Abstract
Introduction: Chronic Granulomatous Disease (CGD) is a genetic disorder of the Nox2 enzyme system in phagocytic cells. Neutrophils and monocytes from patients with CGD are deficient in Nox2 components, unable to produce superoxide anion and other reactive oxygen species (ROS), and cannot kill certain types of bacteria and fungi. Patients with CGD suffer from recurrent, life-threatening infections. Treatment includes prophylaxis with antibiotic and antifungal agents and therapy with IFN-γ. Results from clinical trials of IFN-γ in patients with CGD have demonstrated decreased incidence and severity of infections. The exact mechanism(s) by which IFN-γ achieves its clinical effects is not clear. Previous studies from our laboratory in healthy adult volunteers demonstrated that administration of IFN-γ induced dramatic changes in the neutrophil phenotype with significant functional implications. To investigate IFN-γ induced changes in neutrophils further we initiated a study in CGD patients.
Clinical Trial and Methods: Patients with CGD were enrolled on a study protocol approved by the COMIRB at the University of Colorado Denver. Patients between 5 and 60 years with defined genetic variants of CGD, but without recent infections or medical complications were entered onto the study. IFN-γ was stopped for a week to allow a drug washout period; prophylactic antibiotics and antifungal medications were continued. Blood samples were obtained from patients off IFN-γ, 12 hours after the first dose, and after the fourth dose of IFN-γ (50 mcg./m2 given on a standard schedule three times a week). Neutrophils were isolated by Dextran sedimentation, Ficoll Hypaque density centrifugation, and hypotonic lysis of red blood cells. RNA was isolated by standard technique and gene expression completed by RNAseq analysis (results pending). Ingestion was measured with fluorescently labeled S. aureus, bactericidal activity against S. aureus with 1:1 ratio of neutrophils to bacteria in 10% normal human serum by standard technique, and chemotaxis to fMLF and C5a with calcein-AM labeled neutrophils across fluorescence blocking PET membrane in Corning HTS Fluroblock plates. Superoxide anion was measured as SOD inhibitable cytochrome c reduction in response to standard agonists, CD11b expression and F-actin assembly in response to PMA and fMLF by flow cytometry, MHC Class II antigens by flow cytometry, nitric oxide (NO) levels in neutrophil lystes using a colorimetric assay to measure the combined nitrate and nitrite levels, and apoptosis determined as the combined caspase 3 and caspase 7 activity in isolated lysates using the Caspase-Glo 3/7 Assay from Promega.
Results: We report here preliminary results on the first three patients studied; all are gp91phox deficient X-linked CGD. As expected, superoxide anion was not detected from patient neutrophils off INF-γ or during treatment. Ingestion and cell motility were not affected by IFN-γ administration. In all three subjects, caspase activity was decreased during IFN-γ treatment compared to before. In all three patients bactericidal activity against S. aureus was deficient; in 2/3 patients, administration of IFN was associated with a small but specific increase in killing, without change in ROS production. Strikingly, in two patients who showed improved bactericidal activity, NO production in lysates was increased after IFN-γ treatment compared to before and showed a further increase when cells were stimulated with fMLF. CD11b expression and F-actin assembly did not appear to be altered by IFN-γ treatment. Expression of HLA Class II markers (HLADRA and CD 274) appeared on neutrophils and increased in expression after initiation of IFN treatment. This change also was seen with CD 40.
Summary: IFN-γ appeared to have dramatic effects on neutrophils from patients with CGD including possible improved bactericidal activity, the generation of NO which could be bactericidal and compensate for the deficiency of generation of ROS, and expression of MHC Class II antigens with possible association to antigen processing and enhanced interaction with adaptive immune function.
Conclusion: Changes in the neutrophil phenotype induced by IFN-γ may provide alternative strategies compensating for the genetic deficiency of CGD. Defining how IFN-γ enhances neutrophil function may expand the use of this drug to other medical disorders in which innate immune response is involved.
Disclosures
Ambruso: Horizon Pharma Ireland Ltd.: Consultancy.
Metabolic abnormalities in G6PC3-deficient human neutrophils result in severe functional defects