Anton Gossner

Differential expression of pattern recognition receptors in the three pathological forms of sheep paratuberculosis.

Paratuberculosis is a chronic inflammatory disease of the gut caused by Mycobacterium avium subspecies paratuberculosis. Three forms have been described in sheep--paucibacillary, multibacillary and asymptomatic. The pauci- and multibacillary forms are characterized by type 1 and type 2 immune responses respectively; asymptomatic animals have no clinical signs or pathology. What determines this polarization is unknown, although pattern recognition receptors (PRR) have been implicated in other mycobacterial diseases. To investigate this in sheep paratuberculosis we used real-time RT-PCR to quantify the expression of fifteen PRR and adaptor genes from forty infected and nine control animals. These data show that there is a relationship between the different pathological forms and PRR transcript profiles. Nine PRRs were up-regulated in asymptomatic animals; with TLR9 being significantly raised in relation to the other three groups. Comparison of the three infected groups showed increases in many PRRs, with CARD15 and Dectin-2 being particularly high in both diseased groups. Significant differences between the pauci- and multibacillary animals included TLR2, CD14 and Dectin-1. Sequence analysis of TLR2 exon 2 and CARD15 exon 11 in the forty animals failed to identify any relationship between SNPs and pathological form.

Relationship between susceptibility of Blackface sheep to Teladorsagia circumcincta infection and an inflammatory mucosal T cell response

Teladorsagia circumcincta is the most economically important gastrointestinal (abomasal) nematode parasite of sheep in cool temperate regions, to which sheep show genetically-varying resistance to infection. Lambs, from parents with genetic variation for resistance, were trickle infected with L3 larvae over 12 weeks. 45 lambs were identified with a range of susceptibilities as assessed by: adult worm count at post mortem, faecal egg count (FEC) and IgA antibody levels. This project investigated the correlation of T cell cytokine expression and resistance to infection at the mature stage of response, when the resistant lambs had excluded all parasites.Histopathology showed only minor changes in resistant animals with a low level lymphocyte infiltration; but in susceptible lambs, major pathological changes were associated with extensive infiltration of lymphocytes, eosinophils and neutrophils.Absolute quantitative RT-qPCR assays on the abomasal lymph node (ALN) revealed a significant positive correlation between IL6, IL21 and IL23A transcript levels with adult worm count and FEC. IL23A was also negatively correlated with IgA antibody levels. Significantly positive correlation of TGFB1 levels with adult worm count and FEC were also seen in the abomasal mucosa. These data are consistent with the hypothesis that the inability to control L3 larval colonization, adult worm infection and egg production is due to the activation of the inflammatory Th17 T cell subset.

The Effects of Host Age on the Transport of Complement-Bound Complexes to the Spleen and the Pathogenesis of Intravenous Scrapie Infection

ABSTRACT Infections with variant Creutzfeldt-Jakob disease (vCJD) have almost exclusively occurred in young patients, but the reasons for this age distribution are uncertain. Our data suggest that the pathogenesis of many peripherally acquired transmissible spongiform encephalopathy (TSE) agents is less efficient in aged individuals. Four vCJD cases linked to transfusion of vCJD-contaminated blood or blood products have been described. Three cases occurred in elderly patients, implying that intravenous exposure is more efficient in aged individuals than other peripheral routes. To test this hypothesis, young (6 to 8 weeks old) and aged (600 days old) mice were injected intravenously with a TSE agent. In aged and young mice, the intravenous route was more efficient than other peripheral routes of TSE agent exposure. However, in aged mice, disease pathogenesis was significantly reduced. Although most aged mice failed to develop clinical disease during their life spans, many showed histopathological signs of TSE disease in their brains. Thus, the effects of age on intravenous TSE pathogenesis may lead to significant levels of subclinical disease in the population. After peripheral exposure, many TSE agents accumulate upon follicular dendritic cells (FDCs) in lymphoid tissues before they infect the brain. In aged spleens, PrPC expression and TSE agent accumulation upon FDCs were reduced. Furthermore, the splenic marginal zone microarchitecture was substantially disturbed, adversely affecting the delivery of immune complexes to FDCs. This study is the first to suggest that the effects of aging on the microarchitecture and the function of the splenic marginal zone significantly influence the pathogenesis of an important pathogen.

