RESUMO
Myocarditis is characterized by an influx of inflammatory cells, predominantly of myeloid lineage. The progression of myocarditis to a dilated cardiomyopathy is markedly influenced by TGF-ß signalling. Here, we investigate the role of TGF-ß signalling in inflammatory cardiac macrophages in the development of myocarditis and post-inflammatory fibrosis. Experimental autoimmune myocarditis (EAM) was induced in the LysM-Cre × R26-stop-EYFP × Tgfbr2-fl/fl transgenic mice showing impaired TGF-ß signalling in the myeloid lineage and the LysM-Cre × R26-stop-EYFP control mice. In EAM, immunization led to acute myocarditis on day 21, followed by cardiac fibrosis on day 40. Both strains showed a similar severity of myocarditis and the extent of cardiac fibrosis. On day 21 of EAM, an increase in cardiac inflammatory macrophages was observed in both strains. These cells were sorted and analysed for differential gene expression using whole-genome transcriptomics. The analysis revealed activation and regulation of the inflammatory response, particularly the production of both pro-inflammatory and anti-inflammatory cytokines and cytokine receptors as TGF-ß-dependent processes. The analysis of selected cytokines produced by bone marrow-derived macrophages confirmed their suppressed secretion. In conclusion, our findings highlight the regulatory role of TGF-ß signalling in cytokine production within inflammatory cardiac macrophages during myocarditis.
Assuntos
Doenças Autoimunes , Citocinas , Macrófagos , Camundongos Transgênicos , Miocardite , Transdução de Sinais , Fator de Crescimento Transformador beta , Animais , Miocardite/metabolismo , Miocardite/imunologia , Miocardite/patologia , Miocardite/etiologia , Fator de Crescimento Transformador beta/metabolismo , Camundongos , Macrófagos/metabolismo , Macrófagos/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Citocinas/metabolismo , Modelos Animais de Doenças , Miocárdio/metabolismo , Miocárdio/patologia , Miocárdio/imunologia , Fibrose , MasculinoRESUMO
OBJECTIVES: In SSc, gastrointestinal tract (GIT) involvement is a major concern, with no disease-modifying and limited symptomatic therapies available. Faecal microbiota transplantation (FMT) represents a new therapeutic option for GIT-affliction in SSc, showing clinical promise in a recent controlled pilot trial. Here, we aim to investigate effects of FMT on duodenal biopsies collected from SSc patients by immunohistochemistry and transcriptome profiling. METHODS: We analysed duodenal biopsies obtained pre-intervention (week 0) and post-intervention (weeks 2 and 16) from nine SSc patients receiving an intestinal infusion of FMT (n = 5) or placebo (n = 4). The analysis included immunohistochemistry (IHC) with a selected immune function and fibrosis markers, and whole biopsy transcriptome profiling. RESULTS: In patients receiving FMT, the number of podoplanin- and CD64-expressing cells in the mucosa were lower at week 2 compared with baseline. This decline in podoplanin- (r = 0.94) and CD64-positive (r = 0.89) cells correlated with improved patient-reported lower GIT symptoms. Whole biopsy transcriptome profiling from week 2 showed significant enrichment of pathways critical for cellular and endoplasmic reticulum stress responses, microvillus and secretory vesicles, vascular and sodium-dependent transport, and circadian rhythm. At week 16, we found enrichment of pathways mandatory for binding activity of immunoglobulin receptors, T cell receptor complexes, and chemokine receptors, as well as response to zinc-ions. We found that 25 genes, including Matrix metalloproteinase-1 were upregulated at both week 2 and week 16. CONCLUSION: Combining selective IHC and unbiased gene expression analyses, this exploratory study highlights the potential for disease-relevant organ effects of FMT in SSc patients with GIT involvement. TRIAL REGISTRATION: ClinicalTrials.gov, http://clinicaltrials.gov, NCT03444220.
Assuntos
Transplante de Microbiota Fecal , Escleroderma Sistêmico , Humanos , Transplante de Microbiota Fecal/efeitos adversos , Método Duplo-Cego , Intestinos , Mucosa Intestinal , Escleroderma Sistêmico/terapia , Escleroderma Sistêmico/etiologia , Resultado do TratamentoRESUMO
Endothelial injury and dysfunction (ED) represent a link between cardiovascular risk factors promoting hypertension and atherosclerosis, the leading cause of death in Western populations. High-density lipoprotein (HDL) is considered antiatherogenic and known to prevent ED. Using HDL from children and adults with chronic kidney dysfunction (HDL(CKD)), a population with high cardiovascular risk, we have demonstrated that HDL(CKD) in contrast to HDL(Healthy) promoted endothelial superoxide production, substantially reduced nitric oxide (NO) bioavailability, and subsequently increased arterial blood pressure (ABP). We have identified symmetric dimethylarginine (SDMA) in HDL(CKD) that causes transformation from physiological HDL into an abnormal lipoprotein inducing ED. Furthermore, we report that HDL(CKD) reduced endothelial NO availability via toll-like receptor-2 (TLR-2), leading to impaired endothelial repair, increased proinflammatory activation, and ABP. These data demonstrate how SDMA can modify the HDL particle to mimic a damage-associated molecular pattern that activates TLR-2 via a TLR-1- or TLR-6-coreceptor-independent pathway, linking abnormal HDL to innate immunity, ED, and hypertension.
