The therapeutic effects of labetalol in chronic stress- and immuno-agonist-induced cough hypersensitivity and lung inflammation involve reducing interferon-γ-producing T lymphocytes

Graphical abstract

Chronic stress leads to neurogenic inflammation by activating the sympathetic nervous system to release neurotransmitters. IFN-γ: interferon-γ.

Abstract

Background

Psychological stress correlates with chronic cough, which is often triggered by respiratory viruses. Chronic stress promotes sympathetic nerves to release norepinephrine and neuropeptide Y. Labetalol blocks sympathetic adrenergic receptors. Norepinephrine and neuropeptide Y can upregulate interferon-γ (IFN-γ)-producing lymphocytes and increase IFN-γ levels in vivo. IFN-γ-producing lymphocytes and IFN-γ contribute to chronic stress-induced colonic inflammation and influenza virus-caused pulmonary inflammation. IFN-γ can also enhance cough sensitivity. Administration of haemagglutinin and R848 (immuno-agonists) can induce a virus-infection-like illness in animal models.

Methods

We investigated the effects and underlying mechanisms of labetalol on chronic stress and immuno-agonist-induced cough hypersensitivity and lung inflammation.

Results

Chronic stress and immuno-agonists induced cough hypersensitivity and pulmonary inflammation in guinea pigs, which were alleviated by labetalol. In mice, chronic stress triggered the release of norepinephrine and neuropeptide Y, leading to IFN-γ-producing T lymphocytosis and pulmonary inflammation. Additionally, chronic stress and immuno-agonists increased IFN-γ in the lung of guinea pigs and mice. Labetalol effectively reduced chronic stress-induced and immuno-agonist-induced IFN-γ-producing T lymphocytosis and lung inflammation in mice. Labetalol dose-dependently inhibited the increase in IFN-γ-producing T lymphocytes induced by immuno-agonists and norepinephrine in vitro.

Conclusions

Chronic stress can stimulate the release of sympathetic neurotransmitters, which may increase IFN-γ-producing T lymphocytes and lead to cough hypersensitivity and pulmonary inflammation by activating the adrenergic receptors. Immuno-agonists may increase IFN-γ-producing T lymphocytes, exacerbating chronic stress-induced cough hypersensitivity and pulmonary inflammation. The therapeutic effects of labetalol in chronic stress and immuno-agonist-induced cough hypersensitivity and pulmonary inflammation possibly occur by reducing IFN-γ-producing T lymphocytes.

Shareable abstract

Chronic stress causes cough hypersensitivity and pulmonary inflammation by activating adrenergic receptors. Immuno-agonists increase IFN-γ-producing T lymphocytes and exacerbate inflammation. Labetalol exerts therapeutic effects by reducing T lymphocytes. https://bit.ly/45JXSob

Introduction

Stress, anxiety and depression frequently occur in patients with chronic cough [1, 2] and are positively correlated with chronic cough [3–5]. The prevalence of anxiety is significantly higher in patients with chronic refractory cough compared to in those who have been successfully treated [6]. Excessive anxiety, depression and other stress-related psychological issues contribute to the pathogenesis of somatic cough syndrome [7, 8]. Both chronic refractory cough and somatic cough syndrome are often triggered by a viral respiratory infection [8, 9].

Chronic social stressors contribute to the onset of anxiety and depression disorders in humans [10, 11]. Chronic stressors (including restraint, swimming and cold stress) induce depressive and anxiety-like behaviours in experimental animals [12]. Although chronic social stressors in human beings are different from imposed chronic stressors in experimental animals, they can lead to sustained activation of the sympathetic nervous system and promote the release of several sympathetic neurotransmitters, including norepinephrine (NE) and neuropeptide Y (NPY), in both [13–15]. Both NE and NPY are considered strong markers of sympathetic nervous system activation [14, 15]. The lung and spleen are richly innervated by sympathetic fibres [16, 17]. NE enhances interferon-γ (IFN-γ) secretion via adrenergic receptors [18], and increases IFN-γ-producing cluster of differentiation 8⁺ (CD8⁺) T lymphocyte responses and inflammation [14]. NPY receptor-deficient mice have reduced numbers of splenic CD8⁺ T lymphocytes and lower serum IFN-γ levels in response to delayed-type hypersensitivity reaction [19]. Chronic stress (CS) can directly induce lung inflammation in rats [20]. Labetalol, a blocker of both α and β adrenergic receptors, can suppress a paroxysmal sympathetic storm [21]. The expression of adrenergic receptors in T lymphocytes is well established [22]. Labetalol at a dose of 5 mg·kg⁻¹ effectively blocks the sympathetic nervous system in rats [23]. Based on conversion factors for body surface area, labetalol doses of 4 mg·kg⁻¹ for guinea pigs and 10 mg·kg⁻¹ for mice were used in this study. The in vitro 50% inhibitory concentration of labetalol as an antioxidant is 13.2 μg·mL⁻¹ [24]. Therefore, in our in vitro study, we used the following gradient doses of labetalol: 0.365, 2.19 and 13.14 μg·mL⁻¹.

