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1.
Front Immunol ; 10: 2058, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31555283

RESUMEN

Patients with rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) demonstrate increased circulating microparticles (MP). These vesicles, primarily those that form immune complexes (MP-IC), may activate monocytes. We evaluated the effect of MP and MP-IC in the differentiation of monocytes to macrophages (monocyte-derived macrophages; MDM) and for consequences in autologous lymphocyte activation. Monocytes from healthy controls (HC) and patients with RA and SLE that differentiated into MDM in the presence of MP-IC showed a proinflammatory (M1-like) profile, which was more evident using MP-IC from patients with RA than those from patients with SLE. Notably, MDM from HC and patients with RA that differentiated with MP-IC were more prone to M1-like profile than those from patients with SLE. In HC and patients with RA, monocyte differentiation using MP-IC decreased the frequency of MDM that bound/internalized latex beads. The M1-like profile did not completely revert following IL-4 treatment. The effect of M1-like MDM on T lymphocytes stimulated with phytohemagglutinin was further evaluated. MDM differentiated with MP enhanced the proliferation of T cells obtained from patients with RA compared with those differentiated with MP-IC or without vesicles. Neither MP nor MP-IC induced interferon (IFN)-γ+ and tumor necrosis factor (TNF)-α+ T cells in patients with RA. Conversely, unlike MDM differentiated with or without MP, MP-IC enhanced the proliferation and increased the frequencies of IFN-γ+CD4+ T, TNF-α+CD4+ T, and IFN-γ+CD8+ T cells in patients with SLE. The co-culture of B cells with MDM obtained from patients with RA and SLE and differentiated with MP-IC increased the expression of B-cell activation markers and prevented B lymphocyte death. Strikingly, only for patients with SLE, these responses seemed to be associated with a significant increase in B-cell activating factor levels, high plasmablast frequency and immunoglobulin production. These results showed that MP-IC from patients with systemic autoimmune diseases favored the polarization of MDM into a proinflammatory profile that promotes T-cell activation, and additionally induced B-cell activation and survival. Therefore, the effect of MP-IC in mononuclear phagocytes may be an important factor for modulating adaptive responses in systemic autoimmune diseases.

2.
Ann Rheum Dis ; 78(10): 1379-1387, 2019 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-31405848

RESUMEN

OBJECTIVES: Myofibroblasts are key effector cells in the extracellular matrix remodelling of systemic sclerosis-associated interstitial lung disease (SSc-ILD); however, the diversity of fibroblast populations present in the healthy and SSc-ILD lung is unknown and has prevented the specific study of the myofibroblast transcriptome. We sought to identify and define the transcriptomes of myofibroblasts and other mesenchymal cell populations in human healthy and SSc-ILD lungs to understand how alterations in fibroblast phenotypes lead to SSc-ILD fibrosis. METHODS: We performed droplet-based, single-cell RNA-sequencing with integrated canonical correlation analysis of 13 explanted lung tissue specimens (56 196 cells) from four healthy control and four patients with SSc-ILD, with findings confirmed by cellular indexing of transcriptomes and epitopes by sequencing in additional samples. RESULTS: Examination of gene expression in mesenchymal cells identified two major, SPINT2hi and MFAP5hi, and one minor, WIF1hi, fibroblast populations in the healthy control lung. Combined analysis of control and SSc-ILD mesenchymal cells identified SPINT2hi, MFAP5hi, few WIF1hi fibroblasts and a new large myofibroblast population with evidence of actively proliferating myofibroblasts. We compared differential gene expression between all SSc-ILD and control mesenchymal cell populations, as well as among the fibroblast subpopulations, showing that myofibroblasts undergo the greatest phenotypic changes in SSc-ILD and strongly upregulate expression of collagens and other profibrotic genes. CONCLUSIONS: Our results demonstrate previously unrecognised fibroblast heterogeneity in SSc-ILD and healthy lungs, and define multimodal transcriptome-phenotypes associated with these populations. Our data indicate that myofibroblast differentiation and proliferation are key pathological mechanisms driving fibrosis in SSc-ILD.

