Respiratory Research

RationaleIdentification and validation of circulating biomarkers for lung function decline in COPD remains an unmet need. ObjectiveIdentify prognostic and dynamic plasma protein biomarkers of COPD progression. MethodsWe measured plasma proteins using SomaScan from two COPD-enriched cohorts, the Subpopulations and Intermediate Outcomes Measures in COPD Study (SPIROMICS) and Genetic Epidemiology of COPD (COPDGene), and one population-based cohort, Multi-Ethnic Study of Atherosclerosis (MESA) Lung. Using SPIROMICS as a discovery cohort, linear mixed models identified baseline proteins that predicted future change in FEV1 (prognostic model) and proteins whose expression changed with change in lung function (dynamic model). Findings were replicated in COPDGene and MESA-Lung. Using the COPD-enriched cohorts, Gene Set Enrichment Analysis (GSEA) identified proteins shared between COPDGene and SPIROMICS. Metascape identified significant associated pathways. Measurements and Main ResultsThe prognostic model found 7 significant proteins in common (p < 0.05) among all 3 cohorts. After applying false discovery rate (adjusted p < 0.2), leptin remained significant in all three cohorts and growth hormone receptor remained significant in the two COPD cohorts. Elevated baseline levels of leptin and growth hormone receptor were associated with slower rate of decline in FEV1. Twelve proteins were nominally but not FDR significant in the dynamic model and all were distinct from the prognostic model. Metascape identified several immune related pathways unique to prognostic and dynamic proteins. ConclusionWe identified leptin as the most reproducible COPD progression biomarker. The difference between prognostic and dynamic proteins suggests disease activity signatures may be different from prognosis signatures.

BackgroundA relationship between asthma and the risk of having cancer has been identified in several studies. However, these studies have used different methodologies, been primarily cross-sectional in nature, and the results have been contradictory. Population-level analyses are required to determine if a relationship truly exists. MethodsWe developed a novel machine learning tool to infer associations, Causal Inference using the Composition of Transactions (CICT). Two all payers claim datasets of over two hundred million hospitalization encounters from the US-based Healthcare Cost and Utilization Project (HCUP) were used for discovery and validation. Associations between asthma and neoplasms were discovered in data from the State of Florida. Validation was conducted on eight cohorts of patients with asthma, and seven subtypes of asthma and COPD using datasets from the State of California. Control groups were matched by gender, age, race, and history of tobacco use. Odds ratio analysis with Bonferroni-Holm correction measured the association of asthma and COPD with 26 different benign and malignant neoplasms. ICD9CM codes were used to identify exposures and outcomes. FindingsCICT identified 17 associations between asthma and the risk of neoplasia in the discovery dataset. In the validation studies, 208 case-control analyses were conducted between subtypes of Asthma (N= 999,370, male= 33%, age= 50) and COPD (N=715,971, male = 50%, age=69) with the corresponding matched control groups (N=8,400,004, male= 42%, age= 47). Allergic asthma was associated with benign neoplasms of the meninges, salivary, pituitary, parathyroid, and thyroid glands (OR:1.52 to 2.52), and malignant neoplasms of the breast, intrahepatic biliary system, hematopoietic, and lymphatic system (OR: 1.45 to 2.05). COPD was associated with malignant neoplasms in the lung, bladder, and hematopoietic systems. InterpretationThe combined use of machine learning methods for knowledge discovery and epidemiological methods shows that allergic asthma is associated with the development of neoplasia, including in glandular organs, ductal tissues, and hematopoietic systems. Also, our findings differentiate the pattern of neoplasms between allergic asthma and obstructive asthma. This suggests that inflammatory pathways that are active in asthma also contribute to neoplastic transformation in specific organ systems such as secretory organs. FundingNone At a Glance CommentaryOver the past three decades, studies have suggested that asthma could increase the risk of developing cancer, but a consensus has not been reached. The debate persists because the current evidence has been derived using cross-sectional statistical designs, limited datasets, and small cohorts and conflicting results. In addition, the mechanism by which allergic airway inflammation contributes to neoplastic transformation is postulated but not proven. Here, we present the largest study to date on this association in patients with asthma or COPD. A knowledge discovery method was used for hypothesis generation that, when combined with epidemiological reasoning tools, identified associations between airway disease and neoplasia. The results reveal novel relationships between allergic asthma and benign glandular tumors and confirm the well-known connections between COPD and lung cancer. Further, we identified a novel association between COPD and asthma with hematological malignancies. These findings rectify contradictory results from other studies and demonstrate more specifically that the types of neoplasms associated with asthma compared to COPD that infers mechanistic plausibility.

