Unveiling the Cutting-Edge Impact of Polarized Macrophage-Derived Extracellular Vesicles and MiRNA Signatures on TGF-ß Regulation within Lung Fibroblasts.

Depending on local cues, macrophages can polarize into classically activated (M1) or alternatively activated (M2) phenotypes. This study investigates the impact of polarized macrophage-derived Extracellular Vesicles (EVs) (M1 and M2) and their cargo of miRNA-19a-3p and miRNA-425-5p on TGF-ß production in lung fibroblasts. EVs were isolated from supernatants of M0, M1, and M2 macrophages and quantified using nanoscale flow cytometry prior to fibroblast stimulation. The concentration of TGF-ß in fibroblast supernatants was measured using ELISA assays. The expression levels of miRNA-19a-3p and miRNA-425-5p were assessed via TaqMan-qPCR. TGF-ß production after stimulation with M0-derived EVs and with M1-derived EVs increased significantly compared to untreated fibroblasts. miRNA-425-5p, but not miRNA-19a-3p, was significantly upregulated in M2-derived EVs compared to M0- and M1-derived EVs. This study demonstrates that EVs derived from both M0 and M1 polarized macrophages induce the production of TGF-ß in fibroblasts, with potential regulation by miRNA-425-5p.

Do Circulating Extracellular Vesicles Strictly Reflect Bronchoalveolar Lavage Extracellular Vesicles in COPD?

Cell-derived extracellular vesicles (EVs) found in the circulation and body fluids contain biomolecules that could be used as biomarkers for lung and other diseases. EVs from bronchoalveolar lavage (BAL) might be more informative of lung abnormalities than EVs from blood, where information might be diluted. To compare EVs' characteristics in BAL and blood in smokers with and without COPD. Same-day BAL and blood samples were obtained in 9 nonsmokers (NS), 11 smokers w/o COPD (S), and 9 with COPD (SCOPD) (FEV1: 59 ± 3% pred). After differential centrifugation, EVs (200-500 nm diameter) were identified by flow cytometry and labeled with cell-type specific antigens: CD14 for macrophage-derived EVs, CD326 for epithelial-derived EVs, CD146 for endothelial-derived EVs, and CD62E for activated-endothelial-derived EVs. In BAL, CD14-EVs were increased in S compared to NS [384 (56-567) vs. 172 (115-282) events/µL; p = 0.007] and further increased in SCOPD [619 (224-888)] compared to both S (p = 0.04) and NS (p < 0.001). CD326-EVs were increased in S [760 (48-2856) events/µL, p < 0.001] and in SCOPD [1055 (194-11,491), p < 0.001] when compared to NS [15 (0-68)]. CD146-EVs and CD62E-EVs were similar in the three groups. In BAL, significant differences in macrophage and epithelial-derived EVs can be clearly detected between NS, S and SCOPD, while these differences were not found in plasma. This suggests that BAL is a better medium than blood to study EVs in lung diseases.

The Emerging Role of Extracellular Vesicles Detected in Different Biological Fluids in COPD.

The pathogenesis of chronic obstructive pulmonary disease (COPD) is characterized by complex cellular and molecular mechanisms, not fully elucidated so far. It involves inflammatory cells (monocytes/macrophages, neutrophils, lymphocytes), cytokines, chemokines and, probably, new players yet to be clearly identified and described. Chronic local and systemic inflammation, lung aging and cellular senescence are key pathological events in COPD development and progression over time. Extracellular vesicles (EVs), released by virtually all cells both as microvesicles and exosomes into different biological fluids, are involved in intercellular communication and, therefore, represent intriguing players in pathobiological mechanisms (including those characterizing aging and chronic diseases); moreover, the role of EVs as biomarkers in different diseases, including COPD, is rapidly gaining recognition. In this review, after recalling the essential steps of COPD pathogenesis, we summarize the current evidence on the roles of EVs collected in different biological mediums as biomarkers in COPD and as potential players in the specific mechanisms leading to disease development. We will also briefly review the data on EV as potential therapeutic targets and potential therapeutic agents.

