Introduction Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease involving injury and a dysregulated repair response of an ageing epithelium. Here we investigate the contribution of senescence and telomere shortening to bleomycin-induced injury in alveolar epithelial cells (EpCs) derived from lung parenchymal tissue of patients with pulmonary fibrosis (PF), predominantly IPF, in vitro . Methods Injury responses of EpCs in air–liquid interface (ALI) culture were assessed by ELISA, qPCR and/or immunofluorescence at 48 and 96 h post-bleomycin (30 μg·mL −1 ) treatment. Telomere length was evaluated by qPCR and corroborated by fluorescence in situ hybridisation. Results Bleomycin treatment increased markers of inflammation ( i.e. interleukin (IL)-6 and IL-36γ) and senescence ( i.e. p16 and p21) in EpCs from PF patients (PF EpCs) (p<0.05); responses markedly higher than control EpCs (Ctrl EpCs). Conversely, the effects of bleomycin on the expression of type I and type II alveolar EpC markers were supressed or downregulated in cultures of PF EpCs (when compared with Ctrl EpCs), whereas the percentage of keratin 17-positive type I and II intermediate EpCs were increased (p<0.05). Telomere length was also significantly shorter in PF than Ctrl EpCs at baseline and further reduced post-bleomycin treatment (p<0.05). Induction of telomere shortening by cell passaging augmented bleomycin-induced PF EpC injury, whereas pharmacological telomerase activation and selected senotherapeutics were protective. Conclusion This study provides evidence that senescence-associated telomere shortening increases the susceptibility of IPF lung EpCs to injury. Our cell-based lung epithelial injury model has utility as a pre-clinical tool for drug discovery and the evaluation of potential senotherapeutics for IPF treatment.

BACKGROUND AND OBJECTIVE:Idiopathic pulmonary fibrosis (IPF) is a chronic progressive lung disease with a poor prognosis. Antifibrotics slow the decline of pulmonary function after 12-months, but limited studies have examined the role of circulatory biomarkers in antifibrotic treated IPF patients. METHODS:Serum from 98 IPF participants, from the Australian Idiopathic Pulmonary Fibrosis Registry were collected at four time-points over 1 year post-antifibrotic treatment and analysed as two separate cohorts. Patients were stratified as progressive, if they experienced ≥ 10% decline in FVC or ≥ 15% decline in DLCO or were deceased within 1 year of treatment initiation: or otherwise as stable. Ten molecules of interest were measured by ELISAs in patient serum. RESULTS:Baseline MMP7 levels were higher in progressive than stable patients in Cohort 1 (p = 0.02) and Cohort 2 (p = 0.0002). Baseline MMP7 levels also best differentiated progressive from stable patients (Cohort 1, AUC = 0.74, p = 0.02; Cohort 2, AUC = 0.81, p = 0.0003). Regression analysis of the combined cohort showed that elevated MMP7 levels predicted 12-month progression (OR = 1.530, p = 0.010) and increased risk of overall mortality (HR = 1.268, p = 0.002). LASSO regression identified a multi-biomarker panel (MMP7, ICAM-1, CHI3L1, CA125) that differentiated progression more accurately than MMP7 alone. Furthermore, GAP combined with MMP7, ICAM-1, CCL18 and SP-D was more predictive of 3-year mortality than GAP alone. CONCLUSION:MMP7 along with a multi-biomarker and GAP panel can predict IPF progression and mortality, with the potential for optimising management.

