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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Discov Med. Author manuscript; available in PMC Jul 7, 2011.
Published in final edited form as:
Discov Med. Mar 2010; 9(46): 173–178.
PMCID: PMC3131182
NIHMSID: NIHMS308285

mTOR Signaling: A Central Pathway to Pathogenesis in Systemic Lupus Erythematosus?

Abstract

Systemic lupus erythematosus (SLE) is a common autoimmune disease with unclear etiology. Treatments for it often provide inadequate control of disease activity or are limited by side effects. Recent studies have shown that rapamycin can be an effective treatment in both murine lupus models and human SLE. We demonstrated that rapamycin could directly alter molecular abnormalities in SLE T cells related to calcium signaling but not mitochondrial function. However, in light of increased knowledge of the role of mammalian target of rapamycin (mTOR) signaling throughout the immune system, several other potential sites of rapamycin action have been revealed. Specifically, mTOR regulates the production of interferon-α and the maintenance of immune tolerance at the level of the regulatory T cell and the dendritic cell, and can promote Th2 versus Th1 immune responses. Thus mTOR offers a window into diverse facets of lupus pathogenesis as well as a unifying narrative in our understanding of the therapeutic efficacy of rapamycin in SLE.

Introduction

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by the production of autoantibodies against nuclear antigens, and a strong female predominance. It can present with a wide variety of symptoms, affecting virtually any part of the body, most commonly involving the skin, joints, kidneys, and blood vessels. The disease course is marked by disease flares interspersed with periods of relative quiescence (Cervera et al., 1993). The etiology of the disease remains unclear. The focus in our laboratory is the investigation of molecular and metabolic features of T cells in SLE, which are related to many manifestations of the disease.

Ongoing work in our laboratory has shown that a key regulator of metabolic activity, the mammalian target of rapamycin (mTOR), plays a very important role in disease pathogenesis, and the highly specific mTOR inhibitor rapamycin can be an effective and targeted treatment for the disease. mTOR is a key eukaryotic signaling protein conserved from yeast to humans, which regulates protein synthesis and energy expenditure. It is a point of integration of many inputs relaying information about nutritional status of the cell, including mitochondrial potential, oxygen tension, growth signals, amino acids, and ATP. In conditions of nutrient sufficiency, mTOR signaling is active, permitting protein synthesis and increased cell size. In conditions where any of these is lacking, mTOR activity decreases, limiting energy expenditure by inhibiting protein synthesis, decreasing cell size, and preventing cell proliferation. A full discussion of mTOR signaling is beyond the scope of this article, but has been explored extensively (Laplante and Sabatini, 2009). Recent studies of the mTOR pathway illustrate the diversity of effects it has in the immune system, giving greater insight into the potential relevance of this pathway in nearly all aspects of molecular dysfunction of SLE.

Rapamycin Treatment in SLE

We examined the applicability of rapamycin in the context of SLE, given its input on mitochondrial function and metabolic signaling. Building on reports of mTOR's efficacy in treating the MRL/lpr lupus model mice (Warner et al., 1994), we began to apply rapamycin off-label to SLE patients refractory to standard treatments. We found that patients responded well to rapamycin, leading to a decrease in disease activity and prednisone requirement (Fernandez et al., 2006).

Since it proved to be clinically effective, we investigated whether rapamycin treatment would alter any of the defined features of SLE T cells. Previous work in the laboratory established that SLE T cells have both increased baseline cytoplasmic calcium levels and increased influx of calcium following T cell receptor (TCR) stimulation relative to control T cells. We have demonstrated the presence of mitochondrial abnormalities in SLE T cells, including increased mass, ultra-structural damage, and elevated transmembrane potential (Gergely et al., 2002). We demonstrated that nitric oxide plays an important role in physiologic changes in mitochondrial potential and calcium signaling, as treatment of control peripheral blood lymphocytes with nitric oxide led to a recapitulation of the lupus T cell phenotype with regard to both calcium influx and mitochondrial potential. These features are very commonly present in SLE T cells, and are not associated with disease activity or affected by most treatments (Nagy et al., 2004).

We found that baseline calcium levels were decreased in T cells from rapamycin-treated SLE patients. Additionally, we found that calcium influx following TCR stimulation was decreased in the rapamycin-treated cohort of SLE T cells, and was not significantly different from healthy control T cells. Notably, rapamycin treatment did not affect mitochondrial potential, which remained elevated in both SLE groups (Fernandez et al., 2006). This indicates that if interaction between mitochondrial dysfunction and calcium signaling is present as our results with nitric oxide suggest, mitochondrial dysfunction is upstream of the calcium phenotype and mTOR has an intermediary role.

