The Immunopathology of Pulmonary Rejection after Murine Lung Transplantation.

To improve outcomes following lung transplantation, it is essential to understand the immunological mechanisms that result in chronic graft failure. The associated clinical syndrome is termed chronic lung allograft dysfunction (CLAD), which is known to be induced by alloimmune-dependent (i.e., rejection) and alloimmune-independent factors (e.g., infections, reflux and environmental factors). We aimed to explore the alloimmune-related mechanism, i.e., pulmonary rejection. In this study, we use a murine orthotopic left lung transplant model using isografts and allografts (C57BL/6 or BALB/c as donors to C57BL/6 recipients), with daily immunosuppression (10 mg/kg cyclosporin A and 1.6 mg/kg methylprednisolone). Serial sacrifice was performed at days 1, 7 and 35 post-transplantation (n = 6 at each time point for each group). Left transplanted lungs were harvested, a single-cell suspension was made and absolute numbers of immune cells were quantified using multicolor flow cytometry. The rejection process followed the principles of a classic immune response, including innate but mainly adaptive immune cells. At day 7 following transplantation, the numbers of interstitial macrophages, monocytes, dendritic cells, NK cells, NKT cells, CD4+ T cells and CD8+ T and B cells were increased in allografts compared with isografts. Only dendritic cells and CD4+ T cells remained elevated at day 35 in allografts. Our study provides insights into the immunological mechanisms of true pulmonary rejection after murine lung transplantation. These results might be important in further research on diagnostic evaluation and treatment for CLAD.

Mucosal immune alterations at the early onset of tissue destruction in chronic obstructive pulmonary disease.

Rationale: COPD is characterized by chronic airway inflammation, small airways changes, with disappearance and obstruction, and also distal/alveolar destruction (emphysema). The chronology by which these three features evolve with altered mucosal immunity remains elusive. This study assessed the mucosal immune defense in human control and end-stage COPD lungs, by detailed microCT and RNA transcriptomic analysis of diversely affected zones. Methods: In 11 control (non-used donors) and 11 COPD (end-stage) explant frozen lungs, 4 cylinders/cores were processed per lung for microCT and tissue transcriptomics. MicroCT was used to quantify tissue percentage and alveolar surface density to classify the COPD cores in mild, moderate and severe alveolar destruction groups, as well as to quantify terminal bronchioles in each group. Transcriptomics of each core assessed fold changes in innate and adaptive cells and pathway enrichment score between control and COPD cores. Immunostainings of immune cells were performed for validation. Results: In mildly affected zones, decreased defensins and increased mucus production were observed, along CD8+ T cell accumulation and activation of the IgA pathway. In more severely affected zones, CD68+ myeloid antigen-presenting cells, CD4+ T cells and B cells, as well as MHCII and IgA pathway genes were upregulated. In contrast, terminal bronchioles were decreased in all COPD cores. Conclusion: Spatial investigation of end-stage COPD lungs show that mucosal defense dysregulation with decreased defensins and increased mucus and IgA responses, start concomitantly with CD8+ T-cell accumulation in mild emphysema zones, where terminal bronchioles are already decreased. In contrast, adaptive Th and B cell activation is observed in areas with more advanced tissue destruction. This study suggests that in COPD innate immune alterations occur early in the tissue destruction process, which affects both the alveoli and the terminal bronchioles, before the onset of an adaptive immune response.

COVID-19 progression in hospitalized patients using follow-up in vivo CT and ex vivo microCT.

