Evolution of an expanded mannose receptor gene family.

Sequences of peptides from a protein specifically immunoprecipitated by an antibody, KUL01, that recognises chicken macrophages, identified a homologue of the mammalian mannose receptor, MRC1, which we called MRC1L-B. Inspection of the genomic environment of the chicken gene revealed an array of five paralogous genes, MRC1L-A to MRC1L-E, located between conserved flanking genes found either side of the single MRC1 gene in mammals. Transcripts of all five genes were detected in RNA from a macrophage cell line and other RNAs, whose sequences allowed the precise definition of spliced exons, confirming or correcting existing bioinformatic annotation. The confirmed gene structures were used to locate orthologues of all five genes in the genomes of two other avian species and of the painted turtle, all with intact coding sequences. The lizard genome had only three genes, one orthologue of MRC1L-A and two orthologues of the MRC1L-B antigen gene resulting from a recent duplication. The Xenopus genome, like that of most mammals, had only a single MRC1-like gene at the corresponding locus. MRC1L-A and MRC1L-B genes had similar cytoplasmic regions that may be indicative of similar subcellular migration and functions. Cytoplasmic regions of the other three genes were very divergent, possibly indicating the evolution of a new functional repertoire for this family of molecules, which might include novel interactions with pathogens.

A critical role for MAPK signalling pathways in the transcriptional regulation of toll like receptors.

Toll-like Receptors (TLR) are phylogenetically conserved transmembrane proteins responsible for detection of pathogens and activation of immune responses in diverse animal species. The stimulation of TLR by pathogen-derived molecules leads to the production of pro-inflammatory mediators including cytokines and nitric oxide. Although TLR-induced events are critical for immune induction, uncontrolled inflammation can be life threatening and regulation is a critical feature of TLR biology. We used an avian macrophage cell line (HD11) to determine the relationship between TLR agonist-induced activation of inflammatory responses and the transcriptional regulation of TLR. Exposure of macrophages to specific TLR agonists induced upregulation of cytokine and nitric oxide pathways that were inhibited by blocking various components of the TLR signalling pathways. TLR activation also led to changes in the levels of mRNA encoding the TLR responsible for recognising the inducing agonist (cognate regulation) and cross-regulation of other TLR (non-cognate regulation). Interestingly, in most cases, regulation of TLR mRNA was independent of NFκB activity but dependent on one or more of the MAPK pathway components. Moreover, the relative importance of ERK, JNK and p38 was dependent upon both the stimulating agonist and the target TLR. These results provide a framework for understanding the complex pathways involved in transcriptional regulation of TLR, immune induction and inflammation. Manipulation of these pathways during vaccination or management of acute inflammatory disease may lead to improved clinical outcome or enhanced vaccine efficacy.

Generation and characterization of chicken bone marrow-derived dendritic cells.

Dendritic cells (DCs) are bone marrow-derived professional antigen-presenting cells. The in vitro generation of DCs from either bone marrow or blood is routine in mammals. Their distinct morphology and phenotype and their unique ability to stimulate naïve T cells are used to define DCs. In this study, chicken bone marrow cells were cultured in the presence of recombinant chicken granulocyte-macrophage colony-stimulating factor (GM-CSF) and recombinant chicken interleukin-4 (IL-4) for 7 days. The cultured population showed the typical morphology of DCs, with the surface phenotype of major histocompatibility complex (MHC) class II(+) (high), CD11c(+) (high), CD40(+) (moderate), CD1.1(+) (moderate), CD86(+) (low), CD83(-) and DEC-205(-). Upon maturation with lipopolysaccharide (LPS) or CD40L, surface expression of CD40, CD1.1, CD86, CD83 and DEC-205 was greatly increased. Endocytosis and phagocytosis were assessed by fluorescein isothiocyanate (FITC)-dextran uptake and fluorescent bead uptake, respectively, and both decreased after stimulation. Non-stimulated chicken bone marrow-derived DCs (chBM-DCs) stimulated both allogeneic and syngeneic peripheral blood lymphocytes (PBLs) to proliferate in a mixed lymphocyte reaction (MLR). LPS- or CD40L-stimulated chBM-DCs were more effective T-cell stimulators in MLR than non-stimulated chBM-DCs. Cultured chBM-DCs could be matured to a T helper type 1 (Th1)-promoting phenotype by LPS or CD40L stimulation, as determined by mRNA expression levels of Th1 and Th2 cytokines. We have therefore cultured functional chBM-DCs in a non-mammalian species for the first time.

CD40 ligand supports the long-term maintenance and differentiation of chicken B cells in culture.

