Asthmatic Fibroblasts Research Articles

Calprotectin is regulated by IL-17A and induces steroid hyporesponsiveness in asthma.

Calprotectin, a calcium-binding protein, plays a crucial role in inflammation and has been associated with various inflammatory diseases, including asthma. However, its regulation and impact on steroid hyporesponsiveness, especially in severe asthma, remain poorly understood. This study investigated the regulation of calprotectin proteins (S100A8 and S100A9) by IL-17 and its role in steroid hyporesponsiveness using in vitro and in vivo models. Calprotectin expression was assessed in primary bronchial fibroblasts from healthy controls and severe asthmatic patients, as well as in mouse models of steroid hyporesponsive lung inflammation induced by house dust mite (HDM) allergen and cyclic-di-GMP (cdiGMP) adjuvant. The effects of IL-17A stimulation on calprotectin expression and steroid response markers in bronchial epithelial and fibroblast cells were examined. Additionally, the therapeutic potential of paquinimod, a calprotectin inhibitor, in mitigating airway inflammation and restoring steroid response signatures in the mouse model was evaluated. The results demonstrated upregulation of calprotectin expression in asthmatic bronchial fibroblasts compared to healthy controls, as well as in refractory asthma samples compared to non-refractory asthma. IL-17 stimulation induced calprotectin expression and dysregulated glucocorticoid response signatures in lung epithelial and fibroblast cells. Treatment with paquinimod reversed IL-17-induced dysregulation of steroid signatures, indicating the involvement of calprotectin in this process. In the HDM/cdiGMP mouse model, paquinimod significantly attenuated airway inflammation and hyperresponsiveness, and restored steroid response signatures, whereas dexamethasone showed limited efficacy. Mechanistically, paquinimod inhibited MAPK/ERK and NF-κB pathways downstream of calprotectin, leading to reduced lung inflammation. These findings highlight calprotectin as a potential therapeutic target regulated by IL-17 in steroid hyporesponsive asthma. Targeting calprotectin may offer a promising approach to alleviate airway inflammation and restore steroid responsiveness in severe asthma. Further investigations are warranted to explore its therapeutic potential in clinical settings and elucidate its broader implications in steroid mechanisms of action.

Vitamin D mediated mitigation of fibrotic remodeling in asthma is associated with metabolic reprogramming

Vitamin D mediated mitigation of fibrotic remodeling in asthma is associated with metabolic reprogramming Rakhee K. Ramakrishnan1, Zaina Kalaji1, Khuloud Bajbouj1, Rabih Halwani1,2, Qutayba Hamid1,3 1 Sharjah Institute for Medical Research – College of Medicine, University of Sharjah, Sharjah, United Arab Emirates 2 Immunology Research Lab, College of Medicine, King Saud University, Riyadh, Saudi Arabia 3 Meakins-Christie Laboratories, McGill University, Montreal, Quebec, CanadaAsthma is a heterogeneous disorder that is associated with chronic airway inflammation and remodeling of the airways. While vitamin D deficiency has shown correlations with various clinical features of asthma, the mechanisms responsible for this action is poorly understood. This study therefore explored the molecular mechanisms associated with the anti-remodeling potential of vitamin D. We hypothesized that vitamin D induced metabolic reprogramming contributes to its anti-fibrotic activity. To this end, primary lung fibroblasts from asthmatic and non-asthmatic individuals were stimulated with TGF-β1 alone or in combination with the biologically active form of vitamin D, calcitriol. The expression of various fibrotic and metabolic markers was assessed both at the transcript and protein level. While vitamin D receptor (VDR) was constitutively expressed in human lung fibroblasts, calcitriol treatment induced a robust VDR response in asthmatic fibroblasts indicating increased VDR functionality in these cells when compared to normal. TGF-β1 stimulation led to an increase in the expression of several fibrotic markers, including COL1A1, COL3A1, COL5A1, FN1 and ACTA2, which was reversed after calcitriol treatment. These anti-fibrotic effects of calcitriol were associated with dysregulated AMPK signaling and an inhibition of TGF-β1-induced mitochondrial ROS release. Our findings thus, highlight the ability of vitamin D in regulating the cellular mitochondrial metabolism induced by TGF-β1 in lung fibroblasts, suggesting a possible mechanism associated with its anti-fibrotic activity. Since current asthma therapeutics do not effectively target the airway structural changes that are increasingly associated with asthma severity, vitamin D supplementation presents an attractive therapeutic avenue to attenuate subepithelial fibrosis and airway remodeling in asthmatic airways. We would like to acknowledge the funding received from University of Sharjah targeted research grant (ID 2101090295) in conducting this study. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Asthmatic lung fibroblasts promote type 2 immune responses via endoplasmic reticulum stress response dependent thymic stromal lymphopoietin secretion.

