Pyruvate metabolism dictates fibroblast sensitivity to GLS1 inhibition during fibrogenesis.

Fibrosis is a chronic disease characterized by excessive extracellular matrix production, which leads to disruption of organ function. Fibroblasts are key effector cells of this process, responding chiefly to the pleiotropic cytokine transforming growth factor-ß1 (TGF-ß1), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-ß1 transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-ß1 transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-ß1-induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-ß1-induced fibrogenesis, as pyruvate can be used as the substrate for glutamate and alanine production via glutamate dehydrogenase (GDH) and glutamic-pyruvic transaminase 2 (GPT2), respectively. We further show that dual targeting of either GPT2 or GDH in combination with GLS1 inhibition was required to fully block TGF-ß1-induced collagen synthesis. These findings embolden a therapeutic strategy aimed at additional targeting of mitochondrial pyruvate metabolism in the presence of a glutaminolysis inhibitor to interfere with the pathological deposition of collagen in the setting of pulmonary fibrosis and potentially other fibrotic conditions.

Single-cell analysis of bronchoalveolar cells in inflammatory and fibrotic post-COVID lung disease.

Background: Persistent radiological lung abnormalities are evident in many survivors of acute coronavirus disease 2019 (COVID-19). Consolidation and ground glass opacities are interpreted to indicate subacute inflammation whereas reticulation is thought to reflect fibrosis. We sought to identify differences at molecular and cellular level, in the local immunopathology of post-COVID inflammation and fibrosis. Methods: We compared single-cell transcriptomic profiles and T cell receptor (TCR) repertoires of bronchoalveolar cells obtained from convalescent individuals with each radiological pattern, targeting lung segments affected by the predominant abnormality. Results: CD4 central memory T cells and CD8 effector memory T cells were significantly more abundant in those with inflammatory radiology. Clustering of similar TCRs from multiple donors was a striking feature of both phenotypes, consistent with tissue localised antigen-specific immune responses. There was no enrichment for known SARS-CoV-2-reactive TCRs, raising the possibility of T cell-mediated immunopathology driven by failure in immune self-tolerance. Conclusions: Post-COVID radiological inflammation and fibrosis show evidence of shared antigen-specific T cell responses, suggesting a role for therapies targeting T cells in limiting post-COVID lung damage.

Human SARS-CoV-2 challenge uncovers local and systemic response dynamics.

The COVID-19 pandemic is an ongoing global health threat, yet our understanding of the dynamics of early cellular responses to this disease remains limited1. Here in our SARS-CoV-2 human challenge study, we used single-cell multi-omics profiling of nasopharyngeal swabs and blood to temporally resolve abortive, transient and sustained infections in seronegative individuals challenged with pre-Alpha SARS-CoV-2. Our analyses revealed rapid changes in cell-type proportions and dozens of highly dynamic cellular response states in epithelial and immune cells associated with specific time points and infection status. We observed that the interferon response in blood preceded the nasopharyngeal response. Moreover, nasopharyngeal immune infiltration occurred early in samples from individuals with only transient infection and later in samples from individuals with sustained infection. High expression of HLA-DQA2 before inoculation was associated with preventing sustained infection. Ciliated cells showed multiple immune responses and were most permissive for viral replication, whereas nasopharyngeal T cells and macrophages were infected non-productively. We resolved 54 T cell states, including acutely activated T cells that clonally expanded while carrying convergent SARS-CoV-2 motifs. Our new computational pipeline Cell2TCR identifies activated antigen-responding T cells based on a gene expression signature and clusters these into clonotype groups and motifs. Overall, our detailed time series data can serve as a Rosetta stone for epithelial and immune cell responses and reveals early dynamic responses associated with protection against infection.

Age-specific nasal epithelial responses to SARS-CoV-2 infection.

