Immunoglobulin secretion influences the composition of chicken caecal microbiota.

The chicken caecum is colonised by hundreds of different bacterial species. Which of these are targeted by immunoglobulins and how immunoglobulin expression shapes chicken caecal microbiota has been addressed in this study. Using cell sorting followed by sequencing of V3/V4 variable region of 16S rRNA, bacterial species with increased or decreased immunoglobulin coating were determined. Next, we determined also caecal microbiota composition in immunoglobulin knockout chickens. We found that immunoglobulin coating was common and major taxa were coated with immunoglobulins. Similarly, more taxa required immunoglobulin production for caecum colonisation compared to those which became abundant in immunoglobulin-deficient chickens. Taxa with low immunoglobulin coating such as Lactobacillus, Blautia, [Eubacterium] hallii, Megamonas, Fusobacterium and Desulfovibrio all encode S-layer proteins which may reduce interactions with immunoglobulins. Although there were taxa which overgrew in Ig-deficient chickens (e.g. Akkermansia) indicating immunoglobulin production acted to exclude them from the chicken caecum, in most of the cases, immunoglobulin production more likely contributed to fixing the desired microbiota in the chicken caecum.

The highly and perpetually upregulated thyroglobulin gene is a hallmark of functional thyrocytes.

Abnormalities are indispensable for studying normal biological processes and mechanisms. In the present work, we draw attention to the remarkable phenomenon of a perpetually and robustly upregulated gene, the thyroglobulin gene (Tg). The gene is expressed in the thyroid gland and, as it has been recently demonstrated, forms so-called transcription loops, easily observable by light microscopy. Using this feature, we show that Tg is expressed at a high level from the moment a thyroid cell acquires its identity and both alleles remain highly active over the entire life of the cell, i.e., for months or years depending on the species. We demonstrate that this high upregulation is characteristic of thyroglobulin genes in all major vertebrate groups. We provide evidence that Tg is not influenced by the thyroid hormone status, does not oscillate round the clock and is expressed during both the exocrine and endocrine phases of thyrocyte activity. We conclude that the thyroglobulin gene represents a unique and valuable model to study the maintenance of a high transcriptional upregulation.

The Discovery of Chicken Foxp3 Demands Redefinition of Avian Regulatory T Cells.

Since the publication of the first chicken genome sequence, we have encountered genes playing key roles in mammalian immunology, but being seemingly absent in birds. One of those was, until recently, Foxp3, the master transcription factor of regulatory T cells in mammals. Therefore, avian regulatory T cell research is still poorly standardized. In this study we identify a chicken ortholog of Foxp3 We prove sequence homology with known mammalian and sauropsid sequences, but also reveal differences in major domains. Expression profiling shows an association of Foxp3 and CD25 expression levels in CD4+CD25+ peripheral T cells and identifies a CD4-CD25+Foxp3high subset of thymic lymphocytes that likely represents yet undescribed avian regulatory T precursor cells. We conclude that Foxp3 is existent in chickens and that it shares certain functional characteristics with its mammalian ortholog. Nevertheless, pathways for regulatory T cell development and Foxp3 function are likely to differ between mammals and birds. The identification and characterization of chicken Foxp3 will help to define avian regulatory T cells and to analyze their functional properties and thereby advance the field of avian immunology.

The influences of microbial colonisation and germ-free status on the chicken TCRß repertoire.

Microbial colonisation is paramount to the normal development of the immune system, particularly at mucosal sites. However, the relationships between the microbiome and the adaptive immune repertoire have mostly been explored in rodents and humans. Here, we report a high-throughput sequencing analysis of the chicken TCRß repertoire and the influences of microbial colonisation on tissue-resident TCRß+ cells. The results reveal that the microbiome is an important driver of TCRß diversity in both intestinal tissues and the bursa of Fabricius, but not in the spleen. Of note, public TCRß sequences (shared across individuals) make a substantial contribution to the repertoire. Additionally, different tissues exhibit biases in terms of their V family and J gene usage, and these effects were influenced by the gut-associated microbiome. TCRß clonal expansions were identified in both colonised and germ-free birds, but differences between the groups were indicative of an influence of the microbiota. Together, these findings provide an insight into the avian adaptive immune system and the influence of the microbiota on the TCRß repertoire.