Influenza virus infection can induce pulmonary inflammation by increasing lung-resident IFN-γ-producing T lymphocytes [25]. IFN-γ can also enhance the cough sensitivity [26]. IFN-γ-producing lymphocytes and IFN-γ play important roles in CS-induced colonic inflammation and influenza virus-induced pulmonary inflammation [25, 27]. Haemagglutinin, a key antigen of the influenza virus, can directly trigger inflammatory responses [28]. Toll-like receptor 7/8 (TLR7/8) is involved in lung inflammation and the production of proinflammatory cytokines during influenza virus infection [29, 30]. Administration of the TLR7/8 agonist R848 provides a useful model for virus infection, inducing a strong immune response, lung inflammation and virus infection-like illness [31]. Therefore, the combined administration of haemagglutinin and R848 (immuno-agonists (IAs)) can induce virus infection-like illness in animal models.

In this study, we hypothesised that labetalol might alleviate CS-induced and IA-induced cough hypersensitivity and lung inflammation, and that reducing IFN-γ-producing T lymphocytes might play a role in its therapeutic effects. We studied the effects and the underlying mechanisms both in vivo and in vitro. Although guinea pig is a suitable small animal to study cough reflex, there is a lack of commercially available antibodies and test kits for guinea pig research. In this study, guinea pigs were primarily used to investigate the effects of labetalol on the cough reflex and lung inflammation, while mice were mainly used to explore the underlying mechanisms.

Materials and methods

Detailed methods and all compounds and drugs used are outlined in the Methods section in supplement 1.

Animals

Male closed colony Hartley guinea pigs and male specific pathogen-free C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA, USA). Animal experiments received approval (2021122) from the Animal Care and Use Committee of Guangzhou Medical University, and were performed in strict accordance with approved guidelines.

Study design for guinea pigs

In the first experiment, guinea pigs were divided into a control group, IA group (IAs containing 2 μg·kg⁻¹ influenza A H5N1 haemagglutinin protein and 75 μg·kg⁻¹ R848), CS group and CS+IA group. CS was induced by forced cold water swim and restraint. This experimental protocol is presented in figure 1a.

FIGURE 1.

Chronic stress (CS) and immuno-agonists (IAs) induced cough hypersensitivity and pulmonary inflammation in guinea pigs. a) The experimental protocol for the guinea pig models that were induced by CS and IAs. Guinea pigs in the CS and CS+IA groups were exposed to forced cold water swim and restraint for 30 consecutive days. On day 31 (D31), all the guinea pigs were anaesthetised and intratracheally (i.t.) instilled with PBS or IAs dissolved in PBS. Cough sensitivity was detected in all the guinea pigs on D34. All the guinea pigs were killed on D35. b) Body weight changes of guinea pigs in the first experiment. c) The number of cough events within 10 min of exposing the animals to aerosolised citric acid (0.3 M). d) Group data showing the total number of leukocytes in bronchoalveolar lavage fluid (BALF). e) The total number of lymphocytes in BALF. f) Summarised data showing the total protein concentration in the supernatant of BALF. g) Summarised data showing the ratio of interferon-γ (IFN-γ) concentrations to the total protein concentrations in homogenised lung tissues. Guinea pigs in different groups were treated as follows: control group (no stress+i.t. with PBS), IA group (no stress+i.t. with IAs), CS group (CS+i.t. with PBS) and CS+IA group (CS+i.t. with IAs). Data show one representative of three independent experiments. n=9–10 per group. Data are shown as median (interquartile range) in b and f. Otherwise, data are shown as mean±sd.

In the second experiment, guinea pigs were divided into a control group, CS group, CS+IA group, labetalol (Lab)+CS group and Lab+CS+IA group. Between day 20 and day 33, guinea pigs were intraperitoneally injected with the vehicle solution or labetalol (4 mg·kg⁻¹) daily. This experimental protocol is shown in figure 2a.

FIGURE 2.

