Healthy repair of the alveoli requires alveolar stem cells to differentiate into cells designed for gas exchange. In chronic lung fibrotic disease like idiopathic pulmonary fibrosis (IPF), alveolar epithelial cells regenerate abnormally. The cause of this is unknown but its highly cellular, inflamed and structurally altered regenerating niche is likely to be relevant. Here, in unique sets of human lung tissues capturing advancing fibrosis, and with a 33-plex single cell imaging mass cytometry (IMC), we provide a high resolution and comprehensive temporo-spatial cell atlas of the regenerating alveolar niches. Using a suite of mathematical tools, we expose an organized immune network and identify CD206hi alveolar macrophages as a central immune cell in the immune-alveolar epithelial interactome. A spatially-directed receptor-ligand analysis offers an in-silico mechanism by which these macrophages influenced alveolar regeneration. Our study unravels a complex cellular environment and identifies key interactions that influence alveolar regeneration in a fibrotic lung. ### Competing Interest Statement Andrew Fisher declares the following: Grant Award to Newcastle University from Mallinckrodt Pharmaceuticals, Chiesi and Eurofins/TGI Consultancy via Newcastle University with Fibrofind, Mallinckrodt and Sanofi James Shaw declares the following: Scientific Advisory Board for Mogrify (consultancy fee paid to Newcastle University). ### Funding Statement The study is funded by combination of grants from MRC UKRMP Grant MR/S020918/1; NIHR Oxford Biomedical Research Centre grant; University of Oxford Medical Science Division and Newcastle University Flow Core Facility. LPH is supported by MRC (grant CFR01480) and the NIHR Oxford Biomedical Research Centre. AJF is supported by the National Institute for Health and Care Research (NIHR) Blood and Transplant Research Unit in Organ Donation and Transplantation (NIHR203332), a partnership between NHS Blood and Transplant, University of Cambridge and Newcastle University. The views expressed are those of the author(s) and not necessarily those of the NIHR, NHS Blood and Transplant or the Department of Health and Social Care. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: All patients provided informed consent for use of their tissue via the Cellular and Molecular Mechanisms in Chronic Lung Diseases (EXPLANT) study which was approved by the NHS Research Ethics Service (11/NE/0291) and was sponsored by Newcastle Upon Tyne Hospitals NHS Foundation Trust ( R&D ref 5885). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes

Severe lung damage resulting from COVID-19 involves complex interactions between diverse populations of immune and stromal cells. In this study, we used a spatial transcriptomics approach to delineate the cells, pathways, and genes present across the spectrum of histopathological damage in COVID-19–affected lung tissue. We applied correlation network–based approaches to deconvolve gene expression data from 46 areas of interest covering more than 62,000 cells within well-preserved lung samples from 3 patients. Despite substantial interpatient heterogeneity, we discovered evidence for a common immune-cell signaling circuit in areas of severe tissue that involves crosstalk between cytotoxic lymphocytes and pro-inflammatory macrophages. Expression of IFNG by cytotoxic lymphocytes was associated with induction of chemokines, including CXCL9, CXCL10, and CXCL11, which are known to promote the recruitment of CXCR3+ immune cells. The TNF superfamily members BAFF (TNFSF13B) and TRAIL (TNFSF10) were consistently upregulated in the areas with severe tissue damage. We used published spatial and single-cell SARS-CoV-2 data sets to validate our findings in the lung tissue from additional cohorts of patients with COVID-19. The resulting model of severe COVID-19 immune-mediated tissue pathology may inform future therapeutic strategies.

Background: Many studies have described the blood immune profile of severe COVID-19 patients but few have elucidated the cellular composition and organisation in lung tissue. Method: We used tissue-based single cell mass cytometry and a suite of mathematical tools to generate a spatial analytical pipeline which searched for statistically significant cell-level co-location of immune cells and specified lung microstructures, without a priori specification of neighborhood environment. Lung samples from fatal COVID-19 patients (n=12) were stained with a 35-metal-tagged antibody panel and imaged with the Tissue Hyperion system. Results: 677,931 single segmented cells were derived and 38 immune and structural cell types annotated. Monocytes, macrophages and immature neutrophils showed highest abundance across all histopathology stages of injury and repair. A striking cluster of cells was observed, comprising immature neutrophils and cytotoxic CD8 with high expression of Granzyme B, IFN-β and IFN-γ. These were spatially co-located with proliferating alveolar epithelium in areas with diffuse alveolar damage. Network analysis showed that these inflammatory foci connected other immature neutrophil and monocyte subsets across all histopathology stages. Conclusion: The cellular map of fatal COVID-19 lungs is marked by highly active clusters of immature neutrophils and cytotoxic CD8 T cells, spatially linked with alveolar progenitor cells, and temporally with the diffuse alveolar damage stage. These findings provide new insights into how immune cells interact in the lungs of severe COVID-19 disease.

