Hormonal disruptions are associated with poor asthma control in females, yet how these phenomena are linked remains unknown. Here, we investigated distinct allergen-induced immune responses between the sexes during maturation. By 6 weeks of life, female mice exposed to the aeroallergen house dust mite (HDM) from postnatal day 7 exhibited stronger type 2 (T2) immune responses and higher lung interleukin-33 (IL-33) than males. IL-33 administration to HDM-sensitized males was sufficient to augment T2 immunity and up-regulated epidermal growth factor receptor (EGFR) on T helper 2 (T H 2) cells. EGFR inhibition abrogated T2 cytokine production in vitro. In vivo, EGFR inhibition reduced T2 immunity in females only, thereby abolishing any sex differences. 17β-estradiol (E 2 ) heightened lung Il33 expression and T2 responses of HDM-sensitized males, akin to levels in females. EGFR’s ability to drive sex differences in lung T2 responses downstream of E 2 and IL-33 may link hormonal disruptions to poor asthma control.

Background Epithelial–immune cell interactions are crucial in the regulation of pulmonary immune responses. Emerging evidence suggests that cell populations lining the airways may play a pivotal role in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease characterised by progressive scarring of the lung parenchyma. We profiled the cellular landscape of the airway mucosal niche in incident cases of IPF to understand early-stage events contributing to disease development. Methods Single-cell RNA-sequencing was used to explore cellular heterogeneity in proximal airway brushings from seven healthy controls and nine patients with newly diagnosed IPF. In-depth bioinformatics analysis was used to interrogate changes in cell populations and cell–cell communication in IPF patients compared to controls. Results We show a relative increase in the abundance of airway macrophage subsets in IPF compared to healthy controls, and disease-specific changes in their transcriptional profile. Increased frequency of airway macrophages and proliferating macrophages was associated with more extensive disease at baseline quantified by the composite physiological index and radiological severity of traction bronchiectasis. Monocyte-derived macrophages were significantly enriched at baseline in IPF patients who had disease progression at 12 months. Using CellChat we exposed differences in cell–cell communication between airway epithelial cells, airway macrophages and T-cells in IPF. We identified dysregulation in signalling pathways such as SEMA3, ANXA1 and DESMOSOME, which modulate airway epithelial–macrophage interactions, potentially driving disease pathology. Conclusions Airway epithelial cells and macrophages may play a key role in orchestrating the early immunopathology of IPF, and these data support further exploration of novel, airway-focused therapeutic targets in IPF.

Determining spatial location of cells within tissues gives vital insight into the interactions between resident and inflammatory cells and is a critical factor for uncoupling the mechanisms driving disease. Here, we apply single-cell spatial transcriptomics to reveal the airway wall landscape in health and during asthma. We identified proinflammatory cellular ecosystems that exist within discrete spatial niches in healthy and asthma samples. These cellular hubs are characterized by a high level of chemokine and alarmin expression, along with unique combinations of stromal cells. Mechanistically, we demonstrated that receptors, such as ACKR1, retain immune mediators locally, while amphiregulin-expressing mast cells are prominent within these proinflammatory hubs. Despite anti-inflammatory treatments, the asthma airway mucosa exhibited a distinct remodeling program within these cellular ecosystems, marked by increased proximity between key cell types. This study provides an unprecedented view of the topography of the airway wall, revealing distinct, specific ecosystems within spatial niches to target for therapeutic intervention.

T follicular helper cells (TFH) play a central role in orchestrating antibody mediated immunity. Despite the importance of antibody responses, especially allergen-specific IgE, in allergic airway diseases (AAD) such as asthma, the precise role TFH play in AADs has remained elusive. Using a mouse model of chronic allergen induced AAD we now show that germinal centres (GCs) containing TFH and GC B cells accumulate in both the lung draining lymph nodes (dLNs) and the lungs themselves after allergen exposure. The formation of these GCs is dependent on TFH, as is generation of allergen specific IgA, IgG and IgE, with IgG1 and IgE-switched B cells being predominantly found in the dLNs while IgA switched B cells were only found in the lungs. Fitting with this, allergen-induced lung resident TFH and B cells are functionally and transcriptionally distinct from their lymphoid counterparts, with lung GCs providing a unique site of IgA-switch, a process that is partially IL-17A dependent. Finally while TFH deficiency did not worsen allergic airways disease after 3 weeks of aero-allergen exposure, worsened lung function and enhanced TH2-based inflammation in the respiratory tract were seen following 5 weeks of exposure. Overall these data suggest that TFH play a pivotal role in shaping immune responses both in the dLNs and the respiratory tract, and while they can promote key type-2 inflammatory pathways such as IgE production, they can also act to limit prolonged type-2 inflammation.

