LA BACTERICIDIE DES NEUTROPHILES : MECANISMES CELLULAIRES ET MOLECULAIRES ET LEURS DYSFONCTIONS

Microbicidal activity of neutrophils requires the NADPH oxidase dependent reactive oxygen species (ROS) production and myeloperoxidase (MPO)-H2O2-halide system-catalyzed halogenations (oxidative mechanisms) and the release of granular proteins into the phagosomal lumen (non oxidative mechanisms). The long-established direct role of ROS and MPO has been recently questioned. In this work, using our improved method to assess microbicidal activity, we have re-examined the role of the diverse mechanisms proposed to be involved in the antimicrobial activity of neutrophils. We show that the NADPH oxidase activity is indispensable for the killing of certain microorganisms (e.g. Staphylococcus aureus, Candida albicans) but not of others (e.g. Escherichia coli) which are efficiently killed even in the absence of a respiratory burst. Our data also indicate that NADPH oxidase-dependent killing of S. aureus and C. albicans is largely dependent on MPO. Alkalinization of phagosomal pH and K+ fluxes do not appear to significantly contribute to the killing of these microorganisms. K+ channels activity may account for the partial recovery of killing observed in MPO-deficient neutrophils at long times of incubation.We have also investigated the neutrophils’ microbicidal mechanisms in a cellular model, the human myelomonoblastic cellular line inducible in granulocytes, PLB-985. The ROS production in PLB-985 cells stimulated by opsonized microorganisms is very weak compared to human neutrophils, but sustains a NADPH oxidase dependent killing activity of S. aureus and C. albicans similar to PMN after short times of incubation (<10 min). However, the killing activity of the PLB cells is abolished after long times of incubation. NADPH oxidase independent killing of E. coli is also defective. The ROS overproduction of the mutant DloopNox4-Nox2 (in these cells, the D-loop of Nox2 was replaced by the D-loop of its homolog Nox4) after soluble and particulate agonists dependent activation does not increase their microbicidal power. The weak killing activity of the PLB-985 cellular line can be explained by the faint amounts of cytochrome b558 and by the more or less lack of granular proteins content (MPO, elastase, cathepsine G, lactoferrine, lysozyme, MMP8 and MMP9) involved in the microbicidal event. In conclusion, in PLB-985 DMF-differentiated cells, the oxygen dependent and independent mechanisms are both defective. A lack of NADPH oxidase complex results in a rare inherited disorder, the chronic granulomatose disease (CGD). We characterized an atypical and extremely rare case of CGD: the X91- variant: the new identified point mutation in the CYBB gene promoter (insertion of a T at position -54T/-56T) appeared to be related to the loss of association of ets transcription factors within this region, prevent the normally functional expression of the gene and finally, correlate with the low NADPH oxidase activity. The residual respiratory burst supported no killing of S. aureus and C. albicans, in vitro, and is not sufficient to protect the patient against severe and recurrent infections.L’élimination des microorganismes par les neutrophiles (PMN) est mediée par l’activité des formes réactives de l’oxygène (FRO), produites suite à l’activation de la NADPH oxydase, dont l’efficacité est sensiblement augmentée par la myélopéroxydase, MPO, (mécanismes oxydatifs) et par l’action des protéines bactéricides contenues dans les granules cytoplasmiques (mécanismes non oxydatifs). L’implication directe des FRO et de la MPO dans la bactéricidie des PMN a été récemment remise en question. Nous nous sommes proposés de réexaminer les mécanismes impliqués dans la bactéricidie des neutrophiles sur la base des nouvelles hypothèses récemment proposées en appliquant une méthode de mesure du « killing » des microorganismes que nous avons récemment mise au point et qui permet une estimation plus correcte de la bactéricidie. Nous avons démontré que l’activité NADPH oxydase est indispensable pour le « killing » de certains microorganismes (S. aureus et C. albicans) mais pas pour d’autres (E. coli) qui sont efficacement éliminés même en l’absence du burst respiratoire. Les flux d’ions K+ et l’alcalinisation du pH intraphagosomale, induits par l’activation du complexe oxydase, ne sont pas nécessaires au killing de S. aureus et C. albicans, microorganismes dont la bactéricidie NADPH oxydase dépendante est mediée presque exclusivement par la MPO. Les courants des ions K+, à travers des canaux BKCa-like, semblent être responsables du killing résiduel des PMN MPO déficients, après une longue incubation. Nous avons aussi étudié les mécanismes impliqués dans la bactéricidie des PMN en utilisant comme modèle cellulaire la lignée PLB-985 différentiable en « pseudo-neutrophiles ». Nous avons prouvé que la réponse de ces cellules aux stimuli particulaires est sensiblement plus faible par rapport aux PMN. Ce burst respiratoire support e une bactéricidie comparable à celles des PMN pour des temps courts d’incubation (<10 min) vis-à-vis des microorganismes sensibles aux mécanismes de « killing » oxygène-dépendants (S. aureus et C. albicans); toutefois, en prolongeant les temps d’incubation, le pouvoir bactéricides ce ces cellules est abolit. Le « killing » NADPH oxydase indépendant d’E. coli est aussi partiellement déficitaire. La superproduction de FRO mise en évidence dans les cellules mutantes PLB-985 DloopNox4-Nox2 (où la deuxième boucle intracellulaire de Nox2 a été remplacée par celle de l’oxydase homologue Nox4) en réponse aux stimuli solubles et particulaires n’est pas accompagnée par une augmentation du « killing ». Le faible pouvoir bactéricide de cette lignée cellulaire est du au contenu réduit en cytochrome b558 (un dixième par rapport aux PMN) et aux défauts plus ou moins importants des protéines granulaires (MPO, elastase, cathepsine G, lactoferrine, lysozyme, MMP8 et MMP9) impliquées dans le processus de bactéricidie: dans les cellules PLB-985 différentiées au DMF, les mécanismes bactéricides oxygènes dépendants et indépendants sont donc tous deux compromis.Le dysfonctionnement du complexe NADPH oxydase est à la base d’une maladie génétique rare, la granulomatose septique chronique (CGD). Nous nous sommes intéressés à la caractérisation d’un cas atypique de CGD, la forme X91-. Nous avons démontré que la nouvelle mutation identifiée (insertion d’un T en position -54T/-56T) compromet l’association des facteurs de transcription ets avec la région promotrice, empêche, par conséquence, l’expression normale du gène dans les neutrophiles et détermine une activité oxydase réduite. Ce burst respiratoire résiduel (6% du control) n’est pas suffisante pour soutenir, in vitro, une activité de « killing » vis-à-vis de S. aureus et C. albicans ni pour protéger le patient des infections sévères et récidivantes

