A central role for G9a and EZH2 in the epigenetic silencing of cyclooxygenase-2 in idiopathic pulmonary fibrosis

Selective silencing of the cyclooxygenase-2 (COX-2) gene with the loss of the antifibrotic mediator PGE2 contributes to the fibrotic process in idiopathic pulmonary fibrosis (IPF). This study explored the role of G9a- and EZH2-mediated methylation of histone H3 lysine 9 (H3K9me3) and 27 (H3K27me3) in COX-2 silencing in IPF. Chromatin immunoprecipitation (ChIP) and Re-ChIP assays demonstrated marked increases in H3K9me3, H3K27me3 and DNA methylation, together with their respective modifying enzymes G9a, EZH2 and DNA methyltransferases (Dnmts) and respective binding proteins heterochromatin protein 1 (HP1), polycomb protein complex 1 (PRC1) and MeCP2, at the COX-2 promoter in lung fibroblasts from IPF patients (F-IPF) compared with fibroblasts from non-fibrotic lungs (F-NL). HP1, EZH2 and MeCP2 in turn were associated with additional repressive chromatin modifiers in F-IPF. G9a and EZH2 inhibitors and siRNAs and Dnmt1 inhibitor markedly reduced H3K9me3 (49-79%), H3K27me3 (44-81%) and DNA methylation (61-97%) at the COX-2 promoter. This was correlated with increased histone H3 and H4 acetylation, resulting in COX-2 mRNA and protein re-expression in F-IPF. Our results support a central role for G9a- and EZH2-mediated histone hypermethylation and a model of bidirectional, mutually reinforcing and interdependent crosstalk between histone hypermethylation and DNA methylation in COX-2 epigenetic silencing in IPF

Presence of a prothrombotic state in people with idiopathic pulmonary fibrosis: a population-based case-control study

BACKGROUND: Laboratory studies suggest that the clotting cascade is activated in fibrotic lungs. Since humans vary in their tendency to clot due to a variety of inherited or acquired defects, we investigated whether a prothrombotic state increases the chance of developing idiopathic pulmonary fibrosis (IPF) and/or worsens the prognosis of IPF. METHODS: We recruited 211 incident cases of IPF and 256 age-and sex-matched general population controls and collected data on medical history, medication, smoking habit, blood samples as well as lung function and high-resolution CT scans done as part of routine clinical care. A prothrombotic state was defined as the presence of at least one inherited or acquired clotting defect or marker of fibrinolytic dysfunction. We used logistic regression to quantify the association between a prothrombotic state and IPF adjusted for age, sex, smoking habit and highly sensitive C reactive protein. Cox regression was used to determine the influence of a prothrombotic state on survival. RESULTS: Cases were more than four times more likely than controls to have a prothrombotic state (OR 4.78, 95% CI 2.93 to 7.80; p<0.0001). Cases with a prothrombotic state were also likely to have more severe disease (forced vital capacity <70% predicted) at presentation (OR 10.79, 95% CI 2.43 to 47.91) and had a threefold increased risk of death (HR 3.26, 95% CI 1.09 to 9.75). CONCLUSIONS: People with IPF are more likely to have a prothrombotic state than general population controls and the presence of a prothrombotic state has an adverse impact on survival

Interplay between EZH2 and G9a regulates CXCL10 gene repression in idiopathic pulmonary fibrosis

