Abstract
In recent years, porcine dendritic cells (DCs) have been identified from pig tissues. However, studying the interaction of porcine DCs with pathogens is still difficult due to the scarcity of DCs in tissues. In the present work, the Flt3-ligand (Flt3L)-based in vitro derivation system was further characterized and compared with other cytokine derivation models using a combination of factors: stem cell factor (SCF), GM-CSF, and IL-4. The method using Flt3L alone or combined with SCF supported the development of pig bone marrow hematopoietic cells into in vivo equivalent conventional DCs (cDCs). The equivalent cDC1 (the minor population in the cultures) were characterized as CADM1+CD14–MHC-II+CD172a–/loCD1–CD163– DEC205+CD11R3loCD11R1+CD33+CD80/86+. They expressed high levels of FLT3, ZBTB46, XCR1, and IRF8 mRNA, were efficient in endocytosing dextran and in proliferating allogenic CD4+CD8+ T cells, but were deficient in phagocyting inactivated Staphylococcus aureus (S. aureus). Also, after poly I:C stimulation, they predominantly produced IL-12p40a and matured as indicated by the increase of MHC-I, MHC-II, and CD80/86. The equivalent cDC2 (the main population) were CADM1+CD14–MHC-II+C D172a+CD1+CD163–/loDEC205loCD11R3+CD11R1+CD33+CD80/86+; meanwhile, they overexpressed FcεR1α and IRF4 mRNA. They showed high efficiency in the endocytosis of dextran, but weak in phagocytosing bacteria. They supported allogenic CD4+CD8–/CD4+CD8+ T cell proliferation and were high producers of IL-12p40 (upon TLR7 stimulation) and IL-10 (upon TLR7 stimulation). TLR ligand stimulation also induced their maturation. In addition, a CD14+ population was identified with the phenotype CADM1+CD14+MHC-II+CD172a+ CD1+CD163+DEC205–CD11R3+CD11R1+CD33–/loCD80/86+. They shared some functional similarities with cDC2 and were distinguishable from macrophages. This CD14+ population was efficient in phagocyting S. aureus but showed less maturation upon TLR ligand stimulation than cDC1 or cDC2. The alternative methods of DC derivation including GM-CSF and/or IL-4 produced mostly CADM1– cells that did not fulfill the canonical phenotype of bona fide porcine DCs. Our study provides an exhaustive characterization of Flt3L-derived DCs with different methods that can help the in vitro study of the interaction of DCs with porcine-relevant pathogens.
Highlights
Pig farming is an important source of animal protein
There are different types of Dendritic cells (DCs) that are recognized based on their ontogeny [5, 6]: the monocyte-derived DCs, which are generated from activated monocytes in the tissues [7], and the conventional or bona fide DCs that are generated from Flt3 (CD135)-expressing precursors [8] and can be subdivided into classical/conventional (c) cDC1 and cDC2, which can be differentiated by gene expression and cytokine profiles [5, 6] and their ability to cross-present antigens [9, 10]
The major one (52.9 ± 4.0%, among cells of CADM1+) presented a phenotype of CD14−MHC-IIhiCD172a+ that was compatible with cDC2
Summary
Pig farming is an important source of animal protein. Pigs are epidemiologically important hosts of pathogens that can affect humans such as the H1N1 “swine flu” pandemic in 2009 [2]. Pigs are models for studying several human diseases [3] and potential donors for xenotransplantation [4]. In all those aspects, a better understanding of the pig immune system and of the interaction of pathogens with it is scientifically relevant. Dendritic cells (DCs) are sentinels of the immune system, essential for the activation of naïve T cells to promote the initiation of the adaptive immune response. There are plasmacytoid DCs (pDCs), specialized in producing high amounts of type I interferons (especially IFN-α) during antiviral responses [11, 12], and Langerhans cells are a special type of DCs generated from erythro-myeloid progenitors [6]
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