Evidence for Interleukin-17C governing interleukin-17A pathogenicity and promoting asthma endotype switching in bronchiectasis

Domain: biology
Published: 2025-12-26
Editorial outcome: major_revision

Abstract
The management of bronchiectasis-asthma overlap (BAO) is an important clinical issue to be addressed. Little is known regarding the endotype of BAO. Here we recruit patients with a primary diagnosis of bronchiectasis and co-existing asthma. The levels of interleukin (IL)-17C are positively correlated with the levels of IL-17A or group 3 innate lymphoid cells (ILC3s) in peripheral blood samples from patients with BAO. An in vivo mouse model of Pseudomonas aeruginosa chronic lower respiratory tract infection followed by ovalbumin-induced asthma shows that IL-17C potentiates IL-17A expression via interacting with IL-17 receptor E in ILC3s. Additionally, ablation of Il17re in mice attenuates ILC3 responses and IL-17A-mediated asthma endotype switching towards neutrophilic asthma driven by P. aeruginosa chronic lower respiratory tract infection. Lastly, impaired epithelial barrier integrity by P. aeruginosa exposure is associated with IL-17C production in vitro. Collectively, our study implicates evidence for IL-17C governing IL-17A pathogenicity and promoting asthma endotype switching in bronchiectasis, implicating IL-17C as a potential therapeutic target for individuals with BAO.

Introduction
Non-cystic fibrosis bronchiectasis and asthma exhibit the same degree of heterogeneity in terms of clinical manifestations and outcomes1–4. Based on the related immune-inflammatory pathways, asthma endotypes broadly cover three subgroups: type 2-high, type 2-low, and mixed endotypes. Type 2-high asthma is characterized by a predominance of eosinophils, while neutrophilic asthma is typical of type 2-low asthma5, where with release of cytokines such as interferon (IFN)-γ, interleukin (IL)-17 from T helper (Th)1, Th17 cells, group 3 innate lymphoid cells (ILC3s) to recruit neutrophils and dampen the acquisition of eosinophils6–9, type 1 and type 3 immune responses predominate, leading to the suppression of type 2 immune responses10. In clinical practice, bronchiectasis and asthma frequently coexist, however, it is uncertain whether there is a cause-effect relationship between these two disorders. The overlap between bronchiectasis and asthma can be viewed as asthma-bronchiectasis overlap (ABO), and bronchiectasis-asthma overlap (BAO) with asthma accounting for 3-8% of bronchiectasis cases11–14, which are both clinical issues. The endotype of BAO has yet to be deciphered, and identifying the underlying mechanisms is therefore of key importance in precision medicine. Moreover, managing patients with BAO poses challenges, particularly when it comes to the use of corticosteroids. Inhaled corticosteroids and conventional asthma therapy are not regularly recommended for treating bronchiectasis, as the potential inhibition of host immunity and further increased risk of infections1,15. The conflicting treatment approach poses a significant challenge for healthcare providers. Literature on BAO is still scarce16,17, and the endotype of BAO remains to be fully defined, which has become a barrier to treatment decision-making. Furthermore, the lack of a mature animal model mimicking BAO has limited the availability of basic research data. Notably, Pseudomonas aeruginosa is the most common pathogen in the lower respiratory tract among bronchiectasis patients2,18, and it is also an independent risk factor for acute exacerbation in bronchiectasis16. And P. aeruginosa chronic infection model has been widely used in a repertoire of research on bronchiectasis19–21. Recent studies have indicated that colonization of nontypeable Haemophilus influenzae promotes the development of Th17-related neutrophilic asthma with glucocorticoid resistance22,23. Both P. aeruginosa and nontypeable H. influenzae belong to Gram-negative bacteria, but the precise asthma endotype which P. aeruginosa chronic lower respiratory tract infection drives remains unclear.

Innate lymphoid cells (ILCs) comprise group 1 ILCs (ILC1s), group 2 ILCs (ILC2s), and ILC3s, share the same features as three major effector CD4+ Th subsets, including Th1, Th2, and Th17 in the expression of transcription factors and cytokines, and act as pre-primed effector cells without de novo antigen-triggered effector cells differentiation24–26. The IL-17 family consists of six structurally related members (IL-17A, -17B, -17C, -17D, -17E, -17F). IL-17A and IL-17F are released by immune cells, mostly including ILC3, γδ T17, Th17 cells, and signal through IL-17 receptor A (RA)/IL-17RC and IL-17RA/IL-17RD complexes, and thus participate in host defense, inflammatory diseases, and autoimmune disorders partly by mediating neutrophil accumulation6,7. Other IL-17 members -IL-17B, IL-17C, IL-17D, IL-17E- contribute to regulating tissue inflammation by interacting with receptor complexes in epithelial cells or specific lymphocyte subsets27–30. However, the involvement of IL-17A, IL-17C, and ILC3s in the induction and development of endotypes within the context of BAO has not been deciphered.

In this work, we report a model of P. aeruginosa chronic lower respiratory tract infection followed by ovalbumin-induced experimental asthma mimicking BAO to investigate the underlying mechanisms involved in the endotype of BAO. This study indicates evidence for IL-17C governing IL-17A pathogenicity and promoting asthma endotype switching in bronchiectasis, implicating IL-17C as a potential intervention target for individuals with BAO.

Results
Exposure to P. aeruginosa chronic lower respiratory tract infection drives asthma endotype switching towards neutrophilic asthma associated with Th17 cells and ILC3s
In the present study, 20 healthy controls (HC), 31 bronchiectasis patients, 30 asthmatics, and 31 patients with BAO were enrolled. And as shown in Supplementary Table 1, there was no obvious significance concerning the sex distribution and the percentages of participants with smoking history across the groups (P=0.1643, P=0.4304). Patients with BAO had worse lung function, as indicated by lower FEV1, FVC, FVC% pred values, lower scores on Asthma Control Test (ACT), lower levels of eosinophils and higher percentages of IgE to be below 100 compared to asthmatic patients, but there was no significance of FEV1% pred, FEV1/FVC, fractional exhaled nitric oxide (FeNO) values in patients with BAO (Supplementary Table 2). These characteristics suggested that BAO may tend to be type 2-low asthma with poor control.

As described in Fig. 1a, consistent with patients with a primary diagnosis of bronchiectasis and coexisting asthma within the context of BAO, we established a model of P. aeruginosa chronic lower respiratory tract infection through intratracheal (i.t.) instillation with P. aeruginosa-laden agar beads, followed by ovalbumin (OVA)-induced asthma to explore the specific endotype of BAO. Considering P. aeruginosa clinical strains are more efficient in building chronic infection compared with the PAO1 laboratory strain31, we confirmed a slower growth rate of 52D5 compared to another three existing clinical strains isolated from bronchiectasis patients in our lab (Supplementary Fig. 1a), and therefore adopting the 52D5 clinical strain in the subsequent experiments. To improve the efficiency of airway colonization with P. aeruginosa, we constructed the agar beads of diameter ranging between 100 and 200 μm31. Additionally, compared to sterile agar beads, P. aeruginosa-laden beads seemed denser as shown by visible single bacterial cells interiorly (Supplementary Fig. 1b). The establishment of chronic infection was successful as determined by recovery of more than 1000 colony forming units (CFUs) of P. aeruginosa from lung cultures32 at different time points post infection (Supplementary Fig. 1c). There was no significance of survival rates post infection between P. aeruginosa and P. aeruginosa/OVA (PA/OVA) group (Supplementary Fig. 1d).

Hematoxylin & eosin (HE) staining showed more marked peribronchial infiltration of inflammatory cells in treatment with OVA, PA, and PA/OVA compared to controls (Fig. 1b, c). Moreover, airway resistance was pronouncedly augmented in mice exposed to PA/OVA compared to mice exposed to phosphate-buffered saline/OVA (PBS/OVA) (Fig. 1d). And the levels of serum IgE and Th2-related cytokines IL-5, IL-13 in bronchoalveolar lavage fluid (BALF) were reduced in mice exposed to PA/OVA compared to PBS/OVA, whereas the concentration of IL-17A was increased (Fig. 1e–h). Consistent with pathological presentations, the total cell numbers in BALF were elevated in mice exposed to OVA, PA, and PA/OVA (Fig. 1i). Furthermore, as depicted in Fig. 1j, the cell percentages and numbers of eosinophils whether in BALF or in lung tissues from PA/OVA group were consistently reduced compared to PBS/OVA group, whereas the cell percentages and numbers of neutrophils were elevated and the cell counts of alveolar macrophages (AM) were comparable (Fig. 1k–p). These findings suggested that exposure to P. aeruginosa chronic infection drove the asthma endotype switching towards neutrophilic asthma.

It is known that Th2 cells, together with ILC2s, contribute to eosinophilic asthma by producing IL-5 and IL-1333,34, while IL-17A-producing cells such as Th17, γδ T17, and ILC3 cells contribute to airway neutrophilia via IL-17A6,7,35,36. Consistently, compared to PBS/OVA exposure, PA/OVA exposure resulted in mitigated Th2 cell responses and upregulated Th17 cell responses (Fig. 2a–d). Additionally, through the assessment of ILCs based on the expression of transcription factors or cytokines, we found that the cell percentages and counts of ILC2s were consistently reduced in mice administered with PA/OVA in comparison with PBS/OVA-exposed mice, whereas ILC3s elevated (Fig. 2e–l). Meanwhile, with the determination of ILC2s based on cellular surface receptors, exposure to PBS/OVA increased the levels of ILC2s, whereas exposure to PA/OVA decreased (Supplementary Fig. 2a–c). The percentages of IL-17A-producing cells among CD3+CD4- lymphocytes which might be regarded as γδ T17 cells were increased in PA/OVA-treated mice compared to PBS/OVA-treated mice, but the cell numbers weren’t affected (Supplementary Fig. 2d). Additionally, mRNA level of Il17a was also significantly upregulated in lungs of mice administered with PA/OVA compared to PBS/OVA (Fig. 2m). Notably, we found no pairwise significance of the levels of Th1 and ILC1 cells among four groups (Supplementary Fig. 2e–g). Consistently, human data also indicated that patients with BAO presented higher levels of ILC3s compared to asthmatic patients or healthy participants (Supplementary Fig. 2h). Additionally, in line with the reported link of P. aeruginosa infection to Th2 responses37,38, our data showed raised TSLP in P. aeruginosa-infected patients with bronchiectasis or BAO, whereas no significance of CCL-17 levels was observed (Supplementary Fig. 2i, j). Thus, exposure to P. aeruginosa chronic lower respiratory tract infection led to a shift in asthma endotype towards neutrophilic asthma associated with IL-17A-producing Th17 cells and ILC3s.

IL-17A mediates asthma endotype switching towards neutrophilic asthma driven by P. aeruginosa chronic exposure
To understand the role of IL-17A in asthma endotype switching, an IL-17A neutralizing antibody (anti-IL17A) was administered to determine whether P. aeruginosa exposure-driven asthma endotype could be rescued as depicted in Fig. 3a. The administration of anti-IL17A attenuated peribronchial infiltrates of inflammatory cells and epithelium thickening induced by PA/OVA exposure, however, the effect was not observed in OVA group (Fig. 3b–d). Furthermore, IL-17A neutralization led to a mild increase in the levels of IgE, IL-5, IL-13, eosinophils, Th2 and ILC2 cells, whereas a significant decrease in the levels of IL-17A, total BALF cells, neutrophils, Th17 and ILC3 cells in the PA/OVA group (Fig. 3e–n and Supplementary Fig. 3d, f). Moreover, although the administration of anti-IL17A augmented the ratio of AM from mice treated with PA/OVA, the total numbers of AM, Th1, and ILC1 cells weren’t affected (Supplementary Fig. 3a–c, e). Taken together, these findings demonstrated that IL-17A mediated a shift in asthma endotype towards neutrophilic asthma driven by P. aeruginosa chronic lower respiratory tract infection.