LA BACTERICIDIE DES NEUTROPHILES : MECANISMES CELLULAIRES ET MOLECULAIRES ET LEURS DYSFONCTIONS

Microbicidal activity of neutrophils requires the NADPH oxidase dependent reactive oxygen species (ROS) production and myeloperoxidase (MPO)-H2O2-halide system-catalyzed halogenations (oxidative mechanisms) and the release of granular proteins into the phagosomal lumen (non oxidative mechanisms). The long-established direct role of ROS and MPO has been recently questioned. In this work, using our improved method to assess microbicidal activity, we have re-examined the role of the diverse mechanisms proposed to be involved in the antimicrobial activity of neutrophils. We show that the NADPH oxidase activity is indispensable for the killing of certain microorganisms (e.g. Staphylococcus aureus, Candida albicans) but not of others (e.g. Escherichia coli) which are efficiently killed even in the absence of a respiratory burst. Our data also indicate that NADPH oxidase-dependent killing of S. aureus and C. albicans is largely dependent on MPO. Alkalinization of phagosomal pH and K+ fluxes do not appear to significantly contribute to the killing of these microorganisms. K+ channels activity may account for the partial recovery of killing observed in MPO-deficient neutrophils at long times of incubation.We have also investigated the neutrophils’ microbicidal mechanisms in a cellular model, the human myelomonoblastic cellular line inducible in granulocytes, PLB-985. The ROS production in PLB-985 cells stimulated by opsonized microorganisms is very weak compared to human neutrophils, but sustains a NADPH oxidase dependent killing activity of S. aureus and C. albicans similar to PMN after short times of incubation (<10 min). However, the killing activity of the PLB cells is abolished after long times of incubation. NADPH oxidase independent killing of E. coli is also defective. The ROS overproduction of the mutant DloopNox4-Nox2 (in these cells, the D-loop of Nox2 was replaced by the D-loop of its homolog Nox4) after soluble and particulate agonists dependent activation does not increase their microbicidal power. The weak killing activity of the PLB-985 cellular line can be explained by the faint amounts of cytochrome b558 and by the more or less lack of granular proteins content (MPO, elastase, cathepsine G, lactoferrine, lysozyme, MMP8 and MMP9) involved in the microbicidal event. In conclusion, in PLB-985 DMF-differentiated cells, the oxygen dependent and independent mechanisms are both defective. A lack of NADPH oxidase complex results in a rare inherited disorder, the chronic granulomatose disease (CGD). We characterized an atypical and extremely rare case of CGD: the X91- variant: the new identified point mutation in the CYBB gene promoter (insertion of a T at position -54T/-56T) appeared to be related to the loss of association of ets transcription factors within this region, prevent the normally functional expression of the gene and finally, correlate with the low NADPH oxidase activity. The residual respiratory burst supported no killing of S. aureus and C. albicans, in vitro, and is not sufficient to protect the patient against severe and recurrent infections.L’élimination des microorganismes par les neutrophiles (PMN) est mediée par l’activité des formes réactives de l’oxygène (FRO), produites suite à l’activation de la NADPH oxydase, dont l’efficacité est sensiblement augmentée par la myélopéroxydase, MPO, (mécanismes oxydatifs) et par l’action des protéines bactéricides contenues dans les granules cytoplasmiques (mécanismes non oxydatifs). L’implication directe des FRO et de la MPO dans la bactéricidie des PMN a été récemment remise en question. Nous nous sommes proposés de réexaminer les mécanismes impliqués dans la bactéricidie des neutrophiles sur la base des nouvelles hypothèses récemment proposées en appliquant une méthode de mesure du « killing » des microorganismes que nous avons récemment mise au point et qui permet une estimation plus correcte de la bactéricidie. Nous avons démontré que l’activité NADPH oxydase est indispensable pour le « killing » de certains microorganismes (S. aureus et C. albicans) mais pas pour d’autres (E. coli) qui sont efficacement éliminés même en l’absence du burst respiratoire. Les flux d’ions K+ et l’alcalinisation du pH intraphagosomale, induits par l’activation du complexe oxydase, ne sont pas nécessaires au killing de S. aureus et C. albicans, microorganismes dont la bactéricidie NADPH oxydase dépendante est mediée presque exclusivement par la MPO. Les courants des ions K+, à travers des canaux BKCa-like, semblent être responsables du killing résiduel des PMN MPO déficients, après une longue incubation. Nous avons aussi étudié les mécanismes impliqués dans la bactéricidie des PMN en utilisant comme modèle cellulaire la lignée PLB-985 différentiable en « pseudo-neutrophiles ». Nous avons prouvé que la réponse de ces cellules aux stimuli particulaires est sensiblement plus faible par rapport aux PMN. Ce burst respiratoire support e une bactéricidie comparable à celles des PMN pour des temps courts d’incubation (<10 min) vis-à-vis des microorganismes sensibles aux mécanismes de « killing » oxygène-dépendants (S. aureus et C. albicans); toutefois, en prolongeant les temps d’incubation, le pouvoir bactéricides ce ces cellules est abolit. Le « killing » NADPH oxydase indépendant d’E. coli est aussi partiellement déficitaire. La superproduction de FRO mise en évidence dans les cellules mutantes PLB-985 DloopNox4-Nox2 (où la deuxième boucle intracellulaire de Nox2 a été remplacée par celle de l’oxydase homologue Nox4) en réponse aux stimuli solubles et particulaires n’est pas accompagnée par une augmentation du « killing ». Le faible pouvoir bactéricide de cette lignée cellulaire est du au contenu réduit en cytochrome b558 (un dixième par rapport aux PMN) et aux défauts plus ou moins importants des protéines granulaires (MPO, elastase, cathepsine G, lactoferrine, lysozyme, MMP8 et MMP9) impliquées dans le processus de bactéricidie: dans les cellules PLB-985 différentiées au DMF, les mécanismes bactéricides oxygènes dépendants et indépendants sont donc tous deux compromis.Le dysfonctionnement du complexe NADPH oxydase est à la base d’une maladie génétique rare, la granulomatose septique chronique (CGD). Nous nous sommes intéressés à la caractérisation d’un cas atypique de CGD, la forme X91-. Nous avons démontré que la nouvelle mutation identifiée (insertion d’un T en position -54T/-56T) compromet l’association des facteurs de transcription ets avec la région promotrice, empêche, par conséquence, l’expression normale du gène dans les neutrophiles et détermine une activité oxydase réduite. Ce burst respiratoire résiduel (6% du control) n’est pas suffisante pour soutenir, in vitro, une activité de « killing » vis-à-vis de S. aureus et C. albicans ni pour protéger le patient des infections sévères et récidivantes

