Céline Urien

Existence of CD8α-Like Dendritic Cells with a Conserved Functional Specialization and a Common Molecular Signature in Distant Mammalian Species

The mouse lymphoid organ-resident CD8α+ dendritic cell (DC) subset is specialized in Ag presentation to CD8+ T cells. Recent evidence shows that mouse nonlymphoid tissue CD103+ DCs and human blood DC Ag 3+ DCs share similarities with CD8α+ DCs. We address here whether the organization of DC subsets is conserved across mammals in terms of gene expression signatures, phenotypic characteristics, and functional specialization, independently of the tissue of origin. We study the DC subsets that migrate from the skin in the ovine species that, like all domestic animals, belongs to the Laurasiatheria, a distinct phylogenetic clade from the supraprimates (human/mouse). We demonstrate that the minor sheep CD26+ skin lymph DC subset shares significant transcriptomic similarities with mouse CD8α+ and human blood DC Ag 3+ DCs. This allowed the identification of a common set of phenotypic characteristics for CD8α-like DCs in the three mammalian species (i.e., SIRPlo, CADM1hi, CLEC9Ahi, CD205hi, XCR1hi). Compared to CD26− DCs, the sheep CD26+ DCs show 1) potent stimulation of allogeneic naive CD8+ T cells with high selective induction of the Ifnγ and Il22 genes; 2) dominant efficacy in activating specific CD8+ T cells against exogenous soluble Ag; and 3) selective expression of functional pathways associated with high capacity for Ag cross-presentation. Our results unravel a unifying definition of the CD8α+-like DCs across mammalian species and identify molecular candidates that could be used for the design of vaccines applying to mammals in general.

Bluetongue virus targets conventional dendritic cells in skin lymph.

ABSTRACT Bluetongue virus (BTV) is the etiological agent of bluetongue, a hemorrhagic disease of ruminants (particularly sheep), which causes important economic losses around the world. BTV is transmitted primarily via the bites of infected midges, which inject the virus into the ruminants skin during blood feeding. The virus initially replicates in the draining lymph node and then disseminates to secondary organs where it induces edema, hemorrhages, and necrosis. In this study, we show that ovine conventional dendritic cells (cDCs) are the primary targets of BTV that contribute to the primary dissemination of BTV from the skin to draining lymph nodes. Lymph cDCs support BTV RNA and protein synthesis, as well as the production of infectious virus belonging to several different BTV serotypes, regardless of their level of attenuation. Afferent lymph cell subsets, other than cDCs, showed only marginal levels of BTV protein expression. BTV infection provoked a massive recruitment of cDCs to the sheep skin and afferent lymph, providing cellular targets for infection. Although BTV productively infects cDCs, no negative impact on their physiology was detected. Indeed, BTV infection and protein expression in cDCs enhanced their survival rate. Several serotypes of BTV stimulated the surface expression of the CD80 and CD86 costimulatory molecules on cDCs as well as the mRNA synthesis of cytokines involved in inflammation and immunity, i.e., interleukin-12 (IL-12), IL-1β, and IL-6. BTV-infected cDCs stimulated antigen-specific CD4 and CD8 proliferation as well as gamma interferon production. BTV initially targets cDCs while preserving their functional properties, reflecting the optimal adaptation of the virus to its host cells for its first spread.

Characterization of Dendritic Cells Subpopulations in Skin and Afferent Lymph in the Swine Model

Transcutaneous delivery of vaccines to specific skin dendritic cells (DC) subsets is foreseen as a promising strategy to induce strong and specific types of immune responses such as tolerance, cytotoxicity or humoral immunity. Because of striking histological similarities between human and pig skin, pig is recognized as the most suitable model to study the cutaneous delivery of medicine. Therefore improving the knowledge on swine skin DC subsets would be highly valuable to the skin vaccine field. In this study, we showed that pig skin DC comprise the classical epidermal langerhans cells (LC) and dermal DC (DDC) that could be divided in 3 subsets according to their phenotypes: (1) the CD163neg/CD172aneg, (2) the CD163highCD172apos and (3) the CD163lowCD172apos DDC. These subtypes have the capacity to migrate from skin to lymph node since we detected them in pseudo-afferent lymph. Extensive phenotyping with a set of markers suggested that the CD163high DDC resemble the antibody response-inducing human skin DC/macrophages whereas the CD163negCD172low DDC share properties with the CD8+ T cell response-inducing murine skin CD103pos DC. This work, by showing similarities between human, mouse and swine skin DC, establishes pig as a model of choice for the development of transcutaneous immunisation strategies targeting DC.

