The effect of PrP(Sc) accumulation on inflammatory gene expression within sheep peripheral lymphoid tissue.

Accumulation of the misfolded prion protein, PrP(Sc) in the central nervous system (CNS) is strongly linked to progressive neurodegenerative disease. For many transmissible spongiform encephalopathies (TSEs), peripheral lymphoid tissue is an important site of PrP(Sc) amplification but without gross immunological consequence. Susceptible VRQ homozygous New Zealand Cheviot sheep were infected with SSBP/1 scrapie by inoculation in the drainage area of the prescapular lymph nodes. The earliest time that PrP(Sc) was consistently detected by immunohistology in these nodes was D50 post infection. This transcriptomic study of lymph node taken before (D10) and after (D50) the detection of PrP(Sc), aimed to identify the genes and physiological pathways affected by disease progression within the nodes as assessed by PrP(Sc) detection. Affymetrix Ovine Gene arrays identified 75 and 80 genes as differentially-expressed at D10 and D50, respectively, in comparison with control sheep inoculated with uninfected brain homogenate. Approximately 70% of these were repressed at each time point. RT-qPCR analysis of seven genes showed statistically significant correlation with the array data, although the results for IL1RN and TGIF were different between the two technologies. The ingenuity pathway analysis (IPA) and general low level of repression of gene expression in lymphoid tissue, including many inflammatory genes, contrasts with the pro-inflammatory and pro-apoptotic events that occur within the CNS at equivalent stages of disease progression as assessed by PrP(Sc) accumulation.

Transcriptome analysis of CNS immediately before and after the detection of PrP(Sc) in SSBP/1 sheep scrapie.

Sheep scrapie is a transmissible spongiform encephalopathy (TSE), progressive and fatal neurodegenerative diseases of the central nervous system (CNS) linked to the accumulation of misfolded prion protein, PrP(Sc). New Zealand Cheviot sheep, homozygous for the VRQ genotype of the PRNP gene are most susceptible with an incubation period of 193 days with SSBP/1 scrapie. However, the earliest time point that PrP(Sc) can be detected in the CNS is 125 days (D125). The aim of this study was to quantify changes to the transcriptome of the thalamus and obex (medulla) at times immediately before (D75) and after (D125) PrP(Sc) was detected. Affymetrix gene arrays were used to quantify gene expression in the thalamus and Illumina DGE-tag profiling for obex. Ingenuity Pathway Analysis was used to help describe the biological processes of scrapie pathology. Neurological disease and Cancer were common Bio Functions in each tissue at D75; inflammation and cell death were major processes at D125. Several neurological receptors were significantly increased at D75 (e.g. CHRNA6, GRM1, HCN2), which might be clues to the molecular basis of psychiatric changes associated with TSEs. No genes were significantly differentially expressed at both D75 and D125 and there was no progression of events from earlier to later time points. This implies that there is no simple linear progression of pathological or molecular events. There seems to be a step-change between D75 and D125, correlating with the detection of PrP(Sc), resulting in the involvement of different pathological processes in later TSE disease.

The immunology and genetics of resistance of sheep to Teladorsagia circumcincta.

Teladorsagia circumcincta is one of the most economically important gastrointestinal nematode parasites of sheep in cool temperate regions, to which sheep show genetically-varying resistance to infection. This is a very common parasite and viable sheep production requires the extensive use of anthelmintic drugs. However, the emergence of drug-resistant parasites has stimulated the search for alternative control strategies to curb production losses. Lambs become infected soon after weaning and begin to control parasite burden within 8-10 weeks of continual infection. This control is an acquired characteristic mediated by the development of parasite-specific antibodies. This paper describes the immunology associated with resistance and susceptibility, focussing on differential T cell activation that regulates the production of specific effector mechanisms. It continues by summarizing the methods used to identify genes that could be exploited as molecular markers of selection for resistance. In particular it focusses on the link between understanding the molecular immunology of infection and the identification of candidate genes for selection.

Expression of sheep interleukin 23 (IL23A, alpha subunit p19) in two distinct gastrointestinal diseases.