A longitudinal study of C1q and anti-C1q autoantibodies in homologous and heterologous pregnancies for predicting pre-eclampsia

C1q, the recognition molecule of the classical pathway of the complement system, plays a central role in pregnancy. Lack of C1q is characterized by poor trophoblast invasion and pregnancy failure. C1q can be the target of an antibody response: anti-C1q autoantibodies (anti-C1q) are present in several infectious and autoimmune diseases. The presence of these autoantibodies has been detected also in 2-8% of the general population. Recent evidence indicates that women who undergo assisted reproductive technology (ART) have an increased risk of developing pre-eclampsia (PE), particularly oocyte donation (OD) pregnancies. The aim of this study was to characterize the levels of C1q and anti-C1q in PE gestations, in healthy spontaneous, homologous and heterologous ART pregnancies. Serum of the following four groups of women, who were followed throughout two or three trimesters, were collected: PE, patients diagnosed with PE; OD, oocyte donation recipients; HOM, homologous ART women; Sp, spontaneous physiological pregnancy. Our results indicate that PE patients have lower levels of anti-C1q. In ART pregnant women, the trend of C1q and anti-C1q levels were similar to PE patients, even though these women did not develop PE-like symptoms during pregnancy. This finding suggests an immunological dysfunction at the foetal-maternal interface in ART pregnancies, a hypothesis confirmed by the observation of C1q deposition in placentae derived from OD, comparable to PE. Since significantly lower levels of anti-C1q were detected in PE compared to healthy control sera, we hypothesize the possible binding on placental syncytiotrophoblast microvesicles (STBM), which are increased in the circulation of PE mothers. Furthermore, the characterization of the binding-epitope of anti-C1q revealed that "physiological" autoantibodies were mainly directed against C1q globular domain. We concluded that anti-C1q could have a physiological role in pregnancy: during the healthy spontaneous pregnancy the raised levels of these autoantibodies can be important for the clearance of STBM. In PE and in pathological pregnancies (but also in OD pregnancies), the increase in syncytiotrophoblast apoptosis and consequent increase of the circulating STMB levels lead to a consumption of C1q and anti-C1q

A novel point mutation in the CYBB gene promoter leading to a rare X minus chronic granulomatous disease variant — Impact on the microbicidal activity of neutrophils

AbstractThis article reports an atypical and extremely rare case of X-linked CGD in an Italian family characterized by a low expression of gp91phox (X91− CGD). A novel point mutation in the CYBB gene's promoter (insertion of a T at position −54T to −56T) appeared to prevent the full expression of this gene in the patient's neutrophils and correlated with a residual oxidase activity in the whole cells population. The expression and functional activity of the oxidase in eosinophils appeared to be almost normal. Gel shift assays indicated that the mutation led to decreased interactions with DNA-binding proteins. The total O2− production in the patient's granulocytes (5–7% of normal) supported no microbicidal power after 45 min and 60 min of contact with S. aureus and C. albicans, respectively. Despite this residual oxidase activity, the patients suffered from severe and life-threatening infections. It was concluded that in these X91− CGD neutrophils, the O2− production per se was not sufficient to protect the patient against severe infections