BACKGROUNDAsthma is recognized as an inflammatory disease of the airways, but inflammation may also affect the parenchyma and pulmonary vasculature. Hyperpolarized 129Xe MRI and MR spectroscopy (MRS) provide a way to quantify the transfer of gas from the airways through the alveolar membrane and its binding to hemoglobin in the red blood cells (RBC) of the pulmonary microvasculature. The vast majority of 129Xe MRS studies have investigated interstitial lung disease and the ratio of 129Xe binding to the RBC and 129Xe present in the alveolar membrane, (RBC:membrane) which is a surrogate of oxygen gas-transfer to the blood. We wondered if 129Xe RBC:membrane would differ in asthma patients as compared to healthy volunteers because of recent work showing abnormally diminished pulmonary vascular small-vessel structure in severe-asthma. RESEARCH QUESTIONDo 129Xe MRI gas-transfer measurements differ significantly in patients with moderate-severe asthma? STUDY DESIGN AND METHODSIn this retrospective study, healthy (NCT02484885) and asthma (NCT04651777; NCT02351141) participants were evaluated who provided written informed consent. RESULTSThirty-one participants with asthma (mean age=55 years {+/-}18; 22 females) and 32 healthy volunteers (mean age=31 years {+/-}14; 12 females) with 129Xe MRS were evaluated. FEV1, VDP and DLCO/KCO were significantly different in asthma compared to healthy participants. Age-corrected 1RBC:membrane was significantly different in moderate-severe asthma (0.32{+/-}0.09) as compared to healthy participants (0.47{+/-}0.12, P=.01). RBC:membrane was significantly related to pulse-oximetry hemoglobin estimates ({rho}=.29; P=.04) and DLCO ({rho}=.71; P<.001). Significant relationships between 129Xe RBC:membrane and age were observed in healthy ({rho}=-.55; P=.002) and asthma participants ({rho}=-0.49; P=.006), adjusted for sex. A significant ANCOVA model also revealed the influence of age (P=.002), sex (P<.001), hemoglobin (P=.003) and asthma status (P=.02) on RBC:membrane. INTERPRETATION129Xe RBC:membrane values were significantly different in moderate-severe asthma compared to healthy volunteers and were explained by age, sex, hemoglobin, and asthma status.

BackgroundElexacaftor/Tezacaftor/Ivacaftor (ETI) has significantly improved clinical outcomes for people with cystic fibrosis (pwCF), but the molecular effects on airway inflammation remains incompletely understood. This study aimed to characterise longitudinal changes in airway inflammation and sputum proteomes following ETI treatment, and to correlate proteomic shifts to changes in inflammatory cytokines. Patients and methodsSputum from pwCF (n=30) were collected before start of ETI and after 3 and 9-12 months of treatment. Sputum from healthy control subjects (n=7) were included for comparison. Samples were analysed for total proteome content using data independent acquisition liquid chromatography tandem mass spectrometry (DIA LC-MS/MS), and inflammatory cytokines using Mesoscale assays. Protein expression trends were analysed using k-means clustering, and correlations between airway proteomes and inflammatory cytokines were performed using Pearson correlation and enrichment analysis. ResultsETI therapy resulted in significant changes in the airway proteome, mainly related to decreased neutrophil degranulation and an increase in anti-proteases. Levels of IL-1{beta}, IL-8, and TNF decreased with ETI therapy, which correlated with proteins involved in neutrophil degranulation. In contrast, IL-6 levels increased and correlated with proteins involved in O-glycosylation of mucins. Despite these improvements, proteomic and cytokine profiles remained distinct from healthy controls after 9-12 months. ConclusionETI leads to broad shifts in airway protein expression in pwCF with reduced neutrophilic inflammation and restored protease/antiprotease balance. Despite these changes, there is still increased airway inflammation compared to healthy control sputum. This dataset provides a valuable resource for further exploration of CF airway biology under ETI therapy.

IntroductionEicosanoids are bioactive lipids with roles in airway remodeling, smooth muscle hypertrophy, emphysema and pulmonary fibrosis via inflammatory pathways. Specific eicosanoids have been associated with diseases like asthma and pulmonary fibrosis, yet their broader associations with lung function remain unclear. We investigated associations of eicosanoids and related metabolites with early changes in lung function and structure. MethodsWe comprehensively profiled >250 eicosanoids and eicosanoid-related metabolites using directed non-targeted mass spectrometry in the Multi-Ethnic Study of Atherosclerosis (MESA) Lung Study with independent validation in the Framingham Heart Study (FHS). We performed cross-sectional analysis of associations between metabolites and lung function as assessed by spirometry and quantitative computed tomography (CT) measures. ResultsAmong 3384 MESA Lung participants (mean age 63{+/-}10 years, 51% women), 51 metabolites were associated with lung function (22 with % predicted FEV1, 18 with % predicted FVC, and 25 with FEV1/FVC), with 24 validated in FHS. Of these, 27 were associated with obstructive physiology, including linoleic acid derivatives (9-HODE) and other long-chain fatty acids (LCFAs, hydroxyhexadecanoic and hydroxyoctadecanoic acids) associated with higher odds. Fourteen metabolites were associated with restrictive physiology, including LTB3 and its analog associated with lower odds, and omega-3 fatty acids (EPA, stearidonic acid) associated with higher odds. ConclusionsSpecific eicosanoids and eicosanoid-related metabolites including linoleic acid derivatives and LCFAs were associated with obstructive, and leukotrienes and omega-3 fatty acids with restrictive physiology. These findings highlight bioactive lipids involved in pro- and anti-inflammatory pathways as potential influencers of lung function and may be future therapeutic targets. Take Home MessageWe identified eicosanoid metabolites associated with pulmonary function testing measurements and several that are associated with altered odds of obstructive and restrictive lung physiologies in a cohort MESA participants, with validation in FHS.

