Cytokine antibody array analysis in brain and periphery of scrapie-infected Tg338 mice

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Abstract

Scrapie is a prion-associated transmissible spongiform encephalopathy (TSE) of sheep and goats, and frequently serves as a comparative model for other prion diseases, such as chronic wasting disease and bovine spongiform encephalopathy. TSEs are unique neurologic disorders that do not appear to be accompanied by robust systemic immunologic responses. mRNA data suggest that cytokines are involved in scrapie progression. In this study, brain tissue, mesenteric lymph nodes, splenic tissue and serum from ovinized mice were screened for 62 cytokine and cytokine-related proteins at pre-clinical and clinical points of infection. Expression patterns were compared to brain histology and clinical presentation. Increased cytokine expression in the brain and periphery were noted in scrapie-positive animals before histologic changes or clinical signs were evident. Of the 62 proteins examined, only IL-10 and TIMP-1 were consistently expressed at increased levels in the serum throughout infection. These cytokines could suggest future targets for biomarkers of infection and may, as well, provide insight into the biologic dynamics of prion-associated neurologic diseases.

Introduction

The transmissible spongiform encephalopathies (TSEs) are inevitably fatal neurodegenerative disorders [1]. Human TSEs include a diverse group of iatrogenic, sporadic, familial and acquired disorders, including Creutzfeldt-Jakob Disease and Kuru. TSEs of livestock include chronic wasting disease (CWD) of elk, mule deer, white-tailed deer and moose, bovine spongiform encephalopathy (BSE) of cattle, and scrapie disease of sheep and goats [2]. A disease-associated isoform of the prion protein (e.g., PrPSc) is thought to be the etiologic agent of the TSEs [3]. The cellular isoform (PrPc) is expressed in brain and peripheral tissues of mammals, although its function is still not fully understood. Disease occurs when PrPc is induced to misfold into PrPSc, resulting in a conformation with increased beta-sheets. Histologically, TSEs ultimately present with neuronal apoptosis, astrogliosis and spongiform degeneration or neuropil vacuolization without distinct inflammatory cell accumulation [4]. The mechanisms by which accumulation of PrPSc results in neurodegenerative changes are not entirely understood. Clinically, TSEs present with progressive neurodegenerative signs, often including ataxia, severe disorientation or behavioral changes, and myoclonic twitching. Disease incubation is long and clinical signs are usually not obvious until the terminal stages of infection.

TSEs are unique neurologic disorders in which measurable systemic immune responses do not seem to be evoked by the causative agent [1], [5]. Attempts to demonstrate antibody to the scrapie agent by neutralization, complement fixation, immunoprecipitation and immunofluorescence have been unsuccessful, and scrapie-positive immunodeficient mice depleted of T-lymphocytes show disease course similar to that of immunocompetent scrapie positive animals [6]. Brain inflammation has been noted in conjunction with TSEs, but in some model systems appears to consist primarily of astrocyte and microglia activation [4], suggesting that these resident central nervous system cells are responsible for increased cytokine expression and subsequent neurodegeneration [5], [7]. Increases in cytokine gene expression in murine models of scrapie infection have been reported. Campbell [7] reported a marked increase in tumor necrosis factor alpha (TNFα), Interleukin-1 alpha (IL-1α) and IL-1 beta (IL-1β) mRNA in the terminal stages of murine scrapie. Similar increases in mRNA for pro-inflammatory cytokines in the brain have been found by other groups in several scrapie murine models [8], [9]. Gene expression profiling technologies that allow large panels of genes to be analyzed at one time have allowed for a large amount of information to be collected on cytokine gene expression levels throughout the course of scrapie infection [10]. However, protein levels do not always correlate with mRNA expression levels and protein level analyses may be useful for identification of diagnostic markers and treatment targets [11]. Immunostaining procedures can be employed to explore protein expression, but often the process lacks quantification and is laborious since only one or a few proteins are probed at one time. Relatively new antibody array technology has improved upon this situation and made simultaneous measurement of a large panel of proteins possible. Such arrays could be informative in the ongoing study of the role of cytokines in prion disease pathogenesis.

In this study, brain tissue, mesenteric lymph nodes, splenic tissue and serum from ovinized transgenic mice infected with scrapie (inoculated intracerebrally with scrapie-positive sheep brain) were screened for 62 different cytokine and cytokine-related proteins at pre-clinical and clinical time points during infection. Protein expression assays were used to determine whether cytokine and cytokine-related protein levels in the brain or periphery increased or decreased during infection and whether such changes were associated with brain histopathology and clinical presentation. A global analysis of cytokine protein expression at preclinical and clinical scrapie time points in brain and peripheral tissues could contribute critical information to the pathogenesis of prion infection and/or identify candidate biomarkers for infection or progression of scrapie or similar agents.

Section snippets

Animals

Thirty-five 8–12 week old homozygous Tg338 mice transgenic for the sheep prion gene (PRNP) VRQ allele on a mouse PrP−/− background were used for these studies. The Tg338 mice were maintained and bred at the University of Washington. The mice originated from the laboratory of H. Laude (Jouy-en-Josas, France) and were developed as previously described [12], [13]. PrP expression level in the brain of Tg338 animals is 8–10 fold that in sheep [12]. All treatments and procedures were approved by the

Clinical appearance

Preclinical mice were euthanized at 100 or 160 days after intracerebral (i.c.) inoculation with scrapie-positive sheep brain. These time points were selected based on historical knowledge of the course of scrapie in Tg338 mice. At these points, infected mice were clinically indistinguishable from un-inoculated control mice. Clinical mice were euthanized when signs compatible with those reported by Campbell [7] first presented. Clinical signs consisted of severe ataxia with or without hind limb

Discussion

The appearance of clinical signs and histological evaluation of brain tissue are the most common means of diagnosing scrapie in a sheep herd. Similar to other prion diseases, clinical signs of scrapie infection do not present until the end stage of infection. Histological changes in the brain also occur late and lack a robust inflammatory cell component. Work using mRNA as a determinant suggests that, at least at the end-stage of infection in the brain, cytokine secretion is amplified [7], [8],

Acknowledgements

We thank Hubert Laude for providing the Tg338 mice. We also thank Gina Kiske for her expert technical assistance and colony management. This research was supported in part by USDA SCA #58-5348-7-466, and USDA CRIS #5348-32000-021-00D.

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