Abstract
IL-13 plays a key role during protective type 2 immune responses at mucosal sites, such as during infection with nematodes. However, dysregulation of IL-13 can also contribute to the pathogenesis of atopic and fibrotic diseases such as allergic asthma. Matrix remodelling is an important component of repair processes in the lung but also a hallmark of chronic conditions involving fibrosis. Hence, understanding the role of IL-13 in tissue remodelling has important clinical implications. Since IL-13 shares receptors and signalling pathways with IL-4, disentangling the relative contributions of these type 2 cytokines has been challenging. Additionally, little is known about the singular role of IL-13 following acute tissue injury. In this study, we used Nippostrongylus brasiliensis infection as a model of acute lung tissue damage comparing responses between WT and IL-13-deficient mice, in which IL-4 signalling is intact. Importantly, we found that IL-13 played a critical role in limiting tissue injury and haemorrhaging in the lung following infection. Through proteomic and transcriptomic profiling, we identified IL-13-dependent changes in matrix and associated regulators. We further showed that IL-13 is required for the induction of epithelial-derived type 2 effector molecules such as RELM-α and surfactant protein D. Pathway analyses predicted that IL-13 was heavily involved in the induction of cellular stress responses and regulation of lung epithelial cell differentiation by suppression of Foxa2 pathways. Thus, we propose that IL-13 has tissue-protective functions during lung injury and regulates epithelial cell responses during type 2 immunity in this acute setting.
Introduction
IL-13 is a central effector cytokine with diverse roles during both protective and pathogenic type 2 immune responses. During anti-parasitic immunity, IL-13 is critical for goblet cell hyperplasia and mucus production at mucosal sites (1). These responses are particularly essential for the expulsion of gastrointestinal worms from the host (2). However, the same mucus hypersecretion response is a hallmark of pathogenicity in asthmatic patients (3). Similarly, IL-13 can induce cytoprotective cytokines such as vascular endothelial growth factor to protect from acute lung injury (4), yet drive airway smooth muscle cell contraction leading to broncho-constrictive effects during asthma pathogenesis (5). However, IL-13 and IL-4 have overlapping signalling pathways and both utilize IL-4Rα. IL-4 signals through the type I receptor (IL-4Rα paired with the common γ chain) and the type II receptor (IL-4Rα paired with IL-13Rα1) whereas IL-13 only signals through the type II receptor. However, IL-13 can also ligate IL-13Rα2, which serves primarily as a non-signalling decoy receptor but may have signalling functions distinct from IL-13Rα1 (6, 7). Consequently, disentangling the relative roles of each cytokine in specific cell types has been challenging (8). Whilst IL-13 has been a therapeutic target for asthma with ongoing clinical trials (9), dupilumab, anti-IL-4Rα which inhibits both IL-4 and IL-13 signalling, has been a front-runner treatment showing efficacy in severe asthma patients (10). Thus, understanding the individual roles of these two cytokines has important clinical implications.
Collagen deposition following tissue injury is an important aspect of wound healing and repair. However, in asthma and other chronic inflammatory conditions, dysregulated extracellular matrix (ECM) remodelling and fibrosis leads to many pathological features of disease, with increased deposition of collagen and basement membrane thickening leading to a significant decline in airway function (11, 12). In such disease settings, a pro-fibrotic role for IL-13 is evidenced by its ability to activate pulmonary fibroblasts and stimulate fibrillar collagen synthesis (13–16). Further, the role of IL-13 in promoting liver fibrosis, such as during schistosomiasis and other pathologies, is well-characterised (6, 17, 18). However, there is an apparent context-dependent role for IL-13 in promoting pulmonary fibrosis. For instance, IL-13 is required for fibrotic changes in the lung following Schistosoma mansoni egg challenge but not in the bleomycin model of pulmonary fibrosis (19). While these studies implicate IL-13 in regulating ECM components such as fibrous collagens during a variety of chronic inflammatory conditions, less is known about how IL-13 may affect the ECM and associated regulators as a whole and in acute contexts of lung injury.
