α4 Nicotinic Acetylcholine Receptor Signaling Exacerbates Type 2 Airway Inflammation

To the Editor,

1.

Type 2 (T2) airway inflammation, such as allergic asthma and eosinophilic chronic rhinosinusitis (ECRS), is characterized by marked eosinophil accumulation at the inflamed site and increased Th2 cytokines [1]. Inhaled anticholinergic medications, as therapeutic agents for asthma, exert their effects by blocking muscarinic receptors while having minimal effect on nicotinic acetylcholine receptors (nAChR) [2]. α7 (CHRNA7) subunit is well known for its anti‐inflammatory role in T2 airway inflammation [3], whereas the roles of other nAChRs remain less defined. Recently, emerging evidence suggests that the α4 (CHRNA4) subunit of nAChRs contributes to inflammatory responses such as promoting T‐cell proliferation and cytokine production, inducing calcium influx and inflammatory signaling in metabolic dysfunction‐associated steatohepatitis, and enhancing lung injury via proinflammatory cytokine upregulation [4, 5, 6]. However, the functional role of CHRNA4 in T2 inflammation remains poorly understood.

To investigate CHRNA4 expression in eosinophilic airway inflammation, we assessed nasal polyps from patients with ECRS and mouse lungs in a house dust mite (HDM)‐induced allergic airway inflammation (AAI) model. In humans, CHRNA4 was significantly higher in ECRS nasal polyps than in non‐ECRS and control tissues (Figure 1A, Figure S1A,C,D). Consistently, HDM challenge markedly upregulated Chrna4 mRNA and protein expression in mouse lungs compared with PBS controls (Figure 1B, Figure S1B,E).

FIGURE 1.

CHRNA4 contributes to exacerbating T2 airway inflammation. (A) Representative immunofluorescence staining of CHRNA4 in non‐ECRS, and ECRS nasal polyp; MFI ratio of CHRNA4 in ECRS relative to non‐ECRS. (B) Representative immunofluorescence staining of CHRNA4 in lung of PBS and HDM groups; MFI ratio of CHRNA4 in HDM relative to PBS control. CHRNA4 signals are shown in red, nuclei counter‐stained with Hoechst (cyan). (C) Lung resistance and compliance of wild‐type (α4 ^+/+ ) and Chrna4‐deficient mice (α4 ^−/− ). (D) Total and differential BALF cell counts. (E) Histology of lung sections. Black arrows indicate eosinophilic infiltration. (F) Th2 cytokines and epithelial‐derived cytokines in BALF. (G) Total serum IgE. Data are presented as mean ± SEM (n = 4–6 per group). *p < 0.05, **p < 0.01 vs. α4 ^+/+ /HDM group. Data in (A) were analyzed using Mann–Whitney U test, Student's t‐test was used in (B) and one‐way ANOVA with Dunnett's multiple‐comparisons test was used in (C, D, F, G).

To further clarify the potential role of CHRNA4 in T2 airway inflammation, we generated Chrna4‐deficient mice (Chrna4 ^−/− ) by removing exon 5 and showed no obvious abnormalities in breeding efficiency, mortality, and immune‐cell populations compared with wild‐type littermates (Figure S1F–K). In AAI model, HDM increased airway resistance and decreased compliance in Chrna4 ^+/+ mice, reflecting airway hyperresponsiveness (AHR), whereas these changes were partially attenuated in Chrna4 ^−/− mice (Figure 1C). Inflammatory cell recruitment into bronchoalveolar lavage fluid (BALF) and lung histological inflammation were also significantly reduced in HDM‐challenged Chrna4 ^−/− mice (Figure 1D,E, Figure S2A). Moreover, Th2 cytokines (IL‐4, IL‐5, and IL‐13) and epithelial‐derived cytokines (IL‐25, IL‐33, and TSLP) in BALF and serum IgE were significantly decreased in Chrna4 ^−/− mice upon AAI induction (Figure 1F,G, Figure S2B). Together, these data suggest that Chrna4 contributes to the development of T2 airway inflammation by enhancing cellular infiltration, Th2 and epithelial‐derived cytokines, and IgE production.

