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

Anti-IL-5 treatment, but not neutrophil interference, attenuates inflammation in a mixed granulocytic asthma mouse model, elicited by air pollution | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anti-IL-5 treatment, but not neutrophil interference, attenuates inflammation in a mixed granulocytic asthma mouse model, elicited by air pollution Joyceline De Volder, Annelies Bontinck, Valerie Haelterman, Louis Boon, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4691862/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 28 Jan, 2025 Read the published version in Respiratory Research → Version 1 posted 7 You are reading this latest preprint version Abstract Introduction: Diesel exhaust particles (DEP) have been proven to aggravate asthma pathogenesis. We previously demonstrated that 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. 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 & 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. 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. Pollutant-aggravated allergic asthma eosinophils neutrophils house dust mite diesel exhaust particles Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 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 with allergic asthma being 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), Thelper (Th) cells, eosinophils and mast cells ( 5 , 6 ). 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 ( 7 ). 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 ( 8 ). These patients also have higher levels of neutrophil-recruiting chemokines (CXCL1, CXCL2 and CXCL8) in their BALF ( 9 ). Neutrophilic asthma is thought to be driven by type 3 immunity (IL17A production from Th17 cells and type 3 innate lymphoid cells) ( 10 ), 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 ( 11 ). 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 ( 12 ). 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 ( 13 ). DEP have been proven to worsen asthma pathogenesis ( 14 ) 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 ( 15 ). Moreover, combined HDM + DEP exposure also led to higher neutrophils numbers which associated with higher production of neutrophil-attracting chemokines, neutrophil mediators and NET formation ( 16 ), suggesting a role for neutrophils in pollutant-aggravated allergic asthma, mostly dominated by a type 2 immune response. In current manuscript, we identified eosinophil subsets in our murine pollutant-aggravated asthma model and inhibited IL-5 signalling 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. MATERIAL AND METHODS Mice experiments Female mice (C57BL6/J, 6–8 weeks old) were purchased from Jackson Laboratory. 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 ( 15 , 16 ). In most experiments, mice were sacrificed by a lethal dose of pentobarbital via intraperitoneal injection two days after the last exposure. To characterize the dynamics of the inflammation, mice were killed either 24, 48 or 72 hours after the last intranasal HDM + DEP exposure. 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) in PBS intraperitoneally on day 8 and 15 one hour before intranasal instillation with saline or HDM + DEP, similar to publication ( 7 ). For therapeutic inhibition of neutrophil elastase, mice were intraperitoneally treated with PBS or 100 mg/kg sivelestat (MedChem Express) on days 14, 15 and 16. 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. Treatment schemes and concentrations were based on publications ( 12 , 17 ) and are included in the Figures. Bronchoalveolar lavage A cannula was inserted in the trachea and BALF was collected by instillation of 3 x 300 µl HBSS supplemented with 1% BSA and 6 x 500 µl HBSS supplemented with EDTA. Total amount of BAL cells was counted using a Bürker chamber. 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 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 small 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) The forced oscillation technique (Flexivent System, Canada) was applied to determine AHR towards carbachol (dose range 0-640 µg/kg), as described previously ( 15 ). 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 1. Identification of eosinophil subsets upon subacute HDM + DEP exposure We first investigated the presence of the eosinophil subsets (CD101- hEOS and CD101 + iEOS ( 7 )) in different compartments -i.e. blood, lung tissue and BALF- of our pollutant-aggravated asthma model ( Fig. 1 A 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. 1 B-C ) . In lung tissue, both eosinophil subsets were significantly increased upon combined HDM + DEP exposure compared to saline exposure ( Fig. 1 D-E ) . In BALF, the iEOS subset was predominantly present and significantly increased upon HDM + DEP exposure compared to saline control (data not shown). 2. 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. 2 A ) . Blood eosinophils significantly decreased in anti-IL-5-treated mice, whereas blood neutrophils were unaffected ( Fig. 2 B-C ) . In BALF, the total cell number increased upon HDM + DEP exposure and tended to decrease after anti-IL-5 ( Fig. 2 D ) . The total number of BAL eosinophils significantly decreased ( Fig. 2 E ) while BAL neutrophils remained unaffected ( Fig. 2 F ) upon IL-5 inhibition in HDM + DEP exposed mice. Notably, both eosinophil subsets significantly decreased in lung tissue upon anti-IL-5 treatment ( Fig. 2 G-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. 2 I-L ) . On tissue sections, the HDM + DEP induced peribronchial eosinophil and neutrophil numbers were both significantly decreased after anti-IL-5 treatment ( Fig. 3 A-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. 3 C-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. 3 E ) . 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. 3. 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 hours) after the last intranasal HDM + DEP exposure ( Fig. 4 A ) . Total BAL cell numbers were high (above 10 6 ) and did not significantly differ between the time points ( Fig. 4 B ) . Neutrophil numbers in BALF were highest at 24 hours after last HDM + DEP exposure and significantly decreased at later time points, while BAL eosinophils tended to increase from 24 to 48 hours ( Fig. 4 C-D ) . In lung, peribronchial neutrophils were highest 24 hours after last HDM + DEP exposure and decreased thereafter ( Fig. 4 E, 4 G ) . Interestingly, peribronchial eosinophils in lung show the same trend as neutrophils ( Fig. 4 F ) . Also the percentages of neutrophils in lung single cell suspensions and in blood were highest at 24 hours after the last exposure and decrease over time (Supplemental Fig. 5A-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. 4 H-I ) . In blood, the hEOS subset is predominant and increases significantly 48 hours after last HDM + DEP exposure ( Fig. 4 J ) . 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 hours after last HDM + DEP exposure and decreased thereafter ( Fig. 5 A-C ) . The protein level of CXCL1 in BALF showed the same trend ( Fig. 5 D ) . Also both NET components (NE and dsDNA) in BALF were present at high concentrations at 24 hours and decreased over time ( Fig. 5 E-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 hours after the last HDM + DEP exposure and decreased thereafter (Fig. 5 G-I ) . 4. 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. 6 A ) significantly decreased total BAL cells in mice co-exposed to HDM and DEP ( Fig. 6 B ) . The elevated BAL eosinophil and neutrophil numbers observed upon HDM + DEP exposure tended to decrease after therapeutic sivelestat administration ( Fig. 6 C-D ) . Moreover, BAL DCs significantly decreased after sivelestat injection in HDM + DEP exposed mice ( Fig. 6 E ). Notably, there was no major impact of sivelestat on dsDNA and NE levels ( Fig. 6 F-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. 6 H-N ) . Notably, therapeutic inhibition of NE in mice exposed to saline led to a small, but significant increase in BAL neutrophils and dsDNA ( Fig. 6 C, 6 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) . 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 ( 7 ) and in human samples including blood and induced sputum of asthma patients ( 18 , 19 ). Mesnil et al. provided evidence that lung hEOS and iEOS represent distinct terminally differentiated eosinophils ( 7 ), while another study supported the concept that hEOS can become iEOS during inflammatory processes ( 20 ). 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 ( 7 ). 