Single cell spatial interrogation of the immune-structural interactions in COVID -19 lungs is challenging, mainly because of the marked cellular infiltrate and architecturally distorted microstructure. To address this, we develop a suite of mathematical tools to search for statistically significant co-locations amongst immune and structural cells identified using 37-plex imaging mass cytometry. This unbiased method reveals a cellular map interleaved with an inflammatory network of immature neutrophils, cytotoxic CD8 T cells, megakaryocytes and monocytes co-located with regenerating alveolar progenitors and endothelium. Of note, a highly active cluster of immature neutrophils and CD8 T cells, is found spatially linked with alveolar progenitor cells, and temporally with the diffuse alveolar damage stage. These findings offer further insights into how immune cells interact in the lungs of severe COVID-19 disease. We provide our pipeline [Spatial Omics Oxford Pipeline (SpOOx)] and visual-analytical tool, Multi-Dimensional Viewer (MDV) software, as a resource for spatial analysis.

Mϕs are the main innate immune cells in the lung at homeostasis, with important roles in host defence and immune modulation. Alveolar Mϕs (AMs) and interstitial Mϕs (IMs) are the two lung Mϕ subsets, so called according to the sites they reside in. These subsets are also defined by their origins and immunological microenvironment, which endow these cells with distinct features and plasticity. This review summarizes the latest definitions and functions of lung Mϕs during homeostasis and provides exemplar of their divergent roles in lung fibrosis.

Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. Circulating monocytes have been implicated in immune pathology in IPF but their phenotype is unknown. In this work, we determined the immune phenotype of monocytes in IPF using multi-colour flow cytometry, RNA sequencing and corresponding serum factors, and mapped the main findings to amount of lung fibrosis and single cell transcriptomic landscape of myeloid cells in IPF lungs. We show that monocytes from IPF patients displayed increased expression of CD64 (FcγR1) which correlated with amount of lung fibrosis, and an amplified type I IFN response ex vivo . These were accompanied by markedly raised CSF-1 levels, IL-6, and CCL-2 in serum of IPF patients. Interrogation of single cell transcriptomic data from human IPF lungs revealed increased proportion of CD64 hi monocytes and “transitional macrophages” with higher expression of CCL-2 and type I IFN genes. Our study shows that monocytes in IPF patients are phenotypically distinct from age-matched controls, with a primed type I IFN pathway that may contribute to driving chronic inflammation and fibrosis. These findings strengthen the potential role of monocytes in the pathogenesis of IPF.

Idiopathic pulmonary fibrosis (IPF) is the most severe form of chronic lung fibrosis. It is unclear how monocytes contribute to fibrosis in IPF. Here, we show that levels of circulating monocytes correlated directly with the extent of fibrosis in IPF lungs, as measured by CT imaging. IPF monocytes were phenotypically distinct, displayed increased expression of CD64 (FcgR1), a type 1 IFN gene expression signature by RNA sequencing of monocytes, and an amplified type 1 IFN response ex vivo. CD64 levels correlated with levels of IFN-stimulated genes in monocytes. These abnormalities were accompanied by markedly raised CSF-1 levels in the serum, prolonged survival of monocytes with impaired differentiation to macrophages, and increased numbers of monocytes in lung tissue. Interrogation of single cell transcriptomic data from human IPF lung explants revealed a increased proportion of monocytes and 9transitional macrophages9 with high CCL-2 expression; and reduced mature macrophages. The transitional macrophages in IPFs also expressed higher CD64 and were enriched for type I IFN gene sets. Our study defines the key monocytic abnormalities in IPF, proposing type 1 IFN-primed monocytes as a potential driver of chronic lung fibrosis. It provides a clear biological rationale for targeting monocytes therapeutically in IPF.

Idiopathic pulmonary fibrosis (IPF) is the most severe form of lung fibrosis. It is progressive, and has an extremely poor outcome and limited treatment options. The disease exclusively affects the lungs, and thus less attention has been focused on blood-borne immune cells. which could be a more effective therapeutic target than lung-based cells. Here, we questioned if circulating monocytes, which has been shown to be increased in IPF, bore abnormalities that might contribute to its pathogenesis. We found that levels of circulating monocytes correlated directly with the extent of fibrosis in the lungs, and increased further during acute clinical deterioration. Monocytes in IPF were phenotypically distinct, displaying increased expression of CD64, a type 1 IFN gene expression signature and a greater magnitude of type 1 IFN response when stimulated. These abnormalities were accompanied by markedly raised CSF-1 levels in the serum, prolonged survival of monocytes ex vivo , and increased numbers of monocytes in lung tissue. Our study defines the key monocytic abnormalities in IPF, proposing type 1 IFN-primed monocytes as a potential driver of an aberrant repair response and fibrosis. It provides a rationale for targeting monocytes and identifies monocytic CD64 as a potential specific therapeutic target for IPF.