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease-19 (COVID-19) which can lead to acute respiratory distress syndrome (ARDS) and evolve to pulmonary fibrosis. Computed tomography (CT) is used to study disease progression and describe radiological patterns in COVID-19 patients. This study aimed to assess disease progression regarding lung volume and density over time on follow-up in vivo chest CT and give a unique look at parenchymal and morphological airway changes in "end-stage" COVID-19 lungs using ex vivo microCT. Methods: Volumes and densities of the lung/lobes of three COVID-19 patients were assessed using follow-up in vivo CT and ex vivo whole lung microCT scans. Airways were quantified by airway segmentations on whole lung microCT and small-partition microCT. As controls, three discarded healthy donor lungs were used. Histology was performed in differently affected regions in the COVID-19 lungs. Results: In vivo, COVID-19 lung volumes decreased while density increased over time, mainly in lower lobes as previously shown. Ex vivo COVID-19 lung volumes decreased by 60% and all lobes were smaller compared to controls. Airways were more visible on ex vivo microCT in COVID-19, probably due to fibrosis and increased airway diameter. In addition, small-partition microCT showed more deformation of (small) airway morphology and fibrotic organization in severely affected regions with heterogeneous distributions within the same lung which was confirmed by histology. Conclusions: COVID-19-ARDS and subsequent pulmonary fibrosis alters lung architecture and airway morphology which is described using in vivo CT, ex vivo microCT, and histology.

Proteolytic inactivation of CXCL12 in the lungs and circulation of COVID-19 patients.

The human chemokine stromal cell-derived factor-1 (SDF-1) or CXCL12 is involved in several homeostatic processes and pathologies through interaction with its cognate G protein-coupled receptor CXCR4. Recent research has shown that CXCL12 is present in the lungs and circulation of patients with coronavirus disease 2019 (COVID-19). However, the question whether the detected CXCL12 is bioactive was not addressed. Indeed, the activity of CXCL12 is regulated by NH2- and COOH-terminal post-translational proteolysis, which significantly impairs its biological activity. The aim of the present study was to characterize proteolytic processing of CXCL12 in broncho-alveolar lavage (BAL) fluid and blood plasma samples from critically ill COVID-19 patients. Therefore, we optimized immunosorbent tandem mass spectrometry proteoform analysis (ISTAMPA) for detection of CXCL12 proteoforms. In patient samples, this approach uncovered that CXCL12 is rapidly processed by site-specific NH2- and COOH-terminal proteolysis and ultimately degraded. This proteolytic inactivation occurred more rapidly in COVID-19 plasma than in COVID-19 BAL fluids, whereas BAL fluid samples from stable lung transplantation patients and the non-affected lung of lung cancer patients (control groups) hardly induced any processing of CXCL12. In COVID-19 BAL fluids with high proteolytic activity, processing occurred exclusively NH2-terminally and was predominantly mediated by neutrophil elastase. In low proteolytic activity BAL fluid and plasma samples, NH2- and COOH-terminal proteolysis by CD26 and carboxypeptidases were observed. Finally, protease inhibitors already approved for clinical use such as sitagliptin and sivelestat prevented CXCL12 processing and may therefore be of pharmacological interest to prolong CXCL12 half-life and biological activity in vivo.

The MUC5B Promoter Polymorphism is Not Associated With Non-ILD Chronic Respiratory Diseases or Post-transplant Outcome.

The MUC5B promoter polymorphism (rs35705950) has been associated with interstitial lung disease (ILD) and with prolonged pre-transplant survival in idiopathic pulmonary fibrosis (IPF), but no information is available regarding its prevalence in other respiratory diseases and its influence on post-transplant outcome. We included the Leuven lung transplantation cohort between 1991 and 2015 (n = 801). We assessed the minor allele frequency (MAF) of the MUC5B variant in the entire study cohort and investigated the influence of recipient MUC5B promoter polymorphism on post-transplant outcome in patients who were transplanted after 2004. MUC5B was successfully genotyped in 746 patients. The MAF was significantly higher in ILD (17.6%) compared to chronic obstructive pulmonary disease (COPD)/emphysema (9.3%), cystic fibrosis (CF)/bronchiectasis (BRECT) (7.5%) and pulmonary hypertension (PHT) (7.4%) (p < 0.001). No association was observed between rs35705950 and chronic lung allograft dysfunction (CLAD)/graft loss in the ILD population [CLAD: HR 1.37 95% CI (0.70-2.68); graft loss: HR 1.02 95% CI (0.55-1.89)], nor the entire study cohort [CLAD: HR 0.96 95% CI (0.69-1.34); graft loss: HR 0.97 95% CI (0.70-1.35)]. The MUC5B promoter polymorphism is a very specific predictive factor for the presence of pulmonary fibrosis as it is only associated with pulmonary fibrosis and not with other chronic respiratory diseases. While the MUC5B promoter variant is associated with better pre-transplant survival among IPF patients, recipient MUC5B promoter variant does not play a role in post-transplant outcome.

