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）转导信号的细菌毒素，与多种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TLR-4发挥作用；而脂磷壁酸（lipoteichoic acid, LTA）以及布氏脂蛋白（Braun lipoprotein, BLP）这类细菌脂蛋白，或是其合成类似物Pam3CSK4，则通过TLR-2介导其生物学效应。据认为，TLR介导的部分致病效应，源自内源性生物合成的血小板活化因子（platelet-activating factor, PAF）——这是一种强效的炎症性磷脂，可通过血小板活化因子受体（PAF-receptor, PAF-R）发挥作用。然而，PAF在内毒素血症等炎症性疾病中的作用仍存在争议。为了探究PAF在TLR介导的致病过程中的直接作用，我们向存在或不存在细菌毒素的Wistar白化小鼠腹腔注射PAF。以5 μg/只的剂量向小鼠腹腔注射PAF会导致小鼠猝死；若同时或提前给予高剂量细菌毒素处理，则可延缓小鼠的死亡，这一现象被称为内毒素交叉耐受。细菌毒素诱导的对PAF的耐受可通过提高PAF的浓度得以逆转，这表明交叉耐受具有可逆性。我们利用同时表达经典型PAF-R和TLRs的人源血小板开展了类似实验。尽管细菌毒素无法诱导人源血小板聚集，但它们能够以可逆的方式抑制PAF诱导的血小板聚集。我们利用对PAF高度敏感的兔血小板开展实验，发现细菌毒素（LPS与LTA）以及Pam3CSK4可通过PAF-R依赖的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直接的物理相互作用。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发生。