Lung fibroblasts contribute to asthma pathology partly through modulation of the immune environment in the airway. Tumor necrosis factor-α (TNFα) expression is upregulated in asthmatic lungs. How asthmatic lung fibroblasts respond to TNFα stimulation and subsequently regulate immune responses is not well understood. Endoplasmic reticulum (ER) stress and unfolded protein responses (UPR) play important roles in asthma, but their functional roles are still under investigation. In this study, we investigated TNFα-induced cytokine production in primary lung fibroblasts from asthmatic vs. non-asthmatic human subjects, and downstream effects on type 2 immune responses. TNFα significantly upregulated IL-6, IL-8, C-C motif chemokine ligand 5 (CCL5), and thymic stromal lymphopoietin (TSLP) mRNA expression and protein secretion by lung fibroblasts. Asthmatic lung fibroblasts secreted higher levels of TSLP which promoted IL-33-induced IL-5 and IL-13 production by peripheral blood mononuclear cells. TNFα exposure enhanced expression of ER stress/UPR pathways in both asthmatic and non-asthmatic lung fibroblasts, especially inositol-requiring protein 1α in asthmatics. ER stress/UPR inhibitors decreased IL-6, CCL5, and TSLP protein secretion by asthmatic lung fibroblasts. Our data suggest that TNFα and lung fibroblasts form an important axis in asthmatic lungs to promote asthmatic inflammation that can be attenuated by inhibiting ER stress/UPR pathway.

Leptin-producing monocytes in the airway submucosa may contribute to asthma pathogenesis

BackgroundObesity leads to an increase in the incidence and severity of asthma. Adipokines, such as leptin, secreted by adipocytes induce systemic inflammation, causing airway inflammation. We previously reported that leptin activates both inflammatory and structural cells, including lung fibroblasts. However, little is known about the differential leptin expression and responsiveness to leptin in asthmatic individuals and healthy controls (HC). In this study, we investigated the expression and origin of leptin in asthmatic airways. We also compared the effect of leptin on asthmatic and HC fibroblasts. MethodsLung specimens from asthmatic and non-asthmatic patients were analyzed by immunohistochemical staining using anti-leptin and anti-CD163 antibodies. Leptin mRNA and protein levels in human monocytes were detected by real-time PCR and western blotting and ELISA, respectively. We used flow cytometry to analyze asthmatic and HC lung fibroblasts for leptin receptor (Ob-R) expression. Further, we determined cytokine levels using cytometric bead array and ELISA and intracellular phosphorylation of specific signaling molecules using western blotting. ResultsAsthma specimens displayed accumulation of leptin-positive inflammatory cells, which were also positive for CD163, a high-affinity scavenger receptor expressed by monocytes and macrophages. Leptin expression was observed at both transcript and protein levels in human blood-derived monocytes. No significant differences were observed between asthmatic and HC lung fibroblasts in Ob-R expression, cytokine production, and intracellular phosphorylation of p38 mitogen-activated protein kinase. ConclusionsOur findings reveal similar responsiveness of control and asthmatic fibroblasts to leptin. However, the accumulation of inflammatory leptin-producing monocytes in the airway may contribute to the pathogenesis of asthma.

Modulation of Arginase-2 mRNA Levels by ω-3 PUFAs and Aspirin in Asthmatic Human Lung Fibroblasts.

Airway remodeling (AR) increases disease severity, and morbidity of asthmatic patients by contributing to irreversible airflow obstruction and progressive declines in lung function. Arginase isoenzymes and the downstream enzymes ornithine decarboxylase (ODC) and ornithine aminotransferase (OAT) have been implicated in the hyperplastic and fibrotic changes of AR, respectively. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs) and resolvin metabolites have anti-AR effects, but whether they are mediated through the arginase pathway is unclear. Our study intended to determine the effects of the ω-3 PUFAs eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), resolvin D1 (RvD1), TH1 cytokines, acetylsalicylic acid (ASA), cAMP, and dexamethasone (DEX) on the expression of arginase isoenzymes arginase 1 (ARG1) and arginase 2 (ARG2), ODC, and OAT in human lung fibroblasts (HLF) from normal (NHLF) and diseased (DHLF) asthmatic donors using reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR). Our data showed that EPA and EPA+DHA downregulated ARG2 mRNA 2-fold in both types of HLF. DHA, RvD1, and DEX did not alter mRNA levels for any of the genes studied. EPA lowered the ARG2 protein levels in DHLF, but did not affect those levels in NHLF. ASA upregulated ARG2 mRNA 5-fold and 7-fold in NHLF and DHLF, respectively, TH1 cytokines downregulated ARG2, ODC, and OAT mRNA in DHLF 10-fold, 2-fold, and 2.5-fold, respectively, and cAMP downregulated ARG2 mRNA 2-fold in DHLF. These results are the first to show a direct effect of ω-3 PUFAs on ARG2 mRNA levels and provide further evidence for a role of ω-3 PUFAs in AR.

Bcl10 Regulates Lipopolysaccharide-Induced Pro-Fibrotic Signaling in Bronchial Fibroblasts from Severe Asthma Patients.