Repertoire analysis of γδ T cells in the chicken enables functional annotation of the genomic region revealing highly variable pan-tissue TCR gamma V gene usage as well as identifying public and private repertoires.

BACKGROUND: Despite increasing interest in γδ T cells and their non-classical behaviour, most studies focus on animals with low numbers of circulating γδ T cells, such as mice and humans. Arguably, γδ T cell functions might be more prominent in chickens where these cells form a higher proportion of the circulatory T cell compartment. The TCR repertoire defines different subsets of γδ T cells, and such analysis is facilitated by well-annotated TCR loci. γδ T cells are considered at the cusp of innate and adaptive immunity but most functions have been identified in γδ low species. A deeper understanding of TCR repertoire biology in γδ high and γδ low animals is critical for defining the evolution of the function of γδ T cells. Repertoire dynamics will reveal populations that can be classified as innate-like or adaptive-like as well as those that straddle this definition. RESULTS: Here, a recent discrepancy in the structure of the chicken TCR gamma locus is resolved, demonstrating that tandem duplication events have shaped the evolution of this locus. Importantly, repertoire sequencing revealed large differences in the usage of individual TRGV genes, a pattern conserved across multiple tissues, including thymus, spleen and the gut. A single TRGV gene, TRGV3.3, with a highly diverse private CDR3 repertoire dominated every tissue in all birds. TRGV usage patterns were partly explained by the TRGV-associated recombination signal sequences. Public CDR3 clonotypes represented varying proportions of the repertoire of TCRs utilising different TRGVs, with one TRGV dominated by super-public clones present in all birds. CONCLUSIONS: The application of repertoire analysis enabled functional annotation of the TCRG locus in a species with a high circulating γδ phenotype. This revealed variable usage of TCRGV genes across multiple tissues, a pattern quite different to that found in γδ low species (human and mouse). Defining the repertoire biology of avian γδ T cells will be key to understanding the evolution and functional diversity of these enigmatic lymphocytes in an animal that is numerically more reliant on them. Practically, this will reveal novel ways in which these cells can be exploited to improve health in medical and veterinary contexts.

Early-Life Immune System Maturation in Chickens Using a Synthetic Community of Cultured Gut Bacteria.

The gut microbiome is crucial for both maturation of the immune system and colonization resistance against enteric pathogens. Although chicken are important domesticated animals, the impact of their gut microbiome on the immune system is understudied. Therefore, we investigated the effect of microbiome-based interventions on host mucosal immune responses. Increased levels of IgA and IgY were observed in chickens exposed to maternal feces after hatching compared with strict hygienic conditions. This was accompanied by increased gut bacterial diversity as assessed by 16S rRNA gene amplicon sequencing. Cultivation work allowed the establishment of a collection of 43 bacterial species spanning 4 phyla and 19 families, including the first cultured members of 3 novel genera and 4 novel species that were taxonomically described. This resource is available at www.dsmz.de/chibac A synthetic community consisting of nine phylogenetically diverse and dominant species from this collection was designed and found to be moderately efficient in boosting immunoglobulin levels when provided to chickens early in life.IMPORTANCE The immune system plays a crucial role in sustaining animal health. Its development is markedly influenced by early microbial colonization of the gastrointestinal tract. As chicken are fully dependent on environmental microbes after hatching, extensive hygienic measures in production facilities are detrimental to the microbiota, resulting in low colonization resistance against pathogens. To combat enteric infections, antibiotics are frequently used, which aggravates the issue by altering gut microbiota colonization. Intervention strategies based on cultured gut bacteria are proposed to influence immune responses in chicken.

The dominantly expressed class II molecule from a resistant MHC haplotype presents only a few Marek's disease virus peptides by using an unprecedented binding motif.