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 be more harmful because both eosinophils subsets are depleted while mepolizumab (anti-IL-5) would only affect the iEOS subset ( 21 ). 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 ( 22 ). 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 ( 7 , 23 , 24 ) 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 ( 25 ), 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 ( 26 ), 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 ( 27 ). However, results are controversial since another study observed concurrent increased neutrophil sputum counts in severe asthma patients treated with benralizumab ( 28 ). 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 ( 29 ). Also studies with eosinophil-deficient mice have shown that type 2 inflammation remodelling and lung function are not dependent on eosinophils ( 30 , 31 ). 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 FEV 1 , measures of type 2 inflammation and lung function respectively) despite decreased eosinophil levels and exacerbation rate ( 32 , 33 ). Upon subacute HDM + DEP exposure, neutrophils were highly present 24 hours 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 ( 34 ), whereas neutrophil depletion in an Alternaria alternata asthma model led to a significant reduction of eosinophils in BALF ( 35 ). 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 ( 36 ). 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 ( 37 ). 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 ( 38 ). 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 ( 16 ). 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 ( 39 ). 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 ( 39 ). 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 ( 40 ). 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 ( 39 ). Moreover, sivelestat functions extracellularly thus only inhibiting NE released into the extracellular space ( 39 , 41 ). These challenges could explain the partial decrease of BAL inflammation in our asthma mouse model. CONCLUSIONS In conclusion, our data suggest no significant role for neutrophils in mice co-exposed to particulates and allergen (i.e. HDM) since NE inhibition and neutrophil depletion did not affect eosinophilic responses. 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 ) . List Of 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 Declarations Ethics approval 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). Consent for publication Not applicable. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests 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. Funding The Department of Respiratory Medicine (Ghent University) was funded by Scientific Research in Flanders (FWO Vlaanderen, FWO041819N, and FWO-EOS projects G0G2318N, G0H1222N, G025123N) and a Ghent University Grant (BOF/GOA 01G00819). Author’s 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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Interleukin-5 receptor alpha (CD125) expression on human blood and lung neutrophils. Ann Allergy Asthma Immunol. 2022;128(1):53–60. Jorssen J, Van Hulst G, Mollers K, Pujol J, Petrellis G, Baptista A, et al. Single-cell proteomics and transcriptomics capture eosinophil development and identify the role of IL-5 in their lineage transit amplification. Immunity. 2024;57:1–18. Busse W, Katial R, Gossage D, Sari S, Wang B, Kolbeck R, et al. Safety profile, pharmacokinetics, and biologic activity of MEDI-563, an anti-IL-5 receptor antibody, in a phase I study of subjects with mild asthma. J Allergy Clin Immunol. 2010;125(6):1237–44. Schleich F, Moermans C, Seidel L, Kempeneers C, Louis G, Rogister F et al. Benralizumab in severe eosinophilic asthma in real life: confirmed effectiveness and contrasted effect on sputum eosinophilia versus exhaled nitric oxide fraction - PROMISE. ERJ open Res. 2023;9(6). Scott G, Asrat S, Allinne J, Lim WK, Nagashima K, Birchard D et al. IL-4 and IL-13, not eosinophils, drive type 2 airway inflammation, remodeling and lung function decline. Cytokine. 2023. Denzler K, Borchers M, Crosby J, Cieslewicz G, Hines E, Justice J, et al. Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation. J Immunol. 2001;167(3):1672–82. Denzler K, Farmer S, Crosby J, Borchers M, Cieslewicz G, Larson K et al. Eosinophil major basic protein-1 does not contribute to allergen-induced airway pathologies in mouse models of asthma. J Immunol. 2000;165(10). Pavord ID, Korn S, Howarth P, Bleecker ER, Buhl R, Keene ON, et al. Mepolizumab for severe eosinophilic asthma (DREAM): a multicentre, double-blind, placebo-controlled trial. Lancet. 2012;380(9842):651–9. Haldat P, Brightling C, Hargadon B, Gupta S, Monteiro W, Sousa A, et al. Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med. 2009;360(10):973–84. Patel D, Peiro T, Bruno N, Vuononvirta J, Akthar S, Puttur F et al. Neutrophils restrain allergic airway inflammation by limiting ILC2 function and monocyte-dendritic cell antigen presentation. Sci Immunol. 2019;4(41). Van Nevel S, van Ovost J, Holtappels G, De Ruyck N, Zhang N, Braun H et al. Neutrophils affect IL-33 processing in response to the respiratory allergen alternaria alternata. Front Immunol. 2021;12. Boivin G, Faget J, Ancey PB, Ghasti A, Mussard J, Engblom C, et al. Durable and controlled depletion of neutrophils in mice. Nat Comm. 2020;11(1):2762. Thierry A. anti-protease treatments targeting plasmin(ogen) and neutrophil elastase may be beneficial in fighting COVID-19. Physiol Rev. 2020;100(4):1597–8. Koga H, Miyahara N, Fuchimoto Y, Ikeda G, Waseda K, Ono K, et al. Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses. Respir Res. 2013;14(1):8. Okeke E, Louttit C, Fry C, Najafabadi AH, Han K, Nemzek J et al. Inhibition of neutrophil elastase prevents neutrophil extracellular trap formation and rescues mice from endotoxic shock. Biomaterials. 2020;238. Zeiher B, Artigas A, Vincent JL, Dmitrienko A, Jackson K, Thompson BT, et al. Neutrophil elastase inhibition in acute lung injury: results of the STRIVE study. Crit Care Med. 2004;32(8):1695–702. Aikawa N, Ishizaka A, Hirasawa H, Shimazaki S, Yamamoto Y, Sugimoto H, et al. Reevaluation of the efficacy and safety of the neutrophil elastase inhibitor, sivelestat, for the treatment of acute lung injury associated with systemic inflammatory response syndrome: A phase IV study. Pulm Pharmacol Ther. 2011;24(5):549–54. Additional Declarations Competing interest reported. 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. Supplementary Files SupplementalFigures.pdf FIGURELEGENDSSUPPLEMENT.docx Cite Share Download PDF Status: Published Journal Publication published 28 Jan, 2025 Read the published version in Respiratory Research → Version 1 posted Editorial decision: Revision requested 08 Oct, 2024 Reviews received at journal 17 Aug, 2024 Reviewers agreed at journal 09 Aug, 2024 Reviewers invited by journal 08 Aug, 2024 Editor assigned by journal 08 Jul, 2024 Submission checks completed at journal 08 Jul, 2024 First submitted to journal 05 Jul, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4691862","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":329091865,"identity":"4d33fb18-518a-47f8-b69c-a5309c9d267b","order_by":0,"name":"Joyceline De Volder","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Joyceline","middleName":"","lastName":"De Volder","suffix":""},{"id":329091868,"identity":"6193d89e-898f-4b10-b434-056ec42409a7","order_by":1,"name":"Annelies Bontinck","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Annelies","middleName":"","lastName":"Bontinck","suffix":""},{"id":329091870,"identity":"ef17629a-c04f-48e6-afd6-88dad5f5276b","order_by":2,"name":"Valerie Haelterman","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Valerie","middleName":"","lastName":"Haelterman","suffix":""},{"id":329091871,"identity":"a829fa6a-22eb-4f7f-9983-a323e014ff45","order_by":3,"name":"Louis Boon","email":"","orcid":"","institution":"JJP Biologics","correspondingAuthor":false,"prefix":"","firstName":"Louis","middleName":"","lastName":"Boon","suffix":""},{"id":329091872,"identity":"eb0f50aa-c243-4f3d-9df2-e1973150f8d8","order_by":4,"name":"Guy F Joos","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guy","middleName":"F","lastName":"Joos","suffix":""},{"id":329091873,"identity":"de327adc-f655-4907-b030-0586297fa7c3","order_by":5,"name":"Guy G Brusselle","email":"","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":false,"prefix":"","firstName":"Guy","middleName":"G","lastName":"Brusselle","suffix":""},{"id":329091874,"identity":"f507f4f3-3eb2-4aa4-b583-6dd626c26db1","order_by":6,"name":"Tania