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CTSS contributes to airway neutrophilic inflammation in mixed granulocytic asthma

Respiratory Research volume 25, Article number: 441 (2024) Cite this article

Abstract

Background

Mixed granulocytic asthma (MGA) is usually associated with poor response to corticosteroid therapy and a high risk of severe asthma. Cathepsin S (CTSS) has been found to play an important role in various inflammatory diseases. This study was aimed to investigate the role of CTSS in MGA.

Methods

Induced sputum was obtained from healthy subjects and asthma patients. Two murine models of MGA were established using either TDI (toluene diisocyanate) alone or OVA emulsified in CFA. LY3000328, a specific antagonist of CTSS, was therapeutically given to BALB/c mice after airway challenge with TDI or OVA. The effects of recombinant CTSS was tested in vivo, and Akt inhibition was used to explore a possible mechanism for CTSS-induced airway inflammation.

Results

MGA patients have a significant higher sputum CTSS level than the health and subjects with other inflammatory phenotypes, which was positively correlated with sputum level of soluble E-cadherin (sE-cadherin), sputum neutrophils, FeNO, FEF25-75% and glucocorticoid dosage. Allergen exposure markedly increased CTSS level and pharmacological antagonism of CTSS with LY3000328 decreased airway hyperresponsiveness, airway neutrophil accumulation, as well as the release of IL-17 and sE-cadherin in murine models of MGA, yet had no effects on eosinophilic inflammation nor type 2 inflammatory cytokines (IL-4 and IL-5). In addition, intratracheal instillation of recombinant CTSS leads to neutrophil recruitment and overproduction of sE-cadherin in the lung tissues, which could be attenuated by inhibition of Akt signaling.

Conclusion

Our data suggested that CTSS contributes to airway neutrophilic inflammation in MGA through an Akt-dependent pathway.

Introduction

Asthma is a chronic and heterogeneous airway disease, affecting nearly 400 million individuals worldwide, and its prevalence and burden are increasing despite the development of novel treatments. A variety of phenotypes as well as endotypes have been identified according to clinical, physiological, inflammatory, and comorbidity factors [1, 2]. Among these, the inflammatory phenotype is attracting the most attention. Based on the inflammatory cell types in induced sputum, asthma can be categorized into the following four phenotypes: eosinophilic asthma (EA), mixed granulocytic asthma (MGA), paucigranulocytic asthma (PGA), and neutrophilic asthma (NA) [3]. Over the past years, MGA is gaining increasing attention as it’s usually associated with poor responses to corticosteroid treatment and represents a severe form of asthma with a mixed proportion of neutrophils/eosinophils and Th2/Th17 cells [4]. We’ve previously discovered that MGA patients tend to have a higher fractional exhaled nitric oxide (FeNO) level, higher risks of severe asthma, worse lung function and more pronounced small airway dysfunction than the other inflammatory phenotypes [5]. A series of peer researches also revealed that MGA patients had the poorest lung function and most severe symptoms, and accounted for the largest part of health care costs [6, 7], suggesting that MGA is the most “troublesome” type of asthma. Therefore, elucidating the molecular mechnisms underlying airway inflammation of MGA would be beneficial for better control of the disease.

Cathepsin S (CTSS) is a lysosomal protease, belonging to a family of cysteine cathepsin proteases that promote degradation of damaged proteins in the endolysosomal pathway [8]. CTSS attracts particular interest due to its unique properties including relatively restricted expression profile, inducible upregulation and ability to retain catalytic activity at a broad pH range when compared with other proteases [8]. Increased expression of CTSS was found in a broad spectrum of diseases, including pulmonary cystic fibrosis, COPD and ARDS [8, 9]. Besides, it is also significantly upregulated in experimental murine model of asthma induced by OVA [10]. Single nucleotide polymorphisms of CTSS was also found to be associated with the risk of asthma [11, 12], proposing it as a potential regulator in allergic airway inflammation. In this study, we aimed to assess the role of CTSS in the development of MGA and to investigate the underlying mechanisms.

