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Anti-IL-5 treatment, but not neutrophil interference, attenuates inflammation in a mixed granulocytic asthma mouse model, elicited by air pollution

Respiratory Research volume 26, Article number: 43 (2025) Cite this article

Abstract

Introduction

Diesel exhaust particles (DEP) have been proven to aggravate asthma pathogenesis. We previously demonstrated that concurrent exposure to house dust mite (HDM) and DEP in mice increases both eosinophils and neutrophils in bronchoalveolar lavage fluid (BALF) and also results in higher levels of neutrophil-recruiting chemokines and neutrophil extracellular trap (NET) formation compared to sole HDM, sole DEP or saline exposure. We aimed to evaluate whether treatment with anti-IL-5 can alleviate the asthmatic features in this mixed granulocytic asthma model. Moreover, we aimed to unravel whether neutrophils modulate the DEP-aggravated eosinophilic airway inflammation.

Material and methods

Female C57BL6/J mice were intranasally exposed to saline or HDM and DEP for 3 weeks (subacute model). Interference with eosinophils was performed by intraperitoneal administration of anti-IL-5 (TRFK5), which neutralizes IL-5. Interference with neutrophils and neutrophil elastase was performed by intraperitoneal anti-Ly6G and sivelestat administration, respectively. Outcome parameters included eosinophils subsets (homeostatic EOS and inflammatory EOS), proinflammatory cytokines, goblet cell hyperplasia and airway hyperresponsiveness.

Results

The administration of anti-IL-5 significantly decreased eosinophilic responses, affecting both inflammatory and homeostatic eosinophil subsets, upon subacute HDM + DEP exposure while BAL neutrophils, NET formation and other asthma features remained present. Neutrophils were significantly reduced after anti-Ly6G administration in BALF, lung and blood without affecting the eosinophilic inflammation upon HDM + DEP exposure. Sivelestat treatment tended to decrease BALF inflammation, including eosinophils, upon HDM + DEP exposure, but did not affect lung inflammation.

Conclusion

Inhibition of IL-5 signalling, but not neutrophil interventions, significantly attenuates eosinophilic inflammation in a mouse model of mixed granulocytic asthma, elicited by air pollution exposure.

Key messages

  • Anti-IL-5 reduces homeostatic and inflammatory eosinophils in mice exposed to allergen and pollution

  • Anti-IL-5 does not affect neutrophil extracellular trap formation, goblet cells and airway hyperresponsiveness

  • No impact of neutrophil interventions on eosinophilic inflammation in allergen and pollution-exposed mice.

Introduction

Diesel exhaust particles (DEP) are the main component of outdoor traffic-related air pollution and can have severe effects on human respiratory health, contributing to diseases such as asthma [1]. In healthy individuals, respiratory exposure to DEP induces oxidative stress and epithelial cell damage, leading to the production of proinflammatory cytokines and chemokines (IL-6, IL-1β, CXCL8) in the lungs [2]. Moreover, allergic individuals exposed to DEP demonstrated increased type 2 cytokine (IL-4, IL-5, IL-13) production, airway eosinophilia and IgE levels [3]. Multiple studies have confirmed the influence of particulate matter -including DEP- on asthma incidence, severity and exacerbations [4].

Asthma is a heterogeneous disease, which can be classified into eosinophilic, neutrophilic, mixed granulocytic or paucigranulocytic, based on induced sputum counts [5]. Eosinophilic asthma is often defined by the presence of ≥ 3% sputum eosinophils, whereas the threshold for defining neutrophilic inflammation in asthma varies between ≥ 40% and ≥ 76% sputum neutrophils. In mixed granulocytic asthma, both cell counts % are above these thresholds [6]. Allergic eosinophilic asthma is the best known phenotype [5]. Allergic asthma is characterized by type 2 immune responses including eosinophilia and allergen specific IgE production that drive airway hyperresponsiveness (AHR) and remodelling leading to asthma symptoms. Inflammatory cells that participate in allergic asthma pathogenesis include dendritic cells (DCs), T helper (Th) cells, eosinophils and mast cells [5, 7]. Recently, two eosinophil subsets were identified with distinct CD101 expression, localization and transcriptional signatures. CD101− tissue-resident or homeostatic eosinophils (hEOS) were predominantly located in the lung vascular niche with mostly homeostatic functions whereas CD101+ inflammatory eosinophils (iEOS) were predominantly present in bronchoalveolar lavage fluid and extravascular lung during inflammation [8]. These eosinophil subsets are not yet examined in a pollutant-aggravated asthma mouse model. Moreover, little is known about the role of neutrophils in asthma pathophysiology. Neutrophil numbers are particularly increased in sputum, bronchoalveolar lavage fluid (BALF) and bronchial biopsies from severe asthma patients who remain symptomatic despite using inhaled corticosteroids [9]. These patients also have higher levels of neutrophil-recruiting chemokines (CXCL1, CXCL2 and CXCL8) in their BALF [10]. Neutrophilic inflammation in asthma is thought to be driven by type 3 immunity (IL17A production from Th17 cells and type 3 innate lymphoid cells) [11], however, multiple studies also suggest that neutrophils contribute to allergic asthma, dominated by type 2 immunity, as well. For example, prophylactically impairing neutrophil recruitment decreases the type 2 immune responses in a pollen-induced allergic asthma mouse model [12]. Moreover, neutrophils release neutrophil extracellular traps (NETs), web-like structures containing chromatin, citrullinated histones and neutrophil elastase (NE). Inhibition of NET formation in a murine rhinovirus-asthma exacerbation model resulted in decreased type 2 immunopathology [13]. Also in humans, increased extracellular DNA production was observed in sputum from severe asthma patients and was positively linked with sputum neutrophils, asthma disease severity and exacerbation risk [14]. The presence of mixed eosinophilic/neutrophilic (granulocytic) inflammation is considered a biomarker of the most severe forms of asthma [15,16,17].