Innate lymphoid cells (ILCs) fulfill critical roles in maintenance of tissue-specific homeostasis but have also been implicated in disease pathology when dysregulated. Although they are broadly classified into three core subsets, it is increasingly apparent that ILCs exhibit plasticity in response to microenvironmental factors. Accurate and holistic evaluation of the ILC landscape is critical to understanding the contribution of ILCs to disease pathology. Using high-parameter flow cytometry, we comprehensively interrogated the phenotypic and functional diversity of ILCs in healthy volunteers and patients with severe asthma (SA), assessing the reciprocity between peripheral blood and airway compartments and dissecting the impact of anti-IL-5/5Rα biologics on these responses. We identified substantial heterogeneity and putative plasticity in human ILC responses, highlighting inherent limitations of conventional enumeration strategies. Deep phenotypic and functional profiling demonstrated a distinct sexual dimorphism in ILC responses in patients with SA. Females displayed an elevated abundance of circulating ILC progenitors, ILC1s, and ILC3s, whereas males presented with diminished ILC2s compared with respective healthy controls. Circulating ILC progenitors inversely correlated with testosterone concentrations. Moreover, we identified a reciprocal influx of all core ILC subsets into the airways of patients with SA, with unbiased multisource clustering identifying a relationship between elevated airway ILC2s and reduced lung function. Last, we showed that anti-IL-5/5Rα biologics largely ablated airway ILC type 2 cytokine production without affecting core ILC subset abundance in the peripheral blood or airways, identifying a potential mechanism whereby anti-IL-5/5Rα biologics alleviate clinical disease in patients with SA.

The lungs represent a dynamic microenvironment where airway macrophages (AMs) are the major lung-resident macrophages. AMs dictate the balance between tissue homeostasis and immune activation and thus have contradictory functions by maintaining tolerance and tissue homeostasis, as well as initiating strong inflammatory responses. Emerging evidence has highlighted the connection between macrophage function and cellular metabolism. However, the functional importance of these processes in tissue-resident specialized macrophage populations such as those found in the airways, remain poorly elucidated. Here, we reveal that glycolysis is a fundamental pathway in AMs which regulates both lung homeostasis and responses to inhaled allergen. Using macrophage specific targeting in vivo, and multi-omics approaches, we determined that glycolytic activity in AMs is necessary to restrain type 2 (T2) immunity during homeostasis. Exposure to a range of common aeroallergens, including house dust mite (HDM), drove AM-glycolysis and furthermore, AM-specific inhibition of glycolysis altered inflammation in the airways and HDM-driven airway metabolic adaptations in vivo. Additionally, allergen sensitised asthmatics had profound metabolic changes in the airways, compared to non-sensitised asthmatic controls. Finally, we found that allergen driven AM-glycolysis in mice was TLR2 dependent. Thus, our findings demonstrate a direct relationship between glycolysis in AMs, AM-mediated homeostatic processes, and T2 immune responses in the lungs. These data suggest that glycolysis is essential for the plasticity of AMs. Depending on the immunological context, AM-glycolysis is required to exert homeostatic activity but once activated by allergen, AM-glycolysis influences inflammatory responses. Thus, precise modulation of glycolytic activity in AMs is essential for preserving lung homeostasis and regulating airway inflammation.