Children infected with SARS-CoV-2 rarely progress to respiratory failure. However, the risk of mortality in infected people over 85 years of age remains high. Here we investigate differences in the cellular landscape and function of paediatric (<12 years), adult (30-50 years) and older adult (>70 years) ex vivo cultured nasal epithelial cells in response to infection with SARS-CoV-2. We show that cell tropism of SARS-CoV-2, and expression of ACE2 and TMPRSS2 in nasal epithelial cell subtypes, differ between age groups. While ciliated cells are viral replication centres across all age groups, a distinct goblet inflammatory subtype emerges in infected paediatric cultures and shows high expression of interferon-stimulated genes and incomplete viral replication. In contrast, older adult cultures infected with SARS-CoV-2 show a proportional increase in basaloid-like cells, which facilitate viral spread and are associated with altered epithelial repair pathways. We confirm age-specific induction of these cell types by integrating data from in vivo COVID-19 studies and validate that our in vitro model recapitulates early epithelial responses to SARS-CoV-2 infection.

Pulmonary fibrosis: Emerging diagnostic and therapeutic strategies.

Fibrosis is the concluding pathological outcome and major cause of morbidity and mortality in a number of common chronic inflammatory, immune-mediated and metabolic diseases. The progressive deposition of a collagen-rich extracellular matrix (ECM) represents the cornerstone of the fibrotic response and culminates in organ failure and premature death. Idiopathic pulmonary fibrosis (IPF) represents the most rapidly progressive and lethal of all fibrotic diseases with a dismal median survival of 3.5 years from diagnosis. Although the approval of the antifibrotic agents, pirfenidone and nintedanib, for the treatment of IPF signalled a watershed moment for the development of anti-fibrotic therapeutics, these agents slow but do not halt disease progression or improve quality of life. There therefore remains a pressing need for the development of effective therapeutic strategies. In this article, we review emerging therapeutic strategies for IPF as well as the pre-clinical and translational approaches that will underpin a greater understanding of the key pathomechanisms involved in order to transform the way we diagnose and treat pulmonary fibrosis.

Residual Lung Abnormalities after COVID-19 Hospitalization: Interim Analysis of the UKILD Post-COVID-19 Study.

Rationale: Shared symptoms and genetic architecture between coronavirus disease (COVID-19) and lung fibrosis suggest severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may lead to progressive lung damage. Objectives: The UK Interstitial Lung Disease Consortium (UKILD) post-COVID-19 study interim analysis was planned to estimate the prevalence of residual lung abnormalities in people hospitalized with COVID-19 on the basis of risk strata. Methods: The PHOSP-COVID-19 (Post-Hospitalization COVID-19) study was used to capture routine and research follow-up within 240 days from discharge. Thoracic computed tomography linked by PHOSP-COVID-19 identifiers was scored for the percentage of residual lung abnormalities (ground-glass opacities and reticulations). Risk factors in linked computed tomography were estimated with Bayesian binomial regression, and risk strata were generated. Numbers within strata were used to estimate posthospitalization prevalence using Bayesian binomial distributions. Sensitivity analysis was restricted to participants with protocol-driven research follow-up. Measurements and Main Results: The interim cohort comprised 3,700 people. Of 209 subjects with linked computed tomography (median, 119 d; interquartile range, 83-155), 166 people (79.4%) had more than 10% involvement of residual lung abnormalities. Risk factors included abnormal chest X-ray (risk ratio [RR], 1.21; 95% credible interval [CrI], 1.05-1.40), percent predicted DlCO less than 80% (RR, 1.25; 95% CrI, 1.00-1.56), and severe admission requiring ventilation support (RR, 1.27; 95% CrI, 1.07-1.55). In the remaining 3,491 people, moderate to very high risk of residual lung abnormalities was classified at 7.8%, and posthospitalization prevalence was estimated at 8.5% (95% CrI, 7.6-9.5), rising to 11.7% (95% CrI, 10.3-13.1) in the sensitivity analysis. Conclusions: Residual lung abnormalities were estimated in up to 11% of people discharged after COVID-19-related hospitalization. Health services should monitor at-risk individuals to elucidate long-term functional implications.

Understanding the burden of interstitial lung disease post-COVID-19: the UK Interstitial Lung Disease-Long COVID Study (UKILD-Long COVID).

