Patgaonkar Mandar S, Sathe Ameya, Selvaakumar C, Reddy K V R
Division of Molecular Immunology, National Institute for Research in Reproductive Health (NIRRH), Indian Council of Medical Research (ICMR), Jehangir Merwanji Street, Parel, Mumbai 400 012, India.
Clin Vaccine Immunol. 2011 Oct;18(10):1632-43. doi: 10.1128/CVI.00411-10. Epub 2011 Aug 24.
Antimicrobial peptides (AMPs) constitute a phylogenetically ancient form of innate immunity that provides host defense at various mucosal surfaces, including the vagina. Recently, we have identified one such AMP, rabbit vaginal fluid hemoglobin alpha peptide (RVFHbαP), from the vaginal lavage of rabbits (Oryctolagus cuniculus). The recent demonstration of a protective role of this peptide in erythrocytes and vaginal cells led us to investigate (i) the lipopolysaccharide (LPS) interactive domain in RVFHbαP and (ii) whether RVFHbαP of rabbit origin modulates the cellular immune responses of another species (humans) in vitro. HeLa-S3, a human vaginal epithelial cell line (hVEC), was exposed to LPS alone (10 μg/ml for 6 h), or LPS-induced cells were treated with RVFHbαP (70.45 μM for 1 h) and cultured for 24 h, and the results obtained were compared with the medium control. We show here that RVFHbαP exerts an anti-inflammatory activity in hVECs, as suggested by the prevention of LPS-induced production of extracellular (supernatant) and intracellular (lysate) levels of cytokines (interleukin 6 [IL-6] and IL-1α) and chemokines (IL-8 and monocyte chemoattractant protein 1 [MCP-1]). The demonstration of Toll-like receptor 4 (TLR4) and NF-κB expression in hVECs and the observations of RVFHbαP suppression of human β-defensin-1 (hBD1) mRNA expression further support the hypothesis of a genomic activity of RVFHbαP. Confocal microscopy and flow cytometry results demonstrate that RVFHbαP inhibits LPS-induced phagocytosis of Escherichia coli by macrophages. The chemotaxis studies performed using the Boyden chamber Transwell method showed the increased migration of U937 cells when supernatants of LPS-induced hVECs were used, and this effect was inhibited by RVFHbαP. In conclusion, our study proposes a novel explanation for the protective role of RVFHbαP in inflammation-associated infections, which not only may provide the new cellular targets for the screening of RVFHbαP ligands acting in the vaginal tissue but also has the potential to develop RVFHbαP as a therapeutic agent for reproductive tract infections.
抗菌肽（AMPs）是一种进化上古老的固有免疫形式，可在包括阴道在内的各种黏膜表面提供宿主防御。最近，我们从兔（穴兔）的阴道灌洗液中鉴定出了一种这样的抗菌肽，即兔阴道液血红蛋白α肽（RVFHbαP）。最近该肽在红细胞和阴道细胞中的保护作用证明促使我们研究：（i）RVFHbαP中的脂多糖（LPS）相互作用结构域；（ii）兔源的RVFHbαP在体外是否能调节另一个物种（人类）的细胞免疫反应。人阴道上皮细胞系（hVEC）HeLa-S3单独暴露于LPS（10μg/ml，6小时），或者用RVFHbαP（70.45μM，1小时）处理LPS诱导的细胞并培养24小时，将所得结果与培养基对照进行比较。我们在此表明，RVFHbαP在hVEC中发挥抗炎活性，这表现为预防LPS诱导的细胞因子（白细胞介素6 [IL-6]和IL-1α）和趋化因子（IL-8和单核细胞趋化蛋白1 [MCP-1]）细胞外（上清液）和细胞内（裂解物）水平的产生。hVEC中Toll样受体4（TLR4）和NF-κB表达的证明以及RVFHbαP对人β-防御素-1（hBD1）mRNA表达的抑制作用的观察结果进一步支持了RVFHbαP具有基因组活性的假说。共聚焦显微镜和流式细胞术结果表明，RVFHbαP抑制LPS诱导的巨噬细胞对大肠杆菌的吞噬作用。使用Boyden小室Transwell方法进行的趋化性研究表明，当使用LPS诱导的hVEC的上清液时，U937细胞的迁移增加，而这种作用被RVFHbαP抑制。总之，我们的研究为RVFHbαP在炎症相关感染中的保护作用提出了一种新的解释，这不仅可能为筛选在阴道组织中起作用的RVFHbαP配体提供新的细胞靶点，而且有潜力将RVFHbαP开发为生殖道感染的治疗药物。
