METABOLIC PATHWAYS MEDIATE PATHOGENESIS AND OFFER TARGETS FOR TREATMENT IN RHEUMATIC DISEASES

Abstract

Purpose of Review

The etiology of autoimmune diseases remains incompletely understood. Here, we highlight recent advances in the role of pro-inflammatory metabolic pathways in autoimmune disease, including treatment with antioxidants and mTOR inhibitors.

Recent Findings

Recent studies show that mTOR pathway activation, glucose utilization, mitochondrial oxidative phosphorylation, and antioxidant defenses play critical roles in the pathogenesis of autoimmune diseases, including rheumatoid arthritis, immune thrombocytopenia, Sjögren’s syndrome, large vessel vasculitis, and systemic lupus erythematosus. mTOR activity leads to Th1 and Th17 cell proliferation, Treg depletion, plasma cell differentiation, macrophage dysfunction, and increased antibody and immune complex production, ultimately resulting in tissue inflammation. mTOR also affects the function of connective tissue cells, including fibroblast-like synoviocytes, endothelial cells, and podocytes. mTOR inhibition via rapamycin and N-acetylcysteine, and blockade of glucose utilization show clinical efficacy in both mouse models and clinical trials, such as systemic lupus erythematosus.

Summary:

The mTOR pathway is a central regulator of growth and survival signals, integrating environmental cues to control cell proliferation and differentiation. Activation of mTOR underlies inflammatory lineage specification, and mTOR blockade-based therapies show promising efficacy in several autoimmune diseases.

Introduction

The ability to control the immune system promises the secret of health and longevity. As a complex web of cytokine signaling, inter-cell communication, and ubiquitous existence in almost every tissue of the body, the immune system may just be the critical mediator of host physiology and pathology. Increasingly, the role of the mechanistic Target of Rapamycin (mTOR) pathway has been identified as a critical mediator of immune function.

Discovered after an expedition to the island of Rapa Nui (Easter Island), rapamycin (known clinically as sirolimus) was isolated from soil Streptomyces hygroscopicus. Rapamycin was noted for its many properties, including antifungal and immunosuppressive effects(1, 2). These effects were found to be due to rapamycin forming a complex with FKBP12 and exerting its function on the aptly named mechanistic Target of Rapamycin (mTOR)(3–5). mTOR is a serine/threonine kinase that acts as a central regulator of growth and metabolic signals, integrating environmental inputs and controlling cellular machinery(6).

mTOR exists within two separate complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2) (Figure 1A). mTORC1 functions primarily to stimulate cell growth downstream of growth factors, nutrients, and energy. mTORC1 accomplishes growth promotion by upregulating mRNA transcription, protein synthesis, and metabolic changes that include lipid and nucleotide synthesis(6). mTORC2 signaling is less characterized but promotes cell survival and proliferation downstream of certain growth factors. mTORC2-regulated pathways include apoptosis, glucose metabolism, cell migration, cytoskeleton arrangement, and ion transport to ensure survival of the cell (Figure 1A)(6).

Figure 1.

mTOR activity contributes to autoimmune disease. A) mTORC1 and mTORC2 are distinct complexes that integrate environmental cues to shape cellular phenotype. mTORC1 senses a variety of signals including amino acid sufficiency, glucose sufficiency, cytokines, growth hormones, and cellular energy, and controls mRNA transcription, protein, nucleotide, and lipid synthesis, and metabolic changes within the cell. mTORC2 signaling is less characterized, but activation occurs downstream of the PI3K/AKT pathway. mTORC2 has roles primarily in cytoskeletal reorganization, although it also has roles in apoptosis, ion transport, and glucose metabolism, among others. B) The mTOR pathway is implicated in a variety of autoimmune diseases via controlling cellular development. mTOR activity skews immune cells towards a pro-inflammatory phenotype, as well promoting proliferation in other cell lineages, ultimately promoting disease pathogenesis. TKR = Tyrosine kinase receptor. ROS = Reactive oxidative species. RAPA = Rapamycin. PPP = Pentose phosphate pathway. DC = Dendritic cell. M0 = Macrophage. FLS = Fibroblast-like synoviocyte. MKC = Megakaryocyte. EC = Endothelial cell. SLE = Systemic lupus erythematosus. APS = Antiphospholipid syndrome. PsA = Psoriatic arthritis. AS = Ankylosing spondylitis. RA = Rheumatoid arthritis. ITP = Immune thrombocytopenia.