Epithelial barrier integrity is impaired in a P. aeruginosa-exposed asthma model
Previous studies have reported the defective epithelial barrier integrity among patients with asthma39,40. Consistent with the mouse model, following pre-stimulation with P. aeruginosa, A549 cells were treated with OVA or IL-13 to resemble the airway epithelium within the context of BAO. We found that PA/OVA or P. aeruginosa/IL-13 (PA/IL13) exposure resulted in marked morphological changes in A549 cells from a typical multilateral paving stone-like appearance to a round or fusiform appearance accompanied by protrusions (Fig. 4a and Supplementary Fig. 4a). Furthermore, exposure to PA/OVA or PA significantly increased the intercellular permeability compared to OVA treatment (Fig. 4b). Moreover, the mRNA and protein levels of tight junction (TJ) markers, ZO-2, ZO-3, Claudin-18 and adhesion junction (AJ) marker, E-cadherin were remarkably decreased after PA/OVA or PA/IL13 exposure compared to OVA or IL-13 exposure (Fig. 4c–l).

Airway epithelium, as the first line of the physical barrier in the host, produces several alarmins such as TSLP41. Here we observed that exposure to PA/OVA or PA/IL13 augmented Tslp expression compared to the administration of OVA or IL-13 (Fig. 4m), which corresponds to the upregulated TSLP expression in human corneal epithelial cells exposed to P. aeruginosa infection42. In addition, the expression of ZO-3 and Claudin-18 located at the most apical part of neighboring epithelial cells were both decreased in the lungs of mice exposed to PA/OVA compared to OVA or their control (Fig. 4n, o and Supplementary Fig. 4b, c). Altogether, P. aeruginosa exposure resulted in a loss in epithelial barrier integrity characterized by the increased intercellular permeability and the decreased cell-cell adhesions.

The level of IL-17C positively correlates with IL-17A and is associated with the defective epithelial barrier integrity
Given the critical role of IL-17A in the model, we further explored the potential upstream regulator of IL-17A expression. Previous researches have demonstrated that IL-17C or IL-17B can regulate the activation of Th17 cells via a direct or indirect manner27,29,43,44. Moreover, IL-17B can inhibit eosinophilic inflammation by antagonizing IL-17E (IL-25) expression associated with type 2-high asthma45,46. We then examined the expression of IL-17 family members in P. aeruginosa chronic infection-driven asthma model. We found that Il17c expression was significantly upregulated in lung tissues of PA/OVA group compared with OVA group, whereas no significance of Il17b, Il17d, and Il25 mRNA expression was detected (Fig. 5a–d). Meanwhile, we observed significantly elevated expression of Il17c followed by remarkable upregulation in Il17a expression at the indicated time points in lung tissues of mice initially exposed to P. aeruginosa infection compared to their control (Fig. 5e, f). Furthermore, compared with OVA exposure, PA/OVA exposure elevated the concentration of IL-17C in BALF (Fig. 5g). Consistently, we found more productions of IL-17A and IL-17C in serum from patients with BAO than asthmatics (Fig. 5h, i). Notably, there was a consistently positive correlation between IL-17C and IL-17A levels (Fig. 5j). More importantly, IL-17C levels were also positively correlated with ILC3 levels in patients with BAO (Fig. 5k).

In line with animal and human data, IL-17C expression was upregulated in both BEAS-2B and A549 cells exposed to medium with a certain concentration of OVA following P. aeruginosa pretreatment (Supplementary Fig. 5a, b). Additionally, exposure to PA/OVA or PA/IL13 induced significantly elevated the expression of IL-17C compared to the treatment with OVA or IL-13 (Fig. 5l–o). MMP-9, secreted partly from neutrophils, is reported to modulate epithelial junction integrity47. Interestingly, exposure to PA/OVA or PA/IL13 expressed more Mmp9 than OVA or IL-13 administration (Fig. 5p, q). Notably, the pretreatment of MMP-9 inhibitor HY attenuated the impaired epithelial integrity and the upregulation of IL-17C expression induced by PA/OVA or PA/IL13 exposure (Supplementary Fig. 5c, d and Fig. 5r–u). Together, MMP-9 could modulate epithelial barrier integrity and thus regulating IL-17C expression.

Collectively, these data indicated that the levels of IL-17C positively correlated with IL-17A or ILC3 levels. Defective epithelial barrier integrity was associated with IL-17C production via MMP-9-dependent manner.

Ablation of Il17re inhibits IL-17A production in neutrophilic asthma driven by chronic infection with P. aeruginosa
It is known that IL-17C signals through IL-17RA/IL-17RE complex48. To investigate the potential role of IL-17C in asthma endotype switching, airway inflammation was evaluated in Il17re-/- mice. Exposure to PA/OVA exhibited milder peribronchial inflammatory infiltrates in Il17re-/- mice than wild-type (WT) littermates, but ablation of Il17re didn’t affect inflammatory infiltrates in the OVA group (Fig. 6a–c). Compared to WT littermates, CFU counts in the whole lung were markedly decreased in Il17re-/- mice exposed to PA/OVA or PA (Fig. 6d). Moreover, ablation of Il17re reduced the levels of IL-17A, total cell numbers, the cell counts of neutrophils, Th17 and ILC3 cells in mice exposed to PA/OVA, but failed to impact the levels of IL-5, IL-13, eosinophils, Th2 and ILC2 cells other than a mild increase in IgE levels (Fig. 6e–p and Supplementary Fig. 6b, c). In addition, total numbers of AM were not affected by ablation of Il17re (Supplementary Fig. 6a). Lastly, we found that interfering Il17c expression amplified the upregulated expression of Tslp in exposure to PA/OVA (Fig. 6q). Collectively, these data indicated that ablation of Il17re attenuated neutrophilic airway inflammation driven by P. aeruginosa chronic infection by preventing IL-17A production derived from Th17 cells and ILC3s.

IL-17C binds directly to ILC3s through interacting with IL-17RA-IL-17RE complex in lung tissues and potentiates IL-17A expression
To investigate the mechanisms by which IL-17C regulates IL-17A expression in ILC3s, we initially searched for the potential signaling targets in ILC3s. Interestingly, according to published RNA-seq data49, a significant increase in the expression of Il17re and Il17ra was supported in ILC3s of small intestine from WT mice, with Cd93 and Vipr2 expression being positive controls, but small amounts of transcripts expression of other IL-17R family members Il17rb, Il17rc, and Il17rd were observed (Fig. 7a). We observed that IL-17RE was situated on the surface of ILC3s and 98.9% among sorted ILC3s were RORγt+ (Fig. 7b–d). Next, we validated the binding capacity of IL-17A or IL-17C to ILC3s and identified more IL-17C or His labeling IL-17C binding to ILC3s, whereas ablation of Il17re attenuated the binding capacity (Fig. 7d, e). Furthermore, we found that following IL-17C treatment, ILC3s from C57BL/6 mice, compared to those from PBS-injected control mice, showed the elevated expression of IL-17A, whereas the comparable expression of the other transcripts associated with ILC3 function (Fig. 7f–h and Supplementary Fig. 7a–c). Moreover, we discovered that IL-17RE expression was comparable among ILC3 cell populations, whereas IL-17RE+ ILC3s expressed more IL-17A than IL-17RE- ILC3s in lung tissues (Supplementary Fig. 7d and Fig. 7i, j). Lastly, we found that IL-17RE was mostly expressed in T cells, macrophages/dendritic cells (MΦ/DC), and ILC3s (Supplementary Fig. 7e). Collectively, these findings indicated that IL-17C can interact with IL-17RE in ILC3s and potentiate ILC3 responses.

Methods
Patients’ samples collection
Humans samples were collected from healthy participants, patients with asthma, bronchiectasis, or BAO in Shanghai Pulmonary Hospital. The diagnosis of asthma was based on the Global Initiative for Asthma (GINA) guidelines64. Asthma control was assessed using the ACT score. The diagnosis of idiopathic bronchiectasis was referred to the non-cystic fibrosis bronchiectasis guideline published by the British Thoracic Society in 201965. Patients with BAO met one of the following diagnostic criteria: (1) Bronchiectasis was clinically reported prior to asthma; (2) Asthma was newly diagnosed according to the bronchodilator reversibility test on the basis of a primary diagnosis of bronchiectasis. Patients with cystic fibrosis or allergic bronchopulmonary aspergillosis (ABPA) were excluded from the study. Participants without respiratory symptoms and an indication of acute, active, as well as chronic pulmonary lesions were recruited as healthy controls. The clinical characteristics of the patients and healthy controls are shown in Supplementary Table 1. Study protocols for research related to human samples, including informed consent, publication of demographic data, and associated corresponding source data, were approved by the ethics committees of Shanghai Pulmonary Hospital (approval number K21-318). This study was conducted according to the Declaration of Helsinki principles, and signed informed consent was obtained from all participants.

Mice strains and animal experiments
Il17re-/- mice (Il17retm1Lex/Mmucd) on B6N.129S5 background were provided by Prof. Youcun Qian, Chinese Academy of Sciences. C57BL/6 mice were bought from Shanghai SLRC Laboratory Animal Co., Ltd. (Shanghai, China). All mice were maintained in specific pathogen-free animal facilities with a 12-h light and dark circle, temperature of 20–25°C, and relative humidity of 50% at the Laboratory Animal Center of Tongji University. All animals were age-matched and sex-matched, and then randomized into different groups. The age for all strains of mice was 6–8 weeks. OVA-induced asthma is known to result in eosinophilic inflammation and induction of Th2 cytokines in BALF. To construct a model of P. aeruginosa chronic lower respiratory tract infection followed by experimental asthma mimicking BAO, following i.t. instillation with P. aeruginosa-laden agar beads (2×106 CFUs in 35 μl PBS) to establish chronic infection with P. aeruginosa31, 5 days later Il17re-/- mice and wild type (WT) littermates or C57BL/6 mice entered into the establishment of an experimental asthma model66, in which intraperitoneal injection of 20 μg of OVA (Sigma-Aldrich) mixed with 2 mg of alum (Thermo Scientific) was given, followed 6 days later by continuous three airway challenges with 1% OVA. For IL-17A neutralization, 30 μg of Anti-IL17A (BE0173, BioXcell) or IgG1 isotype control (BE0083, BioXcell) was intratracheally given into the four groups, that is, PBS, OVA, PA, and PA/OVA at 2 h before airway challenge with 1% OVA. For IL-17C administration, 1 μg of IL-17C (2306-ML, R&D) or PBS was intratracheally instilled into C57BL/6 mice every 2 days for a total of five times. All mice were sacrificed 24 h post the last airway challenge or the last instillation via isoflurane inhalation, and samples, including serum, BALF, and lung tissues, were collected for further analysis. Apart from the quantification of IgE in serum and the assessment of indicators, including bacterial load, IL-17 expression, immune and inflammatory cell infiltrates in lung tissues, the levels of IL-5, IL-13, IL-17C, IL-17A, and cell typing were determined in BALF. Experiments were conducted according to the Declaration of Helsinki conventions for the use and care of animals and were approved by the Institutional Animal Care and Use Committee of Tongji University (approval number TJBE00120101).

Measurement of AHR
In PA/OVA-induced airway inflammation model, changes in AHR after allergen exposure were determined by invasive measurement using R&C system (EMMS). At 9 days post OVA sensitization, the trachea was intubated, and then the lungs were mechanically ventilated. Airway resistance was regarded as an indicator of AHR and measured after increasing doses (0, 16, 32, 64, 256 mg/ml) of aerosolized acetylcholine chloride (Ach, A6625, Sigma-Aldrich).

Cell culture and treatment
The A549 lung epithelial cell line (SCSP-503, Chinese Academy of Sciences) and BEAS-2B bronchial epithelial cells (SCSP-5067, Chinese Academy of Sciences) obtained from the Chinese Academy of Sciences, were both cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) containing 10% (v/v) fetal bovine serum (FBS, Gibco). Unless otherwise indicated, P. aeruginosa clinical strain 52D5 was employed in the subsequent experiments. Following pretreatment with live 52D5 (multiplicity of infection (MOI)=1) for 5 h, the previous medium was replaced by medium containing OVA at various concentrations (50, 100, 250 μg/ml) or added with exogenous IL-13 (PeproTech, 100 ng/ml). Finally, cells and supernatants were both collected for further analysis after incubation at 37°C for 24 h. Il17c small interfering RNA (siRNA) used in the experiment was synthesized by GenePharma, and the sequence (5′–3′) was as follows: GCGAGACAGCUGCGCUCAACU; UUGAGCGCAGCUGUCUCGCGG. For Il17c siRNA transfection in A549 cells, GP-transfection-Mate (G04008, GenePharma) was used following the manufacturer’s instructions. For the inhibition of MMP-9 in A549 cells, cells were primed with the inhibitor (HY-135232, MCE, 50 μM) for 2 h before P. aeruginosa stimulation.

