Impact of the New Generation Reconstituted Surfactant CHF5633 on Human CD4+ Lymphocytes

BackgroundNatural 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.AimThe 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.MethodsPurified 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.ResultsNeither 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.ConclusionFor 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.

背景
天然表面活性剂制剂通常从猪或牛肺中分离提取，临床用于治疗早产儿呼吸窘迫综合征。除具备生物物理功效外，多项研究还证实其具有额外的免疫调节特性。在不久的将来，合成表面活性剂制剂或可成为极具前景的替代方案。CHF5633是一款成分明确的新一代重组合成表面活性剂制剂，其组分包含二棕榈酰磷脂酰胆碱（dipalmitoyl-phosphatidylcholine）、棕榈酰油酰磷脂酰甘油（palmitoyl-oleoyl-phosphatidylglycerol）以及表面活性剂蛋白（surfactant protein, SP）B与SP-C的合成类似物。尽管其生物物理功效已在体外与体内实验中得到验证，但其潜在的免疫调节能力目前仍未明确。

研究目的
本研究旨在明确CHF5633及其单一成分对人CD4+淋巴细胞促炎与抗炎细胞因子应答的潜在影响。

研究方法
采用抗CD3/CD28抗体激活纯化的人CD4+ T细胞，将其分别暴露于CHF5633、其组分以及经典的动物源性表面活性剂猪肺磷脂注射液（Poractant alfa，商品名Curosurf®）中。采用流式细胞术与甲基噻唑基二苯基四唑溴化物比色法评估细胞增殖应答与细胞活力。采用定量聚合酶链反应（quantitative PCR，qPCR）检测干扰素γ（IFNγ）、白细胞介素（IL）-2、IL-17A、IL-22、IL-4及IL-10的mRNA表达水平，同时通过流式细胞术检测细胞内蛋白表达水平。

研究结果
在本实验设定的条件下，无论是CHF5633本身，还是其含或不含SP-B、SP-C类似物的磷脂组分，均未对CD4+淋巴细胞的增殖能力与细胞活力产生任何影响。与未暴露的对照组相比，经CHF5633或其组分处理的未激活与激活态CD4+淋巴细胞中，IFNγ、IL-2、IL-17A、IL-22、IL-4及IL-10的mRNA水平，以及IFNγ、IL-2、IL-4与IL-10的蛋白水平均未出现显著变化。然而，与Curosurf®组相比，经CHF5633处理的CD4+淋巴细胞中，抗炎因子IL-4与IL-10的mRNA表达水平显著升高。

结论
本研究首次评估了CHF5633对CD4+淋巴细胞的免疫调节能力。CHF5633未对CD4+细胞产生任何细胞毒性。此外，本研究的体外实验数据表明，CHF5633不会对未激活及激活态CD4+ T细胞产生意外的促炎效应。就抗炎细胞因子而言，与动物源性表面活性剂相比，CHF5633可能不具备全面的抗炎因子下调能力，或可使促炎与抗炎细胞因子应答维持平衡。