The immunosuppressive effects of rapamycin led to its approval by the FDA in 1999 for anti-rejection therapy after kidney transplantation. Similarly, rapamycin showed promising results in alleviating inflammatory autoimmune diseases, including mouse models of lupus and multiple sclerosis(7–9). Due to its successful application in such therapies, mTOR signaling in the immune system has become of great interest. mTOR regulates many facets of immune cell development, including control over whether T cells become regulatory- or effector-type cells(10–13). In particular, dysregulation of mTOR, along with altered immune cell characteristics, prominently appears in the initiation and progression of autoimmune diseases (Figure 1B and Table 1) (14). This dysregulation has been identified as a central biomarker of disease and target of treatment, and in-depth understanding of the mTOR pathway may unlock powerful treatment options in a variety of diseases(14). Here, current advances in the understanding of the mTOR pathway in select diseases, as well as therapeutic targeting of mTOR, will be reviewed.

Table 1.

Pharmacological blockade of the mTOR pathway in autoimmune diseases. mTOR blockade has shown to be beneficial in autoimmune diseases by inhibiting mTOR activity, thereby downregulating inflammation and stabilizing disease activity.

Disease	Organ	Target cell/Antigen	Effector	mTOR blockade
SLE	Systemic	Podocyte/Kidney Neuron/Brain Hepatocyte/Liver Endothelium/Vessel Epithelium/Lung/Pleura/Pericardium	DN/Th17 T cells Treg/EMT contraction B cells Immune complexes	Rapa(52, 57, 65) NAC(66), Metformin(67)
APS	Systemic	Vascular endothelium	T cells B cell Macrophages DCs Hepatocytes	Rapa(62, 63, 65)
GN	Kidney	Podocytes	T cells B cells Macrophages DCs	Rapa(60)
RA	Joint Synovium	Chondrocytes	FLS T cells B cells Macrophages DCs	Everolimus(22), NAC(68, 69)
OA	Joint	Chondrocytes	Chondrocytes	Rapa(70, 71)
PsA	Skin, Joint	Keratinocytes, Chondrocytes	T cells B cells Macrophages DCs	Rapa(72)
SSc	Systemic	Fibroblasts	T cells B cells Macrophages DCs	Rapa(73, 74)
PHTN	Lung	Vascular endothelium, Smooth muscle cells	T cells B cells Macrophages DCs	Everolimus(75)
Vasculitis	Blood Vessels	Endothelial cells	Endothelial cells T cells Macrophages	Rapa(47, 48)
SjS	Exocrine Glands	Glandular epithelium	T cells B cells DCs	Rapa(44), NAC(76)
ITP	Blood vessels	Megakaryocytes/Platelets	T cells B cells Macrophages DCs	Rapa(33, 34)
AIHA	Bone Marrow	RBCs	T cells B cells	Rapa(77)

SLE = Systemic lupus erythematosus. APS = Antiphospholipid syndrome. GN = Glomerulonephritis. RA = Rheumatoid arthritis. OA = Osteoarthritis. PsA = Psoriatic arthritis. SSc = Systemic sclerosis. PHTN = Pulmonary hypertension. SjS = Sjögren’s syndrome. ITP = Immune thrombocytopenia. AIHA = Autoimmune hemolytic anemia. RBC = Red blood cells. EMT = Effector memory T cell. DCs = Dendritic cells. FLS = Fibroblast-like synoviocytes. Rapa = Rapamycin/Sirolimus. NAC = N-acetylcysteine.

mTOR controls immune cell development

mTOR has a profound role in T cell lineage development. Part of the adaptive immune system, T cells are critical to raising host defenses and maintaining immunocompetence. T cells participate in both upregulating and downregulating the immune response by differentiating into effector T cells and regulatory T cells (Tregs), respectively. T effector cells can activate other members of the immune system, while Tregs inhibit cytokine production and cell proliferation. mTOR activation is critical for the development of effector T cells, as mTOR activity leads to STAT activation and transcription of genes, such as STAT3-mediated induction of IL-17 and IFN-γ(15). T cell activation and differentiation into effector subtypes require promotion of glycolysis and flux through the PPP, both of which are regulated by mTOR(16*). Specifically, proinflammatory Th1 and Th17 cell differentiation require activation of mTORC1, while Th2 cell differentiation requires mTORC2(15). mTORC1 induction of Th17 differentiation is mediated by mTORC1 activation of S6K1 and a related kinase, S6K2(17). S6K2 activation targets nuclear translocation of RORγ, a transcription factor required for Th17 differentiation, while S6K1 inhibits Gfi1, a negative regulator of Th17 differentiation(17).