Assuntos
Aterosclerose/imunologia , Hipertensão/imunologia , Nefropatias/imunologia , Lipoproteínas HDL/metabolismo , Receptor 2 Toll-Like/metabolismo , Adulto , Animais , Arginina/análogos & derivados , Arginina/química , Pressão Arterial , Criança , Endotélio , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Lipoproteínas HDL/química , Camundongos , Camundongos Endogâmicos C57BL , Óxido Nítrico/metabolismo , Transdução de Sinais , Superóxidos/metabolismo , Receptor 2 Toll-Like/genética , CicatrizaçãoRESUMO
AIMS: Nuclear receptors and their cofactors regulate key pathophysiological processes in atherosclerosis development. The transcriptional activity of these nuclear receptors is controlled by the nuclear receptor corepressors (NCOR), scaffolding proteins that form the basis of large corepressor complexes. Studies with primary macrophages demonstrated that the deletion of Ncor1 increases the expression of atherosclerotic molecules. However, the role of nuclear receptor corepressors in atherogenesis is unknown. METHODS AND RESULTS: We generated myeloid cell-specific Ncor1 knockout mice and crossbred them with low-density lipoprotein receptor (Ldlr) knockouts to study the role of macrophage NCOR1 in atherosclerosis. We demonstrate that myeloid cell-specific deletion of nuclear receptor corepressor 1 (NCOR1) aggravates atherosclerosis development in mice. Macrophage Ncor1-deficiency leads to increased foam cell formation, enhanced expression of pro-inflammatory cytokines, and atherosclerotic lesions characterized by larger necrotic cores and thinner fibrous caps. The immunometabolic effects of NCOR1 are mediated via suppression of peroxisome proliferator-activated receptor gamma (PPARγ) target genes in mouse and human macrophages, which lead to an enhanced expression of the CD36 scavenger receptor and subsequent increase in oxidized low-density lipoprotein uptake in the absence of NCOR1. Interestingly, in human atherosclerotic plaques, the expression of NCOR1 is reduced whereas the PPARγ signature is increased, and this signature is more pronounced in ruptured compared with non-ruptured carotid plaques. CONCLUSIONS: Our findings show that macrophage NCOR1 blocks the pro-atherogenic functions of PPARγ in atherosclerosis and suggest that stabilizing the NCOR1-PPARγ binding could be a promising strategy to block the pro-atherogenic functions of plaque macrophages and lesion progression in atherosclerotic patients.
Assuntos
Aterosclerose , Macrófagos , Correpressor 1 de Receptor Nuclear , PPAR gama , Animais , Aterosclerose/genética , Aterosclerose/prevenção & controle , Células Espumosas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Correpressor 1 de Receptor Nuclear/genética , PPAR gama/genética , Receptores de LDLRESUMO
Cardiac fibrosis is a pathological process associated with the development of heart failure. TGF-ß and WNT signaling have been implicated in pathogenesis of cardiac fibrosis, however, little is known about molecular cross-talk between these two pathways. The aim of this study was to examine the effect of exogenous canonical WNT3a and non-canonical WNT5a in TGF-ß-activated human cardiac fibroblasts. We found that WNT3a and TGF-ß induced a ß-catenin-dependent response, whereas WNT5a prompted AP-1 activity. TGF-ß triggered profibrotic signatures in cardiac fibroblasts, and co-stimulation with WNT3a or co-activation of the ß-catenin pathway with the GSK3ß inhibitor CHIR99021 enhanced collagen I and fibronectin production and development of active contractile stress fibers. In the absence of TGF-ß, neither WNT3a nor CHIR99021 exerted profibrotic responses. On a molecular level, in TGF-ß-activated fibroblasts, WNT3a enhanced phosphorylation of TAK1 and production and secretion of IL-11 but showed no effect on the Smad pathway. Neutralization of IL-11 activity with the blocking anti-IL-11 antibody effectively reduced the profibrotic response of cardiac fibroblasts activated with TGF-ß and WNT3a. In contrast to canonical WNT3a, co-activation with non-canonical WNT5a suppressed TGF-ß-induced production of collagen I. In conclusion, WNT/ß-catenin signaling promotes TGF-ß-mediated fibroblast-to-myofibroblast transition by enhancing IL-11 production. Thus, the uncovered mechanism broadens our knowledge on a molecular basis of cardiac fibrogenesis and defines novel therapeutic targets for fibrotic heart diseases.
Assuntos
Fibroblastos/metabolismo , Interleucina-11/metabolismo , Miocárdio/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Colágeno/química , Colágeno/metabolismo , Fibrose/patologia , Coração/fisiologia , Humanos , MAP Quinase Quinase Quinases/metabolismo , Miofibroblastos/metabolismo , RNA-Seq , Transdução de Sinais , Fibras de Estresse/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Proteína Wnt-5a/metabolismo , Proteína Wnt3A/metabolismoRESUMO
BACKGROUND: Pathological activation of cardiac fibroblasts is a key step in development and progression of cardiac fibrosis and heart failure. This process has been associated with enhanced autophagocytosis, but molecular mechanisms remain largely unknown. METHODS AND RESULTS: Immunohistochemical analysis of endomyocardial biopsies showed increased activation of autophagy in fibrotic hearts of patients with inflammatory cardiomyopathy. In vitro experiments using mouse and human cardiac fibroblasts confirmed that blockade of autophagy with Bafilomycin A1 inhibited fibroblast-to-myofibroblast transition induced by transforming growth factor (TGF)-ß. Next, we observed that cardiac fibroblasts obtained from mice overexpressing transcription factor Fos-related antigen 2 (Fosl-2tg) expressed elevated protein levels of autophagy markers: the lipid modified form of microtubule-associated protein 1A/1B-light chain 3B (LC3BII), Beclin-1 and autophagy related 5 (Atg5). In complementary experiments, silencing of Fosl-2 with antisense GapmeR oligonucleotides suppressed production of type I collagen, myofibroblast marker alpha smooth muscle actin and autophagy marker Beclin-1 in cardiac fibroblasts. On the other hand, silencing of either LC3B or Beclin-1 reduced Fosl-2 levels in TGF-ß-activated, but not in unstimulated cells. Using a cardiac hypertrophy model induced by continuous infusion of angiotensin II with osmotic minipumps, we confirmed that mice lacking either Fosl-2 (Ccl19CreFosl2flox/flox) or Atg5 (Ccl19CreAtg5flox/flox) in stromal cells were protected from cardiac fibrosis. CONCLUSION: Our findings demonstrate that Fosl-2 regulates autophagocytosis and the TGF-ß-Fosl-2-autophagy axis controls differentiation of cardiac fibroblasts. These data provide a new insight for the development of pharmaceutical targets in cardiac fibrosis.
