RESUMO

OBJECTIVE: To evaluate whether colchicine treatment was associated with the inhibition of NLRP3 inflammasome activation in patients with COVID-19. METHODS: We present a post hoc analysis from a double-blinded placebo-controlled randomized clinical trial (RCT) on the effect of colchicine for the treatment of COVID-19. Serum levels of NOD-like receptor protein 3 (NLRP3) inflammasome products-active caspase-1 (Casp1p20), IL-1ß, and IL-18-were assessed at enrollment and after 48-72 h of treatment in patients receiving standard-of-care (SOC) plus placebo vs. those receiving SOC plus colchicine. The colchicine regimen was 0.5 mg tid for 5 days, followed by 0.5 mg bid for another 5 days. RESULTS: Thirty-six patients received SOC plus colchicine, and thirty-six received SOC plus placebo. Colchicine reduced the need for supplemental oxygen and the length of hospitalization. On Days 2-3, colchicine lowered the serum levels of Casp1p20 and IL-18, but not IL-1ß. CONCLUSION: Treatment with colchicine inhibited the activation of the NLRP3 inflammasome, an event triggering the 'cytokine storm' in COVID-19. TRIAL REGISTRATION NUMBERS: RBR-8jyhxh.

Assuntos

COVID-19 , Inflamassomos , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Interleucina-18 , Proteínas NLR , Colchicina/uso terapêutico , Interleucina-1beta/metabolismo

RESUMO

Osteoclasts play a key role in the regulation of bone mass and are highly active metabolically. Here we show that a metabolic reprogramming toward the hexosamine biosynthetic pathway (HBP) is required not only for osteoclast differentiation but also to determine the bone resorption mode during physiological and pathological bone remodeling. We found that pharmacological inhibition of O-GlcNAc transferase (OGT) significantly reduced protein O-GlcNAcylation and osteoclast differentiation. Accordingly, genetic deletion of OGT also inhibited osteoclast formation and downregulated critical markers related to osteoclasts differentiation and function (NFATc1, αvintegrin, cathepsin K). Indeed, cells treated with OSMI-1, an OGT inhibitor, also reduced nuclear translocation of NFATc1. Furthermore, the addition of exogenous N-acetylglucosamine (GlcNAc) strongly increased osteoclast formation and demineralization ability. Strikingly, our data show for the first time that O-GlcNAcylation facilitates an aggressive trench resorption mode in human cells. The incubation of osteoclasts with exogenous GlcNAc increases the percentage of erosion by trench while having no effect on pit resorption mode. Through time-lapse recording, we documented that osteoclasts making trenches moving across the bone surface are sensitive to GlcNAcylation. Finally, osteoclast-specific Ogt-deficient mice show increased bone density and reduced inflammation-induced bone loss during apical periodontitis model. We show that osteoclast-specific Ogt-deficient mice are less susceptible to develop bacterial-induced periapical lesion. Consistent with this, Ogt-deleted mice showed a decreased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site. In summary, here we describe a hitherto undiscovered role of the HBP/O-GlcNAcylation axis tuning resorption mode and dictating bone resorption outcome.

Assuntos

Reabsorção Óssea , Periodontite Periapical , Camundongos , Humanos , Animais , Hexosaminas/metabolismo , Vias Biossintéticas , Reabsorção Óssea/metabolismo , Osteoclastos/metabolismo , Fatores de Transcrição/metabolismo

