COPD is a chronic lung disease that affects millions of people, declining their lung function and impairing their life quality. Despite years of research and drug approvals, we are still not capable of halting progression or restoring normal lung function. Mesenchymal stem cells (MSC) are cells with extraordinary repair capacity, and MSC-based therapy brings future hope for COPD treatment, although the best source and route of administration are unclear. MSC from adipose tissue (AD-MSC) represents an option for autologous treatment; however, they could be less effective than donor MSC. We compared in vitro behavior of AD-MSC from COPD and non-COPD individuals by migration/proliferation assay, and tested their therapeutic potential in an elastase mouse model. In addition, we tested intravenous versus intratracheal routes, inoculating umbilical cord (UC) MSC and analyzed molecular changes by protein array. Although COPD AD-MSC have impaired migratory response to VEGF and cigarette smoke, they were as efficient as non-COPD in reducing elastase-induced lung emphysema. UC-MSC reduced lung emphysema regardless of the administration route and modified the inflammatory profile in elastase-treated mice. Our data demonstrate equal therapeutic potential of AD-MSC from COPD and non-COPD subjects in the pre-clinical model, thus supporting their autologous use in disease.

Mesenchymal stem cells (MSC) play a role in tissue repair. However, the role of lung MSC (LMSC) in idiopathic pulmonary fibrosis (IPF) remains to be elucidated. Preliminary results revealed that IPF vs non-IPF LMSC present an impaired repair ability and, under TGF-β stimulus, an increased migration activity and decreased proliferation. Moreover, microarray gene expression analysis showed that oxidative phosphorylation is the most altered pathway in the IPF LMSC. Here, we aimed to analyse mitochondrial function-related features, the repair activity in pro-fibrotic conditions, and the underlying molecular mechanisms elicited by TGF-β in IPF LMSC. RT-PCR was used to validate gene expression differences. Mitochondria number was estimated by DNA quantification using ddPCR. Mitochondrial integrity was assessed by TMRM assay. Scratch assays were performed in both direct and indirect coculture with A549 cells. As compared to non-IPF MSC, IPF LMSC presented a lower COX-IV and PINK1 expression and there were no differences in mitochondria number and integrity. However, TGF-β decreased mitochondrial integrity in IPF LMSC. IPF LMSC showed a delayed repair activity and, in the indirect coculture, TGF-β preincubation stimulated wound healing more potently in the IPF group. TGF-β-treated IPF LMSC presented lower COX-IV, ATP6 and PINK1 mRNA levels than non-IPF LMSC. TGF-β treatment induced the expression of the migration marker VEGF. The impaired repair activity in IPF LSMC was associated with signs of mitochondrial dysfunction. IPF LMSC seem to be more sensitive to TGF-β. TGF-β, in turn, induces mitochondrial dysfunction in IPF MSC. Collectively, these results suggest that the overproduction of TGF-β in IPF may alter LMSC function by promoting mitochondrial dysfunction.

Introduction: Mesenchymal stem cells (MSCs) are multipotent cells with an impressive potential for regeneration and immunomodulation. The niche of resident MSCs in the lung is believed to play an important role in pulmonary disease; however, most of the studies are focused on MSC from other origin as bone marrow. Here, we performed a comprehensive study of the characteristics of lung MSCs in both donor and in idiopathic pulmonary fibrosis (IPF) patients. Results and methods: MSCs were isolated from donor and IPF human lung tissue and mesenchymality was confirmed by flow cytometry (Stemflow hMSC Analysis) and Rohart MSC in silico test. The differentiation capacity was assessed using the Human MSC Functional Identification kit and both conditions were able to differentiate into osteoblasts and adipocytes after 21 days, although less potential was observed in IPF lung MSCs for the last one. Moreover, functional analyses were performed for migration and proliferation by real-time monitoring cell assay. Notably, profibrotic stimulation by TGF-ß induced a significant change by increasing migration and decreasing proliferation in the IPF-lung MSCs when compared to donor. Furthermore, the regenerative ability to close an epithelial wound was significantly reduced in lung MSCs from IPF patients as tested by scratch assay in indirect and direct co-culture. Conclusions: Lung MSCs were successfully isolated from both donor and IPF and could differentiate into other cell types. However, IPF lung MSCs displayed a decreased proliferation and an increased migration profile in response to fibrotic stimulus possibly due to an increased sensibility to profibrotic factors. Moreover, IPF lung MSCs had reduced regenerative potential which might be involved in the impaired repair in IPF pathogenesis.