Autoimmunity in thymic epithelial tumors: a not yet clarified pathologic paradigm associated with several unmet clinical needs

Thymic epithelial tumors (TETs) are rare mediastinal cancers originating from the thymus, classified in two main histotypes: thymoma and thymic carcinoma (TC). TETs affect a primary lymphoid organ playing a critical role in keeping T-cell homeostasis and ensuring an adequate immunological tolerance against “self”. In particular, thymomas and not TC are frequently associated with autoimmune diseases (ADs), with Myasthenia Gravis being the most common AD present in 30% of patients with thymoma. This comorbidity, in addition to negatively affecting the quality and duration of patients’ life, reduces the spectrum of the available therapeutic options. Indeed, the presence of autoimmunity represents an exclusion criteria for the administration of the newest immunotherapeutic treatments with checkpoint inhibitors. The pathophysiological correlation between TETs and autoimmunity remains a mystery. Several studies have demonstrated the presence of a residual and active thymopoiesis in adult patients affected by thymomas, especially in mixed and lymphocytic-rich thymomas, currently known as type AB and B thymomas. The aim of this review is to provide the state of art in regard to the histological features of the different TET histotype, to the role of the different immune cells infiltrating tumor microenvironments and their impact in the break of central immunologic thymic tolerance in thymomas. We discuss here both cellular and molecular immunologic mechanisms inducing the onset of autoimmunity in TETs, limiting the portfolio of therapeutic strategies against TETs and greatly impacting the prognosis of associated autoimmune diseases