Selective repression of the antifibrotic gene CXCL10 contributes to tissue remodelling in idiopathic pulmonary fibrosis (IPF). We have previously reported that histone deacetylation and histone H3 lysine 9 (H3K9) methylation are involved in CXCL10 repression. This study explored the role of H3K27 methylation and the interplay between the two histone lysine methyltransferases, Enhancer of Zest Homolog 2 (EZH2) and G9a, in CXCL10 repression in IPF. By applying chromatin immunoprecipitation (ChIP), Re-ChIP and proximity ligation assays, we demonstrated that, like G9a-mediated H3K9 methylation, EZH2-mediated H3K27me3 was significantly enriched at the CXCL10 promoter in fibroblasts from IPF lungs (F-IPF) compared with fibroblasts from non-fibrotic lungs (F-NL) and that EZH2 and G9a physically interacted with each other. EZH2 knockdown reduced not only EZH2 and H3K27me3 but also G9a and H3K9me3 and G9a knockdown reduced not only G9 and H3K9me3 but also EZH2 and H3K27me3. Depletion and inhibition of EZH2 and G9a also reversed histone deacetylation and restored CXCL10 expression in F-IPF. Furthermore, treatment of F-NL with the profibrotic cytokine TGF-β1 increased EZH2, G9a, H3K27me3, H3K9me3 and histone deacetylation at the CXCL10 promoter, similar to that observed in F-IPF, which was correlated with CXCL10 repression and was prevented by EZH2 and G9a knockdown. These findings suggest that a novel and functionally interdependent interplay between EZH2 and G9a regulates histone methylation-mediated epigenetic repression of the antifibrotic CXCL10 gene in IPF. This interdependent interplay may prove to be a target for epigenetic intervention to restore the expression of CXCL10 and other anti-fibrotic genes in IPF

Safety and tolerability of nintedanib in patients with progressive fibrosing interstitial lung diseases: data from the randomized controlled INBUILD trial

Background: In the INBUILD trial in patients with progressive fibrosing interstitial lung diseases (ILDs), nintedanib reduced the rate of decline in forced vital capacity compared with placebo, with side-effects that were manageable for most patients. We used data from the INBUILD trial to characterize further the safety and tolerability of nintedanib. Methods: Patients with fibrosing ILDs other than idiopathic pulmonary fibrosis (IPF), who had experienced progression of ILD within the 24 months before screening despite management deemed appropriate in clinical practice, were randomized to receive nintedanib 150 mg twice daily or placebo. To manage adverse events, treatment could be interrupted or the dose reduced to 100 mg twice daily. We assessed adverse events and dose adjustments over the whole trial. Results: A total of 332 patients received nintedanib and 331 received placebo. Median exposure to trial drug was 17.4 months in both treatment groups. Adverse events led to treatment discontinuation in 22.0% of patients treated with nintedanib and 14.5% of patients who received placebo. The most frequent adverse event was diarrhea, reported in 72.3% of patients in the nintedanib group and 25.7% of patients in the placebo group. Diarrhea led to treatment discontinuation in 6.3% of patients in the nintedanib group and 0.3% of the placebo group. In the nintedanib and placebo groups, respectively, 48.2% and 15.7% of patients had >= 1 dose reduction and/or treatment interruption. Serious adverse events were reported in 44.3% of patients in the nintedanib group and 49.5% of patients in the placebo group. The adverse event profile of nintedanib was generally consistent across subgroups based on age, sex, race and weight, but nausea, vomiting and dose reductions were more common among female than male patients. Conclusions: The adverse event profile of nintedanib in patients with progressive fibrosing ILDs other than IPF is consistent with its established safety and tolerability profile in patients with IPF and characterized mainly by gastrointestinal events, particularly diarrhea. Management of adverse events using symptomatic therapies and dose adjustment is important to minimize the impact of adverse events and help patients remain on therapy

Loss of epithelial Gq and G11 signaling inhibits TGFβ production but promotes IL-33–mediated macrophage polarization and emphysema

Heterotrimeric guanine nucleotide–binding protein (G protein) signaling is a ubiquitous signaling system that links hundreds of G protein–coupled receptors (GPCRs) with four G protein signaling pathways. Two of these pathways, one mediated by Gq and G11 and the other by G12 and G13, are implicated in the force-dependent activation of transforming growth factor–β (TGFβ) in lung epithelial cells. Reduced TGFβ activation in alveolar cells leads to emphysema, whereas enhanced TGFβ activation promotes acute lung injury, and idiopathic pulmonary fibrosis, therefore precise control of alveolar TGFβ activation is essential for alveolar homeostasis. Here, we investigated whether the Gq/G11 or G12/G13 pathways in epithelial cells are required to generate TGFβ and suppress alveolar inflammation. Mice deficient in both Gαq and Gα11 developed inflammation primarily due to alternatively activated (M2-polarized) macrophages, enhanced production of matrix metalloprotease 12 (MMP12), and age-related alveolar airspace enlargement consistent with emphysema. We found that mice with impaired Gq/G11 signaling had reduced stretch-mediated generation of TGFβ by epithelial cells and elevated macrophage MMP12 synthesis, but were protected from the effects of ventilator-induced lung injury. Furthermore, synthesis of the pleiotropic cytokine interleukin-33 (IL-33), was increased in these alveolar epithelial cells resulting in the M2-type polarization of alveolar macrophages independently of the effect on TGFβ. Our results suggest that alveolar Gq/G11 signaling maintains alveolar homeostasis and is likely to independently upregulate mechanotransduced epithelial TGFβ activation and downregulate epithelial IL-33 synthesis. Together, these findings suggest that disruption of Gq/G11 signaling promotes inflammatory emphysema, but protects against mechanostransduced lung injury