Patients with pulmonary fibrosis (PF) often experience exacerbations of their disease, characterised by a rapid, severe deterioration in lung function that is associated with high mortality. Whilst the pathobiology of such exacerbations is poorly understood, virus infection is a trigger. The present study investigated virus-induced injury responses of alveolar and bronchial epithelial cells (AECs and BECs, respectively) from patients with PF and age-matched controls (Ctrls). Air–liquid interface (ALI) cultures of AECs, comprising type I and II pneumocytes or BECs were inoculated with influenza A virus (H1N1) at 0.1 multiplicity of infection (MOI). Levels of interleukin-6 (IL-6), IL-36γ and IL-1β were elevated in cultures of AECs from PF patients (PF-AECs, n = 8–11), being markedly higher than Ctrl-AECs (n = 5–6), 48 h post inoculation (pi) (P<0.05); despite no difference in H1N1 RNA copy numbers 24 h pi. Furthermore, the virus-induced inflammatory responses of PF-AECs were greater than BECs (from either PF patients or controls), even though viral loads in the BECs were overall 2- to 3-fold higher than AECs. Baseline levels of the senescence and DNA damage markers, nuclear p21, p16 and H2AXγ were also significantly higher in PF-AECs than Ctrl-AECs and further elevated post-infection. Senescence induction using etoposide augmented virus-induced injuries in AECs (but not viral load), whereas selected senotherapeutics (rapamycin and mitoTEMPO) were protective. The present study provides evidence that senescence increases the susceptibility of AECs from PF patients to severe virus-induced injury and suggests targeting senescence may provide an alternative option to prevent or treat the exacerbations that worsen the underlying disease.

Bronchoconstriction is the main physiological event in asthma, which leads to worsened clinical symptoms and generates mechanical stress within the airways. Virus infection is the primary cause of exacerbations in people with asthma, however, the impact of bronchoconstriction on host antiviral responses and viral replication is unknown. We developed two disease models, in vitro, and found suppressed IFN response from cells following the application of compression and RV-A1 infection. This explains why people with asthma have deficient IFN response.

Abstract Background Knowledge translation (KT) is a key competency for trainees (graduate students and post-doctoral fellows), the new generation of researchers who must learn how to synthesize, disseminate, exchange, and ethically apply knowledge to improve patient and health system services, products, and outcomes. KT training is a key enabler to support KT competency development. Yet, there is a dearth of research on the design, delivery, and evaluation of KT training for trainees. Methods The study applied a QUAN(qual) mixed methods approach with an embedded experimental model design. A heart and lung patient was also recruited to participate as a partner and researcher in the study. A multi-faceted KT intervention for trainees was designed, delivered, and evaluated. Data were collected using surveys and focus groups. Quantitative data were analyzed using descriptive and inferential statistics in R Studio and MS Excel. Qualitative data were analyzed in NVivo using thematic analysis. Results Participation in each KT intervention varied, with 8–42 participants attending KT webinars, 61 attendees in the Three Minute Thesis (3MT) Competition Heat, and 31 participants in the Patient & Public Forum. In total, 27 trainees and 4 faculty participated in at least one of the KT webinars. Trainee participants reported satisfaction, as well as statistically significant increases in 10/13 KT competencies after receiving one or more components of the KT intervention. Additionally, participating faculty, patients, and the public were satisfied with the intervention components they participated in. Several challenges and facilitators were also identified to improve the KT intervention. Conclusions The KT intervention is a promising initiative that can be adopted and adapted across various post-secondary settings to support trainees’ competency development in KT. This evaluation demonstrates that trainees will respond to opportunities for KT training and that capacity for KT competencies can be advanced through a multi-faceted intervention that involves trainees, faculty, patients, and health system collaborators in its design and delivery. This evaluation study contributes the design and results of a novel KT intervention for multi-stakeholders. Trial registration N/A.