In this study, we examined the function of IL-13 during the early stages of infection with the lung-migrating nematode parasite Nippostrongylus brasiliensis. We found that IL-13 was required for the full induction of airway eosinophilia and for limiting lung injury, independent from the other type 2 cytokines IL-4 and IL-5. IL-13 did not have a major effect on collagen dynamics during the early phase of infection. However, IL-13 was critical for the upregulation of type 2 effector molecules involved in collagen regulation and tissue repair, such as epithelial-derived resistin-like molecule alpha (RELM-α). Through both proteomic and transcriptomic approaches, we provide new insight into the contribution of IL-13 to pulmonary helminth infections, in particular suggesting a broader role for IL-13 in overall type 2 immunity during acute lung injury.
Results
Lung injury and vascular damage is exacerbated in the absence of IL-13
Upon infection in the skin with the nematode parasite N. brasiliensis, larvae migrate into the circulation and by day 2 post-infection (D2pi) burst through the lung capillaries and the airways causing extensive tissue damage and haemorrhaging (20). To evaluate the role of IL-13 we used Il13tm3.1Anjm (IL-13 eGFP knock-in) mice, which are deficient for IL-13 when bred as homozygotes (henceforth referred to as Il13-/-). Following infection of Il13-/-, we assessed acute bleeding as well the cumulative clearance of blood (Figure 1A). We first measured the bronchoalveolar lavage (BAL) fluid absorbance at 540 nm, which correlates with the increased presence of haemoglobin due to bleeding (21). In the absence of IL-13, infected mice had an increased level of airway haemorrhage on D2pi relative to WT mice (Figure 1A). Efferocytosis of red blood cells has been shown to occur in the N. brasiliensis model (22). Therefore, as an additional measure of bleeding, we assessed the accumulated uptake of red blood cells over time by measurement of haemosiderin within airway macrophages. Consistent with exaggerated bleeding, infected Il13-/- mice had increased numbers of haemosiderin-laden macrophages in the lung compared to controls, as determined by Prussian blue staining on D6pi (Figure 1A). To assess airway damage following infection, we evaluated H&E-stained lung sections with lacunarity analysis to determine gaps in alveolar structures (23). By D2pi, Il13-/- mice had increased lacunarity scores relative to WT (Figure 1B). Upon gross inspection of lung sections from D2pi, it was evident that infected Il13-/- lungs had larger gaps in the alveoli, presumably in areas proximal to where larvae burst through the tissue (Figure 1C). However, by D6pi lacunarity was comparable between Il13-/- and WT mice (Figure 1B). These data strongly suggest an early host tissue-protective role for IL-13 in partially limiting airway haemorrhaging and tissue damage immediately after N. brasiliensis entry into the lung.
IL-13 is required for airway eosinophilia during N. brasiliensis infection
Neutrophilia is a major contributor to acute lung injury and haemorrhage during helminth infection (24). At D2pi, neutrophils are the predominant infiltrating granulocyte and contribute to worm killing but at the expense of host tissue injury (24, 25). We hypothesised that the exacerbated bleeding and damage in Il13-/- mice was due to the requirement for IL-13 to suppress the early neutrophilia. Thus, we aimed to characterise the role of IL-13 during the early granulocyte response in the lungs. Using N. brasiliensis infection of Il13-/- mice, we found that the proportion of infiltrating neutrophils in the BAL was not significantly altered in the absence of IL-13 when compared to infected WT mice on D2pi (Figure 2A). In terms of absolute numbers, Il13-/- mice only exhibited a slight trend for increased neutrophils compared to controls on D2pi and D4pi (Figure 2B). Between D4-6pi the parasite larvae have exited the lungs, en route to the small intestine and the granulocyte response shifts towards eosinophilia in the airways. As early as D4pi, Il13-/- mice had a major reduction in the proportion and number of eosinophils in the BAL relative to infected WT mice and this response was sustained at D6pi (Figure 2A, 2C). This major reduction in eosinophil proportions in infected Il13-/- mice accounted for the apparent increase in neutrophil percentage at D4pi and D6pi. Together, these results show that IL-13 is required for the full induction of airway eosinophilia following N. brasiliensis infection.