Subepithelial fibrosis is a characteristic hallmark of airway remodeling in asthma. Current asthma medications have limited efficacy in treating fibrosis, particularly in patients with severe asthma, necessitating a deeper understanding of the fibrotic mechanisms. The NF-κB pathway is key to airway inflammation in asthma, as it regulates the activity of multiple pro-inflammatory mediators that contribute to airway pathology. Bcl10 is a well-known upstream mediator of the NF-κB pathway that has been linked to fibrosis in other disease models. Therefore, we investigated Bcl10-mediated NF-κB activation as a potential pathway regulating fibrotic signaling in severe asthmatic fibroblasts. We demonstrate here the elevated protein expression of Bcl10 in bronchial fibroblasts and bronchial biopsies from severe asthmatic patients when compared to non-asthmatic individuals. Lipopolysaccharide (LPS) induced the increased expression of the pro-fibrotic cytokines IL-6, IL-8 and TGF-β1 in bronchial fibroblasts, and this induction was associated with the activation of Bcl10. Inhibition of the Bcl10-mediated NF-κB pathway using an IRAK1/4 selective inhibitor abrogated the pro-fibrotic signaling induced by LPS. Thus, our study indicates that Bcl10-mediated NF-κB activation signals increased pro-fibrotic cytokine expression in severe asthmatic airways. This reveals the therapeutic potential of targeting Bcl10 signaling in ameliorating inflammation and fibrosis, particularly in severe asthmatic individuals.

TARGETING THE HISTONE DEMETHYLASE JMJD2B/KDM4B AS A NOVEL THERAPEUTIC APPROACH IN IL-13-MEDIATED FIBROSIS IN AIRWAY FIBROBLASTS OF SEVERE ASTHMATICS

TYPE: Abstract TOPIC: Allergy and Airway PURPOSE: Asthma is a heterogeneous disorder characterized by chronic inflammation and remodeling of the airways. Asthma responses are driven mainly by type 2 immune responses, where IL-13 plays a key role in asthma pathogenesis. We previously identified JMJD2B as an IL-13-regulated epigenetic modifier in asthmatic airway fibroblasts. Therefore, this study aimed to target JMJD2B to potentially alleviate IL-13-mediated fibrosis in asthma. METHODS: Bronchial fibroblasts isolated from asthmatic and healthy individuals were stimulated with IL-13 and treated with JIB-04, a pan-selective inhibitor of histone demethylase(s). Gene and protein expression of extracellular matrix markers were assessed using qRT-PCR, western blotting and matrix metalloproteinases (MMPs) activity assay. Chromatin immunoprecipitation (ChIP) assay was used to determine the bindig of JMJD2B to promoter region of Tissue inhibitors of matrix metalloproteinases (TIMPs). RESULTS: Here we show that JIB-04 inhibited JMJD2B activity by reducing the demethylation of its downstream target, H3K36me3, in asthmatic fibroblasts. Inhibition of JMJD2B significantly affected the viability of the bronchial fibroblasts at 48 hours. JMJD2B inhibition was further associated with the downregulation of extracellular matrix (ECM) proteins such as MMP-2, MMP-9, collagen-1, and fibronectin, and upregulation of TIMP-2, at both the gene and protein levels. This was accompanied by inhibition of IL-13 mediated MMPs activity. JIB-04 further prevented the association of JMJD2B on TIMP-2 promoter region. CONCLUSIONS: Our results demonstrate the potential of JMJD2B inhibition in attenuating IL-13-mediated fibrosis in asthma. CLINICAL IMPLICATIONS: Therapeutic targeting of JMJD2B using JIB-04 is a promising candidate to alleviate IL-13-mediated responses in chronic disorders such as asthma. DISCLOSURE: Nothing to declare. KEYWORD: Asthma

Derangement of cell cycle markers in peripheral blood mononuclear cells of asthmatic patients as a reliable biomarker for asthma control