Bacterial growth curve
Growth rates of P. aeruginosa strains were assessed as described by Montanari67. Briefly, P. aeruginosa strains were grown overnight in Luria-Bertani (LB) broth (12780052, Invitrogen), and each culture was diluted at 1:200 and standardized to an OD600 in the range of 0.01 to 0.02. Samples were taken every 0.5 or 1 h and the OD600 was determined. Growth curves were plotted according to the elapsed time and the corresponding OD600 value.

Bacterial culture and preparation
The P. aeruginosa clinical strain 52D5 was thawed from −80°C, cultured in LB broth overnight, and then re-grown for 8 h at a diluted factor of 50 to reach the mid-log phase. Finally, Bacteria were washed and resuspended in PBS for cell pre-stimulation. P. aeruginosa-laden agar-beads were made as described previously31. Briefly, a 1:9 (v/v) mixture of bacterial suspension and melting broth was added to heavy mineral oil (330760, Sigma-Aldrich) that was equilibrated at 55°C, rapidly stirred for 6 min at room temperature, then cooled 35 min at 4°C, followed by washing six times in PBS. CFUs were enumerated on an aliquot of homogenized bead slurry via a 10-fold serial dilution. Sterile beads were prepared using PBS.

Quantification of bacterial loads in lung tissues
The whole lung tissue was removed and then homogenized in 1 ml of sterile PBS. And 0.1 ml of the homogenate was transferred for a 10-fold serial dilution, followed by inoculation on the LB agar plates. Finally, bacterial colonies were counted after incubation at 37°C for 24 h.

Lung histology
Lung lobes were fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned, followed by HE staining. An individual blinded to experimental conditions assessed airway inflammation. The average of histological scoring on airway inflammation by randomly selecting five visual fields of each lung section from at least three mice per group under a light microscope (Olympus) was used for further analysis.

Quantitative real-time PCR
Total RNA was extracted from cells or lung tissues using TRIzol reagent (Invitrogen) according to the instructions, followed by reverse transcription using a cDNA synthesis kit (Yeasen). qPCR analysis was performed with a SYBR RT-PCR kit (Yeasen) on LightCycler 480 (Roche). Gapdh was used as an internal reference gene for data normalization. The primer sequences were shown in the Supplementary Table 3.

Western blotting
The samples were separated by electrophoresis through a 4–12% Precast Bis-Tris gel (Tanon) and electrically transferred to PVDF membranes (Millipore). The membranes were blocked with 5% fat-free milk, followed by incubation with the primary antibodies at 4°C and secondary antibodies at room temperature, as well as repeated washes with TBST solution (0.1% Tris, 0.1% Tween-20). And finally, proteins were visualized using Tanon imager.

ELISA
IL-17A, IL-17C, TSLP, CCL17 in human samples and IgE, IL-5, IL-13, IL-17A, IL-17C in mouse samples were measured using an ELISA kit as shown in the Supplementary Table 4. Assays were referred to the manufacturer’s protocols.

BALF collection
The lavage fluid was collected with 0.4 ml sterile PBS injected into the whole lung each time for a total of three times, immediately stored in ice, and then centrifuged at 350×g for 7 min. The supernatants were maintained at −80°C for cytokine assays. The cell pellets were removed of red blood cells, re-suspended in PBS, and then taken out an aliquot for cell number counting. Finally, the remaining cell suspension was prepared for flow cytometry analysis.

Peripheral blood mononuclear cells (PBMCs) isolation
PBMCs were isolated from the peripheral blood samples collected from the recruited participants with Ficoll Paque PLUS (GE healthcare) using density gradient centrifugation layering according to manufacturer’s protocols. Cells were suspended with a centrifuge at 350×g and then stored in −80°C for further flow cytometry analysis. Human sera were available by centrifuging at 3000 rpm and were also stored in −80°C for cytokine assays.

Flow cytometry analysis
Lung tissues of the indicated mice were prepared into a single cell suspension with the digestion buffer (sterile PBS supplemented with 2.5 mg/mL type 2 collagenase, 1% DNase I). After red blood cell lysis and filtering, cells were centrifuged, and subsequently pellets were suspended with 30% (v/v) Percoll solution (GE healthcare). After centrifuging, cell pellets were washed and re-suspended in sterile PBS containing 3% (v/v) FBS, 0.05 mM EDTA, 100 U/ml penicillin, and streptomycin. An aliquot of suspension was used for cell number counts, and the remaining cells were stimulated with a cell activation cocktail with Brefeldin A (Biolegend) for 4 h prior to staining for flow cytometry analysis by CytoFLEX LX (BECKMAN COULTER). Additionally, fluorescein-conjugated antibodies against CD45 (30-F11, 103116), Lineage (17A2; RB6-8C5; RA3-6B2; Ter-119; M1/70, 133310), CD90.2 (30-H12, 105305), RORγt (AFKJS-9, 12-6988-82), GATA3 (16E10A23, 653806), T-bet (4B10, 644806), EOMES (W17001A, 157703), CD127 (A7R34, 135022), CD3 (17A2, 100218), CD4 (GK1.5, 100443), IL-5 (TRFK5, 504306), IL-13 (W17010B, 159403), IL-17A (TC11-18H10.1, 506930), IFN-γ (XMG1.2, 505806), IL-17RE (944904, FAB7997R), His (J095G46, 362605), IL-17C (311522, IC23061R) were purchased from Biolegend, eBioscience or R&D. Cell surface staining was carried out by incubating cells with the antibodies for 30 min at 4°C after blocking with Fc antibody. GATA3, RORγt, T-bet, EOMES, IL-17A, IL-5, IL-13, and IFN-γ staining was performed using the intracellular transcription factor kit (Invitrogen) or the cytokine staining kit (BD Biosciences). For flow cytometry sorting of ILC3s, FACSAria III (BD Biosciences) was employed in the experiment. The detailed reagents were shown in Supplementary Table 4. Flow cytometry data were analyzed with FlowJo software (v10.7.2)

Immunohistochemistry Staining
Lung sections were completely immersed into Tris-EDTA (pH=8.0, Servicebio) or citric acid (pH=6.0, Servicebio) antigen retrieval solution and then heated with a microwave for 15–20 min, followed by blocked by 3% hydrogen peroxide for 15–20 min. After 5% bovine serum albumin (BSA) blocking, the sections were incubated with antibodies against ZO-3 (Invitrogen) or Claudin-18 (Abcam) at 4°C overnight, followed by colorimetric detection using DAB staining and hematoxylin counterstaining. The sections were examined using a light microscope and quantified by the Image-Pro Plus 6.0. An individual blinded to experimental conditions performed a semiquantified analysis.

Immunofluorescence microscope
Sorted ILC3s from lymphoid cells were cultured on poly-L-lysine-coated plates and were then fixed and blocked with 5% (v/v) goat serum (Yeasen) in PBS for 1 h at room temperature, followed by incubation with an antibody against IL-17RE (bioss) at 4°C overnight. Subsequently, the cells were stained with DAPI (Beyotime) for 5 min at room temperature. Cells were rinsed with PBS for three times, 5 min each time. Images were captured under a Zeiss fluorescence microscope (Zeiss).

Binding assay
For the binding assay, the sorted ILC3s ex vivo were treated with exogenous IL-17A (Biolegend), IL-17C (R&D), IL-17C-His (ACRObiosystems), or His (Beyotime) individually for 30 min, followed by staining with the indicated antibodies conjugated with proper fluorescein for flow cytometry analysis.

Epithelial permeability measurement
A549 cells were grown in 24-well Transwell (Corning Costar) inserts until 90% confluence, and then paracellular flux across the epithelium was measured using fluorescein isothiocyanate (FITC)-labeled 4-kDa dextran (Sigma-Aldrich, 0.5 mg/ml) added to the upper chamber followed by incubation for 2 h at 37°C. Finally, intercellular permeability was indicated by fluorescence intensity of FITC-dextran.