Assuntos
Fibroblastos/metabolismo , Antígeno 2 Relacionado a Fos/metabolismo , Regulação da Expressão Gênica , Insuficiência Cardíaca/metabolismo , Miocárdio/metabolismo , Fator de Transcrição AP-1/metabolismo , Idoso , Animais , Feminino , Fibroblastos/patologia , Fibrose , Antígeno 2 Relacionado a Fos/genética , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Miocárdio/patologia , Fator de Transcrição AP-1/genéticaRESUMO
AIMS: Sirtuin 6 (Sirt6) is a NAD+-dependent deacetylase that plays a key role in DNA repair, inflammation and lipid regulation. Sirt6-null mice show severe metabolic defects and accelerated aging. Macrophage-foam cell formation via scavenger receptors is a key step in atherogenesis. We determined the effects of bone marrow-restricted Sirt6 deletion on foam cell formation and atherogenesis using a mouse model. METHODS AND RESULTS: Sirt6 deletion in bone marrow-derived cells increased aortic plaques, lipid content and macrophage numbers in recipient Apoe-/- mice fed a high-cholesterol diet for 12 weeks (n = 12-14, p < .001). In RAW macrophages, Sirt6 overexpression reduced oxidized low-density lipoprotein (oxLDL) uptake, Sirt6 knockdown enhanced it and increased mRNA and protein levels of macrophage scavenger receptor 1 (Msr1), whereas levels of other oxLDL uptake and efflux transporters remained unchanged. Similarly, in human primary macrophages, Sirt6 knockdown increased MSR1 protein levels and oxLDL uptake. Double knockdown of Sirt6 and Msr1 abolished the increase in oxLDL uptake observed upon Sirt6 single knockdown. FACS analyses of macrophages from aortic plaques of Sirt6-deficient bone marrow-transplanted mice showed increased MSR1 protein expression. Double knockdown of Sirt6 and the transcription factor c-Myc in RAW cells abolished the increase in Msr1 mRNA and protein levels; c-Myc overexpression increased Msr1 mRNA and protein levels. CONCLUSIONS: Loss of Sirt6 in bone marrow-derived cells is proatherogenic; hereby macrophages play an important role given a c-Myc-dependent increase in MSR1 protein expression and an enhanced oxLDL uptake in human and murine macrophages. These findings assign endogenous SIRT6 in macrophages an important atheroprotective role.
Assuntos
Aterosclerose/metabolismo , Aterosclerose/patologia , Medula Óssea/patologia , Deleção de Genes , Receptores Depuradores Classe A/metabolismo , Sirtuínas/genética , Sirtuínas/metabolismo , Animais , Aorta/patologia , Apolipoproteínas E/deficiência , Apolipoproteínas E/metabolismo , Transplante de Medula Óssea , Regulação para Baixo , Técnicas de Silenciamento de Genes , Hematopoese , Homozigoto , Humanos , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Modelos Biológicos , Placa Aterosclerótica/metabolismo , Placa Aterosclerótica/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Células RAW 264.7RESUMO
Heart-specific CD4+ T cells have been implicated in development and progression of myocarditis in mice and in humans. Here, using mouse models of experimental autoimmune myocarditis (EAM) we investigated the role of heart non-specific CD4+ T cells in the progression of the disease. Heart non-specific CD4+ T cells were obtained from DO11.10 mice expressing transgenic T cell receptor recognizing chicken ovalbumin. We found that heart infiltrating CD4+ T cells expressed exclusively effector (Teff) phenotype in the EAM model and in hearts of patients with lymphocytic myocarditis. Adoptive transfer experiments showed that while heart-specific Teff infiltrated the heart shortly after injection, heart non-specific Teff effectively accumulated during myocarditis and became the major heart-infiltrating CD4+ T cell subset at later stage. Restimulation of co-cultured heart-specific and heart non-specific CD4+ T cells with alpha-myosin heavy chain antigen showed mainly Th1/Th17 response for heart-specific Teff and up-regulation of a distinct set of extracellular signalling molecules in heart non-specific Teff. Adoptive transfer of heart non-specific Teff in mice with myocarditis did not affect inflammation severity at the peak of disease, but protected the heart from adverse post-inflammatory fibrotic remodelling and cardiac dysfunction at later stages of disease. Furthermore, mouse and human Teff stimulated in vitro with common gamma cytokines suppressed expression of profibrotic genes, reduced amount of α-smooth muscle actin filaments and decreased contraction of cardiac fibroblasts. In this study, we provided a proof-of-concept that heart non-specific Teff cells could effectively contribute to myocarditis and protect the heart from the dilated cardiomyopathy outcome.