Maes","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4klEQVRIiWNgGAWjYJCCAxCK+ZjEDxAtQUA5D0ILW5pkD0gDMVpgTDNpBmK02LOfPXjgB8M2eX7+M9+kGSru1DFINx/AbwtPXsLBHobbhjNn5G6TZjjzTIJB5lgCAYflGBzgYbjNuOEG7zZp2bbDQIflGODXwv/G4OAfhtv2+8+feSbN+w+kJf8Dfi1AMw8DbUncwJDDJs3bALYFrw4GnhtvDA7LGNxOnnEjzdiw59hhyTaZY/gdxt6fY/zxTcVt2/7+ww8f/Kg5zM8v3fwAvzVggGwsGxHqR8EoGAWjYBQQAAC5QUXaAgtG9wAAAABJRU5ErkJggg==","orcid":"","institution":"Ghent University Hospital","correspondingAuthor":true,"prefix":"","firstName":"Tania","middleName":"","lastName":"Maes","suffix":""}],"badges":[],"createdAt":"2024-07-05 11:14:19","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4691862/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4691862/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12931-024-03082-9","type":"published","date":"2025-01-28T15:56:55+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61487742,"identity":"ffec4856-b926-4de4-a21c-24566d7a1f3b","added_by":"auto","created_at":"2024-07-31 10:01:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":331149,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIdentification of eosinophil subsets upon subacute HDM+DEP exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale C57BL6/J mice were intranasally exposed to saline or HDM+DEP for 3 weeks. \u003cstrong\u003eA, \u003c/strong\u003eschematic representation of the experiment. \u003cstrong\u003eB, \u003c/strong\u003egating strategy for eosinophil subsets starting from CD45+ CD11c- CD11b+ population in blood. \u003cstrong\u003eC, \u003c/strong\u003epercentage homeostatic eosinophils (hEOS) and inflammatory eosinophils (iEOS) of CD45+ cells in blood. \u003cstrong\u003eD, \u003c/strong\u003egating strategy for eosinophil subsets starting from CD45+ CD11c- CD11b+ population in lung single cell suspensions. \u003cstrong\u003eE, \u003c/strong\u003epercentage 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 \u0026lt; 0.05 **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/5e984774adb08036ab6f4746.png"},{"id":61488435,"identity":"bf379168-3b41-464c-8e15-88fada3f8298","added_by":"auto","created_at":"2024-07-31 10:09:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":300206,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-5 inhibition reduces eosinophilic, but not neutrophilic, inflammation in BALF after subacute HDM+DEP exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale 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 second and last HDM+DEP exposure. \u003cstrong\u003eA, \u003c/strong\u003eschematic representation of the experiment. \u003cstrong\u003eB, \u003c/strong\u003epercentage eosinophils of CD45+ cells in blood. \u003cstrong\u003eC, \u003c/strong\u003epercentage neutrophils of CD45+ cells in blood. \u003cstrong\u003eD, \u003c/strong\u003etotal cell number in BALF. \u003cstrong\u003eE, \u003c/strong\u003eeosinophil numbers in BALF (CD45+ CD11c- CD11b+ SiglecF+ Ly6G-).\u003cstrong\u003e F,\u003c/strong\u003e neutrophil numbers in BALF (CD45+ CD11c- CD11b+ SiglecF\u003csup\u003edim\u003c/sup\u003e, Ly6C+ and Ly6G+). \u003cstrong\u003eG, \u003c/strong\u003epercentage hEOS (CD45+ CD11c- CD11b+ SiglecF+ CD101-) of CD45+ cells in lung tissue. \u003cstrong\u003eH, \u003c/strong\u003epercentage iEOS (CD45+ CD11c- CD11b+ SiglecF+ CD101+) of CD45+ cells in lung tissue. \u003cstrong\u003eI, \u003c/strong\u003enumbers of SiglecF+ neutrophils (CD45+ CD11c- CD11b+ SiglecF+ Ly6G+ Ly6C+) in BALF. \u003cstrong\u003eJ, \u003c/strong\u003epercentage SiglecF+ neutrophils (CD45+ CD11c- CD11b+ SiglecF+ Ly6G+ Ly6C+) in lung tissue. \u003cstrong\u003eK, \u003c/strong\u003econcentration of double-stranded DNA (ng/ml) in BAL supernatant. \u003cstrong\u003eL, \u003c/strong\u003eprotein level of neutrophil elastase (pg/ml) in BALF supernatant determined by ELISA.\u003cstrong\u003e \u003c/strong\u003eResults are expressed as mean ± SEM. n = 7-8 mice/group. *p \u0026lt; 0.05 **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/6bea77aefc15e008ce1687d7.png"},{"id":61488436,"identity":"31e10021-ead3-4820-8c13-d549fc414627","added_by":"auto","created_at":"2024-07-31 10:09:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1423881,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIL-5 inhibition reduces eosinophilic and neutrophilic inflammation in lung tissue upon subacute HDM+DEP exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA, \u003c/strong\u003equantification of congo-stained peribronchial eosinophils in lung tissue and representative photomicrographs. \u003cstrong\u003eB, \u003c/strong\u003equantification of MPO-stained peribronchial neutrophils in lung tissue and representative photomicrographs. \u003cstrong\u003eC, \u003c/strong\u003erelative muc5ac mRNA expression in lung tissue determined by RT-qPCR. \u003cstrong\u003eD, \u003c/strong\u003equantification of PAS-positive goblet cells in lung tissue. \u003cstrong\u003eE, \u003c/strong\u003eairway hyperresponsiveness measured in response to increasing doses of carbachol. Results are expressed as mean ± SEM. n = 7-8 mice/group. *p \u0026lt; 0.05 **p \u0026lt; 0.01\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/877bdbb5798074c364163d19.png"},{"id":61487745,"identity":"5b3bd546-371f-4d04-831a-6d1323539feb","added_by":"auto","created_at":"2024-07-31 10:01:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":787425,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutrophils accumulate rapidly after subacute HDM+DEP exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale 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. \u003cstrong\u003eA,\u003c/strong\u003e schematic overview of the experiment. \u003cstrong\u003eB, \u003c/strong\u003etotal cell number in BALF. \u003cstrong\u003eC, \u003c/strong\u003eneutrophil numbers in BALF (CD45+ CD11c- CD11b+ SiglecF\u003csup\u003edim\u003c/sup\u003e, Ly6C+ and Ly6G+). \u003cstrong\u003eD,\u003c/strong\u003e eosinophil numbers in BALF (CD45+ CD11c- CD11b+ SiglecF+ Ly6G-). \u003cstrong\u003eE, \u003c/strong\u003equantification of peribronchial Congo-Red stained eosinophils in lung tissue. \u003cstrong\u003eF-G,\u003c/strong\u003e quantification of peribronchial MPO-stained neutrophils in lung tissue with representative photomicrographs. \u003cstrong\u003eH-I, \u003c/strong\u003epercentage hEOS \u003cstrong\u003e(H)\u003c/strong\u003e and iEOS \u003cstrong\u003e(I) \u003c/strong\u003eof CD45+ cells in lung tissue. \u003cstrong\u003eJ, \u003c/strong\u003epercentage hEOS of CD45+ cells in blood. Results are expressed as mean ± SEM. n = 8-10 mice/group. *p \u0026lt; 0.05 **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/84e51bb5b856795ea6a48946.png"},{"id":61487748,"identity":"6a94e7a3-896d-4095-83d5-43f72341f91a","added_by":"auto","created_at":"2024-07-31 10:01:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":288356,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eNeutrophil-attracting chemokines and NET formation after subacute HDM+DEP exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale 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. \u003cstrong\u003eA-C, \u003c/strong\u003erelative mRNA expression of neutrophil-recruiting chemokines CXCL1 \u003cstrong\u003e(A)\u003c/strong\u003e, CXCL2 \u003cstrong\u003e(B)\u003c/strong\u003eand CXCL5 \u003cstrong\u003e(C) \u003c/strong\u003ein lung tissue.\u003cstrong\u003e D,\u003c/strong\u003e CXCL1 protein levels (pg/ml) in BAL supernatant determined by ELISA. \u003cstrong\u003eE, \u003c/strong\u003econcentration of double-stranded DNA (ng/ml) in BAL supernatant. \u003cstrong\u003eF, \u003c/strong\u003elevels of neutrophil elastase (pg/ml) in BAL supernatant determined via ELISA. \u003cstrong\u003eG-I, \u003c/strong\u003erelative mRNA expression of the type 2 cytokines IL-5 \u003cstrong\u003e(G) \u003c/strong\u003eand IL-13 \u003cstrong\u003e(H) \u003c/strong\u003eand the eosinophil chemoattractant CCL11 \u003cstrong\u003e(I)\u003c/strong\u003e in lung tissue.\u003cstrong\u003e \u003c/strong\u003eResults are expressed as mean ± SEM. n = 8-10 mice/group. *p \u0026lt; 0.05 **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/b8f6f401e7c731e0ab04318c.png"},{"id":61488437,"identity":"b3205392-08cd-46c1-bde5-2c0cf01c8a53","added_by":"auto","created_at":"2024-07-31 10:09:56","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":341995,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eTherapeutic inhibition of neutrophil elastase tends to decrease inflammation upon subacute HDM+DEP exposure.