Statistical analysis
The Shapiro-Wilk test was employed for analyzing the distribution of quantitative variables. Normally distributed data were presented as mean (SD), or mean ± SEM, and non-normally distributed data were presented as median (IQR). Categorical variables were presented as frequencies and percentages, and the Chi-square test was adopted for the comparisons. Spearman correlation was used to investigate the relationship between the level of IL-17C and IL-17A or ILC3s in serum from patients. Log-rank test was performed to evaluate the difference in the comparison of survival curves. The statistical differences in both groups were compared by using Mann-Whitney test, paired or unpaired Student’s t-test, whereas for the comparison of more than two groups, one-way ANOVA, two-way ANOVA, or Kruskal-Wallis test followed by Tukey’s, Dunn’s, or LSD multiple comparisons test was applied. All statistical analysis was performed by the GraphPad Prism 9.0 (San Diego, CA, USA). Each value in the statistical graph represents an individual biological sample, where technical replicates have been averaged. Two-sided significance level of 0.05 was chosen for all tests due to uncertainty in the preset results of experiments relevant with the study (in which identifying a > b or a < b was absolutely correct).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "This well-written paper, authored by an experienced group, appears to introduce a novel connection between IL-17C and the modulation of IL-17A, leading to the shift from eosinophilic to neutrophilic asthma driven by Pseudomonas aeruginosa. While IL-17A's role in inflammation is recognized, IL-17C's contribution to this process in bronchiectasis with asthma may indeed represent a new and underexplored aspect, giving this research its novelty. Overall, a well structured paper, which is clearly presented. The findings are of potential importance and interest to the pulmonary scientific community and helps clinicians to understand the pathophysiology of neutrophilic driven asthma. S\nHowever, there are a few key points that the authors should address before publication:\n1. Tables 1/2:\na. It is important to clarify whether HRCT data are available for the listed patients.\nb. Include FEV1 values to enhance the characterization of pulmonary function in the patient population.\nc. How did you sample for microbiology results? Induced sputum?\n2. FVC and FEV1/FVC Findings:\na. Clarify why patients with bronchiectasis and asthma (BA) show lower FVC but have a similar FEV1/FVC ratio when compared to asthmatic individuals. This raises questions about specific lung mechanics in BA patients that require further exploration.\n3. Pseudomonas Aeruginosa and Th2 Endotype:\na. The concept of Pseudomonas aeruginosa driving a Th2 endotype is intriguing, but several points need addressing:\ni. The majority of the supporting evidence stems from murine models, which calls for stronger human-based validation. Performing assays with human cells, such as ILC3s, would provide more relevant translational insight.\nii. It would be beneficial to include data on Pseudomonas aeruginosa colonization in the studied patient subsets and analyze correlations with Th2-associated markers (e.g., TSLP, CCL17). This would strengthen the manuscript’s human relevance and address the weakness in linking P. aeruginosa to Th2 responses in humans.\niii. Consider referencing previous studies that examine the relationship between P. aeruginosa and Th2 responses in humans, such as this work: https://pubmed.ncbi.nlm.nih.gov/16387607/\nand, this work:\nhttps://pubmed.ncbi.nlm.nih.gov/38888974/\n4. Discussion:\na. The current discussion is somewhat basic and lacks a strong translational perspective. The manuscript should address whether patients colonized by Pseudomonas aeruginosa should be treated differently based on their Th2 responses.\nb. Lessons from cystic fibrosis (CF) management could offer valuable insights into how Pseudomonas aeruginosa infection might be treated in these patients. This could be an important avenue for further research and discussion.", "reviewer_2": "This study by Zhang et al. addresses current challenges in the treatment of patients with coexisting bronchiectasis and asthma. The authors sought to explore the asthma endotype in patients with non-cystic fibrosis bronchiectasis with asthma (BA) to identify new therapeutic targets. Using mouse models, the authors report a pathogenic interaction between IL-17C and ILC3s that potentiates IL-17A responses and switching to a neutrophilic asthma endotype. The study is interesting and warrants investigation, however, there are some major limitations.\n- The authors’ rationale for inducing P. aeruginosa infection followed by allergic sensitisation to Ova in their murine model lacks convincing justification. While individuals of all ages can have bronchiectasis, it is most common in the elderly. The EMBARC study explored this overlap between asthma and bronchiectasis and identified that asthma could be reported as an etiology of bronchiectasis. This is supported by the British thoracic society guideline for non-cystic fibrosis bronchiectasis recommendation for considering asthma as the cause of bronchiectasis in the absence of other etiological explanations. Others show that asthma severity is an independent risk factor for bronchiectasis, that there is a high percentage of bronchiectasis in patients with severe asthma, and that age of asthma onset and exacerbations are independently associated with the occurrence of bronchiectasis.\n- Does P. aeruginosa infection alone adequately model the diverse immunopathological repertoire and pathophysiology of patients with bronchiectasis? This must be established to convince readers that the reported observations are not simply readouts of host:pathogen interactions e.g. the effects of an active infectious challenge on shaping allergic sensitisation and the immunological profile of the recall response.\n- The potential influence of smoking history on the reported data is not discussed further than reported rates of “Smoking, %” in Table 1.\n- The authors identify the paucity of data on asthma endotypes in the context of bronchiectasis-asthma overlap as a barrier to treatment decision-making, but do not present data on sputum inflammometry, which is a global benchmark for characterization and management guidance. This limits the translational applicability of the findings from the mouse experiments.\n- The authors report a positive correlation between peripheral (serum) IL-17A and IL-17C in patients with bronchiectasis with or without asthma. It would be more informative to perform these analyses in patients with bronchiectasis alone, asthma alone, and with both bronchiectasis and asthma. The specific relevance of these findings in the context of ILC3 responses should then be demonstrated in patients with both bronchiectasis and asthma to support the contention that “IL-17C could serve as a potential therapeutic target for individuals of BA”.", "reviewer_3": "MANUSCRIPT NCOMMS-24-60979\nGENERAL COMMENTS.\nThis manuscript presents a clinical-translational study that addresses how bacterial colonization in patients with bronchiectasis and asthma alters immune signaling via the archetype Type 3 signaling pathway involving interleukin (IL)-17 cytokines, especially IL-17A and IL-17C. The data sets presented are extensive, consisting of data from humans, from cell and animal models. Taken together, the scientific basis for the manuscript and the presented conclusions is solid. Nevertheless, the results presented are important for the (improved) understanding of archetype immune signaling in patients with chronic airway disease and bacterial colonization, with future implications beyond asthma.\nThe scientific criticism relates mainly to the present focus on ILC 3 cells, a focus that leaves the expert reader with several unanswered questions regarding the role of Th17 and Tc17 cells. The mechanistic rationale for the critical role of IL-17A per se also deserves to be stated in a more precise manner, given its importance for this field of research (see also Specific comments below). Additional and important criticism relates to how the presentation of the underlying study is made, from a linguistic point-of-view. Key entities need to be handled in a more consistent manner than is now the case. Moreover, as the main text now stands, it is in need of editing by an expert in English. Once it is optimized in these respects, the manuscript is likely to become important for future research on chronic inflammatory airway disorders.\nSPECIFIC COMMENTS.\nA) Title: The title emerges as too definitive. Maybe \"Evidence for IL-17C governing IL-17A pathogenicity...\" would be better given that the focus is set mainly on ILC3 cells and not on additional sources of IL-17A? Another reason for this is that important evidence originates from experimental models and not humans in vivo.\nB) Summary: The authors should make clear what evidence originates from humans and what evidence originates from experimental models. Apart from this, The scientific contents are fine but linguistic editing is needed.\nC) Introduction:\n1. Para 1 and 2: The text would benefit from the addition of information on Type 1 and Type 3-signaling and how these pathways relate to neutrophilic and eosinophilic inflammation. Here, key entities must be introduced and used in a more accurate and consistent manner than is now the case.\n2. The entire study behind this manuscript study is based upon the role of IL-17A in mediating neutrophil accumulation in the lungs. Given this, a brief presentation of the basic evidence is appropriate, published in the original studies from Linden et al and from Kolls et al (summarized in Linden & Kolls, Immunity 2004; and in Linden & Dahlen, ERJ 2014). The original studies are also relevant for comments in the the results and the discussion (see below).\nD) Results:\n1. Table 2: Data sets on FEV1 and on ACT for healthy controls iare missing.\n2. Text line 108: All FENO values are within the normal range, so the comment on a trend here doe s not seem meaningful.\n3. Text line 165: These references are not adequate. See point C.2 for appropriate references.\n4. Text line 281: The data set on MMP-9 emerges as important, not least given that this proteinase originates in part from neutrophils. The latter can be emphasized in the text here (as well as in the discussion and in the principal overview in Fig 8).\n5. Apart from the above, the figures and tables seem adequate.\nE) Discussion:\n1. A general comment is that the text does not really make clear why the emphasis is set on ILC3 cells and not on other sourcces for IL-17A (such as Th17 or Tc17 cells).\n2. Another general comment is that the discussion need to address the (potential) involvement of Type 1 signaling (via ILC 1 cells or via Th1 or Tc1 cells).\n3. Text line 383: Figure 8 need to include MMP-9 given the current data and its relevance in neutrophilic inflammation in general (see also above).\n4. Text line 413: Although this reviewer share the forwarded conclusions re. the current status of clinical evidence on inhibiting IL-17A signaling in asthma, the text here must be revised to make it easy to understand.\n5. Text line 423: The presentation of published evidence on IL-17C i snow somewhat confusing and all publications on this may not be relevant for the current setting. This text can preferably be restricted to the most relevant publications.\n6. Text line 460: The limitations of this study include the points forwarded as points E.1 and E.2 above.\n7. Text line 469: The conclusion section should include comments on MP-9 as well (see above).\nF) Methods:\n1. Text line 610: The authors need to present the specific provider for each utilized assay, to make it possible for interested readers to reproduce data from this potentially important study.\n2. Text line 644: The rationale for always using \"two-sided significance\" (regardless of pre-existing data and hypotheses) seem unclear and a rationale should be explained.\n3. Except for the above, the methods seem adequately described."}, "author_response": "Point-by-point responses\nREVIEWER COMMENTS\nReviewer #1(Remarks to the Author)\nThis well-written paper, authored by an experienced group, appears to introduce\na novel connection between IL-17C and the modulation of IL-17A, leading to the\nshift from eosinophilic to neutrophilic asthma driven by Pseudomonas aeruginosa.\nWhile IL-17A's role in inflammation is recognized, IL-17C's contribution to this\nprocess in bronchiectasis with asthma may indeed represent a new and\nunderexplored aspect, giving this research its novelty. Overall, a well structured\npaper, which is clearly presented. The findings are of potential importance and\ninterest to the pulmonary scientific community and helps clinicians to understand\nthe pathophysiology of neutrophilic driven asthma.\nHowever, there are a few key points that the authors should address before\npublication:\n1.Table 1/2:\na. It is important to clarify whether HRCT data are available for the listed patients.\nReply:\nThank you for your valuable comments. A HRCT scan is required to make the diagnosis\nof bronchiectasis, so it is important for the listed patients in the present study applying\na HRCT scan to distinguish bronchiectasis with asthma from asthma alone. Of all 92\npatients, most patients including bronchiectasis patients (30/31), asthmatics (29/30),\npatients of bronchiectasis with asthma (29/31) received the chest HRCT scan and\nHRCT data can be retrieved from Picture archiving and Communication System (PACS)\nin Shanghai Pulmonary Hospital, whereas remaining 4 patients underwent the chest\nHRCT scan in other hospitals within the last 1 month.\nb. Include FEV1 values to enhance the characterization of pulmonary function in\nthe patient population.\nReply:\nThank you for your constructive remark and the data on FEV1, FEV1% pred, FVC\nvalues have been supplemented in Table 2 of the revised manuscript as shown below.