In asthma, most of the identified biomarkers pertain to the Th2 phenotype and no known biomarkers have been verified for severe asthmatics. Therefore, identifying biomarkers using the integrative phenotype-genotype approach in severe asthma is needed. The study aims to identify novel biomarkers as genes or pathways representing the core drivers in asthma development, progression to the severe form, resistance to therapy, and tissue remodeling regardless of the sample cells or tissues examined. Comprehensive reanalysis of publicly available transcriptomic data that later was validated in vitro, and locally recruited patients were used to decipher the molecular basis of asthma. Our in-silicoanalysis revealed a total of 10 genes (GPRC5A, SFN, ABCA1, KRT8, TOP2A, SERPINE1, ANLN, MKI67, NEK2, and RRM2) related to cell cycle and proliferation to be deranged in the severe asthmatic bronchial epithelium and fibroblasts compared to their healthy counterparts. In vitro, RT qPCR results showed that (SERPINE1 and RRM2) were upregulated in severe asthmatic bronchial epithelium and fibroblasts, (SFN, ABCA1, TOP2A, SERPINE1, MKI67, and NEK2) were upregulated in asthmatic bronchial epithelium while (GPRC5A and KRT8) were upregulated only in asthmatic bronchial fibroblasts. Furthermore, MKI76, RRM2, and TOP2A were upregulated in Th2 high epithelium while GPRC5A, SFN, ABCA1 were upregulated in the blood of asthmatic patients. SFN, ABCA1 were higher, while MKI67 was lower in severe asthmatic with wheeze compared to nonasthmatics with wheezes. SERPINE1 and GPRC5A were downregulated in the blood of eosinophilic asthmatics, while RRM2 was upregulated in an acute attack of asthma. Validation of the gene expression in PBMC of locally recruited asthma patients showed that SERPINE1, GPRC5A, SFN, ABCA1, MKI67, and RRM2 were downregulated in severe uncontrolled asthma. We have identified a set of biologically crucial genes to the homeostasis of the lung and in asthma development and progression. This study can help us further understand the complex interplay between the transcriptomic data and the external factors which may deviate our understanding of asthma heterogeneity.

Wnt Signaling Is Deranged in Asthmatic Bronchial Epithelium and Fibroblasts.

Both canonical and non-canonical Wnt signaling pathway alterations have been documented in pulmonary disease pathogenesis and progression; therefore, they can be an attractive target for pharmaceutical management of severe asthma. Wnt/β-catenin signaling was shown to link early embryonic lung development impairment to later in life asthmatic airway remodeling. Here we explored the changes in Wnt signaling associated with asthma initiation and progression in epithelial and fibroblasts using a comprehensive approach based on in silico analysis and followed by in vitro validation. In summary, the in silico analysis showed that the bronchial epithelium of severe asthmatic patients showed a deranged balance between Wnt enhancer and Wnt inhibitors. A Th2-high phenotype is associated with upregulated Wnt-negative regulators, while inflammatory and neutrophilic severe asthmatics showed higher canonical Wnt signaling member enrichment. Most of these genes are regulators of healthy lung development early in life and, if disturbed, can make people susceptible to developing asthma early in life and prone to developing a severe phenotype. Most of the Wnt members are secreted, and their effect can be in an autocrine fashion on the bronchial epithelium, paracrine on nearby adjacent structural cells like fibroblasts and smooth muscles, or systemic in blood. Our results showed that canonical Wnt signaling is needed for the proper response of cells to proliferative stimuli, which puts cells under stress. Cells in response to this proliferative stress will activate the senescence mechanism, which is also dependent on Wnt signaling. Inhibition of Wnt signaling using FH535 inhibits both proliferation and senescence markers in bronchial fibroblasts compared to DMSO-treated cells. In fibroblasts from asthmatic patients, inhibition of Wnt signaling did not show that effect as the Wnt signaling is deranged besides other pathways that might be non-functional.

Responsiveness of human bronchial fibroblasts and epithelial cells from asthmatic and non-asthmatic donors to the transforming growth factor-\u03b21 in epithelial-mesenchymal trophic unit model

BackgroundThe asthma-related airway wall remodeling is associated i.a. with a damage of bronchial epithelium and subepithelial fibrosis. Functional interactions between human bronchial epithelial cells and human bronchial fibroblasts are known as the epithelial-mesenchymal trophic unit (EMTU) and are necessary for a proper functioning of lung tissue. However, a high concentration of the transforming growth factor-β1 (TGF-β1) in the asthmatic bronchi drives the structural disintegrity of epithelium with the epithelial-to-mesenchymal transition (EMT) of the bronchial epithelial cells, and of subepithelial fibrosis with the fibroblast-to-myofibroblast transition (FMT) of the bronchial fibroblasts. Since previous reports indicate different intrinsic properties of the human bronchial epithelial cells and human bronchial fibroblasts which affect their EMT/FMT potential beetween cells derived from asthmatic and non-asthmatic patients, cultured separatelly in vitro, we were interested to see whether corresponding effects could be obtained in a co-culture of the bronchial epithelial cells and bronchial fibroblasts. In this study, we investigate the effects of the TGF-β1 on the EMT markers of the bronchial epithelial cells cultured in the air-liquid-interface and effectiveness of FMT in the bronchial fibroblast populations in the EMTU models.ResultsOur results show that the asthmatic co-cultures are more sensitive to the TGF-β1 than the non-asthmatic ones, which is associated with a higher potential of the asthmatic bronchial cells for a profibrotic response, analogously to be observed in '2D' cultures. They also indicate a noticeable impact of human bronchial epithelial cells on the TGF-β1-induced FMT, stronger in the asthmatic bronchial fibroblast populations in comparison to the non-asthmatic ones. Moreover, our results suggest the protective effects of fibroblasts on the structure of the TGF-β1–exposed mucociliary differentiated bronchial epithelial cells and their EMT potential.ConclusionsOur data are the first to demonstrate a protective effect of the human bronchial fibroblasts on the properties of the human bronchial epithelial cells, which suggests that intrinsic properties of not only epithelium but also subepithelial fibroblasts affect a proper condition and function of the EMTU in both normal and asthmatic individuals.