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFemale C57BL6/J mice were intranasally exposed to saline or HDM+DEP for 3 weeks. Sivelestat or PBS was therapeutically administered by intraperitoneal injection. \u003cstrong\u003eA,\u003c/strong\u003e schematic overview of the experiment. \u003cstrong\u003eB, \u003c/strong\u003etotal cell number in BALF. \u003cstrong\u003eC, \u003c/strong\u003eneutrophil numbers in BALF (CD45+ CD11c- CD11b+ SiglecF\u003csup\u003edim\u003c/sup\u003e, Ly6C+ and Ly6G+). \u003cstrong\u003eD,\u003c/strong\u003e eosinophil numbers in BALF (CD45+ CD11c- CD11b+ SiglecF+ Ly6G-). \u003cstrong\u003eE,\u003c/strong\u003e numbers of dendritic cells in BALF (CD45+ CD11c+ MHCII+). \u003cstrong\u003eF,\u003c/strong\u003e concentration of double-stranded DNA (ng/ml) in BAL supernatant. \u003cstrong\u003eG, \u003c/strong\u003eneutrophil elastase concentration (pg/ml) in BAL supernatant.\u003cstrong\u003e H, \u003c/strong\u003equantification of congo-stained peribronchial eosinophils in lung tissue. \u003cstrong\u003eI, \u003c/strong\u003equantification of MPO-stained neutrophils in lung tissue. \u003cstrong\u003eJ, \u003c/strong\u003equantification of PAS-stained goblet cells in lung tissue. \u003cstrong\u003eK-N, \u003c/strong\u003erelative mRNA expression of muc5ac \u003cstrong\u003e(K)\u003c/strong\u003e, IL-33 \u003cstrong\u003e(L)\u003c/strong\u003e, IL-5 \u003cstrong\u003e(M) \u003c/strong\u003eand IL-13 \u003cstrong\u003e(N)\u003c/strong\u003e in lung tissue. Results are expressed as mean ± SEM. n = 6-8 mice/group. *p \u0026lt; 0.05 **p \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/0c32f241bd4e1dfbe220dec2.png"},{"id":61487747,"identity":"adebfb3a-13c4-42d1-921b-499d127f2fe8","added_by":"auto","created_at":"2024-07-31 10:01:55","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":337897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAnti-IL-5 treatment, but not neutrophil interference, attenuates inflammation in a pollutant-aggravated allergic asthma mouse model.\u003c/strong\u003e\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/b4fcd5743167d80fafea8f5a.png"},{"id":75351146,"identity":"793e3696-fd86-462b-9276-6dd5dcffd42c","added_by":"auto","created_at":"2025-02-03 16:05:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5021357,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/ec0ea35e-cf51-4d97-8759-44cf9b8eb51a.pdf"},{"id":61487743,"identity":"9656a56d-39ac-4f95-af6d-97658e3eb4e8","added_by":"auto","created_at":"2024-07-31 10:01:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":586171,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalFigures.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/f2e2c138c52d26bda25f1fce.pdf"},{"id":61487741,"identity":"adfe5d97-47c8-4c46-ba02-877494560906","added_by":"auto","created_at":"2024-07-31 10:01:55","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":15740,"visible":true,"origin":"","legend":"","description":"","filename":"FIGURELEGENDSSUPPLEMENT.docx","url":"https://assets-eu.researchsquare.com/files/rs-4691862/v1/a7c1142972a51282259888b0.docx"}],"financialInterests":"Competing interest reported. 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.","formattedTitle":"Anti-IL-5 treatment, but not neutrophil interference, attenuates inflammation in a mixed granulocytic asthma mouse model, elicited by air pollution","fulltext":[{"header":"Key Messages","content":"\u003cul\u003e\n \u003cli\u003eAnti-IL-5 reduces homeostatic and inflammatory eosinophils in mice exposed to allergen and pollution\u003c/li\u003e\n \u003cli\u003eAnti-IL-5 does not affect neutrophil extracellular trap formation, goblet cells and airway hyperresponsiveness\u003c/li\u003e\n \u003cli\u003eNo impact of neutrophil interventions on eosinophilic inflammation in allergen and pollution-exposed mice\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"INTRODUCTION","content":"\u003cp\u003eDiesel 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 (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). 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 (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Moreover, allergic individuals exposed to DEP demonstrated increased type 2 cytokine (IL-4, IL-5, IL-13) production, airway eosinophilia and IgE levels (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Multiple studies have confirmed the influence of particulate matter -including DEP- on asthma incidence, severity and exacerbations (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAsthma is a heterogeneous disease with allergic asthma being the best known phenotype (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). 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), Thelper (Th) cells, eosinophils and mast cells (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Recently, two eosinophil subsets were identified with distinct CD101 expression, localization and transcriptional signatures. CD101\u003csup\u003e\u0026minus;\u003c/sup\u003e tissue-resident or homeostatic eosinophils (hEOS) were predominantly located in the lung vascular niche with mostly homeostatic functions whereas CD101\u003csup\u003e+\u003c/sup\u003e inflammatory eosinophils (iEOS) were predominantly present in bronchoalveolar lavage fluid and extravascular lung during inflammation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). 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 (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). These patients also have higher levels of neutrophil-recruiting chemokines (CXCL1, CXCL2 and CXCL8) in their BALF (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Neutrophilic asthma is thought to be driven by type 3 immunity (IL17A production from Th17 cells and type 3 innate lymphoid cells) (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e), 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 (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). 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 (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). 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 (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDEP have been proven to worsen asthma pathogenesis (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e) 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 (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Moreover, combined HDM\u0026thinsp;+\u0026thinsp;DEP exposure also led to higher neutrophils numbers which associated with higher production of neutrophil-attracting chemokines, neutrophil mediators and NET formation (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), suggesting a role for neutrophils in pollutant-aggravated allergic asthma, mostly dominated by a type 2 immune response. In current manuscript, we identified eosinophil subsets in our murine pollutant-aggravated asthma model and inhibited IL-5 signalling 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\u0026thinsp;+\u0026thinsp;DEP exposure.\u003c/p\u003e"},{"header":"MATERIAL AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMice experiments\u003c/h2\u003e \u003cp\u003eFemale mice (C57BL6/J, 6\u0026ndash;8 weeks old) were purchased from Jackson Laboratory. All \u003cem\u003ein vivo\u003c/em\u003e experiments were approved by the Animal Ethical Committee of the Faculty of Medicine and Health Science, Ghent University (ECD19-40, ECD19-22, ECD23-50).\u003c/p\u003e \u003cp\u003eFor the pollutant-aggravated allergic asthma mouse model, isoflurane anesthetized mice (8/group) were intranasally exposed to saline or the combination of 1 \u0026micro;g HDM (\u003cem\u003eDermatophagoides pteronyssinus\u003c/em\u003e, Greer Laboratories) and 25 \u0026micro;g DEP (SRM2975, NIST) on day 1, 8 and 15 as described previously (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). In most experiments, mice were sacrificed by a lethal dose of pentobarbital via intraperitoneal injection two days after the last exposure. To characterize the dynamics of the inflammation, mice were killed either 24, 48 or 72 hours after the last intranasal HDM\u0026thinsp;+\u0026thinsp;DEP exposure. A scheme of each experimental set-up is included in the Figures.