Reversible cross-tolerance to platelet-activating factor signaling by bacterial toxins

Bacterial toxins signaling through Toll-like receptors (TLRs) are implicated in the pathogenesis of many inflammatory diseases. Among the toxins, lipopolysaccharide (LPS) exerts its action via TLR-4 while lipoteichoic acid (LTA) and bacterial lipoproteins such as Braun lipoprotein (BLP) or its synthetic analogue Pam3CSK4 act through TLR-2. Part of the TLR mediated pathogenicity is believed to stem from endogenously biosynthesized platelet-activating factor (PAF)- a potent inflammatory phospholipid acting through PAF-receptor (PAF-R). However, the role of PAF in inflammatory diseases like endotoxemia is controversial. In order to test the direct contribution of PAF in TLR-mediated pathogenicity, we intraperitoneally injected PAF to Wistar albino mice in the presence or absence of bacterial toxins. Intraperitoneal injection of PAF (5 μg/mouse) causes sudden death of mice, that can be delayed by simultaneously or pre-treating the animals with high doses of bacterial toxins- a phenomenon known as endotoxin cross-tolerance. The bacterial toxins- induced tolerance to PAF can be reversed by increasing the concentration of PAF suggesting the reversibility of cross-tolerance. We did similar experiments using human platelets that express both canonical PAF-R and TLRs. Although bacterial toxins did not induce human platelet aggregation, they inhibited PAF-induced platelet aggregation in a reversible manner. Using rabbit platelets that are ultrasensitive to PAF, we found bacterial toxins (LPS and LTA) and Pam3CSK4 causing rabbit platelet aggregation via PAF-R dependent way. The physical interaction of PAF-R and bacterial toxins is also demonstrated in a human epidermal cell line having stable PAF-R expression. Thus, we suggest the possibility of direct physical interaction of bacterial toxins with PAF-R leading to cross-tolerance.

通过Toll样受体（Toll-like receptors, TLRs）进行信号转导的细菌毒素，与诸多炎症性疾病的发病机制密切相关。在这类毒素中，脂多糖（lipopolysaccharide, LPS）通过TLR4发挥生物学效应，而脂磷壁酸（lipoteichoic acid, LTA）、布氏脂蛋白（Braun lipoprotein, BLP）等细菌脂蛋白及其合成类似物Pam3CSK4，则通过TLR2介导信号通路。
目前认为，TLR介导的致病效应部分源自内源性合成的血小板活化因子（platelet-activating factor, PAF）——一种可通过PAF受体（PAF-receptor, PAF-R）发挥作用的强效炎症性磷脂。然而，PAF在内毒素血症等炎症性疾病中的作用尚存争议。
为验证PAF在TLR介导的致病过程中的直接贡献，我们在存在或不存在细菌毒素的情况下，向Wistar白化小鼠腹腔注射PAF。单次腹腔注射5μg/只的PAF可导致小鼠猝死，而同时或提前给予动物大剂量细菌毒素可延缓小鼠死亡，这一现象被称为内毒素交叉耐受。升高PAF浓度可逆转细菌毒素诱导的PAF耐受，这提示交叉耐受具有可逆性。
我们使用同时表达经典PAF-R和TLRs的人体血小板开展了类似实验。尽管细菌毒素无法直接诱导人体血小板聚集，但它们可通过可逆方式抑制PAF诱导的血小板聚集。
利用对PAF高度敏感的兔血小板进行实验，我们发现细菌毒素（LPS与LTA）以及Pam3CSK4可通过PAF-R依赖途径诱导兔血小板聚集。
在稳定表达PAF-R的人表皮细胞系中，我们也证实了PAF-R与细菌毒素之间存在物理相互作用。
综上，我们提出细菌毒素可与PAF-R发生直接物理相互作用，进而介导交叉耐受的可能性。