Immortalization and Characterization of Porcine Macrophages That Had Been Transduced with Lentiviral Vectors Encoding the SV40 Large T Antigen and Porcine Telomerase Reverse Transcriptase

Takenouchi, Takato; Kitani, Hiroshi; Suzuki, Shunichi; Nakai, Michiko; Fuchimoto, Dai-ichiro; Tsukimoto, Mitsutoshi; Shinkai, Hiroki; Sato, Mitsuru; Uenishi, Hirohide

Inflammasomes are large intracellular multiprotein complexes that function during inflammatory immune responses (21) . The activation of inflammasomes triggers caspase-1 activation followed by the maturation and secretion of IL-1β in macrophages. To verify the functional expression of the inflammasome system in IPKM, we assessed the effects of nigericin on the production and secretion of mature IL-1β in these cells. Nigericin is a potassium ionophore and is recognized as an activator of NLRP3 inflammasomes in humans and rodents (22) . Nigericin triggered the production and secretion of mature IL-1β in the IPKM in a dose-dependent manner ( Figure 5A ). This suggests that IPKM are useful for studying the porcine NLRP3 inflammasome system. Extracellular ATP is also known to be an activator of NLRP3 inflammasomes, and the P2X7 purinergic receptor (P2X7R), an ATP-gated cation channel, plays a critical role in NLRP3 inflammasome activation by ATP (23) . In contrast to rodent macrophages, it has previously been demonstrated that the ATP/ P2X7R pathway is impaired in primary PKM (12) . Therefore, the effects of extracellular ATP on inflammasome activation were assessed in IPKM. As expected, extracellular ATP (in the millimolar range) failed to induce the production or secretion of mature IL-1β in IPKM, as was observed in the primary PKM ( Figure 5B) . This supports the notion that the IPKM originated from porcine macrophages and did not arise due to the contamination of the mouse macrophage cell line that was previously established in our laboratory (12, 17) .