Microvesicles in bronchoalveolar lavage as a potential biomarker of COPD.

Microvesicles (MVs) released from almost all cells are recognized as cell communication tools. MVs have been investigated in several inflammatory diseases but poorly in biological fluids like bronchoalveolar lavage (BAL) of smokers. The purpose of this study was to investigate the presence and source of MVs in BAL of smokers with and without chronic obstructive pulmonary disease (COPD) compared with nonsmoking controls. Using flow cytometry in BAL, we detected endothelial and alveolar macrophage (AM)-derived MVs and found a higher number of AM-MVs in the BAL of smokers with COPD than in smokers without COPD and nonsmokers, which correlated with the pack-years (r = 0.46; P = 0.05) and with the degree of airway obstruction measured by the forced expiratory volume in 1 s percent predicted (r = -0.56; P = 0.01). Endothelial and alveolar macrophage-derived MVs are present and measurable in human BAL fluid. In response to smoking and to the development of COPD, inflammatory signals in AM-derived MVs can be quantified, and their numbers are related to the pack-years and the decrease in lung function. These results open the opportunity for future investigation of these microvesicles as biomarkers and possible mechanistic guides in COPD.

Prognostic role of MUC5B rs35705950 genotype in patients with idiopathic pulmonary fibrosis (IPF) on antifibrotic treatment.

BACKGROUND: A common variant located in the promoter region of MUC5B (rs35705950) is the strongest risk factor for sporadic and familiar IPF, as well as a predictor of outcome. However, there are no data on the effect of MUC5B rs35705950 genotype on the prognosis of IPF patients on antifibrotic treatment. The aim of this study is to determine, in a phenotypically well-characterized population of patients with IPF treated with antifibrotics, the impact of MUC5B rs35705950 genotype on disease progression and survival. METHODS: 88 IPF patients on antifibrotic treatment were followed-up from 2014 until transplantation, death or end of follow-up (December 2019). Disease progression was defined as a forced vital capacity (FVC) loss ≥ 5% per year. All patients were genotyped for MUC5B rs35705950 by PCR amplification and Sanger sequencing. RESULTS: Out of 88 patients, 61 (69%) carried the mutant T allele (TT or TG) and 27 (31%) did not (GG). Carriage of the MUC5B rs35705950 T allele was not associated with a faster decline in FVC. Conversely, at the end of the follow-up, overall survival in carriers of the TT/TG genotype was longer compared to that of the GG genotype carriers. FVC (L) at baseline and time to respiratory failure at rest were independent predictors of worse prognosis. CONCLUSIONS: In IPF patients on antifibrotic treatment, carriage of the MUC5B rs35705950 T allele is associated with longer survival, highlighting the usefulness of MUC5B genetic data in clinical decision making.

Low-Blood Lymphocyte Number and Lymphocyte Decline as Key Factors in COPD Outcomes: A Longitudinal Cohort Study.

BACKGROUND: Smokers with and without chronic obstructive pulmonary disease (COPD) are at risk of severe outcomes like exacerbations, cancer, respiratory failure, and decreased survival. The mechanisms for these outcomes are unclear; however, there is evidence that blood lymphocytes (BL) number might play a role. OBJECTIVE: The objective of this study is to investigate the relationship between BL and their possible decline over time with long-term outcomes in smokers with and without COPD. METHODS: In 511 smokers, 302 with COPD (COPD) and 209 without COPD (noCOPD), followed long term, we investigated whether BL number and BL decline over time might be associated with long-term outcomes. Smokers were divided according to BL number in high-BL (≥1,800 cells/µL) and low-BL (<1,800 cells/µL). Clinical features, cancer incidence, and mortality were recorded during follow-up. BL count in multiple samples and BL decline over time were calculated and related to outcomes. RESULTS: BL count was lower in COPD (1,880 cells/µL) than noCOPD (2,300 cells/µL; p < 0.001). 43% of COPD and 23% of noCOPD had low-BL count (p < 0.001). BL decline over time was higher in COPD than noCOPD (p = 0.040). 22.5% of the whole cohort developed cancer which incidence was higher in low-BL subjects and in BL decliners than high-BL (31 vs. 18%; p = 0.001) and no decliners (32 vs. 19%; p = 0.002). 26% in the cohort died during follow-up. Furthermore, low-BL count, BL decline, and age were independent risk factors for mortality by Cox regression analysis. CONCLUSION: BL count and BL decline are related to worse outcomes in smokers with and without COPD, which suggests that BL count and decline might play a mechanistic role in outcomes deterioration. Insights into mechanisms inducing the fall in BL count could improve the understanding of COPD pathogenesis and point toward new therapeutic measures.