There is significant evidence that these effects of rapamycin are related to alteration of signaling through the TCR, which may affect the inappropriate activation of autoreactive T cells in SLE. The constellation of signaling proteins present at the TCR complex is different in SLE versus healthy control T cells, and it has been shown that the alterations in calcium influx can be related to changes in accessory proteins at the TCR. There is a decrease in CD3ζ, Lck, and CD4, with replacement by FcεRIγ, which recruits Syk instead of ZAP-70 for further downstream signaling (Enyedy et al., 2001). Forcing expression of CD3ζ in T cells from SLE patients normalizes calcium fluxing and IL-2 production, indicating that these processes are downstream from altered signaling at the T cell receptor (Nambiar et al., 2003).

We demonstrated that mTOR activity is increased in human SLE. mTOR regulates the association of these atypical proteins with the TCR, and rapamycin-treated patients exhibited increased levels of CD3ζ and Lck, reflective of the normal signaling complex. We found further that mTOR affects the levels of Rab family members, and that alteration of receptor recycling through these proteins, in particular HRES-1/Rab4, could mediate the changes in TCR signaling seen in SLE (Fernandez et al., 2009).

While these results are very promising in terms of offering new insights into disease pathogenesis, recent reports indicate that the effects of rapamycin in SLE may be more widespread. The mTOR pathway is involved in many aspects of T cell differentiation and function. mTOR has very significant effects on the cells of the innate immune system, particularly on monocytes and dendritic cells, and influences the cytokine milieu present during immune responses.

mTOR and Immune Regulation

Rapamycin treatment, and by extension mTOR signaling, has numerous effects on T cell function and development. Of particular relevance in the context of SLE is the effect of mTOR signaling on the presence of anti-inflammatory regulatory T cells. The preponderance of studies in SLE shows a decrease in either the number of regulatory T cells or their ability to suppress the proliferation of other T cells, or both (Miyara et al., 2005; Valencia et al., 2007). Whether this represents altered generation of regulatory T cells or a failure to maintain them is unclear.

The maintenance of regulatory T cells in SLE may be an issue. IL-2 is essential for the maintenance and function of regulatory T cells, and underproduction of IL-2 is a very consistent feature of T cells from SLE patients (Alcocer-Varela and Alarcon-Segovia, 1982). There is some evidence that low levels of IL-2 contribute to a pro-inflammatory environment in an SLE mouse model due to the corollary effects on regulatory T cell number and function (Humrich et al., 2009). It is possible that rapamycin treatment resulted in recovery of IL-2 production along with normalization of calcium signaling, creating a cytokine milieu more favorable to regulatory T cell function.

Rapamycin may be acting to increase the numbers of regulatory T cells as well. It has been demonstrated that stimulating T cells through the TCR in the presence of rapamycin in vitro promotes the generation of anti-inflammatory regulatory T cells (Battaglia et al., 2005). Whether this would occur in rapamycin-treated SLE patients is not known, but is an area of ongoing investigation in our laboratory. Apparently, there is a role for the related kinase Akt, as inhibition of Akt or mTOR in the context of early cessation of TCR signaling can stimulate differentiation into regulatory T cells (Sauer et al., 2008), while expression of constitutively active Akt in T cells severely limits their potential to differentiate into Foxp3+ regulatory T cells (Haxhinasto et al., 2008).

Genetic studies involving deletion of mTOR have been able to give some mechanistic insight into how rapamycin achieves its effects in SLE patients. Deletion of mTOR at the double-positive thymocyte stage in mice did not affect the generation of regulatory T cells from the thymus, and T cells had normal IL-2 production upon initial stimulation of the TCR despite relatively poor proliferative capacity. However, differentiation into Th1, Th2, or Th17 subsets in polarizing medium was impaired, with corresponding decrements in STAT4/T-bet, STAT6/GATA-3, and STAT3/RORγt signaling, respectively. In contrast, Treg expansion was markedly enhanced, and could be increased further with concomitant exposure to TGF-β (Delgoffe et al., 2009).