3.
Artículo en Inglés | MEDLINE | ID: mdl-31432710

RESUMEN

Idiopathic pulmonary fibrosis (IPF) is the most common and devastating of the interstitial lung diseases. Epithelial dysfunction is thought to play a prominent role in disease pathology and we sought to characterize secreted signals that may contribute to disease pathology. Transcriptional profiling of senescent type II alveolar epithelial cells (AEC2s) from mice with epithelial-specific telomere dysfunction identified the TGFß family member, growth and differentiation factor 15 (Gdf15), as the most significantly upregulated secreted protein. Gdf15 expression is induced in response to telomere dysfunction and bleomycin challenge in mice. Gdf15 mRNA is expressed by lung epithelial cells, and protein can be detected in peripheral blood and bronchoalveolar lavage following bleomycin challenge in mice. In patients with IPF, GDF15 mRNA expression in lung tissue is significantly increased and correlates with pulmonary function. Single cell RNA-sequencing of human lungs identifies epithelial cells as the primary source of GDF15 and circulating concentrations of GDF15 are markedly elevated and correlate with disease severity and survival in multiple independent cohorts. Our findings suggest that GDF15 is an epithelial-derived secreted protein that may be a useful biomarker of epithelial stress and identifies IPF patients with poor outcomes.

4.
Respirology ; 2019 Jul 30.
Artículo en Inglés | MEDLINE | ID: mdl-31364255

RESUMEN

BACKGROUND AND OBJECTIVE: Mesenchymal stem cells (MSC) have been shown to ameliorate the deleterious effects of bleomycin in murine models. However, the mechanism responsible for protection from pulmonary fibrosis by stem cell therapy is still poorly understood, especially in terms of endoplasmic reticulum (ER) stress. We hypothesized that during bleomycin-induced lung injury, markers of ER stress, specifically the activation of the unfolded protein response (UPR), increase during injury, resembling the kinetics of collagen deposition in the lung described for the bleomycin model. We aimed to elucidate the possible role of MSC in ER stress modulation. METHODS: To determine the kinetics of ER stress in aged mice, the expression of ER stress markers after bleomycin lung injury was measured in old mice at different time points (days 0, 3, 7, 14 and 21). To evaluate the consequences of systemic delivery of MSC on lung ER stress in the bleomycin model, we evaluated changes in body weight, lung histology and protein expression of ER stress markers. RESULTS: The level of expression of UPR transcription factor XBP-1 and its regulator BiP was elevated at day 7 and progressively increased up to day 21. MSC inhibited BiP expression in bleomycin-induced ER stress, attenuating ER stress via the protein kinase RNA-like ER kinase (PERK)-Nrf2 pathway. The expression levels of other ER stress markers were not perturbed by MSC. CONCLUSION: Our data suggest that MSC operate on ER stress via several pathways, but the PERK-Nrf2 pathway revealed to be the main functioning pathway in our bleomycin model.

5.
Eur Respir J ; 54(2)2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31221805

RESUMEN

A comprehensive understanding of the changes in gene expression in cell types involved in idiopathic pulmonary fibrosis (IPF) will shed light on the mechanisms underlying the loss of alveolar epithelial cells and development of honeycomb cysts and fibroblastic foci. We sought to understand changes in IPF lung cell transcriptomes and gain insight into innate immune aspects of pathogenesis.We investigated IPF pathogenesis using single-cell RNA-sequencing of fresh lung explants, comparing human IPF fibrotic lower lobes reflecting late disease, upper lobes reflecting early disease and normal lungs.IPF lower lobes showed increased fibroblasts, and basal, ciliated, goblet and club cells, but decreased alveolar epithelial cells, and marked alterations in inflammatory cells. We found three discrete macrophage subpopulations in normal and fibrotic lungs, one expressing monocyte markers, one highly expressing FABP4 and INHBA (FABP4hi), and one highly expressing SPP1 and MERTK (SPP1hi). SPP1hi macrophages in fibrotic lower lobes showed highly upregulated SPP1 and MERTK expression. Low-level local proliferation of SPP1hi macrophages in normal lungs was strikingly increased in IPF lungs.Co-localisation and causal modelling supported the role for these highly proliferative SPP1hi macrophages in activation of IPF myofibroblasts in lung fibrosis. These data suggest that SPP1hi macrophages contribute importantly to lung fibrosis in IPF, and that therapeutic strategies targeting MERTK and macrophage proliferation may show promise for treatment of this disease.