We next examined CHRNA4 signaling in immune cells during T2 airway inflammation. Chrna4 expression was significantly increased in eosinophils upon HDM stimulation, with only marginal upregulation in lymphocytes and macrophages (Figure 2A). To further determine whether Chrna4 modulates eosinophil function and inflammatory cytokines, eosinophils isolated from spleens of HDM‐sensitized hypereosinophilic mice (IL‐5 Tg) were treated with TC‐2559 (CHRNA4 agonist) and/or DHβE (CHRNA4 antagonist). DHβE inhibited the increase in eosinophil Th2 cytokines mRNA, whereas CHRNA4 activation significantly induced them (Figure 2B), suggesting that CHRNA4 signaling promotes eosinophil‐mediated T2 immune responses. Similarly, in human bronchial epithelial cells (BEAS‐2B cell line), HDM stimulation, in the presence of TC‐2559, enhanced epithelial alarmin expression (IL‐25, IL‐33, and TSLP), whereas DHβE reduced these responses (Figure 2C), indicating that CHRNA4 also promotes epithelial activation during T2 inflammation.

FIGURE 2.

CHRNA4 signaling in immune cells and its modulation of T2 airway inflammation. (A) Chrna4 expression in immune cell subsets in the steady state and AAI, shown as fold change relative to naïve B cells (normalized to 1). **p < 0.01 vs. naïve, n = 4. (B) Eosinophils treated with CHRNA4 agonist (TC‐2559) and/or antagonist (DHβE), Th2 cytokine mRNA measured by qPCR. *p < 0.05, **p < 0.01 vs. TC‐2559 alone. Data are presented as mean ± SEM from four independent experiments. (C) IL‐33, IL‐25, and TSLP mRNA in BEAS‐2B cells after exposure to HDM in the presence or absence of TC‐2559 and DHβE. *p < 0.05, **p < 0.01 vs. HDM alone. Data are presented as mean ± SEM from four independent experiments. The dashed line represents the control level (PBS). (D) Th2 cytokines in lung cells following nicotine stimulation in the presence of CHRNA4 inhibition (DHβE) and/or CHRNA7 inhibition (MLA). *p < 0.05, **p < 0.01 vs. nicotine alone. Data are presented as mean ± SEM from five independent experiments; NS, Not significant. Data in (A) were analyzed using Student's t‐test, and one‐way ANOVA with Dunnett's multiple‐comparisons test was used in (B–D).

To clarify the complex effects of nAChR subtypes in T2 airway inflammation, we used nicotine as a broad nAChR agonist and pharmacological antagonists preferentially targeting CHRNA7 and/or CHRNA4‐containing nAChRs. In inflamed lung cells, the CHRNA4 antagonist reduced nicotine‐induced Th2 cytokines, while the CHRNA7 antagonist enhanced these responses (Figure 2D). Co‐treatment with these two antagonists did not influence nicotine‐induced Th2 cytokine levels (Figure 2D). These results indicate opposing roles of nAChR subtypes, with CHRNA4 promoting and CHRNA7 attenuating T2 airway inflammation. Although the pharmacological tools used here do not fully distinguish among CHRNA4‐containing receptor assemblies and off‐target effects cannot be excluded, these results support a role for nAChR subtype‐dependent signaling in allergic airway inflammation and highlight CHRNA4‐containing nAChRs as a potential modulatory pathway in allergic airway disease.

In conclusion, our study first uncovered the role of CHRNA4 signaling in promoting T2 airway inflammation. Genetic deficiency of CHRNA4 and pharmacological inhibition of CHRNA4 effectively attenuated T2 responses in the AAI model and in vitro, indicating that CHRNA4 blocking is a potential therapeutic target for T2 airway inflammation. These findings might be a paradigm shift in the concept of cholinergic regulation in airway immunity, with the selective modulation of nAChR subtypes, rather than broad cholinergic intervention, representing a more precise strategy for treating these diseases.