DEP have been proven to worsen asthma pathogenesis [18] and we already showed that concomitant exposure to house dust mite (HDM) and DEP increased type 2 immune responses including BAL eosinophils, type 2 cytokine production, goblet cell metaplasia and AHR compared to sole exposures to HDM or DEP[19]. Moreover, compared to these control groups, combined HDM + DEP exposure also led to higher neutrophil numbers which associated with higher production of neutrophil-attracting chemokines, neutrophil mediators and NET formation [20]. The inflammatory responses in this pollutant-aggravated allergic asthma model -that mimics human mixed granulocytic asthma- suggest a role for neutrophils in the type 2 immune response. In current manuscript, we identified eosinophil subsets in our murine pollutant-aggravated asthma model and inhibited IL-5 signalling using an IL-5 neutralizing antibody (TRFK5) to investigate the effect on inflammation, goblet cell hyperplasia and AHR. Additionally, we monitored the dynamics of the airway inflammatory response and investigated the contribution of neutrophils in the type 2 inflammation induced by HDM + DEP exposure. We focused on interventions in the HDM + DEP group and not the sole DEP or HDM exposure groups since these have a very mild and variable inflammation (due to the low doses used in the model, [20]), which would compromise interpretation of the intervention experiments.

Material and methods

Mice experiments

Female mice (C57BL6/J, 6–8 weeks old) were purchased from Jackson Laboratory. Female mice were chosen because they fight less and are easier to manipulate than males, however, our model works in both female and male mice (unpublished data). All in vivo experiments were approved by the Animal Ethical Committee of the Faculty of Medicine and Health Science, Ghent University (ECD19-40, ECD19-22, ECD23-50).

For the pollutant-aggravated allergic asthma mouse model, isoflurane anesthetized mice (8/group) were intranasally exposed to saline or the combination of 1 µg HDM (Dermatophagoides pteronyssinus, Greer Laboratories) and 25 µg DEP (SRM2975, NIST) on day 1, 8 and 15 as described previously [19, 20]. Inflammation in the mice was assessed at 48 h after the last exposure, except in the time course experiment, where 3 time points (24, 48 or 72 h after the last intranasal exposure) were used to characterize the dynamics of the inflammaton. Mice were sacrificed by a lethal dose of pentobarbital via intraperitoneal injection. A scheme of each experimental set-up is included in the Figures.

For inhibition of IL-5 in the HDM + DEP model, mice received 100 µg isotype control (clone GL113) or anti-IL-5 (clone TRFK5 [21], a rat-anti mouse antibody that binds IL-5 and has a neutralizing effect, similar to mepolizumab or reslizumab) in PBS intraperitoneally on day 8 and 15 one hour before intranasal instillation with saline or HDM + DEP, similar to publication [8]. For therapeutic inhibition of neutrophil elastase (NE), mice were intraperitoneally treated with PBS or 100 mg/kg sivelestat (MedChem Express), which is a highly specific and potent inhibitor of NE, on days 14, 15 and 16. Sivelestat exerts its effects by binding to the active site of the enzyme [22]. For neutrophil depletion experiments, mice received 100 µg isotype control (rat IgG2a, BioXCell) or anti-Ly6G antibody (clone 1A8, BioXCell) intraperitoneally the day before and the day after intranasal instillation, both in a prophylactic (on days 0, 2, 7, 9, 14 and 16) and a therapeutic (on days 14 and 16) way. Anti-Ly6G mediates neutrophil killing through Fc-dependent macrophage opsonization [23]. Treatment schemes and concentrations were based on publications [13, 24] and are included in the Figures.

Bronchoalveolar lavage

A cannula was inserted in the trachea and BALF was collected by instillation of 3 × 300 µl HBSS supplemented with 1% BSA (for cytokine and chemokine detection using ELISA) and 6 × 500 µl HBSS supplemented with EDTA. The cells from the lavage fractions were pooled and the total amount of BAL cells was counted using a Bürker chamber, as described previously [25].

Lungs

After the BALF collection, the pulmonary circulation was rinsed with saline + EDTA to eliminate the intravascular cells. The inferior lobe of the right lung was digested for flow cytometric analysis, as described previously [19, 25]. The middle lobe of the right lung was stored for RNA extraction and RT-qPCR. The left lung was used for histological analysis.