Methods

Patients

A total of 153 asthma patients from the Department of Allergy and Clinical Immunology at the First Affiliated Hospital of Guangzhou Medical University were enrolled. Healthy subjects had no history of respiratory symptoms and all had normal spirometric results. The demographic and clinical characteristics of participants have been reported previously [5]. Supernatants of the induced sputum samples were used for detection of CTSS and sE-cadherin by commercially available ELISA kits (Elabscience Biotechnology Co.,Ltd., Wuhan, China).

Animal models of MGA

Male BALB/c mice (6–8 weeks of age, 20–25 g of weight) were obtained from Guangdong Medical Laboratory Animal Center and housed in specific pathogen-free environment (23 ± 2℃; 12:12 light-dark cycle; and 40%~70% humidity).

Two different allergens, TDI and OVA, were used to phenocopy mixed granulocytic airway inflammation as described elsewhere [5]. LY3000328 (Selleck, #E2886; Shanghai, China), at the dose of 30 mg/kg [13], was intraperitoneally given to the mice 30 min after each challenge. Sham mice received the same volume of vehicle as control.

Measurement of airway hyper-responsiveness (AHR)

Mice were anesthetized with pentobarbital by intraperitoneal injection 24 h after the last challenge. They were intubated and mechanically ventilated before measurement of lung resistance with the Buxco FinePoint System (Buxco Electronics, Troy, NY, USA). A baseline value was first recorded after acclimatization for 3 min. Then, the mice were challenged with increasing doses of nebulized methacholine (6.25, 12.5, 25 mg/mL). Recordings were taken and averaged for 3 min after each nebulization. Percentages of baseline value were calculated.

Bronchoalveolar lavage

Bronchoalveolar lavage (BAL) was performed immediately after measurement of lung resistance and the BAL fluid (BALF) was collected. The number of total inflammatory cells was counted for each BALF sample as previously described [14], as well as differential cell count calculation. The recovered BALF samples were centrifuged at 500 rpm for 10 min at room temperature, and supernatants were stored at -80℃ until further use for quantitative detection of IL-4, IL-5, IL-17 A, CTSS and sE-cadherin by ELISA according to the manufacturer’s instructions (Elabscience Biotechnology Co.,Ltd., Wuhan, China).

Lung histology

For histopathological evaluation, the left lungs were harvested, fixed with paraformaldehyde and embedded with paraffin. Lung Sect. (4 μm) were prepared and stained with hematoxylin and eosin (HE). Semi-quantification of airway inflammation was performed as previously described [14].

Western blot

For Western blot analyses, whole lung homogenates were subjected to 10% SDS-PAGE. After electrophoresis, proteins were transferred onto PVDF membranes and then probed with indicated dilutions of primary antibodies against CTSS (Abclonal, #A13482, 1:1000), Akt (Abclonal, #A18675, 1:1000) and Phospho-Akt-S473 (Abclonal, #AP0637, 1:1000) at 4℃ overnight. The next day, the membranes were incubated with a IRDye® 800WC-conjugated secondary antibody (LI-COR Biosciences, #P/N 926-32219). Immunoreactive bands were exposed to Odyssey® CLx Imager for image capture. Data analysis was done with Image J software.

In vivo treatments of recombinant CTSS and AKT signaling antagonism

Recombinant mouse CTSS (elabsciense,#PKSM041202,China) was diluted in PBS, and delivered to the mice via intratracheal instillation (5 µg/mouse, in 50 µL PBS). The Akt signaling antagonist MK-2206 2HCl (100 kg/kg, #S1078, Selleck, Shanghai, China) was diluted in PBS before intraperitoneal injection. MK-2206 2HCl was administered in a single dose 30 min after CTSS. 24 h after CTSS treatment, lung tissue and BALF samples were collected for further analysis.

Statistics.