Assuntos
Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/fisiologia , Miocardite/imunologia , Miocárdio/patologia , Animais , Fibrose/imunologia , Humanos , Camundongos , Miocárdio/imunologiaRESUMO
WNT signaling plays an important role in fibrotic processes in the heart. Recently, exosomes have been proposed as novel extracellular transporters for WNT proteins. In this study, we analyzed whether WNT3a and WNT5a carried by exosomes could activate downstream molecular pathways in human cardiac fibroblasts. Exosomes were isolated from conditioned medium of control, WNT3a- and WNT5a-producing L cells by differential ultracentrifugations. Obtained exosomes showed size ranging between 20â»150 nm and expressed exosomal markers ALG-2-interacting protein X (ALIX) and CD63. Treatment with WNT3a-rich exosomes inhibited activity of glycogen synthase kinase 3ß (GSK3ß), induced nuclear translocation of ß-catenin, and activated T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors as well as expression of WNT/ß-catenin responsive genes in cardiac fibroblasts, but did not coactivate extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and activator protein 1 (AP-1) signaling pathways. In contrast, exosomes produced by WNT5a-producing L cells failed to activate ß-catenin-dependent response, but successfully triggered phosphorylation of ERK1/2 and JNK and stimulated IL-6 production. In conclusion, exosomes containing WNT proteins can functionally contribute to cardiac fibrosis by activating profibrotic WNT pathways on cardiac fibroblasts and may represent a novel mechanism of spreading profibrotic signals in the heart.
Assuntos
Exossomos/metabolismo , Fibroblastos/metabolismo , Miocárdio/metabolismo , Via de Sinalização Wnt , Proteína Wnt-5a/metabolismo , Proteína Wnt3A/metabolismo , Biomarcadores , Linhagem Celular , Suscetibilidade a Doenças , Vesículas Extracelulares/metabolismo , Humanos , Miocárdio/citologiaRESUMO
BACKGROUND: Axial spondyloarthritis (axSpA) is characterized by significant bone loss caused by dysregulation of physiological bone turnover, possibly resulting from intensified differentiation of osteoclasts. The aim of this study was to reevaluate the levels of osteoclastogenesis-mediating factors: soluble RANKL, M-CSF, OPG and other cytokines in sera of untreated, with sDMARDs and/or bDMARDs, axSpA patients and to test whether these sera influence differentiation of healthy monocytes towards osteoclast lineage. METHODS: Bone remodeling molecules (RANKL, M-CSF, OPG, IL-6, OSM, IL-17A, TGFß, and TNFα) were evaluated in 27 patients with axSpA and 23 age and sex-matched controls. Disease activity (BASDAI, ASDAS) and inflammatory markers (ESR, CRP) were assessed. Monocytes obtained from healthy individuals were cultured in vitro in presence of sera from 11 randomly chosen axSpA patients and 10 controls, with addition of exogenous M-CSF and/or RANKL or without. Osteoclastic differentiation was assessed analyzing osteoclast markers (cathepsin K and RANK at mRNA level) and with osteoclast-specific staining. RESULTS: axSpA patients' sera levels of soluble RANKL were significantly lower and M-CSF, IL-6, OSM, IL-17A and TNFα significantly higher in comparison to controls, whereas of OPG and TGFß were comparable in both groups. Numbers of generated in vitro osteoclasts and cathepsin K mRNA levels did not differ between cultures supplemented with sera of healthy and axSpA patients, both in the absence and presence of M-CSF. Instead, addition of exogenous RANKL boosted osteoclastogenesis, which was significantly higher in cultures with axSpA sera. Furthermore, sera from axSpA patients induced substantially higher levels of RANK mRNA, independently of M-CSF and RANKL stimulation. CONCLUSION: We show that, paradoxically, serum levels of soluble RANKL observed in axSpA are in fact significantly lower in comparison to healthy blood donors. Our results indicate that sera of axSpA patients - in contrary to healthy subjects - contain circulating, soluble factors (presumably IL-6, OSM, IL-17A, TNFα and others) able to stimulate healthy monocytes responsiveness to even relative low RANKL serum levels, by inducing high RANK mRNA expression and - as a net effect - boosting their osteoclastogenic potential. We suggest also that locally produced RANKL in axSpA may induce overactive osteoclasts from their precursors.