6.
PLoS One ; 14(6): e0218003, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31170232

RESUMEN

We have previously shown that endoplasmic reticulum stress (ER stress) represses the PTEN inducible kinase 1 (PINK1) in lung type II alveolar epithelial cells (AECII) reducing mitophagy and increasing the susceptibility to lung fibrosis. Although increased circulating mitochondrial DNA (mtDNA) has been reported in chronic lung diseases, the contribution of mitophagy in the modulation of mitochondrial DAMP release and activation of profibrotic responses is unknown. In this study, we show that ER stress and PINK1 deficiency in AECII led to mitochondrial stress with significant oxidation and damage of mtDNA and subsequent extracellular release. Extracellular mtDNA was recognized by TLR9 in AECII by an endocytic-dependent pathway. PINK1 deficiency-dependent mtDNA release promoted activation of TLR9 and triggered secretion of the profibrotic factor TGF-ß which was rescued by PINK1 overexpression. Enhanced mtDNA oxidation and damage were found in aging and IPF human lungs and, in concordance, levels of circulating mtDNA were significantly elevated in plasma and bronchoalveolar lavage (BAL) from patients with IPF. Free mtDNA was found elevated in other ILDs with low expression of PINK1 including hypersensitivity pneumonitis and autoimmune interstitial lung diseases. These results support a role for PINK1 mediated mitophagy in the attenuation of mitochondrial damage associated molecular patterns (DAMP) release and control of TGF-ß mediated profibrotic responses.

7.
Am J Respir Crit Care Med ; 200(2): 199-208, 2019 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-31034279

RESUMEN

Rationale: Several common and rare genetic variants have been associated with idiopathic pulmonary fibrosis, a progressive fibrotic condition that is localized to the lung. Objectives: To develop an integrated understanding of the rare and common variants located in multiple loci that have been reported to contribute to the risk of disease. Methods: We performed deep targeted resequencing (3.69 Mb of DNA) in cases (n = 3,624) and control subjects (n = 4,442) across genes and regions previously associated with disease. We tested for associations between disease and 1) individual common variants via logistic regression and 2) groups of rare variants via sequence kernel association tests. Measurements and Main Results: Statistically significant common variant association signals occurred in all 10 of the regions chosen based on genome-wide association studies. The strongest risk variant is the MUC5B promoter variant rs35705950, with an odds ratio of 5.45 (95% confidence interval, 4.91-6.06) for one copy of the risk allele and 18.68 (95% confidence interval, 13.34-26.17) for two copies of the risk allele (P = 9.60 × 10-295). In addition to identifying for the first time that rare variation in FAM13A is associated with disease, we confirmed the role of rare variation in the TERT and RTEL1 gene regions in the risk of IPF, and found that the FAM13A and TERT regions have independent common and rare variant signals. Conclusions: A limited number of common and rare variants contribute to the risk of idiopathic pulmonary fibrosis in each of the resequencing regions, and these genetic variants focus on biological mechanisms of host defense and cell senescence.

8.
Am J Respir Cell Mol Biol ; 61(1): 21-30, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-30965013

RESUMEN

Senescence is a cell fate decision characterized by irreversible arrest of proliferation accompanied by a senescence-associated secretory phenotype. Traditionally, cellular senescence has been recognized as a beneficial physiological mechanism during development and wound healing and in tumor suppression. However, in recent years, evidence of negative consequences of cellular senescence has emerged, illuminating its role in several chronic pathologies. In this context, senescent cells persist or accumulate and have detrimental consequences. In this review, we discuss the possibility that in chronic obstructive pulmonary disease, persistent senescence impairs wound healing in the lung caused by secretion of proinflammatory senescence-associated secretory phenotype factors and exhaustion of progenitor cells. In contrast, in idiopathic pulmonary fibrosis, chronic senescence in alveolar epithelial cells exacerbates the accumulation of senescent fibroblasts together with production of extracellular matrix. We review how cellular senescence may contribute to lung disease pathology.