Rapamycin blocks the differentiation of Th17 cells, underscoring the necessity of mTORC1 signaling in the development of proinflammatory cell lineages(17). Likewise, inhibition or depletion of mTORC1 and mTORC2 together lead to an expansion of regulatory T cells (Tregs)(15). This dual expansion of Tregs and contraction of T effector cells highlight an important mechanism behind mTOR’s role in promoting the inflammatory process, as metabolic shifts mediated by mTOR activity direct the lineage development of T cells(16*).

Aside from T cells, mTOR also controls the fate of other immune cells. B cell proliferation and survival occurs via the mTOR pathway, as rapamycin has shown the ability to attenuate B cell activating factor (BAFF) stimulation(18, 19*). In macrophages, rapamycin prevents the development of M2 macrophages (canonically anti-inflammatory), without having such an effect on M1 macrophages. mTOR activity appears crucial for M2 macrophage development(20). mTOR is critical in the development of multiple immune cell lineages. In turn, aberrations in mTOR signaling can change the immune profile of the body, leading to pathological autoimmunity.

Rheumatoid Arthritis

Joint destruction in rheumatoid arthritis (RA) is seen as the end result of synovitis mediated by cytokine and autoantibody production, as well as cartilage damage mediated by intra-articular cells(21). Previous research has shown efficacy of mTOR blockade in reducing joint inflammation in RA(22). This effect is seen despite any significant changes in T cell mTOR activity within RA patients(23). However, RA does exhibit increased mTORC1 activity in fibroblast-like synoviocytes (FLS) and osteoclasts, with inhibition of mTOR showing promise in targeting these intra-articular cells(24). Recent research has highlighted the role of mTOR in TNF-mediated pro-inflammatory functions(25*).

In RA-FLSs, TNF is shown to activate mTOR via AKT. In turn, mTOR antagonizes nF-κB (via IκB-α), while also promoting transcription of interferon-regulated genes through STAT1 activation(25*). mTOR’s inhibition of nF-κB may point to why use of rapalogues increases inflammatory markers in RA(22). However, overall this novel pathway shows mTOR’s environmental sensing ability can be coupled to pro-inflammatory cytokine and chemokine production, and therapeutic targeting of mTOR in this pathway may be an efficacious strategy in RA treatment.

The JAK/STAT pathway may also be a target for RA treatment. Signal transducer and activator of transcription 3 (STAT3) has been identified in mouse models as a critical mediator of rheumatoid arthritis and potential therapeutic target(26, 27). Since mTOR and STAT3 have complex crosstalk between the two(15, 28), targeted inhibition of the JAK/STAT pathway as well as the mTOR pathway are potentially valid approaches to therapy.

mTOR and thrombocytopenia

Immune thrombocytopenia (ITP) is a common autoimmune-mediated platelet destruction syndrome thought to be due to abnormal T cell function(29). ITP is marked by an increase in proinflammatory Th17 cells and a decrease in Tregs, similar to other autoimmune diseases, covered here, suspected to be under regulation of mTOR(30). However, how mTOR regulates T cell function specifically in the context of ITP remains unknown. One study has shown that indirubin, a drug with potentially therapeutic effect in ITP murine models, ameliorates disease via increased expression of PD1 and PTEN, leading to the suppression of mTOR in CD4+ T cells(31),(32). This work highlights a potential role for mTOR targeting. Indeed, inhibition of mTOR has been used clinically to positive effects for ITP. Rapamycin, when used in combination with the corticosteroid prednisone, appears to have promising potential in ITP therapy(33). Combination therapy exhibited low toxicity and was shown to lead to a sustained increase in platelets along with an increase in Treg levels(33). More recent reporting showed that sirolimus alone, instead of in combination with prednisone, can also be effective for ITP(34). Sirolimus alone achieved complete response in many cases, making it an attractive option for sparing steroid use and limiting toxicity of ITP therapy(34).

Interestingly, aside from T cell abnormalities, recent work highlights the role of the mTOR pathway and autophagy within megakaryocytes in ITP(35*). Not only does ITP involve autoimmune destruction of platelets, but also dysfunctional platelet production by megakaryocytes(36). Proliferation of megakaryocytes and proper platelet production is thought to be under the control of autophagy pathways(37, 38). However, while these catabolic signals are crucial for maintaining megakaryopoiesis and highlight the potential benefit of autophagy-stimulating rapamycin treatment, it appears, paradoxically, that mTOR signaling is also required for platelet production and that induction of autophagy may actually be a pathogenic factor of ITP(39–41). While rapamycin appears to be beneficial in ameliorating the immune response and clinical disease, its effect on megakaryocyte function, helpful or harmful, requires further research. Overall, targeting autophagy with rapamycin in ITP may be a promising therapeutic pathway to reduce inflammatory destruction of platelets(42).