Primary bronchial epithelial cells (pBECs) obtained from donors have limited proliferation capacity. Recently, conditional reprogramming (CR) technique has overcome this and has provided the potential for extended passaging and subsequent differentiation of cells at air-liquid interface (ALI). However, there has been no donor-specific comparison of cell morphology, baseline gene expression, barrier function, and antiviral responses compared with their “parent” pBECs, especially cells obtained from donors with asthma. We, therefore, collected and differentiated pBECs at ALI from mild donors with asthma ( n = 6) for the parent group. The same cells were conditionally reprogrammed and later differentiated at ALI. Barrier function was measured during the differentiation phase. Morphology and baseline gene expression were compared at terminal differentiation. Viral replication kinetics and antiviral responses were assessed following rhinovirus (RV) infection over 96 h. Barrier function during the differentiation phase and cell structural morphology at terminal differentiation appear similar in both parent and CR groups, however, there were elongated cell structures superficial to basal cells and significantly lower FOXJ1 expression in CR group. IFN gene expression was also significantly lower in CR group compared with parent asthma group following RV infection. The CR technique is a beneficial tool to proliferate pBECs over extended passages. Considering lower FOXJ1 expression, viral replication kinetics and antiviral responses, a cautious approach should be taken while choosing CR cells for experiments. In addition, as lab-to-lab cell culture techniques vary, the most appropriate technique must be utilized to best match individual cell functions and morphologies to address specific research questions and experimental reproducibility across the labs.

Oncostatin M (OSM) is a multifunctional cytokine secreted as a 28 kDa glycoprotein primarily from activated T lymphocytes and macrophages. OSM belongs to the interleukin-6 family that also includes interleukin-11, leukemia inhibitory factor, ciliary neurotrophic factor, cardiotrophin-1, "novel neurotrophin-1/B cell-stimulating factor-3′/cardiotrophin-like cytokine," IL-27, and IL-31. These cytokines are grouped together on the basis of weak sequence homology and shared use of the signal transducing subunit gp130 as part of their receptor complexes. Accordingly, oncostatin M shares properties with all members of this cytokine family, but is most closely related in structure and function to leukemia inhibitory factor. Oncostatin M acts on a variety of cells and displays functions involved in regulating gene activation, cell growth, and inflammatory processes. Despite these functions, investigations into its role in respiratory disease have been limited. Those studies that have been performed suggest that oncostatin M is a potent inducer of anti-proteases, is anti-inflammatory, an immune modifier and promotes the deposition of extracellular matrix proteins. Thus, it is likely that in the lung, oncostatin M may play a prominent role in normal wound healing as well as remodeling and pathological fibrosis.

In fibrosis remodelling of ECM leads to changes in composition and stiffness. Such changes can have a major impact on cell functions including proliferation, secretory profile and differentiation. Several studies have reported that fibrosis is characterised by increased senescence and accumulating evidence suggests that changes to the ECM including altered composition and increased stiffness may contribute to premature cellular senescence. This study investigated if increased stiffness could modulate markers of senescence and/or fibrosis in primary human lung fibroblasts. Using hydrogels representing stiffnesses that fall within healthy and fibrotic ranges, we cultured primary fibroblasts from non-diseased lung tissue on top of these hydrogels for up to 7 days before assessing senescence and fibrosis markers. Fibroblasts cultured on stiffer (±15 kPa) hydrogels showed higher Yes-associated protein-1 (YAP) nuclear translocation compared to soft hydrogels. When looking at senescence-associated proteins we also found higher secretion of receptor activator of nuclear factor kappa-B ligand (RANKL) but no change in transforming growth factor-β1 (TGF-β1) or connective tissue growth factor (CTGF) expression and higher decorin protein deposition on stiffer matrices. With respect to genes associated with fibrosis, fibroblasts on stiffer hydrogels compared to soft had higher expression of smooth muscle alpha (α)-2 actin (ACTA2), collagen (COL) 1A1 and fibulin-1 (Fbln1) and higher Fbln1 protein deposition after 7 days. Our results show that exposure of lung fibroblasts to fibrotic stiffness activates genes and secreted factors that are part of fibrotic responses and part of the Senescence-associated secretory phenotype (SASP). This overlap may contribute to the creation of a feedback loop whereby fibroblasts create a perpetuating cycle reinforcing progression of a fibrotic response.