\nIt is shown that in terms of FEV1 and FVC values, there were a remarkable decline in\npatients of bronchiectasis with asthma compared to asthmatics, whereas no significance\nwas detected in terms of FEV1% pred among all the patients.\nTable 2. Clinical characteristics of all subjects\nHealthy controls (n = Bronchiectasis (n = Bronchiectasis with\nVariables Asthma (n = 30) P value\n20) 31) asthma (n = 31)\nLung function NA\nFEV1 1.34 (0.94-2.01) 1.6 (1.21-2.33) 1.24 (0.80-1.64) △ 0.0321\nFEV1% pred 59.09 (23.25) 66.19 (20.69) 54.16 (20.86) 0.1062\nFVC 2.1 (1.53-2.66) 2.4 (1.98-2.99) 1.75 (1.22-2.15) 0.0007\n△△△\nFVC% pred 71.09 (20.94) 79.2 (14.36) 62.92 (17.52) △△ 0.0028\nFEV1/FVC, % 66.28 (13.69) 66.98 (13.01) 68.53 (15.5) 0.8487\nFeNO NA 10 (6.75-15.25) △ 25 (12-112) 17 (9-27) 0.0143\nACT score NA 17 (14-20) 16 (14-17) 0.0367\nPeripheral blood\n1.6 (0.73-2.3) 1.7 (0.9-2.7) △△△△ 7.1 (3.3-9.9) **** 1.9 (1.3-2.8) △△△ <0.0001\neosinophils, %\nPeripheral blood 0.10 (0.06-0.14) △△△\n0.085 (0.053-0.15) 0.39 (0.20-0.66) **** 0.11 (0.07-0.17) △△△ <0.0001\neosinophils ×109·L-1 △\nSerum IgE NA △△ △△△△ <0.0001\n<100 21 (70.0%) 7 (23.3%) 28 (90.3%)\n100-200 4 (13.3%) 8 (26.7%) 1 (3.2%)\n>200 5 (16.7%) 15 (50.0%) 2 (6.5%)\nNote: Quantitative data are summarized as mean (SD) for normally distribute variables or median\n(IQR) for non-normally distribute variables and qualitative data are presented as n (percentage).\nP value: overall comparison among existing groups.\n＊＊＊＊P < 0.0001 compared with healthy control group.\nP < 0.05, P < 0.01, P < 0.001, P < 0.0001 compared with asthma group.\n△ △△ △△△ △△△△\nAbbreviations: FEV1, Forced Expiratory Volume in 1 s; FVC, Forced Vital Capacity; FeNO:\nFractional exhaled nitric oxide; ACT: Asthma Control Test; Ig: immunoglobulin; NA, not available.\nc. How did you sample for microbiology results? Induced sputum?\nReply:\nIn the present study, induced sputum was not involved, so spontaneously expectorated\nsputum samples were lacking in all healthy subjects and the small minority of asthmatic\npatients (1/30).And microbiology results were available according to microbiological\ncultures of sputum or bronchoalveolar lavage fluid (BALF). Notably, among asthmatic\npatients, P. aeruginosa has been confirmed negative by microbiological culture in only\nsputum samples (12/30), only BALF (3/30) or both (12/30), whereas the remaining 3\nasthmatics were absent for microbiological results due to the lacking of BALF or\nsputum samples and the delay in sputum sample delivery. Additionally, among all the\npatients with the isolation of P. aeruginosa including bronchiectasis patients (16/31),\npatients of bronchiectasis with asthma (11/31), only sputum cultures from\nbronchiectasis patients (6/16), patients of bronchiectasis with asthma (3/11) were\npositive for P. aeruginosa, while only BALF cultures from bronchiectasis patients\n(5/16), patients of bronchiectasis with asthma (4/11) and both sputum and BALF\ncultures from bronchiectasis patients (5/16), patients of bronchiectasis with asthma\n(4/11) were positive.\n2. FVC and FEV1/FVC Findings:\na. Clarify why patients with bronchiectasis and asthma (BA) show lower FVC but\nhave a similar FEV1/FVC ratio when compared to asthmatic individuals. This\nraises questions about specific lung mechanics in BA patients that require further\nexploration.\nReply:\nGiven that FEV1 and FVC values were both lower in patients of bronchiectasis with\nasthma in comparison with asthmatics (Table 2), FEV1/FVC value, as the ratio of FEV1\nto FVC, might be comparable among all the patients. Notably, there is limited\nknowledge regarding the comparison for clinical characteristics especially the lung\nfunction between asthmatics and patients of bronchiectasis with asthma.\nCollectively,\nnon-significant FEV1/FVC value raises questions about specific lung mechanics in\npatients with bronchiectasis with asthma and requires further exploration.\n3. Pseudomonas Aeruginosa and Th2 Endotype:\na. The concept of Pseudomonas aeruginosa driving a Th2 endotype is intriguing,\nbut several points need addressing:\ni. The majority of the supporting evidence stems from murine models, which calls\nfor stronger human-based validation. Performing assays with human cells, such\nas ILC3s, would provide more relevant translational insight.\nReply:\nThanks to the efforts from the journal editor for instantly sharing the review report that\nthey have received with us in early November, we were able to plan relevant\nexperiments as soon as possible and conscientiously completed all scheduled scientific\nexperiments as suggested above. In the revised manuscript, we supplemented the ratio\nof ILC3s among lymphocytes and total numbers in peripheral blood mononuclear cells\n(PMBCs) among healthy subjects, asthmatics, patients with bronchiectasis with or\nwithout asthma by flow cytometry analysis, yielding extra data on Supplementary Fig.\n2h. Our data implied that patients of BA presented higher ratio and cell counts of ILC3s\ncompared to asthmatic patients or healthy subjects. The finding corresponded to more\nabundant ILC3s in mice exposed to PA/OVA, laid the basis for investigating IL-17C\nmodulating ILC3 response in asthma endotype switching within the context of\nbronchiectasis with asthma and provided more relevant translational insight.\nSupplementary Figure 2\nii. It would be beneficial to include data on Pseudomonas aeruginosa colonization\nin the studied patient subsets and analyze correlations with Th2-associated\nmarkers (e.g., TSLP, CCL17). This would strengthen the manuscript’s human\nrelevance and address the weakness in linking P. aeruginosa to Th2 responses in\nhumans.\niii. Consider referencing previous studies that examine the relationship between P.\naeruginosa and Th2 responses in humans, such as this work:\nhttps://pubmed.ncbi.nlm.nih.gov/16387607/ and, this\nwork: https://pubmed.ncbi.nlm.nih.gov/38888974/.\nReply:\nThank you for the above suggestion. According to previous studies from Hartl et al. and\nRehman et al. (summarized in Hartl et al, J Allergy Clin Immunol 2006; and in Rehman\net al, JCI Insight 2024) listed in the References section of the revised manuscript (Ref.\n37, 38), we examined the serum levels of Th2-associated markers, TSLP and CCL-17\namong healthy subjects, asthmatics, P. aeruginosa-infected, noninfected patients with\nbronchiectasis with or without asthma by ELISA analysis and added extra data on\nSupplementary Figure 2 of the revised manuscript to establish the link of P. aeruginosa\ninfection to Th2 responses in human. Our data showed raised TSLP in P. aeruginosa-\ninfected patients with bronchiectasis with or without asthma, whereas no significance\nin CCL-17 levels was observed (Supplementary Fig. 2i, j).\nSupplementary Figure 2\n4. Discussion:\na. The current discussion is somewhat basic and lacks a strong translational\nperspective. The manuscript should address whether patients colonized by\nPseudomonas aeruginosa should be treated differently based on their Th2\nresponses.\nb. Lessons from cystic fibrosis (CF) management could offer valuable insights into\nhow Pseudomonas aeruginosa infection might be treated in these patients. This\ncould be an important avenue for further research and discussion.\nReply:\nThank you for your valuable comments. Following the suggestion, we expanded the\nDiscussion section in Para 3 as follows: Our findings linked P. aeruginosa infection to\nTh2 responses. P. aeruginosa infection is correlated with significantly higher levels of\nTh2 cells and CCL-17 in part of cystic fibrosis patients (summarized in Hartl et al, J\nAllergy Clin Immunol 2006; and in Rehman et al, JCI Insight 2024). Th2-endotype of\nbronchiectasis characterized by raised blood eosinophils or FeNO has been\nincreasingly recognized (summarized in Martins et al, Pulmonology 2023). The\nevidence of elevated TSLP in P. aeruginosa-infected patients with bronchiectasis with\nor without asthma might facilitate us to provide precision medicine approach based on\nTh2 responses for these patients. Lessons from cystic fibrosis management could offer\nvaluable insights into the treatment for P. aeruginosa infection in these patients. This\ncould be an important avenue for further research.\nReviewer #2(Remarks to the Author)\nThis study by Zhang et al. addresses current challenges in the treatment of patients\nwith coexisting bronchiectasis and asthma. The authors sought to explore the\nasthma endotype in patients with non-cystic fibrosis bronchiectasis with asthma\n(BA) to identify new therapeutic targets. Using mouse models, the authors report\na pathogenic interaction between IL-17C and ILC3s that potentiates IL-17A\nresponses and switching to a neutrophilic asthma endotype. The study is\ninteresting and warrants investigation, however, there are some major limitations.\n- The authors’ rationale for inducing P. aeruginosa infection followed by allergic\nsensitisation to Ova in their murine model lacks convincing justification. While\nindividuals of all ages can have bronchiectasis, it is most common in the elderly.\nThe EMBARC study explored this overlap between asthma and bronchiectasis\nand identified that asthma could be reported as an etiology of bronchiectasis. This\nis supported by the British thoracic society guideline for non-cystic fibrosis\nbronchiectasis recommendation for considering asthma as the cause of\nbronchiectasis in the absence of other etiological explanations. Others show that\nasthma severity is an independent risk factor for bronchiectasis, that there is a\nhigh percentage of bronchiectasis in patients with severe asthma, and that age of\nasthma onset and exacerbations are independently associated with the occurrence\nof bronchiectasis.\nReply:\nThank you for your valuable comments. As shown above, previous studies and\nguideline have indicated that asthma is a potential cause of bronchiectasis and the\nseverity can be recognized as an independent risk factor for bronchiectasis. Notably, in\nthe present study, to identify the difference in clinical characteristics of patients with\ncoexisting bronchiectasis and asthma from patients with bronchiectasis alone or asthma\nalone, we recruited patients with primary diagnosis of bronchiectasis and coexisting\nasthma. Therefore, we established a mouse model of ovalbumin-induced asthma within\nthe context of P. aeruginosa chronic lower respiratory tract infection to investigate\nunderlying mechanisms involving in the endotype of bronchiectasis and asthma overlap.\n- Does P. aeruginosa infection alone adequately model the diverse\nimmunopathological repertoire and pathophysiology of patients with\nbronchiectasis? This must be established to convince readers that the reported\nobservations are not simply readouts of host:pathogen interactions e.g. the effects\nof an active infectious challenge on shaping allergic sensitisation and the\nimmunological profile of the recall response.\nReply:\nGlobally, the lack of mature animal models in analogy with bronchiectasis has become\na barrier to exploring the pathogenesis of bronchiectasis and potential effective\nmanagements. Bronchiectasis is a complex and heterogeneous condition manifesting\nunknown etiology and highly variable clinical presentation, so there are no animal\nmodels representing bronchiectasis. During the design phase, we found that in\nnumerous researches such as previous studies from Martin et al., Ward et al., Collin et\nal. and Frija et al. (summarized in Martin et al, Eur Respir J 2011; Ward et al, Eur Respir\nJ 2011; Collin et al, EBioMedicine 2020; Frija-Masson et al, Eur Respir J 2017), P.\naeruginosa chronic lower respiratory tract infection has been widely employed to\nexplore the underlying mechanisms involved in immunopathological repertoire and\npathophysiology of bronchiectasis. Consequently, by referring to previous work from\nFacchini et al., Rossi et al. and Codagnone et al., (summarized in Facchini et al, J Vis\nExp 2014; Rossi et al, J Cyst Fibros 2023; Codagnone et al, Mucosal Immunol 2018),\nwe established P. aeruginosa chronic lower respiratory tract infection through\nintratracheal (i.t.) instillation with P. aeruginosa-laden beads. Undoubtedly, the model\nthat we established has yet to be optimized, which is the ongoing work of our team.\nImportantly, we explained the purpose of the model we used in the last paragraph of the\nIntroduction section in the revised manuscript.\n- The potential influence of smoking history on the reported data is not discussed\nfurther than reported rates of “Smoking, %” in Table 1.\nReply:\nThank you for your constructive remark. And in the present study, smoking status was\ndefined as never smokers, ex-smokers and current smokers as depicted in the table\nbelow and in Table 1 of the revised manuscript.\nTable 1. Baseline information of healthy controls, bronchiectasis patients, asthmatic patients\nand patients of bronchiectasis with asthma\nBronchiectasis with\nHC Bronchiectasis Asthma\nasthma\nSubjects 20 31 30 31\nAge (y) 40.5 (36.25-46.75) 57 (54-68) 59.5 (50.75-66) 61 (53-69)\nSex\nMale 8 (40%) 12 (38.7%) 11 (36.7%) 5 (16.1%)\nFemale 12 (60%) 19 (61.3%) 19 (63.3%) 26 (83.9%)\nSmoking status\nNever smokers 16 (80%) 26 (83.9%) 24 (80%) 29 (93.5%)\nEx-smokers 0 (0%) 5 (16.1%) 2 (6.7%) 0 (0%)\nCurrent smokers 4 (20%) 0 (0%) 4 (13.3%) 2 (6.5%)\nDuration of symptoms\nNA 12 (5-30) 4 (2-10.25) 30 (5-40)\n(y)\nPA isolation, % NA 51.6 0 35.5\n- The authors identify the paucity of data on asthma endotypes in the context of\nbronchiectasis-asthma overlap as a barrier to treatment decision-making, but do\nnot present data on sputum inflammometry, which is a global benchmark for\ncharacterization and management guidance. This limits the translational\napplicability of the findings from the mouse experiments.