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Chagas disease, the parasitic infection caused by Trypanosoma cruzi, afflicts about 6 million people in Latin America. Here, we investigated the hypothesis that T. cruzi may fuel heart parasitism by activating B1R, a G protein-coupled (brady) kinin receptor whose expression is upregulated in inflamed tissues. Studies in WT and B1R-/- mice showed that T. cruzi DNA levels (15 days post infection-dpi) were sharply reduced in the transgenic heart. FACS analysis revealed that frequencies of proinflammatory neutrophils and monocytes were diminished in B1R-/- hearts whereas CK-MB activity (60 dpi) was exclusively detected in B1R+/+ sera. Since chronic myocarditis and heart fibrosis (90 dpi) were markedly attenuated in the transgenic mice, we sought to determine whether a pharmacological blockade of the des-Arg9-bradykinin (DABK)/B1R pathway might alleviate chagasic cardiomyopathy. Using C57BL/6 mice acutely infected by a myotropic T. cruzi strain (Colombian), we found that daily treatment (15-60 dpi) with R-954 (B1R antagonist) reduced heart parasitism and blunted cardiac injury. Extending R-954 treatment to the chronic phase (120-160 dpi), we verified that B1R targeting (i) decreased mortality indexes, (ii) mitigated chronic myocarditis, and (iii) ameliorated heart conduction disturbances. Collectively, our study suggests that a pharmacological blockade of the proinflammatory KKS/DABK/B1R pathway is cardioprotective in acute and chronic Chagas disease.
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Introduction: Pulmonary fibrosis is a destructive, progressive disease that dramatically reduces life quality of patients, ultimately leading to death. Therapeutic regimens for pulmonary fibrosis have shown limited benefits, hence justifying the efforts to evaluate the outcome of alternative treatments. Methods: Using a mouse model of bleomycin (BLM)-induced lung fibrosis, in the current work we asked whether treatment with pro-resolution molecules, such as pro-resolving lipid mediators (SPMs) could ameliorate pulmonary fibrosis. To this end, we injected aspirin-triggered resolvin D1 (7S,8R,17R-trihydroxy-4Z,9E,11E,13Z,15E19Z-docosahexaenoic acid; ATRvD1; i.v.) 7 and 10 days after BLM (intratracheal) challenge and samples were two weeks later. Results and discussion: Assessment of outcome in the lung tissues revealed that ATRvD1 partially restored lung architecture, reduced leukocyte infiltration, and inhibited formation of interstitial edema. In addition, lung tissues from BLM-induced mice treated with ATRvD1 displayed reduced levels of TNF-α, MCP-1, IL-1-ß, and TGF-ß. Of further interest, ATRvD1 decreased lung tissue expression of MMP-9, without affecting TIMP-1. Highlighting the beneficial effects of ATRvD1, we found reduced deposition of collagen and fibronectin in the lung tissues. Congruent with the anti-fibrotic effects that ATRvD1 exerted in lung tissues, α-SMA expression was decreased, suggesting that myofibroblast differentiation was inhibited by ATRvD1. Turning to culture systems, we next showed that ATRvD1 impaired TGF-ß-induced fibroblast differentiation into myofibroblast. After showing that ATRvD1 hampered extracellular vesicles (EVs) release in the supernatants from TGF-ß-stimulated cultures of mouse macrophages, we verified that ATRvD1 also inhibited the release of EVs in the bronco-alveolar lavage (BAL) fluid of BLM-induced mice. Motivated by studies showing that BLM-induced lung fibrosis is linked to angiogenesis, we asked whether ATRvD1 could blunt BLM-induced angiogenesis in the hamster cheek pouch model (HCP). Indeed, our intravital microscopy studies confirmed that ATRvD1 abrogates BLM-induced angiogenesis. Collectively, our findings suggest that treatment of pulmonary fibrosis patients with ATRvD1 deserves to be explored as a therapeutic option in the clinical setting.
Assuntos
Fibrose Pulmonar , Humanos , Fibrose Pulmonar/induzido quimicamente , Fibrose Pulmonar/tratamento farmacológico , Fibrose Pulmonar/metabolismo , Aspirina/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Pulmão/patologia , Bleomicina/farmacologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
OBJECTIVES: The aim of this study was to evaluate the potential protective effect of Chromobacterium violaceum and violacein against periodontitis, in experimental models. MATERIALS AND METHODS: A double-blind experimental study on the exposure to C. violaceum or violacein in experimentally ligature-induced periodontitis, as preventive factors against alveolar bone loss by periodontitis. Bone resorption was assessed by morphometry. Antibacterial potential of violacein was assessed in an in vitro assay. Its cytotoxicity and genotoxicity were evaluated using the Ames test and SOS Chromotest assay, respectively. RESULTS: The potential of C. violaceum to prevent/limit bone resorption by periodontitis was confirmed. Daily exposure to 106 cells/ml in water intake since birth and only during the first 30 days of life significantly reduced bone loss from periodontitis in teeth with ligature. Violacein extracted from C. violaceum was efficient in inhibiting or limiting bone resorption and had a bactericidal effect against Porphyromonas gingivalis in the in vitro assay. CONCLUSIONS: We conclude that C. violaceum and violacein have the potential to prevent or limit the progression of periodontal diseases, in an experimental model. CLINICAL RELEVANCE: The effect of an environmental microorganism with potential action against bone loss in animal models with ligature-induced periodontitis represents the possibility of understanding the etiopathogenesis of periodontal diseases in populations exposed to C. violaceum and the possibility of new probiotics and antimicrobials. This would imply new preventive and therapeutic possibilities.