BACKGROUND:Asthma and chronic obstructive pulmonary disease (COPD) are common chronic respiratory diseases, and some patients have overlapping disease features, termed asthma-COPD overlap (ACO). Patients characterized with ACO have increased disease severity; however, the mechanisms driving this have not been widely studied.OBJECTIVES:This study sought to characterize the phenotypic and transcriptomic features of experimental ACO in mice induced by chronic house dust mite antigen and cigarette smoke exposure.METHODS:Female BALB/c mice were chronically exposed to house dust mite antigen for 11 weeks to induce experimental asthma, cigarette smoke for 8 weeks to induce experimental COPD, or both concurrently to induce experimental ACO. Lung inflammation, structural changes, and lung function were assessed. RNA-sequencing was performed on separated airway and parenchyma lung tissues to assess transcriptional changes. Validation of a novel upstream driver SPI1 in experimental ACO was assessed using the pharmacological SPI1 inhibitor, DB2313.RESULTS:Experimental ACO recapitulated features of both asthma and COPD, with mixed pulmonary eosinophilic/neutrophilic inflammation, small airway collagen deposition, and increased airway hyperresponsiveness. Transcriptomic analysis identified common and distinct dysregulated gene clusters in airway and parenchyma samples in experimental asthma, COPD, and ACO. Upstream driver analysis revealed increased expression of the transcription factor Spi1. Pharmacological inhibition of SPI1 using DB2313, reduced airway remodeling and airway hyperresponsiveness in experimental ACO.CONCLUSIONS:A new experimental model of ACO featuring chronic dual exposures to house dust mite and cigarette smoke mimics key disease features observed in patients with ACO and revealed novel disease mechanisms, including upregulation of SPI1, that are amenable to therapy.

Idiopathic pulmonary fibrosis (IPF) is an interstitial lung disease associated with chronic inflammation and tissue remodelling leading to fibrosis, reduced pulmonary function, respiratory failure and death. Bleomycin (Blm)-induced lung fibrosis in mice replicates several clinical features of human IPF, including prominent lymphoid aggregates of predominantly B-cells that accumulate in the lung adjacent to areas of active fibrosis. We have shown previously a requirement for B-cells in the development of Blm-induced lung fibrosis in mice. To determine the therapeutic potential of inhibiting B-cell function in pulmonary fibrosis, we examined the effects of anti-CD20 B-cell ablation therapy to selectively remove mature B-cells from the immune system and inhibit Blm-induced lung fibrosis. Anti-CD20 B-cell ablation did not reduce fibrosis in this model; however, immune phenotyping of peripheral blood and lung resident cells revealed that anti-CD20-treated mice retained a high frequency of CD19+ CD138+ plasma cells. Interestingly, high levels of CD138+ cells were also identified in the lung tissue of patients with IPF, consistent with the mouse model. Treatment of mice with bortezomib, which depletes plasma cells, reduced the level of Blm-induced lung fibrosis, implicating plasma cells as important effector cells in the development and progression of pulmonary fibrosis.

IL-25 is implicated in the pathogenesis of viral asthma exacerbations. However, the effect of IL-25 on antiviral immunity has yet to be elucidated. We observed abundant expression and colocalization of IL-25 and IL-25 receptor at the apical surface of uninfected airway epithelial cells and rhinovirus infection increased IL-25 expression. Analysis of immune transcriptome of rhinovirus-infected differentiated asthmatic bronchial epithelial cells (BECs) treated with an anti-IL-25 monoclonal antibody (LNR125) revealed a re-calibrated response defined by increased type I/III IFN and reduced expression of type-2 immune genes CCL26, IL1RL1 and IL-25 receptor. LNR125 treatment also increased type I/III IFN expression by coronavirus infected BECs. Exogenous IL-25 treatment increased viral load with suppressed innate immunity. In vivo LNR125 treatment reduced IL-25/type 2 cytokine expression and increased IFN-β expression and reduced lung viral load. We define a new immune-regulatory role for IL-25 that directly inhibits virus induced airway epithelial cell innate anti-viral immunity.