\nReply:\nAs shown above, the lack of sputum cytology does indeed limit the translational\napplicability of the findings from the mouse experiments, which has been stated in the\nlimitations in the Discussion section of the manuscript. Neutrophils in sputum can be\nassessed, however, the patients listed in the study are situated across the country, and it\nis pretty difficult to recruit them again for collecting sputum samples and further sputum\ncytology. Notably, previous study from Kupczyk et al. (summarized in Kupczyk et al,\nAllergy 2014) suggested that biomarkers such as sputum inflammometry are often\nunstable over time and it is insufficient to determine asthma phenotype according to\nsputum cytology. Additionally, existing reports from O'Byrne et al. and Chung\n(summarized in O'Byrne et al, Lancet Respir Med 2016; Chung, Lancet Respir Med\n2016) have identified neutrophilic asthma by blood inflammometry among patient with\nsevere asthma. Interestingly, previous work from Ma et al. (summarized in Ma et al,\nArch Bronconeumol 2024) showed that neutrophilic inflammation is the primary\nphenotype in patients with the overlap between asthma and bronchiectasis, which may\nbe suggestive for our study exploring the asthma endotype within the context of\nbronchiectasis-asthma overlap. And if sputum samples are available in the future, we\nwould conduct sputum cytology in these patients.\n- The authors report a positive correlation between peripheral (serum) IL-17A and\nIL-17C in patients with bronchiectasis with or without asthma. It would be more\ninformative to perform these analyses in patients with bronchiectasis alone,\nasthma alone, and with both bronchiectasis and asthma. The specific relevance of\nthese findings in the context of ILC3 responses should then be demonstrated in\npatients with both bronchiectasis and asthma to support the contention that “IL-\n17C could serve as a potential therapeutic target for individuals of BA”.\nReply:\nThank you for your valuable comments and we redrew the figure 5j with the supplement\nof Figure 5k as suggested above. As shown in the figure below and Fig. 5j, k, we\nperformed the correlation analysis between IL-17A and IL-17C in patients with\nbronchiectasis alone, asthma alone, and with both bronchiectasis and asthma, indicating\na positive correlation between serum IL-17C levels and IL-17A levels among these\npatients (Fig. 5j). Importantly, to investigate the specific relevance of these findings in\nthe context of ILC3 responses, given ILC3s as the source of IL-17A and augmented\nnumbers of ILC3 in peripheral blood of patients with bronchiectasis with asthma\n(Supplementary Fig. 2h in the revised manuscript), we identified the correlation\nbetween ILC3s ratios or counts and IL-17C levels in peripheral blood of patients with\nbronchiectasis with asthma and also found a positive correlation between these\nindicators (Fig. 5k), which contributed to support the role of IL-17C as a potential\ntherapeutic target in patient with bronchiectasis with asthma.\nFigure 5\nReviewer #3 (Remarks to the Author):\nMANUSCRIPT NCOMMS-24-60979\nGENERAL COMMENTS.\nThis manuscript presents a clinical-translational study that addresses how\nbacterial colonization in patients with bronchiectasis and asthma alters immune\nsignaling via the archetype Type 3 signaling pathway involving interleukin (IL)-\n17 cytokines, especially IL-17A and IL-17C. The data sets presented are extensive,\nconsisting of data from humans, from cell and animal models. Taken together, the\nscientific basis for the manuscript and the presented conclusions is solid.\nNevertheless, the results presented are important for the (improved)\nunderstanding of archetype immune signaling in patients with chronic airway\ndisease and bacterial colonization, with future implications beyond asthma.\nThe scientific criticism relates mainly to the present focus on ILC3 cells, a focus\nthat leaves the expert reader with several unanswered questions regarding the role\nof Th17 and Tc17 cells. The mechanistic rationale for the critical role of IL-17A\nper se also deserves to be stated in a more precise manner, given its importance for\nthis field of research (see also Specific comments below). Additional and important\ncriticism relates to how the presentation of the underlying study is made, from a\nlinguistic point-of-view. Key entities need to be handled in a more consistent\nmanner than is now the case. Moreover, as the main text now stands, it is in need\nof editing by an expert in English. Once it is optimized in these respects, the\nmanuscript is likely to become important for future research on chronic\ninflammatory airway disorders.\nSPECIFIC COMMENTS.\nA) Title: The title emerges as too definitive. Maybe \"Evidence for IL-17C\ngoverning IL-17A pathogenicity...\" would be better given that the focus is set\nmainly on ILC3 cells and not on additional sources of IL-17A? Another reason for\nthis is that important evidence originates from experimental models and not\nhumans in vivo.\nReply:\nThank you for your valuable suggestion and we modified the title of the revised\nmanuscript as follows: Evidence for IL-17C governing IL-17A pathogenicity and\norchestrating asthma endotype switching in bronchiectasis.\nB) Summary: The authors should make clear what evidence originates from\nhumans and what evidence originates from experimental models. Apart from this,\nThe scientific contents are fine but linguistic editing is needed.\nReply:\nThank you for your valuable comments. As instructed above, inadequacies have been\nmodified in the Summary section of the revised manuscript.\nC) Introduction:\n1. Para 1 and 2: The text would benefit from the addition of information on Type\n1 and Type 3-signaling and how these pathways relate to neutrophilic and\neosinophilic inflammation. Here, key entities must be introduced and used in a\nmore accurate and consistent manner than is now the case.\nReply:\nThank you for your constructive comments. The information of type 1 and type 3-\nsignaling and the association with neutrophilic and eosinophilic inflammation have\nbeen supplemented in Para 1 of the revised manuscript.\nKey entities such as type-2 high (T2), type-2 low (non-T2) in Para 1 and nontypeable\nHaemophilus influenzae (NTHi) in Para 2 have been revised in the manuscript. And\nkey entities such as bronchiectasis with asthma (BA) in Para 2 were introduced with\nreference to previous studies from Ma et al. and Polverino et al. (summarized in Ma et\nal, Arch Bronconeumol 2024;Polverino et al,J Allergy Clin Immunol2024).\n2. The entire study behind this manuscript study is based upon the role of IL-17A\nin mediating neutrophil accumulation in the lungs. Given this, a brief presentation\nof the basic evidence is appropriate, published in the original studies from Linden\net al and from Kolls et al (summarized in Linden & Kolls, Immunity 2004; and in\nLinden & Dahlen, ERJ 2014). The original studies are also relevant for comments\nin the the results and the discussion (see below).\nReply:\nIn the present study, as indicated above, it is imperative to stress the role of IL-17A.\nGiven this, we referred to the original studies from Linden et al and from Kolls et al\n(summarized in Linden & Kolls, Immunity 2004; and in Linden & Dahlen, ERJ 2014)\nlisted in the Reference section of the revised manuscript (Ref. 6, 7) and supplement a\nbrief presentation of the evidence supporting the role of IL-17A in mediating neutrophil\naccumulation in Para 3 of the Introduction section, the Results section and the\nDiscussion section.\nD) Results:\n1. Table 2: Data sets on FEV1 and on ACT for healthy controls are missing.\nReply:\nIn the present study, the assessment of FEV1 and ACT was for individuals manifesting\nclinical symptoms and these ones were willing to receive inspections such as lung\nfunction test and ACT. Notably, the healthy controls that we recruited lacked clinical\nsymptoms, and therefore identifying FEV1 and ACT values of these subjects within\nnormal range.\n2. Text line 108: All FENO values are within the normal range, so the comment on\na trend here does not seem meaningful.\nReply:\nAs suggested above, all the medians of FeNO values were withinthe normal range and\nmore importantly, no significance was detected for FeNO values in patients of BA in\ncomparison with asthmatic patients. Accordingly, we have made correction in the\nrevised manuscript.\n3. Text line 165: These references are not adequate. See point C.2 for appropriate\nreferences.\nReply:\nThank you for your suggestion and we have supplemented the references in the revised\nmanuscript as instructed in point C.2 (Ref. 6, 7).\n4. Text line 281: The data set on MMP-9 emerges as important, not least given that\nthis proteinase originates in part from neutrophils. The latter can be emphasized\nin the text here (as well as in the discussion and in the principal overview in Fig 8).\nReply:\nThank you for your constructive remark. As indicated above, we have supplemented\nneutrophil-derived MMP-9 in the Result section, Para 6 and Para 8 of the Discussion\nsection and the principal overview of Figure 8.\n5. Apart from the above, the figures and tables seem adequate.\nReply:\nWe have comprehensively revised the inadequacies in the manuscript according to the\nabove comments.\nE) Discussion:\n1. A general comment is that the text does not really make clear why the emphasis\nis set on ILC3 cells and not on other sources for IL-17A (such as Th17 or Tc17\ncells).\nReply:\nThank you for your valuable comment and we have made relevant modifications to\nPara 5, Para 6 and Para 7 of the Discussion section. Although the focus of present study\nis the modulation of ILC3 responses by IL-17C, the role of Th17 cells in asthma\nendotype within the context of P. aeruginosa chronic infection has been investigated in\nthe study. PA/OVA exposure increased the cell numbers of Th17 cells (Fig. 2c, d).\nNotably, the modulation of Th17 cells by IL-17C has been widely discussed in\nnumerous researches such as previous study from Chang et al. (summarized in Chang\net al, Immunity 2011), which coincides with that ablation of Il17re reduced the levels\nof Th17 cells in lung tissues from mice exposed to PA/OVA (Fig. 6n). Inspired by this\nstudy, when our preliminary results indicated that ablation of Il17re attenuated ILC3\nresponse, we asked a question that whether IL-17C may regulate ILC3 responses, which\npromoted further exploration and consequently discovered that IL-17C directly bound\nto ILC3s by interacting with IL-17RE and subsequently potentiated IL-17A expression.\nHowever, as indicated above, given that IL-17C governed IL-17A pathogenicity, we\ndidn’t rule out the potential role of Tc17 cells which can secret IL-17A as the same as\nTh17 cells and ILC3s in experimental asthma model within the context of P. aeruginosa\nchronic lower respiratory tract infection. Such inadequacy has been stressed in the fifth\nlimitation in the Discussion section of the revised manuscript, and the conclusion that\nIL-17C governs IL-17A pathogenicity by interacting with ILC3s should be interpreted\ncautiously due to this limitation.\n2. Another general comment is that the discussion need to address the (potential)\ninvolvement of Type 1 signaling (via ILC 1 cells or via Th1 or Tc1 cells).\nReply:\nThank you for your suggestion and we made relevant modifications to Para 4 and Para\n7 of the Discussion section. In the Results section of the manuscript, we investigated\nthe potential involvement of ILC1, Th1 cells associated with type 1 signaling and our\ndata suggested that P. aeruginosa chronic lower respiratory tract infection failed to\naffect ILC1 and Th1 responses in mice exposed to PA/OVA (Supplementary Fig. 2e-g),\nwhich was stressed in Para 4 of the Discussion section. However, apart from ILC1s and\nTh1 cells, we didn’t rule out the potential role of Tc1 cells in experimental asthma\nmodels within the context of P. aeruginosa chronic infection. Such inadequacy has been\nemphasized in the fifth limitation of the Discussion section in the revised manuscript.\n3. Text line 383: Figure 8 need to include MMP-9 given the current data and its\nrelevance in neutrophilic inflammation in general (see also above).\nReply:\nThank you for the above suggestion and we introduced neutrophil-derived MMP-9 to\nFigure 8 of the revised manuscript.\n4. Text line 413: Although this reviewer share the forwarded conclusions re. the\ncurrent status of clinical evidence on inhibiting IL-17A signaling in asthma, the\ntext here must be revised to make it easy to understand.\nReply:\nWe have re-written the part in Para 4 of the Discussion section in the revised manuscript\naccording to the above suggestion.\n5. Text line 423: The presentation of published evidence on IL-17C is now\nsomewhat confusing and all publications on this may not be relevant for the\ncurrent setting. This text can preferably be restricted to the most relevant\npublications.\nReply:\nThank you for your constructive suggestion. To better convey the idea that the role of\nIL-17C in regulating tissue inflammation has been controversial, the removal, addition\nand a brief introduction of the most relevant publications have been made in Para 5 of\nthe Discussion section in the revised manuscript.\n6. Text line 460: The limitations of this study include the points forwarded as points\nE.1 and E.2 above.\nReply:\nAs indicated in E.1 and E.2, we have listed the potential involvement of type 1 and type\n3 signaling as the fifth limitation in the revised manuscript.\n7. Text line 469: The conclusion section should include comments on MMP-9 as\nwell (see above).\nReply:\nAs indicated in D.4, we have introduced neutrophil-derived MMP-9 and its role in\nregulating IL-17C production to the conclusion section of the revised manuscript.\nF) Methods:\n1. Text line 610: The authors need to present the specific provider for each utilized\nassay, to make it possible for interested readers to reproduce data from this\npotentially important study.\nReply:\nThank you for your valuable remark. In the flow cytometry analysis section, due to\nnumerous antibodies for use in the experiment, we briefly introduced the providers of\nthese antibodies. Consequently, we have provided the specific providers of each utilized\nassay in Supplementary Table 2 and indicated it at the end of the paragraph.\n2. Text line 644: The rationale for always using \"two-sided significance\"\n(regardless of pre-existing data and hypotheses) seem unclear and a rationale\nshould be explained.