IL-4 and IL-5 do not compensate in the absence of IL-13 after N. brasiliensis infection
Previous studies using the N. brasiliensis model in IL-4Rα-deficient animals or IL-4/IL-13-double-deficient mice did not distinguish between the relative roles of IL-4 and IL-13 during infection (26–28). Unlike those settings, in IL-13 cytokine-deficient mice which have intact type I and II IL-4 receptors, IL-4 may compensate for IL-13-deficiency. To evaluate the potential role of IL-4, we first assessed overall susceptibility to N. brasiliensis in the small intestine which is known to be dependent on both IL-4Rα and IL-13Rα1 (i.e. type I and II IL-4 receptors) (26, 29). Where WT mice had largely cleared their parasites, all Il13-/- mice harboured adult intestinal worms on D6pi (Figure 3A). However, IL-13 can play a role in the differentiation of Th2 cells (30) and it was therefore possible that increased worm burden in IL-13 deficient mice was due to impaired Th2 cell activation following infection. We therefore assessed lung CD4+ T cells on D6pi by ex-vivo stimulation and measurement of intracellular type 2 cytokine expression. We confirmed that lung CD4+ T cells in our Il13-/- mouse strain expressed eGFP in lieu of functional IL-13 after infection with N. brasiliensis when compared to controls (Figure 3B). Importantly, in the absence of IL-13 expression, neither IL-4 nor IL-5 cytokine expression was changed in CD4+ T cells following infection (Figure 3C). Furthermore, measurement of type 2 cytokine mRNA from whole lung also showed no change in the expression of Il4 and Il5 between infected Il13-/- and WT mice (Figure 3D). Thus, IL-13 cytokine-deficient mice become fully susceptible to N. brasiliensis infection with no evidence that altered susceptibility is due to reduced IL-4/IL-5 during the adaptive type 2 response.
Lung proteomic analysis following N. brasiliensis infection
Our results thus far showed that IL-13 has a role in limiting lung injury and enabling eosinophil recruitment into the airways following N. brasiliensis infection. Whilst gross changes in lung structure were not evident at day 6 between WT and Il13-/- mice (Figure 1C), it is feasible that changes to physical lung injury in the absence of IL-13, as was seen on D2 post-infection (Figure 1B & 1C), could have profound effects on the way in which the lung repairs compared to WT animals. To directly address this possibility at the whole tissue level, we performed mass spectrometry on H&E-stained lung sections at D6pi. In infected WT mice, 648 proteins were significantly changed (adjusted P value < 0.05) relative to uninfected mice with most of these proteins being upregulated (Figure 4A). We then specifically analysed changes in the ECM and matrix-related proteins (defined from the Matrisome Project (31)), performing hierarchical clustering across groups after N. brasiliensis infection (Figure 4B). Several collagen types (notably collagens I, III, and VI) clustered together, with proteins reduced in WT mice after infection, an effect not replicated in Il13-/- mice. Such differences may relate to an already lower level of expression of these collagens under basal conditions in Il13-/- vs. WT mice. Evaluation of the relative abundance of collagen types across groups confirmed dysregulated collagen levels in the absence of IL-13, with N. brasiliensis infection altering collagen expression in WT but not Il13-/- mice (Figure 4C). Fibrillar collagens I and III, while already low in Il13-/- mice, were decreased in infected WT mice and basement membrane-associated collagens IV and VI were also dysregulated in Il13-/- mice. To visualise total collagen deposition, Masson’s trichrome staining was performed but did not show any major differences between infected WT and Il13-/- mice at D6pi (Figure S1). Further, hydroxyproline levels were measured as a proxy for total collagen in the lung and confirmed no gross differences between the groups (Figure S1). To gain further