\u003c/p\u003e \u003cp\u003eFor inhibition of IL-5 in the HDM\u0026thinsp;+\u0026thinsp;DEP model, mice received 100 \u0026micro;g isotype control (clone GL113) or anti-IL-5 (clone TRFK5) in PBS intraperitoneally on day 8 and 15 one hour before intranasal instillation with saline or HDM\u0026thinsp;+\u0026thinsp;DEP, similar to publication (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). For therapeutic inhibition of neutrophil elastase, mice were intraperitoneally treated with PBS or 100 mg/kg sivelestat (MedChem Express) on days 14, 15 and 16. For neutrophil depletion experiments, mice received 100 \u0026micro;g 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. Treatment schemes and concentrations were based on publications (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e) and are included in the Figures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eBronchoalveolar lavage\u003c/h2\u003e \u003cp\u003eA cannula was inserted in the trachea and BALF was collected by instillation of 3 x 300 \u0026micro;l HBSS supplemented with 1% BSA and 6 x 500 \u0026micro;l HBSS supplemented with EDTA. Total amount of BAL cells was counted using a B\u0026uuml;rker chamber.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eFlow cytometry\u003c/h2\u003e \u003cp\u003eCell 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) \u003cb\u003e(Supplemental Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and S2\u003c/b\u003e 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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eHistology\u003c/h2\u003e \u003cp\u003eAfter 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\u0026amp;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\u0026thinsp;\u0026lt;\u0026thinsp;800 \u0026micro;m or \u0026gt;\u0026thinsp;2000 \u0026micro;m were excluded from the analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eProtein measurements\u003c/h2\u003e \u003cp\u003eCommercially available ELISA kits from R\u0026amp;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.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eQuantitative RT-PCR\u003c/h2\u003e \u003cp\u003eRNA from the small lobe of the right lung was isolated using the miRNeasy mini kit (Qiagen). mRNA expression relative to the housekeeping genes \u003cem\u003eGAPDH\u003c/em\u003e and \u003cem\u003eHPRT\u003c/em\u003e, was analysed using Taqman gene expression assays (Thermofisher Scientific) with a Lightcycler 96 system (Roche).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eAirway hyperresponsiveness (AHR)\u003c/h2\u003e \u003cp\u003eThe forced oscillation technique (Flexivent System, Canada) was applied to determine AHR towards carbachol (dose range 0-640 \u0026micro;g/kg), as described previously (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Intravenously injecting pancuronium bromide (dose 1 mg/kg) induced neuromuscular blockade. Resistance of the whole respiratory system was measured.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eSPSS (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\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were regarded as significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n \u003ch2\u003e1. Identification of eosinophil subsets upon subacute HDM\u0026thinsp;+\u0026thinsp;DEP exposure\u003c/h2\u003e\n \u003cp\u003eWe first investigated the presence of the eosinophil subsets (CD101- hEOS and CD101\u0026thinsp;+\u0026thinsp;iEOS (\u003cspan class=\"CitationRef\"\u003e7\u003c/span\u003e)) in different compartments -i.e. blood, lung tissue and BALF- of our pollutant-aggravated asthma model \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA \u003cstrong\u003eand Supplemental Figure \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e for gating strategy)\u003c/strong\u003e. In blood, the hEOS subset was predominant and increased upon HDM\u0026thinsp;+\u0026thinsp;DEP exposure compared to saline control, while barely any iEOS could be detected \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB-C\u003cstrong\u003e)\u003c/strong\u003e. In lung tissue, both eosinophil subsets were significantly increased upon combined HDM\u0026thinsp;+\u0026thinsp;DEP exposure compared to saline exposure \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eD-E\u003cstrong\u003e)\u003c/strong\u003e. In BALF, the iEOS subset was predominantly present and significantly increased upon HDM\u0026thinsp;+\u0026thinsp;DEP exposure compared to saline control (data not shown).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n \u003ch2\u003e2. Inhibition of IL-5 reduces eosinophilic inflammation upon subacute HDM\u0026thinsp;+\u0026thinsp;DEP exposure\u003c/h2\u003e\n \u003cp\u003eSince 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 \u003cstrong\u003e(Supplemental Fig.\u0026nbsp;3A-B)\u003c/strong\u003e. However, the inflammation in BALF and lung tissue upon HDM\u0026thinsp;+\u0026thinsp;DEP exposure did not differ between anti-IL-5 and isotype control-treated mice \u003cstrong\u003e(Supplemental Fig.\u0026nbsp;3C-G)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eSince a single anti-IL-5 treatment may be insufficient to eliminate the extreme eosinophilic inflammation in our HDM\u0026thinsp;+\u0026thinsp;DEP model, we next treated mice with anti-IL-5 before the second and last intranasal exposure \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. Blood eosinophils significantly decreased in anti-IL-5-treated mice, whereas blood neutrophils were unaffected \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB-C\u003cstrong\u003e)\u003c/strong\u003e. In BALF, the total cell number increased upon HDM\u0026thinsp;+\u0026thinsp;DEP exposure and tended to decrease after anti-IL-5 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cstrong\u003e)\u003c/strong\u003e. The total number of BAL eosinophils significantly decreased \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e while BAL neutrophils remained unaffected \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eF\u003cstrong\u003e)\u003c/strong\u003e upon IL-5 inhibition in HDM\u0026thinsp;+\u0026thinsp;DEP exposed mice. Notably, both eosinophil subsets significantly decreased in lung tissue upon anti-IL-5 treatment \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eG-H\u003cstrong\u003e)\u003c/strong\u003e. The SiglecF\u0026thinsp;+\u0026thinsp;neutrophil subset (in BALF and lung) and NET components in BALF increased upon HDM\u0026thinsp;+\u0026thinsp;DEP exposure, but were not significantly influenced by anti-IL-5 therapy \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eI-L\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eOn tissue sections, the HDM\u0026thinsp;+\u0026thinsp;DEP induced peribronchial eosinophil and neutrophil numbers were both significantly decreased after anti-IL-5 treatment \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA-B\u003cstrong\u003e)\u003c/strong\u003e. Notably, the HDM\u0026thinsp;+\u0026thinsp;DEP induced \u003cem\u003emuc5ac\u003c/em\u003e 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 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC-D\u003cstrong\u003e)\u003c/strong\u003e. AHR was higher in HDM\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;DEP exposure \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE\u003cstrong\u003e)\u003c/strong\u003e. Anti-IL-5 treatment of HDM\u0026thinsp;+\u0026thinsp;DEP exposed mice significantly increased \u003cem\u003eIL-33\u003c/em\u003e mRNA expression compared to isotype control, while mRNA expression of \u003cem\u003eCCL11 (eotaxin)\u003c/em\u003e, \u003cem\u003eIL-13\u003c/em\u003e and \u003cem\u003eCXCL1\u003c/em\u003e, \u003cem\u003eCXCL2\u003c/em\u003e and \u003cem\u003eCXCL5\u003c/em\u003e remained unaffected \u003cstrong\u003e(Supplemental Fig.\u0026nbsp;4)\u003c/strong\u003e. 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\u0026thinsp;+\u0026thinsp;DEP model.\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e3. Neutrophils and neutrophil extracellular traps accumulate rapidly in the bronchoalveolar lavage fluid after subacute HDM\u0026thinsp;+\u0026thinsp;DEP exposure\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003eTo determine the dynamics of the accumulation of immune cells in the airways in our HDM\u0026thinsp;+\u0026thinsp;DEP model, we analysed BALF cell composition at multiple time points (24, 48 and 72 hours) after the last intranasal HDM\u0026thinsp;+\u0026thinsp;DEP exposure \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e. Total BAL cell numbers were high (above 10\u003csup\u003e6\u003c/sup\u003e) and did not significantly differ between the time points \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. Neutrophil numbers in BALF were highest at 24 hours after last HDM\u0026thinsp;+\u0026thinsp;DEP exposure and significantly decreased at later time points, while BAL eosinophils tended to increase from 24 to 48 hours \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eC-D\u003cstrong\u003e)\u003c/strong\u003e. In lung, peribronchial neutrophils were highest 24 hours after last HDM\u0026thinsp;+\u0026thinsp;DEP exposure and decreased thereafter \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eE, \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eG\u003cstrong\u003e)\u003c/strong\u003e. Interestingly, peribronchial eosinophils in lung show the same trend as neutrophils \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eF\u003cstrong\u003e)\u003c/strong\u003e. Also the percentages of neutrophils in lung single cell suspensions and in blood were highest at 24 hours after the last exposure and decrease over time \u003cstrong\u003e(Supplemental Fig.