Children with severe therapy-resistant asthma (STRA) have poor control despite maximal treatment, while those with difficult asthma (DA) have poor control from failure to implement basic management, including adherence to therapy. Although recognised as clinically distinct, the airway molecular phenotype, including the role of innate lymphoid cells (ILCs) and their response to steroids in DA and STRA is unknown.Immunophenotyping of sputum and blood ILCs and T-cells from STRA, DA and non-asthmatic controls was undertaken. Leukocytes were analysed longitudinally pre- and post-intramuscular triamcinolone in children with STRA. Cultured ILCs were evaluated to assess steroid responsiveness in vitroAirway eosinophils, type 2 T-helper (Th2) cells and ILC2s were significantly higher in STRA patients compared to DA and disease controls, while IL-17+ lymphoid cells were similar. ILC2s and Th2 cells were significantly reduced in vivo following intramuscular triamcinolone and in vitro with steroids. Furthermore, asthma attacks and symptoms reduced after systemic steroids despite persistence of steroid-resistant IL-17+ cells and eosinophils.Paediatric STRA and DA have distinct airway molecular phenotypes with STRA characterised by elevated type-2 cells. Systemic corticosteroids, but not maintenance inhaled steroids resulted in improved symptom control and exacerbations concomitant with a reduction in functional ILC2s despite persistently elevated IL-17+ lymphoid cells.

The human lung is constantly exposed to the environment and potential pathogens. As the interface between host and environment, the respiratory epithelium has evolved sophisticated sensing mechanisms as part of its defense against pathogens. In this review, we examine how the respiratory epithelium senses and responds to influenza A virus, the biggest cause of respiratory viral deaths worldwide.

Group 2 innate lymphoid cells (ILC2s) are enriched in mucosal tissues (e.g., lung) and respond to epithelial cell-derived cytokines initiating type 2 inflammation. During inflammation, ILC2 numbers are increased in the lung. However, the mechanisms controlling ILC2 trafficking and motility within inflamed lungs remain unclear and are crucial for understanding ILC2 function in pulmonary immunity. Using several approaches, including lung intravital microscopy, we demonstrate that pulmonary ILC2s are highly dynamic, exhibit amoeboid-like movement, and aggregate in the lung peribronchial and perivascular spaces. They express distinct chemokine receptors, including CCR8, and actively home to CCL8 deposits located around the airway epithelium. Within lung tissue, ILC2s were particularly motile in extracellular matrix-enriched regions. We show that collagen-I drives ILC2 to markedly change their morphology by remodeling their actin cytoskeleton to promote environmental exploration critical for regulating eosinophilic inflammation. Our study provides previously unappreciated insights into ILC2 migratory patterns during inflammation and highlights the importance of environmental guidance cues in the lung in controlling ILC2 dynamics.