Critical Review of the Evolution of Extracellular Vesicles' Knowledge: From 1946 to Today.

Extracellular vesicles (EVs) are a family of particles/vesicles present in blood and body fluids, composed of phospholipid bilayers that carry a variety of molecules that can mediate cell communication, modulating crucial cell processes such as homeostasis, induction/dampening of inflammation, and promotion of repair. Their existence, initially suspected in 1946 and confirmed in 1967, spurred a sharp increase in the number of scientific publications. Paradoxically, the increasing interest for EV content and function progressively reduced the relevance for a precise nomenclature in classifying EVs, therefore leading to a confusing scientific production. The aim of this review was to analyze the evolution of the progress in the knowledge and definition of EVs over the years, with an overview of the methodologies used for the identification of the vesicles, their cell of origin, and the detection of their cargo. The MISEV 2018 guidelines for the proper recognition nomenclature and ways to study EVs are summarized. The review finishes with a "more questions than answers" chapter, in which some of the problems we still face to fully understand the EV function and potential as a diagnostic and therapeutic tool are analyzed.

Innate lymphoid cells in isocyanate-induced asthma: role of microRNA-155.

BACKGROUND: Occupational asthma, induced by workplace exposures to low molecular weight agents such as toluene 2,4-diisocyanate (TDI), causes a significant burden to patients and society. Little is known about innate lymphoid cells (ILCs) in TDI-induced asthma. A critical regulator of ILC function is microRNA-155, a microRNA associated with asthma. OBJECTIVE: To determine whether TDI exposure modifies the number of ILCs in the lung and whether microRNA-155 contributes to TDI-induced airway inflammation and hyperresponsiveness. METHODS: C57BL/6 wild-type and microRNA-155 knockout mice were sensitised and challenged with TDI or vehicle. Intracellular cytokine expression in ILCs and T-cells was evaluated in bronchoalveolar lavage (BAL) fluid using flow cytometry. Peribronchial eosinophilia and goblet cells were evaluated on lung tissue, and airway hyperresponsiveness was measured using the forced oscillation technique. Putative type 2 ILCs (ILC2) were identified in bronchial biopsies of subjects with TDI-induced occupational asthma using immunohistochemistry. Human bronchial epithelial cells were exposed to TDI or vehicle. RESULTS: TDI-exposed mice had higher numbers of airway goblet cells, BAL eosinophils, CD4+ T-cells and ILCs, with a predominant type 2 response, and tended to have airway hyperresponsiveness. In TDI-exposed microRNA-155 knockout mice, inflammation and airway hyperresponsiveness were attenuated. TDI exposure induced IL-33 expression in human bronchial epithelial cells and in murine lungs, which was microRNA-155 dependent in mice. GATA3+CD3- cells, presumably ILC2, were present in bronchial biopsies. CONCLUSION: TDI exposure is associated with increased numbers of ILCs. The proinflammatory microRNA-155 is crucial in a murine model of TDI asthma, suggesting its involvement in the pathogenesis of occupational asthma due to low molecular weight agents.

SYMPTOMATIC AND ASYMPTOMATIC SMOKERS WITHOUT AIRFLOW OBSTRUCTION: A NEW CLINICAL ENTITY?

