Mitochondrial Dysfunction in Lung Resident Mesenchymal Stem Cells from Idiopathic Pulmonary Fibrosis Patients.

Idiopathic pulmonary fibrosis (IPF) is characterized by an aberrant repair response with uncontrolled turnover of extracellular matrix involving mesenchymal cell phenotypes, where lung resident mesenchymal stem cells (LRMSC) have been supposed to have an important role. However, the contribution of LRMSC in lung fibrosis is not fully understood, and the role of LRMSC in IPF remains to be elucidated. Here, we performed transcriptomic and functional analyses on LRMSC isolated from IPF and control patients (CON). Both over-representation and gene set enrichment analyses indicated that oxidative phosphorylation is the major dysregulated pathway in IPF LRMSC. The most relevant differences in biological processes included complement activation, mesenchyme development, and aerobic electron transport chain. Compared to CON LRMSC, IPF cells displayed impaired mitochondrial respiration, lower expression of genes involved in mitochondrial dynamics, and dysmorphic mitochondria. These changes were linked to an impaired autophagic response and a lower mRNA expression of pro-apoptotic genes. In addition, IPF TGFß-exposed LRMSC presented different expression profiles of mitochondrial-related genes compared to CON TGFß-treated cells, suggesting that TGFß reinforces mitochondrial dysfunction. In conclusion, these results suggest that mitochondrial dysfunction is a major event in LRMSC and that their occurrence might limit LRMSC function, thereby contributing to IPF development.

Alveolar epithelial cells are competent producers of interstitial extracellular matrix with disease relevant plasticity in a human in vitro 3D model.

Alveolar epithelial cells (AEC) have been implicated in pathological remodelling. We examined the capacity of AEC to produce extracellular matrix (ECM) and thereby directly contribute towards remodelling in chronic lung diseases. Cryopreserved type 2 AEC (AEC2) from healthy lungs and chronic obstructive pulmonary disease (COPD) afflicted lungs were cultured in decellularized healthy human lung slices for 13 days. Healthy-derived AEC2 were treated with transforming growth factor ß1 (TGF-ß1) to evaluate the plasticity of their ECM production. Evaluation of phenotypic markers and expression of matrisome genes and proteins were evaluated by RNA-sequencing, mass spectrometry and immunohistochemistry. The AEC2 displayed an AEC marker profile similar to freshly isolated AEC2 throughout the 13-day culture period. COPD-derived AECs proliferated as healthy AECs with few differences in gene and protein expression while retaining increased expression of disease marker HLA-A. The AEC2 expressed basement membrane components and a complex set of interstitial ECM proteins. TGF-ß1 stimuli induced a significant change in interstitial ECM production from AEC2 without loss of specific AEC marker expression. This study reveals a previously unexplored potential of AEC to directly contribute to ECM turnover by producing interstitial ECM proteins, motivating a re-evaluation of the role of AEC2 in pathological lung remodelling.

Dipeptidyl peptidase 4 expression is not associated with an activated fibroblast phenotype in idiopathic pulmonary fibrosis.

Dipeptidyl peptidase 4 (DPP4) has been proposed as a marker for activated fibroblasts in fibrotic disease. We aimed to investigate whether a profibrotic DPP4 phenotype is present in lung tissue from patients with idiopathic pulmonary fibrosis (IPF). The presence of DPP4+ fibroblasts in normal and IPF lung tissue was investigated using flow cytometry and immunohistology. In addition, the involvement of DPP4 in fibroblast activation was examined in vitro, using CRISPR/Cas9 mediated genetic inactivation to generate primary DPP4 knockout lung fibroblasts. We observed a reduced frequency of primary DPP4+ fibroblasts in IPF tissue using flow cytometry, and an absence of DPP4+ fibroblasts in pathohistological features of IPF. The in vivo observations were supported by results in vitro showing a decreased expression of DPP4 on normal and IPF fibroblasts after profibrotic stimuli (transforming growth factor ß) and no effect on the expression of activation markers (α-smooth muscle actin, collagen I and connective tissue growth factor) upon knockout of DPP4 in lung fibroblasts with or without activation with profibrotic stimuli.

Plasma proteome changes linked to late phase response after inhaled allergen challenge in asthmatics.