A disintegrin and metalloprotease (ADAM)33 is a susceptibility gene for asthma and bronchial hyperresponsiveness (BHR). ADAM33 is a transmembrane protein, but a soluble protein containing the metalloprotease domain (sADAM33) has previously been identified in bronchoalveolar lavage fluid (BALF) from subjects with asthma and its levels significantly and negatively correlated with FEV1, suggesting a role in the development of airflow obstruction.1Lee J.Y. Park S.W. Chang H.K. Kim H.Y. Rhim T. Lee J.H. et al.A disintegrin and metalloproteinase 33 protein in patients with asthma: relevance to airflow limitation.Am J Respir Crit Care Med. 2006; 173: 729-735Crossref PubMed Scopus (99) Google Scholar We have shown that sADAM33 in asthmatic BALF is enzymatically active and that the sADAM33 metalloprotease causes angiogenesis and myogenesis in vivo or ex vivo.2Davies E.R. Kelly J.F. Howarth P.H. Wilson D.I. Holgate S.T. Davies D.E. et al.Soluble ADAM33 initiates airway remodeling to promote susceptibility for allergic asthma in early life.JCI Insight. 2016; 1 (e87632)Crossref Scopus (29) Google Scholar, 3Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar Furthermore, in vivo allergen challenge causes shedding of enzymatically active sADAM33 into BALF of wild-type mice, and in a transgenic mouse model, lung-specific sADAM33 expression causes airway remodeling, which enhances eosinophil recruitment with associated BHR following allergen sensitization and challenge.2Davies E.R. Kelly J.F. Howarth P.H. Wilson D.I. Holgate S.T. Davies D.E. et al.Soluble ADAM33 initiates airway remodeling to promote susceptibility for allergic asthma in early life.JCI Insight. 2016; 1 (e87632)Crossref Scopus (29) Google Scholar Although TGF-β is a trigger for sADAM33 release in vitro,3Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar there is no mechanistic information or evidence of in vivo relevance. Because it is not possible to test directly the importance of TGF-β in ectodomain shedding in human asthma, we characterized the mechanism(s) of the TGF-β–induced ectodomain shedding of murine ADAM33 and determined its importance for shedding of ADAM33 in vivo. Detailed methodology is provided in the Methods section in this article's Online Repository at www.jacionline.org. Initially, we confirmed that murine ADAM33 was similar to human ADAM33 in its sensitivity to TGF-β–induced ectodomain shedding.3Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar As expected, TGF-β treatment caused a dose-dependent increase in sADAM33 in supernatants of Cos-7 cells expressing murine ADAM33 (see Fig E1, A and B, in this article's Online Repository at www.jacionline.org). The main band had a molecular weight of around 102 kDa, indicating that the entire ectodomain had been shed; however, further processing was also evident, suggesting loss of the inhibitory prodomain. Consistent with this, there was a significant increase in ADAM33 enzymatic activity in cell-free supernatants following TGF-β treatment (Fig E1, C). Pericellular proteolysis is frequently mediated by members of the ADAM or MT-MMP families that are sensitive to the broad-spectrum hydroxamic acid–based inhibitor, GM6001; however, we found that it did not affect the shedding (see Fig E2, A and B, in this article's Online Repository at www.jacionline.org) or activity (Fig E2, C) of ADAM33. We also confirmed that GM6001 had no effect on the activity of purified recombinant ADAM33 (Fig E2, D). Because ADAM33 has a unique substrate-binding site and its catalytic activity is insensitive to GM6001,4Orth P. Reichert P. Wang W. Prosise W.W. Yarosh-Tomaine T. Hammond G. et al.Crystal structure of the catalytic domain of human ADAM33.J Mol Biol. 2004; 335: 129-137Crossref PubMed Scopus (74) Google Scholar we postulated that ADAM33 ectodomain shedding was autocatalytic. Consistent with this, mutation of E347A in the catalytic site suppressed shedding of sADAM33 both at baseline and in response to TGF-β (Fig 1, A) and this was accompanied by a significant reduction in enzymatic activity in cell-free supernatants (Fig 1, B). These data suggest that a substantial component of the shedding of sADAM33 is autocatalytic. TGF-β activates multiple signals including mothers against decapentaplegic homolog (SMAD) and non-SMAD pathways. Inhibition of SMAD signaling using SB431452 significantly suppressed TGF-β1–induced shedding of immunoreactive and enzymatically active ADAM33 (Fig 1, C and D). In contrast, the MAP2K/mitogen-activated protein kinase kinase inhibitor PD98059 dose-dependently increased shedding of immunoreactive and enzymatically active forms of sADAM33 (Fig 1, E and F). These data suggest a complex effect of TGF-β signaling, with SMAD activation being stimulatory for ADAM33 shedding, whereas mitogen-activated protein kinase (MAPK) activation has a negative regulatory effect. The latter effect may be mediated via TIMP-3, a known inhibitor of ADAM33 enzymatic activity,5Zou J. Zhu F. Liu J. Wang W. Zhang R. Garlisi C.G. et al.Catalytic activity of human ADAM33.J Biol Chem. 2004; 279: 9818-9830Crossref PubMed Scopus (94) Google Scholar which is reported to be induced by TGF-β via MAPK signaling.6Qureshi H.Y. Sylvester J. El Mabrouk M. Zafarullah M. TGF-beta-induced expression of tissue inhibitor of metalloproteinases-3 gene in chondrocytes is mediated by extracellular signal-regulated kinase pathway and Sp1 transcription factor.J Cell Physiol. 2005; 203: 345-352Crossref PubMed Scopus (71) Google Scholar Thus, inhibiting MAPK activation with PD98059 may inhibit TIMP3 expression and release ADAM33 from the effects of this natural inhibitor leading to increased autocatalytic shedding and augmented enzyme activity. Although ectodomain shedding can be regulated by natural protein inhibitors, other mechanisms including membrane trafficking, protein maturation, and substrate presentation have also been shown to be important for the regulation of other sheddases.7Lichtenthaler S.F. Lemberg M.K. Fluhrer R. Proteolytic ectodomain shedding of membrane proteins in mammals—hardware, concepts, and recent developments.EMBO J. 2018; 37 (e99456)Crossref Scopus (138) Google Scholar Whether release of ADAM33 is preceded by its membrane trafficking or maturation and/or by assembly into higher order complexes requiring additional protein interactions to exosites located either on the ADAM33 or adapter proteins remains to be determined. Epithelial injury leads to release of TGF-β, and we have postulated that this is the source of the TGF-β that drives the increase in sADAM33 observed in response to allergen challenge in vivo.2Davies E.R. Kelly J.F. Howarth P.H. Wilson D.I. Holgate S.T. Davies D.E. et al.Soluble ADAM33 initiates airway remodeling to promote susceptibility for allergic asthma in early life.JCI Insight. 2016; 1 (e87632)Crossref Scopus (29) Google Scholar To test this hypothesis, we used BALF from mice in which lung epithelial Tgfb1 was conditionally deleted in bronchial epithelial club cells before intranasal administration of either 25 μg house dust mite extract or recombinant murine IL-33.8Denney L. Byrne A.J. Shea T.J. Buckley J.S. Pease J.E. Herledan G.M. et al.Pulmonary epithelial cell-derived cytokine TGF-beta1 is a critical cofactor for enhanced innate lymphoid cell function.Immunity. 2015; 43: 945-958Abstract Full Text Full Text PDF PubMed Google Scholar After house dust mite challenge, lower levels of sADAM33 could be detected in the BALF of Tgfb1−/− mice compared with littermate controls (Fig 2, A) and it also contained less sADAM33 enzymatic activity (Fig 2, B). In the same way, when mice were challenged with IL-33, BALF from Tgfb1−/− mice had a lower level of sADAM33 immunoreactive protein (Fig 2, C) and enzymatic activity (Fig 2, D). Of note, exogenous TGF-β alone was ineffective at stimulating shedding of ADAM33 in vivo. This might be explained either by the fact that TGF-β could not pass through the intact epithelium of unchallenged mice and/or by the low dose and short half-life of the growth factor, which did not allow sufficient time for it to pass through the epithelium to reach the subepithelial mesenchymal cells where ADAM33 is expressed.9Haitchi H.M. Powell R.M. Shaw T.J. Howarth P.H. Wilson S.J. Wilson D.I. et al.ADAM33 expression in asthmatic airways and human embryonic lungs.Am J Respir Crit Care Med. 2005; 171: 958-965Crossref PubMed Scopus (91) Google Scholar Epithelial-derived IL-33 induces rapid release of TGF-β into the airways to enhance migration of innate lymphoid cells (ILCs) and development of a robust ILC2 response that initiates allergic immunity.9Haitchi H.M. Powell R.M. Shaw T.J. Howarth P.H. Wilson S.J. Wilson D.I. et al.ADAM33 expression in asthmatic airways and human embryonic lungs.Am J Respir Crit Care Med. 2005; 171: 958-965Crossref PubMed Scopus (91) Google Scholar Because we found that epithelial-derived TGF-β is also required for ectodomain shedding of ADAM33, it may be speculated that sADAM33 can contribute to the recruitment or activation of ILCs or other innate immune cells, either by affecting matrix turnover or by promoting growth factor or chemokine shedding. Of note, polymorphisms in TGFB, IL33, and ADAM33 genes have each been associated with asthma susceptibility, yet each has a small overall effect on disease development. The involvement of 3 susceptibility gene products in epithelial responses to allergens highlights how they may cooperate to amplify the downstream asthmatic responses. Identification of the involvement of TGF-β in ectodomain shedding of ADAM33 in an in vivo model strengthens the case for exploring how human polymorphic variation in the ADAM33 gene is linked