RationaleChronic obstructive pulmonary disease (COPD) and idiopathic pulmonary fibrosis (IPF) are debilitating diseases associated with divergent histopathological changes in the lungs. At present, due to cost and technical limitations, profiling cell types is not practical in large epidemiology cohorts (n>1000). Here, we used computational deconvolution to identify cell types in COPD and IPF lungs whose abundances and cell type-specific gene expression are associated with disease diagnosis and severity. MethodsWe analyzed lung tissue RNA-seq data from 1026 subjects (COPD, n=465; IPF, n=213; control, n=348) from the Lung Tissue Research Consortium. We performed RNA-seq deconvolution, querying thirty-eight discrete cell-type varieties in the lungs. We tested whether deconvoluted cell-type abundance and cell type-specific gene expression were associated with disease severity. ResultsThe abundance score of twenty cell types significantly differed between IPF and control lungs. In IPF subjects, eleven and nine cell types were significantly associated with forced vital capacity (FVC) and diffusing capacity for carbon monoxide (DLCO), respectively. Aberrant basaloid cells, a rare cells found in fibrotic lungs, were associated with worse FVC and DLCO in IPF subjects, indicating that this aberrant epithelial population increased with disease severity. Alveolar type 1 and vascular endothelial (VE) capillary A were decreased in COPD lungs compared to controls. An increase in macrophages and classical monocytes was associated with lower DLCO in IPF and COPD subjects. In both diseases, lower non-classical monocytes and VE capillary A cells were associated with increased disease severity. Alveolar type 2 cells and alveolar macrophages had the highest number of genes with cell type-specific differential expression by disease severity in COPD and IPF. In IPF, genes implicated in the pathogenesis of IPF, such as matrix metallopeptidase 7, growth differentiation factor 15, and eph receptor B2, were associated with disease severity in a cell type-specific manner. ConclusionUtilization of RNA-seq deconvolution enabled us to pinpoint cell types present in the lungs that are associated with the severity of COPD and IPF. This knowledge offers valuable insight into the alterations within tissues in more advanced illness, ultimately providing a better understanding of the underlying pathological processes that drive disease progression.

BackgroundDuring the development of idiopathic pulmonary fibrosis (IPF), elastin is broken down and replaced with a stiffer substrate which interferes with normal breathing. This remodeling process releases the amino acids desmosine and isodesmosine and elastin degradation products (EDP), collectively known as elastokines, into the circulation and may act as a surrogate for the degree of matrix turnover. We examined the association between circulating and urinary elastokine concentrations, lung function, and transplant-free survival. Research questionWhat are the associations between circulating and urinary elastokine concentrations (a marker of mature elastin turnover) with disease progression, as measured by transplant-free survival, in IPF? Study Design and MethodsConcentrations of desmosine and isodesmosine (elastokines) were measured via enzyme linked immunosorbent assay (ELISA) in the urine and serum of healthy volunteers and those with IPF. Samples were obtained from the University of Rochester Medical Center Interstitial Lung Disease (ILD) registry/biorepository (n = 81 with IPF and 24 healthy volunteers). We used linear and logistic regression modeling to determine associations between changes in urine EDP concentrations and lung function. Secondary analyses included the effect of anti-fibrotic medications on EDP concentration and assessment of associations between EDP concentrations at the time of diagnosis, lung function, and clinical outcomes. ResultsPatients with IPF were older, more likely to be male, had ever smoked, and had worse lung function compared to healthy volunteers (p value <0.02 for all parameters). Patients with IPF had higher concentrations of elasotkines (p <0.001), and among those with IPF, higher elastokine concentrations were associated with reduced forced vital capacity (FVC, p = 0.026), and decreased three-year transplant-free survival (p=0.0005). InterpretationCirculating and urinary elastokines are biomarkers of matrix turnover and are associated with relevant clinical outcomes in patients with IPF.