\nReply:\nAs shown above, the rationale for always using “two-sided significance” weren’t be\nclearly explained in the manuscript and we have supplemented the rationale in the\nstatistical analysis section of the revised version. Our study mainly concerns the\ncorrelation analysis, one-way ANOVA, two-way ANOVA in terms of statistical analysis.\nAnd the null hypotheses we establish are that there is no significant correlation between\ntwo variables and all the medians or means are equal. Considering the paucity of pre-\nexisting data on the endotype of bronchiectasis and asthma overlap, we are unable to\ndetermine the preset results of experiments (in which identifying a>b or a<b was\nabsolutely correct) and therefore adopting two-sided test and recognizing two-sided\nsignificance level as 0.5. Moreover, more importantly, in the same situation, the power\nof one-sided test is higher, whereas two-sided test is comparatively conserve, which\nmeans that in case of one-sided test, false positive conclusions would be drawn, so two-\nsided test should be adopted in the present study to avoid false positives and gain\nreliable results.\n3. Except for the above, the methods seem adequately described.\nReply:\nWe have made corrections accordingly in the Methods section of the revised manuscript\nin light of the above suggestions."}, {"round": 2, "reviewer_comments": {"reviewer_1": "My questions and comments have been satisfactorily addressed with the support of new data. At this time, I have no further questions and would like to thank the authors for their detailed responses.", "reviewer_2": "The authors’ response and rationale for inducing P. aeruginosa infection followed by allergic sensitisation to Ova in their murine model remains insufficiently justified. There has also been no attempt to examine the effects of the sequence of exposures experimentally where the authors could have investigated whether P. aeruginosa infection after the establishment of Ova-induced experimental asthma results in similar pathogenic interactions between IL-17C and ILC3s that potentiate IL-17A responses and a switch to the neutrophilic asthma endotype.\nThe additional information presented in Table 1 of the revised manuscript is informative, however, the potential influence of smoking history on the reported outcomes remains undiscussed.\nThere remains insufficient data to claim the study of asthma endotypes in bronchiectasis-asthma overlap. The absence of sputum inflammometry remains a critical limitation of the current study. This is inadequately justified by the authors’ and their reference to previous work showing that neutrophilic inflammation is the primary phenotype in asthma-bronchiectasis overlap, and the unscientific statement that those data are suggestive for the current study’s exploration of asthma endotypes in this overlap.\nNew data of correlations between serum IL-17A AND IL-17C in patients with bronchiectasis alone, asthma alone, and with both bronchiectasis and asthma are informative. However, these data show that positive correlations between IL-17A and IL-17C are also present in patients with asthma or bronchiectasis alone, yielding the same output across all groups in this comparison and weakening the authors’ contention that pathogenic IL-17C uniquely governs IL-17A pathogenicity and asthma endotype switching in bronchiectasis. Therefore, sputum inflammometry is required to provide some insight into the mechanistic disease endotype present in the patients with asthma alone. This also highlights the unsuitability of PBS/OVA mice to model the ‘asthma alone’ cohort in this study as these animals did not exhibit allergic induction of IL-17.", "reviewer_3": "MAJOR COMMENTS\nThe authors have addressed most of the criticism in an adequate manner, and the manuscript i snow substantially improved. There is only one issu ethat remain to be clarified (see below).\nSPECIFIC COMMENTS\nD. Results. Table 2: Data sets on FEV1 and ACT for healthy controls are missing. The authors need to clarify whether they have access to the requested data or not. If the data exists, the authors need to include the data sets in the table and address it in the results."}, "author_response": "Point-by-point responses\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nMy questions and comments have been satisfactorily addressed with the support\nof new data. At this time, I have no further questions and would like to thank the\nauthors for their detailed responses.\nReply:\nThank you for your professional review work on our manuscript. Meanwhile, we were\nvery pleased that our responses have addressed your concerns and met with approval.\nReviewer #2(Remarks to the Author)\nThe authors’ response and rationale for inducing P. aeruginosa infection followed\nby allergic sensitisation to Ova in their murine model remains insufficiently\njustified. There has also been no attempt to examine the effects of the sequence of\nexposures experimentally where the authors could have investigated whether P.\naeruginosa infection after the establishment of Ova-induced experimental asthma\nresults in similar pathogenic interactions between IL-17C and ILC3s that\npotentiate IL-17A responses and a switch to the neutrophilic asthma endotype.\nReply:\nPrevious work from Mao et al. and Chen et al. (summarized in Mao et al, Eur Respir J\n2016; Chen et al, J Thorac Dis 2016) indicated that asthma can be reported as a\ncomorbidity of bronchiectasis. Meanwhile, previous studies from Ma et al. and Coman\net al. (Ma et al, Arch Bronconeumol 2024; Coman et al, Ann Allergy Asthma Immunol\n2018) showed that bronchiectasis is one of comorbidities in severe asthma. Notably,\nbronchiectasis-asthma overlap and asthma-bronchiectasis overlap are both clinical\nissues. At this time, in the present study, we focused on the former and investigated the\nasthma endotype within the context of bronchiectasis-asthma overlap. Accordingly, we\nrecruited the patients with primary bronchiectasis and coexisting asthma. Considering\nP. aeruginosa chronic lower respiratory tract infection has been widely employed to\nexplore the underlying mechanisms involved in bronchiectasis, we established a model\nof P. aeruginosa infection followed by Ova-induced asthma to investigate the\nunderlying mechanisms involving in the asthma endotype within the context of\nbronchiectasis-asthma overlap.\nIndeed, asthma-bronchiectasis overlap is a pivotal and interesting clinical issue. To\nexplore potential role of IL-17C in the endotype of asthma-bronchiectasis overlap, we\nconstructed an alternative model of Ova-induced experimental asthma followed by P.\naeruginosa infection (Fig. a). As depicted below, the preliminary results indicated that\nP. aeruginosa infection didn’t alter the levels of Il17c, Il17a mRNA and IL-17A as well\nas the cell counts of AM, neutrophils, ILC3. Airway inflammation, eosinophils, ILC2\ntogether with the levels of IgE, IL-5, IL-13 and total cell numbers were elevated from\nOVA/PA-exposed mice compared with their control mice, whereas comparable between\nOVA/PA-exposed mice and OVA/PBS-exposed mice (Fig. b-o). Furthermore, ablation\nof Il17re didn’t affect eosinophilic inflammation and ILC3 counts in mice exposed to\nOVA/PA or OVA (Fig. d-p). However, potential factors affecting the asthma endotype\nin an experimental asthma model followed by P. aeruginosa infection could be the time\ninterval between the last challenge and i.t. infection with P. aeruginosa accompanied\nby duration of infection. Thus, the interesting scientific issue remains to be further\ninvestigated in the future work.\nFigure\na Schematic diagram of OVA treatment by i.p. delivery and airway challenge followed by i.t.\ninstillation with P. aeruginosa-laden agar beads at Day 9 in a model and BALF and lung tissues were\ncollected at Day 14. b, c mRNA levels of Il17c and Il17a in lungs from mice exposed to OVA/PBS\n(n = 4), OVA/PA (n = 6) and their control (n = 4) were determined. d-f Semiquantified inflammation\nof lungs and representative images of H&E-stained lung sections from wild-type (WT; PBS, n = 3;\nOVA/PBS, n = 3; OVA/PA, n = 5) and Il17re-/- mice (PBS, n = 3; OVA/PBS, n = 4; OVA/PA, n = 5)\nin the model (e Scale bars, 500 μm; f Scale bars, 50 μm). g-j Protein levels of IgE, IL-5, IL-13, and\nIL-17A in serum or BALF from WT (PBS, n = 3; OVA/PBS, n = 4; OVA/PA, n = 6) and Il17re-/-\nmice (PBS, n = 3; OVA/PBS, n = 4; OVA/PA, n = 6) were assessed. k-p Quantification of total cells,\nAM, eosinophils and neutrophils in BALF together with total numbers of ILC2s, and ILC3s in lungs\nfrom WT (PBS, n = 3; OVA/PBS, n = 3; OVA/PA, n = 6) and Il17re-/- mice (PBS, n = 3; OVA/PBS,\nn = 4; OVA/PA, n = 6) as determined by flow analysis. Data are shown as mean ± SEM. Significance\nwas calculated by one-way ANOVA (b-d,g-p).\nThe additional information presented in Table 1 of the revised manuscript is\ninformative, however, the potential influence of smoking history on the reported\noutcomes remains undiscussed.\nReply:\nIn the present study, the aim is to investigate the asthma endotype in patients of\nbronchiectasis with asthma and airway inflammation is identified as an important\nindicator for evaluation.\nIn the revised version of the manuscript, we supplemented P value of smoking rates\nacross the four groups in para 1 of the Result section, and further discussion concerning\nthe potential influence of smoking history on the reported outcomes was inserted into\npara 2 of the Discussion section as follows: Notably, another potential factor that could\ninfluence asthma endotype might be smoking history being associated with airway\ninflammation. Nevertheless, the percentages of subjects with smoking history were\ncomparable across the groups involved in the study (P=0.4304). With limited sample\nsize, we couldn’t perform subgroup analysis of reported outcomes based on smoking\nhistory among the patients of bronchiectasis with asthma.\nThere remains insufficient data to claim the study of asthma endotypes in\nbronchiectasis-asthma overlap. The absence of sputum inflammometry remains a\ncritical limitation of the current study. This is inadequately justified by the authors’\nand their reference to previous work showing that neutrophilic inflammation is\nthe primary phenotype in asthma-bronchiectasis overlap, and the unscientific\nstatement that those data are suggestive for the current study’s exploration of\nasthma endotypes in this overlap.\nReply:\nThank you for comments. In our study, the asthma endotype in patients of\nbronchiectasis with asthma may tend to be type 2-low asthma characterized by lower\neosinophil counts, higher levels of IL-17A, ILC3s and difficultly controlling asthma\nsymptoms (Table 1, Supplementary Fig. 2h and Fig. 5h). Consistently, the data from\nanimal model used for investigating the asthma endotype in bronchiectasis-asthma\noverlap indicated neutrophilic asthma with airway hyperreactivity, lower levels of IL-\n5, IL-13, IgE and abundant IL-17A (Fig. 1b-n). Indeed, sputum inflammometry is very\nimportant for identifying the asthma endotype among the patients of bronchiectasis-\nasthma overlap. And unavailable direct data on the asthma endotype using sputum\ninflammometry involving sample collection, storage, procession and cell isolation\namong the patients across the country have remained a limitation of the present study,\nwhich remain to be the focus of the future work. However, the findings of BALF\ncytology from mouse experiments revealed that P. aeruginosa chronic lower respiratory\ntract infection drove asthma endotype switching towards neutrophilic asthma, which\ncan be an indirect indication for the asthma endotype of bronchiectasis-asthma overlap.\nImportantly, as mentioned above, we misquoted previous work based on the patients of\nasthma-bronchiectasis overlap for supporting the contention that neutrophilic\ninflammation could be the primary phenotype in bronchiectasis-asthma overlap.\nNew data of correlations between serum IL-17A AND IL-17C in patients with\nbronchiectasis alone, asthma alone, and with both bronchiectasis and asthma are\ninformative. However, these data show that positive correlations between IL-17A\nand IL-17C are also present in patients with asthma or bronchiectasis alone,\nyielding the same output across all groups in this comparison and weakening the\nauthors’ contention that pathogenic IL-17C uniquely governs IL-17A\npathogenicity and asthma endotype switching in bronchiectasis. Therefore,\nsputum inflammometry is required to provide some insight into the mechanistic\ndisease endotype present in the patients with asthma alone. This also highlights\nthe unsuitability of PBS/OVA mice to model the ‘asthma alone’ cohort in this study\nas these animals did not exhibit allergic induction of IL-17.\nReply:\nThank you for comments. The positive correlations between IL-17A and IL-17C were\nuniversally applied for patients with asthma alone, bronchiectasis alone, and\nbronchiectasis-asthma overlap, however, precise modulation of IL-17C on IL-17A and\nasthma endotype switching was on the basis of the findings from mouse experiments in\nthe present study. Notably, according to the findings from animal study, we didn’t\nobserve airway hyperreactivity reflecting asthma feature in mice exposed to P.\naeruginosa infection alone with abundant IL-17C. Moreover, ablation of Il17re purely\naffected IL-17A-mediated inflammation and asthma endotype switching in PA/OVA-\nexposed mice rather than OVA-exposed mice, indicating the role of IL-17C in\ngoverning IL-17A pathogenicity and orchestrating asthma endotype driven by P.\naeruginosa infection. Together, there remain insufficient human data and therefore\nrevising the title as follows: Evidence for IL-17C governing IL-17A pathogenicity and\norchestrating asthma endotype switching in bronchiectasis.\nReviewer #3 (Remarks to the Author):\nMAJOR COMMENTS\nThe authors have addressed most of the criticism in an adequate manner, and the\nmanuscript i snow substantially improved. There is only one issu ethat remain to\nbe clarified (see below).\nSPECIFIC COMMENTS\nD. Results. Table 2: Data sets on FEV1 and ACT for healthy controls are missing.\nThe authors need to clarify whether they have access to the requested data or not.