insight into global IL-13-dependent changes in the lung, we performed pathway analysis on differentially expressed proteins (by non-adjusted P value < 0.05) comparing infected WT and Il13-/- mice (Figure 4D). IL-13-dependent canonical pathway analysis showed a downregulation (blue) in pathways relating to protein synthesis and cellular stress after infection, with 71% of endoplasmic reticulum stress pathway-associated proteins being regulated by IL-13. Additionally, mTOR signalling and antigen presentation pathways appeared to be altered in the absence of IL-13. Whilst proteomic analysis of lungs from infected Il13-/- mice did not reveal any differences in matrisome components compared to infected WT mice, RELM-α and surfactant protein D (SP-D), two proteins heavily implicated in type 2 immunity (32, 33), were significantly decreased in infected Il13-/- lungs amongst the total proteome (Figure S2). Several other key proteins were found to be significantly downregulated in Il13-/- mice based on relative abundance when compared to infected WT mice including AMCase, BRP39, and Ym1 (Figure 4E) molecules also strongly associated with pulmonary type 2 immunity (25, 34). These data suggest that IL-13 may not directly regulate ECM remodelling following acute lung injury on D6pi following N. brasiliensis infection. However, IL-13 is critical for the induction of type 2 effector molecules which may determine the subsequent tissue repair responses.
IL-13 is required for induction of epithelial cell-derived RELM-α
The proteomic analysis led us to further characterise the contribution of IL-13 to RELM-α expression in the lung. RELM-α is an important type 2-associated effector molecule implicated in repair processes that have been previously described in the skin and lungs (35, 36). We measured RELM-α protein in the BAL fluid by ELISA (i.e. release into the airways) and consistent with our proteomics data found decreased RELM-α levels in Il13-/- mice infected with N. brasiliensis relative to infected WT mice (Figure 5A). Similarly, quantification of mRNA in the whole lung showed that the induction of Retnla expression on D4pi and D6pi was reduced in the absence of IL-13 (Figure 5B). In addition, we performed immunofluorescence staining in lung sections and found that the airways, which were highly RELM-α+ following infection in WT mice, appeared largely diminished in RELM-α expression in infected Il13-/- mice (Figure 5C). To directly quantify this cellular RELM-α expression, lung cell suspensions were analysed by flow cytometry for intracellular RELM-α. In the absence of IL-13, CD45−EpCAM+ epithelial cells had significantly impaired expression of RELM-α as early as D2pi and was still muted by D6pi when compared to WT controls (Figure 5D). Consistent with previous findings (36, 37), epithelial cells were the major source of RELM-α at these time points whilst other cellular sources such as alveolar macrophages, neutrophils, and eosinophils, were relatively unchanged in the absence of IL-13 following infection (data not shown). As a complementary experiment, equimolar amounts of systemic IL-4 and IL-13 were each delivered by intraperitoneal injection into WT mice and 18 h later epithelial RELM-α expression was measured in the lungs. In contrast to IL-4 which had no apparent effect, systemic IL-13 delivery was able to potently drive RELM-α expression in CD45−EpCAM+ epithelial cells (Figure 5E). However, we hypothesized that the differences in response to comparable doses of IL-4 vs. IL-13 may be due to bioavailability in the lung when delivered systemically. Therefore, we directly administered the cytokines to the lung via intranasal instillation and found that both IL-4 and IL-13 induced epithelial cell derived-RELM-α (Figure 5F). Taken together, these results show that IL-13 is both necessary and sufficient to stimulate lung epithelial cell RELM-α and suggest that lung epithelial cells may be more sensitive to IL-13 relative to IL-4.