LA BACTERICIDIE DES NEUTROPHILES : MECANISMES CELLULAIRES ET MOLECULAIRES ET LEURS DYSFONCTIONS

Microbicidal activity of neutrophils requires the NADPH oxidase dependent reactive oxygen species (ROS) production and myeloperoxidase (MPO)-H2O2-halide system-catalyzed halogenations (oxidative mechanisms) and the release of granular proteins into the phagosomal lumen (non oxidative mechanisms). The long-established direct role of ROS and MPO has been recently questioned. In this work, using our improved method to assess microbicidal activity, we have re-examined the role of the diverse mechanisms proposed to be involved in the antimicrobial activity of neutrophils. We show that the NADPH oxidase activity is indispensable for the killing of certain microorganisms (e.g. Staphylococcus aureus, Candida albicans) but not of others (e.g. Escherichia coli) which are efficiently killed even in the absence of a respiratory burst. Our data also indicate that NADPH oxidase-dependent killing of S. aureus and C. albicans is largely dependent on MPO. Alkalinization of phagosomal pH and K+ fluxes do not appear to significantly contribute to the killing of these microorganisms. K+ channels activity may account for the partial recovery of killing observed in MPO-deficient neutrophils at long times of incubation.We have also investigated the neutrophils’ microbicidal mechanisms in a cellular model, the human myelomonoblastic cellular line inducible in granulocytes, PLB-985. The ROS production in PLB-985 cells stimulated by opsonized microorganisms is very weak compared to human neutrophils, but sustains a NADPH oxidase dependent killing activity of S. aureus and C. albicans similar to PMN after short times of incubation (<10 min). However, the killing activity of the PLB cells is abolished after long times of incubation. NADPH oxidase independent killing of E. coli is also defective. The ROS overproduction of the mutant DloopNox4-Nox2 (in these cells, the D-loop of Nox2 was replaced by the D-loop of its homolog Nox4) after soluble and particulate agonists dependent activation does not increase their microbicidal power. The weak killing activity of the PLB-985 cellular line can be explained by the faint amounts of cytochrome b558 and by the more or less lack of granular proteins content (MPO, elastase, cathepsine G, lactoferrine, lysozyme, MMP8 and MMP9) involved in the microbicidal event. In conclusion, in PLB-985 DMF-differentiated cells, the oxygen dependent and independent mechanisms are both defective. A lack of NADPH oxidase complex results in a rare inherited disorder, the chronic granulomatose disease (CGD). We characterized an atypical and extremely rare case of CGD: the X91- variant: the new identified point mutation in the CYBB gene promoter (insertion of a T at position -54T/-56T) appeared to be related to the loss of association of ets transcription factors within this region, prevent the normally functional expression of the gene and finally, correlate with the low NADPH oxidase activity. The residual respiratory burst supported no killing of S. aureus and C. albicans, in vitro, and is not sufficient to protect the patient against severe and recurrent infections.L’élimination des microorganismes par les neutrophiles (PMN) est mediée par l’activité des formes réactives de l’oxygène (FRO), produites suite à l’activation de la NADPH oxydase, dont l’efficacité est sensiblement augmentée par la myélopéroxydase, MPO, (mécanismes