9.
J Equine Vet Sci ; 72: 8-15, 2019 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-30929788

RESUMEN

Maintaining the integrity of equine sperm subjected to preservation protocols is essential for the successful development of assisted reproduction procedures. The aim of this study was to assess the mitochondrial membrane potential, lipid peroxidation, and DNA integrity of equine sperm subjected to freezing, vitrification, and freeze-drying. Eight ejaculates obtained from four Colombian Creole horses were subjected to programmable freezing, vitrification, and freeze-drying. After thawing or rehydration, sperm motility and kinetics were assessed through a CASA system. The mitochondrial membrane potential (ΔΨM), lipid peroxidation (LPO), and DNA fragmentation index (DFI) of the spermatozoa were assessed by flow cytometry using the DiOC6 (3), C11-Bodipy 581/591, and propidium iodide (PI) fluorescent dyes. The statistical analysis was conducted via generalized linear models, mean comparisons via the Duncan test, and a principal component analysis. A higher rate of spermatozoa with a high ΔΨM was found for freeze-drying (40.26 ± 7.79%) compared with freezing (21.82 ± 5.38%) and vitrification (5.32 ± 1.17%) (P < .05). Likewise, a higher rate of nonperoxidized viable spermatozoa (Bodipy-/PI-) was found for freeze-drying (35.98 ± 7.01%) in relation to frozen (10.34 ± 2.69%) and vitrified (7.07 ± 2.00%) sperm (P < .05). The DFI of vitrified spermatozoa (0.12 ± 0.04%) was higher when compared with the frozen (0.03 ± 0.01%) and freeze-dried (0.02 ± 0.01%) samples (P < .05). The researchers conclude that vitrification generates greater sperm alterations than freeze-drying and freezing, whereas freeze-drying produces lower LPO and higher ΔΨM for equine spermatozoa.

11.
Cell Signal ; 58: 9-19, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30840855

RESUMEN

There is evidence that B cells from patients with Systemic Lupus Erythematosus (SLE) could be hyperactivated due to changes in their lipid rafts (LR) composition, leading to altered BCR-dependent signals. This study aimed to characterize possible alterations in the recruitment of protein tyrosine kinases (PTK) into B cells LR from SLE patients. Fifteen patients with SLE and ten healthy controls were included. Circulating B cells were isolated by negative selection and stimulated with goat Fab´2 anti-human IgM/IgG. LR were isolated with a non-ionic detergent and ultracentrifuged on 5-45% discontinuous sucrose gradients. Proteins from each fraction were analyzed by Western Blot. Total levels of Lyn, Syk, and ZAP-70 in resting B cells were similar in SLE patients and healthy controls. Upon BCR activation, Lyn, Syk and ZAP-70 recruitment into LR increased significantly in B cells of healthy controls and patients with inactive SLE. In contrast, in active SLE patients there was a great heterogeneity in the recruitment of signaling molecules and the recruitment of ZAP-70 was mainly observed in patients with decreased Syk recruitment into LR of activated B cells. The reduction in Flotilin-1 and Lyn recruitment in SLE patients seem to be associated with disease activity. These findings suggest that in SLE patients the PTK recruitment into B cell LR is dysregulated and that B cells are under constant activation through BCR signaling. The decrease of Lyn and Syk, the expression of ZAP-70 by B cells and the increase in Calcium fluxes in response to BCR stimulation in active SLE patients, further support that B cells from SLE patients are under constant activation through BCR signaling, as has been proposed.