Construction of a rabbit bacterial artificial chromosome (BAC) library: application to the mapping of the major histocompatibility complex to position 12q1.1

In the past five years, large DNA fragment libraries in Bacterial Artificial Chromosomes (BAC) (Shizuya et al. 1992) have permitted remarkable advances in the analysis of major domestic species of agronomic interest. Thus, BAC libraries representing from 3 to 10 haploid genome coverage are now available for a number of domestic animals. The success of the BAC libraries is mainly owing to the DNA fidelity of the genomic inserts and its low level of chimerism. In addition, BAC clones are amenable to readily build physical maps representing the whole genome and direct sequencing for efficient chromosome walking and large-scale sequencing projects. The rabbit ( Oryctolagus cuniculus ) genome has been poorly studied thus far, despite the use of this species in multiple physiological and immunological studies as well as transgenic experiments. Furthermore, a number of laboratories and private companies are interested in improving production as well as reproductive performances in rabbit, which requires the development of molecular tools. In this context, a BAC library would be of great value. Since no rabbit BAC library has yet been made available, to our knowledge, we constructed a library that currently represents three haploid genome equivalents. Despite a limited genome coverage, this kind of library has already proved its efficiency to start working on infrequently studied species. In farm animals, cytogenetic, physical, and comparative maps were developed with 1.5 and 3 genome equivalent BAC libraries for horse and ruminants, respectively (Mariat et al. 2000; Schibler et al. 1998). High-molecular-weight DNA was prepared from white blood cells isolated from a New Zealand sire rabbit which was homozygous DRAdd-DQAbb (Han et al. 1992) for the class II region of the rabbit RLA major histocompatibility complex (MHC). The procedure to construct the BAC library in the pBeloBAC11 vector by using theHindIII cloning site was identical to the one used to build our pig BAC library (Rogel-Gaillard et al. 1999). The whole BAC library comprised 84,480 clones with an average insert size of 100–110 kilobases (kb; Fig. 1), representing a theoretical threefold coverage of the haploid genome. The BAC library was stored in 96-well microtiter plates and was organized in a threedimension pooling system to prepare DNA ready to use for a PCR screening. Each of the 44 superpools corresponds to 20 plates of 96 clones, that is, a total of 1920 clones. The recovery of clones for a specific primer pair requires a two-step process. The first step corresponds to the screening of 44 superpool DNAs and three controls, that is, 47 PCRs, while the second step corresponds to the screening of the pools for the rows, columns, and plates, that is, 44 PCRs with controls per positive superpool. The library was screened upon request from different European and one American laboratory with primer pairs specific for 46 distinct sequences. Thirty-seven out of the 46 primer sets yielded between 1 and 10 clones with an average of 3.3 clones. No clone was recovered for the remaining 9 primer sets. In addition, the library was also screened for genes from each of the three MHC class I, II and III regions (Table 1). Primers for genes of the class I region were derived from the RLA classical class I R19 gene (Marche et al. 1985) and from a single-copy gene corresponding to R27 (Rebiere et al. 1987). Primers for genes of the RLA class II region were derived from the rabbit DRA, DRB, DQA, DQB, DMA, DMB, DPA, DPB genes and the human TAP1 gene (Table 1). Finally, the screening for class III genes was performed by using primers derived from the rabbit TNFAgene, the pigHSP70 gene, and the human TNX and NOTCH4 genes (Table 1). All the PCR products obtained with the different primer sets were sequenced, and nucleic acid similarity searches were conducted with a BLASTN program (Altschul et al., 1997) to assess the specificity of the sequences. The rabbit nucleotide sequences for NOTCH4, TNX, and TAP1 genes were submitted to EMBL/GenBank database and were given the accession numbers AJ297382, AJ297381, and AJ297383, respectively. Two distinct sequences were obtained Correspondence to:C. Rogel-Gaillard; E-mail: rogel@biotec.jouy. inra.fr Fig. 1. Mammalian Genome 12, 253–255 (2001). DOI: 10.1007/s003350010260

