AT and other cholesterol-lowering statins have been shown to be clinically beneficial in various models of T-cell-mediated autoimmune disease. These studies have consistently reported that statin treatment inhibits proinflammatory Th1 cytokine production. Despite the use of various statins and differences in dose or route of administration, many – but not all [3, 6, 23] – of these studies attributed the benefit of statin treatment in autoimmune models to development of Th2 cells [2, 5, 33, 34]. Here we used IL4-reporter mice and STAT6-deficient mice as tools to further explore the involvement anti-inflammatory Th2 cells in the anti-inflammatory effects of AT. We found that dosing of AT in vivo failed to promote detectable frequencies of Th2 cells, but T cells isolated from AT-treated mice did produce IL-4 in increased amounts after re-stimulation in culture in the absence of AT. Similar results were obtained when myelin-specific T cells were treated with AT in vitro; pre-incubation of naïve T cells prior to activation led to a dose-dependent development of Th2 cells, whereas continuous exposure at higher doses inhibited T-cell activation and differentiation. The anti-proliferative effect may thus be a dominant effect of AT. Furthermore, we found that despite their inability to generate IL-4 producing T cells, STAT6-deficient mice were fully protected from development of proinflammatory Th1 and Th17 cells and clinical EAE by AT treatment. Our results clearly demonstrate that Th2 polarization is not required for the in vivo benefit of statin treatment in CNS autoimmune disease. Finally, we provide strong evidence that the anti-proliferative effect of AT probably accounts for a good proportion of its effects in dampening CNS autoimmunity.
By inhibiting HMG-CoA reductase, statins interfere with the rate-limiting step in the mevalonate pathway that generates cholesterol and isoprenoid derivatives, including farnesyl-pyrophosphate and all-trans geranylgeranyl-pyrophosphate. When studying T-cell differentiation in vitro using farnesyl-pyrophosphate and all-trans geranylgeranyl-pyrophosphate, as well as selective antagonists, we previously demonstrated that farnesylation of the small GTP binding protein, Ras, is required for Th1 differentiation and that geranylgeranylation of RhoA is necessary for T-cell proliferation . As statins prevent production of both farnesyl-pyrophosphate and all-trans geranylgeranyl-pyrophosphate, they can inhibit both T-cell differentiation and proliferation, respectively. As we did not observe Th1, Th2 or Th17 differentiation in vivo during AT treatment, we questioned whether AT also exerted a potent anti-proliferative effect in vivo. Here, we observed that 1 mg/kg oral AT treatment, a dose approximately equivalent (weight/weight) to the 80 mg dose administered in recent clinical MS trials, nearly shut down T-cell proliferation in vivo in mice. These results, along with the therapeutic effect in STAT6-deficient mice and our inability to demonstrate Th2 polarization at the time of AT treatment, suggest that the cytostatic effect of statin treatment may provide the predominant mechanism responsible for observed benefits in MS clinical trials.
In our investigation, we observed that AT treatment inhibited T-cell proliferation upon re-stimulation in vitro, and did so in an antigen nonselective manner. Our data are consistent with the work of Aktas and colleagues , who demonstrated that AT mediated inhibition of T-cell proliferation in vitro, which was linked to downregulation of cyclin-dependent kinase 4. Interestingly, a predominant anti-proliferative effect rather than induction of Th2 cells could also explain the benefit of statin treatment in Th2-mediated and B-cell-mediated models of allergic asthma  and systemic lupus erythematosus , respectively.
While our data demonstrate that Th2 polarization is not required for the beneficial immunomodulatory effect of statin treatment, they do not exclude the contribution of statin-induced anti-inflammatory cytokines. In this regard, the clinical benefit mediated by AT treatment was associated with an increased secretion of IL-10. There are a number of immune cell populations that express IL-10, including B regulatory cells and anti-inflammatory macrophages. Future studies will address which immune cell(s) other than T cells may also be producers of IL-10 in AT-treated mice. Interestingly, our results are reminiscent of findings from one of the first monotherapy MS trials. In that MS trial, high-dose AT treatment led to a reduction in the number and volume of newly emerging CNS lesions, and was associated with an enhanced secretion of IL-10, but not IL-4 .
Data indicate that there is a reduction in Treg number and function in MS. Restoration of the Treg balance appears to be an important mechanism of action of GA in MS [29, 30, 37, 38]. Interestingly, there are only limited data suggesting that statins may promote expansion of Tregs [39–41]. We found that neither treatment initiated prior to disease induction nor following EAE onset altered the frequency of CD4+CD25+Foxp3+ Tregs in diseased mice. A potential increase in Tregs is therefore unlikely to significantly contribute to the anti-inflammatory effect of statins in CNS autoimmune disease.
How does this new information impact the potential use of statins in MS ? At first glance, nonspecific inhibition of proinflammatory T-cell differentiation in MS may be viewed less advantageously than the active induction of an anti-inflammatory T-cell phenotype [17, 18, 43]. Interestingly, a recent clinical trial in secondary progressive MS further suggests that simvastatin may slow progression and that this effect also occurs largely independent of peripheral immune modulation . Taken together, these trials demonstrate that statin treatment is beneficial at various stages in the disease. One strategy may be to use statins in combination with approved MS drugs. Several recent trials have investigated the combination of statins with IFNβ [13–16]. While results are not unequivocal , two of these studies suggested that addition of oral statin to IFNβ therapy may result in a paradoxical increase of MS activity [13–16]. In general, for two agents to exert synergistic benefit, distinct mechanisms of action are desirable, while employing the same mechanism may increase the risk of antagonism . Statins and GA have complementary mechanisms of action . Whereas GA promotes induction of both Th2 cells and Tregs [29, 30, 37], statins have been associated with a Th2 bias in treatment of CNS autoimmunity and, as we have shown in this study, a prominent anti-proliferative effect. In EAE, this combination had a synergistic beneficial effect . However, clinical trials will be necessary to determine whether such a combination will elicit a clinical beneficial effect in MS.
One may exercise caution when testing statins in combination with more recently approved MS therapeutic agents. In this regard, it is important to recognize that by inhibiting de novo pyrmidine synthesis, teriflunomide has a primary cytostatic effect on proliferating lymphocytes [48, 49]. While the clinical benefit of dimethyl fumarate has been attributed to its activation of the antioxidant transcription factor (erythroid-derived 2)-related factor (Nrf2) pathway [49, 50], it also exerts anti-proliferative activity . Given the strong in vivo anti-proliferative effects of AT, one might envisage testing a short course of high-dose oral statin as an adjunct to, or in lieu of, high-dose intravenous or oral steroids for MS exacerbations.