INTRODUCTION: The COVID-19 pandemic has led to over 100 million cases worldwide. The UK has had over 4 million cases, 400 000 hospital admissions and 100 000 deaths. Many patients with COVID-19 suffer long-term symptoms, predominantly breathlessness and fatigue whether hospitalised or not. Early data suggest potentially severe long-term consequence of COVID-19 is development of long COVID-19-related interstitial lung disease (LC-ILD). METHODS AND ANALYSIS: The UK Interstitial Lung Disease Consortium (UKILD) will undertake longitudinal observational studies of patients with suspected ILD following COVID-19. The primary objective is to determine ILD prevalence at 12 months following infection and whether clinically severe infection correlates with severity of ILD. Secondary objectives will determine the clinical, genetic, epigenetic and biochemical factors that determine the trajectory of recovery or progression of ILD. Data will be obtained through linkage to the Post-Hospitalisation COVID platform study and community studies. Additional substudies will conduct deep phenotyping. The Xenon MRI investigation of Alveolar dysfunction Substudy will conduct longitudinal xenon alveolar gas transfer and proton perfusion MRI. The POST COVID-19 interstitial lung DiseasE substudy will conduct clinically indicated bronchoalveolar lavage with matched whole blood sampling. Assessments include exploratory single cell RNA and lung microbiomics analysis, gene expression and epigenetic assessment. ETHICS AND DISSEMINATION: All contributing studies have been granted appropriate ethical approvals. Results from this study will be disseminated through peer-reviewed journals. CONCLUSION: This study will ensure the extent and consequences of LC-ILD are established and enable strategies to mitigate progression of LC-ILD.

Fibrometabolism-An emerging therapeutic frontier in pulmonary fibrosis.

Fibrosis is the final pathological outcome and major cause of morbidity and mortality in many common and chronic inflammatory, immune-mediated, and metabolic diseases. Despite the growing incidence of fibrotic diseases and extensive research efforts, there remains a lack of effective therapies that improve survival. The application of omics technologies has revolutionized our approach to identifying previously unknown therapeutic targets and potential disease biomarkers. The application of metabolomics, in particular, has improved our understanding of disease pathomechanisms and garnered a wave of scientific interest in the role of metabolism in the biology of myofibroblasts, the key effector cells of the fibrogenic response. Emerging evidence suggests that alterations in metabolism not only are a feature of but also may play an influential role in the pathogenesis of fibrosis, most notably in idiopathic pulmonary fibrosis (IPF), the most rapidly progressive and fatal of all fibrotic conditions. This review will detail the role of key metabolic pathways, their alterations in myofibroblasts, and the potential this new knowledge offers for the development of antifibrotic therapeutic strategies.

Transcriptome analysis of IPF fibroblastic foci identifies key pathways involved in fibrogenesis.

INTRODUCTION: Fibroblastic foci represent the cardinal pathogenic lesion in idiopathic pulmonary fibrosis (IPF) and comprise activated fibroblasts and myofibroblasts, the key effector cells responsible for dysregulated extracellular matrix deposition in multiple fibrotic conditions. The aim of this study was to define the major transcriptional programmes involved in fibrogenesis in IPF by profiling unmanipulated myofibroblasts within fibrotic foci in situ by laser capture microdissection. METHODS: The challenges associated with deriving gene calls from low amounts of RNA and the absence of a meaningful comparator cell type were overcome by adopting novel data mining strategies and by using weighted gene co-expression network analysis (WGCNA), as well as an eigengene-based approach to identify transcriptional signatures, which correlate with fibrillar collagen gene expression. RESULTS: WGCNA identified prominent clusters of genes associated with cell cycle, inflammation/differentiation, translation and cytoskeleton/cell adhesion. Collagen eigengene analysis revealed that transforming growth factor ß1 (TGF-ß1), RhoA kinase and the TSC2/RHEB axis formed major signalling clusters associated with collagen gene expression. Functional studies using CRISPR-Cas9 gene-edited cells demonstrated a key role for the TSC2/RHEB axis in regulating TGF-ß1-induced mechanistic target of rapamycin complex 1 activation and collagen I deposition in mesenchymal cells reflecting IPF and other disease settings, including cancer-associated fibroblasts. CONCLUSION: These data provide strong support for the human tissue-based and bioinformatics approaches adopted to identify critical transcriptional nodes associated with the key pathogenic cell responsible for fibrogenesis in situ and further identify the TSC2/RHEB axis as a potential novel target for interfering with excessive matrix deposition in IPF and other fibrotic conditions.