Flow cytometry

Cell suspensions were preincubated with anti-CD16/CD32 (2.4G2) to minimize non-specific binding, followed by labelling with fluorochrome-conjugated antibodies targeting CD45 (30-F11), CD11c (N418), MHCII (M5/114.15.2), SiglecF (E50-2440), CD11b (M1/70), Ly6C (AL-21), Ly6G (1A8), GR1 (RB6-8C5), CD62L (MEL-14) and CD101 (Moushi101) (Supplemental Figure S1 and S2 for gating strategies). Data acquisition was performed on a LSR Fortessa cytometer (running DiVa software). Analysis was conducted using FlowJo software based on FMO controls.

Histology

After fixation (4% paraformaldehyde) and embedding (paraffin) of the left lung, tissue sections were stained with congo red or periodic acid-Schiff (PAS) to visualise eosinophils and mucus-producing goblet cells, respectively. To identify neutrophils, de-paraffinized lung tissue sections were incubated with anti-myeloperoxidase (R&D Systems, AF3667) overnight. Slides were then incubated with a biotinylated antibody and streptavidin horse radish peroxidase (HRP) and colored using diaminobenzidine (DAB + , All Dako). Quantitative measurements were performed on Axiovision software and airways < 800 µm or > 2000 µm were excluded from the analysis.

Protein measurements

Commercially available ELISA kits from R&D Systems were used for CXCL1 and NE measurement in BALF. Quant-iT PicoGreen dsDNA assay kit (Thermofisher, Invitrogen) was used to determine dsDNA levels in BALF.

Quantitative RT-PCR

RNA from the middle lobe of the right lung was isolated using the miRNeasy mini kit (Qiagen). mRNA expression relative to the housekeeping genes GAPDH and HPRT, was analysed using Taqman gene expression assays (Thermofisher Scientific) with a Lightcycler 96 system (Roche).

Airway hyperresponsiveness (AHR)

In addition to the experiments to assess the inflammatory responses, we performed independent lung function experiments. Therefore, the forced oscillation technique (Flexivent System, Canada) was applied to determine AHR towards carbachol (dose range 0–640 µg/kg), as described previously [19]. Intravenously injecting pancuronium bromide (dose 1 mg/kg) induced neuromuscular blockade. Resistance of the whole respiratory system was measured.

Statistical analysis

SPSS (version 27.0 IBM) and Graphpad Prism 6.0 software was used to perform statistical analysis. Groups were compared using nonparametric tests (Kruskall-Wallis and Mann–Whitney U) and p values < 0.05 were regarded as significant.

Results

Identification of eosinophil subsets upon subacute HDM+DEP exposure

We first investigated the presence of the eosinophil subsets (CD101- hEOS and CD101 + iEOS [8]) in different compartments -i.e. blood, lung tissue and BALF- of our pollutant-aggravated asthma model (Fig. 1A and Supplemental Figure S1 for gating strategy). In blood, the hEOS subset was predominant and increased upon HDM + DEP exposure compared to saline control, while barely any iEOS could be detected (Fig. 1B, C). In lung tissue, both eosinophil subsets were significantly increased upon combined HDM + DEP exposure compared to saline exposure (Fig. 1D, E). In BALF, the iEOS subset was predominantly present and significantly increased upon HDM + DEP exposure compared to saline control (data not shown).

Fig. 1
figure 1

Identification of eosinophil subsets upon subacute HDM + DEP exposure. Female C57BL6/J mice were intranasally exposed to saline or HDM + DEP for 3 weeks. A schematic representation of the experiment. B gating strategy for eosinophil subsets starting from CD45 + CD11c- CD11b + population in blood. C percentage homeostatic eosinophils (hEOS) and inflammatory eosinophils (iEOS) of CD45 + cells in blood. D gating strategy for eosinophil subsets starting from CD45 + CD11c- CD11b + population in lung single cell suspensions. E percentage hEOS and iEOS of CD45 + cells in lung single cell suspensions. Full gating strategies are shown in Supplemental Figure S1 and S2. Results are expressed as mean ± SEM. n = 8 mice/group. *p < 0.05 **p < 0.01

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Inhibition of IL-5 reduces eosinophilic inflammation upon subacute HDM+DEP exposure

Since mepolizumab (anti-IL-5) is an FDA approved biological to treat severe eosinophilic asthma, we evaluated whether anti-IL-5 treatment would be effective in the mixed granulocytic phenotype of our pollutant-aggravated asthma model. Treatment with anti-IL-5 once on day 15 led to a significant reduction of the hEOS in blood (Supplemental Fig. 3A, B). However, the inflammation in BALF and lung tissue upon HDM + DEP exposure did not differ between anti-IL-5 and isotype control-treated mice (Supplemental Fig. 3C–G).

Since a single anti-IL-5 treatment may be insufficient to eliminate the extreme eosinophilic inflammation in our HDM + DEP model, we next treated mice with anti-IL-5 before the second and last intranasal exposure (Fig. 2A). Blood eosinophils significantly decreased in anti-IL-5-treated mice, whereas blood neutrophils were unaffected (Fig. 2B, C). In BALF, the total cell number increased upon HDM + DEP exposure and tended to decrease after anti-IL-5 (Fig. 2D). The total number of BAL eosinophils significantly decreased (Fig. 2E) while BAL neutrophils remained unaffected (Fig. 2F) upon IL-5 inhibition in HDM + DEP exposed mice. Notably, both eosinophil subsets significantly decreased in lung tissue upon anti-IL-5 treatment (Fig. 2G, H). The SiglecF + neutrophil subset (in BALF and lung) and NET components in BALF increased upon HDM + DEP exposure, but were not significantly influenced by anti-IL-5 therapy (Fig. 2I–L).