Assuntos
Monócitos/fisiologia , Osteogênese/fisiologia , Ligante RANK/sangue , Espondilartrite/sangue , Adulto , Biomarcadores/sangue , Catepsina K/sangue , Diferenciação Celular , Células Cultivadas , Citocinas/sangue , Feminino , Humanos , Interleucina-17/sangue , Fator Estimulador de Colônias de Macrófagos/sangue , Masculino , Osteoclastos/citologia , Osteoprotegerina/sangue , RNA Mensageiro/sangue , Fosfatase Ácida Resistente a Tartarato/sangue , Fator de Crescimento Transformador beta/sangue , Fator de Necrose Tumoral alfa/sangue , Regulação para CimaRESUMO
AIMS: Myocardial fibrosis critically contributes to cardiac dysfunction in inflammatory dilated cardiomyopathy (iDCM). Activation of transforming growth factor-ß (TGF-ß) signalling is a key-step in promoting tissue remodelling and fibrosis in iDCM. Downstream mechanisms controlling these processes, remain elusive. METHODS AND RESULTS: Experimental autoimmune myocarditis (EAM) was induced in BALB/c mice with heart-specific antigen and adjuvant. Using heart-inflammatory precursors, as well as mouse and human cardiac fibroblasts, we demonstrated rapid secretion of Wnt proteins and activation of Wnt/ß-catenin pathway in response to TGF-ß signalling. Inactivation of extracellular Wnt with secreted Frizzled-related protein 2 (sFRP2) or inhibition of Wnt secretion with Wnt-C59 prevented TGF-ß-mediated transformation of inflammatory precursors and cardiac fibroblasts into pathogenic myofibroblasts. Inhibition of T-cell factor (TCF)/ß-catenin-mediated transcription with ICG-001 or genetic loss of ß-catenin also prevented TGF-ß-induced myofibroblasts formation. Furthermore, blocking of Smad-independent TGF-ß-activated kinase 1 (TAK1) pathway completely abrogated TGF-ß-induced Wnt secretion. Activation of Wnt pathway in the absence of TGF-ß, however, failed to transform precursors into myofibroblasts. The critical role of Wnt axis for cardiac fibrosis in iDCM is also supported by elevated Wnt-1/Wnt-5a levels in human samples from hearts with myocarditis. Accordingly, and as an in vivo proof of principle, inhibition of Wnt secretion or TCF/ß-catenin-mediated transcription abrogated the development of post-inflammatory fibrosis in EAM. CONCLUSION: We identified TAK1-mediated rapid Wnt protein secretion as a novel downstream key mechanism of TGF-ß-mediated myofibroblast differentiation and myocardial fibrosis progression in human and mouse myocarditis. Thus, pharmacological targeting of Wnts might represent a promising therapeutic approach against iDCM in the future.
Assuntos
Doenças Autoimunes/etiologia , Miocardite/etiologia , Miocárdio/patologia , Fator de Crescimento Transformador beta/fisiologia , Proteínas Wnt/metabolismo , Animais , Benzenoacetamidas/farmacologia , Diferenciação Celular/fisiologia , Progressão da Doença , Fibrose/fisiopatologia , Humanos , MAP Quinase Quinase Quinases/metabolismo , MAP Quinase Quinase Quinases/fisiologia , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Miofibroblastos/fisiologia , Piridinas/farmacologia , Transdução de Sinais/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição TCF/metabolismo , Disfunção Ventricular/fisiopatologia , Proteína Wnt-5a/metabolismo , Proteína Wnt1/metabolismo , beta Catenina/metabolismoRESUMO
Pathologic accumulation of myofibroblasts in asthmatic bronchi is regulated by extrinsic stimuli and by the intrinsic susceptibility of bronchial fibroblasts to transforming growth factor-ß (TGF-ß). The specific function of gap junctions and connexins in this process has remained unknown. Here, we investigated the role of connexin43 (Cx43) in TGF-ß-induced myofibroblastic differentiation of fibroblasts derived from bronchoscopic biopsy specimens of patients with asthma and donors without asthma. Asthmatic fibroblasts expressed considerably higher levels of Cx43 and were more susceptible to TGF-ß1-induced myofibroblastic differentiation than were their nonasthmatic counterparts. TGF-ß1 efficiently up-regulated Cx43 levels and activated the canonical Smad pathway in asthmatic cells. Ectopic Cx43 expression in nonasthmatic (Cx43low) fibroblasts increased their predilection to TGF-ß1-induced Smad2 activation and fibroblast-myofibroblast transition. Transient Cx43 silencing in asthmatic (Cx43high) fibroblasts by Cx43 small interfering RNA attenuated the TGF-ß1-triggered Smad2 activation and myofibroblast formation. Direct interactions of Smad2 and Cx43 with ß-tubulin were demonstrated by co-immunoprecipitation assay, whereas the sensitivity of these interactions to TGF-ß1 signaling was confirmed by Förster Resonance Energy Transfer analyses. Furthermore, inhibition of the TGF-ß1/Smad pathway attenuated TGF-ß1-triggered Cx43 up-regulation and myofibroblast differentiation of asthmatic fibroblasts. Chemical inhibition of gap junctional intercellular communication with 18 α-glycyrrhetinic acid did not affect the initiation of fibroblast-myofibroblast transition in asthmatic fibroblasts but interfered with the maintenance of their myofibroblastic phenotype. Collectively, our data identified Cx43 as a new player in the feedback mechanism regulating TGF-ß1/Smad-dependent differentiation of bronchial fibroblasts. Thus, our observations point to Cx43 as a novel profibrotic factor in asthma progression.