Labetalol reduced chronic stress (CS)-induced and immuno-agonist (IA)-induced cough hypersensitivity and pulmonary inflammation in guinea pigs. a) Scheme of the experimental protocol. Except for the control group, guinea pigs were exposed to forced cold water swim and restraint for 30 consecutive days. Between day 20 (D20) and D33, guinea pigs were intraperitoneally (i.p.) injected with the vehicle solution or labetalol (4 mg·kg⁻¹) daily. On D31, all the guinea pigs were anaesthetised and intratracheally (i.t.) instilled with PBS or IAs dissolved in PBS. Cough sensitivity was detected in all guinea pigs on D34. All the guinea pigs were killed on D35. b) Body weight changes of guinea pigs in the second experiment. c) The number of cough events within 10 min of exposing the animals to aerosolised citric acid (0.3 M). d) Group data showing the total number of leukocytes in bronchoalveolar lavage fluid (BALF). e) Summarised data showing the total protein concentration in supernatant of BALF. f–j) Representative figures of pathological changes in haematoxylin and eosin-stained lung sections from the different groups. Scale bar: 100 μm. k) Summarised data showing the total lung pathological scores of guinea pigs. Guinea pigs in different groups were treated as follows: control group (no stress+i.t. with PBS+i.p. with PBS), CS group (CS+i.t. with PBS+i.p. with PBS), CS+IA group (CS+i.t. with IAs+i.p. with PBS), Lab+CS group (CS+i.t. with PBS+i.p. with labetalol) and Lab+CS+IA group (CS+i.t. with IAs+i.p. with labetalol). Data show one representative of three independent experiments. n=6–7 per group. Data are shown as median (interquartile range) in k. Otherwise, data are shown as mean±sd.

In the third experiment, guinea pigs were divided into a control group and Lab group. The guinea pigs were not subjected to any stress during the experimental procedures. Between day 1 and day 7, guinea pigs were intraperitoneally injected with the vehicle solution or labetalol (4 mg·kg⁻¹) daily. This experimental protocol is shown in supplement 2: figure S1A.

Study design for mice

Mice were divided into a control group, CS group, CS+IA group, Lab+CS group (labetalol, 10 mg·kg⁻¹) and Lab+CS+IA group (labetalol, 10 mg·kg⁻¹). CS was induced by forced cold water swim and restraint. On day 31, every mouse was anaesthetised and intranasally instilled with PBS or IAs (50 μg·kg⁻¹ influenza A H5N1 haemagglutinin protein and 2 mg·kg⁻¹ R848). This experimental protocol is shown in figure 3a.

FIGURE 3.

Labetalol reduced chronic stress (CS)-induced and immuno-agonist (IA)-induced pulmonary inflammatory markers in mice. a) Scheme of the experimental protocol. Except for the control group, mice were exposed to forced cold water swim and restraint for 30 consecutive days. Between day 20 (D20) and D33, mice were intraperitoneally (i.p.) injected with the vehicle solution or labetalol (10 mg·kg⁻¹) daily. On D31, all mice were anaesthetised and intratracheally (i.t.) instilled with PBS or IAs dissolved in PBS. All mice were killed on D35. b) Body weight changes of mice. Values greater than zero represent weight gain, and values less than zero represent weight loss. c) Group data showing the total number of leukocytes in bronchoalveolar lavage fluid (BALF). d) The total number of lymphocytes in BALF. e) Summarised data showing the lactate dehydrogenase (LDH) activity in BALF. f) Summarised data showing the ratio of interferon-γ (IFN-γ) concentration to total protein concentration in homogenised lung tissues. Mice in different groups were treated as follows: control group (no stress+i.t. with PBS+i.p. with PBS), CS group (CS+i.t. with PBS+i.p. with PBS), CS+IA group (CS+i.t. with IAs+i.p. with PBS), Lab+CS group (CS+i.t. with PBS+i.p. with labetalol) and Lab+CS+IA group (CS+i.t. with IAs+i.p. with labetalol). Data show one representative of three independent experiments. n=6–7 per group. Data are shown as median (interquartile range) in b. Otherwise, data are shown as mean±sd.

Effects of labetalol on IA-induced and NE-induced changes in IFN-γ-producing T lymphocytes in vitro

Primary spleen mononuclear cells were extracted from the spleen of untreated C57BL/6 mice. Primary spleen mononuclear cells were pretreated with low-dose, medium-dose and high-dose labetalol (0.365, 2.19 and 13.14 μg·mL⁻¹, respectively) for 24 h. The old cell medium was then removed, followed by exposure to new cell medium containing IA (5 μg·mL⁻¹ R848 and 0.1 μg·mL⁻¹ influenza A H5N1 haemagglutinin protein) and 0.425 μg·mL⁻¹ NE for 24 h.

Statistical analysis

Normality was assessed using Shapiro–Wilk and Kolmogorov–Smirnov tests. For normally distributed data, ANOVA followed by Bonferroni's multiple comparison tests or unpaired t tests (two-tailed) were applied to evaluate the difference between groups. Post hoc tests were conducted only if F achieved p<0.05 and there was no significant variance inhomogeneity. For non-normally distributed data, Kruskal–Wallis tests or Mann–Whitney tests were used to evaluate the difference between groups. Values of p<0.05 were considered statistically significant.