Sjögren’s Syndrome

Sjögren’s syndrome (SjS) is defined by autoimmune destruction of exocrine glands, as well inflammatory damage to multiple organ systems(43). Previous research has shown that mTOR is implicated in the pathogenesis of SjS, as rapamycin was an effective therapy in a mouse model (44). This effect was due to inhibition of mTOR-mediated T cell activation(44). mTOR may also play a role in human SjS, raising the potential of mTOR-based therapy in this disease(45*). Using peripheral blood mononuclear cells (PBMC) and salivary gland tissue. Interestingly, circulating B cells appear to have decreased expression of mTOR, while B cells within the salivary glands show increased mTOR expression. Plasma cells and T cells within the salivary gland also showed mTOR activation(45*).

Vasculitis

Takayasu arteritis (TA) and giant cell arteritis (GCA) are two large vessel vasculitides whose etiology remains elusive(46). However, the mTOR pathway increasingly appears to play a significant part in the pathogenesis of disease(47*, 48*). Within endothelial cells of TA patients, both mTORC1 and mTORC2 are shown to be activated(47*, 48*). mTORC1 activity in GCA is contrasted by recent work, as it may not be activated in GCA endothelial cells(47*), or possibly have an intermediate activity between that of TA patients and healthy controls(48*).

Further, mTORC1 potentially drives T cell inflammation in large vessel vasculitis, as rapamycin treatment increases Treg and decreases effector T cell populations from cultured patient PBMCs(48*). Interestingly, patient sera and purified IgG is shown to activate mTORC1 and induce endothelial cell proliferation, potentially by targeting a 60–65 kDa antigen on endothelial cells(47*, 48*). Identification of this antigen, as well as further research into the mTOR pathway in vasculitides, is a promising avenue of therapy.

Systemic Lupus Erythematosus

Systemic Lupus Erythematosus (SLE) is a chronic inflammatory disease with potentially life-threatening consequences(49). Multi-organ involvement, variable clinical course, and incompletely understood pathogenic mechanisms create a challenge for pharmacological management(50). However, abnormal activation of T cells appears central to SLE pathogenesis, and the role of mTOR in mediating these effects is critical. mTOR activation follows T cell receptor (TCR) signal transduction and controls increased calcium fluxing in SLE, while rapamycin normalizes this signaling defect(51–53*),. mTORC1 senses a variety of stress signals including the accumulation of amino acids during autophagy, such as leucine and isoleucine, kynurenine, and glutamine(24). Glutaminolysis may enhance mitochondrial oxidative stress and mTORC1 activation in Th17 cells (54*) (2). Deficiency of mRNA splicing also promotes activation of mTORC1 (55*).Further, mTOR activation in SLE T cells leads to the expansion of IL-4 producing double-negative T cells, expansion of Th17 cells, and contraction of Tregs, which contribute to the proinflammatory profile of SLE(56), (57). Other immune cells also contribute to SLE pathogenesis. B cells are antibody-secreting cells responsible for autoantibody production in SLE. Evidence indicates that autoimmune B cells are dependent on mTOR signaling (58). SLC15A4, a lysosomal histidine transporter, is necessary for B cell production of autoantibodies and type I interferon downstream of Toll-Like Receptor 7 (a sensor of single-stranded RNA) stimulation(58). This pathway is mediated by mTORC1, as loss of SLC15A4, as well as direct inhibition of mTOR, lead to disruption of mTORC1 signaling and ablation of autoantibody production(58).

Paradoxically, mTOR appears to mediate an anti-inflammatory M2 phenotype of macrophages, rather than proinflammatory M1 phenotype and rapamycin treatment actually promotes a shift towards M1 macrophages(20). Interestingly, M2 macrophages are a dominant subpopulation in lupus nephritis biopsies, but whether these macrophages are there to rescue the disease or contribute to pathogenesis is unclear(59). The possibility remains that the ‘canonical’ roles of M1/M2 macrophages are reversed in lupus nephritis.

mTOR prominently features in specific organ involvement of SLE. Kidney damage is a potentially fatal consequence of SLE (49). In a murine model of complex-mediated glomerulonephritis, rapamycin treatment is beneficial in treating disease and prolonging survival(60). Leukocyte infiltration, including B cells, T cells, and macrophages, is a hallmark of glomerulonephritis, and this infiltration appears to be under the control of mTOR and treatable by rapamycin(60). Glomerular architecture is also important in lupus nephritis. Podocytes maintain the slit-diaphragm filtration system of the kidney and are known to be injured in lupus nephritis(61*). In lupus nephritis, these podocytes exhibit increased autophagy(61*). Inhibiting this autophagic process further aggravated podocyte damage, while induction of autophagy ameliorated disease, raising the possibility that podocytes upregulate autophagy in a protective attempt(61*).