TNF family members play crucial roles in mammalian B-cell differentiation and function, many of which have not been demonstrated in other species. To investigate the avian CD40/CD40L system, a chicken CD40 cDNA, obtained by expression screening, was used to raise monoclonal antibodies showing that CD40 was expressed on chicken B cells, monocytes and macrophages, like mammalian CD40. CD40 ligand fusion protein supported the proliferation of B cells in culture for up to 3 weeks, during which they differentiated towards a plasma cell phenotype. CD40L-activated B cells from immunised birds secreted antigen-specific IgM and IgG. These results showed important conserved functions of CD40 and its ligand in mammals and birds. CD40L provides a means for maintenance and differentiation of untransformed chicken B cells in culture, for the first time, allowing new approaches to study of post-bursal B cell biology and host-pathogen interactions with B cell tropic viruses.

The BAFF-Interacting receptors of chickens.

The TNF superfamily cytokine BAFF has crucial roles in homoeostatic regulation of B cell populations in mammals. Similar effects on peripheral B cells have been reported for chicken as for mammalian BAFF. Unlike mammalian BAFF, chicken BAFF is produced by B cells, implying an autocrine loop and consequent differences in regulation of B cell homoeostasis. Understanding of these mechanisms requires investigation of BAFF-binding receptors in chickens. We identified and characterised chicken receptors BAFFR and TACI, but found that the gene encoding the third BAFF-binding receptor, BCMA, was disrupted, implying differences in mechanisms for maintenance of long-lived antibody responses. A BAFFR-Ig fusion protein expressed in vivo lowered B cell numbers, showing that it was functional under physiological conditions. We found changes in the ratio of BAFFR and TACI mRNAs in the bursa after hatch that may account for the altered requirements for B cell survival at this stage of development.

Transcriptional profiling reveals a possible role for the timing of the inflammatory response in determining susceptibility to a viral infection.

Using a novel cDNA microarray prepared from sources of actively responding immune system cells, we have investigated the changes in gene expression in the target tissue during the early stages of infection of neonatal chickens with infectious bursal disease virus. Infections of two lines of chickens previously documented as genetically resistant and sensitive to infection were compared in order to ascertain early differences in the response to infection that might provide clues to the mechanism of differential genetic resistance. In addition to major changes that could be explained by previously described changes in infected tissue, some differences in gene expression on infection, and differences between the two chicken lines, were observed that led to a model for resistance in which a more rapid inflammatory response and more-extensive p53-related induction of apoptosis in the target B cells might limit viral replication and consequent pathology. Ironically, the effect in the asymptomatic neonatal infection is that more-severe B-cell depletion is seen in the more genetically resistant chicken. Changes of expression of many chicken genes of unknown function, indicating possible roles in the response to infection, may aid in the functional annotation of these genes.

A genomic analysis of chicken cytokines and chemokines.

As most mechanisms of adaptive immunity evolved during the divergence of vertebrates, the immune systems of extant vertebrates represent different successful variations on the themes initiated in their earliest common ancestors. The genes involved in elaborating these mechanisms have been subject to exceptional selective pressures in an arms race with highly adaptable pathogens, resulting in highly divergent sequences of orthologous genes and the gain and loss of members of gene families as different species find different solutions to the challenge of infection. Consequently, it has been difficult to transfer to the chicken detailed knowledge of the molecular mechanisms of the mammalian immune system and, thus, to enhance the already significant contribution of chickens toward understanding the evolution of immunity. The availability of the chicken genome sequence provides the opportunity to resolve outstanding questions concerning which molecular components of the immune system are shared between mammals and birds and which represent their unique evolutionary solutions. We have integrated genome data with existing knowledge to make a new comparative census of members of cytokine and chemokine gene families, distinguishing the core set of molecules likely to be common to all higher vertebrates from those particular to these 300 million-year-old lineages. Some differences can be explained by the different architectures of the mammalian and avian immune systems. Chickens lack lymph nodes and also the genes for the lymphotoxins and lymphotoxin receptors. The lack of functional eosinophils correlates with the absence of the eotaxin genes and our previously reported observation that interleukin- 5 (IL-5) is a pseudogene. To summarize, in the chicken genome, we can identify the genes for 23 ILs, 8 type I interferons (IFNs), IFN-gamma, 1 colony-stimulating factor (GM-CSF), 2 of the 3 known transforming growth factors (TGFs), 24 chemokines (1 XCL, 14 CCL, 8 CXCL, and 1 CX3CL), and 10 tumor necrosis factor superfamily (TNFSF) members. Receptor genes present in the genome suggest the likely presence of 2 other ILs, 1 other CSF, and 2 other TNFSF members.

Infectious bursal disease virus-induced immunosuppression in the chick is associated with the presence of undifferentiated follicles in the recovering bursa.