Fig. 2
figure 2

IL-5 inhibition reduces eosinophilic, but not neutrophilic, inflammation in BALF after subacute HDM + DEP exposure. Female C57BL6/J mice were intranasally exposed to saline or HDM + DEP for 3 weeks. Mice were intraperitoneally injected with anti-IL-5 or isotype control 1 h before the second and last HDM + DEP exposure. A schematic representation of the experiment. B percentage eosinophils of CD45 + cells in blood. C percentage neutrophils of CD45 + cells in blood. D total cell number in BALF. E, eosinophil numbers in BALF (CD45 + CD11c- CD11b + SiglecF + Ly6G-). F neutrophil numbers in BALF (CD45 + CD11c- CD11b + SiglecFdim, Ly6C + and Ly6G +). G percentage hEOS (CD45 + CD11c- CD11b + SiglecF + CD101-) of CD45 + cells in lung tissue. H percentage iEOS (CD45 + CD11c- CD11b + SiglecF + CD101 +) of CD45 + cells in lung tissue. I numbers of SiglecF + neutrophils (CD45 + CD11c- CD11b + SiglecF + Ly6G + Ly6C +) in BALF. J percentage SiglecF + neutrophils (CD45 + CD11c- CD11b + SiglecF + Ly6G + Ly6C +) in lung tissue. K concentration of double-stranded DNA (ng/ml) in BAL supernatant. L protein level of neutrophil elastase (pg/ml) in BALF supernatant determined by ELISA. Results are expressed as mean ± SEM. n = 7–8 mice/group. *p < 0.05 **p < 0.01

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On tissue sections, the HDM + DEP induced peribronchial eosinophil and neutrophil numbers were both significantly decreased after anti-IL-5 treatment (Fig. 3A-B). Notably, the HDM + DEP induced muc5ac mRNA expression levels further increased by anti-IL-5 therapy, however, goblet cell numbers were similar between anti-IL-5 treated and isotype control mice (Fig. 3C, D). AHR was higher in HDM + DEP exposed mice than in saline exposed mice, both in isotype control and anti-IL-5 groups. Interestingly, anti-IL-5 treatment only tended to reduce AHR upon HDM + DEP exposure (Fig. 3E). Anti-IL-5 treatment of HDM + DEP exposed mice significantly increased IL-33 mRNA expression compared to isotype control, while mRNA expression of CCL11 (eotaxin), IL-13 and CXCL1, CXCL2 and CXCL5 remained unaffected (Supplemental Fig. 4). In summary, anti-IL-5 treatment attenuates eosinophilic responses and peribronchial neutrophilic inflammation, but not NET formation, BAL neutrophilia, mucus overproduction and AHR in the HDM + DEP model.

Fig. 3
figure 3

IL-5 inhibition reduces eosinophilic and neutrophilic inflammation in lung tissue upon subacute HDM + DEP exposure. A quantification of congo-stained peribronchial eosinophils in lung tissue and representative photomicrographs. B quantification of MPO-stained peribronchial neutrophils in lung tissue and representative photomicrographs. C relative muc5ac mRNA expression in lung tissue determined by RT-qPCR. D quantification of PAS-positive goblet cells in lung tissue. E airway hyperresponsiveness measured in response to increasing doses of carbachol. Results are expressed as mean ± SEM. n = 7–8 mice/group. *p < 0.05 **p < 0.01

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Fig. 4
figure 4

Neutrophils accumulate rapidly after subacute HDM + DEP exposure. Female C57BL6/J mice were intranasally exposed to HDM + DEP for 3 weeks. Mice were sacrificed either 24, 48 or 72 h after last HDM + DEP exposure. A schematic overview of the experiment. B total cell number in BALF. C, neutrophil numbers in BALF (CD45 + CD11c- CD11b + SiglecFdim, Ly6C + and Ly6G +). D eosinophil numbers in BALF (CD45 + CD11c- CD11b + SiglecF + Ly6G-). E quantification of peribronchial Congo-Red stained eosinophils in lung tissue. F-G quantification of peribronchial MPO-stained neutrophils in lung tissue with representative photomicrographs. H-I percentage hEOS (H) and iEOS (I) of CD45 + cells in lung tissue. J percentage hEOS of CD45 + cells in blood. Results are expressed as mean ± SEM. n = 8–10 mice/group. *p < 0.05 **p < 0.01

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Neutrophils and neutrophil extracellular traps accumulate rapidly in the bronchoalveolar lavage fluid after subacute HDM+DEP exposure

To determine the dynamics of the accumulation of immune cells in the airways in our HDM + DEP model, we analysed BALF cell composition at multiple time points (24, 48 and 72 h) after the last intranasal HDM + DEP exposure (Fig. 4A). Total BAL cell numbers were high (above 106) and did not significantly differ between the time points (Fig. 4B). Neutrophil numbers in BALF were highest at 24 h after last HDM + DEP exposure and significantly decreased at later time points, while BAL eosinophils tended to increase from 24 to 48 h (Fig. 4C-D). In lung, peribronchial neutrophils were highest 24 h after last HDM + DEP exposure and decreased thereafter (Fig. 4E, G). Interestingly, peribronchial eosinophils in lung show the same trend as neutrophils (Fig. 4F). Also the percentages of neutrophils in lung single cell suspensions and in blood were highest at 24 h after the last exposure and decrease over time (Supplemental Fig. 5, B). Evaluation of the eosinophil subsets in lung tissue at different time points demonstrated that the percentage hEOS decreases over time while the percentage iEOS increases (Fig. 4H–I). In blood, the hEOS subset is predominant and increases significantly 48 h after last HDM + DEP exposure (Fig. 4J).