Data were analysed using GraphPad Prism V 8.4.3 statistical software. Multiple comparisons between groups were analysed with an appropriate test for normally distributed data with analysis of variance, while for data not normally distributed, the KruskalWallis test followed by appropriate post hoc analysis (Dunnett’s or Dunn’s test) was performed. Correlations between clinical parameters and sputum sE-cadherin were analysed using Spearman’s rank correlation tests. p < 0.05 was considered significant.

Results

Increased release of CTSS was observed in MGA patients

We have previously reported the demographic and clinical characteristics of the enrolled subjects [5]. Here we detected the sputum level of CTSS in those patients. As shown in Fig. 1A, the level of CTSS in patients with MGA was significantly higher than the healthy controls. While CTSS levels in patients with the other three inflammatory phenotypes did not differ from control. And no significant differences were observed among the four phenotype groups. Similar results were seen for soluble E-cadherin (sE-cadherin) in the sputum (Fig. 1, B). In addition, sputum CTSS level was found to be positively correlated with inhaled corticosteroid (ICS) dosage required for daily control, FeNO, sputum neutrophil, and the level of sE-cadherin, and negatively correlated with the spirometry parameter FEF25 − 75 (Fig. 2, A ~ E).

Fig. 1
figure 1

CTSS was increased in MGA patients. Sputum CTSS (A) and sE-cadherin (B) levels were detected in healthy control (HC), and patients with mixed granulocyte asthma (MGA), paucigranulocytic asthma (PGA), eosinophilic asthma (EA) and neutrophilic asthma (NA). The data were analyzed using Kruskal-Wallis test. No significant differences of CTSS and sE-cadherin were observed among the four phenotype groups

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Fig. 2
figure 2

CTSS was associated with neutrophilic airway inflammation and lung function in asthma. A ~ D, Significant positive correlations were detected between sputum CTSS and the dose of inhaled corticosteroids (ICS), sputum sE-cadherin, neutrophils (Neu), FeNO. E, CTSS level was negatively correlated with the lung function parametre FEF25 − 75. The data were analyzed using nonparametric Spearman correlation

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CTSS expression is upregulated in experimental MGA models

We established two murine models of MGA induced by either TDI or OVA/CFA to study the role of CTSS. The expression pattern of CTSS was first assessed in these mice. As can be seen in Fig. 3, TDI sensitization and challenge led to dramatically increased release of CTSS in BALF (Fig. 3, A). The total protein expression of CTSS in TDI-exposed mice was also higher than that of vehicle control (Fig. 3, B ~ C). Both could be inhibited by treatment with LY3000328, a specific antagonist of CTSS (Fig. 3, A ~ C). Similar results were found in another model induced by OVA/CFA (Fig. 3, D ~ F).

Fig. 3
figure 3

CTSS was upregulated in experimental models of MGA. BALF CTSS levels was increased in both TDI-esposed (A) and OVA/CFA-exposed (D) mice. n = 6 ~ 10. Whole lung homogenates from TDI (B, C) and OVA/CFA (E, F) models were subjected to western blot for analysis of CTSS expression. Its relative expression was normalized to GAPDH. n = 6. The allergen-induced upregulation of CTSS could be inhibited by LY3000328 (LY), which is a sellective antagonist of CTSS

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Pharmacological inhibition of CTSS attenuated airway inflammation in a TDI-induced MGA model

We evaluated the effects of CTSS inhibition on a TDI-induced MGA model. TDI elicited mixed infiltration of neutrophils and eosinophils in the airway, accompanied by increased airway hyperreactivity, as well as elevated production of type 2 and non-type 2 cytokines (Fig. 4). Initially, we tested the effects of 10 mg/kg of LY300028 but found no obvious changes of airway inflammation nor airway hyperresponsiveness (data not shown). Then we tried to increase its dose. These time, we found that treatment with LY3000328 at the dose of 30 mg/kg per time after each TDI challenge for a total of 3 times resulted in dramatically attenuated airway inflammation and hyperresponsiveness, fewer neutrophils infiltrating in the airway lumen, as well as decreased levels of IL-17 and sE-cadherin in BALF. Yet the increased number of eosinophils induced by TDI was not affected, as well as the levels of type 2 inflammatory cytokines IL-4 and IL-5 (Fig. 4, ).