Viral diseases pose major threats to humans and other animals, including the billions of chickens that are an important food source as well as a public health concern due to zoonotic pathogens. Unlike humans and other typical mammals, the major histocompatibility complex (MHC) of chickens can confer decisive resistance or susceptibility to many viral diseases. An iconic example is Marek's disease, caused by an oncogenic herpesvirus with over 100 genes. Classical MHC class I and class II molecules present antigenic peptides to T lymphocytes, and it has been hard to understand how such MHC molecules could be involved in susceptibility to Marek's disease, given the potential number of peptides from over 100 genes. We used a new in vitro infection system and immunopeptidomics to determine peptide motifs for the 2 class II molecules expressed by the MHC haplotype B2, which is known to confer resistance to Marek's disease. Surprisingly, we found that the vast majority of viral peptide epitopes presented by chicken class II molecules arise from only 4 viral genes, nearly all having the peptide motif for BL2*02, the dominantly expressed class II molecule in chickens. We expressed BL2*02 linked to several Marek's disease virus (MDV) peptides and determined one X-ray crystal structure, showing how a single small amino acid in the binding site causes a crinkle in the peptide, leading to a core binding peptide of 10 amino acids, compared to the 9 amino acids in all other reported class II molecules. The limited number of potential T cell epitopes from such a complex virus can explain the differential MHC-determined resistance to MDV, but raises questions of mechanism and opportunities for vaccine targets in this important food species, as well as providing a basis for understanding class II molecules in other species including humans.

Molecular cloning and characterisation of chicken IL-18 binding protein.

The human IL-1 receptor family is comprised of 11 membrane bound or soluble receptors and the IL-18 binding protein (IL-18BP). These receptors are dispersed across seven genomic loci, with the majority at a single locus. Direct orthologues were identified in the chicken at conserved genomic loci; however, the IL-18BP remained absent from the first four builds of the chicken genome sequence. Subsequent assemblies identified the gene at a locus syntenic with mammals; however, these predicted sequences differed between genome builds and contained multiple errors. A partial IL-18BP-like sequence in the NCBI EST database was used to clone the full-length cDNA. A splice variant, which lacks the exon that encodes part of the signal peptide, was also cloned. Human IL-18BP is differentially spliced to produce a number of variants, which are all secreted. By contrast, the spliced chicken isoform was predicted to be intracellular, and we identified similar variants with the same exon missing in a limited number of divergent vertebrate species. Mammalian and viral IL-18BPs inhibit IL-18 activity by directly binding to this cytokine. Full-length and intracellular chicken IL-18BPs were equally effective at inhibiting IL-18-mediated IFN-γ release from an avian B-cell line. Analysis of the predicted chIL-18BP protein sequence revealed two crucial residues, which account for 50% of the binding affinity between human IL-18 and IL-18BP, are conserved in the chicken and a fowlpox-encoded homologue, fpv214. This suggests specific fowlpox viruses used in humans as a vaccine vector have the potential to dampen anti-viral host immune responses.

In and Out of the Bursa-The Role of CXCR4 in Chicken B Cell Development.

In contrast to mammals, early B cell differentiation and diversification of the antibody repertoire in chickens do not take place in the bone marrow but in a specialized gut associated lymphoid tissue (GALT), the bursa of Fabricius. During embryonic development, B cell precursors migrate to the bursa anlage, where they proliferate and diversify their B cell receptor repertoire. Around hatch these diversified B cells start to emigrate from the bursa of Fabricius and populate peripheral lymphoid organs, but very little is known how the migratory processes are regulated. As CXCL12 (syn. SDF-1) and CXCR4 were shown to be essential for the control of B cell migration during the development of lymphoid tissues in mammals, we analyzed expression and function of this chemokine/chemokine-receptor pair in the chicken bursa. We found a strong variation of mRNA abundance of CXCL12 and CXCR4 in different stages of bursa development, with high abundance of CXCL12 mRNA in the bursa anlage at embryonic day 10 (ED10). In situ hybridization demonstrated disseminated CXCL12 expression in the early bursa anlage, which condensed in the developing follicles and was mainly restricted to the follicle cortex post-hatch. Flow cytometric analysis detected CXCR4 protein already on early B cell stages, increasing during bursal development. Post-hatch, a subpopulation with the hallmarks of emigrating B cells became detectable, which had lower CXCR4 expression, suggesting that downregulation of CXCR4 is necessary to leave the CXCL12-high bursal environment. In vivo blockade of CXCR4 using AMD3100 at the time of B cell precursor immigration strongly inhibited follicle development, demonstrating that CXCL12 attracts pre-bursal B cells into the bursal anlage. Altogether, we show that CXCL12 and its receptor CXCR4 are important for both populating the bursa with B cells and emigration of mature B cells into the periphery post hatch, and that CXCR4 function in primary B cell organs is conserved between mammals and birds.