Rapamycin has been demonstrated to promote an anti-inflammatory environment by affecting the behavior of dendritic cells. Rapamycin treatment of immature myeloid dendritic cells induces a “tolerogenic” phenotype, with poor ability to stimulate conventional T cells but significant ability to stimulate the differentiation of regulatory T cells. When used postoperatively in mice, rapamycin treatment was associated with preservation of a heart transplant for an extended period without vascular evidence of rejection (Turnquist et al., 2007). A similar result was seen in a model of graft-versus-host disease (GvHD); rapamycin-treated dendritic cells downregulated costimulatory molecules, promoted regulatory T cell proliferation, and led to reduced GvHD severity (Reichardt et al., 2008). Thus mTOR inhibition leads to complementary changes in myeloid dendritic cells and T cells leading to the production of regulatory T cells.

mTOR and Th1/Th2 Differentiation Bias

Interestingly, rapamycin treatment does not simply generate a tolerogenic environment, but actively promotes certain immune responses. While T cells treated with rapamycin during TCR stimulation did have increased propensity to differentiate into regulatory T cells, it was shown that both regulatory T cells and CD4+CD25-cells increased STAT3 and STAT5 phosphorylation (Strauss et al., 2009). In keeping with these findings, rapamycin treatment enhanced differentiation of CD8+ T cells into memory cells in mice inoculated with lymphocytic choriomeningitis virus (Araki et al., 2009). While these results seem to contradict some of the findings of the knockout mouse studies, they may simply reveal that pharmacologic inhibition of the pathway has different features than genetic ablation of the mTOR kinase.

Studies of the innate immune system provide more support for a Th1-promoting effect of rapamycin treatment. Rapamycin has a profound effect on monocytes, resulting in decreased STAT3 activity, decreased IL-10 secretion, and increased IL-12 and TNF-α activities in response to lipopolysaccharide or Staphylococcus aureus cells, driving immune responses towards a Th1 or Th17 pathway. Importantly, T cells cocultured with these monocytes were more likely to produce high levels of IL-17 and IFN-γ. This shift in cytokines was also associated with decreased lethality of the Th1-targeted pathogen Listeria monocytogenes in susceptible Balb/c mice treated with rapamycin (Weichhart et al., 2008).

mTOR and Interferon-α

Another key feature of the innate immune system in SLE is the production of interferon-α (IFN-α). It has been known for decades that interferon-α levels were increased in SLE patients during disease flares, but only recently has an “interferon signature” of gene expression been elucidated in SLE, as well as description of specific polymorphisms in proteins like IRF-5 and STAT4 that affect interferon signaling in patients (Kariuki et al., 2009; Niewold et al., 2008; Sigurdsson et al., 2008). This substantiates an important and potentially primary role for interferon in the pathogenesis of SLE.

There is evidence that inhibition of mTOR in plasmacytoid dendritic cells limits their production of type I interferons in response to TLR-9 specific stimuli, which is another means by which T cell responses might be modulated. mTOR mediates this effect by altering the composition of signaling proteins on endosomes where TLR-9 is present, preventing the association of the key signaling protein IRF-7 (Cao et al., 2008; Colina et al., 2008).

We have not yet investigated whether IFN-α levels are affected in SLE, but this represents another point where rapamycin may improve an important mediator of the disease. Additionally, the mechanism of action of mTOR in this setting seems to corroborate our finding that mTOR affects the composition of signaling complexes, specifically at the level of the endosome.

Conclusion

At this point, we are able to generate a limited model of mTOR signaling as it relates to SLE pathogenesis (Figure 1). mTOR signaling is increased in SLE T cells, and inhibition of mTOR signaling with rapamycin has been shown to be effective in the treatment of human SLE. SLE patients treated with rapamycin demonstrate lowered baseline calcium levels and decreased calcium influx following TCR stimulation, but do not show a change in mitochondrial function, indicating the specificity of rapamycin treatment on this manifestation of the disease.

Figure 1
mTOR impacts diverse processes that may be involved in the promotion of autoimmunity in SLE. Rapamycin treatment can directly limit many of these processes in isolation, and some or all of those effects in vivo may explain the efficacy of the drug in ...

Several other aspects of mTOR signaling with clear implications for SLE exist, although direct investigation of their impact on SLE has not occurred. Rapamycin promotes regulatory T cell and tolerogenic dendritic cell expansion, which might limit the proliferation and activity of autoreactive T cells. It limits proinflammatory IFN-α production by plasmacytoid dendritic cells. In some studies it has been shown to promote a Th1-focused immune response, which may limit the T cell stimulation of autoreactive B cells in SLE.

Acknowledgements

This work was supported in part by grants AI 048079, AI 061066, AI 072648, AT004332, and AR056957 from the National Institutes of Health, the Alliance for Lupus Research, and the Central New York Community Foundation.

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