Lung Microenvironments and Disease Progression in Fibrotic Hypersensitivity Pneumonitis.

Rationale: Fibrotic hypersensitivity pneumonitis (fHP) is an interstitial lung disease caused by sensitization to an inhaled allergen. Objectives: To identify the molecular determinants associated with progression of fibrosis. Methods: Nine fHP explant lungs and six unused donor lungs (as controls) were systematically sampled (4 samples/lung). According to microcomputed tomography measures, fHP cores were clustered into mild, moderate, and severe fibrosis groups. Gene expression profiles were assessed using weighted gene co-expression network analysis, xCell, gene ontology, and structure enrichment analysis. Gene expression of the prevailing molecular traits was also compared with idiopathic pulmonary fibrosis (IPF). The explant lung findings were evaluated in separate clinical fHP cohorts using tissue, BAL samples, and computed tomography scans. Measurements and Main Results: We found six molecular traits that associated with differential lung involvement. In fHP, extracellular matrix and antigen presentation/sensitization transcriptomic signatures characterized lung zones with only mild structural and histological changes, whereas signatures involved in honeycombing and B cells dominated the transcriptome in the most severely affected lung zones. With increasing disease severity, endothelial function was progressively lost, and progressive disruption in normal cellular homeostatic processes emerged. All six were also found in IPF, with largely similar associations with disease microenvironments. The molecular traits correlated with in vivo disease behavior in a separate clinical fHP cohort. Conclusions: We identified six molecular traits that characterize the morphological progression of fHP and associate with in vivo clinical behavior. Comparing IPF with fHP, the transcriptome landscape was determined considerably by local disease extent rather than by diagnosis alone.

Local expression profiles of vitamin D-related genes in airways of COPD patients.

Treatment of Chronic Obstructive Pulmonary Disease (COPD) is based on bronchodilation, with inhaled corticosteroids or azithromycin associated when frequent exacerbations occur. Despite the proven benefits of current treatment regimens, the need for new interventions in delineated subgroups remains. There is convincing evidence for oral vitamin D supplementation in reducing exacerbations in COPD patients severely deficient for circulating vitamin D. However, little is known about local vitamin D metabolism in the airways and studies examining expression of the vitamin D receptor (VDR), the activating enzyme (CYP27B1) and inactivating enzyme (CYP24A1) of vitamin D in lung tissue of COPD patients are lacking. Therefore, the expression and localization of key enzymes and the receptor of the vitamin D pathway were examined in tissue of 10 unused donor lungs and 10 COPD explant lungs. No differences in the expression of CYP27B1 and CYP24A1 were found. Although protein expression of VDR was significantly lower in COPD explant tissue, there was no difference in downstream expression of the antimicrobial peptide cathelicidin. Whereas CYP27B1 and CYP24A1 were present in all layers of the bronchial epithelium, VDR was only expressed at the apical layer of a fully differentiated bronchial epithelium with no expression in vascular endothelial cells. By contrast, CYP24A1 expression was highly present in lung endothelial cells suggesting that systemic vitamin D can be inactivated before reaching the epithelial compartment and the tissue immune cells. These data support the idea of exploring the role of vitamin D inhalation in patients with COPD.