Results

CS and IAs induced cough hypersensitivity and pulmonary inflammation in guinea pigs

The body weights of guinea pigs were significantly decreased in the IA and CS+IA groups compared to the control group (figure 1b). Guinea pigs in the CS and CS+IA groups exhibited significantly increased sensitivity to citric acid-induced coughing compared to the control group (figure 1c).

Total leukocyte count and lymphocyte count in bronchoalveolar lavage fluid (BALF) were markedly increased in both the CS and CS+IA groups compared to the control group (figure 1d, e). The total protein concentration in the supernatant of BALF was significantly increased in the IA, CS and CS+IA groups compared to the control group (figure 1f), which indicated airway inflammation [32]. CS in combination with IAs significantly increased IFN-γ levels observed in homogenised lung tissues (figure 1g).

CS and IAs induced splenic changes in guinea pigs

Compared with the control group, the ratio of spleen weight to body weight of guinea pigs was markedly elevated in the IA, CS and CS+IA groups (supplement 2: figure S3A). CS significantly increased the ratio of white pulp area to the total spleen area in guinea pigs (supplement 2: figure S3B–F). Lymphocytes accumulate in the white pulp of the spleen [32]. These results imply that CS might increase the number of lymphocytes in the spleen of guinea pigs.

Labetalol reduced CS-induced and IA-induced cough hypersensitivity and pulmonary inflammation in guinea pigs

Compared with the control group, CS in combination with IAs significantly reduced the body weight of guinea pigs (figure 2b). Labetalol markedly reduced cough hypersensitivity induced by CS and IAs (figure 2c). Compared with the control group, guinea pigs in the CS and CS+IA groups had significantly increased total leukocyte and lymphocyte count in BALF, and total lung pathological scores, which were considerably reduced by labetalol treatment (figure 2d–k). Major pathological changes in lungs included pulmonary oedema, inflammatory cell infiltration, pulmonary congestion and haemorrhage, and excessive mucus secretion.

Labetalol did not alter cough sensitivity to citric acid in the control guinea pigs

In the absence of CS, labetalol treatment did not produce significant changes in cough reflex sensitivity to citric acid compared to the control group (supplement 2: figure S1B). Labetalol treatment did not significantly alter total leukocyte count, lymphocyte count or the total protein concentration in BALF (supplement 2: figure S1C–E).

Labetalol reduced CS-induced and IA-induced pulmonary inflammation in mice

CS in combination with IAs significantly reduced the body weight of mice compared to the control group (figure 3b). Compared with the control group, total leukocyte count in BALF was significantly increased in the CS and CS+IA groups. CS in combination with IAs markedly increased lymphocyte count in BALF. Labetalol significantly reduced total leukocyte count and lymphocyte count in BALF of mice (figure 3c, d). The lactate dehydrogenase (LDH) activity level in BALF, a biomarker of airway inflammation [25], was notably increased in the CS and CS+IA groups when compared with the control group. Treatment with labetalol significantly reduced the LDH activity level (figure 3e).

Compared with the control group, the CS and CS+IA groups had notably increased IFN-γ levels in homogenised lung tissues, which were significantly reduced by labetalol (figure 3f). Compared with the control group, CS in combination with IAs markedly increased total lung pathological scores, which were significantly alleviated by labetalol treatment (figure 4). The main lung abnormalities in mice included pulmonary oedema, inflammatory cell infiltration, pulmonary congestion and haemorrhage, and excessive mucus secretion.

FIGURE 4.

Labetalol reduced chronic stress (CS)-induced and immuno-agonist (IA)-induced pulmonary pathological changes in mice. a–e) Representative figures of pathological changes in haematoxylin and eosin-stained lung sections from the different groups. Scale bar: 100 μm. f) Summarised data showing the total lung pathological scores of mice. Mice in different groups were treated as follows: control group (no stress+i.t. with PBS+i.p. with PBS), CS group (CS+i.t. with PBS+i.p. with PBS), CS+IA group (CS+i.t. with IAs+i.p. with PBS), Lab+CS group (CS+i.t. with PBS+i.p. with labetalol) and Lab+CS+IA group (CS+i.t. with IAs+i.p. with labetalol). Data show one representative of three independent experiments. n=5–7 per group. Data are shown as median (interquartile range).

Changes in spleen weight and levels of sympathetic neurotransmitters in the spleen and lungs of mice

The ratio of spleen weight to body weight in mice was substantially reduced by CS, but was significantly increased by IAs (figure 5a).

FIGURE 5.