Fig. 5
figure 5

Neutrophil-attracting chemokines and NET formation after subacute HDM + DEP exposure. Female C57BL6/J mice were intranasally exposed to HDM + DEP for 3 weeks. Mice were sacrificed either 24, 48 or 72 h after last HDM + DEP exposure. A-C relative mRNA expression of neutrophil-recruiting chemokines CXCL1 (A), CXCL2 (B) and CXCL5 (C) in lung tissue. D, CXCL1 protein levels (pg/ml) in BAL supernatant determined by ELISA. E concentration of double-stranded DNA (ng/ml) in BAL supernatant. F levels of neutrophil elastase (pg/ml) in BAL supernatant determined via ELISA. G-I relative mRNA expression of the type 2 cytokines IL-5 (G) and IL-13 (H) and the eosinophil chemoattractant CCL11 (I) in lung tissue. Results are expressed as mean ± SEM. n = 8–10 mice/group. *p < 0.05 **p < 0.01

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We further examined the expression of CXCL1, CXCL2 and CXCL5 in the dynamics of this inflammatory response. The lung mRNA expression of these chemokines was highest at 24 h after last HDM + DEP exposure and decreased thereafter (Fig. 5A–C). The protein level of CXCL1 in BALF showed the same trend (Fig. 5D). Also both NET components (NE and dsDNA) in BALF were present at high concentrations at 24 h and decreased over time (Fig. 5E, F). These data suggest that neutrophils may participate in the pathogenesis of pollutant-aggravated allergic asthma dominated by a type 2 eosinophilic response. The mRNA expression of the type 2 cytokines IL-5 and IL-13 and the eosinophil chemoattractant CCL11 were also mostly expressed at 24 h after the last HDM + DEP exposure and decreased thereafter (Fig. 5G–I).

Therapeutic inhibition of neutrophil elastase tends to decrease inflammation after subacute HDM+DEP exposure

NE is a serine protease expressed in neutrophils and present in high concentrations on NETs. Therefore, we investigated the role of NE in our subacute HDM + DEP model using sivelestat, which is a highly specific and potent inhibitor of NE. Sivelestat treatment (Fig. 6A) significantly decreased total BAL cells in mice co-exposed to HDM and DEP (Fig. 6B). The elevated BAL eosinophil and neutrophil numbers observed upon HDM + DEP exposure tended to decrease after therapeutic sivelestat administration (Fig. 6C, D). Moreover, BAL DCs significantly decreased after sivelestat injection in HDM + DEP exposed mice (Fig. 6E). Notably, there was no major impact of sivelestat on dsDNA and NE levels (Fig. 6F, G). In lung tissue of HDM + DEP exposed mice, no differences in peribronchial eosinophils and neutrophils, goblet cells and muc5ac, IL-5, IL-13 and IL-33 mRNA expression were seen after neutrophil elastase inhibition (Fig. 6H–N). Notably, therapeutic inhibition of NE in mice exposed to saline led to a small, but significant increase in BAL neutrophils and dsDNA (Fig. 6C, F). Sivelestat treatment did not affect the eosinophil subsets in lung tissue of HDM + DEP exposed mice (Supplemental Fig. 6A-B). Prophylactic inhibition of NE (sivelestat from day 1) and neutrophil depletion by anti-Ly6G (prophylactic or therapeutic setup) did not induce differences in inflammation upon subacute HDM + DEP exposure (Supplemental Fig. 7, 8).

Fig. 6
figure 6

Therapeutic inhibition of neutrophil elastase tends to decrease inflammation upon subacute HDM + DEP exposure. Female C57BL6/J mice were intranasally exposed to saline or HDM + DEP for 3 weeks. Sivelestat or PBS was therapeutically administered by intraperitoneal injection. A schematic overview of the experiment. B total cell number in BALF. C, neutrophil numbers in BALF (CD45 + CD11c- CD11b + SiglecFdim, Ly6C + and Ly6G +). D eosinophil numbers in BALF (CD45 + CD11c- CD11b + SiglecF + Ly6G-). E numbers of dendritic cells in BALF (CD45 + CD11c + MHCII +). F concentration of double-stranded DNA (ng/ml) in BAL supernatant. G neutrophil elastase concentration (pg/ml) in BAL supernatant. H quantification of congo-stained peribronchial eosinophils in lung tissue. I quantification of MPO-stained neutrophils in lung tissue. J, quantification of PAS-stained goblet cells in lung tissue. K-N relative mRNA expression of muc5ac (K), IL-33 (L), IL-5 (M) and IL-13 (N) in lung tissue. Results are expressed as mean ± SEM. n = 6–8 mice/group. *p < 0.05 **p < 0.01

