Rachel Clare Chambers

Post-COVID-19 residual lung abnormalities (RLA) are associated with persistent respiratory symptoms and radiological changes, yet the underlying mechanisms remain unclear. We performed integrated multi-omic profiling of paired bronchoalveolar lavage and blood samples from patients with post-COVID-19 RLA and healthy controls, combining single-cell RNA sequencing, CITE-seq, single-cell T cell receptor sequencing, bronchoalveolar lavage fluid proteomics and functional fibroblast assays. In post-COVID-19 RLA lungs, we identified an increased abundance of profibrotic SPP1hi monocyte-derived alveolar macrophages, arising from an expanded circulating HLA-DRlowCD163+PDE4Dhi classical monocyte progenitor population, supporting a blood-lung myeloid axis. Cell-cell communication modelling positioned macrophages as central hubs of immune-stromal crosstalk, promoting monocyte recruitment with profibrotic priming, and fibroblast activation. Proteomic analysis of bronchoalveolar lavage fluid from post-COVID-19 RLA and idiopathic pulmonary fibrosis, compared with healthy controls, revealed shared and distinct signatures. These alveolar proteins in post-COVID-19 RLA were predominantly attributed to myeloid cells and predicted to engage fibroblast receptors. Bronchoalveolar lavage fluid induced fibroblast proliferation, differentiation and collagen deposition in vitro, with proliferation attenuated by the antifibrotic drug nintedanib. We also identified compartment-specific lymphoid dysregulation, including depletion of mucosal-associated invariant T (MAIT) cells in both the lung and blood, decreased natural killer (NK) cells with oligoclonal T cell expansion in the lung, and expansion of regulatory and cytotoxic T cells in the blood. These findings support a persistent monocyte-macrophage-fibroblast axis linking immune dysregulation to fibroproliferative remodelling after COVID-19 and highlights candidate therapeutic targets for post-viral lung fibrosis. We provide a publicly available atlas (on publication).

Fibrosis, marked by excess extracellular matrix (ECM) deposition, is the end stage of many diseases. Single-cell studies have highlighted the emergence of disease-specific fibroblast populations, including a high collagen–synthesizing CTHRC1 ⁺ subpopulation. The profibrotic cytokine TGF-β1 promotes fibrogenesis via cooperation between Smad and mTORC1/4E-BP1 signaling axes. Using CRISPR-Cas9 gene editing, we report that more than one-third of TGF-β1–regulated matrisome genes are under mTORC1 control. Mapping the transcriptome of TGF-β1–stimulated fibroblasts revealed similarity to CTHRC1 ⁺ fibroblasts identified in idiopathic pulmonary fibrosis (IPF). This overlap is lost when mTORC1 is disabled. Using the selective mTORC1 inhibitor RMC-5552, we confirm a causal role for mTORC1 in promoting the acquisition of the collagen-high, CTHRC1 ⁺ phenotype in response to TGF-β1 stimulation in fibroblasts derived from patients with either IPF or lung adenocarcinoma. We conclude that mTORC1 plays a key role in shaping the transcriptional identity of these fibroblasts, with implications for therapeutic inhibition of mTORC1 in fibrosis and cancer.

Lymphangioleiomyomatosis (LAM) is a rare disease of women in which TSC2 deficient ‘LAM cells’ with dysregulated mTOR signalling and recruited fibroblasts form nodules causing lung cysts and respiratory failure. We examine if mTOR dysregulation can induce senescence and impair the response to lung injury in LAM. The senescence markers p21, p16 and the SenMayo gene set are increased in LAM lungs and colocalise with alveolar type 2 cells. LAM models induce mTOR dependent senescence in alveolar type 2 cell organoids in vitro and in vivo. IL-6 produced by LAM cells, induces p16 and p21 in alveolar type 2 cells, inhibits epithelial wound resolution and is related to lung function in LAM patients. Rapamycin and the IL-6 receptor antagonist Tocilizumab reduce alveolar type 2 cell organoid p21 accumulation and Tocilizumab enhances epithelial wound repair. Targeting IL-6 signalling in parallel with mTOR inhibition, may reduce lung damage in LAM.

Background: Interstitial lung abnormalities (ILAs) are common incidental findings in lung cancer screening (LCS). However, challenges remain in identifying clinically relevant ILAs as highlighted in a joint statement by a European multidisciplinary task force led by the European Respiratory Society (ERS). To address these challenges, we analysed ILAs identified in one of Europe’s largest LCS studies. Methods: Of 11 635 LCS individuals, 417 screen-detected ILAs were evaluated using a new visual classification system focused on traction bronchiolectasis: non-fibrotic ILA (no traction bronchiolectasis), fibrotic ILA (traction bronchiolectasis in ≤2 lobes); undiagnosed interstitial lung disease (traction bronchiolectasis in >2 lobes). Observer agreement was compared with Fleischner Society ILA classification using Cohen’s Kappa. An age, sex and smoking history-matched control group allowed the examination of associations between baseline ILA/UILD and comorbidities, forced vital capacity (FVC), hospitalisations (Student’s t-tests) and mortality (univariable and multivariable Cox proportional hazards models). Findings: Our visual ILA classification showed superior interobserver agreement (K=0.76) versus the Fleischner ILA classification (K=0.64). ILA/UILD subjects had more prevalent comorbidities, increasing (vs controls) approximately 10 years prior to ILA/UILD diagnosis. Compared with controls, mortality rates were 6-fold higher for UILD participants and 3-fold higher for fibrotic and non-fibrotic ILA subtypes. On multivariable Cox regression analysis, ILA/UILD presence (HR=4.90, 95% CI =2.36 to 10.10, p<0.001) showed stronger independent associations with mortality than baseline FVC (HR=0.98, 95% CI =0.96 to 1.00, p=0.04). Conclusion: We demonstrate a new reproducible classification of clinically important ILA/UILDs in LCS populations. We highlight that FVC shows limited associations with mortality in ILA/UILD subjects. Increased multiorgan comorbidity in ILA/UILD subjects highlights a need for comprehensive early multisystem evaluation.

Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease characterized by excessive extracellular matrix deposition. Current IPF therapies slow disease progression but do not stop or reverse it. The (myo)fibroblasts are thought to be the main cellular contributors to excessive extracellular matrix production in IPF. Here we show that fibrotic alveolar type II cells regulate production and crosslinking of extracellular matrix via the co-transcriptional activator YAP. YAP leads to increased expression of Lysl oxidase (LOX) and subsequent LOX-mediated crosslinking by fibrotic alveolar type II cells. Pharmacological YAP inhibition via verteporfin reverses fibrotic alveolar type II cell reprogramming and LOX expression in experimental lung fibrosis in vivo and in human fibrotic tissue ex vivo. We thus identify YAP-TEAD/LOX inhibition in alveolar type II cells as a promising potential therapy for IPF patients.

Pulmonary fibrosis remains a devastating and often fatal condition due to the lack of effective treatments that halt disease progression. Rodent models of pulmonary fibrosis are crucial to identify candidate targets and novel therapeutic agents. However, the attrition rate of novel drug candidates in clinical trials remains high. This review suggests complementing traditional methods used to evaluate antifibrotic therapies in rodent models, such as histopathological and biochemical markers, and lung function tests, with innovative imaging technologies. These imaging techniques could improve the predictive power and translatability of animal studies in human clinical trials. Notably, previous studies in mice and other rodents have observed compensatory lung enlargement in response to lung injury, questioning whether the conventional view of pulmonary fibrosis as a restrictive disease applies to rodents. By adding longitudinal image-based biomarkers, we aim to better unravel the complexity of lung responses and facilitate more effective drug development for pulmonary fibrosis, ultimately improving patient outcomes.

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

Fibrosis, defined as the abnormal deposition of extracellular matrix (ECM), represents the concluding pathological outcome in a number of inflammatory, immune-mediated and metabolic diseases. Recent single cell RNA-sequencing studies have highlighted the diversity and functional heterogeneity of fibroblast populations in multiple fibrotic conditions. These include a novel pathogenic population of high collagen-producing fibroblasts, characterised by expression of the secreted glycoprotein, collagen triple helix repeat containing 1 (CTHRC1). The cardinal pro-fibrotic mediator TGF-β1 has been widely implicated in promoting fibrogenesis and is a potent inducer of CTHRC1 and COL1A1 expression. In addition to the canonical Smad signalling pathway, TGF-β1-induced collagen I production is under critical regulatory control by the mTORC1/4E-BP1 signalling hub. Using pharmacological inhibition in combination with gene-editing approaches, we now demonstrate that the role of the mTORC1 axis extends to the regulation of over a third of all TGF-β1 regulated matrisome genes. We provide further evidence that the global transcriptome of TGF-β1-stimulated fibroblasts in vitro matches that of a subpopulation of the high collagen expressing CTHRC1 + pathological fibroblast population in the IPF lung. In contrast, the TGF-β1 induced transcriptome of fibroblasts in which mTORC1 signalling is disrupted (by RPTOR gene editing using CRISPR-Cas9) does not map to any fibroblast population present in human control or fibrotic lung. Using the novel and selective mTORC1 inhibitor RMC-5552, we further demonstrate a direct functional link between mTORC1 signalling and the acquisition of key marker genes which define the CTHRC1+ fibroblast population in IPF. These data demonstrate, for the first time, a critical role for the mTORC1 signalling hub in determining the transcriptional identity of the CTHRC1+ pathological fibroblast population and provide strong scientific support for targeting mTORC1 as a therapeutic strategy in IPF and potentially other fibrotic conditions associated with dysregulated TGF-β1 signalling in the fibrotic niche.

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–β ₁ (TGF-β ₁ ), which promotes fibroblast to myofibroblast differentiation. We found that extracellular nutrient availability profoundly influenced the TGF-β ₁ transcriptome of primary human lung fibroblasts and that biosynthesis of amino acids emerged as a top enriched TGF-β ₁ transcriptional module. We subsequently uncovered a key role for pyruvate in influencing glutaminase (GLS1) inhibition during TGF-β ₁ –induced fibrogenesis. In pyruvate-replete conditions, GLS1 inhibition was ineffective in blocking TGF-β ₁ –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-β ₁ –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.