Changes of the spleen weight and sympathetic transmitters in the spleen and lung of mice. a) Group data showing the ratio of spleen weight to the body weight of mice. b) Summarised data showing the ratio of norepinephrine (NE) concentrations to the total protein concentrations in homogenised spleen tissues. c) Group data showing the ratio of neuropeptide Y (NPY) concentrations to the total protein concentrations in homogenised spleen tissues. d) Summarised data showing the ratio of NE concentrations to the total protein concentrations in homogenised lung tissues. e) Group data showing the ratio of NPY concentrations to the total protein concentrations in homogenised lung tissues. Mice in different groups were treated as follows: control group (no stress+i.t. with PBS+i.p. with PBS), CS group (chronic stress (CS)+i.t. with PBS+i.p. with PBS), CS+IA group (CS+i.t. with immuno-agonists (IAs)+i.p. with PBS), Lab+CS group (CS+i.t. with PBS+i.p. with labetalol) and Lab+CS+IA group (CS+i.t. with IAs+i.p. with labetalol). Data show one representative of three independent experiments. n=5–7 per group. Data are shown as median (interquartile range) in c, d and e. Otherwise, data are shown as mean±sd.

Compared with the control group, NE and NPY concentrations in homogenised spleen tissues and lung tissues were markedly increased for both CS and CS+IA groups (figure 5b–e). Compared with the CS+IA group, labetalol treatment markedly reduced NPY concentrations in homogenised lung tissues (figure 5e).

Flow cytometric analysis of lung-resident lymphocytes in mice

Compared with the control group, the proportion of CD8⁺ T lymphocytes and CD8⁺IFN-γ⁺ T lymphocytes in lung-resident T lymphocytes was significantly increased in both the CS and CS+IA groups. Labetalol markedly decreased the proportion of CD8⁺ T lymphocytes and CD8⁺IFN-γ⁺ T lymphocytes in lung-resident T lymphocytes when compared with the CS+IA group (figure 6).

FIGURE 6.

Flow cytometric analysis of lung-resident lymphocytes in mice. a–e) Representative plots showing the proportion of CD8⁺ T lymphocytes in lung-resident T lymphocytes from the different groups. f–j) Representative plots showing the proportion of CD8⁺interferon-γ (IFN-γ)⁺ T lymphocytes in lung-resident T lymphocytes from the different groups. k) Summarised data showing the proportion of CD8⁺ T lymphocytes in lung-resident T lymphocytes. l) Summarised data showing the proportion of CD8⁺IFN-γ⁺ T lymphocytes in lung-resident T lymphocytes. Mice in different groups were treated as follows: control group (no stress+i.t. with PBS+i.p. with PBS), CS group (chronic stress (CS)+i.t. with PBS+i.p. with PBS), CS+IA group (CS+i.t. with immuno-agonists (IAs)+i.p. with PBS), Lab+CS group (CS+i.t. with PBS+i.p. with labetalol) and Lab+CS+IA group (CS+i.t. with IAs+i.p. with labetalol). Data show one representative of three independent experiments. n=6 per group. Data are shown as mean±sd. SSC-A: side scatter area; APC-A: allophycocyanin area; PE-A: phycoerythrin area.

Flow cytometric analysis of blood lymphocytes in mice

Compared with the control group, CS in combination with IAs substantially increased the proportion of CD8⁺ T lymphocytes in blood T lymphocytes (figure 7a). The CS and CS+IA groups had higher proportions of IFN-γ⁺ T lymphocytes, CD8⁺IFN-γ⁺ T lymphocytes, CXCR3⁺IFN-γ⁺ T lymphocytes and Ki67⁺IFN-γ⁺ T lymphocytes in blood T lymphocytes compared to the control group (figure 7b–e). CXCR3 is highly expressed on activated T lymphocytes and Ki67 is a biomarker of cell proliferation [32]. Labetalol was effective in reducing these IFN-γ-producing T lymphocytes in the blood.

FIGURE 7.

Flow cytometric analysis of blood lymphocytes in mice. Summarised data showing the proportion of CD8⁺ T lymphocytes (a), interferon-γ (IFN-γ)⁺ T lymphocytes (b), CD8⁺IFN-γ⁺ T lymphocytes (c), CXCR3⁺IFN-γ⁺ T lymphocytes (d) and Ki67⁺IFN-γ⁺ T lymphocytes (e) in blood T lymphocytes. Mice in different groups were treated as follows: control group (no stress+i.t. with PBS+i.p. with PBS), CS group (chronic stress (CS)+i.t. with PBS+i.p. with PBS), CS+IA group (CS+i.t. with immuno-agonists (IAs)+i.p. with PBS), Lab+CS group (CS+i.t. with PBS+i.p. with labetalol) and Lab+CS+IA group (CS+i.t. with IAs+i.p. with labetalol). Data show one representative of three independent experiments. n=6 per group. Data are shown as median (interquartile) in e. Otherwise, data are shown as mean±sd.

