Regulation and function of macrophage colony-stimulating factor (CSF1) in the chicken immune system.

Macrophage colony-stimulating factor (CSF1) is an essential growth factor to control the proliferation, differentiation and survival of cells of the macrophage lineage in vertebrates. We have previously produced a recombinant chicken CSF1-Fc fusion protein and administrated it to birds which produced a substantial expansion of tissue macrophage populations. To further study the biology of CSF1 in the chicken, here we generated anti-chicken CSF1 antibodies (ROS-AV181 and 183) using CSF1-Fc as an immunogen. The specific binding of each monoclonal antibody was confirmed by ELISA, Western blotting and immunohistochemistry on tissue sections. Using the anti-CSF1 antibodies, we show that chicken bone marrow derived macrophages (BMDM) express CSF1 on their surface, and that the level appears to be regulated further by exogenous CSF1. By capture ELISA circulating CSF1 levels increased transiently in both layer and broiler embryos around the day of hatch. The levels of CSF1 in broilers was higher than in layers during the first week after hatch. Antibody ROS-AV183 was able to block CSF1 biological activity in vitro and treatment of hatchlings using this neutralising antibody in vivo impacted on some tissue macrophage populations, but not blood monocytes. After anti-CSF1 treatment, CSF1R-transgene reporter expressing cells were reduced in the bursa of Fabricius and cecal tonsil and TIM4+ Kupffer cells in the liver were almost completely ablated. Anti-CSF1 treatment also produced a reduction in overall bone density, trabecular volume and TRAP+ osteoclasts. Our novel neutralising antibody provides a new tool to study the roles of CSF1 in birds.

Analysis of the Progeny of Sibling Matings Reveals Regulatory Variation Impacting the Transcriptome of Immune Cells in Commercial Chickens.

There is increasing recognition that the underlying genetic variation contributing to complex traits influences transcriptional regulation and can be detected at a population level as expression quantitative trait loci. At the level of an individual, allelic variation in transcriptional regulation of individual genes can be detected by measuring allele-specific expression in RNAseq data. We reasoned that extreme variants in gene expression could be identified by analysis of inbred progeny with shared grandparents. Commercial chickens have been intensively selected for production traits. Selection is associated with large blocks of linkage disequilibrium with considerable potential for co-selection of closely linked "hitch-hiker alleles" affecting traits unrelated to the feature being selected, such as immune function, with potential impact on the productivity and welfare of the animals. To test this hypothesis that there is extreme allelic variation in immune-associated genes we sequenced a founder population of commercial broiler and layer birds. These birds clearly segregated genetically based upon breed type. Each genome contained numerous candidate null mutations, protein-coding variants predicted to be deleterious and extensive non-coding polymorphism. We mated selected broiler-layer pairs then generated cohorts of F2 birds by sibling mating of the F1 generation. Despite the predicted prevalence of deleterious coding variation in the genomic sequence of the founders, clear detrimental impacts of inbreeding on survival and post-hatch development were detected in only one F2 sibship of 15. There was no effect on circulating leukocyte populations in hatchlings. In selected F2 sibships we performed RNAseq analysis of the spleen and isolated bone marrow-derived macrophages (with and without lipopolysaccharide stimulation). The results confirm the predicted emergence of very large differences in expression of individual genes and sets of genes. Network analysis of the results identified clusters of co-expressed genes that vary between individuals and suggested the existence of trans-acting variation in the expression in macrophages of the interferon response factor family that distinguishes the parental broiler and layer birds and influences the global response to lipopolysaccharide. This study shows that the impact of inbreeding on immune cell gene expression can be substantial at the transcriptional level, and potentially opens a route to accelerate selection using specific alleles known to be associated with desirable expression levels.

Collaboration of MYC and RUNX2 in lymphoma simulates T-cell receptor signaling and attenuates p53 pathway activity.