to asthma pathogenesis. Four single nucleotide polymorphisms (S1, S2, T1, and T2) encode amino acid substitutions in the transmembrane and cytoplasmic domain of ADAM33 and have been associated with asthma.4Orth P. Reichert P. Wang W. Prosise W.W. Yarosh-Tomaine T. Hammond G. et al.Crystal structure of the catalytic domain of human ADAM33.J Mol Biol. 2004; 335: 129-137Crossref PubMed Scopus (74) Google Scholar Although the intracellular domain of ADAM33 is relatively short, it is very rich in prolines, having a putative SH3 binding site where the T2 SNP is located, a casein kinase I/II phosphorylation site, and an MAPK consensus sequence that is likely to be important for regulation of ADAM33 function, especially as we have identified a negative regulatory role for MAPK in our current studies. Further work is required to determine whether this effect is direct and involves ADAM33 phosphorylation or indirect via inhibitors such as TIMP3. Alternatively, one mutation Ala395Val is located within the catalytic domain,4Orth P. Reichert P. Wang W. Prosise W.W. Yarosh-Tomaine T. Hammond G. et al.Crystal structure of the catalytic domain of human ADAM33.J Mol Biol. 2004; 335: 129-137Crossref PubMed Scopus (74) Google Scholar which may directly affect catalytic activity. In summary, we have provided direct evidence that epithelial-derived TGF-β is an important regulator of ectodomain shedding of enzymatically active ADAM33 from the mesenchyme. This process appears largely to be autocatalytic and involves SMAD signaling, but is negatively regulated by MAPK signaling. These findings highlight the importance of epithelial-mesenchymal cross-talk in asthma pathogenesis and underscore the potential for co-operation between different asthma susceptibility genes to drive disease pathogenesis. The Cos-7 cell line, a fibroblast-like cell line, was grown in Dulbecco modified Eagle medium supplemented with 10% FBS, 50 units/mL penicillin, 50 μg/mL streptomycin, 2 mM l-glutamine, 1 mM sodium pyruvate, and 1× nonessential amino acids (Dulbecco modified Eagle medium/FBS) (all from Life Technologies, Paisley, UK). For transfection, cells were placed in non-supplemented Opti-MEM (Life Technologies). Cos-7 cells were transfected with a plasmid encoding full-length murine ADAM33 (MR217277 clone, NM_033615; OriGene, Rockville, Md) or green fluorescence protein using X-treme Gene 9 reagent (Roche, Southampton, UK). After 24 hours, cells were treated with TGF-β1 (Peprotech, London, UK) for 8 hours to assess ectodomain shedding of ADAM33. The TGF-β1 isoform was chosen, as this is the major isoform in adult mice.E1Pelton R.W. Johnson M.D. Perkett E.A. Gold L.I. Moses H.L. Expression of transforming growth factor-beta 1, -beta 2, and -beta 3 mRNA and protein in the murine lung.Am J Respir Cell Mol Biol. 1991; 5: 522-530Crossref PubMed Scopus (108) Google Scholar Where indicated, cells were preincubated for 1 hour with the ALK5/SMAD inhibitor SB431542 (10 μM; Stratech, Ely, UK), the mitogen-activated protein kinase kinase (MAP2K, MEK) inhibitor PD98059 (10 or 25 μM, Sigma, Poole, UK), the broad-spectrum metalloprotease inhibitor GM6001 (10 μM; Merck, Millipore, Watford, UK), or vehicle control before addition of 5 ng/mL TGF-β1. Cell-free supernatants were harvested and cells were lysed into RIPA buffer. Concanavalin A-Sepharose 4B beads (ConA) (Sigma) were used to pull down glycosylated ADAM33 from the supernatants of the transfected Cos-7 cells, as previously described.E2Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar In brief, 1 mL supernatant was adjusted to contain 20 mM Tris HCl, pH 7.4, 0.5 M NaCl, and protease inhibitors (Sigma) before addition of activated ConA beads (20 μL beads per mL sample). After binding overnight at 4°C, the beads were washed twice in 20 mM Tris HCl, pH 7.4, containing 0.5 M NaCl and then twice in 20 mM Tris HCl, pH 7.4, to remove excess salt. The bound protein was then solubilized into SDS sample buffer containing 0.5 M methyl α-d-mannopyranoside for SDS-PAGE and Western blotting. An ADAM33 enzymatic assay was performed using a fluorescence resonance energy transfer peptide cleavage assay as previously described.E3Zou J. Zhang R. Zhu F. Liu J. Madison V. Umland S.P. ADAM33 enzyme properties and substrate specificity.Biochemistry. 2005; 44: 4247-4256Crossref PubMed Scopus (26) Google Scholar All assay measurements were made at 37°C in real time using a Bio-Rad CFX96 machine for detection of carboxyfluorescein reporter dye (FAM) (450-490 nm excitation and 510-530 nm detection), allowing determination of initial rates. Each reaction contained 7 μL neat BALF or cell supernatant mixed with 2 μL 5x-assay buffer (100 mM HEPES, pH 7, 2.5 M NaCl, 50 mM CaCl2, and 1 mg/mL BSA); the assay was initiated by addition of 44 pmol fluorescence resonance energy transfer peptide (DABCYL-YRVAFQKLAE(FAM)K-NH2) (Severn Biotech, Kidderminster, UK) and 100 pmol ZnCl2 to make a final reaction volume of 10 μL. Enzymatic activity was determined by plotting relative fluorescence units against time after the background had been subtracted. The rate of the reaction (relative fluorescence units/min) was determined from the line of best fit in the linear phase of the assay. Control assays used human recombinant soluble active ADAM33 (positive control) and mutant ADAM33 (E365A) (negative control) as previously described.E2Puxeddu I. Pang Y.Y. Harvey A. Haitchi H.M. Nicholas B. Yoshisue H. et al.The soluble form of a disintegrin and metalloprotease 33 promotes angiogenesis: implications for airway remodeling in asthma.J Allergy Clin Immunol. 2008; 121 (1406.e1-4): 1400-1406Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar Solubilized samples from ConA pull down or murine BALF were run on 12.5% acrylamide or 8% to 20% gradient gels (Bio-Rad, Watford, UK) and transferred onto polyvinylidene fluoride (PVDF) membranes. The transferred protein was assessed by Ponceau staining to confirm equivalent protein loading before blocking using 2.5% BSA in TBS/Tween and Western blotting. Membranes were probed with a polyclonal goat antibody against the ectodomain of mouse ADAM33 (AF2434, 1:1000, R&D Systems, Abingdon, UK). Secondary antibody was rabbit antigoat IgG horseradish peroxidase antibody (Merck; 1:5000). The blots were visualized using enhanced chemiluminescence (Clarity ECL; Bio-Rad) with an Amersham Imager 600 (GE Healthcare, Buckinghamshire, UK); densitometric images were semiquantified using ImageJ software (National Institutes of Health, Wis) and data plotted as relative intensity. Mice conditionally lacking lung epithelial Tgfb expression (Ccsp-creTgfb1−/−) were generated as previously described.E4Denney L. Byrne A.J. Shea T.J. Buckley J.S. Pease J.E. Herledan G.M. et al.Pulmonary epithelial cell-derived cytokine TGF-beta1 is a critical cofactor for enhanced innate lymphoid cell function.Immunity. 2015; 43: 945-958Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Mice were pretreated with doxycycline (Sigma) (or vehicle as control) to delete Tgfb1 from the bronchiolar epithelial cells 72 hours before intranasal administration of either 25 μg HDM extract (1 mg/mL protein solution dissolved in PBS) or 25 μL of PBS 5 times a week for 3 weeks. Mice were culled 4 hours after the final challenge. In other experiments, carrier-free recombinant murine IL-33 (1 μg/dose in 25 μL PBS) (eBioscience, Thermo Fisher Scientific, Hemel Hempstead, UK) was administered 3 times a week for 1 week and mice culled 18 hours after the final dose. Where required, 50 ng recombinant TGF-β1 in PBS (R&D Systems) was administered intranasally without or with IL-33. At the point of killing, BALF was collected by washing the airways 3 times with 400 μL PBS. After centrifugation to remove cells, supernatants were stored at −80°C. The protein content of the BALF was quantified (BCA Assay, Thermofisher, Hemel Hempstead, UK) and normalized before use in ADAM33 enzyme assays or solubilization in 2x Laemmli sample buffer (Bio-Rad) for analysis of sADAM33 protein by Western blotting. Mice of both sexes were used between age 7 and 12 weeks, housed in specific-pathogen-free conditions, and given food and water ad libitum. All procedures were conducted in accordance with the Animals (Scientific Procedures) Act 1986. Normal distribution of the numeric data was evaluated, and appropriate parametric or nonparametric statistical tests applied. All data are parametric and plotted as mean with 1 SD. Statistical significance was assessed by using Student t test (unpaired data) with Welch correction if SDs were not equal for comparisons between 2 groups. For comparison of 3 or more groups, a 1-way ANOVA with Dunn's multiple comparison test was used. For comparison of 2 or more groups with 2 independent variables, a 2-way ANOVA with Tukey multiple comparison test was used (*P < .05, **P < .01, ***P < .001).Fig E2The broad-spectrum MMP inhibitor GM6001 does not inhibit ADAM33 shedding. Cos-7 cells transiently transfected with full-length murine ADAM33 were preincubated with GM6001 (0-10 μM) for 1 hour before addition of 5 ng/mL TGF-β for 8 hours. Cell-free supernatants were assessed for soluble ADAM33 by Western blotting (A) with densitometry (arbitrary units, AU) (B) and by fluorescence resonance energy transfer peptide cleavage assay (C). Purified recombinant ADAM33 protein was incubated with GM6001 and tested for activity in a fluorescence resonance energy transfer peptide cleavage assay (D). Activity is presented as a percent of that measured in the absence of GM6001. MW, Molecular weight. Data are expressed as mean + SD and are representative of 4 independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