Method: Resident MSC confer regenerative potential in the diseased lung. Additional application of high numbers of MSC increased this natural regenerative effect, thus highlighting MSC as a potential therapeutic tool in respiratory diseases, however, there is controversy concerning the best administration routine and the appropriate tissue-related source. Recently, MSC in the 3D form of biospheres were found to provide a healthy microenvironment and better maintain cell integrity, increasing the amount of shedded vesicles, thus enhancing the secretion of miRNA and regenerative factors. Results & Methods: We have successfully formed intact BS that are viable over time as monitored by microscopy with Ki67, PDGFR and CD90 expression. Also, an in vitro wound-healing assay demonstrated an increased potential of the BS (3D) vs the cell-suspension (2D) to regenerate the damaged lung epithelium. In addition, pre-staining DON-MSC that intratracheally inoculated BS remained longer in the murine lungs compared to cell-suspension. This supports that administration of MSC in form of BS leads to an increased MSC-survival in the lung, conceivably increasing their potential of a repair and regeneration on damaged lung tissue. Conclusions: The cultivation and subsequent administration of DON-MSC in the form of BS provide a superior cell organization that promotes the regeneration of a lung epithelial wound, furthermore, MSC engrafment was prolonged in the murine lung when applied as BS. Therefore, BS of DON-MSC represent a plausible innovative tool for the treatment of chronic lung diseases like Idiopathic Pulmonary Fibrosis.

BACKGROUND:Bone marrow (BM) produces hematopoietic and progenitor cells that contribute to distant organ inflammation and repair. Chronic obstructive pulmonary disease (COPD) is characterized by defective lung repair. Yet, BM composition has not been previously characterized in COPD patients.METHODS:In this prospective and controlled study, BM was obtained by sternum fine-needle aspiration in 35 COPD patients and 25 healthy controls (10 smokers and 15 never-smokers). BM cell count and immunophenotype were determined by microscopy and flow cytometry, respectively. Circulating inflammatory (C-reactive protein, IL-6, IL-8) and repair markers (HGF, IGF, TGF-β, VEGF) were quantified by ELISA. Results were integrated by multi-level network correlation analysis.RESULTS:We found that: (1) there were no major significant pair wise differences between COPD patients and controls in the BM structural characteristics; (2) multi-level network analysis including patients and controls identifies a relation between immunity, repair and lung function not previously described, that remains in the COPD network but is absent in controls; and (3) this novel network identifies eosinophils as a potential mediator relating immunity and repair, particularly in patients with emphysema.CONCLUSIONS:Overall, these results suggest that BM is activated in COPD with impaired repair capacity in patients with more emphysema and/or higher circulating eosinophils.

Introduction: Peripheral eosinophilia in COPD, defined as an eosinophil count ≥300 cel/microL, is associated with different clinical outcomes and treatment response. Bone marrow (BM) produces progenitor cells responsible for lung repair and hematopoietic cells, including eosinophils. Aims and Objectives: To study the cellular and functional characteristics of BM in patients with COPD and peripheral eosinophilia and its relationship with lung function, Th1 inflammation and repair parameters. Methods: Consecutive patients with COPD were divided into eosinophilic COPD (EO-COPD) or non-eosinophilic COPD (NE-COPD) depending on the cell count greater or less than 300 eosinophils/microL in peripheral blood (pb). Fine-needle BM aspiration was performed. Cellular characteristics were determined by microscopy, cell immunophenotype by flow cytometry, Th1 inflammation (IL-6, IL-8, TNF-alpha) and repair markers (HGF, IGF, TGF-beta, VEGF) by ELISA. These variables were correlated with pulmonary function, inflammatory parameters and repair markers in sputum and BM. Results: We studied 35 COPD patients, 21 NE-COPD and 14 EO-COPD. The EO-COPD group had worse FEV1 and DLCO. There were no differences in Th1 inflammatory markers. BM showed no difference in eosinophil count but a lower percentage of plasma cells and CD34+ckit precursor cells were observed. Statistically significant negative correlations were found between the number of blood eosinophils and FEV1, DLCO, HGF pb and HGF BM. Conclusions: A higher blood eosinophil count is correlated with worse lung function. A worse repair capacity may contribute to explain the worse lung function of these patients.