The promise of mTOR as a therapeutic target pathway in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is characterised by the progressive deposition of excessive extracellular matrix proteins within the lung parenchyma and represents the most rapidly progressive and fatal of all fibrotic conditions. Current anti-fibrotic drugs approved for the treatment of IPF fail to halt disease progression and have significant side-effect profiles. Therefore, there remains a pressing need to develop novel therapeutic strategies for IPF. Mammalian target of rapamycin (mTOR) forms the catalytic subunit of two complexes, mTORC1 and mTORC2. mTORC1 acts as critical cellular sensor which integrates intracellular and extracellular signals to reciprocally regulate a variety of anabolic and catabolic processes. The emerging evidence for a critical role for mTORC1 in influencing extracellular matrix production, metabolism, autophagy and senescence in the setting of IPF highlights this axis as a novel therapeutic target with the potential to impact multiple IPF pathomechanisms.

Targeting coagulation activation in severe COVID-19 pneumonia: lessons from bacterial pneumonia and sepsis.

Novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), has rapidly spread throughout the world, resulting in a pandemic with high mortality. There are no effective treatments for the management of severe COVID-19 and current therapeutic trials are focused on antiviral therapy and attenuation of hyper-inflammation with anti-cytokine therapy. Severe COVID-19 pneumonia shares some pathological similarities with severe bacterial pneumonia and sepsis. In particular, it disrupts the haemostatic balance, which results in a procoagulant state locally in the lungs and systemically. This culminates in the formation of microthrombi, disseminated intravascular coagulation and multi-organ failure. The deleterious effects of exaggerated inflammatory responses and activation of coagulation have been investigated in bacterial pneumonia and sepsis and there is recognition that although these pathways are important for the host immune response to pathogens, they can lead to bystander tissue injury and are negatively associated with survival. In the past two decades, evidence from preclinical studies has led to the emergence of potential anticoagulant therapeutic strategies for the treatment of patients with pneumonia, sepsis and acute respiratory distress syndrome, and some of these anticoagulant approaches have been trialled in humans. Here, we review the evidence from preclinical studies and clinical trials of anticoagulant treatment strategies in bacterial pneumonia and sepsis, and discuss the importance of these findings in the context of COVID-19.

Translational pharmacology of an inhaled small molecule αvß6 integrin inhibitor for idiopathic pulmonary fibrosis.

The αvß6 integrin plays a key role in the activation of transforming growth factor-ß (TGFß), a pro-fibrotic mediator that is pivotal to the development of idiopathic pulmonary fibrosis (IPF). We identified a selective small molecule αvß6 RGD-mimetic, GSK3008348, and profiled it in a range of disease relevant pre-clinical systems. To understand the relationship between target engagement and inhibition of fibrosis, we measured pharmacodynamic and disease-related end points. Here, we report, GSK3008348 binds to αvß6 with high affinity in human IPF lung and reduces downstream pro-fibrotic TGFß signaling to normal levels. In human lung epithelial cells, GSK3008348 induces rapid internalization and lysosomal degradation of the αvß6 integrin. In the murine bleomycin-induced lung fibrosis model, GSK3008348 engages αvß6, induces prolonged inhibition of TGFß signaling and reduces lung collagen deposition and serum C3M, a marker of IPF disease progression. These studies highlight the potential of inhaled GSK3008348 as an anti-fibrotic therapy.

Author Correction: The mTORC1/4E-BP1 axis represents a critical signaling node during fibrogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.