BACKGROUND:Airway remodeling is a prominent pathologic feature in preschool wheeze (PSW) and school-age asthma (SA). Although the relationships between altered lung function, extracellular matrix (ECM) changes, and airway remodeling are described in PSW and SA, the underlying mechanisms remain undefined. OBJECTIVE:We sought to investigate mechanisms resulting in altered airway ECM landscape in PSW (1-5 years of age) and SA (6-16 years of age) and track ECM dynamics in house dust mite (HDM)-exposed neonatal mice. METHODS:We applied spatial transcriptomics, confocal microscopy, and SHG microscopy in PSW and SA endobronchial biopsy specimens and in HDM-exposed neonatal mice lung specimens to reveal transcriptional, phenotypic, and structural ECM-associated changes during allergic airway inflammation. RESULTS:Spatial transcriptomic analysis of the airways of children with PSW and SA revealed increased gene expression for fibrillar collagens I, II, and III and basement membrane collagen VI in fibroblast-rich regions in both diseases. Similarly, increased collagen III and collagen VI deposition with exaggerated collagen fibril disorganization was observed in the peribronchial regions of HDM-exposed neonatal mice. Collagen disorganization was also evident in the airways of children with PSW and SA and was accompanied by increased production of bronchial epithelial cell-derived lumican and increased airway lumican in children with PSW, children with SA, and HDM-exposed neonatal mice. Lumican directly altered primary healthy airway fibroblast function, increasing proliferation and collagen production. CONCLUSIONS:We demonstrate a previously uncharacterized role of collagen-associated phenotypic and geometric changes in early-life airway remodeling and show lumican as a crucial remodeling factor associated with collagen organization in PSW and SA.

Introduction:Up to 11% of patients are left with residual lung abnormalities following COVID-19 infection. It is unclear whether these changes resolve over time or progress to fibrosis. The airway microbiome is altered in interstitial lung disease, potentially contributing to pathogenesis and disease progression. We hypothesised that the airway microbiome in patients with post-COVID-19 residual lung abnormalities may be altered. Methods:The POST COVID-19 interstitial lung DiseasE (POSTCODE) study recruited subjects with post-COVID-19 residual lung abnormalities for bronchoscopy. 16S ribosomal RNA gene amplicon sequencing was performed on DNA extracted from bronchoalveolar lavage fluid and compared with that from patients with idiopathic pulmonary fibrosis, fibrotic hypersensitivity pneumonitis and control subjects. Results:28 subjects with post-COVID-19 residual lung abnormalities were recruited an average of 11 months after infection. No significant associations were found between the lower airway microbiome or bacterial burden and disease severity or trajectory. There was no difference in bacterial burden between post-COVID-19 patients and interstitial lung disease or control subjects. Furthermore, no differences in microbial composition were observed between these patients and those with fibrotic hypersensitivity pneumonitis or controls. However, compared with idiopathic pulmonary fibrosis, there was an increased abundance of Streptococcus and higher α-diversity in subjects with post-COVID-19 residual lung abnormalities. Conclusions:The microbiome and bacterial burden in the lower airways of subjects with post-COVID-19 residual lung abnormalities do not differ from those of controls. The microbiome differs from idiopathic pulmonary fibrosis. This, and the absence of associations between microbial features and disease severity or clinical outcomes, suggests that the microbiome is unlikely to contribute to residual lung abnormalities in patients recovering from COVID-19 infection.

BACKGROUND:Asthma is a chronic, heterogeneous disease characterised by airway remodelling, inflammation, and mucus production. Airway macrophages' functions are underpinned by changes in cellular metabolism. The TCA cycle-derived metabolite itaconic acid (whose synthesis is mediated by aconitate decarboxylase) is a master regulator of macrophage function; however, its role during inhaled allergen challenge is not clear. The objective of this study was to define the role of itaconate during inhaled allergen challenge. METHODS:Sputum metabolite levels were measured in participants with mild allergic asthma undergoing allergen inhalation challenge, and in a second cohort, baseline levels in mild, moderate, and severe asthmatics. Airway inflammation, lung function, and bronchoalveolar lavage metabolite levels were assessed in wild-type and aconitate decarboxylase-deficient mice, or in mice treated with inhaled itaconate. RESULTS:Allergen inhalation in mild asthmatics led to a significant reduction in sputum itaconate. We found no difference in baseline sputum itaconate levels when comparing healthy controls to mild, moderate, or severe asthmatics. Continuous exposure to aeroallergen in wild type and aconitate decarboxylase-deficient mice showed no change in disease phenotype after 48 h, 1, 3, or 5 weeks of allergen exposure. Treatment of house dust mite-exposed mice with inhaled itaconate reduced airway inflammation. CONCLUSION:Levels of itaconate are altered after allergen challenge in mild asthmatics and in murine models of disease. Itaconate deficiency did not alter house dust mite-induced pathology at any of the timepoints tested; however, inhaled itaconate ameliorated inflammatory responses to inhaled allergen.