The respiratory epithelium is the major interface between the environment and the host. Sophisticated barrier, sensing, anti-microbial and immune regulatory mechanisms have evolved to help maintain homeostasis and to defend the lung against foreign substances and pathogens. During influenza virus infection, these specialised structural cells and populations of resident immune cells come together to mount the first response to the virus, one which would play a significant role in the immediate and long term outcome of the infection. In this review, we focus on the immune defence machinery of the respiratory epithelium and briefly explore how it repairs and regenerates after infection.

Airway hyperresponsiveness (AHR) is a critical feature of wheezing and asthma in children, but the initiating immune mechanisms remain unconfirmed. We demonstrate that both recombinant interleukin-33 (rIL-33) and allergen [house dust mite (HDM) or Alternaria alternata] exposure from day 3 of life resulted in significantly increased pulmonary IL-13+CD4+ T cells, which were indispensable for the development of AHR. In contrast, adult mice had a predominance of pulmonary LinnegCD45+CD90+IL-13+ type 2 innate lymphoid cells (ILC2s) after administration of rIL-33. HDM exposure of neonatal IL-33 knockout (KO) mice still resulted in AHR. However, neonatal CD4creIL-13 KO mice (lacking IL-13+CD4+ T cells) exposed to allergen from day 3 of life were protected from AHR despite persistent pulmonary eosinophilia, elevated IL-33 levels, and IL-13+ ILCs. Moreover, neonatal mice were protected from AHR when inhaled Acinetobacter lwoffii (an environmental bacterial isolate found in cattle farms, which is known to protect from childhood asthma) was administered concurrent with HDM. A. lwoffii blocked the expansion of pulmonary IL-13+CD4+ T cells, whereas IL-13+ ILCs and IL-33 remained elevated. Administration of A. lwoffii mirrored the findings from the CD4creIL-13 KO mice, providing a translational approach for disease protection in early life. These data demonstrate that IL-13+CD4+ T cells, rather than IL-13+ ILCs or IL-33, are critical for inception of allergic AHR in early life.

