Rapamycin inhibits relapsing experimental autoimmune encephalomyelitis by both effector and regulatory T cells modulation

https://doi.org/10.1016/j.jneuroim.2010.01.001Get rights and content

Abstract

Rapamycin is an oral immunosuppressant drug previously reported to efficiently induce naturally occurring CD4+CD25+FoxP3+ regulatory T (nTreg) cells re-establishing long-term immune self-tolerance in autoimmune diseases. We investigated the effect of rapamycin administration to SJL/j mice affected by PLP139–151-induced relapsing–remitting experimental autoimmune encephalomyelitis (RR-EAE). We found that oral or intraperitoneal treatment at the peak of disease or at the end of the first clinical attack, dramatically ameliorated the clinical course of RR-EAE. Treatment suspension resulted in early reappearance of disease. Clinical response was associated with reduced central nervous system demyelination and axonal loss. Rapamycin induced suppression of IFN-γ, and IL-17 release from antigen-specific T cells in peripheral lymphoid organs. While CD4+FoxP3+ cells were unaffected, we observed disappearance of CD4+CD45RBhigh effector T (Teff) cells and selective expansion of Treg cells bearing the CD4+CD45RBlowFoxP3+CD25+CD103+ extended phenotype. Finally, the dual action of rapamycin on both Teff and Treg cells resulted in modulation of their ratio that closely paralleled disease course. Our data show that rapamycin inhibits RR-EAE, provide evidence for the immunological mechanisms, and indicate this compound as a potential candidate for the treatment of multiple sclerosis.

Introduction

Relapsing–remitting experimental autoimmune encephalomyelitis (RR-EAE) is a CD4+ TH1 and TH17 T cell-mediated autoimmune, inflammatory and demyelinating disease of the central nervous system (CNS), used as animal model for human multiple sclerosis (MS) (Kroenke and Segal, 2007). RR-EAE is induced in susceptible female SJL/j mice by immunization with the proteolipid protein (PLP139–151)-peptide (Kennedy et al., 1990), and the clinical course is characterized by multiple cycles of relapses and remissions. The immunoregulatory mechanisms responsible for the spontaneous recovery, which may be similar to those responsible for spontaneous remissions seen in patients with relapsing–remitting MS (RRMS), remain to date to be fully elucidated (Duplan et al., 2006, Zhang et al., 2006), but may provide novel therapeutic targets for autoimmunity.

Among the compounds with potent immunosuppressive activity aimed at inducing immune modulation and redirecting awry immune functions is rapamycin. Rapamycin is an oral immunosuppressive drug (Abraham and Wiederrecht, 1996) currently used to prevent rejection in human organ transplantation (Kahan and Camardo, 2001). In mammalian cells, rapamycin forms a complex with the intracellular immunophilin FK506-binding protein-12 (FKBP12), which blocks the activation of a serine/threonine protein kinase called mammalian target of rapamycin (mTOR), that is crucial for cell-cycle progression and protein synthesis, inhibiting antigen-induced T and B cell proliferation (Sehgal, 2003). The efficacy and safety of rapamycin have been previously evaluated in an open-label trial. Patients with clinically definitive RRMS or secondary progressive MS with relapses displayed a significant beneficial effect on the incidence of new enhancing magnetic resonance imaging lesions and number of relapses, with an acceptable risk/benefit profile (Kappos et al., 2005, Neuhaus et al., 2007). In EAE, the effect of this drug has also been examined in rat and mouse models, in which it was highly effective in preventing the onset and severity of disease (Carlson et al., 1993, Branisteanu et al., 1997). Until now, the mechanisms by which rapamycin exerts its beneficial effect both in humans and in preclinical animal models of MS are largely unknown.

