A variety of drugs are now approved for the treatment of MS. However, these drugs have side effects and do not cure the disease. Thus, there is a significant need to develop new and more effective medications to treat MS.
The current study investigated the potential of β-lapachone, as a possible novel treatment for MS. Our studies demonstrate that β-lapachone inhibits LPS induction of the cytokines IL-12 and IL-23 by primary microglia and DCs. IL-12 is critical in the differentiation of Th1 cells and IL-23 is critical in the differentiation of Th17 cells, both of which play significant roles in the development of EAE. This suggests that β-lapachone may alter T cell activation, differentiation, and maintenance through effects on peripheral and CNS APCs. Our data demonstrating that β-lapachone suppression of IL-23 production by microglia in turn suppresses the production of IL-17 by T cells further supports the idea that β-lapachone alters T cell function, at least in part, through effects on microglia. β-lapachone potently suppressed the development of EAE, suggesting that this molecule may be effective in the treatment of MS. β-lapachone suppressed the expression of IL-17 as well as TLR signaling molecules in EAE mice, suggesting mechanisms by which β-lapachone suppresses EAE.
EAE is an established animal model of MS, and demonstrates clinical and histopathological similarities to the human disease (Martin and McFarland, 1995
). EAE is characterized by axonal pathology and demyelination. The disease is principally mediated by CD4+ T lymphocytes. These CD4+
Th cells have traditionally been separated into two classifications (Mosmann and Coffman, 1989
). Th1 cells are characterized by the production of IFN-γ and lymphotoxin. These cells control extracellular pathogens, initiate delayed type hypersensitive reactions and participate in cell mediated immunity. Th2 cells are characterized by the production of IL-4, IL-5, IL-10, and IL-13. These cells control extracellular pathogens, contribute to allergic responses, and mediate humoral immune responses (Abbas et al., 1996
). While Th1 cells contribute to exacerbations of MS and other autoimmune diseases, Th2 cells are protective against these diseases. Previous studies indicated that IFN-γ and IFN-γR knockout mice were actually more susceptible than wild-type mice to the development of autoimmunity (Krakowski and Owens, 1996
). In addition, IL-12 which triggers the differentiation of Th1 cells was shown to not play a sufficient role in EAE, as IL-12 knockout mice were susceptible to development of EAE (Gran et al., 2002
). These studies suggested that although Th1 cells contribute to development of EAE, that other factors are also important in triggering disease. More recently, an additional CD4+ T cell was shown to contribute to the development of EAE. This cell termed Th17 is characterized by expression of IL-17A, IL-17F, IL-21, and IL-22 and was previously demonstrated to clear extracellular pathogens. Th17 cells differentiate in the presence of IL-6 and TGF-β (Bettelli et al., 2006
, Mangan et al., 2006
), although IL-6 and IL-21 can also trigger differentiation of these cells in the absence of TGF-β (Korn et al., 2007
, Nurieva et al., 2007
, Zhou et al., 2007
). In addition to being important in Th17 cell differentiation, IL-21, through a positive feedback mechanism, is important in the amplification of the Th17 cell population (Korn et al., 2007
). Although not directly required for the differentiation of Th17 cells, IL-23 is critical for stabilizing and maintaining this cell population (Langrish et al., 2005
). Several studies support the role of IL-23 and Th17 cells in modulating autoimmunity. Mice deficient in the p19 subunit of IL-23 were demonstrated to be resistant to development of EAE (Cua et al., 2003
). Also, adoptive transfer of Th17 cells induces EAE (Kleinschek et al., 2007
). IL-17 deficient mice exhibit decreased severity of EAE (Komiyama et al., 2006
). Th17 cells are also elevated in MS patients, further supporting a role of these cells in autoimmunity (Lock et al., 2002
). Cells respond to IL-23 through a heterodimeric receptor consisting of a unique IL-23R in association with IL-12Rβ1 which is common to the IL-12R complex, IL-23R is expressed at high levels on T cells and NK cells and at lower levels on DCs and monocytes (Parham et al., 2002
). IL-6 which is critical to Th17 cell differentiation also increases IL-23R expression on T cells which facilitates the response to IL-23 by these cells (Zhou et al., 2007
). IL-17A and IL-17F bind a heterodimeric IL-17R composed of IL-17RA in association with IL-17RC. IL-17RA can also bind other IL-17 family members through association of IL-17R heteromeric partners, although IL-17A and IL-17F are believed to principally mediate IL-17 effects on EAE and MS (Gaffen, 2011
). Our current studies indicate that β-lapachone inhibits IL-23 production by microglia and DCs and suppresses IL-17 production by T cells. The studies also demonstrate that β-lapachone suppresses EAE and that this suppression is associated with decreased expression of IL-17RA and IL-23R expression in the CNS of these mice. Collectively, these studies suggest that β-lapachone modulates EAE, at least in part, through alterations in IL-23 and IL-17 signaling.
TLRs are a family of pattern recognition receptors (PRRs) which recognize pathogen-associated molecular patterns (PAMPs) present on viruses, bacteria, and fungi. TLRs play a critical role in innate immune responses. CD14 is a PRR expressed on cells of the innate immune system including monocytes which recognizes PAMPS including peptidoglycan on the surface of gram-positive bacteria and LPS on the surface of gram-negative bacteria. Peptidoglycan is a ligand for TLR2 while LPS is a ligand for TLR4. Ligand binding to most TLRs triggers activation of the MyD88-dependent signaling pathway. However, TLR3 signals through a MyD88-independent signaling pathway and TLR4 can activate both the MyD88-dependent and – independent signaling pathways. The MyD88-dependent pathway ultimately results in activation of the transcription factor NF-κB which is known to activate the expression of a wide variety of genes encoding proinflammatory molecules. The MyD88-independent pathway leads to activation of NF-κB as well as the transcription factor IRF-3 which activates interferon-responsive genes (Racke and Drew, 2009
). Β-lapachone was previously demonstrated to suppress the activation of NF-κB and the production of inflammatory molecules (Moon et al., 2007
). Our current studies demonstrate that β-lapachone suppression of EAE is associated with decreased CNS expression of TLR2, TLR4, CD14, and MyD88. Collectively, these studies suggest that β-lapachone may suppress EAE, at least in part, through effects on MyD88 signaling.
In summary, the present studies indicate that β-lapachone suppresses the production of IL-12 and IL-23 by microglia and DCs and IL-17 expression by T cells. β-lapachone suppresses EAE and this suppression is associated with decreased expression of molecules critical to IL-23, IL-17, and MyD88-dependent signaling. Collectively, these studies suggest mechanisms by which β-lapachone suppresses EAE and suggest that β-lapachone may be effective in the treatment of MS.