The Double-Stranded RNA Bluetongue Virus Induces Type I Interferon in Plasmacytoid Dendritic Cells via a MYD88-Dependent TLR7/8-Independent Signaling Pathway

ABSTRACT Dendritic cells (DCs), especially plasmacytoid DCs (pDCs), produce large amounts of alpha/beta interferon (IFN-α/β) upon infection with DNA or RNA viruses, which has impacts on the physiopathology of the viral infections and on the quality of the adaptive immunity. However, little is known about the IFN-α/β production by DCs during infections by double-stranded RNA (dsRNA) viruses. We present here novel information about the production of IFN-α/β induced by bluetongue virus (BTV), a vector-borne dsRNA Orbivirus of ruminants, in sheep primary DCs. We found that BTV induced IFN-α/β in skin lymph and in blood in vivo. Although BTV replicated in a substantial fraction of the conventional DCs (cDCs) and pDCs in vitro, only pDCs responded to BTV by producing a significant amount of IFN-α/β. BTV replication in pDCs was not mandatory for IFN-α/β production since it was still induced by UV-inactivated BTV (UV-BTV). Other inflammatory cytokines, including tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and IL-12p40, were also induced by UV-BTV in primary pDCs. The induction of IFN-α/β required endo-/lysosomal acidification and maturation. However, despite being an RNA virus, UV-BTV did not signal through Toll-like receptor 7 (TLR7) for IFN-α/β induction. In contrast, pathways involving the MyD88 adaptor and kinases dsRNA-activated protein kinase (PKR) and stress-activated protein kinase (SAPK)/Jun N-terminal protein kinase (JNK) were implicated. This work highlights the importance of pDCs for the production of innate immunity cytokines induced by a dsRNA virus, and it shows that a dsRNA virus can induce IFN-α/β in pDCs via a novel TLR-independent and Myd88-dependent pathway. These findings have implications for the design of efficient vaccines against dsRNA viruses.

Culicoides Midge Bites Modulate the Host Response and Impact on Bluetongue Virus Infection in Sheep

Many haematophagous insects produce factors that help their blood meal and coincidently favor pathogen transmission. However nothing is known about the ability of Culicoides midges to interfere with the infectivity of the viruses they transmit. Among these, Bluetongue Virus (BTV) induces a hemorrhagic fever- type disease and its recent emergence in Europe had a major economical impact. We observed that needle inoculation of BTV8 in the site of uninfected C. nubeculosus feeding reduced viraemia and clinical disease intensity compared to plain needle inoculation. The sheep that developed the highest local inflammatory reaction had the lowest viral load, suggesting that the inflammatory response to midge bites may participate in the individual sensitivity to BTV viraemia development. Conversely compared to needle inoculation, inoculation of BTV8 by infected C. nubeculosus bites promoted viraemia and clinical symptom expression, in association with delayed IFN- induced gene expression and retarded neutralizing antibody responses. The effects of uninfected and infected midge bites on BTV viraemia and on the host response indicate that BTV transmission by infected midges is the most reliable experimental method to study the physio-pathological events relevant to a natural infection and to pertinent vaccine evaluation in the target species. It also leads the way to identify the promoting viral infectivity factors of infected Culicoides in order to possibly develop new control strategies against BTV and other Culicoides transmitted viruses.

Defining Mononuclear Phagocyte Subset Homology Across Several Distant Warm-Blooded Vertebrates Through Comparative Transcriptomics

Mononuclear phagocytes are organized in a complex system of ontogenetically and functionally distinct subsets, that has been best described in mouse and to some extent in human. Identification of homologous mononuclear phagocyte subsets in other vertebrate species of biomedical, economic, and environmental interest is needed to improve our knowledge in physiologic and physio-pathologic processes, and to design intervention strategies against a variety of diseases, including zoonotic infections. We developed a streamlined approach combining refined cell sorting and integrated comparative transcriptomics analyses which revealed conservation of the mononuclear phagocyte organization across human, mouse, sheep, pigs and, in some respect, chicken. This strategy should help democratizing the use of omics analyses for the identification and study of cell types across tissues and species. Moreover, we identified conserved gene signatures that enable robust identification and universal definition of these cell types. We identified new evolutionarily conserved gene candidates and gene interaction networks for the molecular regulation of the development or functions of these cell types, as well as conserved surface candidates for refined subset phenotyping throughout species. A phylogenetic analysis revealed that orthologous genes of the conserved signatures exist in teleost fishes and apparently not in Lamprey.