BACKGROUND: A subset of individuals with allergic asthma develops a late phase response (LPR) to inhaled allergens, which is characterized by a prolonged airway obstruction, airway inflammation and airway hyperresponsiveness. The aim of this study was to identify changes in the plasma proteome and circulating hematopoietic progenitor cells associated with the LPR following inhaled allergen challenge. METHODS: Serial plasma samples from asthmatics undergoing inhaled allergen challenge were analyzed by mass spectrometry and immunosorbent assays. Peripheral blood mononuclear cells were analyzed by flow cytometry. Mass spectrometry data were analyzed using a linear regression to model the relationship between airway obstruction during the LPR and plasma proteome changes. Data from immunosorbent assays were analyzed using linear mixed models. RESULTS: Out of 396 proteins quantified in plasma, 150 showed a statistically significant change 23 h post allergen challenge. Among the most upregulated proteins were three protease inhibitors: alpha-1-antitrypsin, alpha-1-antichymotrypsin and plasma serine protease inhibitor. Altered levels of 13 proteins were associated with the LPR, including increased factor XIII A and decreased von Willebrand factor. No relationship was found between the LPR and changes in the proportions of classical, intermediate, and non-classical monocytes. CONCLUSIONS: Allergic reactions to inhaled allergens in asthmatic subjects were associated with changes in a large proportion of the measured plasma proteome, whereof protease inhibitors showed the largest changes, likely to influence the inflammatory response. Many of the proteins altered in relation to the LPR are associated with coagulation, highlighting potential mechanistic targets for future treatments of type-2 asthma.

CD105+CD90+CD13+ identifies a clonogenic subset of adventitial lung fibroblasts.

Mesenchymal cells are important components of specified niches in the lung, and can mediate a wide range of processes including tissue regeneration and repair. Dysregulation of these processes can lead to improper remodeling of tissue as observed in several lung diseases. The mesenchymal cells responsible remain poorly described, partially due to the heterogenic nature of the mesenchymal compartment and the absence of appropriate markers. Here, we describe that CD105+CD90+ mesenchymal cells can be divided into two populations based on their expression of CD13/aminopeptidase N (CD105+CD90+CD13- and CD105+CD90+CD13+). By prospective isolation using FACS, we show that both these populations give rise to clonogenic fibroblast-like cells, but with an increased clonogenic and proliferative capacity of CD105+CD90+CD13+ cells. Transcriptomic and spatial analysis pinpoints an adventitial fibroblast subset as the origin of CD105+CD90+CD13+ clonogenic mesenchymal cells in human lung.

Harnessing the ECM Microenvironment to Ameliorate Mesenchymal Stromal Cell-Based Therapy in Chronic Lung Diseases.

It is known that the cell environment such as biomechanical properties and extracellular matrix (ECM) composition dictate cell behaviour including migration, proliferation, and differentiation. Important constituents of the microenvironment, including ECM molecules such as proteoglycans and glycosaminoglycans (GAGs), determine events in both embryogenesis and repair of the adult lung. Mesenchymal stromal/stem cells (MSC) have been shown to have immunomodulatory properties and may be potent actors regulating tissue remodelling and regenerative cell responses upon lung injury. Using MSC in cell-based therapy holds promise for treatment of chronic lung diseases such as idiopathic pulmonary fibrosis (IPF) and chronic obstructive pulmonary disease (COPD). However, so far clinical trials with MSCs in COPD have not had a significant impact on disease amelioration nor on IPF, where low cell survival rate and pulmonary retention time are major hurdles to overcome. Research shows that the microenvironment has a profound impact on transplanted MSCs. In our studies on acellular lung tissue slices (lung scaffolds) from IPF patients versus healthy individuals, we see a profound effect on cellular activity, where healthy cells cultured in diseased lung scaffolds adapt and produce proteins further promoting a diseased environment, whereas cells on healthy scaffolds sustain a healthy proteomic profile. Therefore, modulating the environmental context for cell-based therapy may be a potent way to improve treatment using MSCs. In this review, we will describe the importance of the microenvironment for cell-based therapy in chronic lung diseases, how MSC-ECM interactions can affect therapeutic output and describe current progress in the field of cell-based therapy.