Investigating the effects of nintedanib on biomarkers of extracellular matrix turnover in patients with IPF : design of the randomised placebo-controlled INMARK®trial

Introduction A feature of the pathogenesis of idiopathic pulmonary fibrosis (IPF) is the excess accumulation of extracellular matrix (ECM) in the lungs. Cleavage of the ECM by metalloproteinases (MMPs) generates freecirculating protein fragments known as neoepitopes. The PROFILE study suggested that changes in ECM turnover proteins may be of value as markers of disease progression in patients with IPF. Nintedanib is an approved treatment for IPF that slows disease progression by reducing decline in forced vital capacity (FVC). Methods and analysis The INMARK® trial is evaluating the effect of nintedanib on the rates of change of biomarkers of ECM turnover in patients with IPF, the value of changes in these biomarkers as predictors of disease progression and whether nintedanib affects the associations between changes in these biomarkers and disease progression. Following a screening period, 347 patients with IPF and FVC ≥80% predicted were randomised 1:2 to receive nintedanib 150 mg two times a day or placebo for 12 weeks, followed by an open-label period in which all patients will receive nintedanib for 40 weeks. The primary endpoint is the rate of change in C reactive protein degraded by MMP-1/8 from baseline to week 12. Ethics and dissemination This trial is being conducted in compliance with the protocol, the ethical principles detailed in the Declaration of Helsinki and in accordance with the International Conference on Harmonisation Harmonised Tripartite Guideline for Good Clinical Practice. The results of the trial will be presented at national and international meetings and published in peer-reviewed journals. Trial registration number NCT0278847

A systematic review and meta-analysis of Anakinra, Sarilumab, Siltuximab and Tocilizumab for Covid-19

Background There is accumulating evidence for an overly activated immune response in severe Covid-19, with several studies exploring the therapeutic role of immunomodulation. Through systematic review and meta-analysis, we assess the effectiveness of specific interleukin inhibitors for the treatment of Covid-19.Methods Electronic databases were searched on 7th January 2021 to identify studies of immunomodulatory agents (anakinra, sarilumab, siltuximab and tocilizumab) for the treatment of Covid-19. The primary outcomes were severity on an ordinal scale measured at day 15 from intervention and days to hospital discharge. Key secondary endpoints included overall mortality.Results 71 studies totalling 22,058 patients were included, six were randomised trials. Most explored outcomes in patients who received tocilizumab (59/71). In prospective studies, tocilizumab was associated with improved unadjusted survival (RR 0.83 95%CI 0.72;0.96 I2 = 0.0%), but conclusive benefit was not demonstrated for other outcomes. In retrospective studies, tocilizumab was associated with less severe outcomes on an ordinal scale (Generalised odds ratio 1.34 95%CI 1.10;1.64, I2=98%) and adjusted mortality risk (HR 0.52 95%CI 0.41;0.66, I2 =76.6%). The mean difference in duration of hospitalisation was 0.36 days (95%CI -0.07;0.80, I2 =93.8%). There was substantial heterogeneity in retrospective studies, and estimates should be interpreted cautiously. Other immunomodulatory agents showed similar effects to tocilizumab, but insufficient data precluded meta-analysis by agent.Conclusion Tocilizumab was associated with a lower relative risk of mortality in prospective studies, but effects were inconclusive for other outcomes. Current evidence for the efficacy of anakinra, siltuximab or sarilumab in Covid-19 is insufficient, with further studies urgently needed for conclusive findings