•3D printed SiO2/PTHF/PCL-diCOOH hybrid scaffolds properties are unchanged after sterilisation with 50 kGy γ-irradiation.•Subcutaneous implantation in mice revealed an early resolving inflammatory response.•3D printed hybrid scaffolds support hBM-MSC chondrogenesis in vitro.•Articular cartilage-specific matrix deposition within these scaffolds was enhanced under hypoxic conditions, even without chondrogenic supplements.

Allergic asthma generally starts during early life and is linked to substantial tissue remodeling and lung dysfunction. Although angiogenesis is a feature of the disrupted airway, the impact of allergic asthma on the pulmonary microcirculation during early life is unknown. Here, using quantitative imaging in precision -cut lung slices (PCLSs), we report that exposure of neonatal mice to house dust mite (HDM) extract disrupts endothelial cell/pericyte interactions in adventitial areas. Central to the blood vessel structure, the loss of pericyte coverage was driven by mast cell (MC) proteases, such as tryptase, that can induce pericyte retraction and loss of the critical adhesion molecule N-cadherin. Furthermore, spatial transcriptomics of pediatric asthmatic endobronchial biopsies suggests intense vascular stress and remodeling linked with increased expression of MC activation pathways in regions enriched in blood vessels. These data provide previously unappreciated insights into the pathophysiology of allergic asthma with potential long-term vascular defects.

Aberrant expansion of KRT5 + basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5 + cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5 + cells migrate within the fibrotic lung, navigating regional differences in collagen topography. In vitro, KRT5 + cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry- based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5 + cell migration in vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5 + cell behaviour and function contributing to remodelling events in the fibrotic niche.

Allergic asthma generally starts during early life and is linked to significant tissue remodelling and lung dysfunction. Although angiogenesis is a feature of the disrupted airway, the impact of allergic asthma on the pulmonary microcirculation during early life is unknown. Here, using quantitative imaging in precision-cut lung slices (PCLS), we report that exposure of neonatal mice to house dust mite (HDM) extract disrupts endothelial cell/pericyte interactions in adventitial areas. Central to the blood vessel structure, the loss of pericyte coverage was driven by mast cell (MCs) proteases, such as tryptase, that can induce pericyte retraction and loss of the critical adhesion molecule N-Cadherin. Furthermore, spatial transcriptomics of paediatric asthmatic endobronchial biopsies revealed intense remodelling associated with increased expression of MC proteases in regions enriched in blood vessels. These data provide previously unappreciated insights into the pathophysiology of allergic asthma with potential long-term vascular defects.

BACKGROUND:Early life represents a major risk window for asthma development. However, the mechanisms controlling the threshold for establishment of allergic airway inflammation in early life are incompletely understood. Airway macrophages (AMs) regulate pulmonary allergic responses and undergo TGF-β-dependent postnatal development, but the role of AM maturation factors such as TGF-β in controlling the threshold for pathogenic immune responses to inhaled allergens remains unclear. OBJECTIVE:Our aim was to test the hypothesis that AM-derived TGF-β1 regulates pathogenic immunity to inhaled allergen in early life. METHODS:Conditional knockout (Tgfb1ΔCD11c) mice, with TGF-β1 deficiency in AMs and other CD11c+ cells, were analyzed throughout early life and following neonatal house dust mite (HDM) inhalation. The roles of specific chemokine receptors were determined by using in vivo blocking antibodies. RESULTS:AM-intrinsic TGF-β1 was redundant for initial population of the neonatal lung with AMs, but AMs from Tgfb1ΔCD11c mice failed to adopt a mature homeostatic AM phenotype in the first weeks of life. Evidence of constitutive TGF-β1 signaling was also observed in pediatric human AMs. TGF-β1-deficient AMs expressed enhanced levels of monocyte-attractant chemokines, and accordingly, Tgfb1ΔCD11c mice exposed to HDM throughout early life accumulated CCR2-dependent inflammatory CD11c+ mononuclear phagocytes into the airway niche that expressed the proallergic chemokine CCL8. Tgfb1ΔCD11c mice displayed augmented TH2, group 2 innate lymphoid cell, and airway remodeling responses to HDM, which were ameliorated by blockade of the CCL8 receptor CCR8. CONCLUSION:Our results highlight a causal relationship between AM maturity, chemokines, and pathogenic immunity to environmental stimuli in early life and identify TGF-β1 as a key regulator of this.