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Fig. 7
figure 7

Anti-IL-5 treatment, but not neutrophil interference, attenuates inflammation in a pollutant-aggravated allergic asthma mouse model

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Discussion

In this study, we identified the eosinophil subsets in lung tissue of HDM + DEP exposed mice. Inhibition of IL-5 signalling in HDM + DEP exposed mice reduced both pulmonary eosinophil subsets and peribronchial neutrophilic inflammation, but not NET formation and BAL neutrophilia, mucus production and AHR. We demonstrated that neutrophils accumulate rapidly in BALF upon combined HDM + DEP exposure concomitant with higher expression of neutrophil-recruiting chemokines and NET formation. Therapeutic inhibition of NE (sivelestat) only tended to decrease inflammation in the pollutant-aggravated allergic asthma model. Moreover, neutrophil depletion did not reduce eosinophilic inflammation upon HDM + DEP exposure. None of the neutrophil-interfering treatments affected the eosinophil subsets (Supplemental Fig. 6).

Two subsets of eosinophils -hEOS with homeostatic functions and inflammatory iEOS- have previously been identified by flow cytometry in the mouse lung after allergen exposure [8] and in human samples including blood and induced sputum of asthma patients [26, 27]. Mesnil et al. provided evidence that lung hEOS and iEOS represent distinct terminally differentiated eosinophils [8], while another study supported the concept that hEOS can become iEOS during inflammatory processes [28]. In our experiments, both hEOS and iEOS were present in lung tissue of HDM + DEP exposed mice and increased significantly upon combined HDM + DEP exposure. Notably, after the last HDM + DEP exposure, the percentage hEOS in lung tissue declined over time while the percentage iEOS increased. Moreover, in blood, nearly all eosinophils were hEOS and their numbers also increased with time after the last HDM + DEP exposure. Together, these data suggest that hEOS may transform into iEOS under inflammatory conditions. Anti-IL-5 treatment clearly affected both eosinophil subsets in lung tissue of HDM + DEP exposed mice which is not in accordance with Mesnil et al., who reported that hEOS are not dependent on IL-5 for their presence in lungs and blood after HDM exposure [8]. Differences in experimental design may explain these contrasting data. Our findings however may be of clinical importance since it has been suggested that benralizumab (anti-IL-5R) could induce more adverse events (i.e. parasitic infections) because both eosinophils subsets are depleted while mepolizumab (anti-IL-5) would only affect the iEOS subset [29, 30]. In blood of severe eosinophilic asthma patients, mepolizumab treatment induced a marked reduction of iEOS while the proportion of hEOS increased. Moreover, they also assumed that hEOS and iEOS could be the same cells in different activation states, depending on the cytokine release in the environment [31].

Interestingly, anti-IL-5 therapy significantly decreased peribronchial neutrophils in HDM + DEP exposed mice, without affecting BAL neutrophils and NET formation. The expression of CD125 (IL-5 receptor alpha) was thought to be restricted to eosinophils and basophils in mice, but was also recently found on murine lung neutrophils [8, 32, 33] suggesting that neutrophils may also be influenced by IL-5. Moreover, a recent study has shown that CD125 is also widely expressed on human blood and airway neutrophils [34], suggesting that IL-5 and IL-5R targeting may also affect neutrophilic inflammation. Although the specificity of neutrophil staining with some anti-CD125 clones is debated [35], it is noteworthy that treatment with anti-IL-5R (MEDI-563) in mild atopic asthma patients led to a slight decrease in blood neutrophil number [36]. However, results are controversial since another study observed concurrent increased neutrophil sputum counts in severe asthma patients treated with benralizumab [37].

Notably, in our model also type 2-associated asthma features including mucus production, AHR, chemokine and cytokine production were not significantly affected by anti-IL-5 treatment upon HDM + DEP exposure. These results are in accordance with a recent study in which IL-4/IL-13 blockade (anti-IL-4Rα) and IL-5 inhibition were compared in a HDM-induced asthma murine model. IL-4Rα blockade improved lung function decreased chemokine production and prevented mucus production. IL-5 neutralization however did not significantly impact these changes meaning that reducing eosinophil numbers alone does not influence other inflammatory features [38]. Also studies with eosinophil-deficient mice have shown that type 2 inflammation remodelling and lung function are not dependent on eosinophils [39, 40]. Many biologics targeting type 2 inflammation have been approved for the treatment of asthma leading to reduced asthma exacerbations. However, studies with anti-IL-5, mepolizumab and reslizumab, have reported inconsistent improvements in other secondary endpoints (FeNO and FEV1, measures of type 2 inflammation and lung function respectively) despite decreased eosinophil levels and exacerbation rate [41, 42].

Upon subacute HDM + DEP exposure, neutrophils were highly present 24 h after the exposure and declined thereafter. This early neutrophilic inflammation associated with increased expression of neutrophil-attracting chemokines and NET formation. These data, together with the increase in eosinophils with time, suggested that neutrophils may play a role in the development of eosinophilic responses. To investigate this, we administered anti-Ly6G antibodies to deplete the neutrophils in BAL, lung and blood (Supplemental Fig. 8). This neutrophil depletion did not induce differences in eosinophilic inflammation upon HDM + DEP exposure. Patel et al. demonstrated that neutrophil depletion in a murine HDM asthma model resulted in increased type 2 inflammation, airway remodelling and hyperresponsiveness [43], whereas neutrophil depletion in an Alternaria alternata asthma model led to a significant reduction of eosinophils in BALF [44]. Of note, HDM + DEP exposure in our model induces a very strong eosinophilic inflammation compared to other murine models, which may explain the limited effects of neutrophil depletion. Moreover, anti-Ly6G can induce rapid renewal of neutrophils in the bone marrow which have lower Ly6G membrane expression and thus less susceptible to anti-Ly6G-mediated depletion [23].