Idiopathic pulmonary fibrosis (IPF) is a chronic disease characterised by a dense fibrosing of the lung parenchyma. An association between IPF and cellular senescence is well established and several studies now describe a higher abundance of senescent fibroblasts and epithelial cells in the lungs of IPF patients compared with age-matched controls. The cause of this abnormal accumulation of senescent cells is unknown but evidence suggests that, once established, senescence can be transferred from senescent to non-senescent cells. In this study, we investigated whether senescent human lung fibroblasts (LFs) and alveolar epithelial cells (AECs) could induce a senescent-like phenotype in "naïve" non-senescent LFs in vitro. Primary cultures of LFs from adult control donors (Ctrl-LFs) with a low baseline of senescence were exposed to conditioned medium (CM) from: (i) Ctrl-LFs induced to become senescent using H2O2 or etoposide; (ii) LFs derived from IPF patients (IPF-LFs) with a high baseline of senescence; or (iii) senescence-induced A549 cells, an AEC line. Additionally, ratios of non-senescent Ctrl-LFs and senescence-induced Ctrl-LFs (100:0, 0:100, 50:50, 90:10, 99:1) were co-cultured and their effect on induction of senescence measured. We demonstrated that exposure of naïve non-senescent Ctrl-LFs to CM from senescence-induced Ctrl-LFs and AECs and IPF-LFs increased the markers of senescence including nuclear localisation of phosphorylated-H2A histone family member X (H2AXγ) and expression of p21, IL-6 and IL-8 in Ctrl-LFs. Additionally, co-cultures of non-senescent and senescence-induced Ctrl-LFs induced a senescent-like phenotype in the non-senescent cells. These data suggest that the phenomenon of "senescence-induced senescence" can occur in vitro in primary cultures of human LFs, and provides a possible explanation for the abnormal abundance of senescent cells in the lungs of IPF patients.

The interleukin (IL)-6 family of cytokines and exaggerated signal transducer and activator of transcription (STAT)3 signaling is implicated in idiopathic pulmonary fibrosis (IPF) pathogenesis, but the mechanisms regulating STAT3 expression and function are unknown. Suppressor of cytokine signaling (SOCS)1 and SOCS3 block STAT3, and low SOCS1 levels have been reported in IPF fibroblasts and shown to facilitate collagen production. Fibroblasts and lung tissue from IPF patients and controls were used to examine the mechanisms underlying SOCS1 down-regulation in IPF. A significant reduction in basal SOCS1 mRNA in IPF fibroblasts was confirmed. However, there was no difference in the kinetics of activation, and methylation of SOCS1 in control and IPF lung fibroblasts was low and unaffected by 5′-aza-2′-deoxycytidine’ treatment. SOCS1 is a target of microRNA-155 and although microRNA-155 levels were increased in IPF tissue, they were reduced in IPF fibroblasts. Therefore, SOCS1 is not regulated by SOCS1 gene methylation or microRNA155 in these cells. In conclusion, we confirmed that IPF fibroblasts had lower levels of SOCS1 mRNA compared with control fibroblasts, but we were unable to determine the mechanism. Furthermore, although SOCS1 may be important in the fibrotic process, we were unable to find a significant role for SOCS1 in regulating fibroblast function.

HIF-1 plays an important role in epithelial restitution, selectively inducing integrins α6 and α2 to promote migration and proliferation, respectively. HIF-stabilizing prolyl-hydroxylase inhibitors accelerate intestinal mucosal healing by inducing epithelial integrin expression.

