Ex vivo and in vitro production of pro-inflammatory cytokines in Blau syndrome
Ex vivo and in vitro production of pro-inflammatory cytokines in Blau syndrome
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DOI：
10.4081/reumatismo.2014.772
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发表时间：
2014-01-01
期刊：
影响因子：
1.4
通讯作者：
Punzi, L.
中科院分区：
文献类型：
--
作者：
Galozzi, P.;Negm, O.;Punzi, L.
本研究的目的是研究与BLAU综合征(BS)相关的CARD15/NOD2基因P383K突变患者外周血单个核细胞(PBMC)、3例患者和健康对照的外周血单个核细胞(PBMC)体外和体外分泌促炎细胞因子的情况。外周血单核细胞在有或没有炎症促进剂的情况下培养，如脂多糖(LPS)和胞壁二肽(MDP)。用免疫分析法或芯片法检测IL-1β、IL-6、IL-8、肿瘤坏死因子(TNF)-α和干扰素(干扰素)-γ的水平。为了进行体外研究，将人野生型和P.E383K突变的NOD2基因克隆到表达载体pCMV-Tag2c中，构建了不同的表达载体。稳定转染HEK293细胞，加入或不加入MDP培养，检测其培养上清液中IL-8水平。两项研究均采用非参数检验进行统计分析。体外和体外研究均未发现所分析的促炎细胞因子的分泌显著增加。P.E383K突变的NOD2转基因细胞表达低水平的IL-8。血清和PBMC培养上清液的体外基础水平在患者和对照组中呈现相似的IL-1β、IL-6、TNF-α和干扰素-γ水平。与对照组相比，单独或成对的刺激剂(LPS和MDP)的存在并不会导致患者所有细胞因子浓度的显著增加。综上所述，体外和体外数据表明，携带P.E383K的BS患者并不存在IL-1β和其他促炎细胞因子的主要中介作用。
The objective was to study both ex vivo and in vitro secretion of pro-inflammatory cytokines in patients affected by Blau syndrome (BS) and carrying p.E383K mutation in the CARD15/NOD2 gene associated with the disease.For ex vivo studies, peripheral blood mononuclear cells (PBMCs), serum from three patients and healthy controls have been collected. PBMCs have been cultured in the presence or absence of inflammatory enhancers, such as lipopolysaccharide (LPS) and muramyl dipeptide (MDP). The levels of interleukin (IL)-1 beta, IL-6, IL-8, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were assayed by either immunoassay or array-based system. For in vitro studies, different constructs were created cloning human wild-type and p.E383K-mutated NOD2 cDNA into the expression vector pCMV-Tag2c. HEK293 cell lines were stably transfected, cultured with or without MDP and IL-8 level was assayed in their surnatants. Statistical analysis in both studies was performed using non-parametric tests.Both ex vivo and in vitro studies have not identified a significant increase in secretion of the analyzed pro-inflammatory cytokines. p.E383K-mutated NOD2 transfected cells express low level of IL-8. The ex vivo basal level results from both serum and PBMCs surnatants present similar levels of IL-1 beta, IL-6, TNF-alpha and IFN-gamma in patients and controls. The presence of the stimulant agents (LPS and MDP), either individual or paired, does not lead to significant increases in all cytokines concentrations in patients compared to controls.Taken together, the ex vivo and in vitro data suggest that there is not a primary mediation of IL-1 beta and other pro-inflammatory cytokines in BS patients carrying p.E383K.