A physical map of large segments of pig Chromosome 7q11–q14: comparative analysis with human Chromosome 6p21

The aim of this study was to establish a porcine physical map along the chromosome SSC7q by construction of BAC contigs between microsatellites Sw1409 and S0102. The SLA class II contig, located on SSC7q, was lengthened. Four major BAC contigs and 10 short contigs span a region equivalent to 800 cR measured by IMpRH7000 mapping. The BAC contigs were initiated by PCR screening with primers derived from human orthologous segments, extended by chromosome walking, and controlled and oriented by RH mapping with the two available panels, IMpRH7000Rad and IMNpRH12000Rad. The location of 43 genes was revealed by sequenced segments, either from BAC ends or PCR products from BAC clones. The 220 BAC end sequences (BES) were also used to analyze the different marks of evolution. Comparative mapping analysis between pigs and humans demonstrated that the gene organization on HSA6p21 and on SSC7p11 and q11–q14 segments was conserved during evolution, with the exception of long fragments of HSA6p12 which shuffled and spliced the SLA extended class II region. Additional punctual variations (unique gene insertion/deletion) were observed, even within conserved segments, revealing the evolutionary complexity of this region. In addition, 18 new polymorphic microsatellites have been selected in order to cover the entire SSC7p11–q14 region.

Construction of a cytogenetically anchored microsatellite map in rabbit.

Rabbit (Oryctolagus cuniculus) represents a valuable source of biomedical models and corresponds to a small but active economic sector in Europe for meat and fur. The rabbit genome has not been thoroughly studied until recently, and high-resolution maps necessary for identification of genes and quantitative trait loci (QTL) are not yet available. Our aim was to isolate over 300 new and regularly distributed (TG)n or (TC)n rabbit microsatellites. To achieve this purpose, 164 microsatellite sequences were isolated from gene-containing bacterial artificial chromosome (BAC) clones previously localized by fluorescence in situ hybridization (FISH) on all the rabbit chromosomes. In addition, 141 microsatellite sequences were subcloned from a plasmid genomic library, and for 41 of these sequences, BAC clones were identified and FISH-mapped. TC repeats were present in 62% of the microsatellites derived from gene-containing BAC clones and in 22% of those from the plasmid genomic library, with an average of 42.9% irrespective of the microsatellite origin. These results suggest a higher proportion of (TC)n repeats and a nonhomogeneous distribution of (TG)n and (TC)n repeats in the rabbit genome compared to those in man. Among the 305 isolated microsatellites, 177 were assigned to 139 different cytogenetic positions on all the chromosomes except rabbit Chromosome 21. Sequence similarity searches provided hit locations on the Human Build 35a and hypothetical assignments on rabbit chromosomes for ten additional microsatellites. Taken together, these results report a reservoir of 305 new rabbit microsatellites of which 60% have a cytogenetic position. This is the first step toward the construction of an integrated cytogenetic and genetic map based on microsatellites homogeneously anchored to the rabbit genome.

The respiratory DC/macrophage network at steady-state and upon influenza infection in the swine biomedical model.

Human and mouse respiratory tracts show anatomical and physiological differences, which will benefit from alternative experimental models for studying many respiratory diseases. Pig has been recognized as a valuable biomedical model, in particular for lung transplantation or pathologies such as cystic fibrosis and influenza infection. However, there is a lack of knowledge about the porcine respiratory immune system. Here we segregated and studied six populations of pig lung dendritic cells (DCs)/macrophages (Mθs) as follows: conventional DCs (cDC) 1 and cDC2, inflammatory monocyte-derived DCs (moDCs), monocyte-derived Mθs, and interstitial and alveolar Mθs. The three DC subsets present migratory and naive T-cell stimulation capacities. As observed in human and mice, porcine cDC1 and cDC2 were able to induce T-helper (Th)1 and Th2 responses, respectively. Interestingly, porcine moDCs increased in the lung upon influenza infection, as observed in the mouse model. Pig cDC2 shared some characteristics observed in human but not in mice, such as the expression of FCɛRIα and Langerin, and an intra-epithelial localization. This work, by unraveling the extended similarities of the porcine and human lung DC/Mθ networks, highlights the relevance of pig, both as an exploratory model of DC/Mθ functions and as a model for human inflammatory lung pathologies.