BACKGROUND:Chronic obstructive pulmonary disease (COPD) is an inflammatory disorder partially resistant to glucocorticoids. A reduced histone deacetylase (HDAC) activity has been proposed to explain this resistance. Haemophilus influenzae frequently colonizes the airways of COPD patients, where it enhances inflammation. The effects of Haemophilus influenzae on HDAC activity have not been investigated before.METHODS:The effects of the presence or absence of Haemophilus influenzae ex-vivo and in vitro were studied. To this end, we determined: (1) cytokine release in alveolar macrophages (AM) from 7 patients with COPD, 5 healthy smokers, 6 healthy non-smokers and (2) HDAC activity, nuclear factor kappa B (NF-κB) activation in a macrophage-like cell line (PMA-transformed U937 cells) co-cultured with epithelial cells. Experiments were repeated with dexamethasone (1 μM) and/or the HDAC enhancer theophylline (10 μM).RESULTS:Haemophilus influenzae induced a steroid-resistant inflammatory response in AM from COPD and controls and decreased HDAC activity, activated NF-κB and induced the secretion of several cytokines (IL-6, IL-8, IL-1β, IL-10 and TNF-α) (p < 0.001 for all comparisons) in the macrophage-like cell line. Dexamethasone reduced NF-κB activation but it did not modify HDAC activity. The addition of theophylline to dexamethasone increased HDAC activity and suppressed cytokine release completely, without modifying NF-κB activation.CONCLUSIONS:These results indicate that Haemophilus influenzae reduces HDAC activity and induces a NF-κB mediated inflammatory response that is only partially suppressed by glucocorticoids irrespective of having COPD. Yet, the latter can be fully restored by targeting HDAC activity.

Background: The adaptive immune response contributes to the pathogenesis of Chronic Obstructive Pulmonary Disease (COPD). One manifestation of adaptive immunity in COPD is the presence of pulmonary lymphoid follicles (LFs) containing B-cells. Aims: B-cell activating factor of TNF family (BAFF) regulates B-cell function in health. Increases in BAFF levels promote autoimmune responses, but BAFF9s role in COPD pathogenesis is unclear. Our goal was to fill this knowledge gap. Methods: 150 subjects were studied: COPD patients (GOLD stages I–IV], healthy smokers (SC) and non-smokers (NSC). We quantified and correlated LF number and size with the number of BAFF-positive and apoptotic B-cells in LFs in COPD vs. control lungs. We tested whether: 1) cigarette smoke extract (CSE) induces B-cell apoptosis which is attenuated by treating cells with recombinant BAFF (rBAFF); and 2) rBAFF inhibits CSE-induced B-cell apoptosis by regulating the NF-κB pathway. Results: GOLD stage IV COPD patients had increased numbers and larger pulmonary LFs than GOLD stages I-II COPD patients and SC. We identified two main types of LFs: 1) type A , predominant in GOLD stage I-II COPD and SC lungs, containing abundant A poptotic but few BAFF-positive cells; and 2) type B , mainly in GOLD stage IV COPD lungs, bigger in size than Type A, and containing abundant B AFF-positive cells but few apoptotic cells. rBAFF blocked CSE-induced B-cell apoptosis by inhibiting CS-induced NF-κB activation. Conclusion: We associate BAFF with the formation and expansion of pulmonary LFs in the severe stages of COPD. Thus, we implicate BAFF in a self-perpetuating loop that may contribute to COPD progression by promoting lung B-cell survival and LF expansion.

Introduction: Mesenchymal Stem Cells (MSCs) have been shown to contribute to pulmonary repair and regeneration in lung injury models. However, it is still unknown whether MSCs from patients with chronic diseases have the same regenerative / reparative capacity than MSCs from healthy subjects (H). Aims: To study the functional and molecular response of bone marrow (BM)-MSCs and adipose derived (AD)-MSCs from patients with chronic obstructive pulmonary disease (COPD), idiopathic pulmonary fibrosis (IPF) and H. Methods: We have evaluated: 1) the cellular response of MSCs to VEGFs, PDFGs, tobacco; and, 2) the expression level of some of the associated growth factor receptors (GFR). MSCs were isolated from sternal aspirated BM samples and subcutaneous fat from subjects undergoing thoracic surgery. MSC functional response was real time monitored with the xCELLigence system. The amount of GFR was analyzed by RT-PCR and with LI-COR's Odyssey system. Tyrosine phosphorylation was checked by Western Blot. Results: 1) MSCs from different origins respond to VEGF121, VEGF165, PDGFAA and PDFGBB indistinctive ways; 2) PDGFBB elicits the greatest response in tyrosine phosphorylation; 3) BM-COPD cells and ADSC-IPF cells have a significantly higher PDGFR beta expression than cells from H; and, 4) There seems to be a gradient response to tobacco stimulation, being MSC-COPD cells more sensitive than cells from other origins. Conclusions: MSC-COPD and ADSC-IPF exhibit molecular and functional differences. Additional evaluation of these cells is needed to elucidate their potential involvement in the pathogenesis of these diseases. Supported by PI10-00983, PI12-01152, SEPAR 2011 and 2013.