Interstitial lung disease (ILD) encompasses multiple pulmonary disorders characterized by damaged pulmonary tissue caused by a sequence of inflammation and fibrosis. While much is known about ILD-associated changes within the parenchyma and the pathophysiological processes underpinning the disruption of pulmonary dynamics and gas exchange, less is known about ILD-associated changes of luminal area within the large conducting airways. We aimed to investigate whether luminal area of the large conducting airways is different between patients with ILD and healthy controls. In a retrospective case-control study, luminal areas of seven large conducting airways were measured using three-dimensional reconstructions of computed tomography imaging. Patients with ILD (N=82; 54% female) were compared to control subjects matched for age, sex, and height. Univariate ANOVA tests or Kruskal-Wallis tests were used to analyze group and sex differences. Patients with ILD had greater large conducting airway luminal areas than control subjects for measured large conducting airways, including the trachea (296{+/-}73 vs. 247{+/-}65 mm2, P<0.001), right main bronchus (214{+/-}59 vs. 161{+/-}46 mm2, P<0.001), bronchus intermediate (123{+/-}32 vs. 94{+/-}28 mm2, P<0.001), right upper lobe (81{+/-}22 vs. 61{+/-}20 mm2, P<0.001), left main bronchus (151{+/-}41 vs. 119{+/-}35 mm2, P<0.001), left lower lobe (71{+/-}22 vs. 48{+/-}15 mm2, P<0.001), and left upper lobe (86{+/-}21 vs. 68{+/-}22 mm2, P<0.001). Among patients with ILD, males had 17-34% greater luminal areas (normalized to height) than females depending on the airway segment (all P<0.05). NEW & NOTEWORTHYThis study provides evidence that interstitial lung disease (ILD) is associated with greater large conducting airway luminal area, even if matched for key characteristics (age, sex, and height). Consistent with observations in health, adult males with ILD had greater height-normalized large conducting luminal areas than adult females with ILD.

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with limited treatment options and poor survival. The airway microbiome plays a critical role in IPF, and there is emerging interest in the potential involvement of the gut microbiome through the gut-lung axis. Previous studies have reported genetic associations with IPF, and there is increasing evidence that microbiome composition also has a genetic basis. Previous studies have identified genetic signals involved in lung defence and cell proliferation to IPF risk and progression, but their effects on microbiome composition remain unclear. To uncover potential causal mechanisms linking the microbiome and lung fibrosis, we explored previously reported genetic association signals and their relationship with gut microbiome composition and IPF risk and outcomes. This revealed shared signals between gut microbiome variation and IPF that warrant further investigation. Our findings emphasise the value of further research leveraging genetic associations to improve our understanding of causal disease mechanisms and identify novel therapeutic opportunities.

BackgroundPathogenesis of Coronavirus disease 2019 (Covid-19) is poorly understood. Most histologic studies come from post-mortem analysis, with existing data indicating that histologic features of acute respiratory distress syndrome are typically present in fatal cases. However, this observation may be misleading, due to confounding factors in pre-terminal disease, including injury resulting from prolonged mechanical ventilation. Ante-mortem lung biopsy may provide major pathogenetic insights, potentially providing a basis for novel treatment approaches. AimThis comparative, multicenter, prospective, observational study was planned to identify ante-mortem histological profile and immunohistochemical features of lung tissue in patients with Covid-19 in early and late phases of the disease, including markers of inflammatory cells and major pathways involved in the cytokine storm triggering. MethodsEnrolled patients underwent lung biopsy, according to the study protocol approved by local Ethical Committee, either within 15 days of the first symptoms appearing (early phase) or after >15 days (more advanced disease). Key exclusion criteria were excessive or uncorrectable bleeding risk and cardiovascular disease with heart failure. Lung samples were obtained by conventional transbronchial biopsy, trans-bronchial lung cryobiopsy or surgical lung biopsy. Results23 patients were enrolled: 12 patients underwent lung biopsy within 15 days and 11 patients more than 15 days after the onset of symptoms. Early biopsies were characterized by spots of patchy acute lung injury (ALI) with alveolar type II cells hyperplasia and significant vascular abnormalities (disordered angiogenesis with alveolar capillary hyperplasia, luminal enlargement and thickened walls of pulmonary venules, perivascular CD4-T-cell infiltration), with no hyaline membranes. In the later stages, the alveolar architecture appeared disrupted, with areas of organizing ALI, venular congestion and capillary thromboembolic microangiopathy. Striking phenotypic features were demonstrated in hyperplastic pneumocytes and endothelial cells, including the expression of phospho-STAT3 and molecules involved in immunoinhibitory signals (PD-L1 and IDO-1). Alveolar macrophages exhibited macrophage-related markers (CD68, CD11c, CD14) together with unusual markers, such as DC-Lamp/CD208, CD206, CD123/IL3AR. ConclusionA morphologically distinct "Covid pattern" was identified in the earlier stages of the disease, with prominent epithelial and endothelial cell abnormalities, that may be potentially reversible, differing strikingly from findings in classical diffuse alveolar damage. These observations may have major therapeutic implications, justifying studies of early interventions aimed at mitigating inflammatory organ injury.

