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Background Some COVID-19 survivors present lung function abnormalities during follow-up, particularly reduced carbon monoxide lung diffusing capacity (DLCO). To investigate risk factors and underlying pathophysiology, we compared the clinical characteristics and levels of circulating pulmonary epithelial and endothelial markers in COVID-19 survivors with normal or reduced DLCO 6 months after discharge. Methods Prospective, observational study. Clinical characteristics during hospitalization, and spirometry, DLCO and plasma levels of epithelial (surfactant protein (SP) A (SP-A), SP-D, Club cell secretory protein-16 (CC16) and secretory leukocyte protease inhibitor (SLPI)), and endothelial (soluble intercellular adhesion molecule 1 (sICAM-1), soluble E-selectin and Angiopoietin-2) 6 months after hospital discharge were determined in 215 COVID-19 survivors. Results DLCO was . Levels of epithelial (SP-A, SP-D and SLPI) and endothelial (sICAM-1 and angiopoietin-2) markers were higher in patients with reduced DLCO, particularly in those with DLCO ≤ 50% ref. Circulating SP-A levels were associated with the occurrence of acute respiratory distress syndrome (ARDS), organizing pneumonia and pulmonary embolisms during hospitalization. Conclusions Reduced DLCO is common in COVID-19 survivors 6 months after hospital discharge, especially in those older than 60 years with very severe acute disease. In these individuals, elevated levels of epithelial and endothelial markers suggest persistent lung damage.

A specific T-cell response persists in the majority of COVID-19 patients 6 months after hospital discharge. This response is more prominent in those who required critical care during the acute COVID-19 episode but is reduced in patients with lung sequelae.

Fibroblast-to-myofibroblast transdifferentiation and the acquisition of a senescent phenotype are hallmarks of fibrotic diseases. The study of the localization of senescent myofibroblasts as well as their interactions with other cell types in the fibrotic tissue has been hindered by the lack of methods to detect these cells in vivo. Here, we describe methods to detect tissue localization of senescent myofibroblasts in precision-cut lung slices (PCLS) by combining β-galactosidase staining with immunofluorescence techniques.

Rationale: Idiopathic Pulmonary Fibrosis (IPF) is a lethal disease with a mechanism of onset and progression that is not fully understood. Past genomic studies, focused on fibrotic lungs, provided valuable insights of its pathophysiology, but an important unanswered question remains: what are the cellular and molecular changes in the histologically normal looking parts of IPF lungs? The answer may help understand the early disease stages and unveil biological pathways that precede and likely lead to the histological changes in IPF. Methods: We reanalyzed our published single cell expression data from healthy controls and IPF patients (Morse et al, 2019, ERJ) . We used Seurat to identify cell types and perform all analyses. Results: We identified 20 cell types and further focused on 4 fibroblast sub-clusters. Two of the sub-clusters were significantly enriched for cells originating from the IPF lungs. Analysis of differentially expressed genes in each cluster identified unique expression profiles for IPF/control states. Interestingly, IPF upper non-fibrotic and lower fibrotic lobes didn’t show significant differences in these profiles. This result shows that fibroblast types such as myofibroblasts which have been associated with the IPF state, are present in parts of the lung that are histologically non-fibrotic. We also found an enrichment in multiple copper-binding proteins supporting the role of metal copper in IPF pathogenesis. Conclusion: We showed histologically normal tissue in IPF lungs is a profibrotic environment representing an earlier stage where many disease-associated cellular alterations have not yet occurred. This establishes a more appropriate environment to study early interventions.

Rationale: One of the features of IPF is the accumulation of senescent cells that produce the senescence-associated secretory phenotype (SASP) that modifies the lung microenvironment. We focus on the alteration of the lung immune response by the SASP, specifically on the natural killer (NK) lymphocytes cytotoxicity against senescence cells. Methods: Fresh lung samples were used for scRNAseq and flow. Blood was collected for the study of plasma cytokines and PBMCs. Results: There was decreased proportion of NK cells in the IPF lung, specifically of the circulatory/cytotoxic NK cells. Such NKs are more senescent, there is a higher proportion of IL6 producing cells and reduced expression of the chemokine receptor CCR2, which is involved in NK recruitment to the lung. scRNAseq analysis of the NK cluster in IPF patients revealed an impaired expression in some of the main NK genes and the fibroblast cluster revealed a profound decrease in the lung capacity to produce chemokines. There is an increase in the proportion of NK cells in the IPF blood with an increase of the circulating/cytotoxic NK population. Additionally, healthy blood NK cells have less migration capacity towards conditioned media (CM) from IPF lung fibroblasts and the culture with the IPF-CM reduces their cytotoxic capacity. Conclusions: Our data reveal that IPF patients have reduced numbers an impaired activity of NK cells in the lung, specifically of the circulating/cytotoxic subpopulation due to a deficiency in the lung capacity of the lung to recruit NKs, which results in their accumulation in the blood. This deficiency in the NK activity and senescence status in the lung could be one of the altered mechanisms perpetrating the accumulation of senescent cells in IPF lungs.

