Endogenous Molecules Induced by a Pathogen-Associated Molecular Pattern (PAMP) Elicit Innate Immunity in Shrimp

Invertebrates rely on an innate immune system to combat invading pathogens. The system is initiated in the presence of cell wall components from microbes like lipopolysaccharide (LPS), β-1,3-glucan (βG) and peptidoglycan (PG), altogether known as pathogen-associated molecular patterns (PAMPs), via a recognition of pattern recognition protein (PRP) or receptor (PRR) through complicated reactions. We show herein that shrimp hemocytes incubated with LPS, βG, and PG caused necrosis and released endogenous molecules (EMs), namely EM-L, EM-β, and EM-P, and found that shrimp hemocytes incubated with EM-L, EM-β, and EM-P caused changes in cell viability, degranulation and necrosis of hemocytes, and increased phenoloxidase (PO) activity and respiratory burst (RB) indicating activation of immunity in vitro. We found that shrimp receiving EM-L, EM-β, and EM-P had increases in hemocyte count and other immune parameters as well as higher phagocytic activity toward a Vibrio pathogen, and found that shrimp receiving EM-L had increases in proliferation cell ratio and mitotic index of hematopoietic tissues (HPTs). We identified proteins of EMs deduced from SDS-PAGE and LC-ESI-MS/MS analyses. EM-L and EM-P contained damage-associated molecular patterns (DAMPs) including HMGBa, HMGBb, histone 2A (H2A), H2B, and H4, and other proteins including proPO, Rab 7 GPTase, and Rab 11 GPTase, which were not observed in controls (EM-C, hemocytes incubated in shrimp salt solution). We concluded that EMs induced by PAMPs contain DAMPs and other immune molecules, and they could elicit innate immunity in shrimp. Further research is needed to identify which individual molecule or combined molecules of EMs cause the results, and determine the mechanism of action in innate immunity.无脊椎动物依赖先天免疫系统（innate immune system）抵御入侵病原体。该系统的激活需以微生物细胞壁组分为触发因子，包括脂多糖（lipopolysaccharide, LPS）、β-1,3-葡聚糖（β-1,3-glucan, βG）与肽聚糖（peptidoglycan, PG），这类物质统称为病原相关分子模式（pathogen-associated molecular patterns, PAMPs）；其启动过程依赖模式识别蛋白（pattern recognition protein, PRP）或模式识别受体（pattern recognition receptor, PRR）的识别，并经由一系列复杂反应完成。 本研究证实，经LPS、βG及PG处理的虾血细胞会发生坏死并释放内源性分子（endogenous molecules, EMs），分别命名为EM-L、EM-β与EM-P；进一步实验发现，经EM-L、EM-β及EM-P孵育的虾血细胞会出现细胞活力改变、脱颗粒与坏死现象，同时酚氧化酶（phenoloxidase, PO）活性与呼吸爆发（respiratory burst, RB）水平升高，提示体外免疫激活。 研究还显示，注射EM-L、EM-β及EM-P的虾，其血细胞数量与其他免疫参数均显著升高，且对弧菌（Vibrio）病原体的吞噬活性增强；此外，注射EM-L的虾，其造血组织（hematopoietic tissues, HPTs）的细胞增殖比例与有丝分裂指数均出现上升。 通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）与液相色谱-电喷雾电离串联质谱（LC-ESI-MS/MS）分析，研究人员对EMs的蛋白组分进行了鉴定。结果表明，EM-L与EM-P中含有损伤相关分子模式（damage-associated molecular patterns, DAMPs），包括HMGBa、HMGBb、组蛋白2A（histone 2A, H2A）、H2B与H4，同时还包含酚氧化酶原（proPO）、Rab7 GTP酶、Rab11 GTP酶；而对照组（EM-C，即经虾盐溶液孵育的血细胞）中未检测到上述蛋白。 综上，本研究表明，由PAMPs诱导产生的EMs含有DAMPs与其他免疫分子，可在虾体内诱发先天免疫应答。未来仍需进一步开展研究，以明确EMs中哪些单一分子或组合分子介导了上述效应，并阐明其在先天免疫中的作用机制。