Flow cytometric analysis of spleen lymphocytes in mice

Compared with the control group, the CS and CS+IA groups had increased proportions of CD8⁺ T lymphocytes, IFN-γ⁺ T lymphocytes and CD8⁺IFN-γ⁺ T lymphocytes in spleen T lymphocytes (figure 8a–c). CS in combination with IAs markedly increased the proportion of CXCR3⁺ T lymphocytes in spleen T lymphocytes (figure 8d). Compared with the control group, the CS and CS+IA groups had higher proportions of CXCR3⁺IFN-γ⁺ T lymphocytes and Ki67⁺IFN-γ⁺ T lymphocytes in spleen T lymphocytes (figure 8e, f). Labetalol effectively reduced the proportion of IFN-γ⁺ T lymphocytes, CD8⁺IFN-γ⁺ T lymphocytes, CXCR3⁺ T lymphocytes, CXCR3⁺IFN-γ⁺ T lymphocytes and Ki67⁺IFN-γ⁺ T lymphocytes in spleen T lymphocytes (figure 8b–f).

FIGURE 8.

a–f) Summarised data for flow cytometric analysis of spleen lymphocytes in mice, showing the proportion of a) CD8⁺ T lymphocytes, b) interferon-γ (IFN-γ)⁺ T lymphocytes, c) CD8⁺IFN-γ⁺ T lymphocytes, d) CXCR3⁺ T lymphocytes, e) CXCR3⁺IFN-γ⁺ T lymphocytes and f) Ki67⁺IFN-γ⁺ T lymphocytes in spleen T lymphocytes. Mice in different groups were treated as follows: control group (no stress+i.t. with PBS+i.p. with PBS), CS group (chronic stress (CS)+i.t. with PBS+i.p. with PBS), CS+IA group (CS+i.t. with immuno-agonists (IAs)+i.p. with PBS), Lab+CS group (CS+i.t. with PBS+i.p. with labetalol) and Lab+CS+IA group (CS+i.t. with IAs+i.p. with labetalol). Data show one representative of three independent experiments. n=6 per group. Data are shown as median (interquartile range) in f. Otherwise, data are shown as mean±sd. g–i) Labetalol dose-dependently inhibited IA-induced and norepinephrine (NE)-induced increases of IFN-γ-producing T lymphocyte in vitro. g) Summarised data showing the proportion of CD8⁺ T lymphocytes in primary cultured mouse spleen T lymphocytes. h) Summarised data showing the proportion of IFN-γ⁺ T lymphocytes in primary cultured mouse spleen T lymphocytes. i) Group data showing IFN-γ concentrations in the supernatant of primary cultured mouse spleen mononuclear cells. Cells in different groups were treated as follows: Control group (medium without pretreatment and exposure), Model group (no pretreatment+exposure to IA and NE), low-dose Lab group (pretreatment with 0.365 μg·mL⁻¹ labetalol+exposure to IA and NE), Medium-dose Lab group (pretreatment with 2.19 μg·mL⁻¹ labetalol+exposure to IA and NE), and High-dose Lab group (pretreatment with 13.14 μg·mL⁻¹ labetalol+exposure to IA and NE). Data show one representative of three independent experiments. n=5 per group. Data are shown as mean±sd.

Labetalol dose-dependently inhibited IA-induced and NE-induced increases of IFN-γ-producing T lymphocytes in vitro

IA in combination with NE also significantly increased CD8⁺ T lymphocytes, IFN-γ⁺ T lymphocytes and IFN-γ levels in primary cultured mouse spleen mononuclear cells or the supernatants, which were dose-dependently reduced by labetalol treatment (figure 8g–i).

Discussion

In our study, CS and IAs induced chronic cough and pulmonary inflammation in guinea pigs, both of which were suppressed by labetalol. CS in combination with IAs increased IFN-γ levels in the lung of guinea pigs. CS increased IFN-γ-producing T lymphocytes, IFN-γ levels and pulmonary inflammation in mice. Sympathetic neurotransmitters (NE and NPY) were elevated in the lungs and spleens of mice in both the CS and CS+IA groups. Labetalol effectively reduced CS-induced and IA-induced IFN-γ-producing T lymphocytosis and mouse lung inflammation. Additionally, labetalol dose-dependently inhibited the IA-induced and NE-induced increases of IFN-γ-producing T lymphocytes in vitro.

In the present study, CS in combination with IAs significantly reduced the body weight of guinea pigs and mice. These results suggest that CS and IAs might cause a decline in overall conditions. CS is well known to decrease the body weight of animal models [33]. Intraperitoneally administrated R848 decreases the body weight of mice [34].