Asthma is a highly prevalent lung disease, characterized by airway dysfunction and chronic inflammation. Asthma occurs in both children and adults, but frequently originates in early life. Heterogeneous asthma phenotypes exist, but Th2 cells are key players in a large proportion of cases, while other CD4+ T cell subsets are also implicated in driving and limiting pathology. In this chapter, we describe methods for establishing allergic airway disease to model asthma in adult and neonatal mice, along with protocols for measuring key disease parameters and quantifying and phenotyping CD4+ T cell subtypes.

Lung function deficits established in asthmatic children persist into adulthood, but the mechanisms are not well understood. Airway remodelling is a key pathological feature of paediatric and adult asthma and may contribute to loss of lung function. Epidermal growth factor receptor (EGFR) was shown to be overexpressed in paediatric and adult asthmatics. In addition, several in vivo studies using rodent models of allergic airway disease (AAD) have described a role for EGFR signalling in driving impaired lung function and airway remodelling in adult animals. Here, we aimed to study the role of EGFR in early life AAD. We used flow-sorted murine lung cell populations to investigate EGFR expression at different stages of postnatal development. qPCR analysis revealed high expression of EGFR and ligands primarily in epithelial and structural cells in both neonatal and adult mice and high expression of EGFR on epithelial cells was confirmed by flow cytometry. To assess the role of EGFR during neonatal AAD, an EGFR inhibitor was administered during the last three of five weeks of inhaled allergen exposure. Our findings indicate that EGFR inhibition in neonatal mice resulted in worsened lung function, as measured by a 2-fold increase in airway resistance (area under the curve), whereas lung function in adult mice was unaffected by EGFR inhibition. Interestingly, we did not observe any changes in pulmonary inflammation, allergic sensitisation, mucus production or remodelling-associated gene expression upon EGFR inhibition in either model. These results indicate that EGFR is present in lungs at all stages of life and that, in contrast to its widely described pathogenic contribution to airway remodelling of adult animals, signalling through EGFR may play a protective role during early life AAD.

Idiopathic pulmonary fibrosis (IPF) is a fatal lung disease in which airway macrophages (AMs) play a key role. Itaconate has emerged as a mediator of macrophage function, but its role during fibrosis is unknown. Here, we reveal that itaconate is an endogenous antifibrotic factor in the lung. Itaconate levels are reduced in bronchoalveolar lavage, and itaconate-synthesizing cis-aconitate decarboxylase expression (ACOD1) is reduced in AMs from patients with IPF compared with controls. In the murine bleomycin model of pulmonary fibrosis, Acod1−/− mice develop persistent fibrosis, unlike wild-type (WT) littermates. Profibrotic gene expression is increased in Acod1−/− tissue-resident AMs compared with WT, and adoptive transfer of WT monocyte-recruited AMs rescued mice from disease phenotype. Culture of lung fibroblasts with itaconate decreased proliferation and wound healing capacity, and inhaled itaconate was protective in mice in vivo. Collectively, these data identify itaconate as critical for controlling the severity of lung fibrosis, and targeting this pathway may be a viable therapeutic strategy.

The ontogeny of airway macrophages (AMs) in human lung and their contribution to disease are poorly mapped out. In mice, aging is associated with an increasing proportion of peripherally, as opposed to perinatally derived AMs. We sought to understand AM ontogeny in human lung during healthy aging and after transplant. We characterized monocyte/macrophage populations from the peripheral blood and airways of healthy volunteers across infancy/childhood (2–12 yr), maturity (20–50 yr), and older adulthood (>50 yr). Single-cell RNA sequencing (scRNA-seq) was performed on airway inflammatory cells isolated from sex-mismatched lung transplant recipients. During healthy aging, the proportions of blood and bronchoalveolar lavage (BAL) classical monocytes peak in adulthood and decline in older adults. scRNA-seq of BAL cells from lung transplant recipients indicates that after transplant, the majority of AMs are recipient derived. These data show that during aging, the peripheral monocyte phenotype is consistent with that found in the airways and, furthermore, that the majority of human AMs after transplant are derived from circulating monocytes.