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Perda do Osso Alveolar , Antibacterianos , Periodontite , Animais , Perda do Osso Alveolar/prevenção & controle , Perda do Osso Alveolar/etiologia , Antibacterianos/administração & dosagem , Modelos Animais de Doenças , Periodontite/tratamento farmacológico , Periodontite/prevenção & controle , Periodontite/complicações , Indóis/administração & dosagem , Método Duplo-Cego , Porphyromonas gingivalis/efeitos dos fármacosRESUMO
The severe acute respiratory syndrome coronavirus 2, the agent of the ongoing coronavirus disease 2019 (COVID-19) pandemic, has spread worldwide since it was first identified in November 2019 in Wuhan, China. Since then, progress in pathogenesis linked severity of this systemic disease to the hyperactivation of network of cytokine-driven pro-inflammatory cascades. Here, we aimed to identify molecular biomarkers of disease severity by measuring the serum levels of inflammatory mediators in a Brazilian cohort of patients with COVID-19 and healthy controls (HCs). Critically ill patients in the intensive care unit were defined as such by dependence on oxygen supplementation (93% intubated and 7% face mask), and computed tomography profiles showing ground-glass opacity pneumonia associated to and high levels of D-dimer. Our panel of mediators included HMGB1, ATP, tissue factor, PGE2 , LTB4 , and cys-LTs. Follow-up studies showed increased serum levels of every inflammatory mediator in patients with COVID-19 as compared to HCs. Originally acting as a transcription factor, HMGB1 acquires pro-inflammatory functions following secretion by activated leukocytes or necrotic tissues. Serum levels of HMGB1 were positively correlated with cys-LTs, D-dimer, aspartate aminotransferase, and alanine aminotransferase. Notably, the levels of the classical alarmin HMGB1 were higher in deceased patients, allowing their discrimination from patients that had been discharged at the early pulmonary and hyperinflammatory phase of COVID-19. In particular, we verified that HMGB1 levels above 125.4 ng/ml is the cutoff that distinguishes patients that are at higher risk of death. In conclusion, we propose the use of serum levels of HMGB1 as a biomarker of severe prognosis of COVID-19.
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COVID-19 , Proteína HMGB1 , Humanos , Tromboplastina , COVID-19/diagnóstico , Biomarcadores , Prognóstico , Lipídeos , Trifosfato de AdenosinaRESUMO
Microangiopathy may worsen the clinical outcome of Chagas disease. Given the obstacles to investigating the dynamics of inflammation and angiogenesis in heart tissues parasitized by Trypanosoma cruzi, here we used intravital microscopy (IVM) to investigate microcirculatory alterations in the hamster cheek pouch (HCP) infected by green fluorescent protein-expressing T. cruzi (GFP-T. cruzi). IVM performed 3 days post-infection (3 dpi) consistently showed increased baseline levels of plasma extravasation. Illustrating the reciprocal benefits that microvascular leakage brings to the host-parasite relationship, these findings suggest that intracellular amastigotes, acting from inside out, stimulate angiogenesis while enhancing the delivery of plasma-borne nutrients and prosurvival factors to the infection foci. Using a computer-based analysis of images (3 dpi), we found that proangiogenic indexes were positively correlated with transcriptional levels of proinflammatory cytokines (pro-IL1ß and IFN-γ). Intracellular GFP-parasites were targeted by delaying for 24 h the oral administration of the trypanocidal drug benznidazole. A classification algorithm showed that benznidazole (>24 h) blunted angiogenesis (7 dpi) in the HCP. Unbiased proteomics (3 dpi) combined to pharmacological targeting of chymase with two inhibitors (chymostatin and TY-51469) linked T. cruzi-induced neovascularization (7 dpi) to the proangiogenic activity of chymase, a serine protease stored in secretory granules from mast cells.
