Accordingly, administration of rmTSLP exacerbated disease severity and joint damage significantly, associated with an increased T cell activation 32. (APCs) and exogenous IL-4 16. In addition to eliciting the Dynemicin A proliferation of naive CD4+ T cells, TSLP has been demonstrated to act on CD8+ T cells. Both TSLPR and IL-7R are expressed by CD8+ T cells, and TSLP could act directly on murine CD8+ T cells to activate the STAT-5 and protein kinase B (Akt) signalling pathways in these cells 17. However, the underlying mechanisms and cell signalling pathways mediating these effects on T cells remain unclear. Autoimmune diseases, specifically multiple sclerosis (MS), have been associated with single nucleotide polymorphisms (SNPs) in the IL-7R gene locus 18. Thus, in the present study using TSLP KO mice we examined the role of TSLP for the first time in the experimental autoimmune encephalomyelitis (EAE). EAE, the conventional experimental model for MS, is usually a demyelinating disease of the central nervous system (CNS). In our study, EAE was induced once by immunization with myelin oligodendrocyte glycoprotein peptide 35C55 (MOG35C55), resulting in a monophasic acute form of EAE. In this model interferon (IFN)–producing Th1 and Dynemicin A IL-17-producing Th17 cells are considered to be the main players in the developing CNS inflammation 19. In contrast, regulatory T cells confer significant protection from EAE 20. Keeping in mind that TSLP is usually a potent activator of DCs, which play a key role in T cell differentiation and activation, and that TSLP could also directly activate T cells, we monitored the course of EAE in TSLP KO and TSLP WT mice and investigated its effects on CNS and lymphatic organs at different stages of disease. We found that mice deficient for TSLP showed an amelioration of EAE symptoms accompanied by reduced inflammatory infiltrates in brain and spinal cord. Dynemicin A Interestingly, in CNS of TSLP KO mice the encephalitogenic T cells showed a reduced activation. experiments confirmed that TSLP directly, and not only indirectly via DCs, also activates T cells and that T cells from TSLP KO mice show a reduced antigen-driven activation. Taken together, these results suggest that in mice, TSLP may be involved not only in allergic diseases, but also in inflammatory disorders such as EAE. Materials and methods Animals TSLP KO mice and TSLP WT mice were housed under specific pathogen-free conditions. TSLP KO mice were constructed as described previously 14. TSLP KO and TSLP WT mice were crossed with DEREG (DEpletion of REGulatory T cells) mice, which were kindly provided by Professor T. Sparwasser 27. All animal experiments were performed in accordance with institutional, state and federal guidelines. EAE induction and scoring Male TSLP KO and TSLP WT mice (12C16 weeks aged) were immunized subcutaneously (s.c.) with MOG35C55 (Sigma-Genosys, The Woodlands, TX, USA) at day 0 to induce EAE as described previously 49. EAE paralysis of mice was scored as follows: 0, no disease; 1, tail weakness; 2, paraparesis; 3, paraplegia; 4, paraplegia with forelimb weakness; and 5, moribund or dead animals. Neuropathology For visualization of inflammatory infiltrates, brains and spinal cords were harvested, fixed in liquid nitrogen and stored Dynemicin A at ?80C. Subsequent analyses of sections were performed according to protocols as described previously 49. Histochemistry was performed on 3-m thick paraffin sections. Quantification of demyelinated areas was performed on Luxol fast blue-stained sections. Areas with complete demyelination were identified as lesions. Blinded quantification was performed on serial sections and included three impartial spinal cross-sections per mouse. Immunofluorescence Immunofluorescence images of brain (and spinal cord) have been generated using the multi-epitope ligand cartography (MELC) technique. Test preparation, data acquisition and data analysis have already been performed as described 50 previously. Real time invert transcriptionCpolymerase chain response (RTCPCR) Total RNA was ready from cells or cells using the RNeasy Plus Mini Package and QIAshredder spin columns (Qiagen, Valencia, CA, USA). Traces of genomic DNA had been Rabbit Polyclonal to CRMP-2 (phospho-Ser522) eliminated by DNase digestive function using the RNase-free DNase Arranged (Qiagen). Subsequently, 1?g RNA was reverse-transcribed into sscDNA using the Initial Strand cDNA Synthesis Package (Fermentas, Burlington, ON, Canada), while specified by the product manufacturer. Realtime RTCPCR was performed in the Rotor-Gene Q (Qiagen) using the.