For the first time, CS has been shown to induce cough hypersensitivity and pulmonary inflammation characterised by an increased number of lymphocytes in the lung and spleen in guinea pigs. In addition to the above-mentioned effects, CS in combination with IAs increased pulmonary IFN-γ levels in guinea pigs. Labetalol reduced cough hypersensitivity and pulmonary inflammation in the guinea pig models of CS and CS+IA. In the absence of CS, labetalol administration failed to modulate cough reflex sensitivity to citric acid in guinea pigs. IFN-γ can enhance cough sensitivity [26]. Respiratory viral infections cause airway inflammation that leads to the release of IFN-γ, which has been shown to activate vagal afferents and cause cough [25, 26]. These results imply that CS might cause cough hypersensitivity and pulmonary inflammation by activating the adrenergic receptors. IAs might exacerbate CS-induced cough hypersensitivity and lung inflammation. IFN-γ and lymphocytes in the lungs might play a role in these processes. CS has also been shown to cause lung inflammation and the production of pulmonary cytokines [20, 35]. Maternal stress increases the risk of allergic lung inflammation in mice [36]. Peripheral sympathectomy reduces influenza virus-induced pneumonia, pulmonary lymphocyte infiltration and cytokine responses (including IFN-γ) [37]. R848 administration induces lung inflammation and IFN-γ production in mice [31, 38]. In clinical settings, chronic psychological stress is associated with an increased risk of respiratory illness following virus exposure in adults [39]. IFN-γ-producing T lymphocytes are conserved between the mice and guinea pigs, with guinea pigs also expressing these cells [40].

In this study, CS increased the total leukocyte count and the LDH activity in mouse BALF, indicating pulmonary inflammation. It also increased the level of sympathetic neurotransmitters (NE and NPY), the proportion of CD8⁺ and CD8⁺IFN-γ⁺ T lymphocytes, and IFN-γ concentrations in the lungs of mice. Additionally, CS in combination with IAs increased total lung pathological scores and lymphocyte counts in BALF. Labetalol (10 mg·kg⁻¹) effectively reduced CS-induced and IA-induced lung inflammation, as well as the associated increases in IFN-γ-producing T lymphocytes and IFN-γ concentrations. Furthermore, labetalol inhibited IA-induced and NE-induced increases in IFN-γ-producing T lymphocytes and IFN-γ levels in primary T lymphocytes in a dose-dependent manner. It is well established that adrenergic receptors are expressed on T lymphocytes [22]. Increases in proinflammatory cytokines triggered by CS are diminished in IFN-γ-deficient mice [41]. Repeated stress has been shown to elevate colonic T lymphocytes and IFN-γ levels, thereby increasing colonic permeability in control mice, but not IFN-γ-deficient mice [27]. We previously demonstrated that anti-IFN-γ treatment alleviates influenza virus-induced pulmonary lymphocytic inflammation by reducing IFN-γ-producing lymphocytes [25]. These findings suggest that CS might stimulate the release of sympathetic neurotransmitters, which could increase IFN-γ-producing T lymphocytes in the lungs, leading to pulmonary inflammation by activating adrenergic receptors. IAs might increase IFN-γ-producing T lymphocytes and exacerbate CS-induced pulmonary inflammation. IFN-γ might play an important role in CS-induced and IA-induced pulmonary inflammation.

The therapeutic effects of labetalol on CS-induced and IA-induced pulmonary inflammation are possibly due to reduced levels of IFN-γ-producing T lymphocytes. Consistently, NE and NPY are co-released during sympathetic activation, such as in cold stress [15]. CS prior to influenza viral infection augments pulmonary CD8⁺ T lymphocytes and proinflammatory cytokines (including IFN-γ) in mice [42]. NE can also stimulate IFN-γ production in primary splenic lymphocytes [43]. NPY is essential for the development of allergen-induced airway inflammation [44]. R848 has been shown to promote the proliferation of CD8⁺ T lymphocytes and CD8⁺IFN-γ⁺ T lymphocytes in vitro [45]. Clinically, patients with chronic refractory cough often exhibit elevated levels of CD8⁺IFN-γ⁺ T lymphocytes and IFN-γ in the airways [46]. Chronic cold stress causes lung inflammation in pigs by upregulating IFN-γ and other cytokines [47]. Labetalol (3, 10 and 30 mg·kg⁻¹) dose-dependently alleviates restraint stress-induced gastric mucosal injury in rats [48], and at 5 mg·kg⁻¹, it reduces circulating proinflammatory cytokines under stress conditions [49]. Additionally, phentolamine, an adrenergic receptor blocker, when combined with ambroxol, significantly shortened cough duration in children with pneumonia compared to the routine treatment group [50]. Further clinical studies are needed to evaluate the potential of labetalol in treating stress-related chronic cough.