Background Chronic Obstructive Pulmonary Disease (COPD) is associated with an abnormal pulmonary and systemic immune response to tobacco smoking. Yet, how do immune cells relate within and between these two biological compartments, how the pulmonary infiltrate influences the lung transcriptome, and what is the role of active smoking vs. presence of disease is unclear. Methods To investigate these questions, we simultaneously collected lung tissue and blood from 65 individuals stratified by smoking habit and presence of the disease. The immune cell composition of both tissues was assessed by flow cytometry, whole lung transcriptome was determined with Affymetrix arrays, and we used Weighted Gene Co-expression Network Analysis (WGCNA) to integrate results. Results Main results showed that: (1) current smoking and the presence of COPD were both independently associated with a reduction in the proportion of lung T cells and an increase of macrophages, specifically those expressing CD80 + CD163+; (2) changes in the proportion of infiltrating macrophages, smoking status or the level of airflow limitation were associated to different WGCNA modules, which were enriched in iron ion transport, extracellular matrix and cilium organization gene ontologies; and, (3) circulating white blood cells counts were correlated with lung macrophages and T cells. Conclusions Mild-moderated COPD lung immune infiltrate is associated with the active smoking status and presence of disease; is associated with changes in whole lung tissue transcriptome and marginally reflected in blood. Electronic supplementary material The online version of this article (10.1186/s12931-019-1105-z) contains supplementary material, which is available to authorized users.

Tobacco smoking is the main environmental risk factor for chronic obstructive pulmonary disease (COPD), but not all smokers develop the disease. A population of lung-resident mesenchymal stem cells (LR-MSCs) exist in healthy lungs, but how tobacco smoking affects them and their role in COPD have not been assessed yet. Using a sphere-based culture technique, we isolated LR-MSCs from lung tissue obtained from nonsmokers and current and former smokers with and without COPD (

Acute respiratory distress syndrome (ARDS) is a high-mortality syndrome that develops following an infection or trauma, leading to a dysregulated inflammatory response in the lung that can cause tissue remodeling, pulmonary dysfunction, and death [1]. ARDS is characterized by acute respiratory failure caused by an increase of fluids in the alveolar space. The breakdown of the immune response increases the permeability of the epithelial–endothelial barrier. This results in an increased filtration of protein-rich fluid from the vascular system into the alveolar spaces, with a subsequent lung edema and a decrease in the ability of gas exchange [2].

Background c-Kit + lung stem cells have been described in the human healthy lung. Their potential relation with smoking and/or chronic obstructive pulmonary disease (COPD) is unknown. Methods We characterized and compared c-Kit+ cells in lung tissue of 12 never smokers (NS), 15 smokers with normal spirometry (S) and 44 COPD patients who required lung resectional surgery. Flow cytometry (FACS) was used to characterize c-Kit+ cells in fresh lung tissue disaggregates, and immunofluorescence (IF) for further characterization and to determine their location in OCT- embedded lung tissue. Results We identified 4 c-Kit+ cell populations, with similar proportions in NS, S and COPD: (1) By FACS, c-Kithigh/CD45+ cells (4.03 ± 2.97% (NS), 3.96 ± 5.30% (S), and 5.20 ± 3.44% (COPD)). By IF, these cells were tryptase+ (hence, mast cells) and located around the airways; (2) By IF, c-Kitlow/CD45+/triptase- (0.07 ± 0.06 (NS), 0.03 ± 0.02 (S), and 0.06 ± 0.07 (COPD) cells/field), which likely correspond to innate lymphoid cells; (3) By FACS, c-Kitlow/CD45-/CD34+ (0.95 ± 0.84% (NS), 1.14 ± 0.94% (S) and 0.95 ± 1.38% (COPD)). By IF these cells were c-Kitlow/CD45-/CD31+, suggesting an endothelial lineage, and were predominantly located in the alveolar wall; and, (4) by FACS, an infrequent c-Kitlow/CD45-/CD34- population (0.09 ± 0.14% (NS), 0.08 ± 0.09% (S) and 0.08 ± 0.11% (COPD)) compatible with a putative lung stem cell population. Yet, IF failed to detect them and we could not isolate or grow them, thus questioning the existence of c-Kit+ lung stem-cells. Conclusions The adult human lung contains a mixture of c-Kit+ cells, unlikely to be lung stem cells, which are independent of smoking status and/or presence of COPD. Electronic supplementary material The online version of this article (10.1186/s12890-018-0688-3) contains supplementary material, which is available to authorized users.