IL-13 broadly regulates type 2 immunity in the lung during N. brasiliensis infection
Due to the technical limitations of proteomics not being able to detect low molecular weight cytokines and chemokines, we performed transcriptional profiling of lung tissue from N. brasiliensis-infected WT and Il13-/- mice to better define the role of IL-13 during type 2 immunity in acute injury settings and to highlight potential mechanisms/pathways involved. Using the Nanostring Myeloid Innate Immunity v2 panel, we screened for differentially regulated genes across groups on D6pi. Principal component analysis showed distinct separation between groups based on infection status (PC1) and genotype (PC2) (Figure 6A). Genes were grouped by unsupervised hierarchical clustering and differentially expressed genes were represented as a heatmap to identify expression patterns across all groups (Figure 6B). Notably, various signature type 2 genes were downregulated in infected Il13-/- mice such as Chil3/4, Arg1, Il33, Retnla, and the eotaxins (encoded by Ccl11 and Ccl24). Conversely, a cluster of pro-inflammatory genes that included Mmp8, Il18, and Tlr2 were upregulated in infected IL-13-deficient mice (Figure 6B). Differentially expressed genes were further characterised using Ingenuity Pathway Analysis to identify potential upstream regulators (Figure 6C). Very few regulators were predicted to be upregulated during IL-13 deficiency but included the airway epithelial cell-associated Foxa2 (38) as well as the adenosine A1 receptor, Adora1. In summary, these data demonstrate a broad role for IL-13 in regulating type 2 immunity and epithelial cell function during acute helminth infection in the lung.
Discussion
Despite sharing receptors/signalling components with IL-4, various studies have established a distinct role for IL-13 during type 2 immunity and argued against simple functional redundancy between these two cytokines. For example, IL-4 cytokine-deficient mice, but not IL-4Rα-deficient mice, are able to expel N. brasiliensis from the gut due to intact IL-13 signalling (29). In addition, IL-13-deficient mice fail to clear N. brasiliensis parasites from the gut when compared to IL-4-deficient and WT mice (39). Whilst these studies establish a clear role for IL-13 in mediating type 2 immunity in the small intestine and the establishment of the adaptive immune response, very little is known about the functions of IL-13 in the earlier lung stage of this infection model. Our study reinforces these earlier findings but also reveal a crucial protective role for IL-13 in limiting acute lung injury, promoting airway eosinophilia, and inducing type 2 effector proteins. These lung-specific effects of IL-13 are also consistent with other work showing that pulmonary eosinophilia is impaired in IL-13Rα1-deficient mice during asthma (40, 41).
Our data demonstrates a profound epithelial cell-specific effect of IL-13 during acute lung injury with N. brasiliensis. Expression of the type I and type II IL-4 receptors is restricted based on cell type. Hematopoietic cells predominantly express the type I receptor while non-hematopoietic cells such as epithelial cells primarily express the type II receptor (42). While the type II receptor can be ligated by both IL-4 and IL-13, IL-13 can outcompete IL-4 by more efficiently promoting receptor assembly (43). In terms of epithelial cell expression, a model of mechanical injury shows that IL-13Rα1 expression increases at the wound edge in cultured alveolar epithelial cells (44). As we observed exacerbated airway injury in Il13-/- mice, it is therefore plausible in our model of nematode-induced lung injury that early alveolar epithelial cell function is dependent on the presence of IL-13. Our study also highlights impaired expression of type 2 effector molecules RELM-α and SP-D by lung epithelial cells in the absence of IL-13. RELM-α is an IL-4Rα-dependent protein involved in lung tissue repair and has been shown to mediate collagen crosslinking via lysl hydroxylase 2 (32, 35). Interestingly, SP-D has known roles in promoting immunity to N. brasiliensis and is required for the upregulation of RELM-α in the lung (33). Further, our transcriptional profiling predicted a dysregulation of the transcription factor Foxa2 in the absence of IL-13 after infection. Foxa2 is required for alveolarization and negatively regulates goblet cell hyperplasia (38). In terms of type 2 function, the predicted increased Foxa2 activity in Il13-/- mice is consistent with previous studies showing that IL-13 decreases Foxa2 expression to enable mucin production by airway epithelial cells (45–47). We therefore hypothesize a mechanism of IL-13-mediated suppression of Foxa2 in alveolar epithelial cells that enables the expression and release of type 2 effector molecules such as RELM-α. It is also notable that MMP-8 and IL-18 were upregulated in infected Il13-/- mice. MMP-8 and IL-18 are pro-inflammatory markers associated with chronic obstructive pulmonary disease (COPD) (48, 49). As N. brasiliensis infection eventually results in emphysema (22) that resemble features of COPD, our data thus suggest a complex role for IL-13 in the development emphysema.