IL-13 Augments Histone Demethylase JMJD2B/KDM4B Expression Levels, Activity, and Nuclear Translocation in Airway Fibroblasts in Asthma.

Purpose Asthma is one of the most common obstructive pulmonary diseases worldwide. Epigenetic alterations, including DNA methylation and histone modifications, have been reported to contribute to asthma pathogenesis. Since the inflammation mediator and remodeling trigger, IL-13, is known to play a central role in the pathophysiology of asthma, this study was aimed to identify novel IL-13-regulated epigenetic modifiers in asthma that may contribute to subepithelial fibrosis. Methods Publicly available transcriptomic datasets from Gene Expression Omnibus (GEO) were used to identify differentially expressed genes on an epigenetic level upon IL-13 exposure in lung fibroblasts. Bronchial fibroblasts isolated from healthy and asthmatic individuals were assessed for the gene and protein expression levels of the identified gene at baseline and upon IL-13 treatment using qRT-PCR and western blotting, respectively. Its subcellular localization and tissue distribution were examined in bronchial fibroblasts as well as bronchial biopsies by immunofluorescence and immunohistochemical analysis, respectively. Results Bioinformatic analysis revealed the differential expression of the histone demethylase JMJD2B/KDM4B, a well-known epigenetic modulator that leads to the demethylation of different lysine residues on histones, in IL-13-treated lung fibroblasts. The baseline expression levels of JMJD2B were higher in asthmatic fibroblasts and in bronchial biopsies in comparison to healthy ones. There was also an increase in JMJD2B activity as evidenced by the demethylation of its downstream target, H3K36me3. Furthermore, IL-13 stimulation induced JMJD2B expression and further demethylation of H3K36me3 in asthmatic fibroblasts. This was accompanied by increased translocation of JMJD2B into the nucleus. Conclusion This study highlights the novel pathological involvement of the histone demethylase JMJD2B/KDM4B in asthmatic airway fibroblasts that are regulated by IL-13. Clinical implications. Given that there is no single therapeutic medicine to effectively treat the various subtypes of asthma, this study provides promising insights into JMJD2B as a new therapeutic target that could potentially improve the treatment and management of asthma.

Enhanced mitophagy in bronchial fibroblasts from severe asthmatic patients.

Sub-epithelial fibrosis is a characteristic feature of airway remodeling in asthma which correlates with disease severity. Current asthma medications are ineffective in treating fibrosis. In this study, we aimed to investigate the mitochondrial phenotype in fibroblasts isolated from airway biopsies of non-asthmatic and severe asthmatic subjects by examining mitophagy as a mechanism contributing to fibroblast persistence and thereby, fibrosis in severe asthma. Bioinformatics analysis of publicly available transcriptomic data was performed to identify the top enriched pathways in asthmatic fibroblasts. Endogenous expression of mitophagy markers in severe asthmatic and non-asthmatic fibroblasts was determined using qRT-PCR, western blot and immunofluorescence. Mitophagy flux was examined by using lysosomal protease inhibitors, E64d and pepstatin A. Mitochondrial membrane potential and metabolic activity were also evaluated using JC-1 assay and MTT assay, respectively. Bioinformatics analysis revealed the enrichment of Pink/Parkin-mediated mitophagy in asthmatic fibroblasts compared to healthy controls. In severe asthmatic fibroblasts, the differential expression of mitophagy genes, PINK1 and PRKN, was accompanied by the accumulation of PINK1, Parkin and other mitophagy proteins at baseline. The further accumulation of endogenous LC3BII, p62 and PINK1 in the presence of E64d and pepstatin A in severe asthmatic fibroblasts reinforced their enhanced mitophagy flux. Significantly reduced mitochondrial membrane potential and metabolic activity were also demonstrated at baseline confirming the impairment in mitochondrial function in severe asthmatic fibroblasts. Interestingly, these fibroblasts displayed neither an apoptotic nor senescent phenotype but a pro-fibrotic phenotype with an adaptive survival mechanism triggered by increased AMPKα phosphorylation and mitochondrial biogenesis. Our results demonstrated a role for mitophagy in the pathogenesis of severe asthma where the enhanced turnover of damaged mitochondria may contribute to fibrosis in severe asthma by promoting the persistence and pro-fibrotic phenotype of fibroblasts.

Blood and Salivary Amphiregulin Levels as Biomarkers for Asthma.