oxydatifs) et par l’action des protéines bactéricides contenues dans les granules cytoplasmiques (mécanismes non oxydatifs). L’implication directe des FRO et de la MPO dans la bactéricidie des PMN a été récemment remise en question. Nous nous sommes proposés de réexaminer les mécanismes impliqués dans la bactéricidie des neutrophiles sur la base des nouvelles hypothèses récemment proposées en appliquant une méthode de mesure du « killing » des microorganismes que nous avons récemment mise au point et qui permet une estimation plus correcte de la bactéricidie. Nous avons démontré que l’activité NADPH oxydase est indispensable pour le « killing » de certains microorganismes (S. aureus et C. albicans) mais pas pour d’autres (E. coli) qui sont efficacement éliminés même en l’absence du burst respiratoire. Les flux d’ions K+ et l’alcalinisation du pH intraphagosomale, induits par l’activation du complexe oxydase, ne sont pas nécessaires au killing de S. aureus et C. albicans, microorganismes dont la bactéricidie NADPH oxydase dépendante est mediée presque exclusivement par la MPO. Les courants des ions K+, à travers des canaux BKCa-like, semblent être responsables du killing résiduel des PMN MPO déficients, après une longue incubation. Nous avons aussi étudié les mécanismes impliqués dans la bactéricidie des PMN en utilisant comme modèle cellulaire la lignée PLB-985 différentiable en « pseudo-neutrophiles ». Nous avons prouvé que la réponse de ces cellules aux stimuli particulaires est sensiblement plus faible par rapport aux PMN. Ce burst respiratoire support e une bactéricidie comparable à celles des PMN pour des temps courts d’incubation (<10 min) vis-à-vis des microorganismes sensibles aux mécanismes de « killing » oxygène-dépendants (S. aureus et C. albicans); toutefois, en prolongeant les temps d’incubation, le pouvoir bactéricides ce ces cellules est abolit. Le « killing » NADPH oxydase indépendant d’E. coli est aussi partiellement déficitaire. La superproduction de FRO mise en évidence dans les cellules mutantes PLB-985 DloopNox4-Nox2 (où la deuxième boucle intracellulaire de Nox2 a été remplacée par celle de l’oxydase homologue Nox4) en réponse aux stimuli solubles et particulaires n’est pas accompagnée par une augmentation du « killing ». Le faible pouvoir bactéricide de cette lignée cellulaire est du au contenu réduit en cytochrome b558 (un dixième par rapport aux PMN) et aux défauts plus ou moins importants des protéines granulaires (MPO, elastase, cathepsine G, lactoferrine, lysozyme, MMP8 et MMP9) impliquées dans le processus de bactéricidie: dans les cellules PLB-985 différentiées au DMF, les mécanismes bactéricides oxygènes dépendants et indépendants sont donc tous deux compromis.Le dysfonctionnement du complexe NADPH oxydase est à la base d’une maladie génétique rare, la granulomatose septique chronique (CGD). Nous nous sommes intéressés à la caractérisation d’un cas atypique de CGD, la forme X91-. Nous avons démontré que la nouvelle mutation identifiée (insertion d’un T en position -54T/-56T) compromet l’association des facteurs de transcription ets avec la région promotrice, empêche, par conséquence, l’expression normale du gène dans les neutrophiles et détermine une activité oxydase réduite. Ce burst respiratoire résiduel (6% du control) n’est pas suffisante pour soutenir, in vitro, une activité de « killing » vis-à-vis de S. aureus et C. albicans ni pour protéger le patient des infections sévères et récidivantes

