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There is a rapidly growing interest in how the avian intestine is affected by dietary components and feed additives. The paucity of physiologically relevant models has limited research in this field of poultry gut health and led to an over-reliance on the use of live birds for experiments. The development of complex 3D intestinal organoids or "mini-guts" has created ample opportunities for poultry research in this field. A major advantage of the floating chicken intestinal organoids is the combination of a complex cell system with an easily accessible apical-out orientation grown in a simple culture medium without an extracellular matrix. The objective was to investigate the impact of a commercial proprietary blend of organic acids and essential oils (OA+EO) on the innate immune responses and kinome of chicken intestinal organoids in a Salmonella challengemodel. Tomimic the in vivo prolonged exposure of the intestine to the product, the intestinal organoids were treated for 2 days with 0.5 or 0.25 mg/mL OA+EO and either uninfected or infected with Salmonella and bacterial load in the organoids was quantified at 3 hours post infection. The bacteria were also treated with OA+EOfor 1 day prior to challenge of the organoids to mimic intestinal exposure. The treatment of the organoids with OA+EO resulted in a significant decrease in the bacterial load compared to untreated infected organoids. The expression of 88 innate immune genes was investigated using a high throughput qPCR array, measuring the expression of 88 innate immune genes. Salmonella invasion of the untreated intestinal organoids resulted in a significant increase in the expression of inflammatory cytokine and chemokines as well as genes involved in intracellular signaling. In contrast, when the organoids were treatedwithOA+EO and challenged with Salmonella, the inflammatory responses were significantly downregulated. The kinome array data suggested decreased phosphorylation elicited by the OA+EO with Salmonella in agreement with the gene expression data sets. This study demonstrates that the in vitro chicken intestinal organoids are a new tool to measure the effect of the feed additives in a bacterial challenge model by measuring innate immune and protein kinases responses. [ABSTRACT FROM AUTHOR]

Evidence suggests that susceptibility to avian influenza A virus in chickens is influenced by host genetics, but the mechanisms are poorly understood. A previous study demonstrated that inbred line 0 chickens are more resistant to low-pathogenicity avian influenza (LPAI) infection than line CB.12 birds based on viral shedding, but the resistance was not associated with higher AIV-specific IFNγ responses or antibody titres. In this study, we investigated the proportions and cytotoxic capacity of T-cell subpopulations in the spleen and the early immune responses in the respiratory tract, analysing the innate immune transcriptome of lung-derived macrophages following in vitro stimulation with LPAI H7N1 or the TLR7 agonist R848. The more susceptible C.B12 line had a higher proportion of CD8αβ+ γδ and CD4+CD8αα+ αVβ1 T cells, and a significantly higher proportion of the CD8αβ+ γδ and CD8αβ+ αVβ1 T cells expressed CD107a, a surrogate marker of degranulation. Lung macrophages isolated from line C.B12 birds expressed higher levels of the negative regulator genes TRIM29 and IL17REL, whereas macrophages from line 0 birds expressed higher levels of antiviral genes including IRF10 and IRG1. After stimulation with R848, the macrophages from line 0 birds mounted a higher response compared to line C.B12 cells. Together, the higher proportion of unconventional T cells, the higher level of cytotoxic cell degranulation ex vivo and post-stimulation and the lower levels of antiviral gene expression suggest a potential role of immunopathology in mediating susceptibility in C.B12 birds. [ABSTRACT FROM AUTHOR]

Chicken bone marrow-derived macrophages (BMMF) and dendritic cells (BMDC) are utilized as models to study the mononuclear phagocytic system (MPS). A widely used method to generate macrophages and DC in vitro is to culture bone marrow cells in the presence of colony-stimulating factor-1 (CSF1) to differentiate BMMF and granulocyte-macrophage-CSF (GM-CSF, CSF2) and interleukin-4 (IL-4) to differentiate BMDC, while CSF2 alone can lead to the development of granulocyte-macrophage-CSF-derived DC (GMDC). However, in chickens, the MPS cell lineages and their functions represented by these cultures are poorly understood. Here, we decipher the phenotypical, functional and transcriptional differences between chicken BMMF and BMDC along with examining differences in DC cultures grown in the absence of IL-4 on days 2, 4, 6 and 8 of culture. BMMF cultures develop into a morphologically homogenous cell population in contrast to the BMDC and GMDC cultures, which produce morphologically heterogeneous cell cultures. At a phenotypical level, all cultures contained similar cell percentages and expression levels of MHCII, CD11c and CSF1R-transgene, whilst MRC1L-B expression decreased over time in BMMF. All cultures were efficiently able to uptake 0.5 μm beads, but poorly phagocytosed 1 μm beads. Little difference was observed in the kinetics of phagosomal acidification across the cultures on each day of analysis. Temporal transcriptomic analysis indicated that all cultures expressed high levels of CSF3R, MERTK, SEPP1, SPI1 and TLR4, genes associated with macrophages in mammals. In contrast, low levels of FLT3, XCR1 and CAMD1, genes associated with DC, were expressed at day 2 in BMDC and GMDC after which expression levels decreased. Collectively, chicken CSF2 + IL-4- and CSF2-dependent BM cultures represent cells of the macrophage lineage rather than inducing conventional DC. [ABSTRACT FROM AUTHOR]