Background: Genome-wide association studies have identified the ORM (yeast)-like protein isoform 3 (ORMDL3) gene locus on human chromosome 17q to be a highly significant risk factor for childhood-onset asthma.Objective: We sought to investigate in vivo the functional role of ORMDL3 in disease inception.Methods: An Ormdl3-deficientmousewas generated andthe role of ORMDL3 in the generation of allergic airways disease to the fungal aeroallergen Alternaria alternata was determined. An adenoassociated viral vector was also used to reconstitute ORMDL3 expression in airway epithelial cells of Ormdl3 knockout mice.Results: Ormdl3 knockout mice were found to be protected from developing allergic airways disease and showed a marked decrease in pathophysiology, including lung function and airway eosinophilia induced by Alternaria. Alternaria is a potent inducer of cellular stress and the unfolded protein response, and ORMDL3 was found to play a critical role in driving the activating transcription factor 6-mediated arm of this response through Xbp1 and downstream activation of the endoplasmic reticulum-associated degradation pathway. In addition, ORMDL3 mediated uric acid release, another marker of cellular stress. In the knockout mice, reconstitution of Ormdl3 transcript levels specifically in the bronchial epithelium resulted in reinstatement of susceptibility to fungal allergen-induced allergic airways disease.Conclusions: This study demonstrates that ORMDL3, an asthma susceptibility gene identified by genome-wide association studies, contributes to key pathways that promote changes in airway physiology during allergic immune responses.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.