BackgroundIt is important to understand the spectrum of pulmonary diseases that patients are presenting after recovery from initial SARS-CoV-2 infection. We aim to study small airway disease and changes in Computed Tomography (CT)and pulmonary function tests (PFTs) with time. MethodsThis is retrospective observation study including adult patients with confirmed SARS-CoV-2 infection with at-least two CT scans either during acute (defined as <1 month) or subacute (1-3 months) or chronic (>3months) phase after positive test. Radiological features and follow up PFTs were obtained. Results22 patients met the inclusion criteria with mean age 57.6 years (range 36-83). Out of these,18 (81.81%) were hospitalized. Mean duration of diagnosis to CT and PFT was 192.68(112-385) days and 161.54(31-259) days respectively. On PFTs, restrictive pulmonary physiology was predominant finding during subacute 56.25% (9/16) and chronic phases 47% (7/15). PFTs improved significantly with time {FEV1((p=0.0361), FVC (p=0.0341), FEF 25%-75% (p=0.0259) and DLCO (p=0.0019)}, but there was persistent air trapping in the expiratory chronic phase CT. There was resolution of ground glass opacity, consolidation, and bronchiectasis however air trapping increased with time in 41.61% (10/21) of subacute CTs compared to 81.25% (13/16) in chronic CTs. ConclusionOur study shows evidence of airway as well as parenchymal disease as relatively long-term sequel of SARS-CoV-2 infection. It also highlights the natural course and spontaneous recovery of some radiological and pulmonary function test abnormalities over time with evidence of persistent small airway disease (air trapping) on expiratory CT imaging months after infection. HighlightsO_LILong term pulmonary complication of SARS-CoV-2 infection include small airway disease C_LIO_LIThere is role of inspiratory and expiratory Computed tomography (CT) scan to identify air trapping in patients with persistent respiratory symptoms after SARS-CoV-2 infection C_LIO_LINormal spirometry and normal routine CT may not be sufficient to characterize and identify cause of persistent respiratory complains in patients after COVID-19 C_LIO_LIThis study highlights persistent parenchymal and physiological airway abnormalities more than six months after recovery from initial SARS-CoV-2 infection C_LI

Bronchopulmonary dysplasia (BPD) in infancy is a risk factor for obstructive lung disease in adults. We hypothesized that adults born preterm and diagnosed with BPD have an altered lung architecture which is correlated to lung function impairment. Individuals from the LUNAPRE cohort (clinicaltrials.gov/ct2/show/NCT02923648) were included: preterm born (gestational week <32) with (n=24) or without (n=23) a previous diagnosis of BPD, full term born with allergic asthma (n=22) and healthy volunteers (n=24). Inspiratory and expiratory HRCT scans were performed and interpreted by two expert reviewers in a blinded manner. Structural changes were scored and quantitative density measurements were analysed automatically using a dedicated post-processing workstation. The HRCT scores were significantly higher in the BPD group compared to the other groups (p<0.001) and had highest numbers in subjects diagnosed with severe BPD. Most common HRCT changes were small peripheral opacities. Hypoattenuation during inspiration was only observed in the BPD group. Architectural distortion was observed in 6/24 BPD and 2/23 premature without BPD. HRCT scores correlated to FEV1 in a negative manner for preterm (p<0.001) and BPD (p<0.05) groups. Oxygen supplementation during the neonatal period correlated with HRCT score in a positive manner for preterm group (p<0.001). No differences in lung density were observed between the groups. Young adults previously diagnosed with BPD have structural changes on CT which correlate with airway obstruction. Severity of BPD at the diagnosis was associated with CT abnormalities in adulthood. HRCT changes in adults with BPD were correlated with spirometry findings.

BackgroundIdiopathic pulmonary fibrosis (IPF) is a fibrosing interstitial pneumonia of unknown etiology often leading to respiratory failure. Over half of IPF patients present with discordant features of usual interstitial pneumonia on high-resolution computed tomography at diagnosis which warrants surgical lung biopsy to exclude the possibility of other interstitial lung diseases (ILDs). Therefore, there is a need for non-invasive biomarkers for expediting the differential diagnosis of IPF. MethodsUsing mass spectrometry, we performed proteomic analysis of plasma extracellular vesicles (EVs) in a cohort of subjects with IPF, chronic hypersensitivity pneumonitis, nonspecific interstitial pneumonitis, and healthy subjects (HS). A five-protein signature was identified by lasso regression and was validated in an independent cohort using ELISA. We evaluated the concordance between plasma EV proteome and the lung transcriptome data. Lastly, we compared the molecular pathways overrepresented in IPF by differentially expressed proteins and transcripts from EVs and lung tissues, respectively. ResultsThe five-protein signature derived from mass spectrometry data showed area under the receiver operating characteristic curve of 0.915 (95%CI: 0.819-1.011) and 0.958 (95%CI: 0.882-1.034) for differentiating IPF from other ILDs and from HS, respectively. We also found that the EV protein expression profiles mirrored their corresponding mRNA expressions in IPF lungs. Further, we observed an overlap in the EV proteome- and lung mRNA-associated molecular pathways. ConclusionsWe discovered a plasma EV-based protein signature for differential diagnosis of IPF and validated this signature in an independent cohort. The signature needs to be tested in large prospective cohorts to establish its clinical utility.