Re-establishment of a functional barrier following injury requires successful re-differentiation of airway epithelial cells. A population of resident basal epithelial cells has been identified in human airways that express cytokeratin 5 (Krt5) and 14 (Krt14) and are capable of self-renewal and generate differentiated epithelial cells during repair. Appropriately differentiated airway epithelium protects the lung parenchyma from inhaled particles/pathogens via luminal muco-ciliary clearance and structural barrier conferred by tight junctions (TJ) which are regulated by a network of tissue-specific inter- and intramembrane protein complexes linked to the underlying actin cytoskeleton. Contractility in actin filaments in turn drives junctional biogenesis that supports repair and return to steady state function. In patients with asthma, epithelial barrier is intrinsically compromised with loss of structural integrity perpetuated by a state of aberrant, non-resolving repair. Therefore, understanding the expression and distribution of structural proteins that are required for junction formation will identify novel targets for pharmacological modulation to promote airway epithelial barrier repair in asthma. We have mapped temporal expression of TJ proteins during in vitro differentiation of bronchial epithelial cells from subjects with asthma and healthy donors. We identified disrupted localization of ZO-1, claudin-4 and -7 in early phases of differentiation in bronchial epithelial cells from asthmatic patients that coincided with persistent expression of Krt5+ and Krt14+ in resident Krt5+ basal cells, indicative of unresolved repair. This was associated with dysregulated arrangement in peri-junctional actin cytoskeleton. In summary, we provide evidence that disrupted airway epithelial barrier in asthma is related to loss of F-actin-mediated localisation of TJ proteins and reduced transition of basal cells from a reparative phenotype to differentiated epithelial cells.

Excessive mucus production is a major feature of allergic asthma. Disruption of epithelial junctions by allergens such as house dust mite (HDM) results in the activation of β-catenin signaling, which has been reported to stimulate goblet cell differentiation. β-catenin interacts with various co-activators including CREB binding protein (CBP) and p300, thereby regulating the expression of genes involved in cell proliferation and differentiation, respectively. We specifically investigated the role of the β-catenin/CBP signaling pathway in goblet cell metaplasia in a HDM-induced allergic airway disease model in mice using ICG-001, a small molecule inhibitor that blocks the binding of CBP to β-catenin. Female 6- 8-week-old BALB/c mice were sensitized to HDM/saline on days 0, 1, and 2, followed by intranasal challenge with HDM/saline with or without subcutaneous ICG-001/vehicle treatment from days 14 to 17, and samples harvested 24 h after the last challenge/treatment. Differential inflammatory cells in bronchoalveolar lavage (BAL) fluid were enumerated. Alcian blue (AB)/Periodic acid–Schiff (PAS) staining was used to identify goblet cells/mucus production, and airway hyperresponsiveness (AHR) was assessed using invasive plethysmography. Exposure to HDM induced airway inflammation, goblet cell metaplasia and increased AHR, with increased airway resistance in response to the non-specific spasmogen methacholine. Inhibition of the β-catenin/CBP pathway using treatment with ICG-001 significantly attenuated the HDM-induced goblet cell metaplasia and infiltration of macrophages, but had no effect on eosinophils, neutrophils, lymphocytes or AHR. Increased β-catenin/CBP signaling may promote HDM-induced goblet cell metaplasia in mice.