12.
Circulation ; 139(19): 2238-2255, 2019 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-30759996

RESUMEN

BACKGROUND: Deficiencies of iron-sulfur (Fe-S) clusters, metal complexes that control redox state and mitochondrial metabolism, have been linked to pulmonary hypertension (PH), a deadly vascular disease with poorly defined molecular origins. BOLA3 (BolA Family Member 3) regulates Fe-S biogenesis, and mutations in BOLA3 result in multiple mitochondrial dysfunction syndrome, a fatal disorder associated with PH. The mechanistic role of BOLA3 in PH remains undefined. METHODS: In vitro assessment of BOLA3 regulation and gain- and loss-of-function assays were performed in human pulmonary artery endothelial cells using siRNA and lentiviral vectors expressing the mitochondrial isoform of BOLA3. Polymeric nanoparticle 7C1 was used for lung endothelium-specific delivery of BOLA3 siRNA oligonucleotides in mice. Overexpression of pulmonary vascular BOLA3 was performed by orotracheal transgene delivery of adeno-associated virus in mouse models of PH. RESULTS: In cultured hypoxic pulmonary artery endothelial cells, lung from human patients with Group 1 and 3 PH, and multiple rodent models of PH, endothelial BOLA3 expression was downregulated, which involved hypoxia inducible factor-2α-dependent transcriptional repression via histone deacetylase 1-mediated histone deacetylation. In vitro gain- and loss-of-function studies demonstrated that BOLA3 regulated Fe-S integrity, thus modulating lipoate-containing 2-oxoacid dehydrogenases with consequent control over glycolysis and mitochondrial respiration. In contexts of siRNA knockdown and naturally occurring human genetic mutation, cellular BOLA3 deficiency downregulated the glycine cleavage system protein H, thus bolstering intracellular glycine content. In the setting of these alterations of oxidative metabolism and glycine levels, BOLA3 deficiency increased endothelial proliferation, survival, and vasoconstriction while decreasing angiogenic potential. In vivo, pharmacological knockdown of endothelial BOLA3 and targeted overexpression of BOLA3 in mice demonstrated that BOLA3 deficiency promotes histological and hemodynamic manifestations of PH. Notably, the therapeutic effects of BOLA3 expression were reversed by exogenous glycine supplementation. CONCLUSIONS: BOLA3 acts as a crucial lynchpin connecting Fe-S-dependent oxidative respiration and glycine homeostasis with endothelial metabolic reprogramming critical to PH pathogenesis. These results provide a molecular explanation for the clinical associations linking PH with hyperglycinemic syndromes and mitochondrial disorders. These findings also identify novel metabolic targets, including those involved in epigenetics, Fe-S biogenesis, and glycine biology, for diagnostic and therapeutic development.

13.
J Biol Chem ; 294(13): 5008-5022, 2019 Mar 29.
Artículo en Inglés | MEDLINE | ID: mdl-30709904

RESUMEN

The hormone relaxin is considered a potential therapy for idiopathic pulmonary fibrosis (IPF). We have previously shown that a potential limitation to relaxin-based IPF therapy is decreased expression of a relaxin receptor, relaxin/insulin-like family peptide receptor 1 (RXFP1), in IPF fibroblasts. The mechanism that down-regulates RXFP1 in IPF remains unclear. To determine whether microRNAs (miRs) regulate RXFP1 gene expression, here we employed a bioinformatics approach to identify miRs predicted to target RXFP1 and identified a putative miR-144-3p target site in the RXFP1 mRNA. In situ hybridization of IPF lung biopsies revealed that miR-144-3p is expressed in fibroblastic foci. Furthermore, we found that miR-144-3p is up-regulated in IPF fibroblasts compared with lung fibroblasts from healthy donors. Transforming growth factor ß increased miR-144-3p expression in both healthy and IPF lung fibroblasts in a SMAD family 2/3 (SMAD2/3)-dependent manner, and Jun proto-oncogene AP-1 transcription factor subunit (AP-1) was required for constitutive miR-144-3p expression. Overexpression of an miR-144-3p mimic significantly reduced RXFP1 mRNA and protein levels and increased expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in healthy lung fibroblasts. IPF lung fibroblasts transfected with anti-miR-144-3p had increased RXFP1 expression and reduced α-SMA expression. Of note, a lentiviral luciferase reporter carrying the WT 3' UTR of RXFP1 was significantly repressed in IPF lung fibroblasts, whereas a reporter carrying a mutated miR-144-3p-binding site exhibited less sensitivity toward endogenous miR-144-3p expression, indicating that miR-144-3p down-regulates RXFP1 in IPF lung fibroblasts by targeting its 3' UTR. We conclude that miR-144-3p directly represses RXFP1 mRNA and protein expression.

