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Frazão JB. The effect of interferon-gamma on splicing defects leading to X-linked chronic granulomatous disease [Master Dissertation]. São Paulo: Instituto de Ciências Biomédicas da Universidade de São Paulo; 2009. Phagocytes present a nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) associated to the plasma membrane that generates superoxide and other oxygen reactive intermediates; playing microbicide, tumoricide, and inflammatory activities. Defects on the NADPH oxidase system leads to Chronic Granulomatous Disease (CGD), characterized by recurrent and severe infections and granuloma. Mutations close to splice sites that interfere with mRNA processing leading to deletion of one or more exons are commonly described in the scientific literature. In these cases, the molecular mechanisms causing CGD are not always totally clarified, as well as the effect of IFN-γ on the mRNA processing or on transcripts stability. Having this together, the analysis of the molecular mechanisms that regulate the expression and function of this system is of great importance for the understanding phagocytic cells physiology and their microbicide activity. The aim of this work was to investigate the effect of IFN-γ on preand post-transcriptional events related to CYBB gene expression, enconding the gp91-phox component in patients with X-linked CGD caused by splicing defects. We will examine the NADPH system activity measuring the superoxide release before and after IFN-γ treatment, analyze CYBB transcripts expression on patients with splicing defects and with nonsense mutations before and after the IFN-γ and the effect of IFN-γ on spliceossome proteins. The study of the NADPH oxidase function demonstrates an impairment on superoxide release after PMA stimuli in cells from patients with CGD caused by splicing defects on CYBB gene, with an improvement on superoxide production after IFN-γ stimuli. There was an increase of superoxide production on not stimulated with PMA control cells (PMN without IFN-γ: 1,32; PMN with IFN-γ:: 2,05 – mononuclear cells without IFN-γ: 2,2; mononuclear cells with IFNγ: 6,6 nmol/10/hour) and on the PMA-stimulated control cells (PMN without IFN-γ: 9,5; PMN with IFN-γ: 8,9 mononuclear cells without IFN-γ: 0,0; mononuclear cells with IFN-γ: 8,2 nmol/10/hour). On cells of patient VG there was an improvement on superoxide production by non stimulated cells (PMN without IFN-γ: 0,75; PMN with IFN-γ: 1,7 mononuclear cells without IFN-γ: 0,0; mononuclear cells with IFN-γ: 3,6 nmol/10/hour) as well as on the PMA-stimulated cells (PMN without IFN-γ: 0,26; PMN with IFN-γ: 0,7 – mononuclear cells without IFN-γ: 3,8; mononuclear cells with IFN-γ: 4,9 nmol/10/hour). Improvement on superoxide production after IFN-γ treatment on non stimulated control cells (without IFN-γ: 0,96; with IFN-γ: 1,01 nmol/10/hour) and stimulated with PMA (without IFN-γ: 7,02; with IFN-γ: 7,11 nmol/10/hour), on patient GCG stimulated with PMA (without IFN-γ: 1,09; with IFNγ: 1,16 nmol/10/hour) and on patient MF not stimulated with PMA (without IFN-γ: 0,55; with IFN-γ: 1,01 nmol/10/hour) could be identified. Preand pos-treatment with IFN-γ PCR assays demonstrate differences on amplified products band pattern of region between Exons 2-4 between control and patient MF (higher bands) and patients GCG and RVSS (lower bands) because of its particular molecular characteristics. Relative expression analysis shows that after IFN-γ treatment an increase on gp91-phox expression in normal individuals (without IFN-γ 2,13; with IFN-γ 2,96) and in patients with X-CGD caused by splicing defects (GCG: without IFN-γ 0,44, with IFN-γ 4,15; RVSS: without IFN-γ 0,54, with IFN-γ 4,11) is noted, and on patient MF (without IFNγ 5,11, with IFN-γ 2,94) there is an reduction of this expression. Real-time PCR results indicate a differentiated gene expression between patients with CGD and healthy controls. In polimorfonuclear cells the expression levels found are higher than on those found for mononuclear cells, Thp1 cells and B-EBV lymphocytes. In polimorfonuclear cells, control cells (without IFN-γ: 1,01, with IFN-γ: 35,99) and on VG patient (without IFN-γ: 3,67, with IFN-γ: 5,31) there is an increase on relative expression when treated with IFN-γ, on control this rise is higher than those found on patient VG. On patient MF an decrease of expression (without IFN-γ: 29.96, with IFN-γ: 24,76) was observed. On mononuclear cells, patient VG (without IFN-γ: 0,73, with IFN-γ: 1,2) and patient MF (without IFN-γ: 0,38, with IFN-γ: 0,78) have relative expression increased when treated with IFN-γ, the values found for patient VG are higher than of those found on patient MF. On control cells it was observed a decrease on expression (without IFN-γ: 29.96, with IFN-γ: 24,76). On Thp1 cells and on B-EBV lymphocytes, there was an increase on CYBB expression in all tested cells when they were treated with IFN-γ. Similar levels of gp91-phox expression were found on Thp1 cells (without IFN-γ: 1,02, with IFN-γ: 1,57), control (without IFN-γ: 1,02, with IFN-γ: 1,79), RVSS (without IFN-γ: 0,87, with IFN-γ: 1,53) and MF (without IFN-γ: 0,13, with IFN-γ: 1,47). Patient GCG presented the lowest levels of gene expression levels (without IFN-γ 0,19, with IFN-γ 2,2). Spliceossome proteins labeling showed an increase of fluorescence in addition to modification on the labeling pattern of Hep-2 adherent cells after IFN-γ treatment. On Thp1, IFN-γ treatment also caused an increase on antigen labeling similar to those observed on Hep-2. In B immortalized lymphocytes form healthy individuals and of one CGD patient with splicing defects (IC) and of one CGD patient with non-sense mutation (MF) it was possible to identificate labeling alterations on spliceossoma proteins, they were more evident on Sm-RNPs proteins. On the patient that presented nonsense mutation (MF), the negative labeling patterns, SSA/Ro and Sm-RNP labeling were considerate the same to those found on control group. These results indicate that IFN-γ may influence CYBB expression by pre-transcriptional events. Our study shows that IFN-γ increases the release of superoxide by cells from healthy individuals and as well as cells from the studied patients. This increase of superoxide release was higher by PMN cells. IFN-γ also increases the expression of gp91-phox gene transcripts in cells form healthy individuals and from the studied CGD patients. IFN-γ acts on the pretranscriptional level increasing the quantity of proteins involved on the splicing process. By this manner, the observed results reinforce the importance of the use of IFN-γ on patients with CGD and that present specific splicing defects.

Efeito do interferon-gamma sobre defeitos de "splicing" que levam à doença granulomatosa crônica ligada ao cromossomo X.