activation and its suppression by the TRPA1-antagonist, HC-030031, were quantified from the cell supernatant on a luminometer using the ATP Bioluminescence Assay Kit HS II (Roche). Additional information about study methods are available in the following repository (DOI 10.5281/zenodo.14244289).

Human bronchoepithelial cells (BEAS2B, ATCC), were used as a surrogate for airway epithelium. The effect of P2X3-receptor antagonist, MAF-454, in preventing activation of P2X3 receptors were determined by treating cultured BEAS2B cells with ATP disodium 4 μM (Sigma) stabilised with KOH in the presence and absence of MAF-454 preincubation. Differential gene expression (DEGs) in these samples versus untreated controls were determined using TaqMan rat inflammation array (Thermo Fisher). Pathway analysis, and upstream regulator analysis of the DEGs in the ATP-treated samples, compared to the MAF-454 preincubated ATP-treated samples were performed using the Ingenuity Pathway Analysis platform (Qiagen). As this was an in vitro study and did not require patient serum or patient data, no IRB was required.

Descriptive statistics and t-tests were performed to analyse differences in the average concentration of ATP released between JT010-treated and HC-030031 + JT010-treated DRGN cells. Activation Z-scores were used for analysis of the DEGs to determine the significant canonical pathways, and upstream regulators in the ATP-treated samples, compared to the MAF-454 preincubated ATP-treated samples.

For experiments on DRGNs, the mean ATP concentration in the JT010-treated and HC-030031 + JT010-treated DRGN cells were 0.52 (±0.03) and 0.26 (±0.01) μM; these differences were statistically significant (p < 0.05) (Figure 1B). Functional assays demonstrated significant TRPA1-induced Ca-signalling in the DRGNs (data not shown). For gene expression experiments on ATP-treated BEAS2B, the top DEGs between ATP-treated and MAF-454 plus ATP-treated BEAS2B cells were leukotriene B4 receptor (LTB4R1 activation promotes migration of macrophages and neutrophils into tissue), Caspase 1 (CASP1 proteolytically cleaves and activates the inactive precursor of IL-1 involved in inflammation), histamine receptor 1 (HRH mediates smooth muscle contraction and increases in capillary permeability), adrenoceptor Beta 1 (ADRB1 associated with diseases affecting resting heart rate and variations in sleep), phospholipase C (PLC cleaves phospholipids such as phosphatidylinositol 4,5 biphosphate into diacyl glycerol and inositol 1,4,5-trisphosphate, important second messengers), integrin beta 1 (ITGB1 cell adhesion receptor involved in a variety of biologic processes), and leukotriene A4 hydrolase (LTA4H catalyses LTA4 to LTB4).

Based on activation z-scores, the top canonical pathways predicated to be activated by ATP and inhibited by MAF-454 were FAK signalling, phagosome formation, CREB signalling in neurons and GPCR signalling (eSupplement). IL4 and IL13 were predicted as significant upstream regulators of these pathways, and IL1B as a key biomarker. Additional information about study findings are available in the following repository (DOI 10.5281/zenodo.14244289).

These in vitro experiments provide indirect evidence of a possible interaction between TRPA1-induced ATP release from sensory neurons in the airway epithelium by exogenous triggers that may activate P2X3 receptors on adjacent BECs. Prior antagonism of P2X3 receptors on airway epithelial cells by the P2X3-specific antagonist, MAF-454, can diminish the irritant effect of exogenous trigger-induced ATP release via neuronal TRPA1 channels. Interestingly, the DEG pathways that were downregulated with the P2X3 antagonist were involved in biological processes that promote airway inflammation. For example, these genes have been identified to play a direct or indirect role in cough regulation investigated in the context of diseases manifesting as cough [8]. Furthermore, inhibition of H1 receptors by dexbrompheniramine, has been shown to dose dependently inhibit capsaicin-evoked calcium responses and TRPV1 receptor inhibition [9]. This may potentially explain why first-generation antihistamines like dextrobrompheniramine are effective in chronic cough [9].

In summary, these experiments provide preliminary data that suggests a potential mechanistic role for activation of TRP channels resulting in increased ATP release and activation of P2X3 receptors on adjacent BECs. Specific antagonism of P2X3 receptors on BECs attenuated the activation of these DEGs supporting the promising role of these agents for treatment of UCC.

[Correction added on 24 January 2025, after first online publication: Subsections have been removed.]

Dr. Jonathan A. Bernstein and Dr. Umesh Singh conceptualized and designed the study; Dr. Umesh Singh performed the experiments. Drs. Jonathan A. Bernstein and Umesh Singh analysed the data and wrote this manuscript.

Jonathan A Bernstein is a PI and consultant for Merck and GSK; Umesh Singh has no disclosures.