Rationale: Chronic Obstructive Pulmonary Disease (COPD) is a complex and heterogeneous disease whose pathobiology is not fully understood. Objectives: To characterize and contrast the pulmonary mRNA expression in former smokers with COPD (mild=15; severe-very severe=16) and healthy controls (never smokers=11). Methods: Lung tissue was obtained from individuals undergoing thoracic resectional surgery (mostly because of lung cancer) or lung transplantation explants. Paired end TruSEQ RNASeq libraries were constructed and analyzed with an Illumina HiSeq. Sequencing analysis and differential gene expression was done with DESeq2, and in order to find modules of co-expressed genes related to clinical traits (airflow limitation severity, BMI and PackYear) weighted correlation networks were constructed with the whole matrix. Measurements and Main Results: The number of differentially expressed genes was higher between non-smokers vs. severe COPD (n=675), followed by mild COPD vs. severe COPD (n=391), while the expression profile of non-smokers vs. mild COPD was the most similar. Network weighted co-expression analysis identified 19 modules, 5 of them were associated to the presence of airflow limitation and its severity but not smoking or BMI. Key genes are validated in a second cohort of individuals. Conclusions: Severe airflow limitation represents a distinctive transcriptomic signature in comparison to mild COPD and non-smokers. Transcriptomic changes of mild former smokers with COPD vs. non-smokers are more heterogeneous. Supported, in part, Instituto de Salud Carlos III (PI15/00799), CP (16/00039) and a grant from MSD.

Background Chronic Obstructive Pulmonary Disease (COPD) is characterized by pulmonary and systemic manifestations. Multiple studies have identified airway or blood biomarkers associated with the severity of airflow limitation, but it is not clear whether these biomarkers directly represent the pathobiology in the lung. Objective To identify pathobiological mechanisms shared between different compartments in COPD patients. Methods Weighted Gene Co-expression Network Analysis (WGCNA) was used to meta-analyse four gene expression datasets from Caucasian former smokers with COPD: two lung tissue datasets (n=70 and n=124), and one each of sputum (n=121) and peripheral blood (n=121). Results Eighteen (86%) of the twenty-one co-expression modules identified were preserved between the two lung tissue datasets; seven (33%) of them were preserved also in sputum, and five also (24%) in blood. One module (labelled “yellow”) was preserved across all datasets and associated with FEV 1 % in lung and sputum but not in blood. This module was enriched in ontologies related to mitochondrial function, cell cycle, ubiquitination and RNA processing. Two other modules (brown and magenta) were preserved and associated with FEV 1 % across lung and sputum datasets (but not in blood). These modules included genes related to ion homeostasis, T cell response, cytokine signalling and RNA processing. Conclusions Our study identified several biological functions in mRNA co-expression modules associated with FEV 1 % in lung and sputum but not in blood. These results suggest that the systemic manifestations of COPD likely constitute an independent component of the disease. Funding Unristricted grant form GSK, U.S. NIH P01 HL105339 and FIS PI15/00799.

Chronic obstructive pulmonary disease (COPD) is characterized by chronic airflow limitation caused by a combination of airways disease (bronchiolitis) and parenchymal destruction (emphysema), whose relative proportion varies from patient to patient.To explore and contrast the molecular pathogenesis of emphysema and bronchiolitis in COPD.We used network analysis of lung transcriptomics (Affymetrix arrays) in 70 former smokers with COPD to compare differential expression and gene coexpression in bronchiolitis and emphysema.We observed that in emphysema (but not in bronchiolitis) (1) up-regulated genes were enriched in ontologies related to B-cell homing and activation; (2) the immune coexpression network had a central core of B cell-related genes; (3) B-cell recruitment and immunoglobulin transcription genes (CXCL13, CCL19, and POU2AF1) correlated with emphysema severity; (4) there were lymphoid follicles (CD20(+)IgM(+)) with active B cells (phosphorylated nuclear factor-κB p65(+)), proliferation markers (Ki-67(+)), and class-switched B cells (IgG(+)); and (5) both TNFRSF17 mRNA and B cell-activating factor protein were up-regulated. These findings were by and large reproduced in a group of patients with incipient emphysema and when patients with emphysema were matched for the severity of airflow limitation of those with bronchiolitis.Our study identifies enrichment in B cell-related genes in patients with COPD with emphysema that is absent in bronchiolitis. These observations contribute to a better understanding of COPD pathobiology and may open new therapeutic opportunities for patients with COPD.

Background Tobacco smoking is the main risk factor of chronic obstructive pulmonary disease (COPD) but not all smokers develop the disease. An abnormal pulmonary and systemic inflammatory response to smoking is thought to play a major pathogenic role in COPD, but this has never been tested directly. Methods We studied the systemic biomarker and leukocyte transcriptomic response (Affymetrix microarrays) to smoking exposure in 10 smokers with COPD and 10 smokers with normal spirometry. We also studied 10 healthy never smokers (not exposed to smoking) as controls. Because some aspects of COPD may differ in males and females, and the inflammatory response to other stressors (infection) might be different in man and women, we stratified participant recruitment by sex. Differentially expressed genes were validated by q-PCR. Ontology enrichment was evaluated and interaction networks inferred. Results Principal component analysis identified sex differences in the leukocyte transcriptomic response to acute smoking. In both genders, we identified genes that were differentially expressed in response to smoking exclusively in COPD patients (COPD related signature) or smokers with normal spirometry (Smoking related signature), their ontologies and interaction networks. Conclusions The use of an experimental intervention (smoking exposure) to investigate the transcriptomic response of peripheral leukocytes in COPD is a step beyond the standard case-control transcriptomic profiling carried out so far, and has facilitated the identification of novel COPD and Smoking expression related signatures which differ in males and females.