Neurons and astrocytes of the chicken hypothalamus directly respond to lipopolysaccharide and chicken interleukin-6.

In 4-5-month-old chicken, intravenous injections of bacterial lipopolysaccharide (LPS) induced a dose-dependent fever response and a pronounced increase of circulating interleukin-6 (IL-6). To assess a possible role for IL-6 in the brain of birds, a hypothalamic neuro-glial primary culture from 1-day-old chicken was established. Each well of cultured hypothalamic cells contained some 615 neurons, 1350 astrocytes, and 580 microglial cells on average. Incubation of chicken hypothalamic primary cultures with 10 or 100 µg/ml LPS induced a dose-dependent release of bioactive IL-6 into the supernatant. Populations of hypothalamic neurons (4%) and astrocytes (12%) directly responded to superfusion with buffer containing 10 µg/ml LPS with a transient increase of intracellular calcium, a sign of direct cellular activation. Stimulation of hypothalamic cultures with buffer containing 50 ng/ml chicken IL-6 induced calcium signaling in 11% of neurons and 22% of astrocytes investigated. These results demonstrate that IL-6 is produced in the periphery and in the hypothalamus in response to LPS in chicken. The observed cellular responses of hypothalamic cells to chicken IL-6 indicate that this cytokine may readily be involved in the manifestation of fever in the avian hypothalamus.

IFNα and IFNγ Impede Marek's Disease Progression.

Marek's disease virus (MDV) is an alphaherpesvirus that causes Marek's disease, a malignant lymphoproliferative disease of domestic chickens. While MDV vaccines protect animals from clinical disease, they do not provide sterilizing immunity and allow field strains to circulate and evolve in vaccinated flocks. Therefore, there is a need for improved vaccines and for a better understanding of innate and adaptive immune responses against MDV infections. Interferons (IFNs) play important roles in the innate immune defenses against viruses and induce upregulation of a cellular antiviral state. In this report, we quantified the potent antiviral effect of IFNα and IFNγ against MDV infections in vitro. Moreover, we demonstrate that both cytokines can delay Marek's disease onset and progression in vivo. Additionally, blocking of endogenous IFNα using a specific monoclonal antibody, in turn, accelerated disease. In summary, our data reveal the effects of IFNα and IFNγ on MDV infection and improve our understanding of innate immune responses against this oncogenic virus.

The Long Pentraxin PTX3 Is of Major Importance Among Acute Phase Proteins in Chickens.

The expression level of acute phase proteins (APPs) mirrors the health status of an individual. In human medicine, C-reactive protein (CRP), and other members of the pentraxin family are of significant relevance for assessing disease severity and prognosis. In chickens, however, which represent the most common livestock species around the world, no such marker has yet gained general acceptance. The aim of this study was therefore, to characterize chicken pentraxin 3 (chPTX3) and to evaluate its applicability as a general marker for inflammatory conditions. The mammalian and chicken PTX3 proteins were predicted to be similar in sequence, domain organization and polymeric structure. Nevertheless, some characteristics like certain sequence sections, which have varied during the evolution of mammals, and species-specific glycosylation patterns, suggest distinct biological functions. ChPTX3 is constitutively expressed in various tissues but, interestingly, could not be found in splenic tissue samples without stimulation. However, upon treatment with lipopolysaccharide (LPS), PTX3 expression in chicken spleens increased to 95-fold within hours. A search for PTX3 reads in various publicly available RNA-seq data sets of chicken spleen and bursa of Fabricius also showed that PTX3 expression increases within days after experimental infection with viral and bacterial pathogens. An experimental infection with avian pathogenic E.coli and qPCR analysis of spleen samples further established a challenge dose-dependent significant up-regulation of chPTX3 in subclinically infected birds of up to over 150-fold as compared to untreated controls. Our results indicate the potential of chPTX3 as an APP marker to monitor inflammatory conditions in poultry flocks.

Unraveling the role of B cells in the pathogenesis of an oncogenic avian herpesvirus.