Non-obstructed ever-smokers, with or without symptoms, have generated a great deal of information recently, but few reviews. Even individuals with normal spirometry can present changes in sputum with inflammatory biomarkers (cellular and molecular) and airways and parenchyma with remodeling; when symptomatic (cough, sputum, wheezing, and dyspnea) exacerbations are frequent affecting the individuals' quality of life, there is an increased use of health resources: more medication, emergency visits, and hospital admissions. Non-obstructed smokers may have exercise limitations, increased lung volumes, low diffusion capacity, air entrapment, peripheral airways obstruction, elevated airways resistance, and abnormal multiple breath nitrogen washout, as well as abnormalities in computed tomography studies, such as airway wall thickening, emphysema, or interstitial lung abnormalities. Quitting smoking comprises a first, inexpensive, and often abandoned intervention to arrest respiratory impairment. It is controversial whether or not this population should be treated with other medications. Further studies should be conducted to elucidate the consequences of follow-up and prognosis in this clinical entity.

Clinical and Pathologic Factors Predicting Future Asthma in Wheezing Children. A Longitudinal Study.

Wheeze is a common symptom in infants, but not all wheezers develop asthma. Indeed, up to 50% of wheezing children outgrow their symptoms by school age. How to predict if early wheeze will become asthma is still a matter of vivid debate. In this work, we sought to assess the clinical and pathological factors that might predict the future development of asthma in children. Eighty children (mean age 3.8 ± 1 yr) who underwent a clinically indicated bronchoscopy were followed prospectively for a median of 5 years. At baseline, clinical characteristics with a particular focus on wheezing and its presentation (episodic or multitrigger) were collected, and structural and inflammatory changes were quantified in bronchial biopsies. Follow-up data were available for 74 of the 80 children. Children who presented with multitrigger wheeze were more likely to have asthma at follow-up than those with episodic wheeze (P = 0.04) or without wheeze (P < 0.0001). Children with asthma also had lower birth weights (P = 0.02), a lower prevalence of breastfeeding (P = 0.02), and a trend for increased IgE (P = 0.07) at baseline than those with no asthma. Basement membrane thickness and airway eosinophils at baseline were increased in children who developed asthma at follow-up (P = 0.001 and P = 0.026, respectively). Multivariate analysis showed that among all clinical and pathological factors, multitrigger wheezing, basement membrane thickening, and reduced birth weight were predictive of future asthma development. We conclude that multitrigger wheeze and reduced birth weight are clinical predictors of asthma development. Basement membrane thickening in early childhood is closely associated with asthma development, highlighting the importance of airway remodeling in early life as a risk factor for future asthma.

Dual polarization of human alveolar macrophages progressively increases with smoking and COPD severity.

BACKGROUND: It is known that tissue macrophages derive not only from blood monocytes but also from yolk sac or fetal liver, and the tissue of residence guides their function. When isolated, they lose tissue specific signatures, hence studies of human macrophages should be ideally done directly in the tissue. The aim of this study was to investigate directly in human lung tissue the polarization of alveolar macrophage (AM), classic (M1) or alternative (M2), in health and disease, using COPD as a model. METHODS: Surgical lungs from 53 subjects were studied: 36 smokers whose FEV1 varied from normal to severe COPD, 11 non-smokers and 6 normal donors. iNOS and CD206 immunohistochemistry was used to quantify the percentage of AM polarized as M1 or M2 in lung sections. RESULTS AND DISCUSSION: The percentage of M1 and M2 increased progressively with smoking and COPD severity, from 26% to 84% for M1 and from 7% to 78% for M2. In donors 74% of AM were negative for M1 and 93% for M2. Confocal microscopy showed co-localization of M1 and M2 in the same AM in severe COPD. CONCLUSION: In normal lungs alveolar macrophages were mostly non-polarized. With smoking and COPD severity, M1 and M2 polarization increased significantly and so did the co-expression of M1 and M2 in the same alveolar macrophage.

Alpha-1 Antitrypsin Deficiency Today: New Insights in the Immunological Pathways.