\u0026nbsp;5A-B)\u003c/strong\u003e. 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 \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eH-I\u003cstrong\u003e)\u003c/strong\u003e. In blood, the hEOS subset is predominant and increases significantly 48 hours after last HDM\u0026thinsp;+\u0026thinsp;DEP exposure \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eJ\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n \u003cp\u003eWe 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 hours after last HDM\u0026thinsp;+\u0026thinsp;DEP exposure and decreased thereafter \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA-C\u003cstrong\u003e)\u003c/strong\u003e. The protein level of CXCL1 in BALF showed the same trend \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD\u003cstrong\u003e)\u003c/strong\u003e. Also both NET components (NE and dsDNA) in BALF were present at high concentrations at 24 hours and decreased over time \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eE-F\u003cstrong\u003e)\u003c/strong\u003e. 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 hours after the last HDM\u0026thinsp;+\u0026thinsp;DEP exposure and decreased thereafter (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eG-I\u003cstrong\u003e)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n \u003ch2\u003e4. Therapeutic inhibition of neutrophil elastase tends to decrease inflammation after subacute HDM\u0026thinsp;+\u0026thinsp;DEP exposure\u003c/h2\u003e\n \u003cp\u003eNE 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\u0026thinsp;+\u0026thinsp;DEP model using sivelestat, which is a highly specific and potent inhibitor of NE. Sivelestat treatment \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA\u003cstrong\u003e)\u003c/strong\u003e significantly decreased total BAL cells in mice co-exposed to HDM and DEP \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB\u003cstrong\u003e)\u003c/strong\u003e. The elevated BAL eosinophil and neutrophil numbers observed upon HDM\u0026thinsp;+\u0026thinsp;DEP exposure tended to decrease after therapeutic sivelestat administration \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC-D\u003cstrong\u003e)\u003c/strong\u003e. Moreover, BAL DCs significantly decreased after sivelestat injection in HDM\u0026thinsp;+\u0026thinsp;DEP exposed mice \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eE\u003cstrong\u003e).\u003c/strong\u003e Notably, there was no major impact of sivelestat on dsDNA and NE levels \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF-G\u003cstrong\u003e)\u003c/strong\u003e. In lung tissue of HDM\u0026thinsp;+\u0026thinsp;DEP exposed mice, no differences in peribronchial eosinophils and neutrophils, goblet cells and \u003cem\u003emuc5ac, IL-5, IL-13 and IL-33\u003c/em\u003e mRNA expression were seen after neutrophil elastase inhibition \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eH-N\u003cstrong\u003e)\u003c/strong\u003e. Notably, therapeutic inhibition of NE in mice exposed to saline led to a small, but significant increase in BAL neutrophils and dsDNA \u003cstrong\u003e(\u003c/strong\u003eFig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eF\u003cstrong\u003e)\u003c/strong\u003e. Sivelestat treatment did not affect the eosinophil subsets in lung tissue of HDM\u0026thinsp;+\u0026thinsp;DEP exposed mice \u003cstrong\u003e(Supplemental Fig.\u0026nbsp;6A-B)\u003c/strong\u003e. 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\u0026thinsp;+\u0026thinsp;DEP exposure \u003cstrong\u003e(Supplemental Fig.\u0026nbsp;7, 8)\u003c/strong\u003e.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we identified the eosinophil subsets in lung tissue of HDM\u0026thinsp;+\u0026thinsp;DEP exposed mice. Inhibition of IL-5 signalling in HDM\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;DEP exposure. None of the neutrophil-interfering treatments affected the eosinophil subsets \u003cb\u003e(Supplemental Fig.\u0026nbsp;6)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003eTwo subsets of eosinophils -hEOS with homeostatic functions and inflammatory iEOS- have previously been identified by flow cytometry in the mouse lung after allergen exposure (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) and in human samples including blood and induced sputum of asthma patients (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Mesnil \u003cem\u003eet al.\u003c/em\u003e provided evidence that lung hEOS and iEOS represent distinct terminally differentiated eosinophils (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), while another study supported the concept that hEOS can become iEOS during inflammatory processes (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). In our experiments, both hEOS and iEOS were present in lung tissue of HDM\u0026thinsp;+\u0026thinsp;DEP exposed mice and increased significantly upon combined HDM\u0026thinsp;+\u0026thinsp;DEP exposure. Notably, after the last HDM\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;DEP exposed mice which is not in accordance with Mesnil \u003cem\u003eet al.\u003c/em\u003e, who reported that hEOS are not dependent on IL-5 for their presence in lungs and blood after HDM exposure (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). 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 be more harmful because both eosinophils subsets are depleted while mepolizumab (anti-IL-5) would only affect the iEOS subset (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e). 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 (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInterestingly, anti-IL-5 therapy significantly decreased peribronchial neutrophils in HDM\u0026thinsp;+\u0026thinsp;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 (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) 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 (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e), 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 (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), 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 (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). However, results are controversial since another study observed concurrent increased neutrophil sputum counts in severe asthma patients treated with benralizumab (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNotably, 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\u0026thinsp;+\u0026thinsp;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 (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Also studies with eosinophil-deficient mice have shown that type 2 inflammation remodelling and lung function are not dependent on eosinophils (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). 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 FEV\u003csub\u003e1\u003c/sub\u003e, measures of type 2 inflammation and lung function respectively) despite decreased eosinophil levels and exacerbation rate (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUpon subacute HDM\u0026thinsp;+\u0026thinsp;DEP exposure, neutrophils were highly present 24 hours 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 \u003cb\u003e(Supplemental Fig.\u0026nbsp;8)\u003c/b\u003e. This neutrophil depletion did not induce differences in eosinophilic inflammation upon HDM\u0026thinsp;+\u0026thinsp;DEP exposure. Patel \u003cem\u003eet al.