Background: Amphiregulin (AREG) expression in asthmatic airways and sputum was shown to increase and correlate with asthma. However, no studies were carried out to evaluate the AREG level in blood and saliva of asthmatic patients.Objective: To measure circulating AREG mRNA and protein concentrations in blood, saliva, and bronchial biopsies samples from asthmatic patients.Methods: Plasma and Saliva AREG protein concentrations were measured using ELISA while PBMCs, and Saliva mRNA expression was measured by RT qPCR in non-severe, and severe asthmatic patients compared to healthy controls. Primary asthmatic bronchial epithelial cells and fibroblasts were assessed for AREG mRNA expression and released soluble AREG in their conditioned media. Tissue expression of AREG was evaluated using immunohistochemistry of bronchial biopsies from asthmatic patients and healthy controls. Publicly available transcriptomic databases were explored for the global transcriptomic profile of bronchial epithelium, and PBMCs were explored for AREG expression in asthmatic vs. healthy controls.Results: Asthmatic patients had higher AREG protein levels in blood and saliva compared to control subjects. Higher mRNA expression in saliva and primary bronchial epithelial cells plus higher AREG immunoreactivity in bronchial biopsies were also observed. Both blood and saliva AREG levels showed positive correlations with allergic rhinitis status, atopy status, eczema status, plasma periostin, neutrophilia, Montelukast sodium use, ACT score, FEV1, and FEV1/FVC. In silico analysis showed that severe asthmatic bronchial epithelium with high AREG gene expression is associated with higher neutrophils infiltration.Conclusion: AREG levels measured in a minimally invasive blood sample and a non-invasive saliva sample are higher in non-allergic severe asthma.Clinical ImplicationsThis is the first report to show the higher level of AREG levels in blood and saliva of non-allergic severe asthma.

IL-17 Induced Autophagy Regulates Mitochondrial Dysfunction and Fibrosis in Severe Asthmatic Bronchial Fibroblasts.

The accumulation of fibroblasts, their synthesis of extracellular matrix (ECM) proteins and their innate resistance to apoptosis are characteristics of subepithelial fibrosis observed in severe asthma. Interleukin-17 (IL-17) is an important regulator of airway remodeling in asthma. However, the contribution of IL-17 to the pro-fibrotic phenotype of bronchial fibroblasts is not well-characterized. In this study, we investigated whether IL-17 induced autophagy regulates mitochondrial and pro-fibrotic function in bronchial fibroblasts. The primary cultured bronchial fibroblasts isolated from non-asthmatic (NHBF) and severe asthmatic (DHBF) subjects were treated with IL-17 in order to ascertain its effect on mitochondrial function, mitochondrial quality control, and apoptosis using immunoblotting and flow cytometric analyses. At baseline, DHBF exhibited higher levels of mitophagy and mitochondrial biogenesis compared to NHBF. Immunohistochemical evaluation of bronchial biopsies showed intense PINK1 immunoreactivity in severe asthma than in control. IL-17 intensified the mitochondrial dysfunction and impaired the mitochondrial quality control machinery in NHBF and DHBF. Moreover, IL-17 augmented a pro-fibrotic and anti-apoptotic response in both group of fibroblasts. Inhibition of autophagy using bafilomycin-A1 reduced PINK1 expression in NHBF and restored the IL-17 mediated changes in PINK1 to their basal levels in DHBF. Bafilomycin-A1 also reversed the IL-17 associated fibrotic response in these fibroblasts, suggesting a role for IL-17 induced autophagy in the induction of fibrosis in bronchial fibroblasts. Taken together, our findings suggest that IL-17 induced autophagy promotes mitochondrial dysfunction and fibrosis in bronchial fibroblasts from both non-asthmatic and severe asthmatic subjects. Our study provides insights into the therapeutic potential of targeting autophagy in ameliorating fibrosis, particularly in severe asthmatic individuals.

Bronchial thermoplasty decreases airway remodelling by blocking epithelium-derived heat shock protein-60 secretion and protein arginine methyltransferase-1 in fibroblasts.

Bronchial thermoplasty (BT) is to date the only therapy that provides a lasting reduction in airway wall remodelling. However, the mechanism of action of BT is not well understood. This study aimed to characterise the changes of remodelling regulating signalling pathways by BT in asthma.Bronchoalveolar lavage fluid (BALF) was obtained from eight patients with severe asthma before and after BT. Primary bronchial epithelial cells were isolated from 23 patients before (n=66) and after (n=62) BT. Epithelial cell culture supernatant (Epi.S) was collected and applied to primary fibroblasts.Epithelial cells obtained from asthma patients after BT proliferated significantly faster compared with epithelial cells obtained before BT. In airway fibroblasts, BALF or Epi.S obtained before BT increased CCAAT enhancer-binding protein-β (C/EBPβ) expression, thereby downregulating microRNA-19a. This upregulated extracellular signal-regulated kinase-1/2 (ERK1/2) expression, protein arginine methyltransferase-1 (PRMT1) expression, cell proliferation and mitochondrial mass. BALF or Epi.S obtained after BT reduced the expression of C/EBPβ, ERK1/2, peroxisome proliferator-activated receptor-γ coactivator-1α (PGC1α), PRMT1 and mitochondrial mass in airway fibroblasts. Proteome and transcriptome analyses indicated that epithelial cell-derived heat shock protein-60 (HSP60) is the main mediator of BT effects on fibroblasts. Further analysis suggested that HSP60 regulated PRMT1 expression, which was responsible for the increased mitochondrial mass and α-smooth muscle actin expression by asthmatic fibroblasts. These effects were ablated after BT. These results imply that BT reduces fibroblast remodelling through modifying the function of epithelial cells, especially by reducing HSP60 secretion and subsequent signalling pathways that regulate PRMT1 expression.We therefore hypothesise that BT decreases airway remodelling by blocking epithelium-derived HSP60 secretion and PRMT1 in fibroblasts.