Given the attribution of IL-13 to tissue remodelling and fibrosis in a variety of contexts, we anticipated changes to the matrisome in our acute lung injury model using proteomic analysis. While we saw many changes to the lung matrisome due to infection in WT mice, we did not observe major changes in lung collagens in Il13-/- mice after infection. However, we saw that some major collagens (especially collagen I and III) were reduced in abundance following infection of WT mice. This reduction in the expression of these collagens did not occur in Il13-/- mice, which already had a baseline decrease in these collagens. It is thus possible that IL-13 has a developmental role in collagen organisation that predisposed the Il13-/- mice to enhanced tissue injury. A limitation of our matrisome analysis is that we did not account for potential changes in the localisation of specific collagen types and cannot exclude a role for glycosylation state or other post-translational modifications. Additionally, the precise timing of changes in the IL-13-dependent proteome are to be resolved since we observed both early (D2pi) and late (D6pi) effects in our model. Nevertheless, our observation that Il13-/- mice had enhanced haemorrhaging lead us to hypothesize that there may be compromised endothelial cell integrity (e.g. with respect to basement membrane collagen composition) during lung injury in the absence of IL-13. To the best of our knowledge, no prior study has shown a direct protective effect of IL-13 in mitigating airway haemorrhage, which warrants further investigation mechanistically. Additionally, pathway analyses of the proteomic data predicted a dysregulation in protein synthesis and cellular stress pathways (presumably in epithelial cells) upon acute tissue injury in the absence of IL-13, which will be the subject of future studies.
In conclusion, our study has demonstrated a pivotal role for IL-13 in limiting tissue injury and airway bleeding and suggest broader functions for IL-13 in regulating type 2 immunity, in the context of acute lung damage.
Materials and Methods
Mice and ethics statements
WT C57BL/6J were purchased from Charles River UK. Il13tm3.1Anjm (50) were maintained on a C57BL/6J background and bred in-house at the University of Manchester. Most experiments had a combination of purchased WT mice and littermate controls. Female and male mice were used at age 8-14 weeks old. Animals were housed in individually ventilated cages with food and water provided ad libitum. Experimental mice were randomly assigned to groups. All experiments were carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and under a Project License (70/8548) granted by the Home Office and approved by local Animal Ethics Review Group at the University of Manchester.
N. brasiliensis infection
N. brasiliensis worms were propagated as previously described (51). Infective L3 larvae were isolated and mice were injected with 250 L3s subcutaneously. Mice were culled by overdose of pentobarbitone i.p. and BAL was performed with 10% FBS in PBS and lung lobes were collected. Perfusion was not performed due to the compromised lung vasculature during N. brasiliensis infection. On D2pi, BAL fluid absorbance was measured at 540 nm using a VersaMax microplate reader (Molecular Devices) to assess airway haemorrhage. Lung lobes were either stored in RNAlater (Thermofisher), fixed in 10% neutral-buffered formalin for histology or minced and digested with Liberase TL (Roche) for 30 min at 37°C for analysis of lung epithelial cells by flow cytometry and ex vivo stimulation of lung T cells using cell stimulation cocktail (plus protein transport inhibitors) (eBioscience). On D6pi, adult small intestinal worms were counted using a dissecting microscope following incubation of the small intestine at 37 °C to collect live adult worms that migrate out of the tissue.