Antiphospholipid antibodies (aPL) are a diagnostic criterion of SLE, leading to antiphospholipid syndrome (APS), which can occur in patients with and without SLE. mTORC1 and mTORC2 are activated in APS endothelial cells, associated with intimal hyperplasia, and APS IgG stimulates endothelial cell mTOR activity in vitro(62). Further, sirolimus is clinically beneficial for APS kidney transplant recipients. These patients exhibit reduced renal lesions, stable GFR, and improved graft survival, compared to patients that did not receive sirolimus(62). Further research in mouse models of APS show rapamycin treatment is capable of blocking antiphospholipid antibody production(63).

Targeting mTOR in SLE therapy is promising. In several SLE murine models, inhibition of mTOR decreases production of autoantibodies, decreases proteinuria (a marker of kidney damage), decreases inflammation in multiple organ systems, and even expands lifespan(8, 60, 64),. In human patient populations, a recent phase 1/2 trial studied sirolimus in SLE patients resistant/ intolerant of conventional treatment(65*). After 12 months of treatment, more than half of patients responded to sirolimus therapy, showing decreased disease activity, decreased prednisone usage, sustained antiphospholipid antibody reduction, and restored altered Treg/Th17 ratios(65*). Notably, immunophenotype changes were predictive of responders of non-responders, identifying immunophenotyping as an important biomarker for precision medicine, although the mechanisms underlying differential response require further study.

Concluding Remarks

mTOR signaling, while vastly complex, works to incorporate catabolic and anabolic signals to determine cell fate. By directly and indirectly sensing amino acids, growth hormones, energy, and other factors, mTOR regulates downstream signals of growth, proliferation, and survival. While some deride mTOR for doing everything, it is important to appreciate the role it has in the development and mediation of autoimmunity. mTOR activation and proinflammatory changes lie at the heart of many autoimmune disorders. mTOR is critical to the skewing of Treg/Th17 populations, pathogenesis of other immune cells, and controlling autophagic pathways in multiple cell types in various organs. Targeting this pathway, through both conventional and novel mTOR inhibitors, unlocks a promising therapy in the diseases covered here, among others.

However, some questions remain outstanding for a more complete picture of this therapeutic goal. Primarily, identification of patients that will benefit from treatment and become successful responders is an important goal in the era of precision medicine. While immunophenotyping is predictive of those patients that respond to therapy after initiation, the ability to predict those who will respond to treatment beforehand remains elusive(65*). Similarly, predicting patients that will experience adverse events from conventional mTOR inhibitors, such as sirolimus, is critical for tailoring treatment to populations that will tolerate it. Serious side effects of mTOR inhibition include hyperlipidemia, pneumonia, and sepsis(14). Identification of patients at risk of developing these side effects would be extremely beneficial to therapy goals. In the meantime, combination or alternative therapies that target the mTOR pathway may increase the beneficial effects of sirolimus and reduce side effects. One example of such treatment is N-acetyl cysteine (NAC), a metabolite that shows promising potential in the treatment of SLE via altering the mTOR pathway(66). NAC reduces reactive oxidative species upstream of mTOR activation and may attenuate oxidative damage(66). Combination therapy of NAC and sirolimus could have synergistic effects in therapy, allowing a lower dose and better control of serious side effects. Future work should be directed at successful mitigation of mTOR’s proinflammatory effects, while simultaneously mitigating the off-target effects of mTOR therapy. In pursuit of this, a more complete understanding of the molecular mechanisms behind mTOR signaling can help in the development of new therapeutic targets.

Key Points.

• The mTOR pathway integrates environmental cues to shape and alter cellular phenotypes.

• mTOR activity, particularly in T cells, potentially drives inflammation in a variety of autoimmune diseases.

• Targeted therapy of the mTOR pathway with rapamycin, rapalogues, and novel inhibitors show promising efficacy in the treatment of autoimmune disease.