IFN-γ released by T lymphocytes in the context of airway inflammation can induce cough hypersensitivity by activating airway sensory afferents [25, 26]. Airway inflammation can also increase the production of neuropeptides (including substance P, calcitonin gene-related peptide and neurokinins) in airway sensory afferents and cause airway innervation in the long term [51, 52]. These neuropeptides play an important role in airway inflammation [53]. In this study, airway sensory afferents were possibly activated by IFN-γ to induce cough hypersensitivity. Airway sensory afferents express NPY receptor-Y1 (NPY1R), which can be upregulated following allergic airway inflammation [54]. NPY limits the excitability of airway sensory neurons by interacting with NPY1R [54]. Therefore, NPY might not have directly activated airway sensory afferents in our study.

Intranasally administrated doses of haemagglutinin (50 μg·kg⁻¹) and R848 (2.5 mg·kg⁻¹) in our mouse study were based on both our preliminary experiments and previously published studies [38, 55]. Haemagglutinin (0.1 μg·mL⁻¹) can induce inflammatory responses in vitro [28]. Primary blood dendritic cells were activated with 5 μg·mL⁻¹ R848 [56]. Thus, primary mononuclear cells were exposed to a cell medium containing IAs (5 μg·mL⁻¹ R848 and 0.1 μg·mL⁻¹ haemagglutinin) in this study. In our mouse study, NE concentrations in homogenised lung tissues of the CS and CS+IA groups were 150–500 pg·mg⁻¹, which could be translated to 0.15–0.5 μg·mL⁻¹, if 1 g of homogenised lung tissues is converted to 1 mL body fluid. Primary mononuclear cells were exposed to 0.425 μg·mL⁻¹ NE and IAs in our in vitro study. Therefore, application of 0.425 μg·mL⁻¹ NE in vitro, which corresponds to the in vivo exposures, might mimic the CS in the mouse model of this study.

Notably, although labetalol markedly reduced NPY concentrations, it could not significantly reduce NE concentrations in homogenised lung tissues. Labetalol had no significant effect on NE or NPY concentrations in the spleen. These phenomena might be because labetalol directly blocks the receptors of sympathetic neurotransmitters but does not directly affect the level of sympathetic neurotransmitters.

In our study, CS increased the spleen weight in guinea pigs but decreased the spleen weight in mice. IAs increased the spleen weight in both guinea pigs and mice. Flow cytometric results suggest that CS and IAs increased the IFN-γ-producing T lymphocytes in the spleen and blood, which were effectively reduced by labetalol. Changes in spleen weight in response to CS vary among different animal species [57, 58]. Intraperitoneally administrated R848 can increase the spleen weight of mice [59]. Our previous work demonstrated that 1) intrapulmonary instillation of IFN-γ increases spleen weight and the number of IFN-γ-producing T lymphocytes in the spleen of mice; and 2) these spleen-derived IFN-γ-producing T lymphocytes can migrate into the bloodstream and lungs [32]. Psychological stress has also been shown to elevate IFN-γ⁺ and CD8⁺IFN-γ⁺ T lymphocytes in human blood [60]. In the current study, intranasal administration of IAs increased IFN-γ-producing T lymphocytes and IFN-γ levels in mouse lungs. Therefore, the increase in spleen-derived IFN-γ-producing T lymphocytes caused by CS and IAs may contribute to the elevated pulmonary IFN-γ and IFN-γ-producing T lymphocytes observed in this study.

In addition to activating the sympathetic nervous system, CS also stimulates glucocorticoid secretion through activation of the hypothalamic-pituitary-adrenocortical axis [61]. However, glucocorticoids are well known to suppress inflammation by reducing immune cell activity and cytokine production. Thus, it is unlikely that the proinflammatory effects of CS such as cough hypersensitivity and lung inflammation are mediated through increased glucocorticoid secretion.

This study has some limitations. First, the levels of sympathetic neurotransmitters (NE and NPY) were not measured in guinea pigs. The finding that sympathetic neurotransmitters were increased in the CS model of mice might not directly map onto guinea pigs. Second, although IFN-γ-producing T lymphocytes might play an important role in CS-induced and IA-induced cough hypersensitivity and pulmonary inflammation, the involvement of other inflammatory cells, such as neutrophils, cannot be excluded. Third, besides IFN-γ, other proinflammatory cytokines probably participate in these inflammatory changes. Fourth, IA exposure is not synonymous with a native virus infection.

In conclusion, CS may lead to the release of sympathetic neurotransmitters, which can increase IFN-γ-producing T lymphocytes and contribute to cough hypersensitivity and pulmonary inflammation through the activation of adrenergic receptors. IAs may increase IFN-γ-producing T lymphocytes and exacerbate CS-induced cough hypersensitivity and pulmonary inflammation. The therapeutic effects of labetalol in CS-induced and IA-induced cough hypersensitivity and pulmonary inflammation possibly occur by reducing IFN-γ-producing T lymphocytes.

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Supplementary material

00342-2025.SUPPLEMENT

Supplementary material

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