ABSTRACT

Anti-cytokine therapy is widely acknowledged as an anti-inflammatory technique to treat varied infectious diseases. TNF-α and IL-1β are major cytokines that regulate every aspect of the inflammatory process. However, the effects of single or dual cytokine neutralization on S. aureus mediated CXCL8 secretion and CXCR1 expression in murine peritoneal macrophages remained noninvestigated. Thus we aimed to explore the effects of kinetic-dose dependent neutralization of TNF-α and IL-1β using specific anti-cytokine antibodies and its influential impact on the CXCL8/CXCR1 axis at different stages of S. aureus (30, 60, and 90 min) infection. The murine peritoneal macrophages were isolated and infected with viable S. aureus followed by subsequent addition of anti-TNF-α and anti-IL-1β into the medium. The treated cells were centrifuged and lysate and supernatant collected for various experiments. The ROS generation was measured and cytokine production was estimated by ELISA. The expression of TNFR1, IL-1R, CXCR1, signaling molecules (NF-κB and JNK) were evaluated by Western blot. The role of single or dual cytokine neutralization on intracellular bacterial phagocytosis had also been analyzed by confocal microscopy. Dual cytokine neutralization significantly suppressed ROS, cytokines, CXCL8 secretion, and intracellular bacterial count compared to single cytokine neutralization and it was more apparent at 90 min post S. aureus infection. There was a drastic reduction in TNFR1, IL-1R, and CXCR1 expression on macrophage surface due to reduced expression of downstream signaling molecules, NF-κB and JNK. Hence dual cytokine neutralization was more effectual compared to single cytokine neutralization in the downregulation of S. aureus induced CXCR1 expression.

摘要

抗细胞因子疗法被广泛认为是治疗各种传染病的抗炎技术。TNF-α 和 IL-1β 是调节炎症过程各个方面的主要细胞因子。然而，单一或双重细胞因子中和对鼠腹膜巨噬细胞中金黄色葡萄球菌介导的 CXCL8 分泌和 CXCR1 表达的影响仍未得到研究。因此，我们旨在探索使用特异性抗细胞因子抗体对 TNF-α 和 IL-1β 的动力学剂量依赖性中和的影响及其对金黄色葡萄球菌不同阶段的 CXCL8/CXCR1 轴的影响（30、60 和90 分钟）感染。分离鼠腹膜巨噬细胞并感染金黄色葡萄球菌随后在培养基中加入抗 TNF-α 和抗 IL-1β。将处理过的细胞离心并收集裂解物和上清液用于各种实验。测量了 ROS 的产生，并通过 ELISA 估计了细胞因子的产生。通过蛋白质印迹评估TNFR1、IL-1R、CXCR1、信号分子（NF-κB和JNK）的表达。还通过共聚焦显微镜分析了单一或双重细胞因子中和对细胞内细菌吞噬作用的作用。与单一细胞因子中和相比，双重细胞因子中和显着抑制了 ROS、细胞因子、CXCL8 分泌和细胞内细菌计数，并且在金黄色葡萄球菌感染后 90 分钟更为明显感染。由于下游信号分子 NF-κB 和 JNK 的表达减少，巨噬细胞表面的 TNFR1、IL-1R 和 CXCR1 表达急剧减少。因此，在下调金黄色葡萄球菌诱导的 CXCR1 表达时，双细胞因子中和比单细胞因子中和更有效。