\u003c/em\u003e demonstrated that neutrophil depletion in a murine HDM asthma model resulted in increased type 2 inflammation, airway remodelling and hyperresponsiveness (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e), whereas neutrophil depletion in an \u003cem\u003eAlternaria alternata\u003c/em\u003e asthma model led to a significant reduction of eosinophils in BALF (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Of note, HDM\u0026thinsp;+\u0026thinsp;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 (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eNE 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 (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). 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 (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Therefore, we aimed to target NE in our HDM\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;DEP model in which neutrophils are more prominent (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). 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 (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). 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 (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). 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 (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). 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 (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Moreover, sivelestat functions extracellularly thus only inhibiting NE released into the extracellular space (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). These challenges could explain the partial decrease of BAL inflammation in our asthma mouse model.\u003c/p\u003e"},{"header":"CONCLUSIONS","content":"\u003cp\u003eIn conclusion, our data suggest no significant role for neutrophils in mice co-exposed to particulates and allergen (i.e. HDM) since NE inhibition and neutrophil depletion did not affect eosinophilic responses. Anti-IL-5 treatment however attenuated HDM\u0026thinsp;+\u0026thinsp;DEP-induced inflammation, affecting both eosinophil subsets and neutrophils in lung tissue, but not BAL neutrophilia, NET formation and other asthma features \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003cp\u003eAHR Airway hyperresponsiveness\u003c/p\u003e \u003cp\u003eBALF Bronchoalveolar lavage fluid\u003c/p\u003e \u003cp\u003eCXCL CXC-motif chemokine ligand\u003c/p\u003e \u003cp\u003eDC Dendritic cell\u003c/p\u003e \u003cp\u003eDEP Diesel exhaust particles\u003c/p\u003e \u003cp\u003edsDNA Double-stranded DNA\u003c/p\u003e \u003cp\u003eHDM House dust mite\u003c/p\u003e \u003cp\u003ehEOS Homeostatic eosinophils\u003c/p\u003e \u003cp\u003eiEOS Inflammatory eosinophils\u003c/p\u003e \u003cp\u003eIL Interleukin\u003c/p\u003e \u003cp\u003eNE Neutrophil elastase\u003c/p\u003e \u003cp\u003eNET Neutrophil extracellular trap\u003c/p\u003e \u003cp\u003eOVA Ovalbumin\u003c/p\u003e \u003cp\u003eTh Thelper cell\u003c/p\u003e \u003cp\u003eLPS Lipopolysacharide\u003c/p\u003e "},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll \u003cem\u003ein vivo\u0026nbsp;\u003c/em\u003emouse\u0026nbsp;experiments were approved by the Animal Ethical Committee of the Faculty of Medicine and Health Science, Ghent University (ECD19-40, ECD19-22, ECD23-50).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTM 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.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Department of Respiratory Medicine (Ghent University) was funded by Scientific Research in Flanders (FWO Vlaanderen, FWO041819N, and FWO-EOS projects G0G2318N, G0H1222N, G025123N) and a Ghent University Grant (BOF/GOA 01G00819).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eJDV:\u003c/strong\u003e Investigation, Writing \u0026ndash; Original Draft, Visualization \u003cstrong\u003eAB:\u003c/strong\u003e Investigation, Writing \u0026ndash; Review \u0026amp; Editing \u003cstrong\u003eVH:\u0026nbsp;\u003c/strong\u003eInvestigation, Writing \u0026ndash; Review \u0026amp; Editing \u003cstrong\u003eLB:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; Review \u0026amp; Editing \u003cstrong\u003eGJ:\u003c/strong\u003e Writing \u0026ndash; Review \u0026amp; Editing \u003cstrong\u003eGB:\u0026nbsp;\u003c/strong\u003eWriting \u0026ndash; Review \u0026amp; Editing \u003cstrong\u003eTM:\u0026nbsp;\u003c/strong\u003eSupervision, Writing \u0026ndash; Review \u0026amp; Editing.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe 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.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWorld Health Organisation (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.who.int/\u003c/span\u003e\u003cspan address=\"https://www.who.int/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMurrison LB, Brandt E, Myers J, Hershey G. Environmental exposures and mechanisms in allergy and asthma development. J Clin Invest. 2019;129(4):1504\u0026ndash;15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCarlsten C, Blomberg A, Pui M, Sandstrom T, Wing Wong S, Alexis N, et al. Diesel exhaust augments allergen-induced lower airway inflammation in allergic individuals: a controlled human exposure study. 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J Clin Invest. 2016;126(9):3279\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Volder J, Vereecke L, Joos G, Maes T. Targeting neutrophils in asthma: a therapeutic opportunity? Biochem Pharmacol. 2020.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoleva E, Hauk P, Hall C, Liu A, Riches D, Martin R, et al. Corticosteroid-resistand asthma is associated with classical antimicrobial activation of airway macrophages. J Allergy Clin Immunol. 2008;122(3):550\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBrusselle G, Koppelman G. Biological therapies for severe asthma. N Engl J Med. 2022;389:157\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHosoki K, Aguilera-Aguirre L, Brasier A, Kurosky A, Boldogh I, Sur S. Facilitation of allergic sensitization and allergic airway inflammation by pollen-induced innate neutrophil recruitment. Am J Respir Cell Mol Biol. 2016;54(1):81\u0026ndash;90.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eToussaint M, Jackson D, Swieboda D, Guedan A, Tsourouktsoglou TD, Ching YM, et al. Host DNA released by NETosis promotes rhinovirus-induced type-2 allergic asthma exacerbation. Nat Med. 2017;23(6):681\u0026ndash;91.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAbdo M, Uddin M, Goldmann T, Marwitz S, Bahmer T, Holz O et al. Raised sputum extracellular DNA confers lung function impairment and poor symptom control in an exacerbation-susceptible phenotype of neutrophilic asthma. Respir Res. 2021;22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim J, Natarajan S, Vaickus L, Bouchard J, Beal D, Cruikshank W, et al. Diesel exhaust particulates exacerbate asthma-like inflamation by increasing CXC chemokines. Am J Pathol. 2011;179(6):2730\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Grove K, Provoost S, Hendriks R, McKenzie A, Seys L, Kumar S, et al. Dysregulation of type 2 innate lymphoid cells and Th2 cells impairs pollutant-induced allergic airway responses. J Allergy Clin Immunol. 2017;139(1):246\u0026ndash;57.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDe Volder J, Bontinck A, De Grove K, Dirven I, Haelterman V, Joos G et al. Trajectory of neutrophilic responses in a mouse model of pollutant-aggravated allergic asthma. Environ Pollut. 2023;329.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakemasa A, Ishii Y, Fukuda T. A neutrophil elastase inhibitor prevents bleomycin-induced pulmonary fibrosis in mice. Eur Respir J. 2012;40(6):1475\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatucci A, Nencini F, Maggiore G, Chiccoli F, Accinno M, Vivarelli E, et al. High proportion of inflammatory CD62L low eosinophils in blood and nasal polyps of severe asthma patients. Clin Exp Allergy. 2023;53(1):78\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCurto E, Mateus-Medina E, Crespo-Lessmann A, Osuna-Gomez R, Ujaldon-Miro C, Garcia-Moral A et al. Identification of two eosinophil subsets in induced sputum from patients with allergic asthma according to CD15 abd CD66b expression. Int J Environ Res Public Health. 2022;19(20).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValencia A, Loffredo L, Misharin A, Berdnikovs S. Phenotypic plasticity and targeting of SiglecF high CD11c low eosinophils to the airway in a murine model of asthma. Allergy. 2016;71(2):267\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRothenberg M. A hidden residential cell in the lung. J Clin Invest. 2016;126(9):3185\u0026ndash;7.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVultaggio A, Accinno M, Vivarelli E, Mecheri V, Maggiore G, Cosmi L, et al. Blood CD62Llow inflammatory eosinophils are related to the severity of asthma and reduced by mepolizumab. Allergy. 