| Background and objective:Behcet’s disease (BD) is a chronic, multisystem inflammatory disorder of unknown etiology. The genetic factors, infections, abnormal innate and acquired immunity play important roles in the pathogenesis of BD. Macrophages, an important component of innate immune cells, can differentiate into a pro-inflammatory classically activated subtype (M1) and an anti-inflammatory alternatively activated subtype (M2) according to their micro environment. Here we investigate how BD patients serum may regulate macrophage polarization and function.Methods:1. Thirty active BD patients and healthy volunteers serum were detected using ELISA for estimating cytokines levels.2. PBMCs from peripheral blood of healthy volunteers were separated and monocytes were isolated from PBMC using magnetic activated cell sorting and positive selection with CD 14 MicroBeads. Monocyte-derived macrophages (HMDM) were obtained after 7 days in DMEM medium supplemented with 10% decomplemented fetal calf serum (FCS),100 IU/ml penicillin and 100 ug/ml streptomycin.3. Macrophage were polarized for 48 h to M1 macrophages using LPS/IFN Υ and to M2 macrophages using IL-4. Flow cytometric analysis was performed to detect the expression of CD86, CD 163 and CD206. ELISA was performed to detect the expression of TNF-a and IL12.4.14 active BD patients and 14 gender-and age matched healthy volunteers serum were used to stimulate macrophage polarization, and macrophage polarization was identified according to surface markers and cytokine production.5. Macrophage phagocytic activity was determined by cellular uptake of Dextran using flow cytometric analysis. Macrophages and naive CD4+T cells were cultured at a 1:5 ratio for Thl and Th17 cell differentiation. Cells were analyzed for intracellular production of IFNy and IL17A using flow cytometric analysis.6. The expressions of (p/T)STAT1 and (p/T) NF-icBp65 were detected using western blot.Results:1. Levels of TNFα, IFN Υ,IL8 and IL6 were significantly increased in serum of active BD compared to healthy control (P<0.05). There was no significant difference in the level of IL10 between two groups (p>0.05). Serum IL6 level and ESR level (p=0.012), IL6 level and hCRP level (p=0.009), IL8 level and hCRP level (p=0.005) and IL8 level and hCRP level (p=0.013) were statistically significant.2. Active BD serum promoted macrophage polarization towards the M1-like subtype, which is characterized by high levels of CD86 (MBD 66.67 ± 15.85% vs MO 45.88 ± 9.75%, p=0.024), IL12 [MBD 139.7pg/ml (36.59-1038.17) vs MO 8.48pg/ml (5.27-132.93), p=0.019] and TNF-a (MBD 205.6± 232.37 pg/ml vs MO 56.02 ±45.99 pg/ml, p=0.189), and a marked down regulation of CD 163 (MBBD 2.2 ± 1.57%vs MO 5.69 ± 2.43%, p=0.009) compared to MO phenotype. The healthy control serum induced a MO macrophage.3. Compared to MO phenotype, The M1 phenotype (Ml 51.38 ± 16.83 vs MO 27.03 ± 11.14, p=0.014) and serum from BD patients induced human monocyte-derived macrophages subtype (MBD 50.53 ± 16.93 vs MO 27.03 ± 11.14, p=0.011) showed increased phagocytosis capacity. Phagocytosis capacity of HC serum induced human monocyte-derived macrophages subtype was also increased, but there was no significant difference (MBD 39.26 ± 15.47 vs MO27.03 ±11.14, p=0.134).4. The proliferation and differentiation of Thl cells was increased with high expressions of IFNy after naive CD4+T cells was cultured with M1 phenotype (Ml 19.5 ± 2.62% vs MO11.78 ± 3.45%, p=0.037) and BD patients induced human monocyte-derived macrophages subtype (MBD 18.5 ± 2.29% vs Mo 11.78 ± 3.45%, p=0.048) compared to MO phenotype.5. Compared to MO phenotype, the expression of CD4+IL17A+was increased after naive CD4+T cells was cultured with M1 phenotype and BD serum induced human monocyte-derived macrophages subtype, but there was no significant difference.6. pSTAT1 and pNF-KBp65 were up-regulation in the Ml phenotype and BD serum induced macrophage compared to MO phenotype.Conclusions:Serum from BD patients could promote human monocyte-derived macrophages from healthy donor polarized towards the Ml-like phenotype in vitro. The BD patients serum induced macrophage showed increased phagocytosis capacity, and further promote the proliferation and differentiation of Thl cells, eventually lead to disease onset. BD serum showed high levels of TNFα, IFN Υ, IL8 and IL6 and JAK-STAT and NF-kB signaling activity was significantly upregulated. This study may provide new insights into the pathogenesis of BD and reveal potential new therapeutic targets for BD treatment. |
PDF Full Text Request