Infectious bursal disease virus (IBDV) causes an acute cytolytic infection in chicken B lymphocytes resulting in destruction of the B-cell population. Most severe depletion occurs in the bursa of Fabricius, where the immunoglobulin repertoire is developed by gene conversion. Chicks surviving IBDV infection are immunosuppressed despite repopulation of the bursa with B cells. Here we show that infection of neonatal chicks with a classical virulent IBDV strain (F52/70) causes severe bursal Bcell depletion with recovery after about one week. Two distinct types of bursal follicles developed: large reconstituted follicles and small poorly developed follicles lacking a discernible cortex and medulla. The presence of large numbers of undifferentiated follicles was associated with inability to mount antibody responses to IBDV itself and after immunization with Salmonella Enteritidis bacterin, indicating that B cells in these follicles are unable to produce peripheral B-cells with an effective immunoglobulin repertoire. Additionally a number of inflammatory foci were observed in the recovering bursa. These foci contained few B cells at the margins, but large numbers of CD4(+) and CD8(+) cells, scattered gammadelta(+) T-cells and macrophages, and small central aggregates of dendriticlike cells expressing the CD40 antigen.

Conservation of biological properties of the CD40 ligand, CD154 in a non-mammalian vertebrate.

Signals delivered by the CD40 ligand, CD154, have crucial roles in immune responses in mammals, being required for development of germinal centres, maturation of T-dependent antibody responses, and generation of B-cell memory. To determine whether these functions were conserved in a non-mammalian species, a putative chicken CD 154 cDNA was used to make an oligomeric fusion protein, and to raise monoclonal antibodies. The antibodies detected surface expression on activated T-cells. The fusion protein detected expression of a receptor on B-cells, thrombocytes and macrophages. Biological effects of the fusion protein included induction of NO synthesis in a macrophage cell line, enhancement of splenic B-cell survival, and induction of apoptosis in a bursal lymphoma cell line. These observations demonstrated substantial functional equivalence with mammalian CD 154 and thus provided evidence for the early evolutionary emergence of the set of functions associated with this molecule, and its central role in the vertebrate immune system.

Developmentally programmed expression of AID in chicken B cells.

In mice, activation induced deaminase, AID, is expressed only in germinal center B cells. It is required for the initiation of somatic hypermutation and class switch recombination. In chickens and most mammals immunoglobulin gene rearrangement generates limited diversity and the primary immunoglobulin repertoire depends on subsequent somatic hypermutation or gene conversion. Immunoglobulin gene conversion in chickens starts in the embryonic bursa, before antigen exposure. The demonstrated requirement for AID for gene conversion in the bursal lymphoma cell line, DT40, implies developmental regulation of AID expression. To test this prediction, we examined the timing and location of AID mRNA expression. An abrupt increase in AID mRNA coincided with the onset of extensive Ig gene conversion in the bursa. Expression was also detected at earlier stages, implying either that expression of AID is not the only controlling factor for gene conversion, or that gene conversion can precede the formation of bursal follicles.

Cloning and characterization of chicken IL-10 and its role in the immune response to Eimeria maxima.

We isolated the full-length chicken IL-10 (chIL-10) cDNA from an expressed sequence tag library derived from RNA from cecal tonsils of Eimeria tenella-infected chickens. It encodes a 178-aa polypeptide, with a predicted 162-aa mature peptide. Chicken IL-10 has 45 and 42% aa identity with human and murine IL-10, respectively. The structures of the chIL-10 gene and its promoter were determined by direct sequencing of a bacterial artificial chromosome containing chIL-10. The chIL-10 gene structure is similar to (five exons, four introns), but more compact than, that of its mammalian orthologues. The promoter is more similar to that of Fugu IL-10 than human IL-10. Chicken IL-10 mRNA expression was identified mainly in the bursa of Fabricius and cecal tonsils, with low levels of expression also seen in thymus, liver, and lung. Expression was also detected in PHA-activated thymocytes and LPS-stimulated monocyte-derived macrophages, with high expression in an LPS-stimulated macrophage cell line. Recombinant chIL-10 was produced and bioactivity demonstrated through IL-10-induced inhibition of IFN-gamma synthesis by mitogen-activated lymphocytes. We measured the expression of mRNA for chIL-10 and other signature cytokines in gut and spleen of resistant (line C.B12) and susceptible (line 15I) chickens during the course of an E. maxima infection. Susceptible chickens showed higher levels of chIL-10 mRNA expression in the spleen, both constitutively and after infection, and in the small intestine after infection than did resistant chickens. These data indicate a potential role for chIL-10 in changing the Th bias during infection with an intracellular protozoan, thereby contributing to susceptibility of line 15I chickens.