RESUMO
Essentials Complement, Toll-like receptors and coagulation cross-talk in the process of thromboinflammation. This is explored in a unique human whole-blood model of S. aureus bacteremia. Coagulation is here shown as a downstream event of C5a-induced tissue factor (TF) production. Combined inhibition of C5 and CD14 efficiently attenuated TF and coagulation. SUMMARY: Background There is extensive cross-talk between the complement system, the Toll-like receptors (TLRs), and hemostasis. Consumptive coagulopathy is a hallmark of sepsis, and is often mediated through increased tissue factor (TF) expression. Objectives To study the relative roles of complement, TLRs and TF in Staphylococcus aureus-induced coagulation. Methods Lepirudin-anticoagulated human whole blood was incubated with the three S. aureus strains Cowan, Wood, and Newman. C3 was inhibited with compstatin, C5 with eculizumab, C5a receptor 1 (C5aR1) and activated factor XII with peptide inhibitors, CD14, TLR2 and TF with neutralizing antibodies, and TLR4 with eritoran. Complement activation was measured by ELISA. Coagulation was measured according to prothrombin fragment 1 + 2 (PTF1 + 2 ) determined with ELISA, and TF mRNA, monocyte surface expression and functional activity were measured with quantitative PCR, flow cytometry, and ELISA, respectively. Results All three strains generated substantial and statistically significant amounts of C5a, terminal complement complex, PTF1 + 2 , and TF mRNA, and showed substantial TF surface expression on monocytes and TF functional activity. Inhibition of C5 cleavage most efficiently and significantly inhibited all six markers in strains Cowan and Wood, and five markers in Newman. The effect of complement inhibition was shown to be completely dependent on C5aR1. The C5 blocking effect was equally potentiated when combined with blocking of CD14 or TLR2, but not TLR4. TF blocking significantly reduced PTF1 + 2 levels to baseline levels. Conclusions S. aureus-induced coagulation in human whole blood was mainly attributable to C5a-induced mRNA upregulation, monocyte TF expression, and plasma TF activity, thus underscoring complement as a key player in S. aureus-induced coagulation.
Assuntos
Bacteriemia/microbiologia , Coagulação Sanguínea , Ativação do Complemento , Complemento C5a/metabolismo , Monócitos/microbiologia , Infecções Estafilocócicas/microbiologia , Staphylococcus aureus/imunologia , Tromboplastina/metabolismo , Anticorpos Neutralizantes/farmacologia , Bacteriemia/sangue , Bacteriemia/genética , Bacteriemia/imunologia , Carga Bacteriana , Coagulação Sanguínea/efeitos dos fármacos , Ativação do Complemento/efeitos dos fármacos , Complemento C5a/antagonistas & inibidores , Complemento C5a/genética , Complemento C5a/imunologia , Inativadores do Complemento/farmacologia , Interações Hospedeiro-Patógeno , Humanos , Receptores de Lipopolissacarídeos/antagonistas & inibidores , Receptores de Lipopolissacarídeos/imunologia , Receptores de Lipopolissacarídeos/metabolismo , Viabilidade Microbiana , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Monócitos/metabolismo , Receptor da Anafilatoxina C5a/antagonistas & inibidores , Receptor da Anafilatoxina C5a/sangue , Receptor da Anafilatoxina C5a/imunologia , Transdução de Sinais , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/genética , Infecções Estafilocócicas/imunologia , Tromboplastina/genética , Fatores de Tempo , Receptor 2 Toll-Like/antagonistas & inibidores , Receptor 2 Toll-Like/sangue , Receptor 2 Toll-Like/imunologiaRESUMO
This study aimed to examine whether acute intermittent porphyria (AIP) is associated with systemic inflammation and whether the inflammation correlates with disease activity. A case-control study with 50 AIP cases and age-, sex- and place of residence-matched controls was performed. Plasma cytokines, insulin and C-peptide were analysed after an overnight fast using multiplex assay. Long pentraxin-3 (PTX3) and complement activation products (C3bc and TCC) were analysed using enzyme-linked immunosorbent assay (ELISA). Urine porphobilinogen ratio (U-PBG, µmol/mmol creatinine), haematological and biochemical tests were performed using routine methods. Questionnaires were used to register AIP symptoms, medication and other diseases. All 27 cytokines, chemokines and growth factors investigated were increased significantly in symptomatic AIP cases compared with controls (P < 0·0004). Hierarchical cluster analyses revealed a cluster with high visfatin levels and several highly expressed cytokines including interleukin (IL)-17, suggesting a T helper type 17 (Th17) inflammatory response in a group of AIP cases. C3bc (P = 0·002) and serum immunoglobulin (Ig)G levels (P = 0·03) were increased significantly in cases with AIP. The U-PBG ratio correlated positively with PTX3 (r = 0·38, P = 0·006), and with terminal complement complex (TCC) levels (r = 0·33, P = 0·02). PTX3 was a significant predictor of the biochemical disease activity marker U-PBG in AIP cases after adjustment for potential confounders in multiple linear regression analyses (P = 0·032). Prealbumin, C-peptide, insulin and kidney function were all decreased in the symptomatic AIP cases, but not in the asymptomatic cases. These results indicate that AIP is associated with systemic inflammation. Decreased C-peptide levels in symptomatic AIP cases indicate that reduced insulin release is associated with enhanced disease activity and reduced kidney function.
