Methods in molecular biology (Clifton, N.J.), 2019
The innate immune complement system is a powerful defense cascade against pathogens, but can indu... more The innate immune complement system is a powerful defense cascade against pathogens, but can induce host tissue damage when overactivated. In pathological conditions, mainly but not restricted to renal diseases, such as lupus nephritis, atypical hemolytic uremic syndrome, and C3 glomerulopathies, complement is overactivated or dysregulated by autoantibodies directed against its components and regulators. Among the key autoantibody targets are the initiator of the classical complement pathway C1q, the alternative pathway regulator Factor H, the components of the alternative pathway C3 convertase complex C3 and Factor B and the convertase complex itself. This methodological article describes our experience with a method for detection of anti-complement autoantibodies in real time using surface plasmon resonance-based technology. It allows label-free evaluation of the binding efficacy and stability of the formed antigen-antibody complexes.
In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the ... more In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme...
The complement system is a key component of the innate immunity, playing a role in pathogen elimi... more The complement system is a key component of the innate immunity, playing a role in pathogen elimination and in host homeostasis. The complement system has been considered for long time as an anti-tumoral element. However, recent studies showed a pro-tumoral effect of complement and particularly of the anaphylatoxines C3a and C5a in a large variety of tumor types. Complement proteins act on different levels of tumor progression, affecting the tumor cells, the angiogenesis and the immune microenvironment. The impact of the complement system on tumor progression seems to be cancer type-dependent and this has to be taken into account in the establishment of potential biomarkers and development of therapeutic strategies.
Intravascular erythrocyte destruction, accompanied by the release of pro-oxidative and pro-inflam... more Intravascular erythrocyte destruction, accompanied by the release of pro-oxidative and pro-inflammatory components hemoglobin and heme, is a common event in the pathogenesis of numerous diseases with heterogeneous etiology and clinical features. A frequent adverse effect related to massive hemolysis is the renal injury and inflammation. Nevertheless, it is still unclear whether heme--a danger-associated molecular pattern--and ligand for TLR4 or upstream hemolysis-derived products are responsible for these effects. Well-characterized animal models of hemolysis with kidney impairment are needed to investigate how hemolysis drives kidney injury and to test novel therapeutic strategies. Here, we characterized the pathological processes leading to acute kidney injury and inflammation during massive intravascular hemolysis, using a mouse model of phenylhydrazine (PHZ)-triggered erythrocyte destruction. We observed profound changes in mRNA levels for markers of tubular damage (Kim-1, NGAL)...
Introduction L’heme, en conditions hemolytiques, est un signal de danger qui provoque inflammatio... more Introduction L’heme, en conditions hemolytiques, est un signal de danger qui provoque inflammation et dysfonction cellulaire. Pour reguler ses effets deleteres, il induit l’expression d’heme oxygenase 1 (HO-1), enzyme capable de degrader l’heme et moduler certains genes. Il est responsable de l’hyperactivation du complement a la surface de cellules endotheliales, pouvant contribuer a la pathogenese du syndrome hemolytique et uremique atypique (SHUa). Notre objectif est de caracteriser le role de l’HO-1 dans la protection des cellules endotheliales renales contre l’attaque du complement en situation hemolytique. Materiels et methodes Sur cellules HUVECS et endotheliales glomerulaires, incubees avec des concentrations croissantes d’heme, l’expression d’HO-1 et de regulateurs du complement (MCP, DAF et thrombomoduline) a ete etudiee par RT-qPCR, Western Blot ou cytometrie. Apres rechallenge a l’heme, les depots de C3 (serum normal ou deregule) ont ete etudies en cytometrie. In vivo, des souris C57B6 ont ete injectees par heme ou phenylhydrazine (traitement hemolytique) pour etudier les marquages HO-1, C3 et co-localisations endotheliales dans les reins, cœurs, foies et rates en immuno-histochimie et fluorescence. Resultats Nous mettons en evidence une protection contre l’attaque par le complement sur les HUVECS exprimant l’HO-1, mais pas les cellules endotheliales glomerulaires. L’expression des regulateurs du complement varie avec l’incubation des HUVECs par l’heme. En immuno-histochimie et immunofluorescence, les souris injectees par heme ou phenyl hydrazine ont des depots de C3 glomerulaires et le long de la membrane basale tubulaire. Discussion En conditions hemolytiques in vivo et in vitro, la surexpression d’HO-1 est associee avec des depots de C3 plus faibles. Les depots les plus importants sont intra-glomerulaires ou l’HO-1 n’est pas exprimee. Cet effet protecteur est peut-etre lie a la modulation de l’expression des regulateurs du complement lors d’une surexpression d’HO-1. Conclusion L’absence de protection glomerulaire par l’HO-1 peut expliquer le defaut de protection endotheliale contre l’attaque par le complement, notamment, en cas d’hyperactivation du complement comme dans le SHUa, expliquant potentiellement le tropisme renal du SHUa.
Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Be... more Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Because the complement system plays a critical role in orchestrating inflammatory and immune responses as well as in the clearance of immune complexes, autoreactivity to complement components may have considerable pathological consequences. Autoantibodies against the central complement component C3 have been reported in systemic lupus erythematosus, but their molecular mechanism and functional relevance are not well understood. The objective of this study was to evaluate the frequency and the functional properties of the anti-C3 autoantibodies. Anti-C3 autoantibodies were measured in plasma of 39 LN patients, and identification of their epitopes on the C3 molecule was performed. By using surface plasmon resonance, we analyzed the influence of patient-derived IgG antibodies on the interaction of C3b with Factor B, Factor H, and complement receptor 1. The capacity of these antibodies to dysregulate the C3 convertase on the surface of endothelial cell was measured by flow cytometry. Here we report that the frequency of anti-C3 autoantibodies in LN is ∼30%. They inhibited interactions of the negative complement regulators Factor H and complement receptor 1 with C3b. An enhanced C3 deposition was also observed on human endothelial cells in the presence of C3 autoantibodies. In addition, anti-C3 autoantibody levels correlated with disease activity. In conclusion, the anti-C3 autoantibodies in LN may contribute to the autoimmune pathology by their capacity to overactivate the complement system.
Methods in molecular biology (Clifton, N.J.), 2019
The innate immune complement system is a powerful defense cascade against pathogens, but can indu... more The innate immune complement system is a powerful defense cascade against pathogens, but can induce host tissue damage when overactivated. In pathological conditions, mainly but not restricted to renal diseases, such as lupus nephritis, atypical hemolytic uremic syndrome, and C3 glomerulopathies, complement is overactivated or dysregulated by autoantibodies directed against its components and regulators. Among the key autoantibody targets are the initiator of the classical complement pathway C1q, the alternative pathway regulator Factor H, the components of the alternative pathway C3 convertase complex C3 and Factor B and the convertase complex itself. This methodological article describes our experience with a method for detection of anti-complement autoantibodies in real time using surface plasmon resonance-based technology. It allows label-free evaluation of the binding efficacy and stability of the formed antigen-antibody complexes.
In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the ... more In hemolytic diseases, such as sickle cell disease (SCD), intravascular hemolysis results in the release of hemoglobin, heme, and heme-loaded membrane microvesicles in the bloodstream. Intravascular hemolysis is thus associated with inflammation and organ injury. Complement system can be activated by heme in vitro. We investigated the mechanisms by which hemolysis and red blood cell (RBC) degradation products trigger complement activation in vivo. In kidney biopsies of SCD nephropathy patients and a mouse model with SCD, we detected tissue deposits of complement C3 and C5b-9. Moreover, drug-induced intravascular hemolysis or injection of heme or hemoglobin in mice triggered C3 deposition, primarily in kidneys. Renal injury markers (Kim-1, NGAL) were attenuated in C3-/- hemolytic mice. RBC degradation products, such as heme-loaded microvesicles and heme, induced alternative and terminal complement pathway activation in sera and on endothelial surfaces, in contrast to hemoglobin. Heme...
The complement system is a key component of the innate immunity, playing a role in pathogen elimi... more The complement system is a key component of the innate immunity, playing a role in pathogen elimination and in host homeostasis. The complement system has been considered for long time as an anti-tumoral element. However, recent studies showed a pro-tumoral effect of complement and particularly of the anaphylatoxines C3a and C5a in a large variety of tumor types. Complement proteins act on different levels of tumor progression, affecting the tumor cells, the angiogenesis and the immune microenvironment. The impact of the complement system on tumor progression seems to be cancer type-dependent and this has to be taken into account in the establishment of potential biomarkers and development of therapeutic strategies.