RationaleWhile many studies have examined gene expression in lung tissue, the gene regulatory processes underlying emphysema are still not well understood. Finding efficient non-imaging screening methods and disease-modifying therapies has been challenging, but knowledge of the transcriptomic features of emphysema may help in this effort. ObjectivesOur goals were to identify emphysema-associated biological pathways through transcriptomic analysis of bulk lung tissue, to determine the lung cell types in which these emphysema-associated pathways are altered, and to detect unique and overlapping transcriptomic signatures in blood and lung. MethodsUsing RNA-sequencing data from 456 samples in the Lung Tissue Research Consortium and 2,370 blood samples from the COPDGene study, we examined the transcriptomic features of computed tomography quantified emphysema. We also queried lung single-cell RNA-sequencing data to identify cell types showing COPD-associated differential expression of the emphysema pathways found in the bulk analyses. Measurements and Main ResultsIn the lung, 1,055 differentially expressed genes and 29 dysregulated pathways were significantly associated with emphysema. We observed alternative splicing of genes regulating NF-{kappa}B and cell adhesion and increased activity in the TGF-{beta} and FoxO signaling pathways. Multiple lung cell types displayed dysregulation of epithelial barrier function pathways, and an imbalance between pro-inflammatory M1 and anti-inflammatory M2 macrophages was detected. Lung tissue and blood samples shared 251 differentially expressed genes and two pathways (oxidative phosphorylation and ribosomal function). ConclusionsThis study identified emphysema-related changes in gene expression and alternative splicing, cell-type specific dysregulated pathways, and instances of shared pathway dysregulation between blood and lung. AT A GLANCE COMMENTARYO_ST_ABSScientific Knowledge on the SubjectC_ST_ABSPrior studies have investigated the transcriptomic characteristics of emphysema and its associated biological pathways. However, less is known about alternative splicing mechanisms and cell-type specific transcriptional patterns in emphysema. Additionally, a comparison between dysregulated genes and pathways in blood and lung tissues is needed to better understand the utility of non-invasive diagnostic and prognostic tools for emphysema. What This Study Adds to the FieldUsing lung samples from the Lung Tissue Research Consortium (LTRC) and blood samples from the COPDGene study, we performed differential gene and alternative splicing association analyses for CT-quantified emphysema. We then queried a previously published lung tissue single-cell RNA-sequencing atlas of COPD patients and controls to determine lung cell-type specific expression patterns of the biological pathways identified from the bulk analyses. We demonstrated that multiple pathways, including oxidative phosphorylation and ribosomal function processes, were enriched in both blood and lung tissues. We also observed that in COPD, oxidative phosphorylation was downregulated in pro-inflammatory (M1) macrophages and upregulated in anti-inflammatory (M2) macrophages. Additionally, other immunity-related cell types, including plasma cells, natural killer cells, and T lymphocytes, were linked to epithelial barrier function, such as the Rap1, adherens junction, and TGF-{beta} signaling pathways.

In contrast to early reports of conventional acute respiratory distress syndrome (ARDS) as the underlying pathophysiology of hypoxemic respiratory failure observed in patients with severe COVID-19, more recent findings implicate direct involvement of the pulmonary vasculature in giving rise to these symptoms. In earlier research, we demonstrated that patients with COVID-19 showed markedly reduced pulmonary blood volumes in pulmonary vessels <5 mm2 in cross-sectional area visible on imaging (termed "BV5"), with attendant dilation of larger, more proximal vessels. Here, we present preliminary results in which reduced BV5 is shown to correlate significantly with increased need for supplemental oxygen and abnormal arterial blood gas measurements in hospitalized COVID-19 patients. We suggest a potential mechanistic link between observed clinical, pathological, and imaging findings, and outline how these may be helpful in clinical assessment as well as the development of novel therapies.