Fig. 4
figure 4

Pharmacological inhibition of CTSS with LY3000328 (LY) alleviated neutrophilic inflammation in toluene diisocyanate (TDI) -induced asthma model. A, Representative H&E stained lung sections of mice in vehicle, TDI and TDI + LY groups, as well as semi-quantitative scoring of airway inflammation (B, data analyzed using Kruskal-Wallis test), n = 8 ~ 10. Scale bar = 200 μm. C, Airway hyperresponsiveness was measured by resistance. Results were shown as percentage of baseline value. n = 4 ~ 5. D ~ F, Total cell in BALF were counted, as well as the numbers of eosinophils and neutrophils. n = 8 ~ 10. G ~ J, Levels of IL-4, IL-5, IL-17 and sE-cadherin in BALF were tested. n = 8 ~ 10. *: p < 0.05; ***: p < 0.001; ns: none significance. Data were analysed using One way ANOVA unless otherwise indicated

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Pharmacological inhibition of CTSS attenuated airway inflammation in an OVA/CFA-induced MGA model

In order to confirm the role of CTSS in MGA, we established another murine model induced by OVA emulsified in CFA (complete freund adjuvant), which is also characterized by a mixture of neutrophil and eosinophil accumulation in the airway. In consistent with what was found in the TDI model, blocking CTSS with 30 mg/kg LY3000328 also markedly alleviated hyperresponsiveness and neutrophilic inflammation of the airway induced by OVA/CFA (Fig. 5), but had no effects on the number of eosinophils, as well as levels of IL-4 and IL-5 in BALF. Together, these data suggested that CTSS promotes airway neutrophilic inflammation in MGA.

Fig. 5
figure 5

Pharmacological inhibition of CTSS with LY3000328 (LY) alleviated airway neutrophilic inflammation in OVA/CFA -induced asthma. A, Representative H&E stained lung sections of mice in control, OVA/CFA and OVA/CFA + LY groups, as well as semi-quantitative scoring of airway inflammation (B, data analyzed using Kruskal-Wallis test), n = 8 ~ 10. Scale bar = 200 μm. C, Airway hyperresponsiveness was measured by resistance. Results were shown as percentage of baseline value. n = 4 ~ 5. D ~ F, Total cell in BALF were counted, as well as the numbers of eosinophils and neutrophils. n = 8 ~ 10. G ~ J, Levels of IL-4, IL-5, IL-17 and sE-cadherin in BALF were tested. n = 8 ~ 10. *: p < 0.05; **: p < 0.01; ***: p < 0.001; ns: none significance. Data were analysed using One way ANOVA unless otherwise indicated

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CTSS directly induced neutrophilic lung inflammation in an AKT dependent pathway

Having identified a pro-inflammatory role for CTSS in MGA models, we next sought to investigate its direct effects, by administering recombinant mouse CTSS into the lungs of mice via intratracheal instillation. Despite the unaffected airway responsiveness to methacholine when compared with control (data not shown), intratracheal recombinant CTSS resulted in dramatically increased infiltration of total cells and neutrophils into the lungs after 24 h of treatment, accompanied by increased production of sE-cadherin (Fig. 6, A ~ E). Yet the levels of IL-6, IL1β and TNF-α in BALF were all below detection level (data not shown).

