Development of pig conventional dendritic cells from bone marrow hematopoietic cells in vitro

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-/lo CD1-CD163- DEC205+CD11R3 lo CD11R1+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-/lo DEC205 lo CD11R3+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-/lo CD80/86+. 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.info:eu-repo/semantics/publishedVersio

T Helper Plasticity Is Orchestrated by STAT3, Bcl6, and Blimp-1 Balancing Pathology and Protection in Malaria

Hybrid Th1/Tfh cells (IFN-γ+IL-21+CXCR5+) predominate in response to several persistent infections. In Plasmodium chabaudi infection, IFN-γ+ T cells control parasitemia, whereas antibody and IL-21+Bcl6+ T cells effect final clearance, suggesting an evolutionary driver for the hybrid population. We found that CD4-intrinsic Bcl6, Blimp-1, and STAT3 coordinately regulate expression of the Th1 master regulator T-bet, supporting plasticity of CD4 T cells. Bcl6 and Blimp-1 regulate CXCR5 levels, and T-bet, IL-27Rα, and STAT3 modulate cytokines in hybrid Th1/Tfh cells. Infected mice with STAT3 knockout (KO) T cells produced less antibody and more Th1-like IFN-γ+IL-21−CXCR5lo effector and memory cells and were protected from re-infection. Conversely, T-bet KO mice had reduced Th1-bias upon re-infection and prolonged secondary parasitemia. Therefore, each feature of the CD4 T cell population phenotype is uniquely regulated in this persistent infection, and the cytokine profile of memory T cells can be modified to enhance the effectiveness of the secondary response

El ácido linoleico conjugado aumenta la inmunidad innata de la mucosa intestinal contra el parásito Giardia lamblia en un modelo murino

Giardia lamblia, a protozoan intestinal parasite is able to evade or suppress defense mechanisms, such as innate response. Antigen presenting cells (APC) like dendritic cells may orchestrate an immune response and support a more effective adaptive defense. Conjugated linoleic acid (CLA), a dietary lipid, has shown biological activity on APC. The aim was to evaluate the effect of CLA in intestinal innate response measuring the frequency of mucosal APC populations in Giardia lamblia murine infection. Method The giardiasis infection model was established in C3H/HeN mice (n=32), and parasite load was followed at 0, 2, 6 and 8 days post infection (dpi). APC obtained from small intestine by enzymatic digestion were assessed by flow cytometry. Oral supplementation with CLA (50:50 of cis-9, trans-11-CLA and trans-10, cis-12-CLA isomers) and placebo control begun three days before infection and continued during first week post infection. Results Infection kinetics showed a peak of trophozoites at 6 dpi in acute phase. Parasite load was lower in CLA group in comparison with control infected group (p(0.05). Conjugated linoleic acid stimulates innate immune response, percentage of intestinal CD11chiMHC-IIhi increases after 2 dpi, meanwhile CD11chiMHC-IIhiCD103+ APC was higher after three days of CLA supplementation than control (p(0.05). Also, the CD11c+ F4/80+ population shown a percentage increases after 6 and 8 dpi by the effect of treatment and time of infection (p<0.05). Conclusion In conclusion CLA increased percentages of small intestine APC phenotypes CD11chiMHCIIhi, CD11chiMHCIIhiCD103+ and CD11c+ F4/80+, and reduced G. lamblia parasitic load. Further studies are needed to elucidate potential mechanisms involved in CLA immunomodulatory effect and its contribution in adaptive immune response.Giardia lamblia es un parásito intestinal de prevalencia mundial. Es capaz de evadir los mecanismos de defensa del hospedero. Las células presentadoras de antígeno (CPA), como las células dendríticas y macrófagos, orquestan la respuesta inmune y favorecen el desarrollo de una respuesta adaptativa más robusta y eficiente, y tienen un papel clave en la respuesta inmune de mucosas en intestino. Algunos componentes de la dieta pueden modular la capacidad de respuesta del sistema inmune, como ejemplo el ácido linoleico conjugado, un lípido de origen dietario que presenta actividad biológica sobre células del linaje monocito fagocito. El objetivo del estudio fue evaluar la actividad del CLA sobre la respuesta inmune innata intestinal en un modelo murino de infección con Giardia lamblia. Método El modelo de infección se estableció en los ratones C3H/HeN (n=32). Se diseñó un bioensayo experimental con dos grupos de animales uno suplementado con CLA (mezcla de isómeros 50:50 de 9-cis, 11-trans y 10-trans, 12-cis) y otro control con PBS. La suplementación oral se dio por 11 días, 3 días previo a la infección, y continuó por 8 días más después de la infección. Se sacrificaron cuatro animales de cada grupo a los 0, 2, 6 y 8 días post infección (dpi) para conteo de trofozoítos y obtención de las células presentadoras de antígeno, a partir de digestión enzimática del intestino delgado. La fenotipificación de poblaciones se realizó por citometría de flujo. Resultados La cinética de infección mostró un pico en el número de trofozoítos a los 6 dpi en la fase aguda. La carga parasitaria fue menor en el grupo con CLA comparado con el grupo control (p(0.05). Los porcentajes de las poblaciones de APC intestinales CD11chiMHC-IIhi se incrementaron a los 2 dpi y CD11chiMHC-IIhiCD103+ de forma temprana después de 3 días de suplementación con CLA, comparado con el grupo control (p(0.05). La población de células CD11c+ F4/80+, también mostró un incremento por efecto del tratamiento y del tiempo de infección (p<0.01). Conclusión La suplementación con el CLA redujo la carga parasitaria de Giardia lamblia y estimuló la respuesta innata, incrementando los porcentajes de poblaciones de células presentadoras de antígeno CD11chiMHC-IIhi, CD11chiMHC-IIhiCD103+ y CD11c+ F4/80+ en intestino delgado. Serán necesarios más estudios que permitan dilucidar los potenciales mecanismos involucrados en la inmunomodulación del CLA en la mucosa intestinal y su contribución en la respuesta adaptativa