BackgroundLung oscillometry is an emerging lung function test for assessing obstructive airway disease. Comparisons of oscillometry parameters and their bronchodilator responsiveness (BDR) between bronchial asthma and chronic obstructive pulmonary disease (COPD) patients are limited. Research QuestionDo oscillometry parameters and their BDR differ between stable asthma and COPD patients with similar severity of airflow obstruction? Study Design and MethodsWe included 467 consecutive adult patients with a clinical history of asthma (n=187) or COPD (n=280). Oscillometry, spirometry, and body plethysmography were performed before and after inhaling 400 g of salbutamol. Patients were stratified based on the severity of airflow obstruction in spirometry. The z scores of the oscillometry parameters were used for the comparison. The BDR of oscillometry parameters with other lung function parameters was also compared. ResultsThe average age of the study population was 54.9 years, and 76.4% were male. COPD patients were older, had a greater number of smokers, and had poorer lung function. The magnitude of oscillometry parameters worsened with increasing severity of airflow obstruction, regardless of the underlying disease. Asthma patients, particularly those with moderate and severe airway obstruction, had significantly higher R5 and R19 than COPD patients. The within- and whole-breath X5 of asthma were not different from those of COPD patients with similar severities of airflow obstruction. Expiratory flow limitation at tidal breaths ({Delta}X5 > 0.28 kPa/L/s) was observed in both asthma and COPD patients across all severities of airflow obstruction. The proportion of BDR in oscillometry was significantly lower than that in spirometry for both asthma (35.3% vs. 57.1%; p<0.01) and COPD patients (19.3% vs. 47.1%; p=0.02). InterpretationOscillometry parameters except for R5 and R19 did not differ between asthma and COPD patients with similar severities of airflow obstruction. Similar to spirometry, COPD patients had lower BDR in oscillometry than asthma patients. Take-home PointsO_ST_ABSStudy QuestionC_ST_ABSAre oscillometry parameters and their bronchodilator responsiveness different between bronchial asthma and COPD patients with similar severities of airflow obstruction? ResultsWe compared the FOT between 187 bronchial asthma and 280 COPD patients. Except for R5 and R19, the severity and distribution of high oscillometry parameters did not differ between asthma and COPD patients. InterpretationThe severity of oscillometry abnormalities is primarily determined by the severity of airflow obstruction, not the underlying disease.

BackgroundA significant proportion of patients experience prolonged pulmonary, cardiocirculatory or neuropsychiatric symptoms after Coronavirus disease 2019 (COVID-19), termed post-acute sequelae of COVID (PASC). Lung manifestations of PASC include cough, dyspnea on exertion and persistent radiologic abnormalities and have been linked to viral persistence, ongoing inflammation and immune dysregulation. So far, there is limited data on lung histopathology and tissue-based immune cell subtyping in PASC. Methods51 unvaccinated patients (median age, 40 years; 43% female) with a median of 17 weeks (range, 2-55 weeks) after mild SARS-CoV-2 infection (without hospitalization) underwent full clinical evaluation including high-resolution computed tomography (HR-CT) and transbronchial biopsy. We used RT-PCR/FISH and immunohistochemistry (nucleocapsid/spike/CD3/CD4/CD8) for residual SARS-CoV-2 detection and T lymphocyte subtyping, respectively. We assessed interstitial fibrosis and macrophage profiles by transmission electron microscopy (TEM) and immunofluorescence multiplex staining, while cytokine profiling in bronchoalveolar lavage (BAL) fluid was performed by legendplex immunoassay. ResultsDyspnea on exertion was the leading symptom of pulmonary PASC in our cohort. In 16% and 42.9% of patients, FEV1 and MEF50 were [&le;] 80% and 35.3% showed low attenuation volume (LAV) in >5% of lung area, in line with airflow obstruction. There was a significant correlation between oxygen pulse and time since COVID (p=0.009). Histopathologically, PASC manifested as organizing pneumonia (OP), fibrinous alveolitis and increased CD4+ T cell infiltrate predominantly around airways (bronchiolitis), while the residual virus components were detectable in only a single PASC patient (2%). T cell infiltrates around small airways were inversely correlated with time since COVID, however, this trend failed to reach statistical significance. We identified discrete interstitial fibrosis and a pro-fibrotic macrophage subtype (CD68/CD163/S100A9) as well as significantly elevated interleukin 1{beta} in BAL fluid from PASC patients (p=0.01), but H-scores for fibrotic macrophage population did not correlate with severity of clinical symptoms or T cell infiltration. InterpretationWe show decreased FEV1/MEF50 and increased LAV in line with obstructive lung disease due to CD4+ T cell-predominant bronchiolitis as well as evidence of pro-fibrotic signaling in a subset of unvaccinated PASC patients. Since our results point towards self-limiting inflammation of small airways without detectable viral reservoirs, it remains unclear whether pulmonary symptoms in PASC are SARS-CoV-2-specific or represent a general response to viral infection. Still, evidence of pro-fibrotic signaling should warrant clincal follow-up and further research into possible long-time fibrotic remodeling in PASC patients. Key pointsO_LIDyspnea on exertion is the leading clinical manifestation of PASC in the lung C_LIO_LIa minority of pts have significantly impaired lung function (FVC/TLC[&le;]80% or DLCO[&le;]70%) in spiroergometry and/or radiologic abnormalities, oxygen pulse seems to normalize over time O_LI16% and 42.9% of pts have FEV1 and MEF50[&le;]80% and 35.3% have LAV>5% of lung area, in line with airflow obstruction due to bronchiolitis C_LI C_LIO_LIResidual virus was not detectable in the lung tissue of all but one PASC patient (2%) C_LIO_LIHistologically, PASC may manifest as T cell-mediated bronchiolitis, OP and fibrinous alveolitis C_LIO_LIThere is evidence of fibrotic remodeling (ultrastructural interstitial fibrosis, pro-fibrotic macrophage subpopulation, pro-fibrotic cytokine IL-1{beta} in BAL) but this did not correlate with the degree of T cell infiltrate/bronchiolitis C_LI