La bactéricidie des neutrophiles: mécanismes cellulaires et moléculaires et leurs dysfonctions

2008/2009In questa tesi ci siamo proposti di riesaminare i meccanismi implicati nell’attività microbicida dei neutrofili, alla luce delle nuove ipotesi proposte recentemente, impiegando una metodica corretta di misura del killing microbico da noi recentemente messa a punto. Nel complesso, i risultati da noi ottenuti provano che l’attività NADPH ossidasica è indispensabile per il killing di certi microrganismi (quali S. aureus e C. albicans) ma non per altri (quali E. coli) che sono uccisi efficacemente anche in assenza di burst respiratorio. I flussi di ioni potassio e l’alcalinizzazione del pH intrafagosomale, indotti dall’attivazione del complesso ossidasi, non sono necessari al killing di S. aureus e C. albicans, il cui killing NADPH ossidasi-dipendente è mediato quasi esclsivamente dalla mieloperossidasi. Le correnti di ioni K+ sembrano responsabili dell’attività microbicida residua dei neutrofili MPO-deficienti, a lunghi tempi di incubazione. I meccanismi implicati nell’attività microbicida dei neutrofili sono stati studiati anche ricorrendo ad un modello sperimentale ampiamente utilizzato in letteratura per le ricerche sul funzionamento del complesso NADPH ossidasi: la linea cellulare PLB-985 differenziabile in neutrophils-like. In questo contesto, i risultati da noi ottenuti suggeriscono che la produzione di ROS da parte delle linea cellulare PLB-985 in risposta a stimoli particolati (microrganismi opsonizzati) è sensibilmente più debole rispetto ai PMN. Tale burst respiratorio è responsabile di un’attività microbicida, nei confronti di microrganismi sensibili ai meccanismi di killing ossigeno-dipendenti (S. aureus e C. albicans), paragonabile a quella dei PMN a tempi brevi di incubazione; prolungando i tempi di fagocitosi, il potere microbicida di queste cellule risulta invece abolito. Nei confronti di E. coli, battere sensibile ai processi di killing ossigeno-indipendenti, l’attività microbicida delle cellule PLB-985 risulta parzialmente difettosa se paragonata ai neutrofili La super-produzione di anioni superossido, evidenziata nelle cellule PLB-985 DloopNox4-Nox2 (cellule in cui la seconda ansa intracellulare di No2 é sostituita da quella dell’ossidasi omologa Nox4) in risposta a stimoli solubili e particolati, non è accompagnata da un’aumentata capacità microbicida di queste cellule nei confronti di S. aureus, C. albicans ed E. coli. Nelle cellule PLB-985 i processi microbicidi non ossidativi risultano compromessi, a causa della carenza parziale (MPO, elastasi, catepsina G, β-glucuronidasi, CD11b) o totale (lattoferrina, lisozima, MMP-8, MMP-9) degli enzimi e delle proteine granulari coinvolti nell’attività antimicrobica e a causa della loro difettosa esocitosi.. In particolare, i nostri risultati suggeriscono un difetto a livello della biogenesi dei granuli specifici e terziari. Il ridotto funzionamento del complesso enzimatico NADPH ossidasi è alla base di una rara malattia genetica, la malattia granulomatosa cronica. Parte di questo lavoro di tesi è stata dedicata alla caratterizzazione di un caso atipico di CGD, la forma X91-. I risultati ottenuti ci hanno permesso di dimostrare che la nuova mutazione da noi descritta nel promotore del gene CYBB (inserzione di una T nella posizione da -54T a -57T) é responsabile della ridotta associazione dei fattori di trascrizione ets con la regione promotrice, impedisce una normale espressione del gene nei neutrofili dei pazienti ed é correlata alla ridotta attività ossidasica misurata in tutta la popolazione di granulociti; l’espressione e l’attività funzionale della NADPH ossidasi appaiono invece normali negli eosinofili, suggerendo che l’espressione di gp91phox in queste cellule é regolata da fattori di trascrizione differenti da quelli operanti nei neutrofili. Infine, l’attività ossidasica residua (5-7% del normale) non é di per sé sufficiente a garantire un’attività di killing nei confronti di S. aureus e C. albicans e a proteggere il paziente contro le infezioni .XXI Cicl