RESUMO

We aimed to evaluate whether the administration of riboflavin to septic animals reduces inflammation, oxidative stress, organ dysfunction, and mortality. C57BL/6 mice, 6-8 weeks old, were allocated to the study group (polymicrobial sepsis induced by cecal ligation and puncture (CLP) + antibiotic + iv riboflavin), control (CLP + antibiotic + iv saline), or naïve (non-operated controls). Serum concentrations of alanine aminotransferase (ALT), creatine kinase-MB (CK-MB), urea, and creatinine, and markers of inflammation [interleukin (IL)-6, tumor necrosis factor (TNF)-α, keratinocyte-derived chemokine (KC), and macrophage inflammatory protein (MIP)-2)], and oxidative stress (malondialdehyde (MDA) were measured 12 h after the experiment. Animal survival rates were calculated after 7 days. Means between groups were compared using linear regression models adjusted under the Bayesian approach. No significant difference was observed between control and study groups in serum concentrations of IL-6 (95% credible interval) (-0.35 to 0.44), TNF-α (-15.7 to 99.1), KC (-0.13 to 0.05), MIP-2 (-0.84 to 0.06), MDA (-1.25 to 2.53), or ALT (-6.6 to 11.5). Serum concentrations of CK-MB (-145.1 to -30.1), urea (-114.7 to -15.1), and creatinine (-1.14 to -0.01) were higher in the study group. Survival was similar in both groups (P=0.8). Therefore, the use of riboflavin in mice undergoing sepsis induced by CLP did not reduce inflammation, oxidative stress, organ dysfunction, or mortality compared with placebo.

Assuntos

Antioxidantes , Sepse , Animais , Antibacterianos/uso terapêutico , Anti-Inflamatórios/uso terapêutico , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Teorema de Bayes , Quimiocinas , Creatinina , Inflamação/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Modelos Teóricos , Insuficiência de Múltiplos Órgãos/tratamento farmacológico , Riboflavina/uso terapêutico , Sepse/tratamento farmacológico , Sepse/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Ureia

RESUMO

We aimed to evaluate whether the administration of riboflavin to septic animals reduces inflammation, oxidative stress, organ dysfunction, and mortality. C57BL/6 mice, 6-8 weeks old, were allocated to the study group (polymicrobial sepsis induced by cecal ligation and puncture (CLP) + antibiotic + iv riboflavin), control (CLP + antibiotic + iv saline), or naïve (non-operated controls). Serum concentrations of alanine aminotransferase (ALT), creatine kinase-MB (CK-MB), urea, and creatinine, and markers of inflammation [interleukin (IL)-6, tumor necrosis factor (TNF)-α, keratinocyte-derived chemokine (KC), and macrophage inflammatory protein (MIP)-2)], and oxidative stress (malondialdehyde (MDA) were measured 12 h after the experiment. Animal survival rates were calculated after 7 days. Means between groups were compared using linear regression models adjusted under the Bayesian approach. No significant difference was observed between control and study groups in serum concentrations of IL-6 (95% credible interval) (-0.35 to 0.44), TNF-α (-15.7 to 99.1), KC (-0.13 to 0.05), MIP-2 (-0.84 to 0.06), MDA (-1.25 to 2.53), or ALT (-6.6 to 11.5). Serum concentrations of CK-MB (-145.1 to -30.1), urea (-114.7 to -15.1), and creatinine (-1.14 to -0.01) were higher in the study group. Survival was similar in both groups (P=0.8). Therefore, the use of riboflavin in mice undergoing sepsis induced by CLP did not reduce inflammation, oxidative stress, organ dysfunction, or mortality compared with placebo.