Assuntos
Asma/metabolismo , Asma/patologia , Brônquios/patologia , Diferenciação Celular , Conexina 43/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patologia , Adulto , Diferenciação Celular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Humanos , MAP Quinase Quinase Quinases/metabolismo , Masculino , Pessoa de Meia-Idade , Miofibroblastos/efeitos dos fármacos , Fenótipo , Transdução de Sinais/efeitos dos fármacos , Proteína Smad2/metabolismo , Fator de Crescimento Transformador beta1/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
Experimental autoimmune myocarditis (EAM) is a CD4(+) T-cell-mediated model of human inflammatory dilated cardiomyopathies. Heart-specific CD4(+) T-cell activation is dependent on autoantigens presented by MHC class II (MHCII) molecules expressed on professional APCs. In this study, we addressed the role of inflammation-induced MHCII expression by cardiac nonhematopoietic cells on EAM development. EAM was induced in susceptible mice lacking inducible expression of MHCII molecules on all nonhematopoietic cells (pIV-/- K14 class II transactivator (CIITA) transgenic (Tg) mice) by immunization with α-myosin heavy chain peptide in CFA. Lack of inducible nonhematopoietic MHCII expression in pIV-/- K14 CIITA Tg mice conferred EAM resistance. In contrast, cardiac pathology was induced in WT and heterozygous mice, and correlated with elevated cardiac endothelial MHCII expression. Control mice with myocarditis displayed an increase in infiltrating CD4(+) T cells and in expression of IFN-γ, which is the major driver of nonhematopoietic MHCII expression. Mechanistically, IFN-γ neutralization in WT mice shortly before disease onset resulted in reduced cardiac MHCII expression and pathology. These findings reveal a previously overlooked contribution of IFN-γ to induce endothelial MHCII expression in the heart and to progress cardiac pathology during myocarditis.
Assuntos
Doenças Autoimunes/imunologia , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Miocardite/imunologia , Animais , Autoantígenos , Linfócitos T CD4-Positivos , Modelos Animais de Doenças , Endotélio/imunologia , Inflamação , Interferon gama/imunologia , Ativação Linfocitária , Camundongos , Camundongos Transgênicos , Miocárdio/patologia , Miocárdio/ultraestrutura , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Transativadores/genéticaRESUMO
AIMS: Supernatants of serum-free cultured mononuclear cells (MNC) contain a mix of immunomodulating factors (secretome), which have been shown to attenuate detrimental inflammatory responses following myocardial ischaemia. Inflammatory dilated cardiomyopathy (iDCM) is a common cause of heart failure in young patients. Experimental autoimmune myocarditis (EAM) is a CD4+ T cell-dependent model, which mirrors important pathogenic aspects of iDCM. The aim of this study was to determine the influence of MNC secretome on myocardial inflammation in the EAM model. METHODS AND RESULTS: BALB/c mice were immunized twice with an alpha myosin heavy chain peptide together with Complete Freund adjuvant. Supernatants from mouse mononuclear cells were collected, dialysed, and injected i.p. at Day 0, Day 7, or Day 14, respectively. Myocarditis severity, T cell responses, and autoantibody formation were assessed at Day 21. The impact of MNC secretome on CD4+ T cell function and viability was evaluated using in vitro proliferation and cell viability assays. A single high-dose application of MNC secretome, injected at Day 14 after the first immunization, effectively attenuated myocardial inflammation. Mechanistically, MNC secretome induced caspase-8-dependent apoptosis in autoreactive CD4+ T cells. CONCLUSION: MNC secretome abrogated myocardial inflammation in a CD4+ T cell-dependent animal model of autoimmune myocarditis. This anti-inflammatory effect of MNC secretome suggests a novel and simple potential treatment concept for inflammatory heart diseases.
Assuntos
Doenças Autoimunes/prevenção & controle , Linfócitos T CD4-Positivos/fisiologia , Miocardite/prevenção & controle , Cadeias Pesadas de Miosina/farmacologia , Animais , Anticorpos/farmacologia , Apoptose/fisiologia , Autoanticorpos/metabolismo , Relação CD4-CD8 , Ligante de CD40/imunologia , Linfócitos T CD8-Positivos/fisiologia , Inibidores de Caspase/farmacologia , Proliferação de Células/fisiologia , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/fisiologia , Modelos Animais de Doenças , Proteína Ligante Fas/imunologia , Humanos , Camundongos Endogâmicos BALB C , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/imunologia , Baço/citologiaRESUMO
BACKGROUND: Granulocyte macrophage-colony stimulating factor (GM-CSF) is critically required for the induction of experimental autoimmune myocarditis (EAM), a model of post-inflammatory dilated cardiomyopathy. Its specific role in the progression of myocarditis into end stage heart failure is not known. METHODS AND RESULTS: BALB/c mice were immunized with myosin peptide and complete Freund's adjuvant at days 0 and 7. Heart-infiltrating inflammatory CD133(+) progenitors were isolated from inflamed hearts at the peak of inflammation (day 21). In the presence of GM-CSF, inflammatory CD133(+) progenitors up-regulated integrin, alpha X (CD11c), class II major histocompatibility complex, CD80 and CD86 co-stimulatory molecules reflecting an inflammatory dendritic cell (DC) phenotype. Inflammatory DCs stimulated antigen-specific CD4(+) T cell proliferation and induced myocarditis after myosin peptide loading and adoptive transfer in healthy mice. Moreover, GM-CSF treatment of mice after the peak of disease, between days 21 and 29 of EAM, transiently increased accumulation of inflammatory DCs in the myocardium. Importantly, bone marrow-derived CD11b(+) monocytes, rather than inflammatory CD133(+) progenitors represent the dominant cellular source of heart-infiltrating inflammatory DCs in EAM. In contrast, GM-CSF treatment neither affected numbers of heart-infiltrating CD45(+) and CD3(+) T cells nor the development of post-inflammatory fibrosis. CONCLUSIONS: GM-CSF treatment promotes formation of inflammatory DCs in EAM. In contrast to the active roles of GM-CSF and DCs in EAM induction, GM-CSF-induced inflammatory DCs neither prevent resolution of active inflammation, nor contribute to post-inflammatory cardiac remodelling. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.
