Supplementary Material for: TLR9 dependent activation by inactivated parapoxvirus ovis (iPPVO) in murine bone marrow-derived dendritic cells is associated with specific strain-dependent dendritic cell subsets

Introduction: Inactivated parapoxvirus ovis (iPPVO) exerts strong immunomodulatory effects on innate immune cells, making it an attractive therapeutic candidate. However, little is known about the signaling pathways involvIing in iPPVO-induced immune responses. Methods: In this study, we systematically analyzed how different types of dendritic cells (DCs) react to iPPVO (Zylexis, strain D1710) in both BALB/c and C57BL/c mice by Flow cytometry and ELISAs, and investigated which signaling pathway is related to DC activation by Western blotting and Protein profiling. Results: We demonstrated that bone marrow-derived conventional DCs (BM-cDCs) and bone marrow-derived plasmacytoid DCs (BM-pDCs) matured and secreted IFN-α/β in response to Zylexis stimulation in both mouse strains. Similarly, Zylexis promoted the secretion of IL-12/23p40 and TNF by pDCs. However, IL-12/23p40 and TNF secretion by cDCs was induced in BALB/c mice but not in C57BL/6 mice. Analyzing the underlying signaling pathways revealed that iPPVO-induced maturation of cDCs was TLR9 independent, while the maturation of pDCs partially depended on the TLR9 pathway. Moreover, the production of proinflammatory cytokines by cDCs and the secretion IFN-α/β by pDCs partially depended on the TLR9 pathway in both mouse strains. Therefore, other signaling pathways seem to participate in the response of DCs to iPPVO, supported by protein profiling.Conclusion: Our data provide useful insights into the diversity of iPPVO sensors and their varying effects across different strains and species.