RESUMO

While Treg cells are responsible for self-tolerance and immune homeostasis, pathogenic autoreactive Th17 cells produce pro-inflammatory cytokines that lead to tissue damage associated with autoimmunity, as observed in multiple sclerosis. Therefore, the immunological balance between Th17 and Treg cells may represent a promising option for immune therapy. Statin drugs are used to treat dyslipidemia; however, besides their effects on preventing cardiovascular diseases, statins also have anti-inflammatory effects. Here, we investigated the role of pitavastatin on experimental autoimmune encephalomyelitis (EAE) and the differentiation of Treg and Th17 cells. EAE was induced by immunizing C57BL/6 mice with MOG35-55. EAE severity was determined by analyzing the clinical score and inflammatory parameters in the spinal cord. Naive CD4 T cells were cultured under Treg and Th17-skewing conditions in vitro in the presence of pitavastatin. We found that pitavastatin decreased EAE development, which was accompanied by a reduction of all parameters investigated. Pitavastatin also reduced the expression of IBA1 and pSTAT3 (Y705 and S727) in the spinal cords of EAE mice. Interestingly, the reduction of Th17 cell frequency in the draining lymph nodes of EAE mice treated with pitavastatin was followed by an increase of Treg cells. Indeed, pitavastatin directly affects T cell differentiation in vitro by decreasing Th17 and increasing Treg cell differentiation. Mechanistically, pitavastatin effects are dependent on mevalonate synthesis. Thus, our data show the potential anti-inflammatory effect of pitavastatin on the pathogenesis of the experimental neuroinflammation by modulating the Th17/Treg axis.

Assuntos

Anti-Inflamatórios/farmacologia , Diferenciação Celular/efeitos dos fármacos , Encefalomielite Autoimune Experimental/prevenção & controle , Ácido Mevalônico/metabolismo , Quinolinas/farmacologia , Medula Espinal/efeitos dos fármacos , Linfócitos T Reguladores/efeitos dos fármacos , Células Th17/efeitos dos fármacos , Animais , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalomielite Autoimune Experimental/induzido quimicamente , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Mediadores da Inflamação/metabolismo , Linfonodos/efeitos dos fármacos , Linfonodos/imunologia , Linfonodos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Glicoproteína Mielina-Oligodendrócito , Fragmentos de Peptídeos , Medula Espinal/imunologia , Medula Espinal/metabolismo , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/metabolismo , Células Th17/imunologia , Células Th17/metabolismo

RESUMO

Special attention has emerged towards biomass smoke-induced chronic obstructive pulmonary disease (COPD), providing new knowledge for prevention and therapeutic approach of non-smoker COPD patients. However, the understanding of biomass smoke COPD is still limited and somewhat controversial. The aim of the present study was to compare COPD exclusively caused by tobacco smoking with COPD exclusively caused by environmental or occupational exposures. For this cross-sectional study, COPD patients were recruited from outpatient clinics and formed two groups: non-smoker COPD group (n=16) with exposure to biomass smoke who did not smoke cigarette and tobacco smoker COPD group (n=15) with people who did not report biomass smoke exposure. Subjects underwent pulmonary function tests, thoracic high-resolution computed tomography, 6-min walk test, and sputum induction. The non-smoker COPD group had biomass smoke exposure of 133.3±86 hour-years. The tobacco COPD group smoked 48.5±27.4 pack-years. Women were 62.5 and 66.7%, respectively, of non-smokers and smokers. The non-smoker COPD group showed higher prevalence of dyspnea, lower arterial oxygen tension (PaO2), and lower arterial oxygen saturation (SaO2%) with similar spirometry results, lung volumes, and diffusion capacity. Regarding inflammatory biomarkers, differences were detected in sputum number of lymphomononuclear cells and in sputum concentrations of interleukin (IL)-6 and IL-8 with higher values in the smoker group. Emphysema was more prevalent in the tobacco smoker group, which also showed higher relative bronchial wall thickness and lower lung density by quantitative analysis. Biomass smoke induced more hypoxemia compared to tobacco in COPD patients with similar severity.

Assuntos

Biomassa , Hipóxia/diagnóstico por imagem , Nicotiana/efeitos adversos , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Fumaça/efeitos adversos , Idoso , Estudos Transversais , Exposição Ambiental , Feminino , Humanos , Hipóxia/etiologia , Masculino , Pessoa de Meia-Idade , Doença Pulmonar Obstrutiva Crônica/etiologia , Testes de Função Respiratória , Espirometria , Escarro/química , Tomografia Computadorizada por Raios X