Flow cytometry
Single cell suspensions were washed in PBS and Live/Dead staining (Thermofisher) was performed. Samples were Fc-blocked using α-CD16/32 (2.4G2) (BD Biosciences) and mouse serum (BioRad). Blocking and subsequent surface staining was performed using PBS containing 2 mM EDTA, 2% FBS, and 0.05% NaN3. Antibodies used for staining are listed in Table 1. Following surface staining, cells were incubated with IC fixation buffer (ThermoFisher) prior to permeabilization for intracellular staining. For secondary detection of Ym1 and RELM-α, Zenon goat and rabbit antibody labels (ThermoFisher) were used. For RELM-α intracellular staining, cells were directly stained without stimulation or protein transport inhibition. Lung CD4+ T cells were stimulated ex vivo with cell stimulation cocktail containing protein transport inhibitors (ThermoFisher) for 4 h at 37° C prior to staining. For cell quantification, some samples were spiked with 10 μm polystyrene beads (Sigma-Aldrich) prior to acquisition. Data were acquired on a BD LSRFortessa flow cytometer and analysed using FlowJo v10 software.
RNA extraction and quantitative real-time PCR
Tissue samples stored in RNAlater (Thermo Fisher Scientific) were processed for RNA extraction using a TissueLyser II and QIAzol reagent (Qiagen). Isolated RNA was quantified using a Qubit fluorimeter and RNA BR kit (Qiagen). cDNA was synthesized using Tetro reverse transcription kit (Bioline) and oligo dT 15-mers (Integrated DNA Technologies). Quantitative real-time PCR was performed using SYBR green mix (Agilent Technologies) and a LightCycler 480 II (Roche). A list of primer sequences used are shown in Table 2. Gene expression levels were determined by second derivative maxima using standard curves (LightCycler software) and expressed relative to the housekeeping gene Rpl13a.
Lung proteomic analysis
Samples from slides containing whole lung tissue sections were scraped excluding major blood vessels and processed as previously described (52). Peptides were evaluated by liquid chromatography coupled tandem mass spectrometry using an UltiMate® 3000 Rapid Separation LC system (Dionex Corporation) coupled to a Q Exactive HF mass spectrometer (ThermoFisher). Raw spectra were aligned using MAXQuant software v1.6.17.0 (53) with the variable modifications of proline and methionine oxidation in addition to “matched between runs” being enabled. Raw data was then imported into R for differential analysis with MSqRob (54) using the default pipeline. Heatmaps were plotted using scaled log10-transformed LFQ counts. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (55) partner repository with the dataset identifier PXD021853.
Hydroxyproline assay
Hydroxyproline assay was performed as previously described (56). Whole lungs were incubated overnight in 6 M HCl, in screw-top tubes at 100 °C covered with aluminium foil. Tubes were cooled to RT and centrifuged at 12,000 x g for 3 min. Hydroxyproline standards were prepared (starting at 0.25 mg/ml) and serially diluted with 6 M HCl. Samples and standards (50 μl) were transferred into Eppendorf tubes and 450 μl chloramine T reagent (55.79 mM chloramine T (initially dissolved in 50% N-propanol) in acetate citrate buffer — 0.88 M sodium acetate trihydrate, 294 mM citric acid, 1.2% glacial acetic acid, 0.85 M sodium hydroxide — adjusted to pH 6.5; reagents from Sigma) was added to each tube and incubated at RT for 25 min. Ehrlich’s reagent (500 μl; 1 M 4-dimethylaminobenzaldehyde in N-propanol:perchloric acid (2:1), Sigma) was added to each tube and incubated at 65 °C for 10 min and absorbance at 558 nm was measured.
Histological and immunofluorescence staining
Whole left lung lobes were paraffin embedded and 5 μm sections were prepared for haematoxylin/eosin or Masson’s Trichrome staining. For visualisation of haemosiderin deposits, lung sections were rehydrated and stained with a solution of 5% potassium ferrocyanide with 10% HCl (Prussian blue) for 20 min; sections were then rinsed with dH2O and counterstained with a solution of 1% neutral red and 1% acetic acid for 5 min before being rinsed with dH2O and dehydrated for mounting. Brightfield images were captured using an Olympus slide scanner and analysed using CaseViewer software (3DHISTECH).