MYC and RUNX oncogenes each trigger p53-mediated failsafe responses when overexpressed in vitro and collaborate with p53 deficiency in vivo. However, together they drive rapid onset lymphoma without mutational loss of p53. This phenomenon was investigated further by transcriptomic analysis of premalignant thymus from RUNX2/MYC transgenic mice. The distinctive contributions of MYC and RUNX to transcriptional control were illustrated by differential enrichment of canonical binding sites and gene ontology analyses. Pathway analysis revealed signatures of MYC, CD3, and CD28 regulation indicative of activation and proliferation, but also strong inhibition of cell death pathways. In silico analysis of discordantly expressed genes revealed Tnfsrf8/CD30, Cish, and Il13 among relevant targets for sustained proliferation and survival. Although TP53 mRNA and protein levels were upregulated, its downstream targets in growth suppression and apoptosis were largely unperturbed. Analysis of genes encoding p53 posttranslational modifiers showed significant upregulation of three genes, Smyd2, Set, and Prmt5. Overexpression of SMYD2 was validated in vivo but the functional analysis was constrained by in vitro loss of p53 in RUNX2/MYC lymphoma cell lines. However, an early role is suggested by the ability of SMYD2 to block senescence-like growth arrest induced by RUNX overexpression in primary fibroblasts.

Combination of novel and public RNA-seq datasets to generate an mRNA expression atlas for the domestic chicken.

BACKGROUND: The domestic chicken (Gallus gallus) is widely used as a model in developmental biology and is also an important livestock species. We describe a novel approach to data integration to generate an mRNA expression atlas for the chicken spanning major tissue types and developmental stages, using a diverse range of publicly-archived RNA-seq datasets and new data derived from immune cells and tissues. RESULTS: Randomly down-sampling RNA-seq datasets to a common depth and quantifying expression against a reference transcriptome using the mRNA quantitation tool Kallisto ensured that disparate datasets explored comparable transcriptomic space. The network analysis tool Graphia was used to extract clusters of co-expressed genes from the resulting expression atlas, many of which were tissue or cell-type restricted, contained transcription factors that have previously been implicated in their regulation, or were otherwise associated with biological processes, such as the cell cycle. The atlas provides a resource for the functional annotation of genes that currently have only a locus ID. We cross-referenced the RNA-seq atlas to a publicly available embryonic Cap Analysis of Gene Expression (CAGE) dataset to infer the developmental time course of organ systems, and to identify a signature of the expansion of tissue macrophage populations during development. CONCLUSION: Expression profiles obtained from public RNA-seq datasets - despite being generated by different laboratories using different methodologies - can be made comparable to each other. This meta-analytic approach to RNA-seq can be extended with new datasets from novel tissues, and is applicable to any species.

Analysis of Campylobacter jejuni infection in the gnotobiotic piglet and genome-wide identification of bacterial factors required for infection.

To investigate how Campylobacter jejuni causes the clinical symptoms of diarrhoeal disease in humans, use of a relevant animal model is essential. Such a model should mimic the human disease closely in terms of host physiology, incubation period before onset of disease, clinical signs and a comparable outcome of disease. In this study, we used a gnotobiotic piglet model to study determinants of pathogenicity of C. jejuni. In this model, C. jejuni successfully established infection and piglets developed an increased temperature with watery diarrhoea, which was caused by a leaky epithelium and reduced bile re-absorption in the intestines. Further, we assessed the C. jejuni genes required for infection of the porcine gastrointestinal tract utilising a transposon (Tn) mutant library screen. A total of 123 genes of which Tn mutants showed attenuated piglet infection were identified. Our screen highlighted a crucial role for motility and chemotaxis, as well as central metabolism. In addition, Tn mutants of 14 genes displayed enhanced piglet infection. This study gives a unique insight into the mechanisms of C. jejuni disease in terms of host physiology and contributing bacterial factors.

Campylobacter jejuni 81-176 forms distinct microcolonies on in vitro-infected human small intestinal tissue prior to biofilm formation.