RESUMO

BACKGROUND: Although some glycolytic intermediates have been shown to modulate several cell type formation and activation, the functional role of fructose 1,6-bisphosphate (FBP) on osteoclastogenesis is still unknown. METHODS: Osteoclastogenesis was evaluated on bone marrow preosteoclasts cultured with M-CSF - 30 ng/ml, RANKL - 10 ng/ml, and two concentrations of FBP (100 and 300 µM). TRAP-positive stained cells were counted, and osteoclastogenic marker genes expression were evaluated by qPCR. Osteoclasts resorption capacity was evaluated by the expression of specific enzymes and capacity to resorb a mineralized matrix. The NF-κB activation was detected using RAW 264.7, stably expressing luciferase on the NF-κB responsive promoter. RESULTS: We show that FBP, the product of the first stage of glycolysis, inhibited RANKL-induced osteoclasts differentiation and TRAP activity. The treatment of preosteoclasts with FBP attenuated osteoclast fusion and formation, without affecting cell viability. Moreover, the inhibition of several osteoclastogenic marker genes expression (TRAP, OSCAR, DC-STAMP, Integrin αv, NFATc1) by FBP correlates with a reduction of mineralized matrix resorption capacity. The mechanism underlying FBP-inhibition of osteoclastogenesis involves NF-κB/NFATc1 signaling pathway inhibition. CONCLUSION: Altogether these data show a protective role of a natural glycolytic intermediate in bone homeostasis that may have therapeutic benefit for osteolytic diseases.

Assuntos

Frutosedifosfatos/farmacologia , NF-kappa B/metabolismo , Fatores de Transcrição NFATC/genética , Osteoclastos/efeitos dos fármacos , Osteogênese/efeitos dos fármacos , Ligante RANK/farmacologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Fêmur/citologia , Masculino , Camundongos Endogâmicos C57BL , Osteoclastos/citologia , Tíbia/citologia

RESUMO

Apical periodontitis is an inflammatory disorder that results from the host immune response to microbial infection through the dental pulp, leading to alveolar bone destruction. The nod-like receptor 12 (NLRP12) is an atypical intracellular sensor of the NLR family that is involved in the negative regulation of several inflammatory conditions and also osteoclastogenesis. However, the role of NLRP12 in the regulation of immune response and bone loss induced by bacterial infection remains unclear. Here we investigated the development of apical periodontitis in wild-type (WT) and NLRP12 knockout (NLRP12-/-) mice by using micro-computed tomography together with histological, immunohistochemical, and molecular analyses. We found that NLRP12-/- mice are highly susceptible to apical periodontitis induced by bacterial infection, which is associated with an elevated infiltration of neutrophils and macrophages, periapical lesion extension, and alveolar bone destruction. Furthermore, NLRP12-/- mice showed a high expression of inflammatory cytokines ( Il1b, Il6, and Tnfa) and the osteoclastogenic markers ( Rankl and Acp5) in the periapical tissues. Consistent with this observation, NLRP12-/- mice showed an increased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site, which was associated with augmented expression of the osteoclast effector genes, Ctsk and Mmp9. Mechanistically, NLRP12-deficient preosteoclasts showed elevated IκB-α degradation and p65 phosphorylation when stimulated with receptor activator of nuclear factor (NF)-κB ligand (RANKL). Similarly, increased IκB-α degradation was observed in the periapical tissue of NLRP12-/- mice. Furthermore, our in vitro study showed that preosteoclasts from NLRP12-/- mice exhibited higher RANKL-induced osteoclastogenesis, which was synergistically amplified by interleukin-1ß and tumor necrosis factor α (mimicking an inflammatory periapical milieu). In conclusion, our data show that NLRP12 exhibits a protective role in the periapical bone destruction by attenuating inflammation and osteoclastogenesis through negative regulation of the NF-κB pathway.