Battaglia et al. have shown that rapamycin administration does not block proliferation in all T cell subtypes, but induces selective expansion of the naturally occurring regulatory T (nTreg) cells subpopulation in vitro (Battaglia et al., 2005), and establishes long-term immune self-tolerance in NOD mice affected by type 1 diabetes (Battaglia et al., 2006). A great deal of uncertainty remains, however, about the phenotypic characterization, among the murine CD4+ Treg cells that express the transcription factor Forkhead box P3 (FoxP3) (Fontenot et al., 2003, Hori et al., 2003), of the specialized subsets with effective suppressive function, since recently activated T cells can also express FoxP3 (Zhou et al., 2008). Thus, several additional markers have been used to this aim. They include the historically first proposed Treg marker, interleukin (IL)-2Rα chain (CD25) (Sakaguchi et al., 1995), an activation-induced cytokine receptor component apparently unrelated to the regulatory function because it does not easily discriminate Treg cells from activated T cells. Furthermore, suppressive activity has been demonstrated also in CD25 negative subsets (Annacker et al., 2001, Olivares-Villagomez et al., 2000, Stephens and Mason, 2000). The cytotoxic T lymphocyte-associated antigen (CTLA)-4 (Read et al., 2000) and the integrin αEβ7 (CD103) (Lehmann et al., 2002) have been described as additional markers strictly related to a unique population of Treg cells with highly potent regulatory function into sites of inflammation (Korn et al., 2007), although other studies have excluded a significant role for these molecules (Annacker et al., 2005, Levings et al., 2001, Thornton and Shevach, 1998). Finally, other investigations suggest the use of CD45RB to discriminate Treg from Teff cells (Powrie et al., 1994, Dardalhon et al., 2008), the latter being CD45RBhigh while those required for tolerance induction being CD4+CD45RBlowFoxP3+CD25+ (Fehervari and Sakaguchi, 2004, Mason and Powrie, 1998, Powrie et al., 1993). These data, altogether, suggest that the use of additional markers may allow to distinguish more efficiently effector from regulatory T cells.

In the present study we used rapamycin monotherapy administered to RR-EAE mice to investigate the effect of this immunomodulatory compound on the pool of endogenous Treg cells and its ability to directly expand their number or preserve their suppressive function in vivo. Our data show that rapamycin administration inhibits the induction and the progression of established RR-EAE by effector T (Teff) cells suppression and simultaneously increasing the percentage of CD4+CD45RBlowFoxP3+CD25+CD103+ Treg cells.

Section snippets

Mice

Female SJL/j mice, 6–8 weeks of age, were purchased from Charles River Laboratories (Calco, Italy). Mice were housed in specific pathogen-free conditions, in roomy cages, allowing free access to food and water. All procedures involving animals were performed according to the animal protocol guidelines prescribed by the Institutional Animal Care and Use Committee (IACUC #331) at San Raffaele Scientific Institute (Milan, Italy).

Induction and assessment of EAE

RR-EAE was induced in female SJL/j mice, as previously described (

Rapamycin inhibits clinical signs of RR-EAE independently of administration route and schedule

We first examined the therapeutic efficacy of rapamycin in our model of RR-EAE, comparing oral and intraperitoneal (i.p.) administration routes (Fig. 1). Treatment was initiated 10 days post-immunization (p.i.), to avoid interference with the differentiation of the first wave of encephalitogenic Teff cells. We used a 15 day long induction period where 1 mg/kg rapamycin, the same dosage used in humans, was administered every day, followed by further 20 days where rapamycin was given every three

Discussion

Immunosuppression is widely used in the therapy of autoimmune diseases such as MS. The administration route, needing hospitalization in most cases, the non-specific mode of action, and severe side effects, are the main drawbacks of this treatment. Rapamycin is an orally administered immunosuppressant drug widely used in organ transplantation (Kahan and Camardo, 2001), with a good safety profile, already tested on MS patients in a small scale pilot trial (Kappos et al., 2005), that has been

Acknowledgments

We thank Maria-Grazia Roncarolo, Anna Mondino, and David C. Wraith for fruitful discussion and helpful advice. This work was supported by the Italian Association for Multiple Sclerosis (FISM) and by the Italian Ministry of Research and University (MIUR).

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