Background We assessed whether Toll-like receptor (TLR)2 activation boosts the innate immune response to rhinovirus infection, as a treatment strategy for virus-induced respiratory diseases. Methods We employed treatment with a novel TLR2 agonist (INNA-X) prior to rhinovirus infection in mice, and INNA-X treatment in differentiated human bronchial epithelial cells derived from asthmatic-donors. We assessed viral load, immune cell recruitment, cytokines, type I and III interferon (IFN) production, as well as the lung tissue and epithelial cell immune transcriptome. Results We show, in vivo , that a single INNA-X treatment induced innate immune priming characterised by low-level IFN-λ, Fas ligand, chemokine expression and airway lymphocyte recruitment. Treatment 7 days before infection significantly reduced lung viral load, increased IFN-β/λ expression and inhibited neutrophilic inflammation. Corticosteroid treatment enhanced the anti-inflammatory effects of INNA-X. Treatment 1 day before infection increased expression of 190 lung tissue immune genes. This tissue gene expression signature was absent with INNA-X treatment 7 days before infection, suggesting an alternate mechanism, potentially via establishment of immune cell-mediated mucosal innate immunity. In vitro , INNA-X treatment induced a priming response defined by upregulated IFN-λ, chemokine and anti-microbial gene expression that preceded an accelerated response to infection enriched for nuclear factor (NF)-κB-regulated genes and reduced viral loads, even in epithelial cells derived from asthmatic donors with intrinsic delayed anti-viral immune response. Conclusion Airway epithelial cell TLR2 activation induces prolonged innate immune priming, defined by early NF-κB activation, IFN-λ expression and lymphocyte recruitment. This response enhanced anti-viral innate immunity and reduced virus-induced airway inflammation.

Alveolar epithelial cell (AEC) senescence is implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Mitochondrial dysfunction including release of mitochondrial DNA (mtDNA) is a feature of senescence, which led us to investigate the role of the DNA-sensing guanine monophosphate-adenine monophosphate (GMP-AMP) synthase (cGAS) in IPF, with a focus on AEC senescence. cGAS expression in fibrotic tissue from lungs of patients with IPF was detected within cells immunoreactive for epithelial cell adhesion molecule (EpCAM) and p21, epithelial and senescence markers, respectively. Submerged primary cultures of AECs isolated from lung tissue of patients with IPF (IPF-AECs, n = 5) exhibited higher baseline senescence than AECs from control donors (Ctrl-AECs, n = 5–7), as assessed by increased nuclear histone 2AXγ phosphorylation, p21 mRNA, and expression of senescence-associated secretory phenotype (SASP) cytokines. Pharmacological cGAS inhibition using RU.521 diminished IPF-AEC senescence in culture and attenuated induction of Ctrl-AEC senescence following etoposide-induced DNA damage. Short interfering RNA (siRNA) knockdown of cGAS also attenuated etoposide-induced senescence of the AEC line, A549. Higher levels of mtDNA were detected in the cytosol and culture supernatants of primary IPF- and etoposide-treated Ctrl-AECs when compared with Ctrl-AECs at baseline. Furthermore, ectopic mtDNA augmented cGAS-dependent senescence of Ctrl-AECs, whereas DNAse I treatment diminished IPF-AEC senescence. This study provides evidence that a self-DNA-driven, cGAS-dependent response augments AEC senescence, identifying cGAS as a potential therapeutic target for IPF.

The airway epithelium of children with wheeze is characterized by defective repair that contributes to disease pathobiology. Dysregulation of developmental processes controlled by Notch has been identified in chronic asthma. However, its role in airway epithelial cells of young children with wheeze, particularly during repair, is yet to be determined. We hypothesized that Notch is dysregulated in primary airway epithelial cells (pAEC) of children with wheeze contributing to defective repair. This study investigated transcriptional and protein expression and function of Notch in pAEC isolated from children with and without wheeze. Primary AEC of children with and without wheeze were found to express all known Notch receptors and ligands, although pAEC from children with wheeze expressed significantly lower NOTCH2 (10-fold, p = 0.004) and higher JAG1 (3.5-fold, p = 0.002) mRNA levels. These dysregulations were maintained in vitro and cultures from children with wheeze displayed altered kinetics of both NOTCH2 and JAG1 expression during repair. Following Notch signaling inhibition, pAEC from children without wheeze failed to repair (wound closure rate of 76.9 ± 3.2%). Overexpression of NOTCH2 in pAEC from children with wheeze failed to rescue epithelial repair following wounding. This study illustrates the involvement of the Notch pathway in airway epithelial wound repair in health and disease, where its dysregulation may contribute to asthma development.