2023;78:3154\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorski SA, Hahn Y, Braciale T. Group 2 innate lymphoid cell production of IL-5 is regulated by NKT cells during influenza virus infection. PLoS Pathog 9(9).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGorski SA, Lawrence M, Hinkelman A, Spano M, Steinke J, Borish L et al. Expression of IL-5 receptor alpha by murine and human lung neutrophils. PLoS ONE. 2019;14(8).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLawrence M, Teague G, Feng X, Welch C, Etter E, Negri J, et al. Interleukin-5 receptor alpha (CD125) expression on human blood and lung neutrophils. Ann Allergy Asthma Immunol. 2022;128(1):53\u0026ndash;60.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJorssen J, Van Hulst G, Mollers K, Pujol J, Petrellis G, Baptista A, et al. Single-cell proteomics and transcriptomics capture eosinophil development and identify the role of IL-5 in their lineage transit amplification. Immunity. 2024;57:1\u0026ndash;18.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBusse W, Katial R, Gossage D, Sari S, Wang B, Kolbeck R, et al. Safety profile, pharmacokinetics, and biologic activity of MEDI-563, an anti-IL-5 receptor antibody, in a phase I study of subjects with mild asthma. J Allergy Clin Immunol. 2010;125(6):1237\u0026ndash;44.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchleich F, Moermans C, Seidel L, Kempeneers C, Louis G, Rogister F et al. Benralizumab in severe eosinophilic asthma in real life: confirmed effectiveness and contrasted effect on sputum eosinophilia versus exhaled nitric oxide fraction - PROMISE. ERJ open Res. 2023;9(6).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eScott G, Asrat S, Allinne J, Lim WK, Nagashima K, Birchard D et al. IL-4 and IL-13, not eosinophils, drive type 2 airway inflammation, remodeling and lung function decline. Cytokine. 2023.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenzler K, Borchers M, Crosby J, Cieslewicz G, Hines E, Justice J, et al. Extensive eosinophil degranulation and peroxidase-mediated oxidation of airway proteins do not occur in a mouse ovalbumin-challenge model of pulmonary inflammation. J Immunol. 2001;167(3):1672\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDenzler K, Farmer S, Crosby J, Borchers M, Cieslewicz G, Larson K et al. Eosinophil major basic protein-1 does not contribute to allergen-induced airway pathologies in mouse models of asthma. J Immunol. 2000;165(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePavord ID, Korn S, Howarth P, Bleecker ER, Buhl R, Keene ON, et al. Mepolizumab for severe eosinophilic asthma (DREAM): a multicentre, double-blind, placebo-controlled trial. Lancet. 2012;380(9842):651\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaldat P, Brightling C, Hargadon B, Gupta S, Monteiro W, Sousa A, et al. Mepolizumab and exacerbations of refractory eosinophilic asthma. N Engl J Med. 2009;360(10):973\u0026ndash;84.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel D, Peiro T, Bruno N, Vuononvirta J, Akthar S, Puttur F et al. Neutrophils restrain allergic airway inflammation by limiting ILC2 function and monocyte-dendritic cell antigen presentation. Sci Immunol. 2019;4(41).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Nevel S, van Ovost J, Holtappels G, De Ruyck N, Zhang N, Braun H et al. Neutrophils affect IL-33 processing in response to the respiratory allergen alternaria alternata. Front Immunol. 2021;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBoivin G, Faget J, Ancey PB, Ghasti A, Mussard J, Engblom C, et al. Durable and controlled depletion of neutrophils in mice. Nat Comm. 2020;11(1):2762.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eThierry A. anti-protease treatments targeting plasmin(ogen) and neutrophil elastase may be beneficial in fighting COVID-19. Physiol Rev. 2020;100(4):1597\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoga H, Miyahara N, Fuchimoto Y, Ikeda G, Waseda K, Ono K, et al. Inhibition of neutrophil elastase attenuates airway hyperresponsiveness and inflammation in a mouse model of secondary allergen challenge: neutrophil elastase inhibition attenuates allergic airway responses. Respir Res. 2013;14(1):8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOkeke E, Louttit C, Fry C, Najafabadi AH, Han K, Nemzek J et al. Inhibition of neutrophil elastase prevents neutrophil extracellular trap formation and rescues mice from endotoxic shock. Biomaterials. 2020;238.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZeiher B, Artigas A, Vincent JL, Dmitrienko A, Jackson K, Thompson BT, et al. Neutrophil elastase inhibition in acute lung injury: results of the STRIVE study. Crit Care Med. 2004;32(8):1695\u0026ndash;702.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAikawa N, Ishizaka A, Hirasawa H, Shimazaki S, Yamamoto Y, Sugimoto H, et al. Reevaluation of the efficacy and safety of the neutrophil elastase inhibitor, sivelestat, for the treatment of acute lung injury associated with systemic inflammatory response syndrome: A phase IV study. Pulm Pharmacol Ther. 2011;24(5):549\u0026ndash;54.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Pollutant-aggravated allergic asthma, eosinophils, neutrophils, house dust mite, diesel exhaust particles","lastPublishedDoi":"10.21203/rs.3.rs-4691862/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4691862/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e \u003cp\u003eDiesel exhaust particles (DEP) have been proven to aggravate asthma pathogenesis. We previously demonstrated that 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. 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.\u003c/p\u003e\u003ch2\u003eMaterial \u0026amp; methods\u003c/h2\u003e \u003cp\u003eFemale 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. 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.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe administration of anti-IL-5 significantly decreased eosinophilic responses, affecting both inflammatory and homeostatic eosinophil subsets, upon subacute HDM\u0026thinsp;+\u0026thinsp;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\u0026thinsp;+\u0026thinsp;DEP exposure. Sivelestat treatment tended to decrease BALF inflammation, including eosinophils, upon HDM\u0026thinsp;+\u0026thinsp;DEP exposure, but did not affect lung inflammation.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eInhibition 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.\u003c/p\u003e","manuscriptTitle":"Anti-IL-5 treatment, but not neutrophil interference, attenuates inflammation in a mixed granulocytic asthma mouse model, elicited by air pollution","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-31 10:01:50","doi":"10.21203/rs.3.rs-4691862/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-10-08T17:54:54+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-17T14:21:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"293775219366099896551716639715683625785","date":"2024-08-09T14:08:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-08T17:48:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-08T19:14:01+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-08T05:25:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Respiratory Research","date":"2024-07-05T11:13:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"respiratory-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"rere","sideBox":"Learn more about [Respiratory Research](http://respiratory-research.biomedcentral.com/)","snPcode":"12931","submissionUrl":"https://submission.nature.com/new-submission/12931/3","title":"Respiratory Research","twitterHandle":"@RespiratoryBMC","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a8157ced-f197-4c88-811b-57999516d3fb","owner":[],"postedDate":"July 31st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-02-03T15:58:46+00:00","versionOfRecord":{"articleIdentity":"rs-4691862","link":"https://doi.org/10.1186/s12931-024-03082-9","journal":{"identity":"respiratory-research","isVorOnly":false,"title":"Respiratory Research"},"publishedOn":"2025-01-28 15:56:55","publishedOnDateReadable":"January 28th, 2025"},"versionCreatedAt":"2024-07-31 10:01:50","video":"","vorDoi":"10.1186/s12931-024-03082-9","vorDoiUrl":"https://doi.org/10.1186/s12931-024-03082-9","workflowStages":[]},"version":"v1","identity":"rs-4691862","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4691862","identity":"rs-4691862","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}