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Lawsonia intracellularis is the etiological agent of proliferative enteropathy (PE) in pigs, horses and wide range of mammals. Little is known about the role of innate immune response during L. intracellularis infection. In this study, we investigated the nuclear factor-κB (NF-κB)-regulated immune response against infection of a clinical strain Dkp23 and a live-attenuated Enterisol vaccine strain in PK-15 cells. We found that expression of NF-κB target genes TNF-α, IFN-γ, IL-6 and IL-8 were modulated during the course of infection. At 5 dpi, there was a significant increase in p65 NF-κB activation, including protein nuclear translocation and phosphorylation, synchronous with the induction of IL-6, IFN-γ and IL-8 expression in L. intracellularis infected cells, especially for Enterisol vaccine strain-infected cells. This result suggests that NF-κB signalling level is induced when L. intracellularis bacterial load peaks at 5 dpi. The induction of pro-inflammatory cytokines expression is consistent with the decreased viability of L. intracellularis-infected cells especially that of the vaccine strain. There were no significant changes in NF-κB signalling between vaccine and Dkp23 infection in PK-15 cells, except for moderate levels of differences in NF-κB target genes expression which might be a reflection of differences in intracellular bacterial load. Overall, the data presented here indicate a correlation between the induction of NF-κB signalling and the L. intracellularis bacterial load in PK-15 cells. [ABSTRACT FROM AUTHOR]

Conventional dendritic cells (cDC) are bone marrow‐derived immune cells that play a central role in linking innate and adaptive immunity. cDCs efficiently uptake, process and present antigen to naïve T cells, driving clonal expansion of antigen‐specific T‐cell responses. In chicken, vital reagents are lacking for the efficient and precise identification of cDCs. In this study, we have developed several novel reagents for the identification and characterization of chicken cDCs. Chicken FLT3 cDNA was cloned and a monoclonal antibody to cell surface FLT3 was generated. This antibody identified a distinct FLT3HI splenic subset which lack expression of signature markers for B cells, T cells or monocyte/macrophages. By combining anti‐FLT3 and CSF1R‐eGFP transgenic expression, three major populations within the mononuclear phagocyte system were identified in the spleen. The cDC1 subset of mammalian cDCs express the chemokine receptor XCR1. To characterize chicken cDCs, a synthetic chicken chemokine (C motif) ligand (XCL1) peptide conjugated to Alexa Fluor 647 was developed (XCL1AF647). Flow cytometry staining of XCL1AF647 on splenocytes showed that all chicken FLT3HI cells exclusively express XCR1, supporting the hypothesis that this population comprises bona fide chicken cDCs. Further analysis revealed that chicken cDCs expressed CSF1R but lacked the expression of CSF2R. Collectively, the cell surface phenotypes of chicken cDCs were partially conserved with mammalian XCR1+ cDC1, with distinct differences in CSF1R and CSF2R expression compared with mammalian orthologues. These original reagents allow the efficient identification of chicken cDCs to investigate their important roles in the chicken immunity and diseases. [ABSTRACT FROM AUTHOR]

Conventional dendritic cells (cDCs) are antigen-presenting cells (APCs) that play a central role in linking innate and adaptive immunity. cDCs have been well described in a number of different mammalian species, but remain poorly characterised in the chicken. In this study, we use previously described chicken cDC specific reagents, a novel gene-edited chicken line and single-cell RNA sequencing (scRNAseq) to characterise chicken splenic cDCs. In contrast to mammals, scRNAseq analysis indicates that the chicken spleen contains a single, chemokine receptor XCR1 expressing, cDC subset. By sexual maturity the XCR1 + cDC population is the most abundant mononuclear phagocyte cell subset in the chicken spleen. scRNAseq analysis revealed substantial heterogeneity within the chicken splenic XCR1 + cDC population. Immature MHC class II (MHCII) LOW XCR1 + cDCs expressed a range of viral resistance genes. Maturation to MHCII HIGH XCR1 + cDCs was associated with reduced expression of anti-viral gene expression and increased expression of genes related to antigen presentation via the MHCII and cross-presentation pathways. To visualise and transiently ablate chicken XCR1 + cDCs in situ , we generated XCR1 -iCaspase9-RFP chickens using a CRISPR-Cas9 knockin transgenesis approach to precisely edit the XCR1 locus, replacing the XCR1 coding region with genes for a fluorescent protein (TagRFP), and inducible Caspase 9. After inducible ablation, the chicken spleen is initially repopulated by immature CD1.1 + XCR1 + cDCs. XCR1 + cDCs are abundant in the splenic red pulp, in close association with CD8 + T-cells. Knockout of XCR1 prevented this clustering of cDCs with CD8 + T-cells. Taken together these data indicate a conserved role for chicken and mammalian XCR1 + cDCs in driving CD8 + T-cells responses., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Wu, Shih, Macdonald, Meunier, Hogan, Chintoan-Uta, Gilhooley, Hu, Beltran, Henderson, Sang, Stevens, McGrew and Balic.)