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Megalin-mediated albumin endocytosis plays a critical role in albumin reabsorption in proximal tubule (PT) epithelial cells (PTECs). Some studies have pointed out the modulatory effect of bradykinin (BK) on urinary protein excretion, but its role in PT protein endocytosis has not yet been determined. Here, we studied the possible correlation between BK and albumin endocytosis in PT. Using LLC-PK1 cells, a model of PTECs, we showed that BK specifically inhibited megalin-mediated albumin endocytosis. This inhibitory effect of BK was mediated by B2 receptor (B2R) because it was abolished by HOE140, an antagonist of B2R, but it was not affected by Lys-des-Arg9-BK, an antagonist of B1. BK induced the stall of megalin in EEA1+ endosomes, but not in LAMP1+ lysosomes, leading to a decrease in surface megalin expression. In addition, we showed that BK, through B2R, activated calphostin C-sensitive protein kinase C, which mediated its effect on the surface megalin expression and albumin endocytosis. These results reveal an important modulatory mechanism of PT albumin endocytosis by BK, which opens new possibilities to understanding the effect of BK on urinary albumin excretion.
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Albuminas/metabolismo , Bradicinina/farmacologia , Endocitose/efeitos dos fármacos , Túbulos Renais Proximais/efeitos dos fármacos , Proteína-2 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Túbulos Renais Proximais/metabolismo , Células LLC-PK1 , Proteína Quinase C/metabolismo , Receptor B2 da Bradicinina/metabolismo , SuínosRESUMO
Intestinal barrier is essential for dietary products and microbiota compartmentalization and therefore gut homeostasis. When this barrier is broken, cecal content overflows into the peritoneal cavity, leading to local and systemic robust inflammatory response, characterizing peritonitis and sepsis. It has been shown that IL-1ß contributes with inflammatory storm during peritonitis and sepsis and its inhibition has beneficial effects to the host. Therefore, we investigated the mechanisms underlying IL-1ß secretion using a widely adopted murine model of experimental peritonitis. The combined injection of sterile cecal content (SCC) and the gut commensal bacteria Bacteroides fragilis leads to IL-1ß-dependent peritonitis, which was mitigated in mice deficient in NLRP3 (nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3) inflammasome components. Typically acting as a damage signal, SCC, but not B. fragilis, activates canonical pathway of NLRP3 promoting IL-1ß secretion in vitro and in vivo. Strikingly, absence of fiber in the SCC drastically reduces IL-1ß production, whereas high-fiber SCC conversely increases this response in an NLRP3-dependent manner. In addition, NLRP3 was also required for IL-1ß production induced by purified dietary fiber in primed macrophages. Extending to the in vivo context, IL-1ß-dependent peritonitis was worsened in mice injected with B. fragilis and high-fiber SCC, whereas zero-fiber SCC ameliorates the pathology. Corroborating with the proinflammatory role of dietary fiber, IL-1R-deficient mice were protected from peritonitis induced by B. fragilis and particulate bran. Overall, our study highlights a function, previously unknown, for dietary fibers in fueling peritonitis through NLRP3 activation and IL-1ß secretion outside the gut.
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Infecções por Bacteroides/imunologia , Bacteroides fragilis/imunologia , Fibras na Dieta/efeitos adversos , Inflamassomos/metabolismo , Interleucina-1beta/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/deficiência , Peritonite/imunologia , Animais , Infecções por Bacteroides/microbiologia , Dieta , Fibras na Dieta/administração & dosagem , Modelos Animais de Doenças , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Peritonite/microbiologia , Receptores de Interleucina-1/deficiência , Receptores de Interleucina-1/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologiaRESUMO
The global SARS-CoV-2 pandemic starting in 2019 has already reached more than 2.3 million deaths. Despite the scientific community's efforts to investigate the COVID-19 disease, a drug for effectively treating or curing patients yet needs to be discovered. Hematopoietic stem cells (HSC) differentiating into immune cells for defense express COVID-19 entry receptors, and COVID-19 infection hinders their differentiation. The importance of purinergic signaling in HSC differentiation and innate immunity has been recognized. The metabotropic P2Y14 receptor subtype, activated by UDP-glucose, controls HSC differentiation and mobilization. Thereon, the exacerbated activation of blood immune cells amplifies the inflammatory state observed in COVID-19 patients, specially through the continuous release of reactive oxygen species and extracellular neutrophil traps (NETs). Further, the P2Y14 subtype, robustly inhibits the infiltration of neutrophils into various epithelial tissues, including lungs and kidneys. Here we discuss findings suggesting that antagonism of the P2Y14 receptor could prevent the progression of COVID-19-induced systemic inflammation, which often leads to severe illness and death cases. Considering the modulation of neutrophil recruitment of extreme relevance for respiratory distress and lung failure prevention, we propose that P2Y14 receptor inhibition by its selective antagonist PPTN could limit neutrophil recruitment and NETosis, hence limiting excessive formation of oxygen reactive species and proteolytic activation of the kallikrein-kinin system and subsequent bradykinin storm in the alveolar septa of COVID-19 patients.