The spleen is a major site for the immunological responses to blood-borne antigens that is coordinated by cells of the mononuclear phagocyte system (MPS). The chicken spleen is populated with a number of different macrophages while the presence of conventional dendritic cells (cDC) has been described. However, a detailed characterization of the phenotype and function of different macrophage subsets and cDC in the chicken spleen is limited. Using the CSF1R -reporter transgenic chickens (CSF1R -tg), in which cells of the MPS express a transgene under the control elements of the chicken CSF1R , we carried out an in-depth characterization of these cells in the spleen. Immunohistological analysis demonstrated differential expression of MRC1L-B by periarteriolar lymphoid sheaths (PALS)-associated CSF1R -tg+ cells. In the chicken's equivalent of the mammalian marginal zone, the peri-ellipsoid white-pulp (PWP), we identified high expression of putative CD11c by ellipsoid-associated cells compared to ellipsoid-associated macrophages. In addition, we identified a novel ellipsoid macrophage subset that expressed MHCII, CD11c, MRC1L-B, and CSF1R but not the CSF1R -tg. In flow cytometric analysis, diverse expression of the CSF1R -tg and MHCII was observed leading to the categorization of CSF1R -tg cells into CSF1R -tgdim MHCIIinter−hi, CSF1R -tghi MHCIIhi, and CSF1R -tghi MHCIIinter subpopulations. Low levels of CD80, CD40, MHCI, CD44, and Ch74.2 were expressed by the CSF1R -tghi MHCIIinter cells. Functionally, in vivo fluorescent bead uptake was significantly higher in the CSF1R -tghi MHCIIhi MRC1L-B+ cells compared to the CSF1R -tgdim and CSF1R -tghi MHCIIinter MRC1L-B+ subpopulations while LPS enhanced phagocytosis by the CSF1R -tghi MHCIIinter subpopulation. The analysis of bead localization in the spleen suggests the presence of ellipsoid-associated macrophage subsets. In addition, we demonstrated the functionality of ex vivo derived CSF1R -tg+ MRC1L-Bneg cDC. Finally, RNA-seq analysis of the CSF1R -tg subpopulations demonstrated that separating the CSF1R -tghi subpopulation into CD11chi and CD11cdim cells enriched for cDC and macrophage lineages, respectively, while the CSF1R -tghi MHCIIinter subpopulation was enriched for red pulp macrophages. However, our analysis could not define the cell lineage of the heterogeneous CSF1R -tgdim subpopulation. This detailed overview of the MPS in the chicken spleen will contribute to future research on their role in antigen uptake and presentation. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the response of Harderian gland (HG) cells after in vitro stimulation with class B synthetic oligodeoxyribonucleotides (ODN) containing CpG motifs. This knowledge is of importance for the development of mucosal vaccines for poultry, such as eye-drop or spray vaccines, to determine if class B CpG ODN can act as an vaccine adjuvant or as a prophylactic treatment mainly against respiratory disease viruses. The relative expression of Toll-like receptor 21 (TLR21), interferon (IFN)-γ, interleukin (IL)-1β and IL-10 genes were quantified at 1, 3, 6 and 18 h post-stimulation of HG cells from 5-week-old birds. In addition, it was also investigated if expression of these genes was affected by the age of the birds (differences between 5- and 12-week-old birds), concentrations of ODN or cell preparation method used. Class B CpG ODN induced upregulation of TLR21 and IFN-γ mRNA expression levels at 1 h post-stimulation depending on concentration of ODN used but only in HG cells isolated from young birds. [ABSTRACT FROM AUTHOR]

Toll-like receptor (TLR) ligands (TLR-Ls) are critical activators of immunity and are successfully being developed as vaccine adjuvants in both mammals and birds. In this study, we investigated the synergistic effect of co-stimulation of membrane and endosomal TLRs on the innate immune responses using chicken bone marrow-derived macrophages (BMMs), and studied the effect of age on the induction of innate responses. BMMs from 1 and 4-week-old birds were stimulated with Pam3Cys-SK4 (PCSK; TLR2), synthetic monophosphoryl lipid A (MPLA), Di[3-deoxy- d -manno-octulosonyl]-lipid A ammonium salt (KLA; TLR4), Gardiquimod, Resiquimod (R848; TLR7), CpG class B and C (TLR21). Nitric oxide (NO) production and mRNA levels of IL-1β, IL-10 and IL-12p40 showed macrophages from 4-week-old birds showed more sensitive responses compared to 1-week-old birds. The most potent TLR-Ls, PCSK, MPLA and CpG B were used to study the effect of co-stimulation on macrophages. Co-stimulation with TLR21 and TLR4 synergistically up-regulated inflammatory-related genes, as well as NO production. However, incubation of splenocytes with PCSK, MPLA and CpG B did not induce cell proliferation. Moreover, treatment with CpG B led to significant cell death. [ABSTRACT FROM AUTHOR]