Research Centre for Animal Biologics, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
Department of Veterinary Medicine, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan.
Viruses. 2023 Mar 17;15(3):775. doi: 10.3390/v15030775.
PRRSV infects CD163-positive macrophages and skews their polarization toward an M2 phenotype, followed by T-cell inactivation. In our previous study, we found that recombinant protein A1 antigen derived from PRRSV-2 was a potential vaccine or adjuvant for immunization against PRRSV-2 infection due to its ability to repolarize macrophages into M1 subtype, thereby reducing CD163 expression for viral entry and promoting immunomodulation for Th1-type responses, except for stimulating Toll-like receptor (TLR) activation. The aim of our current study was to evaluate the effects of another two recombinant antigens, A3 (ORF6L5) and A4 (NLNsp10L11), for their ability to trigger innate immune responses including TLR activation. We isolated pulmonary alveolar macrophages (PAMs) from 8- to 12-week-old specific pathogen free (SPF) piglets and stimulated them with PRRSV (0.01 MOI and 0.05 MOI) or antigens. We also investigated the T-cell differentiation by immunological synapse activation of PAMs and CD4 T-cells in the cocultured system. To confirm the infection of PRRSV in PAMs, we checked the expression of , , , and . Our results showed that the expression of , , and were significantly upregulated in PAMs by A3 antigen induction, similar to the extent of PRRSV infection. Gene profile results showed that A3 repolarizes macrophages into the M1 subtype potently, in parallel with A1, as indicated by significant upregulation of proinflammatory genes (, -, - and ). Upon immunological synapse activation, A3 potentially differentiated CD4 T cells into Th1 cells, determined by the expression of IL-12 and IFN-γ secretion. On the contrary, antigen A4 promoted regulatory T cell (T-reg) differentiation by significant upregulation of expression. Finally, we concluded that the PRRSV-2 recombinant protein A3 provided better protection against PRRSV infection, suggested by its capability to reeducate immunosuppressive M2 macrophages into proinflammatory M1 cells. As M1 macrophages are prone to be functional antigen-presenting cells (APCs), they can call for TLR activation and Th1-type immune response within the immunological synapse.
PRRSV 感染 CD163 阳性巨噬细胞，并使其极化向 M2 表型倾斜，随后导致 T 细胞失活。在我们之前的研究中，我们发现来自 PRRSV-2 的重组蛋白 A1 抗原是一种有潜力的疫苗或佐剂，可用于免疫接种 PRRSV-2 感染，因为它能够将巨噬细胞重新极化为 M1 亚型，从而减少病毒进入的 CD163 表达，并促进 Th1 型反应的免疫调节，除了刺激 Toll 样受体 (TLR) 激活。我们目前研究的目的是评估另外两种重组抗原 A3（ORF6L5）和 A4（NLNsp10L11）的作用，以确定它们触发先天免疫反应（包括 TLR 激活）的能力。我们从 8 至 12 周龄的无特定病原体 (SPF) 仔猪中分离出肺泡巨噬细胞 (PAMs)，并用 PRRSV（0.01 MOI 和 0.05 MOI）或抗原刺激它们。我们还研究了 PAMs 和 CD4 T 细胞在共培养系统中的免疫突触激活所导致的 T 细胞分化。为了确认 PAMs 中 PRRSV 的感染，我们检查了 、 、 和 的表达。我们的结果表明，A3 抗原诱导 PAMs 中 、 和 的表达显著上调，与 PRRSV 感染的程度相似。基因谱结果表明，A3 像 A1 一样，将巨噬细胞强烈地重新极化到 M1 亚型，这是由促炎基因（ 、 、 和 ）的显著上调所表明的。在免疫突触激活后，A3 通过 IL-12 的表达和 IFN-γ 的分泌，可能将 CD4 T 细胞分化为 Th1 细胞。相反，抗原 A4 通过 表达的显著上调，促进了调节性 T 细胞（T-reg）的分化。最后，我们得出结论，PRRSV-2 重组蛋白 A3 通过将免疫抑制性 M2 巨噬细胞重新教育为促炎 M1 细胞，为 PRRSV 感染提供了更好的保护。由于 M1 巨噬细胞容易成为功能性抗原呈递细胞 (APC)，因此它们可以在免疫突触内引起 TLR 激活和 Th1 型免疫反应。