Differential expression of Prnp and Sprn in scrapie infected sheep also reveals Prnp genotype specific differences.

The central role for PrP in the pathogenesis of the transmissible spongiform encephalopathies (TSEs) is illustrated by the resistance of Prnp(0/0) mice to disease and by the inverse association of Prnp gene dosage with incubation period. Understanding the role of PrP(C) in TSEs necessitates knowledge of expression levels of the Prnp gene during the development of disease. SSBP/1 scrapie shows a defined pattern of disease progression and here we show that Prnp and shadow of PrP (Sprn) are differentially expressed in different brain areas and lymphoid tissues. Counter-intuitively we found that there is no positive correlation between expression of Prnp or Sprn and patterns of disease progression. Prnp and Sprn expression levels are both influenced by Prnp genotype; although the scrapie-sensitive VRQ/VRQ sheep did not express the highest level of either. In addition, infection with SSBP/1 scrapie seems to have little effect on either PrP or Shadoo expression levels.

Gene Gun-delivered pGM-CSF Adjuvant Induces Enhanced Emigration of two Dendritic Cell Subsets from the Skin

Two subsets of sheep afferent lymph dendritic cells (DC) are defined by the differential expression of CD172a and CD45RA. The majority (∼70%) of CD172a+ subset is CD45RA/CD11c+/CD207+/TLR4+. The CD172a− DC are CD45RA+/CD207− and express low levels of CD11c and CD86. Real‐time RT‐PCR showed that CD172+ DC produce IL‐1β and IL‐10 and high levels of IL‐18 but almost no IL‐12p40; CD172a− DC express IL‐12p40 but no IL‐10 and low levels of IL‐1β and IL‐18. Gene gun‐delivered granulocyte‐macrophage colony‐stimulating factor (pGM‐CSF) caused an early rise in the output of CD172a+ DC, changes to DC phenotype and significant increases in the levels of expression cytokine transcripts. However, pGM‐CSF did not affect any qualitative changes to cytokine expression, CD172a+ DC remained IL‐10+/IL‐12p40− and the CD172− DC remained IL‐10−/IL‐12p40+.

Differential expression of pattern recognition receptors during the development of foetal sheep.

Pattern recognition receptors (PRRs) play a crucial role in the initiation of the adaptive immune response. Immunological competence of foetal lambs occurs progressively throughout gestation, and in order to understand the role played by PRRs in foetal immunological competence, we quantified transcript expression, in the skin and spleen, of the TLRs, key C-type lectins and CARD15 during the critical second trimester. These data show that lambs express the same spectrum of PRRs as the adult but that the level of expression for most is dependent on developmental age. Key findings include: TLR1 and TLR5 are expressed at high levels in the foetus but are low in the adult; in contrast, TLR4, CD14 and CARD15 increase with age. In addition, TLR9 and TLR10 are expressed by the spleen and not the skin, while CARD15 is low in the spleen and high in the skin.

Expression profiling reveals differences in immuno-inflammatory gene expression between the two disease forms of sheep paratuberculosis.

Paratuberculosis is a chronic enteropathy of ruminants caused by Mycobacterium avium subspecies paratuberculosis (MAP); infection of sheep results in two disease forms - paucibacillary (tuberculoid) and multibacillary (lepromatous) associated with the differential polarization of the immune response. In addition the majority of MAP-infected animals show no pathology and remain asymptomatic. Microarray and real-time RT-qPCR analyses were used to compare gene expression in ileum from sheep with the two disease forms and asymptomatic sheep, to further understand the molecular basis of the pathologies. Microarrays identified 36 genes with fold-change of >1.5 and P< or = 0.05 in at least one comparison; eight candidates were chosen for RT-qPCR validation. Sequence analysis of two candidates, CXCR4 and IGFBP6, identified three SNPs in each; five were found in all three forms of disease and showed no significant relationship to pathological type. The IGFBP6 G(3743) A SNP was not detected in asymptomatic sheep. The data show that the two forms of disease are associated with distinct molecular profiles highlighted by the differential expression of chemokine and chemokine receptor transcripts, the protein products of which might be implicated in the different cell infiltrates of the pathologies. The cells within the lesions also show evidence of abnormal activation; they express high levels of cytokine transcripts but have reduced expression levels of transcripts for T cell receptor associated molecules.

