Background Behçet’s Disease (BD) is a systemic inflammatory disorder characterized by vasculitis that is usually accompanied by oral and genital ulcers, skin lesions, uveitis and arthritis. Increased neutrophil activity and hyperresponsiveness to streptococci have been considered to play a role in Behçet’s disease (BD) pathogenesis.
Objectives We previously showed that testosterone (T) may also play a role in BD pathogenesis via increasing neutrophil activity and decreasing neutrophil apoptosis. Here we aimed further to define the effects of testosterone in detail.
Methods Twelve BD patients who were in remission at least 6 months were included in this study. Peripheral blood from patient and 20 controls (10 ankylosing spondylitis/10 healthy donors) groups were drawn into heparinized Vacutainer tubes (BD BioSciences, USA). None of the patients were on any immunosuppressive treatment other than colchicum dispert 1.5gr/d. Neutrophils were incubated with testosterone, CpG DNA (5’-TCG ATC GGG GCG GGG CGA GC-3’, XX IDT) and lipoteichoic acid (LTA) and in combinations of Ts + LTA and Ts + CpG DNA. Activation degree of neutrophils after a 2 hour incubation were analyzed by Dihydrorhodamine 123 conversion to rhodamine as a result of mitochondria activation. Culture supernatants were collected for cytokine measurements. FC500 flow cytometer and CXP software were used for analysis. IL-1, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, INF-gamma, TNF-alpha were assessed by using Th1/Th2 11plex RTU FlowCytomix Kit (BenderMed/e-Bioscience, Austria) on FC500 cytometry.
Results Testosterone caused neutrophil activation in all groups, however, LTA and CpGDNA activated neutrophils in combination with T exclusively in BD (LTA vs LTA/T 66.2±23.7 vs82.3±22.5 p<0.05; CpGDNA vs CpGDNA/T 62.2±23.6 vs 84.7±13.4 p<0.02)When we further analyze the effect of T onto cytokines in different conditions, we observed that T induce IL-10 secretion (unstimulated vs stimulated 9.6±1.01 vs 15.9±4.3 p<0.003)in HC. Whereas, T led to elevated IL-12 levels (BD: 15.1±4.6 vs 21.4±5.4 p<0.002; AS:14.1±2.4 vs20.5±5.9 p<0.006) in both BD and AS and increased INFg only in AS. Interestingly, TNF-a levels were increased with LTA in both BD (18.5±7.0 vs 28.4±16.0 p<0.01) and AS (16.5±1.7 vs 31.6±25.3 p<0.03) compared to HC but significantly elevated IL-4 levels were observed in BD patients (103.2±42.5) compared to AS (19.5±2.8) and HC (17.6±3.4) when stimulation occurred with LTA+T (p<0.0001). Similarly, CpGDNA+T induced significant upregulation of TNF-a (18.5±7.0 vs 57.4±52.1 p<0.02) in BD patients but also led to increase in IL-4 levels in BD (93.1±37.2) compared to AS (18.3±3.5,p<0.0001) and HC (20.1±2.4,p<0.01).
Conclusions Despite immunosuppressive behavior in healthy subjects, T causes TH1 type immune alterations in BD patients and AS patients. The higher amount of IL-4 stimulation seen with uniquely in BD patients might be related to IL-4 gene polymorphism which is suggested to be a susceptibility factor to BD.
Disclosure of Interest None Declared
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