The Immunopathology of Complement Proteins and Innate Immunity in Autoimmune Disease

International audienceThe complement is a powerful cascade of the innate immunity and also acts as a bridge between innate and acquired immune defence. Complement activation can occur via three distinct pathways, the classical, alternative and lectin pathways, each resulting in the common terminal pathway. Complement activation results in the release of a range of biologically active molecules that significantly contribute to immune surveillance and tissue homeostasis. Several soluble and membrane-bound regulatory proteins restrict complement activation in order to prevent complement-mediated autologous damage, consumption and exacerbated inflammation. The crucial role of complement in the host homeostasis is illustrated by association of both complement deficiency and overactivation with severe and life-threatening diseases. Autoantibodies targeting complement components have been described to alter expression and/or function of target protein resulting in a dysregulation of the delicate equilibrium between activation and inhibition of complement. The spectrum of diseases associated with complement autoantibodies depends on which complement protein and activation pathway are targeted, ranging from autoimmune disorders to kidney and vascular diseases. Nevertheless, these autoantibodies have been identified as differential biomarkers for diagnosis or follow-up of disease only in a small number of clinical conditions. For some autoantibodies, a clear relationship with clinical manifestations has been identified, such as anti-C1q, anti-Factor H, anti-C1 Inhibitor antibodies and C3 nephritic factor. For other autoantibodies, the origin and the functional consequences still remain to be elucidated, questioning about the pathophysiological significance of these autoantibodies, such as anti-mannose binding lectin, anti-Factor I, anti-Factor B and anti-C3b antibodies. The detection of autoantibodies targeting complement components is performed in specialized laboratories; however, there is no consensus on detection methods and standardization of the assays is a real challenge. This review summarizes the current panorama of autoantibodies targeting complement recognition proteins of the classical and lectin pathways, associated proteases, convertases, regulators and terminal components, with an emphasis on autoantibodies clearly involved in clinical conditions

Intracellular shunting of O2 12 contributes to charge compensation and preservation of neutrophil respiratory burst in the absence of voltage-gated proton channel activity

Proton efflux via voltage-gated proton channels (Hv1) is considered to mediate the charge compensation necessary to preserve NADPH oxidase activity during the respiratory burst. Using the Hv1 inhibitor Zn(2+), we found that the PMA-induced respiratory burst of human neutrophils is inhibited when assessed as extracellular production of O2(-) and H2O2, in accordance with literature studies, but, surprisingly, unaffected when measured as oxygen consumption or total (extracellular plus intracellular) H2O2 production. Furthermore, we show that inhibiting Hv1 with Zn(2+) results in an increased production of intracellular ROS. Similar results, i.e. decreased extracellular and increased intracellular ROS production, were obtained using a human granulocyte-like cell line with severely impaired Hv1 expression. Acidic extracellular pH, which dampens proton efflux, also augmented intracellular production of H2O2. Zinc caused an increase in the rate but not in the extent of depolarization and cytosolic acidification indicating that mechanisms other than proton efflux take part in charge compensation. Our results suggest a hitherto unpredicted mechanism of charge compensation whereby, in the absence of proton efflux, part of O2(-) generated within gp91(phox) in the plasma membrane is shunted intracellularly down electrochemical gradient to dampen excessive depolarization. This would preserve NADPH oxidase activity under conditions such as the inflammatory exudate in which the acidic pH hinders charge compensation by proton efflux