Fig. 6
figure 6

Exogenous CTSS induce pulmonary neutrophilia through activation of Akt signaling in mice. Recombinant mouse CTSS (5 µg/mouse) were given intratracheally to the mice, with or without the Akt antagonist MK-2206 2HCl. 24 h later, mice were sacrificed for analysis of airway inflammation. A, Representative HE-stained cytospin samples from BALF (left panel, Scale bar = 50 μm) and lung sections from different treatment groups (right panel, Scale bar = 200 μm). Each represented images from 4 ~ 6 fields of at least 8 mice. B, Semi-quantitative scoring of airway inflammation in H&E-stained lung sections. The data were analyzed using Kruskal-Wallis test. n = 8 ~ 10. C ~ D, The numbers of total inflammatory cells, and neutrophils in BALF were determined. n = 8 ~ 10. E, The level of sE-cadherin in BALF were determined. n = 8 ~ 10. F, Phosphor-Akt (Ser473) was detected via western blot analysis by using whole lung homogenates from mice treated with recombinant mouse CTSS. Its relative expression was normalized to total Akt (G). n = 6. The data were analyzed using one-way ANOVA unless otherwise indicated. **: p < 0.01; ***: p < 0.001

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To investigate the underlying signaling pathways of CTSS-induced inflammation, we performed additional mechanistic experiments. As we’ve previously demonstrated activated Akt pathway in the TDI-induced MGA model [15], which is an important regulator of neutrophilic inflammation [16], here we wondered if it is also responsible for CTSS-induced responses in the lung. Western blot analysis revealed a higher level of phosphorylated Akt (Ser473) in CTSS-treated mice when compared with PBS control (Fig. 6, F ang G), indicating CTSS-induced activation of the Akt pathway. Therefore, we set on to treat the sE-cadherin-exposed mice with MK-2206 2HCl, a small molecular antagonist of Akt As expected, administration of MK-2206 2HCl significantly alleviated the neutrophil accumulation in the lung induced by CTSS, and reduced the release of sE-cadherin in BALF (Fig. 6), suggesting that the CTSS-induced inflammatory response in the lung is partly dependent on AKT signaling.

Discussion

Aberrant CTSS expression has been demonstrated in a variety of pathological conditions and disease states, marking it out as both a biomarker and potential therapeutic target [8]. In this study, we found increased release of CTSS both in MGA patients and murine models of MGA, while pharmacological inhibition of CTSS attenuated the development of experimental MGA in mice. Notably, exogenous CTSS could elicit an Akt-dependent neutrophilic inflammation in the lung, shedding new lights on the role of CTSS.

MGA is characterized by the presence of both Th2/Th17 cells and eosinophilic/neutrophilic inflammation [3]. Although MGA only accounts for about 11% of the asthma population [17], it is more common for mixed inflammation to be intertwined in real-world patients [18], as type 2 inflammation would convert to non-type 2 inflammation and vice versa. Dual-positive populations of Th1/Th17 and Th2/Th17 cells accumulating in the airways of patients with asthma were associated with steroid-resistant severe asthma and neutrophil-dominant inflammation in the airways [19, 20]. Both Th2 and Th17 lymphocytes were involved in the induction of severe forms of experimental asthma [21]. Clinically, patients with MGA often respond poorly to corticosteroid therapy, and increased numbers of neutrophils with persistent eosinophilia are often seen in severe asthma and sudden-onset fatal asthma [6], indicating that MGA phenotype is associated with severe asthma. A number of studies revealed that patients with MGA had poorer lung function, more severe and persistent inflammation-related symptoms and more frequent exacerbations despite the use of corticosteroids than patients with neutrophilic asthma and eosinophilic asthma [6, 7], proposing MGA as the most troublesome phenotype. Previously, we also found that MGA patients tend to have a higher fractional exhaled nitric oxide (FeNO) level, higher risks of severe asthma, worse lung function and more pronounced small airway dysfunction than the other phenotypes [5]. All these suggest that MGA is a severe asthma phenotype that deserves further mechanistic investigation.