More than 50 years ago, the observation that absence of the α1 band from protein electrophoresis is associated with severe emphysema established the link between α1-antitrypsin deficiency (AATD) and lung damage. From this discovery, the classic paradigm of protease/antiprotease imbalance was derived, linking lung destruction in patients with AATD to the unopposed effect of proteases. By extension, this paradigm was also applied to patients with 'common' chronic obstructive pulmonary disease, in whom large increases in smoke-induced proteases could overwhelm the antiprotease capability of AAT. However, it has become increasingly evident that AAT has important anti-inflammatory and immunoregulatory activities which, beside its antiprotease function, may be critically involved in lung destruction. From this perspective, we will consider recent evidence, based on epidemiological, clinical and immunopathological studies, suggesting that it is time to move on from the original protease/antiprotease paradigm toward a more complex view of the condition, which embraces its immunomodulating functions. Of importance, the potent immunoregulatory, tolerogenic role of AAT may support its therapeutic use in a number of diseases other than AATD, particularly in immune-related disorders.

Immune activation in α1-antitrypsin-deficiency emphysema. Beyond the protease-antiprotease paradigm.

RATIONALE: α1-Antitrypsin (AAT) is a potent protease inhibitor, deficiency of which is associated with the presence of emphysema. An imbalance of elastase and antielastase, along with innate inflammation in the lung, is believed to cause lung destruction in α1-antitrypsin deficiency (AATD). It is now apparent that AAT has important immune-regulatory roles that would be lost in AATD, yet adaptive immune responses in the lung have not been investigated in patients with AATD. OBJECTIVES: To assess the adaptive immune response in severe AATD emphysema and compare it with that present in "usual" chronic obstructive pulmonary disease (COPD). METHODS: The immune inflammatory response in explanted lungs from 10 subjects with AATD was characterized and quantified, and the results were compared with those of 26 subjects with usual COPD and those of 17 smoking and 11 nonsmoking control subjects with normal lung function. MEASUREMENTS AND MAIN RESULTS: Lymphoid follicles (LFs) in AATD and usual COPD were markedly increased when compared with control groups. Molecular analysis of B lymphocytes in LFs showed predominantly mono/oligoclonality. LF number correlated negatively with FEV1/FVC. B lymphocytes and CD4(+) and CD8(+) T lymphocytes were significantly increased in AATD and usual COPD when compared with control groups. IL-32, an important cytokine in induction of autoimmunity, was markedly up-regulated in AATD and usual COPD. CONCLUSIONS: An important adaptive immune inflammation, comprising B, CD4(+), and CD8(+) lymphocytes, and LFs, is a prominent feature in AATD. These results change the paradigm of the mechanism of AATD-induced emphysema from a pure elastase-antielastase imbalance to a much more complex one involving the adaptive immune system, similarly to what occurs in usual COPD.

Is chronic obstructive pulmonary disease a disease of aging?

Chronic obstructive pulmonary disease (COPD) is a disease that usually presents clinically at an advanced age, after years of smoking cigarettes. It is usually believed that aging and its biological consequences are important mechanisms in the disease pathogenesis. This concept has maintained the focus of studies on COPD in old-age individuals. Here we analyze the possible role of aging from a different point of view and introduce different concepts that might be considered useful additions to the understanding of the disease. Essentially, we propose and show evidence that COPD is a disease of the young susceptible smoker that progresses over time and manifests in older age because we live longer and not so much because of the effect of aging itself; we examine the concept of cell senescence, the basis of tissue aging, and how stressors like the ones produced by smoking can accelerate cell senescence with all of its untoward consequences in COPD. We thus finally suggest that COPD might accelerate aging rather than be a consequence of it. In conclusion, we suggest that COPD could be considered a disease of the predisposed young individual that manifests clinically in old age because we live longer, with all of its consequences.

Suppressor of cytokine signaling-3 expression and its regulation in relation to inflammation in Chronic Obstructive Pulmonary Disease.