Assuntos
Inflamação/sangue , Porfiria Aguda Intermitente/sangue , Biomarcadores/sangue , Peptídeo C/sangue , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Humanos , Imunoglobulina G/sangue , Inflamação/imunologia , Inflamação/metabolismo , Insulina/sangue , Rim/imunologia , Rim/fisiopatologia , Masculino , Pessoa de Meia-Idade , Porfiria Aguda Intermitente/imunologia , Porfiria Aguda Intermitente/metabolismo , Pré-Albumina/metabolismo , Linfócitos T Auxiliares-Indutores/imunologiaRESUMO
Mediterranean spotted fever caused by Rickettsia conorii is a potentially lethal disease characterized by vascular inflammation affecting multiple organs. Studies of R. conorii so far have focused on activation of inflammatory cells and their release of inflammatory cytokines, but complement activation has not been investigated in R. conorii-infected patients. Here, we performed a comprehensive analysis of complement activation markers and the soluble cross-talking co-receptor CD14 (sCD14) in plasma from R. conorii-infected patients. The clinical data were supplemented with ex vivo experiments where the cytokine response was characterized in human whole blood stimulated with R. conorii. Complement activation markers at the level of C3 (C3bc, C3bBbP) and terminal pathway activation (sC5b-9), as well as sCD14, were markedly elevated (p <0.01 for all), and closely correlated (p <0.05 for all), in patients at admission compared with healthy matched controls. All tested markers were significantly reduced to baseline values at time of follow up. Rickettsia conorii incubated in human whole blood was shown to trigger complement activation accompanied by release of the inflammatory cytokines interleukin-1ß (IL-1ß), IL-6, IL-8 and tumour necrosis factor. Whereas inhibition of either C3 or CD14 had only a minor effect on released cytokines, combined inhibition of C3 and CD14 resulted in significant reduction, virtually to baseline levels, of the four cytokines (p <0.05 for all). Our data show that complement is markedly activated upon R. conorii infection and complement activation is, together with CD14, responsible for a major part of the cytokine response induced by R. conorii in human whole blood.
Assuntos
Febre Botonosa/imunologia , Febre Botonosa/metabolismo , Ativação do Complemento/imunologia , Proteínas do Sistema Complemento/imunologia , Citocinas/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Rickettsia conorii/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores , Febre Botonosa/microbiologia , Estudos de Casos e Controles , Citocinas/sangue , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
Both the complement system and tissue factor (TF), a key initiating component of coagulation, are activated in sepsis, and cross-talk occurs between the complement and coagulation systems. C1-inhibitor (C1-INH) can act as a regulator in both systems. Our aim in this study was to examine this cross-talk by investigating the effects of C1-INH on Escherichia coli-induced haemostasis and inflammation. Fresh human whole blood collected in lepirudin was incubated with E. coli or ultrapurified E. coli lipopolysaccharide (LPS) in the absence or presence of C1-INH or protease-inactivated C1-INH. C3 activation was blocked by compstatin, a specific C3 convertase inhibitor. TF mRNA was measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and TF surface expression was measured by flow cytometry. In plasma, the terminal complement complex, prothrombin F1·2 (PTF1·2) and long pentraxin 3 (PTX3) were measured by enzyme-linked immunosorbent assay (ELISA). Cytokines were analysed using a multiplex kit. C1-INH (1·25-5 mg/ml) reduced both LPS- and E. coli-induced coagulation, measured as a reduction of PTF1·2 in plasma, efficiently and dose-dependently (P < 0·05). Both LPS and E. coli induced marked up-regulation of TF mRNA levels and surface expression on whole blood monocytes. This up-regulation was reduced efficiently by treatment with C1-INH (P < 0·05). C1-INH reduced the release of PTX3 (P < 0·05) and virtually all cytokines measured (P < 0·05). Complement activation was inhibited more efficiently with compstatin than with C1-INH. C1-INH inhibited most of the other readouts more efficiently, consistent with additional non-complement-dependent effects. These results indicate that complement plays a role in activating coagulation during sepsis and that C1-INH is a broad-spectrum attenuator of the inflammatory and haemostatic responses.