\nIf the data exists, the authors need to include the data sets in the table and address\nit in the results.\nReply:\nThank you for comments. In terms of data sets on FEV1 and ACT for healthy controls,\nin our study, considering that the assessment of FEV1 and ACT was for individuals\nmanifesting clinical symptoms, the healthy controls that we recruited lacked clinical\nsymptoms, and therefore the requested data being inaccessible."}, {"round": 3, "reviewer_comments": {"reviewer_2": "The authors have satisfactorily addressed my questions and comments with the support of new data, disclosures of the study’s limitations, and textual revision.", "reviewer_3": "GENERAL COMMENTS:\nIn this manuscript, the authors present a range of interesting and potentially important findings on key mechanistic aspects of the involvement of IL-17C in the immunopathology of the combined clinical phenotype of allergic asthma and bronchiectasis with bacterial colonization. In essence, all the needed data sets were in place already in the first version of the manuscript. However, although the authors have improved some of the reasoning, writing and grammar in their most recent revision, there is still need for improved scientific clarity. There is also some concern about the novel data set added (see point C below).\nSPECIFIC COMMENTS:\nResponse and revisions relating to comments from\nA) Reviewer 1: Satisfactory.\nB) Reviewer 2: The authors do not responde to the criotical comments from this reviewer in a satisfactory manner. Reviewer 2 makes an important point regarding the seemingly artificial order of establishing the principal pathogenic events in the mouse model. This “order of events” is yet to be justified with clear and precise reasoning and writing, by the authors. Intuitively, it does seem less feasible that bronchiectasis including colonization with P aeruginosa would precede allergic asthma in real life. It seems more feasible that allergic asthma comes first and bronchiectasis later, as a feasible consequence of impaired immunity and chronic inflammation.\nIn their most recent revision of the manuscript, the authors fail to write about the above related matters in a clear and concise manner. For example, the statement on line 296 could just as well have been written in the opposite manner and be just as true (“Bronchiectasis is commonly reported in asthma patients”). This illustrates a more universal problem with the way the authors seem to reason and write – they fail to argue in a clear and structured manner why the chose the “experimental approach” that they did.\nGiven the above, it does become problematic that authors now have chosen to introduce a new and limited data set that fails to prove a matching impact on the specific immunopathology, using a revised model where the order of establishing principial pathogenic events is seemingly more relevant (ie. allergic asthma precedes bronchiectasis including colonization with P aeruginosa). It is hard to understand why the authors chose to include so few observations (small “n”) in their new data set, since this makes it hard to prove positive outcomes of immunopathological interest, when the alterations in cytokines and/or cells are small or variable. The small size of the novel material also makes it close to impossible to draw negative conclusions (due to risk for Type II error). Thus, by doing so, the authors weaken their own standpoint.\nHowever, and importantly, given the current limitations of the scientific understanding of the clinical phenotype with both disorders (named \"BA\" in the manuscript), addressing the above is mainly a question of improving the way the authors present and discuss their evidence, although doubling the observations in their novel data set would clearly help in the current situation.\nC) Reviewer 3: The response to this reviewer is acceptable but not fully satisfactory. The lack of basic clinical characterization of lung functon(spirometry) and symptoms (ACT) in the group of healthy subjects gives a poor impression in terms of clinical characterization, although it is understood that it is too late now to do anything about it now. Evidently, the authors cannot guarantee that their controls are truly healthy, although it seems feasible that this is the case. This reviewer thinks that this is acceptable, given that the problem is addressed as a “limitation” in the discussion."}, "author_response": "Point-by-point responses\nREVIEWER COMMENTS\nReviewer #2 (Remarks to the Author):\nThe authors have satisfactorily addressed my questions and comments with the\nsupport of new data, disclosures of the study’s limitations, and textual revision.\nReply:\nThank you for your insightful remarks on our manuscript. Meanwhile, we were very\ngrateful that our work has addressed your concerns and met the expectations.\nReviewer #3(Remarks to the Author)\nGENERAL COMMENTS:\nIn this manuscript, the authors present a range of interesting and potentially\nimportant findings on key mechanistic aspects of the involvement of IL-17C in the\nimmunopathology of the combined clinical phenotype of allergic asthma and\nbronchiectasis with bacterial colonization. In essence, all the needed data sets were\nin place already in the first version of the manuscript. However, although the\nauthors have improved some of the reasoning, writing and grammar in their most\nrecent revision, there is still need for improved scientific clarity. There is also some\nconcern about the novel data set added (see point C below).\nSPECIFIC COMMENTS:\nResponse and revisions relating to comments from\nA) Reviewer 1: Satisfactory.\nB) Reviewer 2: The authors do not respond to the critical comments from this\nreviewer in a satisfactory manner. Reviewer 2 makes an important point regarding\nthe seemingly artificial order of establishing the principal pathogenic events in the\nmouse model. This “order of events” is yet to be justified with clear and precise\nreasoning and writing, by the authors. Intuitively, it does seem less feasible that\nbronchiectasis including colonization with P aeruginosa would precede allergic\nasthma in real life. It seems more feasible that allergic asthma comes first and\nbronchiectasis later, as a feasible consequence of impaired immunity and chronic\ninflammation.\nIn their most recent revision of the manuscript, the authors fail to write about the\nabove related matters in a clear and concise manner. For example, the statement\non line 296 could just as well have been written in the opposite manner and be just\nas true (“Bronchiectasis is commonly reported in asthma patients”). This\nillustrates a more universal problem with the way the authors seem to reason and\nwrite – they fail to argue in a clear and structured manner why they chose the\n“experimental approach” that they did.\nGiven the above, it does become problematic that authors now have chosen to\nintroduce a new and limited data set that fails to prove a matching impact on the\nspecific immunopathology, using a revised model where the order of establishing\nprincipial pathogenic events is seemingly more relevant (ie. allergic asthma\nprecedes bronchiectasis including colonization with P aeruginosa). It is hard to\nunderstand why the authors chose to include so few observations (small “n”) in\ntheir new data set, since this makes it hard to prove positive outcomes of\nimmunopathological interest, when the alterations in cytokines and/or cells are\nsmall or variable. The small size of the novel material also makes it close to\nimpossible to draw negative conclusions (due to risk for Type II error). Thus, by\ndoing so, the authors weaken their own standpoint.\nHowever, and importantly, given the current limitations of the scientific\nunderstanding of the clinical phenotype with both disorders (named \"BA\" in the\nmanuscript), addressing the above is mainly a question of improving the way the\nauthors present and discuss their evidence, although doubling the observations in\ntheir novel data set would clearly help in the current situation.\nReply:\nThank you for your valuable comments. Previous work from Mao et al. and Chen et al.\n(summarized in Mao et al, Eur Respir J 2016;Chen et al, J Thorac Dis 2016) indicated\nthat asthma can be reported as a comorbidity of bronchiectasis. Meanwhile, previous\nstudies from Ma et al. and Coman et al. (Ma et al, Arch Bronconeumol 2024; Coman et\nal, Ann Allergy Asthma Immunol 2018) showed that bronchiectasis has been reported\nin 25-80% of patients with severe asthma.\nAs suggested by the reviewer, we have added more details of the rationale behind\nexperimental design on the order of events that P. aeruginosa chronic lower respiratory\ntract infection precedes allergic asthma in the introduction, results, discussion and\nmethods section of the revised manuscript as follows: In clinical practice,\nbronchiectasis and asthma frequently coexist, however it is uncertain whether there is\na cause-effect relationship between these two disorders. The overlap between\nbronchiectasis and asthma can be viewed as asthma-bronchiectasis overlap (ABO), and\nbronchiectasis-asthma overlap (BAO) with asthma accounting for 3-8% of\nbronchiectasis cases, which are both clinical issues. Despite the fact that asthma-\nbronchiectasis overlap might be more prevalent globally, at this time, in the present\nstudy, we focused on the endotype of bronchiectasis-asthma overlap among the patients\nwith primary diagnosis of bronchiectasis and coexisting asthma, and identifying the\nunderlying mechanisms is therefore of key importance in precision medicine. Moreover,\nmanaging patients with bronchiectasis-asthma overlap poses challenges, particularly\nwhen it comes to the use of corticosteroids. Literature on bronchiectasis-asthma overlap\nis still scarce, and the endotype of bronchiectasis-asthma overlap remains to be fully\ndefined, which has become a barrier to treatment decision-making. Furthermore, the\nlack of mature animal model mimicking bronchiectasis-asthma overlap has limited the\navailability of basic research data.\nHere the management of bronchiectasis-asthma overlap has become an important\nclinical issue to be addressed in our study. Considering that P. aeruginosa chronic\ninfection model has been widely used in a repertoire of research on bronchiectasis,\nconsistent with the patients with primary diagnosis of bronchiectasis and coexisting\nasthma, we report a model of P. aeruginosa chronic lower respiratory tract infection\nfollowed by ovalbumin-induced experimental asthma mimicking bronchiectasis-\nasthma overlap to investigate the underlying mechanisms involved in the endotype of\nbronchiectasis-asthma overlap.\nIndeed, asthma-bronchiectasis overlap is also a pivotal and interesting clinical issue. To\nexplore potential role of IL-17C in the endotype of asthma-bronchiectasis overlap, we\nconstructed an alternative model of ovalbumin-induced experimental asthma followed\nby P. aeruginosa infection and conducted repeated experiments (Fig. a). As depicted\nbelow, consistent with our previous animal experiment data, the preliminary results\nindicated that P. aeruginosa infection didn’t alter the levels of Il17c, Il17a mRNA and\nIL-17A together with the cell counts of AM, neutrophils and ILC3. Airway\ninflammation, eosinophils, ILC2 together with the levels of IgE, IL-5, IL-13 and total\ncell numbers were elevated from OVA/PA-exposed mice compared with their control\nmice, whereas comparable between OVA/PA-exposed mice and OVA/PBS-exposed\nmice (Fig. b-o). Furthermore, ablation of Il17re didn’t affect eosinophilic inflammation\nand ILC3 counts in mice exposed to OVA/PA or OVA (Fig. d-p). The interesting\nscientific issue remains to be further investigated and accordingly the relevant data are\nstill unavailable in the revised manuscript.\nFigure\na Schematic diagram of OVA treatment by i.p. delivery and airway challenge followed by i.t.\ninstillation with P. aeruginosa-laden agar beads at Day 9 in a model and BALF and lung tissues were\ncollected at Day 14. b, c mRNA levels of Il17c and Il17a in lungs from mice exposed to OVA/PBS\n(n = 8), OVA/PA (n = 8) and their control (n = 8) were determined. d-f Semiquantified inflammation\nof lungs and representative images of H&E-stained lung sections from wild-type (WT; PBS, n = 8;\nOVA/PBS, n = 8; OVA/PA, n = 8) and Il17re-/- mice (PBS, n = 8; OVA/PBS, n = 8; OVA/PA, n = 8)\nin the model (e Scale bars, 500 μm; f Scale bars, 50 μm). g-j Protein levels of IgE, IL-5, IL-13, and\nIL-17A in serum or BALF from WT (PBS, n = 8; OVA/PBS, n = 8; OVA/PA, n = 8) and Il17re-/-\nmice (PBS, n = 8; OVA/PBS, n = 8; OVA/PA, n = 8) were assessed. k-p Quantification of total cells,\nAM, eosinophils and neutrophils in BALF together with total numbers of ILC2s, and ILC3s in lungs\nfrom WT (PBS, n = 8; OVA/PBS, n = 8; OVA/PA, n = 8) and Il17re-/- mice (PBS, n = 8; OVA/PBS,\nn = 8; OVA/PA, n = 8) as determined by flow analysis. Data are shown as mean ± SEM. Significance\nwas calculated by one-way ANOVA (b-d,g-p).\nC) Reviewer 3: The response to this reviewer is acceptable but not fully satisfactory.\nThe lack of basic clinical characterization of lung functon(spirometry) and\nsymptoms (ACT) in the group of healthy subjects gives a poor impression in terms\nof clinical characterization, although it is understood that it is too late now to do\nanything about it now. Evidently, the authors cannot guarantee that their controls\nare truly healthy, although it seems feasible that this is the case. This reviewer\nthinks that this is acceptable, given that the problem is addressed as a “limitation”\nin the discussion.\nReply:\nWe agree with the reviewer’s comment and have addressed the missing values of\nspirometry and ACT score among the healthy controls as the second limitation in the\nrevised manuscript.\nPoint-by-point responses\nREVIEWER COMMENTS\nComments from reviewer #3 were mediated by in house editors and considered to\nbe addressed.\nReply:\nThank you for your insightful remarks on our manuscript. Meanwhile, we were very\ngrateful that our work has addressed your concerns and met the expectations with the\nsubstantial help from house editors."}]

Decision letter