This paper reports the sequence of sheep interleukin 23A (p19), and shows that it shares 98% identity with bovine IL23A, 85% with human and 76% with mouse IL23A. It also reports the existence of two allelic variants that differ largely within the region encoding the amino terminal polypeptide signal sequence. An optimized RT-qPCR assay was used to quantify IL23A transcripts in sheep infected with two common gastrointestinal pathogens, the intracellular bacterium Mycobacterium avium subspecies paratuberculosis and the parasitic nematode Teladorsagia circumcincta. No differential expression of IL23A was detected in the mesenteric lymph node of sheep with the different pathogenic forms of paratuberculosis, however significantly high levels of IL23A were detected in the ileal mucosa of the paucibacillary form in comparison with the asymptomatic or multibacillary forms. Similarly, significantly high levels were present in the gastric lymph node draining T. circumcincta-infected abomasum in susceptible sheep. High levels of IL23A seem to be associated with lymphocytic infiltration and inflammation in both diseases but not with the macrophage infiltrate of multibacillary paratuberculosis.

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.

Gene expression analysis in distinct regions of the central nervous system during the development of SSBP/1 sheep scrapie.

Rodent scrapie models have been exploited to define the molecular basis for the progression of neuropathological changes in TSE diseases. We aim to assess whether CNS gene expression changes consistently observed in mouse models are of generic relevance, for example to natural TSE diseases, or are TSE strain, host species or brain region specific. Six genes, representing distinct physiological pathways and showing consistent changes in expression levels with disease progression in murine scrapie models were analysed for expression (RT-qPCR) in defined regions of the sheep brain at various times after SSBP/1 scrapie infection. Gene expression was examined in relation to the development of neuropathological changes including PrP(Sc) deposition and vacuolation. Peripheral infection of sheep with SSBP/1 showed consistent progression of neuropathology as assessed by the temporal course of PrP(Sc) deposition and neuropil vacuolation. The first region affected was the medulla (obex), then the thalamus and finally the cerebellum and frontal cortex. In contrast to mouse scrapie, there were few significant changes in transcript expression for any of the six genes and no consistent changes in patterns of expression in relation to brain region, time after infection or neuropathology in sheep SSBP/1. Gene expression changes in mouse TSE models, even changes consistent with the neuropathology, cannot necessarily be extrapolated to species in which disease naturally occurs. This may represent differences in pathological processes of different scrapie strains or across species; and highlights the difficulties in identifying generic molecular pathways associated to the pathogenesis of TSE disease.

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.

Differential expression of pattern recognition receptors in sheep tissues and leukocyte subsets.

The various members of the different pattern recognition receptor families are now recognized as playing a crucial role in the initial interactions between a pathogen and the host. This paper identifies all 10 members of the TLR family in sheep as well as key members of the C-type lectin and NLR families. Our data show that sheep possess the 'human' and not the 'mouse' pattern of TLRs and confirm the high degree of sequence identity between orthologous genes in the different species. In the absence of definitive antibodies, qRT-PCR assays were developed to quantify PRR transcript expression patterns in a range of normal sheep tissues as well as isolated dendritic cell (DC) and leukocyte subsets. These data show that the lymphoid organs (spleen and lymph nodes) express the widest range of PRRs and that organs such as the lung and kidney have distinctive arrays of PRRs that reflect their potential risk of pathogen exposure. In addition we show that the two DC subsets, defined by the differential expression of CD172a/CD45RA and their cytokine expression profiles, have different and characteristic PRR complements again possibly reflecting their distinctive function. These data are important for future studies on the role of PRRs in disease pathogenesis and control.

Patterns of cytokine gene expression of naïve and memory T lymphocytes in vivo.

Large-scale lymphocyte recirculation occurs only at the level of secondary lymphoid tissue. Cells enter lymph nodes via afferent lymph from the tissue and via arterioles from the blood. They exit only via the efferent duct. Afferent and efferent lymphocytes have distinct phenotypes; afferent lymphocytes have a 'memory' phenotype, being CD62L(-)/CD45RA(-) and expressing high levels of CD2 and CD11a; efferent cells are largely 'naïve', being CD62L(+)/CD45RA(+) with low levels of CD2 and CD11a. We will show that functionally the efferent lymphocytes, like cells from the blood and spleen, can be activated in vitro only by dendritic cells. However, afferent lymphocytes are less stringent in their activation requirements and can be stimulated by both macrophages and dendritic cells. To explain these functional differences we have developed a multiprobe RNAase protection assay for 13 sheep cytokines (IL-1beta, IL-2, IL-3, IL-4, IL-6, IL-8, IL-10, IL-12, IL-18, GMCSF, IFNgamma, TGFbeta and TNFalpha) and two housekeeping genes (ATPase and GADPH). We have used this assay to measure the constitutive expression of cytokine mRNA in MACS-purified CD4+ and CD8+ T lymphocytes from both lymphoid compartments.