Introduction : Chronic Obstructive Pulmonary Disease (COPD) is a major health problem. Bone marrow (BM)-derived MSCs have been shown to contribute to pulmonary repair and regeneration in experimental models. However, their role in COPD is unclear. Since COPD is also characterized by systemic inflammation, we hypothesize that BM-MSCs functional capacity and/or regenerative capability can be altered in these patients. Aims : To study the functional capacity of BM-MSCs from COPD patients (BM-COPD) compared to BM-MSCs from controls (BM-C). We have evaluated: 1) the cellular response of MSCs to both tobacco smoke (TS) and VEGF; and, 2) their differentiation capacity. Methods : MSCs were isolated from BM of sternum from subjects who underwent thoracic surgery. After treatments, MSCs were monitored with the xCELLigence system. This system measures real time changes in cellular impedance providing a value named cell index (CI). The differentiation protocol and cellular staining was carried out following RD 2) Both COPD and C cells react to VEGF also in a dose/time dependent manner, but at the higher doses and the longer times, BM-COPD are 4 times less sensitive than BM-C; and, 3) There is also a clear differentiation deficiency in BM-COPD, at least to adipocytes. Conclusions : BM-COPD have abnormal functional capacity compared with BM-C. However, whether BM-MSCs can be involved in the pathogenesis of COPD should be evaluated in further studies. Supported by FIS 10/00983 and SEPAR 2011.

BACKGROUND:Chronic obstructive pulmonary disease (COPD) is characterized by an enhanced and persistent innate and acquired immune response to tobacco smoking. Myeloid-derived suppressor cells (MDSCs) modulate T-cell responses by down-modulating the T cell receptor ζ chain (TCR ζ) through the catabolism of l-arginine. The effects of smoking on MDSCs and their potential participation in COPD immunopathogenesis have not been explored so far.METHODS:To investigate it, we compared the level of circulating Lineage-/HLA-DR-/CD33+/CD11b+ MDSCs, the serum concentration of arginase I (ARG I) and the expression of peripheral T-cell receptor ζ chain (TCR ζ) in never smokers, smokers with normal spirometry and COPD patients. Flow cytometry was used to quantify circulating MDSCs and TCR ζ expression. Serum ARG I levels were determined by ELISA.RESULTS:The main findings of this study were that: (1) current smoking upregulates and activates circulating MDSCs both in smoker controls and COPD patients; and, (2) at variance with the smokers with normal spirometry, in patients with COPD this effect persists after quitting smoking and is accompanied by a significant and specific down-regulation of the TCR ζ chain expression in circulating T lymphocytes.CONCLUSION:Smoking modulates circulating MDSCs. Their regulation appears altered in patients with COPD.

Introduction: Chronic obstructive pulmonary disease (COPD) is characterized by an abnormal inflammatory response of the lungs to noxious particles or gases where both innate and adaptive immunity participate. Polymorphonuclear neutrophils (PMNs) play a key role in the pathogenesis of COPD due to the production of reactive oxygen species and lytic enzymes. Recent evidence shows that PMNs can express MHC class II and activate an adaptive immune response. Material and methods: The expression of HLA-DR (MHC class II) and CD 11b, as a marker of activation, were quantified on peripheral PMNs by flow cytometry in: a) 31 non-smokers controls (NSC) (mean±SD, 60 ±8y., FEV1% pred= 112 ± 14,6); b) 34 smokers with normal lung function (SM) (57±9y., FEV1% pred= 102±16, 30±18 paq/y); and c) 53 COPD patients (64±10y, FEV1% pred=61±20, 54±30 paq/y). Results: The percentage of HLA-DR+PMNs was higher in COPD patients (median [range]: 12.7[1.7-78.3%]) than in NSC (7.6 [1.7-23.8], p=0.003). COPD current smokers showed an increased proportion of HLA-DR+PMNs (14.7 [3.7-78.3]) as compared with current smokers with normal spirometry (10 [2.4-20.6], p=0.02) and COPD former smokers (11.2 [1.7-23.2], p=0.009). The correlation between the expression of CD 11b and HLA-DR was significant (r=0.785, p= Conclusions: The HLA-DR expression on circulating PMNs of COPD patients suggests the participation of activated PMNs in the modulation of the adaptive immune response in COPD.