Assuntos

Periodontite Periapical , Ligante RANK , Animais , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos NOD , Osteoclastos , Microtomografia por Raio-X

RESUMO

Special attention has emerged towards biomass smoke-induced chronic obstructive pulmonary disease (COPD), providing new knowledge for prevention and therapeutic approach of non-smoker COPD patients. However, the understanding of biomass smoke COPD is still limited and somewhat controversial. The aim of the present study was to compare COPD exclusively caused by tobacco smoking with COPD exclusively caused by environmental or occupational exposures. For this cross-sectional study, COPD patients were recruited from outpatient clinics and formed two groups: non-smoker COPD group (n=16) with exposure to biomass smoke who did not smoke cigarette and tobacco smoker COPD group (n=15) with people who did not report biomass smoke exposure. Subjects underwent pulmonary function tests, thoracic high-resolution computed tomography, 6-min walk test, and sputum induction. The non-smoker COPD group had biomass smoke exposure of 133.3±86 hour-years. The tobacco COPD group smoked 48.5±27.4 pack-years. Women were 62.5 and 66.7%, respectively, of non-smokers and smokers. The non-smoker COPD group showed higher prevalence of dyspnea, lower arterial oxygen tension (PaO2), and lower arterial oxygen saturation (SaO2%) with similar spirometry results, lung volumes, and diffusion capacity. Regarding inflammatory biomarkers, differences were detected in sputum number of lymphomononuclear cells and in sputum concentrations of interleukin (IL)-6 and IL-8 with higher values in the smoker group. Emphysema was more prevalent in the tobacco smoker group, which also showed higher relative bronchial wall thickness and lower lung density by quantitative analysis. Biomass smoke induced more hypoxemia compared to tobacco in COPD patients with similar severity.

Assuntos

Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Fumaça/efeitos adversos , Nicotiana/efeitos adversos , Biomassa , Doença Pulmonar Obstrutiva Crônica/diagnóstico por imagem , Hipóxia/diagnóstico por imagem , Testes de Função Respiratória , Espirometria , Escarro/química , Tomografia Computadorizada por Raios X , Estudos Transversais , Doença Pulmonar Obstrutiva Crônica/etiologia , Exposição Ambiental , Hipóxia/etiologia

RESUMO

OBJECTIVES: The aim of this study was to analyse the expression and function of nucleotide-binding oligomerization domain (NOD)1 and NOD2 in isolated cells of patients with rheumatoid arthritis (RA). METHOD: mRNA expression levels of NOD1, NOD2, and receptor-interacting serine/threonine kinase 2 (RIPK2) genes were determined by quantitative polymerase chain reaction (qPCR) in peripheral blood mononuclear cells (PBMCs) and synovial fluid T cells (SFTCs) isolated from RA and osteoarthritis (OA) patients. Cytokines were measured by enzyme-linked immunosorbent assay (ELISA) in plasma and cell culture supernatants. The stimulatory effect of RA SF was assessed by an in-vitro NOD2 activation assay using nuclear factor kappa B (NF-κB) luciferase-transfected cells. RESULTS: A significantly higher level of NOD2 and RIPK2 mRNA expression, but not NOD1, was observed on PBMCs and SFTCs isolated from RA patients compared to the OA control group. In addition, the NOD2 pathway up-regulation was functional, as stimulation of PBMCs with muramyl dipeptide (MDP) induced the production of higher amounts of tumour necrosis factor (TNF)-α, interleukin (IL)-8, and IL-1ß compared with OA PBMCs. Incubation of PBMCs from healthy donors with recombinant TNF-α or RA serum induced the expression of NOD2 mRNA. Finally, SF isolated from RA patients is able to activate the NF-κB signalling pathway in HEK293T-transfected cells in a NOD2-dependent manner. CONCLUSIONS: Our findings suggest that NOD2/RIPK2 signalling is up-regulated in immune cells of RA patients. Moreover, it seems that there is a NOD2 agonist in the SF of RA patients. Therefore, NOD2/RIPK2 activation can modulate the innate immune response and may play a role in the perpetuation of the inflammatory response in RA.