Assuntos
Doenças Autoimunes/patologia , Células Dendríticas/patologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Miocardite/patologia , Miocárdio/patologia , Transferência Adotiva , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Doenças Autoimunes/induzido quimicamente , Doenças Autoimunes/imunologia , Biomarcadores/metabolismo , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Proliferação de Células/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/transplante , Modelos Animais de Doenças , Progressão da Doença , Expressão Gênica/efeitos dos fármacos , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Imunização , Inflamação/induzido quimicamente , Inflamação/imunologia , Inflamação/patologia , Camundongos , Camundongos Endogâmicos BALB C , Monócitos/imunologia , Monócitos/patologia , Miocardite/induzido quimicamente , Miocardite/imunologia , Miocárdio/imunologia , Miosinas , Transdução de Sinais/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
BACKGROUND: Activation of innate pattern-recognition receptors promotes CD4+ T-cell-mediated autoimmune myocarditis and subsequent inflammatory cardiomyopathy. Mechanisms that counterregulate exaggerated heart-specific autoimmunity are poorly understood. METHODS AND RESULTS: Experimental autoimmune myocarditis was induced in BALB/c mice by immunization with α-myosin heavy chain peptide and complete Freund's adjuvant. Together with interferon-γ, heat-killed Mycobacterium tuberculosis, an essential component of complete Freund's adjuvant, converted CD11b(hi)CD11c(-) monocytes into tumor necrosis factor-α- and nitric oxide synthase 2-producing dendritic cells (TipDCs). Heat-killed M. tuberculosis stimulated production of nitric oxide synthase 2 via Toll-like receptor 2-mediated nuclear factor-κB activation. TipDCs limited antigen-specific T-cell expansion through nitric oxide synthase 2-dependent nitric oxide production. Moreover, they promoted nitric oxide synthase 2 production in hematopoietic and stromal cells in a paracrine manner. Consequently, nitric oxide synthase 2 production by both radiosensitive hematopoietic and radioresistant stromal cells prevented exacerbation of autoimmune myocarditis in vivo. CONCLUSIONS: Innate Toll-like receptor 2 stimulation promotes formation of regulatory TipDCs, which confine autoreactive T-cell responses in experimental autoimmune myocarditis via nitric oxide. Therefore, activation of innate pattern-recognition receptors is critical not only for disease induction but also for counterregulatory mechanisms, protecting the heart from exaggerated autoimmunity.
Assuntos
Doenças Autoimunes/fisiopatologia , Células Dendríticas/metabolismo , Tolerância Imunológica/fisiologia , Interferon gama/fisiologia , Miocardite/fisiopatologia , Óxido Nítrico Sintase Tipo II/biossíntese , Óxido Nítrico/biossíntese , Transdução de Sinais , Linfócitos T Auxiliares-Indutores/patologia , Receptor 2 Toll-Like/fisiologia , Animais , Doenças Autoimunes/imunologia , Cardiomiopatia Dilatada/etiologia , Cardiomiopatia Dilatada/imunologia , Cardiomiopatia Dilatada/prevenção & controle , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas/citologia , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/imunologia , Indução Enzimática/efeitos dos fármacos , Células-Tronco Hematopoéticas/enzimologia , Células-Tronco Hematopoéticas/efeitos da radiação , Tolerância Imunológica/imunologia , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Monócitos/citologia , Monócitos/efeitos dos fármacos , Mycobacterium tuberculosis/imunologia , Miocardite/imunologia , NF-kappa B/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Comunicação Parácrina , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Quimera por Radiação , Tolerância a Radiação , Células Estromais/enzimologia , Células Estromais/efeitos da radiação , Linfócitos T Auxiliares-Indutores/imunologia , Miosinas Ventriculares/imunologia , Miosinas Ventriculares/toxicidadeRESUMO
AIMS: Tumour necrosis factor α (TNF-α) represents a classical pro-inflammatory cytokine, and its increased levels positively correlate with the severity of many cardiovascular diseases. Surprisingly, some heart failure patients receiving high doses of anti-TNF-α antibodies showed serious health worsening. This work aimed to examine the role of TNF-α signalling on the development and progression of myocarditis and heart-specific autoimmunity. METHODS AND RESULTS: Mice with genetic deletion of TNF-α (Tnf+/- and Tnf-/-) and littermate controls (Tnf+/+) were used to study myocarditis in the inducible and the transgenic T cell receptor (TCRM) models. Tnf+/- and Tnf-/- mice immunized with α-myosin heavy chain peptide (αMyHC) showed reduced myocarditis incidence, but the susceptible animals developed extensive inflammation in the heart. In the TCRM model, defective TNF-α production was associated with increased mortality at a young age due to cardiomyopathy and cardiac fibrosis. We could confirm that TNF-α as well as the secretome of antigen-activated heart-reactive effector CD4+ T (Teff) cells effectively activated the adhesive properties of cardiac microvascular endothelial cells (cMVECs). Our data suggested that TNF-α produced by endothelial in addition to Teff cells promoted leucocyte adhesion to activated cMVECs. Analysis of CD4+ T lymphocytes from both models of myocarditis showed a strongly increased fraction of Teff cells in hearts, spleens, and in the blood of Tnf+/- and Tnf-/- mice. Indeed, antigen-activated Tnf-/- Teff cells showed prolonged long-term survival and TNF-α cytokine-induced cell death of heart-reactive Teff. CONCLUSION: TNF-α signalling promotes myocarditis development by activating cardiac endothelial cells. However, in the case of established disease, TNF-α protects from exacerbating cardiac inflammation by inducing activation-induced cell death of heart-reactive Teff. These data might explain the lack of success of standard anti-TNF-α therapy in heart failure patients and open perspectives for T cell-targeted approaches.