NE is a neutrophil-associated protease with a very important role in NETosis since it translocates to the nucleus and degrades histones, promoting chromatin decondensation and further NET release [45]. In an OVA-asthma mouse model, treatment with the NE inhibitor sivelestat significantly attenuated allergic airway responses including type 2 cytokine levels and eosinophilia leading to reduced AHR and goblet cell metaplasia [46]. Therefore, we aimed to target NE in our HDM + DEP asthma model. Therapeutic inhibition of NE only tended to decrease BAL inflammation. Of note, BAL neutrophil numbers were higher in the OVA asthma model compared to our HDM + DEP- model, which may explain their better response towards sivelestat. In future studies, it could therefore be of interest to test sivelestat in our previously published chronic HDM + DEP model in which neutrophils are more prominent [20]. Since sivelestat has poor pharmacokinetics, alternative delivery methods could also be considered. Nanocarrier delivery of sivelestat to neutrophils can improve biodistribution and thus its efficacy. For example, in an LPS- mouse model, free sivelestat was not effective, however, vesicles incorporating sivelestat were successfully taken up by neutrophils and prevented NET formation leading to less pulmonary inflammation [47]. Still, it should be pointed out that despite potential advantages of these strategies in the treatment of certain NET-mediated diseases, systemic inhibition of NETosis in animal models increases the susceptibility towards infections [47]. Sivelestat is clinically available in Japan and South Korea for acute lung injury (ALI), however, efforts to use sivelestat in other countries have failed since a multinational clinical trial on ALI patients was unsuccessful [48]. Other challenges in the use of NE inhibitors include the fact that NE bound to extracellular DNA in NETs is resistant to the activity of inhibitors [47]. Moreover, sivelestat functions extracellularly thus only inhibiting NE released into the extracellular space [47, 49]. These challenges could explain the partial decrease of BAL inflammation in our asthma mouse model.

A limitation of our study is the absence of the sole DEP and sole HDP exposure groups in the intervention experiments. In our previous research, we have shown that combined HDM + DEP exposure induces a strong mixed inflammatory response while the sole HDM or DEP exposures only result in a very modest and variable inflammation [20], leading to the decision to not include these additional control groups. However, we cannot exclude that the interventions could have impacted the mild inflammation observed in the sole exposure groups. Another limitation is the exclusive use of female mice since sex differences are important in asthma, with e.g. female mice being be more susceptible to develop type 2 responses compared to males [50].

Conclusions

In conclusion, our data suggest no significant role for neutrophils in eosinophilic responses in mice co-exposed to particulates and allergen (i.e. HDM) since NE inhibition and neutrophil depletion did not affect eosinophil numbers. Anti-IL-5 treatment however attenuated HDM + DEP-induced inflammation, affecting both eosinophil subsets and neutrophils in lung tissue, but not BAL neutrophilia, NET formation and other asthma features (Fig. 7).

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

AHR:

Airway hyperresponsiveness

BALF:

Bronchoalveolar lavage fluid

CXCL:

CXC-motif chemokine ligand

DC:

Dendritic cell

DEP:

Diesel exhaust particles

dsDNA:

Double-stranded DNA

HDM:

House dust mite

hEOS:

Homeostatic eosinophils

iEOS:

Inflammatory eosinophils

IL:

Interleukin

NE:

Neutrophil elastase

NET:

Neutrophil extracellular trap

OVA:

Ovalbumin

Th:

Thelper cell

LPS:

Lipopolysacharide

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Acknowledgements

We thank Greet Barbier, Indra De Borle, Anouck Goethals, Ann Neesen and Katleen De Saedeleer (Department of Respiratory Medicine, Laboratory for Translational Research in Obstructive Pulmonary Diseases, Ghent University Hospital, Ghent, Belgium) for their technical assistance. We also thank the Flowcytometry Core facility and ARTH Core from Ghent University.

Funding

The Department of Respiratory Medicine (Ghent University) was funded by Scientific Research in Flanders (FWO Vlaanderen, FWO041819N and G025123N, and FWO-EOS projects G0G2318N, G0H1222N) and a Ghent University Grant (BOF/GOA 01G00819).

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  1. Department of Respiratory Medicine, Laboratory for Translational Research in Obstructive Pulmonary Diseases, Medical Research Building (MRB) II, Ghent University Hospital, 2 Floor, Corneel Heymanslaan 10, 9000, Ghent, Belgium

    Joyceline De Volder, Annelies Bontinck, Valerie Haelterman, Guy F. Joos, Guy G. Brusselle & Tania Maes

  2. JJP Biologics, Warsaw, Poland

    Louis Boon

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Contributions

JDV: Investigation, Writing – Original Draft, Visualization AB: Investigation, Writing – Review & Editing VH: Investigation, Writing – Review & Editing LB: Writing – Review & Editing GJ: Writing – Review & Editing GB: Writing – Review & Editing TM: Supervision, Writing – Review & Editing.