Defective Fibrillar Collagen Organization by Fibroblasts Contributes to Airway Remodeling in Asthma.

Rationale: Histologic stains have been used as the gold standard to visualize extracellular matrix (ECM) changes associated with airway remodeling in asthma, yet they provide no information on the biochemical and structural characteristics of the ECM, which are vital to understanding alterations in tissue function.Objectives: To demonstrate the use of nonlinear optical microscopy (NLOM) and texture analysis algorithms to image fibrillar collagen (second harmonic generation) and elastin (two-photon excited autofluorescence), to obtain biochemical and structural information on the remodeled ECM environment in asthma.Methods: Nontransplantable donor lungs from donors with asthma (n = 13) and control (n = 12) donors were used for the assessment of airway collagen and elastin fibers by NLOM, and extraction of lung fibroblasts for in vitro experiments.Measurements and Main Results: Fibrillar collagen is not only increased but also highly disorganized and fragmented within large and small asthmatic airways compared with control subjects, using NLOM imaging. Furthermore, such structural alterations are present in pediatric and adult donors with asthma, irrespective of fatal disease. In vitro studies demonstrated that asthmatic airway fibroblasts are deficient in their packaging of fibrillar collagen-I and express less decorin, important for collagen fibril packaging. Packaging of collagen fibrils was found to be more disorganized in asthmatic airways compared with control subjects, using transmission electron microscopy.Conclusions: NLOM imaging enabled the structural assessment of the ECM, and the data suggest that airway remodeling in asthma involves the progressive accumulation of disorganized fibrillar collagen by airway fibroblasts. This study highlights the future potential clinical application of NLOM to assess airway remodeling in vivo.

Connective tissue growth factor regulates transition of primary bronchial fibroblasts to myofibroblasts in asthmatic subjects

Fibroblast to myofibroblast transition (FMT) contributes to bronchial wall remodelling in persistent asthma. Among other numerous factors involved, transforming growth factor type β (TGF-β) plays a pivotal role. Recently it has been demonstrated that connective tissue growth factor (CTGF), a matricellular protein, combines with TGF-β in the pathomechanism of many fibrotic disorders. However, it is not clear whether this interaction takes place in asthma as well.Primary cultures of human bronchial fibroblasts from asthmatic and non-asthmatic subjects were used to investigate the impact of CTGF and TGF-β1 on the fibroblast to myofibroblast transition. The combined activity of TGF-β1 and CTGF resulted in an average of 90% of FMT accomplished in cell lines derived from asthmatics. In this group FMT was highly dependent on the presence of CTGF produced by the cells, as shown by gene silencing experiments with the specific siRNA.Results support the important role of CTGF biosynthesis in the asthmatic bronchi amplifying FMT. This is evidenced by inhibition of TGF-β1-induced FMT following CTGF silencing in asthmatic bronchial fibroblasts. CTGF is produced by fibroblasts and contributes to the FMT phenomenon in positive loop-back, inducing and boosting TGF-β1 triggered FMT. Thus, CTGF is a promising target for pharmacological intervention in secondary prevention of bronchial remodelling in asthma.

Fibroblast-derived exosomes promote epithelial cell proliferation through TGF-β2 signalling pathway in severe asthma.

Bronchial fibroblasts play a key role in airway remodelling in asthma. They regulate epithelial cell functions such as proliferation through growth factors, cytokines, chemokines and exosomes. The role of exosomes in the communication between epithelial cells and fibroblasts by vehiculing these mediators in asthma remains to be determined. To evaluate the role of exosomes released by bronchial fibroblasts on epithelial cell proliferation in severe asthma. Exosomes were obtained from culture media of primary bronchial fibroblasts and characterized using Western blot, electron microscopy and flow cytometry. Uptake profile of fluorescent-labelled exosomes in epithelial cells was assessed by flow cytometry. Exosome cytokine content was analysed by Cytokine Arrays. Bronchial epithelial cell proliferation was evaluated by BrdU incorporation test. Exosome biogenesis/release was blocked using sphingomyelinase inhibitor. Plasmid transfection was used to modulate transforming growth factor beta 2 (TGF-β2) gene expression. We showed that bronchial fibroblasts secreted exosomes, which were internalized by bronchial epithelial cells. Exosomes of severe asthmatic subjects' fibroblasts showed a lower level of TGF-β2 and significantly increased the epithelial cell proliferation of both healthy and severe asthmatic subjects compared to healthy controls' exosomes. Overexpression of TGF-β2 in severe asthmatics' fibroblasts induced enhanced TGF-β2 in exosomes leading to a reduced proliferation of epithelial cells, whereas knockdown of TGF-β2 enhanced epithelial cell proliferation. Our study shows that exosomes are involved in fine-tuning intercellular communication in asthma. Exosomes of severe eosinophilic asthmatics' fibroblasts can contribute to airway remodelling, at least in part, by modulating epithelial cell proliferation observed in severe asthma.