Shared genetic etiology between idiopathic pulmonary fibrosis and COVID-19 severity

Background: Idiopathic pulmonary fibrosis (IPF) is a complex lung disease, characterized by progressive lung scarring. Severe COVID-19 is associated with substantial pneumonitis and has a number of shared major risk factors with IPF. This study aimed to determine the genetic correlation between IPF and severe COVID-19 and assess a potential causal role of genetically increased risk of IPF on COVID-19 severity. Methods: The genetic correlation between IPF and COVID-19 severity was estimated with linkage disequilib-rium (LD) score regression. We performed a Mendelian randomization (MR) study for IPF causality in COVID-19. Genetic variants associated with IPF susceptibility (P Findings: We detected a positive genetic correlation of IPF with COVID-19 severity (rg=0.31 [95% CI 0.04-0.57], P = 0.023). The MR estimates for severe COVID-19 did not reveal any genetic association (OR 1.05, [95% CI 0.92-1.20], P = 0.43). However, outlier analysis revealed that the IPF risk allele rs35705950 at MUC5B had a dif-ferent effect compared with the other variants. When rs35705950 was excluded, MR results provided evidence that genetically increased risk of IPF has a causal effect on COVID-19 severity (OR 1.21, [95% CI 1.06-1.38], P = 4.24 x 10(-3)). Furthermore, the IPF risk-allele at MUC5B showed an apparent protective effect against COVID-19 hospitalization only in older adults (OR 0.86, [95% CI 0.73-1.00], P = 2.99 x 10(-2)) . Interpretation: The strongest genetic determinant of IPF, rs35705950 at MUC5B, seems to confer protection against COVID-19, whereas the combined effect of all other IPF risk loci seem to confer risk of COVID-19 severity. The observed effect of rs35705950 could either be due to protective effects of mucin over-produc-tion on the airways or a consequence of selection bias due to (1) a patient group that is heavily enriched for the rs35705950 T undertaking strict self-isolation and/or (2) due to survival bias of the rs35705950 non-IPF risk allele carriers. Due to the diverse impact of IPF causal variants on SARS-CoV-2 infection, with a possible selection bias as an explanation, further investigation is needed to address this apparent paradox between variance at MUC5B and other IPF genetic risk factors. (C) 2021 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/)Peer reviewe

Complex roles of TGF-beta signaling pathways in lung development and bronchopulmonary dysplasia

As survival of extremely preterm infants continues to improve, there is also an associated increase in bronchopulmonary dysplasia (BPD), one of the most significant complications of preterm birth. BPD development is multifactorial resulting from exposure to multiple antenatal and postnatal stressors. BPD has both short-term health implications and long-term sequelae including increased respiratory, cardiovascular and neurological morbidity. Transforming growth factor beta (TGF-b) is an important signaling pathway in lung development, organ injury and fibrosis and is implicated in the development of BPD. This review provides a detailed account on the role of TGF-b in antenatal and postnatal lung development, the effect of known risk factors for BPD on the TGF-b signaling pathway, and how medications currently in use or under development, for the prevention or treatment of BPD, affect TGF-b signaling

Respiratory sequelae of COVID-19: pulmonary and extrapulmonary origins, and approaches to clinical care and rehabilitation

Although the exact prevalence of post-COVID-19 condition (also known as long COVID) is unknown, more than a third of patients with COVID-19 develop symptoms that persist for more than 3 months after SARS-CoV-2 infection. These sequelae are highly heterogeneous in nature and adversely affect multiple biological systems, although breathlessness is a frequently cited symptom. Specific pulmonary sequelae, including pulmonary fibrosis and thromboembolic disease, need careful assessment and might require particular investigations and treatments. COVID-19 outcomes in people with pre-existing respiratory conditions vary according to the nature and severity of the respiratory disease and how well it is controlled. Extrapulmonary complications such as reduced exercise tolerance and frailty might contribute to breathlessness in post-COVID-19 condition. Non-pharmacological therapeutic options, including adapted pulmonary rehabilitation programmes and physiotherapy techniques for breathing management, might help to attenuate breathlessness in people with post-COVID-19 condition. Further research is needed to understand the origins and course of respiratory symptoms and to develop effective therapeutic and rehabilitative strategies