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Correspondence to Tania Maes.

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All in vivo mouse experiments were approved by the Animal Ethical Committee of the Faculty of Medicine and Health Science, Ghent University (ECD19-40, ECD19-22, ECD23-50).

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TM holds a Chiesi Chair on the Role of Environmental factors in Asthma development and a GSK chair on eosinophilic airway disease. The other authors declare that they have no competing interests.

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Additional file 1. Figure S1. Flow cytometry gating strategy used to distinguish eosinophil subsets in lung tissue of HDM+DEP exposed mice. The markers used for each cell type are as follows: hEOS; iEOS. Gates were set based on FMO controls. Figure S2. Flow cytometry gating strategy used to distinguish cell populations in BAL fluid of HDM+DEP exposed mice. The markers used for each cell type are as follows: Dendritic cells; Eosinophils; Neutrophils; SiglecF+ neutrophils. Gates were set based on FMO controls. Figure S3. One Anti-IL-5 administration before the last HDM+DEP instillation did not attenuate eosinophilic inflammation in BAL and lung tissue. Female C57BL6/J mice were intranasally exposed to saline or HDM+DEP for 3 weeks. Mice were intraperitoneally injected with anti-IL-5 or isotype control 1 hour before the last HDM+DEP exposure. A, schematic representation of the experiment. B, percentage hEOS of CD45+ cells in blood. C, total cell number in BALF. D, eosinophil numbers in BALF. E, neutrophil numbers in BALF. F, percentage hEOSof CD45+ cells in lung tissue. G, percentage iEOSof CD45+ cells in lung tissue. Results are expressed as mean ± SEM. n = 7-8 mice/group. *p < 0.05 **p < 0.01. Figure S4. IL-5 inhibition does not reduce cytokine and chemokine expression in lung tissue upon HDM+DEP exposure. A-C, relative mRNA expression of type-2 associated cytokines IL-33, CCL11and IL-13in lung tissue. D-F, relative mRNA expression of neutrophil-recruiting chemokines CXCL1, CXCL2and CXCL5in lung tissue determined by RT-qPCR. Results are expressed as mean ± SEM. n = 7-8 mice/group. *p < 0.05 **p < 0.01. Figure S5. Neutrophils in blood upon subacute HDM+DEP exposure. Female C57BL6/J mice were intranasally exposed to HDM+DEP for 3 weeks. Mice were sacrificed either 24, 48 or 72 hours after last HDM+DEP exposure. A, percentage neutrophilsof CD45+ cells in lung tissue. B, percentage neutrophils of CD45+ cells in blood. Results are expressed as mean ± SEM. n = 8-10 mice/group. *p < 0.05 **p < 0.01. Figure S6: Neutrophil interference did not affect eosinophil subsets in lung tissue upon subacute HDM+DEP exposure. Female C57BL6/J mice were intranasally exposed to saline or combined HDM+DEP for 3 weeks. Inhibition of neutrophil elastase and neutrophil depletion were performed by intraperitoneal administration of sivelestatand anti-Ly6G antibodies, respectively. Percentage hEOSin lung tissueand percentage iEOSin lung tissue. Results are expressed as mean ± SEM. n = 8 mice/group. *p < 0.05 **p < 0.01. Figure S7: Prophylactic inhibition of neutrophil elastase does not improve eosinophilic inflammation after subacute HDM+DEP exposure. Female C57BL6/J mice were intranasally exposed to saline or HDM+DEP for 3 weeks. Sivelestat or PBS was prophylactically administered by intraperitoneal injection. A, schematic overview of the experiment. B, total cell number in BALF. C, neutrophil numbers in BALF. D, eosinophil numbers in BALF. E, numbers of dendritic cells in BALF. F, concentration of double-stranded DNAin BAL supernatant. G-H, protein level of neutrophil elastaseand IL-13in BALF supernatant determined by ELISA. Results are expressed as mean ± SEM. n = 8 mice/group. *p < 0.05 **p < 0.01. Figure S8: Neutrophil depletion does not improve eosinophilic inflammation upon subacute HDM+DEP exposure. Female C57BL6/J mice were intranasally exposed to HDM+DEP for 3 weeks. Mice were intraperitoneally injected with anti-Ly6G for neutrophil depletion. A, schematic overview of the experiments. B, percentage neutrophils of CD45+ cells in blood. C, percentage neutrophils of CD45+ cells in lung single cell suspensions. D, neutrophil numbers in BALF. E, total cell number in BALF. F, eosinophil numbers in BALF. G, Quantification of congo-stained peribronchial eosinophils. H, double-stranded DNA levelsin BAL supernatant. I, protein level of neutrophil elastasein BALF supernatant determined by ELISA. Results are expressed as mean ± SEM. n = 8 mice/group. *p < 0.05 **p < 0.01

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De Volder, J., Bontinck, A., Haelterman, V. et al. Anti-IL-5 treatment, but not neutrophil interference, attenuates inflammation in a mixed granulocytic asthma mouse model, elicited by air pollution. Respir Res 26, 43 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12931-024-03082-9

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