Synergistic interaction between Toll-like receptor agonists is required for induction of experimental autoimmune encephalomyelitis in Lewis rats☆
Introduction
Experimental autoimmune encephalomyelitis (EAE) in the Lewis rat is an acute T cell-mediated inflammatory disease that can be readily induced with a single injection of myelin basic protein (MBP) or MBP-derived peptides administered in complete Freund's adjuvant (CFA) (reviewed in Swanborg, 2001). In contrast, the injection of MBP or MBP peptides in incomplete Freund's adjuvant fails to elicit EAE in Lewis rats. Rather, this regimen induces tolerance with respect to EAE, which is due, at least in part, to the generation of T suppressor/regulatory cells that prevent the pathogenic T cells from eliciting paralysis (Swierkosz and Swanborg, 1975). These regulatory cells secrete TGF-β, which downregulates the production of proinflammatory cytokines (e.g., IFN-γ) by the encephalitogenic T cells (Karpus and Swanborg, 1991, Miller et al., 1992).
Until recently, the use of CFA and, in some models, other ancillary adjuvants were a requirement for the induction of EAE in rodents (reviewed in Swanborg, 1995). CFA was developed in the 1940s and led to pioneering studies of EAE (Kabat et al., 1947, Morgan, 1947), and other autoimmune diseases. However, the role of adjuvants in EAE was unclear until recently, when Segal et al. (1997) discovered that microbial products including bacterial lipopolysaccharide (LPS) and cytosine-guanine dinucleotide-containing oligodeoxynucleotides (CpG-ODN) could facilitate the induction of EAE by promoting IL-12 production. CpG-ODN is a Toll-like receptor (TLR)-9 agonist that activates APCs through innate immunity to secrete IL-12 and IL-6, which in turn activates the adaptive immune system (Hemmi et al., 2000, Janeway, 2001). LPS is a TLR-4 agonist that activates NF-κB in the nucleus (Medzhitov et al., 1997).
Recently, Ichikawa et al. (2002) reported that unresponsive T cells from SJL mice tolerized to an encephalitogenic peptide fragment of proteolipid protein are converted into pathogenic effector cells that proliferate and transfer EAE when activated in the presence of CpG-ODN. We confirmed their in vitro findings and showed that Lewis rats tolerized against the major dominant self MBP epitope from rat MBP (MBP(68–86)) harbor potentially encephalitogenic T cells that can be activated in vitro to proliferate to the tolerizing peptide by exposure to CpG-ODN (Conant and Swanborg, 2004). In the present report, we employ the peptide-IFA tolerance protocol to demonstrate that selected TLR agonists can replace the mycobacterial component of CFA and induce EAE in Lewis rats.
Section snippets
Peptide synthesis
The major encephalitogenic epitope from rat (self) MBP was synthesized on an Applied Biosystems Synergy model 432A peptide synthesizer (PerkinElmer, Foster City, CA) as previously described (Conant and Swanborg, 2004). The sequence of the peptide was YGSLPQKSQRTQDENPV. This peptide was designated MBP(68–86).
Animals and immunization
Female Lewis rats were immunized s.c. with MBP(68–86) in either incomplete Freund's adjuvant (IFA) or complete Freund's adjuvant (CFA), as previously described (Swanborg and Stepaniak, 1996,
Studies of rats immunized with MBP(68–86) in IFA
Lewis rats do not develop EAE when immunized with MBP or MBP peptides in IFA. Rather, this treatment protects them against EAE, due in part to the generation of suppressor/regulatory T cells (Swierkosz and Swanborg, 1975). Recently we reported that MBP(68–86) + IFA-immunized Lewis rats harbor encephalitogenic T cells. These could be activated in vitro by culturing T cells in the presence of MBP(68–86) and CpG-ODN; T cells cultured only with MBP(68–86) were not activated (Conant and Swanborg, 2004
Discussion
The major finding in the present report is that the addition of CpG-ODN and LPS to emulsions consisting of MBP(68–86) in incomplete Freund's adjuvant renders the emulsions encephalitogenic for Lewis rats. Thus, the two TLR agonists can replace mycobacteria, suggesting that Mycobacterium tuberculosis in CFA functions to activate innate immunity, which in turn, initiates an adaptive immune response to the antigen in the adjuvant emulsion. Neither CpG-ODN nor LPS alone initiated EAE when included
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This study is supported by NIH grants 5-RO1-NS06985–37 and 5-RO1-NS048070–02.