Innate lymphoid cells (ILCs) are characterized by their lymphoid morphology and absence of lymphocyte lineage surface markers.1 Group 2 ILCs (ILC2s) expressing CD127 and CRTH2 are induced by the epithelial cytokines IL-33 and IL-25 and are implicated in the pathogenesis of allergic airways disease in murine models. ILC2s have been identified in cord blood,2 tonsils, and nasal polyps from patients with chronic rhinoosinusitis3 and in peripheral blood and bronchoalveolar lavage (BAL) from adults with asthma.

Epithelial cells orchestrate pulmonary homeostasis and pathogen defense and play a crucial role in the initiation of allergic immune responses. Maintaining the balance between homeostasis and inappropriate immune activation and associated pathology is particularly complex at mucosal sites that are exposed to billions of potentially antigenic particles daily. We demonstrated that epithelial cell-derived cytokine TGF-β had a central role in the generation of the pulmonary immune response. Mice that specifically lacked epithelial cell-derived TGF-β1 displayed a reduction in type 2 innate lymphoid cells (ILCs), resulting in suppression of interleukin-13 and hallmark features of the allergic response including airway hyperreactivity. ILCs in the airway lumen were primed to respond to TGF-β by expressing the receptor TGF-βRII and ILC chemoactivity was enhanced by TGF-β. These data demonstrate that resident epithelial cells instruct immune cells, highlighting the central role of the local environmental niche in defining the nature and magnitude of immune reactions.

BACKGROUND:TH2 cytokines are not responsible for the ongoing symptoms and pathology in children with severe therapy-resistant asthma (STRA). IL-33 induces airway hyperresponsiveness, but its role in airway remodeling and steroid resistance is unknown.OBJECTIVE:We sought to investigate the relationship between IL-33 and airway remodeling in pediatric patients with STRA.METHODS:IL-33 levels were quantified in neonatal mice given inhaled house dust mite (HDM), and the effect of blocking IL-13 on remodeling and IL-33 levels was assessed. HDM-induced allergic airways disease (AAD) in neonatal ST2(-/-) mice lacking the IL-33 receptor was assessed, together with collagen production after IL-33 administration. The effect of steroid therapy on IL-33 levels in patients with neonatal AAD was explored. IL-33 expression was quantified in endobronchial biopsy (EB) specimens from children with STRA and related to remodeling, and collagen production by airway fibroblasts from pediatric patients stimulated with IL-33 and budesonide was quantified.RESULTS:Blocking IL-13 after AAD was established in neonatal mice and did not reduce remodeling or IL-33 levels; airway hyperresponsiveness was only partially reduced. IL-33 promoted collagen synthesis both from asthmatic fibroblasts from pediatric patients and after intranasal administration in mice. Increased cellular expression of IL-33, but not IL-13, was associated with increased reticular basement membrane thickness in EB specimens from children with STRA, whereas remodeling was absent in HDM-exposed ST2(-/-) mice. IL-33 levels were maintained, whereas IL-13 levels were abrogated by steroid treatment in neonatal HDM-exposed mice and in EB specimens from children with STRA.CONCLUSION:IL-33 is a relatively steroid-resistant mediator that promotes airway remodeling in patients with STRA and is an important therapeutic target.

Neuropathology in multiple sclerosis is closely linked to presence of macrophages in the CNS. Both M1 (inflammatory) and M2 (alternatively activated, noninflammatory) macrophages are found in the inflamed CNS and thought to differentiate from infiltrating monocytes. It is unclear whether the balance of M1 and M2 macrophages can be altered and whether this affects disease outcome. We show in this article that Ly6C(hi) inflammatory monocytes are the early and dominant infiltrating cells in the CNS during experimental autoimmune encephalomyelitis, a model for the acute phase of multiple sclerosis. Activation of invariant NKT (iNKT) cells reduced the frequency of Ly6C(hi) monocytes and increased the proportion of M2 macrophages in the CNS with associated improvement in neurologic impairment. In contrast, iNKT-deficient mice showed higher numbers of Ly6C(hi) monocytes, reduced M2, and much more severe disease. Adoptive transfer of M2-enriched cells to iNKT-deficient mice markedly improved neurologic impairment. In vitro and in vivo experiments showed that iNKT cells promote differentiation of monocytes to M2 macrophages in an IL-4 and CD1d-dependent process. These findings indicate that infiltrating Ly6C(hi) inflammatory monocytes are early players in acute neuroinflammation and that their frequency and differentiation can be influenced by activation of iNKT cells with resultant improvement in disease outcome.