Connexin43 Controls the Myofibroblastic Differentiation of Bronchial Fibroblasts from Patients with Asthma.

Pathologic accumulation of myofibroblasts in asthmatic bronchi is regulated by extrinsic stimuli and by the intrinsic susceptibility of bronchial fibroblasts to transforming growth factor-β (TGF-β). The specific function of gap junctions and connexins in this process has remained unknown. Here, we investigated the role of connexin43 (Cx43) in TGF-β-induced myofibroblastic differentiation of fibroblasts derived from bronchoscopic biopsy specimens of patients with asthma and donors without asthma. Asthmatic fibroblasts expressed considerably higher levels of Cx43 and were more susceptible to TGF-β1-induced myofibroblastic differentiation than were their nonasthmatic counterparts. TGF-β1 efficiently up-regulated Cx43 levels and activated the canonical Smad pathway in asthmatic cells. Ectopic Cx43 expression in nonasthmatic (Cx43low) fibroblasts increased their predilection to TGF-β1-induced Smad2 activation and fibroblast-myofibroblast transition. Transient Cx43 silencing in asthmatic (Cx43high) fibroblasts by Cx43 small interfering RNA attenuated the TGF-β1-triggered Smad2 activation and myofibroblast formation. Direct interactions of Smad2 and Cx43 with β-tubulin were demonstrated by co-immunoprecipitation assay, whereas the sensitivity of these interactions to TGF-β1 signaling was confirmed by Förster Resonance Energy Transfer analyses. Furthermore, inhibition of the TGF-β1/Smad pathway attenuated TGF-β1-triggered Cx43 up-regulation and myofibroblast differentiation of asthmatic fibroblasts. Chemical inhibition of gap junctional intercellular communication with 18 α-glycyrrhetinic acid did not affect the initiation of fibroblast-myofibroblast transition in asthmatic fibroblasts but interfered with the maintenance of their myofibroblastic phenotype. Collectively, our data identified Cx43 as a new player in the feedback mechanism regulating TGF-β1/Smad-dependent differentiation of bronchial fibroblasts. Thus, our observations point to Cx43 as a novel profibrotic factor in asthma progression.

Epigenetic modifying enzyme expression in asthmatic airway epithelial cells and fibroblasts

BackgroundRecognition of the airway epithelium as a central mediator in the pathogenesis of asthma has necessitated greater understanding of the aberrant cellular mechanisms of the epithelium in asthma. The architecture of chromatin is integral to the regulation of gene expression and is determined by modifications to the surrounding histones and DNA. The acetylation, methylation, phosphorylation, and ubiquitination of histone tail residues has the potential to greatly alter the accessibility of DNA to the cells transcriptional machinery. DNA methylation can also interrupt binding of transcription factors and recruit chromatin remodelers resulting in general gene silencing. Although previous studies have found numerous irregularities in the expression of genes involved in asthma, the contribution of epigenetic regulation of these genes is less well known. We propose that the gene expression of epigenetic modifying enzymes is cell-specific and influenced by asthma status in tissues derived from the airways.MethodsAirway epithelial cells (AECs) isolated by pronase digestion or endobronchial brushings and airway fibroblasts obtained by outgrowth technique from healthy and asthmatic donors were maintained in monolayer culture. RNA was analyzed for the expression of 82 epigenetic enzymes across 5 families of epigenetic modifying enzymes. Western blot and immunohistochemistry were also used to examine expression of 3 genes.ResultsBetween AECs and airway fibroblasts, we identified cell-specific gene expression in each of the families of epigenetic modifying enzymes; specifically 24 of the 82 genes analyzed showed differential expression. We found that 6 histone modifiers in AECs and one in fibroblasts were differentially expressed in cells from asthmatic compared to healthy donors however, not all passed correction. In addition, we identified a corresponding increase in Aurora Kinase A (AURKA) protein expression in epithelial cells from asthmatics compared to those from non-asthmatics.ConclusionsIn summary, we have identified cell-specific variation in gene expression in each of the families of epigenetic modifying enzymes in airway epithelial cells and airway fibroblasts. These data provide insight into the cell-specific variation in epigenetic regulation which may be relevant to cell fate and function, and disease susceptibility.