Intravascular erythrocyte destruction, accompanied by the release of pro-oxidative and pro-inflam... more Intravascular erythrocyte destruction, accompanied by the release of pro-oxidative and pro-inflammatory components hemoglobin and heme, is a common event in the pathogenesis of numerous diseases with heterogeneous etiology and clinical features. A frequent adverse effect related to massive hemolysis is the renal injury and inflammation. Nevertheless, it is still unclear whether heme--a danger-associated molecular pattern--and ligand for TLR4 or upstream hemolysis-derived products are responsible for these effects. Well-characterized animal models of hemolysis with kidney impairment are needed to investigate how hemolysis drives kidney injury and to test novel therapeutic strategies. Here, we characterized the pathological processes leading to acute kidney injury and inflammation during massive intravascular hemolysis, using a mouse model of phenylhydrazine (PHZ)-triggered erythrocyte destruction. We observed profound changes in mRNA levels for markers of tubular damage (Kim-1, NGAL)...
Introduction L’heme, en conditions hemolytiques, est un signal de danger qui provoque inflammatio... more Introduction L’heme, en conditions hemolytiques, est un signal de danger qui provoque inflammation et dysfonction cellulaire. Pour reguler ses effets deleteres, il induit l’expression d’heme oxygenase 1 (HO-1), enzyme capable de degrader l’heme et moduler certains genes. Il est responsable de l’hyperactivation du complement a la surface de cellules endotheliales, pouvant contribuer a la pathogenese du syndrome hemolytique et uremique atypique (SHUa). Notre objectif est de caracteriser le role de l’HO-1 dans la protection des cellules endotheliales renales contre l’attaque du complement en situation hemolytique. Materiels et methodes Sur cellules HUVECS et endotheliales glomerulaires, incubees avec des concentrations croissantes d’heme, l’expression d’HO-1 et de regulateurs du complement (MCP, DAF et thrombomoduline) a ete etudiee par RT-qPCR, Western Blot ou cytometrie. Apres rechallenge a l’heme, les depots de C3 (serum normal ou deregule) ont ete etudies en cytometrie. In vivo, des souris C57B6 ont ete injectees par heme ou phenylhydrazine (traitement hemolytique) pour etudier les marquages HO-1, C3 et co-localisations endotheliales dans les reins, cœurs, foies et rates en immuno-histochimie et fluorescence. Resultats Nous mettons en evidence une protection contre l’attaque par le complement sur les HUVECS exprimant l’HO-1, mais pas les cellules endotheliales glomerulaires. L’expression des regulateurs du complement varie avec l’incubation des HUVECs par l’heme. En immuno-histochimie et immunofluorescence, les souris injectees par heme ou phenyl hydrazine ont des depots de C3 glomerulaires et le long de la membrane basale tubulaire. Discussion En conditions hemolytiques in vivo et in vitro, la surexpression d’HO-1 est associee avec des depots de C3 plus faibles. Les depots les plus importants sont intra-glomerulaires ou l’HO-1 n’est pas exprimee. Cet effet protecteur est peut-etre lie a la modulation de l’expression des regulateurs du complement lors d’une surexpression d’HO-1. Conclusion L’absence de protection glomerulaire par l’HO-1 peut expliquer le defaut de protection endotheliale contre l’attaque par le complement, notamment, en cas d’hyperactivation du complement comme dans le SHUa, expliquant potentiellement le tropisme renal du SHUa.
Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Be... more Lupus nephritis (LN) is a complication of the autoimmune disease systemic lupus erythematosus. Because the complement system plays a critical role in orchestrating inflammatory and immune responses as well as in the clearance of immune complexes, autoreactivity to complement components may have considerable pathological consequences. Autoantibodies against the central complement component C3 have been reported in systemic lupus erythematosus, but their molecular mechanism and functional relevance are not well understood. The objective of this study was to evaluate the frequency and the functional properties of the anti-C3 autoantibodies. Anti-C3 autoantibodies were measured in plasma of 39 LN patients, and identification of their epitopes on the C3 molecule was performed. By using surface plasmon resonance, we analyzed the influence of patient-derived IgG antibodies on the interaction of C3b with Factor B, Factor H, and complement receptor 1. The capacity of these antibodies to dysregulate the C3 convertase on the surface of endothelial cell was measured by flow cytometry. Here we report that the frequency of anti-C3 autoantibodies in LN is ∼30%. They inhibited interactions of the negative complement regulators Factor H and complement receptor 1 with C3b. An enhanced C3 deposition was also observed on human endothelial cells in the presence of C3 autoantibodies. In addition, anti-C3 autoantibody levels correlated with disease activity. In conclusion, the anti-C3 autoantibodies in LN may contribute to the autoimmune pathology by their capacity to overactivate the complement system.
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