Marek's disease virus (MDV) is a highly oncogenic alphaherpesvirus that causes immunosuppression, paralysis, and deadly lymphomas in chickens. In infected animals, B cells are efficiently infected and are thought to amplify the virus and transfer it to T cells. MDV subsequently establishes latency in T cells and transforms CD4+ T cells, resulting in fatal lymphomas. Despite many years of research, the exact role of the different B and T cell subsets in MDV pathogenesis remains poorly understood, mostly due to the lack of reverse genetics in chickens. Recently, Ig heavy chain J gene segment knockout (JH-KO) chickens lacking mature and peripheral B cells have been generated. To determine the role of these B cells in MDV pathogenesis, we infected JH-KO chickens with the very virulent MDV RB1B strain. Surprisingly, viral load in the blood of infected animals was not altered in the absence of B cells. More importantly, disease and tumor incidence in JH-KO chickens was comparable to wild-type animals, suggesting that both mature and peripheral B cells are dispensable for MDV pathogenesis. Intriguingly, MDV efficiently replicated in the bursa of Fabricius in JH-KO animals, while spread of the virus to the spleen and thymus was delayed. In the absence of B cells, MDV readily infected CD4+ and CD8+ T cells, allowing efficient virus replication in the lymphoid organs and transformation of T cells. Taken together, our data change the dogma of the central role of B cells, and thereby provide important insights into MDV pathogenesis.

Characterization of Chicken Tumor Necrosis Factor-α, a Long Missed Cytokine in Birds.

Tumor necrosis factor-α (TNF-α) is a pleiotropic cytokine playing critical roles in host defense and acute and chronic inflammation. It has been described in fish, amphibians, and mammals but was considered to be absent in the avian genomes. Here, we report on the identification and functional characterization of the avian ortholog. The chicken TNF-α (chTNF-α) is encoded by a highly GC-rich gene, whose product shares with its mammalian counterpart 45% homology in the extracellular part displaying the characteristic TNF homology domain. Orthologs of chTNF-α were identified in the genomes of 12 additional avian species including Palaeognathae and Neognathae, and the synteny of the closely adjacent loci with mammalian TNF-α orthologs was demonstrated in the crow (Corvus cornix) genome. In addition to chTNF-α, we obtained full sequences for homologs of TNF-α receptors 1 and 2 (TNFR1, TNFR2). chTNF-α mRNA is strongly induced by lipopolysaccharide (LPS) stimulation of monocyte derived, splenic and bone marrow macrophages, and significantly upregulated in splenic tissue in response to i.v. LPS treatment. Activation of T-lymphocytes by TCR crosslinking induces chTNF-α expression in CD4+ but not in CD8+ cells. To gain insights into its biological activity, we generated recombinant chTNF-α in eukaryotic and prokaryotic expression systems. Both, the full-length cytokine and the extracellular domain rapidly induced an NFκB-luciferase reporter in stably transfected CEC-32 reporter cells. Collectively, these data provide strong evidence for the existence of a fully functional TNF-α/TNF-α receptor system in birds thus filling a gap in our understanding of the evolution of cytokine systems.

The role of type I interferons (IFNs) in the regulation of chicken macrophage inflammatory response to bacterial challenge.

Mammalian type I interferons (IFNα/ß) are known to modulate inflammatory processes in addition to their antiviral properties. Indeed, virus-induced type I interferons regulate the mammalian phagocyte immune response to bacteria during superinfections. However, it remains unresolved whether type I IFNs similarly impact the chicken macrophage immune response. We first evidenced that IFNα and IFNß act differently in terms of gene expression stimulation and activation of intracellular signaling pathways in chicken macrophages. Next, we showed that priming of chicken macrophages with IFNα increased bacteria uptake, boosted bacterial-induced ROS/NO production and led to an increased transcriptional expression or production of NOS2/NO, IL1B/IL-1ß and notably IFNB/IFNß. Neutralization of IFNß during bacterial challenge limited IFNα-induced augmentation of the pro-inflammatory response. In conclusion, we demonstrated that type I IFNs differently regulate chicken macrophage functions and drive a pro-inflammatory response to bacterial challenge. These findings shed light on the diverse functions of type I IFNs in chicken macrophages.

Infectious bursal disease virus inoculation infection modifies Campylobacter jejuni-host interaction in broilers.