Background: The family of Suppressor of Cytokine Signaling (SOCS) acts as a controller of the duration and intensity of cytokine function by negatively regulating the JAK-STAT signaling pathway. SOCS' role in inflammatory diseases in animal models is well demonstrated. However, its role in the development of human disease is still under investigation. SOCS3 plays an important role in tumor development where its downregulation has been implicated in the pathogenesis of various solid tumors such as triple-negative breast cancer. Aim: The aim of this work was to study (1) the expression of SOCS3 in smokers' lungs and its relation to the degree of inflammation and (2) SOCS3 regulation by microRNA (miRNA) in alveolar-macrophage (AM)-derived extracellular vesicles (EVs) in bronchoalveolar lavage (BAL). Methods: Group A: 35 smokers' [19 with COPD (SC) and 16 without COPD (S)] and 9 nonsmokers (NS); SOCS3, TNFα in AM, and CD8+ T cells were quantified by immunohistochemistry, in lung tissue. Group B: additional 9 SC, 11 S, and 5 NS; AM-EVs expressing SOCS3 (CD14+SOCS3+) and SOCS3 suppressors miRNA-19a-3p and 221-3p in EVs were quantified by flow cytometry and PCR, in BAL. Results: The percentage of SOCS3+ AM was higher in SC [68 (6.6-99)%] and S [48 (8-100)%] than in NS [9.6 (1.9-61)%; p = 0.002; p = 0.03] and correlated with % of TNFα+AM (r = 0.48; p = 0.0009) and CD8+ T cells (r = 0.44; p = 0.0029). In BAL, the CD14+SOCS3+ EVs/µL were increased in SC [33 (21-74)] compared to S [16 (8-37); p = 0.03] and NS [9 (7-21); p = 0.003]. Conversely, miRNA-19a-3p and miRNA-221-3p expression were increased in S when compared to SC [19 (2-53) vs. 3 (0.6-8); p = 0.03 and 3 (0.005-9.6) vs. 0.2 (0.08-0.7); p = 0.05]. Conclusions: The suppressor function of SOCS3 in COPD seems to be overridden by other factors and does not follow the animal-model paradigm. Expression of SOCS3 in BAL macrophage-derived EVs might be useful to assess the degree of inflammation and possible progression of COPD. Downregulation of SOCS3, by miRNA, in smokers without COPD might contribute to the risk of developing cancer in these patients.

Mast cell infiltration discriminates between histopathological phenotypes of chronic obstructive pulmonary disease.

RATIONALE: COPD is a complex disease with heterogeneous manifestations. Attempts have been made to define different phenotypes that could guide toward better disease understanding. We described before that smokers can develop either panlobular (PLE) or centrilobular emphysema (CLE). The latter has worse small airways remodeling and narrowing, which account for the airflow obstruction similar to asthma. OBJECTIVES: Because of the small airways involvement in CLE similar to asthma, we hypothesized a role for mast cells in CLE but not in PLE. Hence, we investigated mast cell infiltration, along with overall inflammation, and their relation with hyperreactivity and emphysema type in COPD. METHODS: We studied lung function, emphysema type, mast cells, and overall inflammation in small airways and alveolar walls, along with alveolar wall thickening in 67 subjects undergoing lung resection (59 smokers, 8 nonsmokers). MEASUREMENTS AND MAIN RESULTS: Twenty-seven smokers had CLE, 24 had PLE, and 8 had no emphysema. Mast cells were significantly increased in CLE compared with PLE and control subjects. Especially relevant was the mast cell increase in airway smooth muscle in CLE, which related significantly to airway hyperreactivity. CD4(+)T cells, neutrophils, and macrophages, but not eosinophils and CD8(+)T cells, were significantly higher in CLE than PLE. Alveolar wall thickness was increased in all smokers, but significantly more in CLE. CONCLUSIONS: The pathological phenotypes of COPD CLE and PLE show important differences in their overall inflammation with a protagonism of mast cells, which are related to airway reactivity. These findings highlight the distinctness of these COPD phenotypes and the role of mast cells in the pathophysiology of COPD.