Transcriptional profiling of peripheral lymphoid tissue reveals genes and networks linked to SSBP/1 scrapie pathology in sheep

Transmissible spongiform encephalopathies (TSEs) are slow and progressive neurodegenerative diseases of humans and animals. The major target organ for all TSEs is the brain but some TSE agents are associated with prior accumulation within the peripheral lymphoid system. Many studies have examined the effects of scrapie infection on the expression of central nervous system (CNS) genes, but this study examines the progression of scrapie pathology in the peripheral lymphoid system and how scrapie infection affects the transcriptome of the lymph nodes and spleen. Infection of sheep with SSBP/1 scrapie resulted in PrP(Sc) deposition in the draining prescapular lymph node (PSLN) by 25 days post infection (dpi) in VRQ/VRQ genotype sheep and 75 dpi in tonsils and spleen. Progression of PrP(Sc) deposition in VRQ/ARR animals was 25 dpi later in the PSLN and 250 dpi later in spleen. Microarray analysis of 75 dpi tissues from VRQ/VRQ sheep identified 52 genes in PSLN and 37 genes in spleen cells that showed significant difference (P ≤ 0.05) between scrapie-infected and mock-infected animals. Transcriptional pathway analysis highlighted immunological disease, cell death and neurological disease as the biological pathways associated with scrapie pathogenesis in the peripheral lymphoid system. PrP(Sc) accumulation of lymphoid tissue resulted in the repression of genes linked to inflammation and oxidative stress, and the up-regulation of genes related to apoptosis.

The effect of gene gun-delivered pGM-CSF on the immunopathology of the vaccinated skin

The aim of this study was to investigate the skin immunopathology of gene gun‐delivered plasmid‐encoded granulocyte‐macrophage colony‐stimulating factor (pGM‐CSF) and hence explore the possible mechanisms of its adjuvant activity. Using sheep as the experimental model, expressible pGM‐CSF was administered to the epidermis and the dermal/epidermal junction and its effects on the skin were assessed by histopathology, immunohistology and quantitative RT‐PCR for a range of pro‐inflammatory and immune response‐polarizing cytokines. Both functional and non‐functional plasmids caused an acute inflammatory response with the infiltration of neutrophils and micro‐abscess formation; however, the response to pGM‐CSF was more severe and was also associated with the accumulation of eosinophils, immature (CD1b−/CD172a−) dendritic cells and B cells. In terms of cytokine expression, an early TNF‐α response was stimulated by gene gun delivery of plasmid‐associated gold beads, which coincided with an immediate infiltration of neutrophils. However, only pGM‐CSF triggered the short‐lived expression of GM‐CSF (peaking at 4 h) and significant long‐term increases in both TNF‐α and IL‐1β. pGM‐CSF did not affect the expression of the immune response‐polarizing cytokines, IL‐10 and IL‐12.

Variations in IL-23 and IL-25 receptor gene structure, sequence and expression associated with the two disease forms of sheep paratuberculosis.

The immunopathology of paucibacillary and multibacillary sheep paratuberculosis is characterized by inflammatory T cell and macrophage responses respectively. IL-23 and IL-25 are key to the development of these responses by interaction with their complex receptors, IL-23R/IL-12RB1 and IL-17RA/IL-17RB. In humans, variations in structure, sequence and/or expression of these genes have been implicated in the different pathological forms of tuberculosis and leprosy, and in gastrointestinal inflammatory disorders such as Crohn’s disease. Sequencing has identified multiple transcript variants of sheep IL23R, IL12RB1 and IL17RB and a single IL17RA transcript. RT-qPCR assays were developed for all the identified variants and used to compare expression in the ileo-caecal lymph node of sheep with paucibacillary or multibacillary paratuberculosis and uninfected animals. With IL-23 receptor, only the IL12RB1v3 variant, which lacks the receptor activation motif was differentially expressed and was significantly increased in multibacillary disease; this may contribute to high Th2 responses. Of the IL17RB variants only full length IL17RB was differentially expressed and was significantly increased in multibacillary pathology; which may also contribute to Th2 polarization. IL17RA expression was significantly increased in paucibacillary disease. The contrast between the IL17RA and IL17RB results may indicate that, in addition to Th1 cells, Th17 T cells are also involved in paucibacillary pathology.