14.
Arthritis Res Ther ; 21(1): 34, 2019 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-30674349

RESUMEN

BACKGROUND: Endothelial activation and damage is commonly observed in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) and is related to development of atherosclerosis and cardiovascular diseases. Different components of the immune system seem to participate in the endothelial injury, such as generation of autoantibodies and formation of immune complexes (ICs). Microparticles (MPs) and their immune complexes (MPs-ICs) are increased in the circulation of patients with SLE and RA; therefore, we propose these extracellular vesicles could interact and modulate the function of endothelial cells. Hence, the effect of MPs and MPs-ICs from patients with SLE and RA in endothelial cells was evaluated. METHODS: Macrovascular and microvascular endothelial cells were exposed to MPs and MPs-ICs from healthy donors and patients with SLE and RA. Vesicles uptake/binding, expression of adhesion molecules, cytokine and chemokine production, monocyte adherence, and alterations of endothelial monolayer were evaluated by flow cytometry and fluorescence microscopy. RESULTS: Endothelial cells internalized MPs and MPs-ICs and increased CD54 and CD102 expression and CCL2, CCL5, and IL-6 production after the treatment with these extracellular vesicles, which led to an increase in the adherence of classic monocytes. These vesicles also induced low expression of VE-cadherin in membrane, depolymerization of actin filaments, and formation of intercellular spaces, which led to endothelial death and increased permeability after MPs and MPs-ICs exposure. CONCLUSIONS: MPs and MPs-ICs from patients with SLE and RA increase adhesion molecules expression, chemokine production, and structural alterations in macrovascular and microvascular endothelial cells. Therefore, high counts of these vesicles in patients would promote endothelial alterations and secondary tissue leukocyte infiltration.

15.
BMC Genomics ; 20(1): 22, 2019 Jan 09.
Artículo en Inglés | MEDLINE | ID: mdl-30626320

RESUMEN

BACKGROUND: Aging is affected by genetic and environmental factors, and cigarette smoking is strongly associated with accumulation of senescent cells. In this study, we wanted to identify genes that may potentially be beneficial for cell survival in response to cigarette smoke and thereby may contribute to development of cellular senescence. RESULTS: Primary human bronchial epithelial cells from five healthy donors were cultured, treated with or without 1.5% cigarette smoke extract (CSE) for 24 h or were passaged into replicative senescence. Transcriptome changes were monitored using RNA-seq in CSE and non-CSE exposed cells and those passaged into replicative senescence. We found that, among 1534 genes differentially regulated during senescence and 599 after CSE exposure, 243 were altered in both conditions, representing strong enrichment. Pathways and gene sets overrepresented in both conditions belonged to cellular processes that regulate reactive oxygen species, proteasome degradation, and NF-κB signaling. CONCLUSIONS: Our results offer insights into gene expression responses during cellular aging and cigarette smoke exposure, and identify potential molecular pathways that are altered by cigarette smoke and may also promote airway epithelial cell senescence.


Asunto(s)
Bronquios/metabolismo , Senescencia Celular/genética , Fumar Cigarrillos/genética , Bronquios/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Fumar Cigarrillos/efectos adversos , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , FN-kappa B/genética , Cultivo Primario de Células , Especies Reactivas de Oxígeno/metabolismo , Análisis de Secuencia de ARN , Transducción de Señal/efectos de los fármacos
16.
Am J Respir Cell Mol Biol ; 60(6): 629-636, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-30543447

RESUMEN

Idiopathic pulmonary fibrosis (IPF) pathogenesis has been postulated to involve a variety of mechanisms associated with the aging process, including loss of protein homeostasis (proteostasis). Heat shock proteins are cellular chaperones that serve a number of vital maintenance and repair functions, including the regulation of proteostasis. Previously published data have implicated heat shock protein 70 (Hsp70) in the development of pulmonary fibrosis in animal models. We sought to identify alterations in Hsp70 expression in IPF lung. Hsp70 mRNA and protein were decreased in primary fibroblasts cultured from IPF versus normal donor lung tissue. In addition to cultured fibroblasts, Hsp70 expression was decreased in intact IPF lung, a stressed environment in which upregulation of protective heat shock proteins would be anticipated. In support of a mechanistic association between decreased Hsp70 and fibrosis, cultured primary lung fibroblasts deficient in Hsp70 secreted increased extracellular matrix proteins. Treatment of primary normal human lung fibroblasts in vitro with either of the profibrotic molecules IGFBP5 (insulin-like growth factor-binding protein 5) or transforming growth factor-ß1 downregulated Hsp70, suggesting Hsp70 is a downstream target in the fibrotic cascade. Hsp70-knockout mice subjected to an inhalational bleomycin model of pulmonary fibrosis demonstrated accelerated fibrosis versus wild-type control animals. We therefore conclude that reduced Hsp70 protein contributes to fibrosis and that interventions aimed at restoring normal expression of Hsp70 represent a novel therapeutic strategy for pulmonary fibrosis.