Assuntos
Doenças Autoimunes , Insuficiência Cardíaca , Miocardite , Animais , Camundongos , Linfócitos T CD4-Positivos , Citocinas/metabolismo , Morte , Células Endoteliais/patologia , Insuficiência Cardíaca/metabolismo , Inflamação/metabolismo , Miocardite/metabolismo , Miocárdio/metabolismo , Inibidores do Fator de Necrose Tumoral , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The etiology of multisystem inflammatory syndrome in children (MIS-C), frequently observed following COVID-19 infection, remains elusive. This study unveils insights derived from cytokine analysis in the sera of MIS-C patients, both before and after the administration of intravenous immunoglobulin (IVIG) and glucocorticosteroids (GCS). In this study, we employed a comprehensive 45-cytokine profile encompassing a spectrum of widely recognized proinflammatory and antiinflammatory cytokines, as well as growth factors, along with other soluble mediators. The analysis delineates three principal cytokine-concentration patterns evident in the patients' sera. Pattern no.1 predominantly features proinflammatory cytokines (IL-6, IL-15, IL-1ra, granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor α (TNFα), C-X-C motif chemokine ligand 10 (CXCL10/ IP-10), and IL-10) exhibiting elevated concentrations upon admission, swiftly normalizing post-hospital treatment. Pattern no. 2 includes cytokines (IL-17 A, IL-33, IFNγ, vascular endothelial growth factor (VEGF), and programmed death ligand (PD-L1)) with moderately elevated levels at admission, persisting over 7-10 days of hospitalization despite the treatment. Pattern no. 3 comprises cytokines which concentrations escalated after 7-10 days of hospitalization and therapy, including IL-1α, IL-1ß, IL-2, IL-13, platelet-derived growth factor AA/BB (PDGF AA/BB). The observed in cytokine profile of MIS-C patients showed a transition from acute inflammation to sustaining inflammation which turned into induction of humoral memory mechanisms and various defense mechanisms, contributing to recovery.
Assuntos
COVID-19 , Citocinas , Síndrome de Resposta Inflamatória Sistêmica , Humanos , Criança , COVID-19/imunologia , COVID-19/sangue , COVID-19/complicações , Síndrome de Resposta Inflamatória Sistêmica/sangue , Síndrome de Resposta Inflamatória Sistêmica/imunologia , Citocinas/sangue , Masculino , Feminino , Pré-Escolar , Adolescente , Imunoglobulinas Intravenosas/uso terapêutico , Lactente , SARS-CoV-2/imunologia , Glucocorticoides/uso terapêutico , Criança HospitalizadaRESUMO
The antioxidant and anti-inflammatory activities of acylated and decarboxylated gomphrenins, as well as Basella alba L. fruit extract, were investigated in relation to gomphrenin, known for its high biological potential. The most abundant natural acylated gomphrenins, namely, 6'-O-E-caffeoyl-gomphrenin (malabarin) and 6'-O-E-4-coumaroyl-gomphrenin (globosin), were isolated from B. alba extract for the studies. In addition, controlled thermal decarboxylation of gomphrenin in the purified B. alba extract at 65-75 °C resulted in the formation of the most prevalent decarboxylated products, including 17-decarboxy-gomphrenin and 2,17-bidecarboxy-gomphrenin, along with their isoforms. The structures of the decarboxylated pigments were confirmed by NMR analyses. Exploring the matrix effect on pigment reactivity revealed a tremendous increase in the stability of all betacyanins after the initial stage of extract purification using a cation exchanger under various conditions. This indicates the removal of a substantial portion of the unfavorable matrix from the extract, which presumably contains reactive species that could otherwise degrade the pigments. Furthermore, the high concentration of citrates played a significant role in favoring the formation of 2-decarboxy-gomphrenin to a considerable extent. In vitro screening experiments revealed that the tested compounds demonstrated strong anti-inflammatory properties in lipopolysaccharide (LPS)-activated human macrophages. This effect encompassed the selective inhibition of cytokine and chemokine release from activated macrophages, modulation of the chemotactic activity of immune cells, and the regulation of tissue remodeling mediators' release.
Assuntos
Betacianinas , Caryophyllales , Humanos , Betacianinas/química , Spinacia oleracea , Frutas/química , Extratos Vegetais/química , Cromatografia Líquida de Alta Pressão/métodos , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/análise , Betalaínas/farmacologia , Betalaínas/químicaRESUMO
Autophagy, a dynamic and complex process responsible for the clearance of damaged cellular components, plays a crucial role in maintaining myocardial homeostasis. In the context of heart failure, autophagy has been recognized as a response mechanism aimed at counteracting pathogenic processes and promoting cellular health. Its relevance has been underscored not only in various animal models, but also in the human heart. Extensive research efforts have been dedicated to understanding the significance of autophagy and unravelling its complex molecular mechanisms. This review aims to consolidate the current knowledge of the involvement of autophagy during the progression of heart failure. Specifically, we provide a comprehensive overview of published data on the impact of autophagy deregulation achieved by genetic modifications or by pharmacological interventions in ischemic and non-ischemic models of heart failure. Furthermore, we delve into the intricate molecular mechanisms through which autophagy regulates crucial cellular processes within the three predominant cell populations of the heart: cardiomyocytes, cardiac fibroblasts, and endothelial cells. Finally, we emphasize the need for future research to unravel the therapeutic potential associated with targeting autophagy in the management of heart failure.