CTSS is a lysosomal and extracellular cysteine protease that is predominantly expressed in antigen-presenting cells and is up-regulated in several antigen-driven lung (and systemic) mouse models of inflammation, suggesting a role in allergic response [8]. In preclinical models of asthma, CTSS was significantly elevated [10, 22]. In line with these, in the present study, we detected an increased level of sputum CTSS in patients of MGA when compared with the healthy volunteers, which exhibited significant positive correlations with ICS dosage, FeNO level and sputum neutrophils, and a negative correlation with lung function. While no significant elevation of sputum CTSS in patients with other inflammatory phenotypes was seen. And when we phenocopied two MGA models in mice, we observed similar results, with increased CTSS in both lung tissue and BALF supernatant of the allergen- sensitized and challenged mice. Interestingly, in the OVA-induced asthma model, though genetic deficiency and prophylactic inhibition of CTSS could attenuate pulmonary eosinophilia [23, 24], the selective CTSS inhibitor given in a therapeutic paradigm failed to offer protective effects in mice, indicating that CTSS does not appear to play a significant role in the downstream effector phase of eosinophilic asthma [24]. Consistent with this, in our study, sputum CTSS levels did not differ between patients with eosinophilic asthma and the health, and no correlations were found between CTSS and sputum or serum eosinophils. Yet CTSS is significantly positively correlated with FeNO level, which strongly correlates with sputum eosinophils [25, 26]. It’s worth to be noted that though FeNO is typically elevated in atopic asthma, it is not always correlated with markers of eosinophilic airway inflammation, and it was also found to positively correlate with the number of sensitizations in nonasthmatic children [27,28,29], which can partly account for the inconsistent correlations found in this cohort.

In order to confirm the role of CTSS in the development of MGA, we established two murine models. Pharmacological inhibition of CTSS with LY300028 dramatically attenuated allergen-induced airway hyperreactivity and neutrophilic inflammation, but did not alleviate eosinophil accumulation and the levels of type 2 inflammatory cytokines in both TDI-induced and OVA/CFA-induced models. These agreed with finding in OVA-induced eosinphilic asthma model [24]. suggesting that CTSS is involved in airway neutrophilic inflammation in MGA.

To confirm its proinflammatory function, recombinant CTSS was intratracheally administrated to wild type mice. Exogenous CTSS supplement elicited a significantly increased number of neutrophils into the airway, which was in line with the findings of Small DM et al. [9]. Yet no increases of airway reactivity were observed, suggesting a dissociation between lung inflammation and airway resistance, similar to the finding of Deschamps K et al. [24]. Indeed, dissociation between antigen-induced airway inflammation and hyperresponsiveness has been frequently observed in preclinical studies of asthma, indicating distinct mechanisms involved [30,31,32,33]. The decrease in airway reactivity after CTSS intervention in the allergen-exposed mice of the present study may be a subsequent effect of reduced inflammation. Nevertheless, these findings suggested that CTSS contributes to asthmatic airway inflammation. Besides, concordant with the positive correlation between sputum CTSS and sE-cadherin level in the asthmatic patients of the current study, we also observed elevated secretion of sE-cadherin induced by CTSS in mice, which is produced through the breakdown of E-cadherin and regarded as a marker of epithelial injury [34]. Indeed, increased release of sE-cadherin was found in both animal and human studies of asthma [35, 36], and we’ve previously demonstrated a potent capacity of driving neutrophilic inflammation in asthma for sE-cadherin [37]. These indicate that CTSS may contribute to the development of MGA by promoting sE-cadherin production. Besides, we also observed increased serine phosphorylation of Akt at the site of 473 in CTSS-treated mice, which is an well-known regulator of neutrophilic inflammation as well as E-cadherin dysfunction [16, 38]. Here we wondered if it is also responsible for CTSS-induced responses in the lung. Inhibition of Akt with a small molecular antagonist MK-2206 2HCl significantly alleviated the neutrophil accumulation in the lung induced by CTSS and the release of sE-cadherin in BALF, proposing that the proinflammatory effect of CTSS depends on Akt signaling pathway.