17.
Eur J Immunol ; 49(2): 323-335, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30537116

RESUMEN

Non-classical monocytes infiltrate the kidney parenchyma and participate in tissue damage in patients with lupus nephritis (LN). Circulating microparticles (MPs) seem to play critical roles in the activation of monocytes in systemic lupus erythematosus (SLE) patients. This study aims to characterize the phenotypes of MPs and monocyte subsets in LN patients and to determine their potential to discriminate between SLE patients with and without LN. Blood and urine samples from SLE patients were collected. In monocyte subsets from whole blood samples several phenotypic markers were evaluated. MPs were isolated from platelet-poor plasma and urine by centrifugation. This phenotypic marker characterization was performed using multiparametric flow cytometry. We observed that patients with active LN have lower counts of non-classical monocytes than do those without renal involvement. All monocyte subsets exhibited lower expression of CX3CR1 and ICAM-1 in LN than in patients without LN. High frequencies of MP-HMGB1+ and MP-HLA-DR+ were detected in circulation and urine of LN patients. Although MP-HMGB1+ , MP-HLA-DR+ , and MP-CX3CR1+ from urine were able to discriminate between patients with and without LN, only urinary MP-HMGB1+ were different between patients with active and inactive LN. Therefore, these vesicles may be useful as biomarkers of LN.

19.
Cell Immunol ; 336: 1-11, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30538031

RESUMEN

Patients with rheumatoid arthritis (RA) have increased amount of platelet-derived microparticles (PMPs) positive for citrullinated peptides (CPs) that form immune complexes (PMPs-ICs). Monocytes are important inflammatory mediators that play a role in the clearance of PMPs-ICs. We aimed to generate PMPs-ICs in vitro and determine its effect on monocytes from patients with RA and healthy individuals (HI). PMPs from patients showed platelet markers, mitochondria content, and phosphatidylserine exposure similar to PMPs from HI. However, patients had a higher frequency of IgG+ and CPs+ vesicles than HI. PMPs-ICs generated in vitro were similar to the circulating vesicles of patients with respect to IgG- and CPs-positivity. PMPs-ICs induced pro-inflammatory cytokines and CX3CR1 expression in monocytes from HI, and IL-10 and CD36 upregulation in monocytes from patients. These results suggest that PMPs-ICs induce activation of monocytes, with a pro-inflammatory response in HI and a more tolerant response in cells of patients with RA.

20.
Sci Rep ; 8(1): 17917, 2018 12 17.
Artículo en Inglés | MEDLINE | ID: mdl-30559453

RESUMEN

Patients with rheumatoid arthritis (RA) and autoantibodies, such as rheumatoid factor and those against cyclic citrullinated peptides, are designated as seropositive and have a more severe disease with worse prognosis than seronegative RA patients. Understanding the factors that participate in systemic inflammation, in addition to articular commitment, would allow better treatment approaches for prevention of RA comorbidities and disease reactivation. We evaluated whether monocyte subsets and extracellular vesicles (EVs) could contribute to this phenomenon. Seropositive patients had higher levels of proinflammatory cytokines than those of seronegative patients and healthy controls (HCs); however, this systemic inflammatory profile was unrelated to disease activity. High frequencies of circulating EVs positive for IgG, IgM, CD41a, and citrulline, together with altered counts and receptor expression of intermediate monocytes, were associated with systemic inflammation in seropositive patients; these alterations were not observed in seronegative patients, which seem to be more similar to HCs. Additionally, the EVs from seropositive patients were able to activate mononuclear phagocytes in vitro, and induced proinflammatory cytokines that were comparable to the inflammatory response observed at the systemic level in seropositive RA patients; therefore, all of these factors may contribute to the greater disease severity that has been described in these patients.

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