The gain-of-function MUC5B promoter variant is the dominant risk factor for the development of idiopathic pulmonary fibrosis (IPF). However, its impact on protein expression in both non-fibrotic control and IPF lung specimens have not been well characterized. Utilizing laser capture microdissection coupled to mass spectrometry (LCM-MS), we investigated the proteomic profiles of airway and alveolar epithelium in non-fibrotic controls (n = 12) and IPF specimens (n = 12), stratified by the presence of the MUC5B promoter variant. Through qualitative and quantitative analyses, as well as pathway analysis and immunohistological validation, we have identified a distinct MUC5B-associated protein profile. Notably, the non-fibrotic control alveoli exhibited substantial MUC5B-associated protein changes, with an increase of IL-3 signaling. Additionally, we found that the epithelial cells overlying IPF fibroblastic foci cluster closely to alveolar epithelia and express proteins associated with cellular stress pathways. In conclusion, our findings suggest that the MUC5B promoter variant leads to protein changes in alveolar and airway epithelium that appears to be associated with the initiation and progression of lung fibrosis.

Pulmonary vascular remodelling is common in patients with chronic obstructive pulmonary disease (COPD). Vascular endothelial growth factors (VEGFs) are key mediators in angiogenesis and vascular remodelling and exist in different isoforms. VEGF-A is the most potent angiogenic member binding to VEGF receptor 2 (VEGFR2). There are, however, few studies on other isoforms, as VEGF-C, and its receptor VEGFR3 in COPD and subsequent impact of cAMP therapies on VEGF isoforms. Our aim was to evaluate the VEGF isoform synthesis in primary distal lung fibroblasts from control subjects (non-smokers (n=6) and ex-smokers (n=4), and COPD subjects with GOLD stage II (n=4) or GOLD stage IV (n=6), and the expression of VEGFR2 and VEGFR3 in human lung tissue. Primary lung fibroblasts were exposed to the cAMP generating therapies formoterol, iloprost, or roflumilast, the adenylyl cyclase activator forskolin or to transforming growth factor (TGF)-b1. VEGF isoforms were evaluated with ELISA. VEGF-C release was not significantly altered by TGF-{beta}1, in contrast to the increased levels of VEGF-A, in all fibroblasts. VEGF-C was significantly decreased by iloprost, forskolin and formoterol, whereas VEGF-A was significantly increased by iloprost and forskolin, with differences in release pattern between and within fibroblasts from control and COPD subjects. Exposure to VEGF-C specifically towards VEGFR3 decreased proliferative rate in human lung fibroblasts and bronchial epithelial cells. VEGFR2 and VEGFR3 were both present in parenchymal lung tissue and VEGFR2 in pulmonary blood vessels. in both healthy and COPD, whereas there was elevated expression of VEGFR3 in bronchial epithelium. In conclusion, TGF-{beta}1 and cAMP generating compounds have significant effects on VEGF-C and VEGF-A synthesis, which appear dysregulated in lung fibroblasts from ex-smokers and patients with COPD. Increased VEGFR3 expression in the bronchial epithelium in lung tissue, and studies into their functional impact, warrants further investigations.

BackgroundCigarette smoking has a significant impact on global health. Although cessation has positive health benefits, some molecular changes to intercellular communications may persist in the lung. In this study we created a framework to generate hypotheses by predicting altered cell-cell communication in smoker lungs using single-cell and spatial transcriptomic data. MethodsWe integrated publicly available lung single-cell transcriptomic data with spatial transcriptomic data from never-smoker and current-smoker lung tissue samples to create spatial transcriptomic data at virtual single-cell resolution by mapping individual cells from our lung scRNA-seq atlas to spots in the spatial transcriptomic data. Cell-cell communications altered in smoking were identified using the virtual single-cell transcriptomic data. ResultsWe identified pathways altered in the three current-smoker samples compared with the three never-smoker samples, including the up-regulated collagen pathway. We observed increased collagen pathway activity involving the ligands COL1A1 and COL1A2 in adventitial fibroblasts and decreased activity involving COL1A2 and COL6A3 in pericytes and myofibroblasts, respectively. We also identified other pathways with structural (e.g. Fibronectin-1), immune-related (e.g. MHC-II), growth factor (e.g. Pleiotrophin) and immunophilin (e.g. Cyclophilin A) roles. ConclusionsIn this study we inferred spatially proximal cell-cell communication between interacting cell types from spatial transcriptomics at virtual single-cell resolution to identify lung intercellular signaling altered in smoking. Our findings further implicate several pathways previously identified, and provide additional molecular context to inform future functional experiments and therapeutic avenues to mitigate pathogenic effects of smoking.