Author Contributions

N.K.T.L. and A.K. were involved in all stages of this investigation. D.V.B., L.T.T.K., Q.D.H. performed experiments. Yu.K., K.T., Yo.K., Y.Y., L.M.N., Q.D.H., K.S., A.K., A.M., A.S., D.V.B., L.T.T.K., R.K., M.M., D.D., H.I., and M.Y. were involved in technical comments and drafting the manuscript. All authors approved the final manuscript and publication.

Funding

This work was supported by funding from the Academic Society for Research in Otolaryngology, Kansai Medical University (KMU); a research grant from the KMU research consortium; Grant‐in‐Aid for Scientific Research (C), 19K09860; the branding program as a world‐leading research university on intractable immune and allergic diseases. DD is supported by grants from the Agence Nationale de la Recherche (ANR) and the European Union: EGID ANR‐10‐LABX‐46 and ANR‐20‐CE15‐0026, Fondation pour la Recherche Médicale (FRM), Nouveaux Défis en Allergologie. All these funding institutions were not involved in study design; in the collection, analysis, and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: Immunofluorescence staining of CHRNA4 and generation of Chrna4‐deficient mice. (A) Representative human nasal polyp and (B) mouse lung sections stained with rabbit IgG isotype control. (C) Representative immunofluorescence staining of CHRNA4 in uncinate tissue (UT) control sections. (D) CHRNA4 expression in UT controls (n = 8), non‐ECRS (n = 7), and ECRS nasal polyps (n = 8). (E) Chrna4 expression in lung tissue of PBS and HDM (n = 4 per group). **p < 0.01. (F) Gene maps of the targeting construct and Chrna4 locus before and after homologous recombination. (G) Genotyping of Chrna4‐deficient mice, WT (α4 ^+/+ ), heterogenous (α4 ^flox/− ) and homogenous (α4 ^−/− ). (H) RT‐PCR analysis confirming the successful deletion of Chrna4 mRNA expression in bone marrow, blood, and lung tissues from α4 ^−/− and α4 ^+/+ mice. (I) Chrna4 mRNA expression in lung tissue from α4 ^+/+ and α4 ^−/− mice. (J) RT‐PCR expression of other nAChR subunits in lung tissue. (K) Differential leukocytes in the blood of α4 ^−/− and α4 ^+/+ mice. Data are expressed as mean ± SEM (n = 3 for each group). ND, not detected. No significant difference compared with α4 ^+/+ mice (p values > 0.05). Data in (D), (E) were analyzed using Mann–Whitney U test, Student's t‐test was used in (K).

Figure S2: The role of CHRNA4 in T2 airway inflammation. (A) Histology of lung sections stained with PAS to visualize mucus production. Orange arrows indicate goblet cells. (B) qPCR analysis of Th1, Th2, Th17, IL‐25, IL‐33, TSLP mRNA expression in lung tissue, *p < 0.05, **p < 0.01 vs. α4 ^+/+ /HDM group. (C) Gating strategy for the identification of cell populations in the mouse lung tissue using flow cytometry. (D) Immunofluorescence staining of BEAS‐2B for CHRNA4 (red), EpCAM (blue), nuclei (cyan). (E) CCL11, CCL26 mRNA expression in eosinophil after treating with TC‐2559 and/or DHβE. *p < 0.05, **p < 0.01 vs. TC‐2559 alone. (F) Schematic of the AAI model and the ex vivo lung‐cell cholinergic stimulation experiment. (G) Relative mRNA expression of IL‐4, IL‐5, and IL‐13 in lung cells with nicotine stimulation in the presence of CHRNA4 inhibition (DHβE) and/or CHRNA7 inhibition (MLA). Data are presented as mean ± SEM from five independent experiments. *Significant difference from nicotine alone: *p < 0.05, **p < 0.01. NS, not significant. All data were analyzed using one‐way ANOVA with Dunnett's multiple‐comparisons test.

Table S1: Demographics of the human nasal polyp and UT samples.

Table S2: Guide RNAs and single strand donor DNAs for producing Chrna4 ^flox mice by CRISPR/Cas9 system.

Table S3: Genotyping information.

Table S4: Amplification primers (5′–3′).

Table S5: List of antibodies.

Data Availability Statement

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.