In summary, our data demonstrated that CTSS contributes to airway neutrophilic inflammation in mixed granulocytic asthma, which is partly dependent on Akt signaling pathway. Therefore, targeting CTSS may well have important clinical implications for the development of new therapeutic avenues in asthma.

Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

We thank the Biobank for Respiratory Diseases in the National Clinical Research Center for Respiratory Disease (BRD-NCRCRD, Guangzhou, Southern China).

Funding

This study was supported by National Natural Science Foundation of China (82161138020, U1801286), Science and Technology Program of Guangzhou (202102010011); Zhongnanshan Medical Foundation of Guangdong Province (ZNSA-2020003, ZNSA-2020013), Natural Science Foundation of Guangdong Province (2022A1515012534), Guangzhou Science and Technology Plan City-School Joint Funding Project (202201020370), the grant of State Key Laboratory of Respiratory Disease (SKLRD-Z-202325), Innovation team of respiratory diseases and regenerative medicine (2021KCXTD028), National Natural Science Foundation of China(82070038).

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Author notes

  1. Haixiong Tang, Zhongli Li, Changyun Yang, Lin Fu and Xiaolong Ji contributed equally to this work.

Authors and Affiliations

  1. Department of Pulmonary and Critical Care Medicine, Zhujiang Hospital, Southern Medical University, Guangzhou, Guangdong, China

    Haixiong Tang, Zhongli Li & Xin Chen

  2. Department of Pulmonary and Critical Care Medicine, Guangzhou Institute of Respiratory Health, National Clinical Research Center for Respiratory Disease, National Center for Respiratory Medicine, State Key Laboratory of Respiratory Diseases, The First Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong, China

    Changyun Yang, Lin Fu, Zemin Chen, Sudan Gan, Shiyue Li & Lihong Yao

  3. Department of Allergy and Clinical Immunology, Guangzhou Institute of Respiratory Health, National Clinical Research Center for Respiratory Disease, State Key Laboratory of Respiratory Disease, the First Affiliated Hospital of Guangzhou Medical University, Guangzhou, China

    Xiaolong Ji, PingAn Zhang & Jing Li

  4. Guangzhou National Laboratory, Guangzhou, China

    Zemin Chen

  5. Department of Pulmonary and Critical Care Medicine, Nanfang Hospital, Southern Medical University, Guangzhou, Guangdong, China

    Hailing Zhang

  6. The Second Clinical College of Southern Medical University, Guangzhou, Guangdong, China

    Wenjun Zhang

Authors
  1. Haixiong Tang
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Contributions

Tang H, Li J and Yao L designed this study. Li J and Chen X supervised the whole study. Tang H, Yang CY and Fu L performed most of the experiments. Tang H and Ji XL analyzed the results and wrote the manuscript. Chen Z helped with the data analysis. Gan S and Zhang WJ helped with the in vitro experiments. Zhang H and Zhang pingAn helped with the animal experiments. Li S helped to collect the clinical data. Li J, Chen X and Yao L revised the manuscript. All the authors agreed that the final approval of the version to be published and ensured questions relating to the accuracy or integrity of any part of the work were appropriately investigated and resolved. All authors reviewed the manuscript.

Corresponding authors

Correspondence to Xin Chen, Lihong Yao or Jing Li.

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Ethics approval and consent to participate

All subjects signed informed consent forms approved by the Ethics Committee of our institution [Medical Ethics Year 2022 (No. 55)]. All animal experiments described here complied with the guidelines of the Committee of Guangzhou Medical University on the use and care of animals and were approved by the Animal Subjects Committee of Guangzhou Medical University.

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Not Applicable.

Competing interests

The authors declare no competing interests.

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Tang, H., Li, Z., Yang, C. et al. CTSS contributes to airway neutrophilic inflammation in mixed granulocytic asthma. Respir Res 25, 441 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-024-03077-6

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Keywords

Respiratory Research

ISSN: 1465-993X

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