Fehrholz Markus, Glaser Kirsten, Seidenspinner Silvia, Ottensmeier Barbara, Curstedt Tore, Speer Christian P, Kunzmann Steffen
University Children's Hospital, University of Wuerzburg, Wuerzburg, Germany.
Department of Molecular Medicine and Surgery, Karolinska Institutet at Karolinska University Hospital, Stockholm, Sweden.
PLoS One. 2016 Apr 14;11(4):e0153578. doi: 10.1371/journal.pone.0153578. eCollection 2016.
Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown.
The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4+ lymphocytes.
Purified human CD4+ T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry.
Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4+ lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4+ lymphocytes.
For the first time, the immunomodulatory capacity of CHF5633 on CD4+ lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4+ cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.
天然表面活性剂制剂通常从猪或牛的肺中分离出来，用于治疗早产儿的呼吸窘迫综合征。除了生物物理有效性外，多项研究还记录了其额外的免疫调节特性。在不久的将来，合成表面活性剂制剂可能是一种有前景的替代品。CHF5633是一种新一代的重组合成表面活性剂制剂，成分明确，含有二棕榈酰磷脂酰胆碱、棕榈酰油酰磷脂酰甘油以及表面活性蛋白（SP-）B和SP-C的合成类似物。虽然其生物物理有效性已在体外和体内得到证实，但其可能的免疫调节能力目前尚不清楚。
本研究的目的是确定CHF5633及其单一成分对人CD4+淋巴细胞中促炎和抗炎细胞因子反应的潜在影响。
使用抗CD3/CD28抗体激活纯化的人CD4+T细胞，并使其暴露于CHF5633、其成分或知名的动物源性表面活性剂固尔苏（Curosurf®）。使用流式细胞术和甲基噻唑基二苯基四氮唑溴盐比色法评估增殖反应和细胞活力。通过定量PCR测量IFNγ、IL-2、IL-17A、IL-22、IL-4和IL-10的mRNA表达，同时通过流式细胞术评估细胞内蛋白表达。
在给定条件下，CHF5633及其任何含或不含SP-B或SP-C类似物的磷脂成分对CD4+淋巴细胞的增殖能力和活力均无影响。与未暴露的对照相比，暴露于CHF5633或其成分后的未激活和激活的CD4+淋巴细胞中，IFNγ、IL-2、IL-17A、IL-22、IL-4和IL-10的mRNA以及IFNγ、IL-2、IL-4和IL-10的蛋白水平均未受影响。然而，与固尔苏相比，暴露于CHF5633的CD4+淋巴细胞中抗炎性IL-4和IL-10的mRNA表达水平显著增加。
首次评估了CHF5633对CD4+淋巴细胞的免疫调节能力。CHF5633对CD4+细胞未显示任何细胞毒性。此外，我们的体外数据表明，CHF5633对未激活和激活的CD4+T细胞不会产生意外的促炎作用。就抗炎细胞因子而言，与动物源性表面活性剂相比，它可能缺乏整体的还原能力，可能使促炎和抗炎细胞因子反应保持平衡。
