Takenouchi Takato, Kitani Hiroshi, Suzuki Shunichi, Nakai Michiko, Fuchimoto Dai-Ichiro, Tsukimoto Mitsutoshi, Shinkai Hiroki, Sato Mitsuru, Uenishi Hirohide
Division of Animal Sciences, Institute of Agrobiological Sciences, National Agriculture and Food Research Organization, Tsukuba, Japan.
Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Japan.
Front Vet Sci. 2017 Aug 21;4:132. doi: 10.3389/fvets.2017.00132. eCollection 2017.
The domestic pig is an important agricultural animal, and thus, infectious diseases that affect pigs can cause severe economic losses in the global swine industry. Various porcine pathogens target macrophages, which are classical innate immune cells. Although macrophages basically protect the host from pathogens, they also seem to contribute to infectious processes. Therefore, cultured macrophages can be used to develop models for studying not only genes associated with porcine innate immunity but also the infectious processes of porcine pathogens. However, the availability of porcine macrophage cell lines is limited. In this study, we describe a novel immortalized porcine kidney-derived macrophage (IPKM) cell line, which was generated by transferring the SV40 large T antigen (SV40LT) and porcine telomerase reverse transcriptase (pTERT) genes into primary porcine kidney-derived macrophages using lentiviral vectors. The IPKM displayed a typical macrophage morphology and was routinely passaged (doubling time: about 4 days). These cells were immunostained for macrophage markers. In addition, they exhibited substantial phagocytosis of polystyrene microbeads and released inflammatory cytokines upon lipopolysaccharide (LPS) stimulation. Furthermore, the maturation and secretion of interleukin-1β were observed after nigericin-induced inflammasome activation in LPS-primed IPKM. These findings suggest that IPKM exhibit the typical inflammatory characteristics of macrophages. By transferring the SV40LT and pTERT genes using lentiviral vectors, we also successfully immortalized macrophages derived from the peripheral blood of a low-density lipoprotein receptor-deficient pig. These results suggest that the co-expression of SV40LT and pTERT is an effective way of immortalizing porcine macrophages.
家猪是一种重要的农业动物，因此，影响猪的传染病会给全球养猪业造成严重经济损失。多种猪病原体靶向巨噬细胞，巨噬细胞是经典的固有免疫细胞。虽然巨噬细胞基本上能保护宿主免受病原体侵害，但它们似乎也参与了感染过程。因此，培养的巨噬细胞不仅可用于开发研究与猪固有免疫相关基因的模型，还可用于研究猪病原体的感染过程。然而，猪巨噬细胞系的可得性有限。在本研究中，我们描述了一种新型的永生化猪肾源巨噬细胞（IPKM）系，它是通过使用慢病毒载体将SV40大T抗原（SV40LT）和猪端粒酶逆转录酶（pTERT）基因导入原代猪肾源巨噬细胞而产生的。IPKM呈现典型的巨噬细胞形态，可常规传代（倍增时间：约4天）。这些细胞用巨噬细胞标志物进行免疫染色。此外，它们对聚苯乙烯微珠表现出大量吞噬作用，并在脂多糖（LPS）刺激下释放炎性细胞因子。此外，在LPS预处理的IPKM中，尼日利亚菌素诱导炎性小体激活后，观察到白细胞介素-1β的成熟和分泌。这些发现表明IPKM表现出巨噬细胞典型的炎性特征。通过使用慢病毒载体转移SV40LT和pTERT基因，我们还成功地使低密度脂蛋白受体缺陷猪外周血来源的巨噬细胞永生化。这些结果表明，SV40LT和pTERT的共表达是使猪巨噬细胞永生化的有效方法。