For immunofluorescence staining, lung sections were rehydrated and subjected to heat-mediated antigen retrieval using citrate buffer (10 mM sodium citrate, 0.05% Tween-20, pH 6.0) followed by primary antibody incubation overnight at 4 °C using biotin-anti-Ym1 and anti-RELM-α (Table 1); sections were then incubated with anti-rabbit FITC (Invitrogen) and streptavidin NL-557 (R&D systems) for 30 min RT before being mounting using Fluoromount-G containing DAPI (SouthernBiotech). Fluorescent slides were imaged using an EVOS™ FL Imaging System (ThermoFisher) and analysed using ImageJ.
Lung lacunarity analysis
Slide-scanned images of H&E-stained lung lobes were processed in a KNIME software workflow to obtain 50 random regions of interests (ROIs) across the whole lung section. ROIs that contained lobe boundaries or extensive artefacts were excluded from the analysis. The ROIs were then converted to binary images and lacunarity was quantified using the FracLac plugin for ImageJ (default settings). Lacunarity values of all the ROIs were averaged to obtain estimates for the entire lobe.
ELISA
BAL supernatants were analysed for RELM-α using commercially available ELISA kits (Peprotech). Analytes were detected using horse radish peroxidase-conjugated streptavidin and TMB substrate (BioLegend) and stopped with 1 M HCl. Final absorbance at 450 nm was measured using a VersaMax microplate reader (Molecular Devices).
IL-4-Fc and IL-13-Fc injections
To extend the half-life of IL-4 and IL-13, fusion proteins were generated of mouse IL-4 and IL-13 with the Fc portion of IgG1 (custom order with Absolute Antibody). Mice were injected intraperitoneally with either PBS, 10 μg IL-4-Fc, or 10 μg IL-13-Fc in 100 μl PBS. In other experiments, mice were anaesthetised using isoflurane inhalation and intranasally instilled with either PBS, 10 μg IL-4-Fc, or 10 μg IL-13-Fc in 40 μl PBS. The following day at 18 h post-treatment, mice were culled for lung cell analysis by flow cytometry.
Nanostring analysis
Quality control was performed on RNA samples with an Agilent 2200 TapeStation system prior to downstream analyses. Samples were diluted and 100 ng of RNA was processed for running on a Nanostring nCounter® FLEX system using the Myeloid Innate Immunity v2 panel. Raw count data was imported into R for analysis using the limma package (57). Internal housekeeping and negative control probes were used to ensure data integrity and set thresholds for minimum expression. Data was normalised using the edgeR package (58) and then differential expression was calculated using the limma package. Figures were generated in R using ggplot and the complexheatmap package. Heatmaps were plotted using scaled normalised counts.
Statistical analyses
Graphpad Prism 8 software was used for all statistical analyses. Data were assessed to be normally distributed by the D’Agostino-Pearson omnibus normality test. Differences between experimental groups were assessed by ANOVA (for normally distributed data) followed by Tukey-Kramer post hoc multiple comparisons test or an unpaired two-tailed Student’s t test. In cases where data were not normally distributed, a Kruskal-Wallis test was used. For gene expression data, values were log2 transformed to achieve normal distribution. Comparisons with a P value of less than 0.05 were considered to be statistically significant.
Disclosure
The authors declare no competing interests.
The authors declare no conflicts of interest.
Acknowledgements
This work was supported by the Wellcome Trust (203128/Z/16/Z, 110126/Z/15/Z and 106898/A/15/Z) and the Medical Research Council UK (MR/K01207X/2). TES was supported by Medical Research Foundation UK joint funding with Asthma UK (MRFAUK-2015-302). We thank Andrew McKenzie (Cambridge) for providing the Il13tm3.1Anjm mice. We further thank the Flow Cytometry, Bioimaging, Genomic Technologies, BioMS and Biological Services core facilities at the University of Manchester.