Illustrations
Figure 1
RAC1 expression levels (A) and LPS-induced IL-1β production (B) in healthy controls and FMF patients in remission or crisis. A) RAC1 expression levels were evaluated by real-time PCR following blood RNA extraction from healthy controls and FMF patients in remission or crisis, in comparison with the reference gene ABL . B) Healthy controls and FMF patients PBMCs were cultured at 1 × 106 cells/mL for 24 hours at 37 °C, 5% CO2 with stimulation of monocytes by E. coli lipopolysaccharide (LPS) (1 μg/mL). Supernatants were collected and IL-1β levels were assayed by Luminex Multiplex Elisa. Data are represented as mean ± SEM. Differences among groups were evaluated using the nonparametric One Way ANOVA test further tested by Tukey's test for post hoc pairwise comparisons. ** P < 0.01; *** P < 0.001.
Figure 1
Figure 2
A) RAC1 gene expression levels in controls and FMF patients in remission with various MEFV genotypes. Subjects indicated by (*) show an overexpression of RAC1 gene (ratio > 1.5), while subjects indicated by (▪) have RAC1 expression levels less than 0.65. B) RAC1 expression levels in FMF patients divided into subsets according to MEFV genotype. Whisker box plot displaying minimum, median, and maximum values of RAC1 expression levels in FMF patients is shown. Patients were divided according to the MEFV genotype as either homozygous for the M694V variant (n = 2), compound heterozygous for M694V (M694V/htz) [M694V/M680I (n = 2), M694V/V726A (n = 1), M694V/M694I (n = 3)] or having other genotypes [M680I/M680I (n = 5), V726A/V726A (n = 1), M694I/M694I (n = 2), M680I/V726A (n = 3), M694I/V726A (n = 4), M694I/E148V (n = 1), V726A/E167D/F479L (n = 1)]. Differences among groups were evaluated using the nonparametric One Way ANOVA test further tested by Tukey's test for post hoc pairwise comparisons. * P < 0,05.
Figure 2
Figure 3
Caspase-1 (A) , IL-1β (B) and IL-6 (C) levels in PBMC culture supernatants of FMF patients and healthy controls.PBMCs were cultured at 1 × 106 cells/mL in 24-well plates. After 1 hour incubation in the presence or absence of 100 μM of the RAC1 inhibitor NSC23766, cells were either left unstimulated or stimulated with E. coli lipopolysaccharide (LPS) (1 μg/mL) and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2 . Supernatants were collected and cytokine levels were assayed. Data are represented as mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001 based on nonparametric Mann-Whitney's U test for differences between FMF patients and healthy controls and Wilcoxon's matched pairs test for values obtained in the presence or absence of RAC1 inhibitor.
Figure 3
Figure 4
MDA levels in PMN culture supernatants of FMF patients and healthy controls. PMNs were cultured at 1 × 106 cells/mL in 24-well plates. After 1 hour incubation in the presence or absence of 100 μM of the RAC1 inhibitor NSC23766, cells were either left unstimulated or stimulated with E. coli lipopolysaccharide (LPS) (1 μg/mL) and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2 . Supernatants were collected and MDA levels were assayed. Data are represented as mean ± SEM. ** P < 0.01 based on nonparametric Mann-Whitney's U test for differences between FMF patients and healthy controls and Wilcoxon's matched pairs test for values obtained in the presence or absence of RAC1 inhibitor.
Figure 4
Figure 5
Catalase levels in PMN culture supernatants of FMF patients and healthy controls. PMNs were cultured at 1 × 106 cells/mL in 24-well plates. After 1 hour incubation in the presence or absence of 100 μM of the RAC1 inhibitor NSC23766, cells were either left unstimulated or stimulated with E. coli lipopolysaccharide (LPS) (1 μg/mL) and incubated 24 h at 37 °C in a humidified atmosphere containing 5% CO2 . Supernatants were collected and catalase levels were assayed. Data are represented as mean ± SEM. * P < 0.05; ** P < 0.01 based on nonparametric Mann-Whitney's U test for differences between FMF patients and healthy controls and Wilcoxon's matched pairs test for values obtained in the presence or absence of RAC1 inhibitor.
Figure 5
Figure 6
Quantification of total (A) and reduced glutathione (GSH) (B) levels and GSH/GSSG ratio (C) in PMN culture supernatants of FMF patients and healthy controls. PMNs were cultured at 1 × 106 cells/mL in 24-well plates. After 1 hour of incubation in the presence or absence of 100 μM of the RAC1 inhibitor NSC23766, cells were either left unstimulated or stimulated with E. coli lipopolysaccharide (LPS) (1 μg/mL) and incubated for 24 h at 37 °C in a humidified atmosphere containing 5% CO2 . Supernatants were collected and levels were assayed. The amount of oxidized glutathione (GSSG) and GSH/GSSG ratio were then calculated. Data are represented as mean ± SEM. * P < 0.05; ** P < 0.01 based on nonparametric Mann-Whitney's U test for differences between FMF patients and healthy controls and Wilcoxon's matched pairs test for values obtained in the presence or absence of RAC1 inhibitor.
Figure 6
Auteurs
1 Lebanese University, Faculty of Sciences II, Department of Life and Earth Sciences, Beirut, Lebanon
2 CHU of Poitiers, University of Poitiers, Laboratoire inflammation, tissus épithéliaux et cytokines (LITEC), EA4331, Poitiers, France
3 Saint Joseph University, Faculty of Medicine, Medical Genetics Unit, Beirut, Lebanon
4 Institut Jérôme Lejeune, Paris, France
* Correspondence: Department of Life and Earth Sciences, Faculty of Sciences II, Lebanese University, Box 90656, Jdeidet El Metn, Fanar, Lebanon.
Objectives Familial Mediterranean fever (FMF) is a recessively inherited autoinflammatory disorder. The caspase-1-dependent cytokine, IL-1β, plays an important role in FMF pathogenesis, and RAC1 protein has been recently involved in IL-1β secretion. This study aims to investigate RAC1 expression and role in IL-1β and caspase-1 production and oxidative stress generation in FMF.