BACKGROUND: Campylobacter jejuni is considered as a chicken commensal. The gut microbiota and the immune status of the host may affect its colonization. Infectious bursal disease virus (IBDV) is an immunosuppressive virus of chickens, which allows secondary pathogens to invade or exacerbates their pathogenesis. To investigate the effect of IBDV-induced immunosuppression on the pathogenesis of C. jejuni, broiler chickens were inoculated with a very virulent (vv) strain of IBDV at 14 days post hatch followed by C. jejuni inoculation at 7 (Experiment A) or 9 (Experiment B) days post virus (IBDV) inoculation. RESULTS: vvIBDV-infection led to a depression in caecal lamina propria B lymphocytes and the anti-C. jejuni-antibody response starting at 14 days post C. jejuni inoculation (pbi). The C. jejuni-colonization pattern was comparable between mono-inoculated groups of both experiments, but it varied for vvIBDV + C. jejuni co-inoculated groups. In Experiment A significant higher numbers of colony forming units (CFU) of C. jejuni were detected in the caecum of co-inoculated birds compared to C. jejuni-mono-inoculated birds in the early phase after C. jejuni-inoculation. In Experiment B the clearance phase was affected in the co-inoculated group with significantly higher CFU at 21 days pbi compared to the mono-inoculated group (P < 0.05). No major differences were seen in numbers local lamina propria T lymphocyte populations between C. jejuni-inoculated groups with or without vvIBDV-infection. Interestingly, both pathogens affected the microbiota composition. The consequences of these microflora changes for the host have to be elucidated further. CONCLUSION: Our data suggests that the timing between viral and bacterial infection might affect the outcome of C. jejuni colonization differently. Our results confirm previous studies that anti-Campylobacter-antibodies may specifically be important for the clearance phase of the bacteria. Therefore, as vvIBDV is widely distributed in the field, it may have a significant impact on the colonization and shedding rate of C. jejuni in commercial poultry flocks. Subsequently, successful IBDV-control strategies may indirectly also benefit the gut-health of chickens.

Propagation and titration of infectious bursal disease virus, including non-cell-culture-adapted strains, using ex vivo-stimulated chicken bursal cells.

Infectious bursal disease virus (IBDV) is a Birnaviridae family member of economic importance for poultry. This virus infects and destroys developing B lymphocytes in the cloacal bursa, resulting in a potentially fatal or immunosuppressive disease in chickens. Naturally occurring viruses and many vaccine strains are not able to grow in in vitro systems without prior adaptation, which often affects viral properties such as virulence. Primary bursal cells, which are the main target cells of lymphotropic IBDV in vivo, may represent an attractive system for the study of IBDV. Unfortunately, bursal cells isolated from bursal follicles undergo apoptosis within hours following their isolation. Here, we demonstrate that ex vivo stimulation of bursal cells with phorbol 12-myristate 13-acetate maintains their viability long enough to allow IBDV replication to high titres. A wide range of field-derived or vaccine serotype 1 IBDV strains could be titrated in these phorbol 12-myristate 13-acetate -stimulated bursal cells and furthermore were permissive for replication of non-cell-culture-adapted viruses. These cells also supported multistep replication experiments and flow cytometry analysis of infection. Ex vivo-stimulated bursal cells therefore offer a promising tool in the study of IBDV.

Gene expression in the chicken caecum is dependent on microbiota composition.

Gut microbiota is of considerable importance for each host. Despite this, germ-free animals can be obtained and raised to sexual maturity and consequences of the presence or absence of gut microbiota on gene expression of the host remain uncharacterised. In this study, we performed an unbiased study of protein expression in the caecum of germ-free and colonised chickens. The major difference between these two groups was in the expression of immunoglobulins which were essentially absent in the germ-free chickens. Microbiota also caused a minor decrease in the expression of focal adhesion and extracellular matrix proteins and an increase in the expression of argininosuccinate synthase ASS1, redox potential sensing, fermentative metabolic processes and detoxification systems represented by sulfotransferases SULT1C3 or SULT1E1. Since we also analysed expression in the caecum of E. coli Nissle and E. faecium DSM7134 mono-associated chickens, we concluded that at least immunoglobulin expression and expression of cystathionine synthase (CBS) was dependent on microbiota composition with E. coli Nissle stimulating more immunoglobulin and PIGR expression and E. faecium DSM7134 stimulating more CBS expression. Gut microbiota and its composition therefore affected protein expression in the chicken caecum though except for immunoglobulin production, the remaining differences were unexpectedly low.