Human small and large intestinal tissue was used to study the interaction of Campylobacter jejuni with its target tissue. The strain used for the study was 81-176 (+pVir). Tissue was processed for scanning and transmission electron microscopy, and by immunohistochemistry for light microscopy. Organisms adhered to the apical surface of ileal tissues at all time points in large numbers, in areas where mucus was present and in distinct groups. Microcolony formation was evident at 1-2 h, with bacteria adhering to mucus on the tissue surface and to each other by flagellar interaction. At later time points (3-4 h), biofilm formation on ileal tissue was evident. Flagellar mutants did not form microcolonies or biofilms in tissue. Few organisms were observed in colonic tissue, with organisms present but not as abundant as in the ileal tissue. This study shows that C. jejuni 81-176 can form microcolonies and biofilms on human intestinal tissue and that this may be an essential step in its ability to cause diarrhoea in man.

Campylobacter jejuni-infected human epithelial cell lines vary in their ability to secrete interleukin-8 compared to in vitro-infected primary human intestinal tissue.

Campylobacter jejuni causes symptoms of acute inflammatory diarrhoea in man. C. jejuni interaction with epithelial cells elicits interleukin-8 (IL-8) production, and IL-8 recruits neutrophils to sites of infection. Cell culture models of bacterial interaction with epithelium are useful to define bacteria-host interaction and are used because it is thought they mimic the same bacteria-host cell interaction in the natural disease. This study looks at the ability of C. jejuni strains to elicit IL-8 production from a variety of cell lines previously used for investigating the interaction of C. jejuni with host cells. A spectrum of IL-8 responses was observed, with minimal IL-8 elicited from Caco-2 cells and more marked responses elicited from HeLa and T84 cells. These in vitro-infected cell line responses were compared to IL-8 production from in vitro C. jejuni-infected human colonic and ileal tissue. The in vitro-infected tissue elicited the highest IL-8 responses and the cytokine was manifested earlier compared to the infected cell lines.

Campylobacter jejuni activates mitogen-activated protein kinases in Caco-2 cell monolayers and in vitro infected primary human colonic tissue.

The mitogen-activated protein kinases (MAPKs) play a central role in many host signalling pathways. These signalling proteins are known to be involved in host responses against invasive bacteria including generation of chemotactic and inflammatory cytokines. It was hypothesized that Campylobacter jejuni may activate MAPKs, as intestinal infection may induce a clinical and pathological picture of acute colonic inflammation. Infection of Caco-2 cell monolayers (human colonic epithelial cell line) and human colonic tissue with C. jejuni in vitro demonstrated increased MAPK activity for ERK 1/2 (p44/42 MAPK), JNK and p38 MAPKs. Kinase activity and phosphorylated forms were increased in infected Caco-2 cells and human colonic explants, suggesting that these pathways are important in inflammatory responses induced by C. jejuni in man.

Campylobacter jejuni inhibits the absorptive transport functions of Caco-2 cells and disrupts cellular tight junctions.

Caco-2 cells are models of absorptive enterocytes. The net transport of fluid from apical to basolateral surfaces results in 'domes' forming in differentiated monolayers. Here, the effect of Campylobacter jejuni on this process has been examined. C. jejuni caused no changes in short-circuit current upon infection of Caco-2 cell monolayers in Ussing chambers. Thus, no active secretory events could be demonstrated using this model. It was therefore hypothesized that C. jejuni could inhibit the absorptive function of enterocytes and that this may contribute to diarrhoeal disease. C. jejuni infection of fluid-transporting ('doming') Caco-2 cells resulted in a significant reduction in dome number, which correlated with a decrease in tight junction integrity in infected monolayers, when measured as transepithelial electrical resistance. Defined mutants of C. jejuni also reduced dome numbers in infected monolayers. C. jejuni also altered the distribution of the tight junction protein occludin within cell monolayers. The addition to monolayers of extracellular gentamicin prevented these changes, indicating the contribution of extracellular bacteria to this process. Thus, tight junction integrity is required for fluid transport in Caco-2 cell monolayers as leaky tight junctions cannot maintain support of transported fluid at the basolateral surface of infected cell monolayers. Inhibition of absorptive cell function, changes in epithelial resistance and rearrangement of tight junctional proteins such as occludin represent a potential diarrhoeal mechanism of C. jejuni.