Metabolic control of lupus pathogenesis: central role for activation of the mechanistic target of rapamycin

Introduction

The pathogenesis of systemic lupus erythematosus (SLE) is attributed to immune cell malfunction that results in the production of antinuclear autoantibodies (ANA). Current therapies mainly rely on cytotoxic agents. B-cell blockades have limited clinical efficacy. ^, Selective depletion of T cells (e.g., CD4 ⁺ cells) blocks lupus, however, such therapy creates a state of severe immunodeficiency similar to that caused by HIV. Although other cell types, such as macrophages, dendritic cells, and neutrophils may not be essential to disease pathogenesis, their production of proinflammatory metabolites, such as nitric oxide (NO) and cytokines, such as type I interferon (IFN), and deficient phagocytosis or killing of infectious organisms also contribute to pathogenesis.

Oxidative stress, that is, the production of reactive oxygen intermediates (ROI), has been long considered only as toxic by-products of aerobic existence. However, ROI are increasingly viewed as essential modulators of various signal-transduction pathways, including epigenetic regulation of gene transcription, mRNA translation, protein folding as well as degradation and recycling of organelles via autophagy. In accordance with these diverse functions, oxidative stress seems to mediate T-cell dysfunction in SLE at multiple levels. Such T-cell defects result in aberrant immune responses and, in concert with oxidative autoantigenesis, elicit the inflammatory pathology, and comorbidities of SLE.

In turn, redox signaling is tightly controlled by intracellular antioxidant systems that mainly rely on the availability of reduced glutathione (GSH). Oxidative stress in SLE was initially revealed by the increased mitochondrial ROI production which is caused by the elevation of mitochondrial transmembrane potential (=ΔΨ) or mitochondrial hyperpolarization (MHP) in T lymphocytes. ROI production is mainly increased by complex I of the mitochondrial electron transport chain (ETC) in lupus T cells. Increased mitochondrial ROI production has been extended to neurophils and implicated in netosis in SLE.

Although increased blood levels of lipid hydroperoxides, malonaldehyde (MDA), hydroxynonenal (HNE), and other reactive aldehydes, provide evidence for an overall increase of oxidative stress in patients with SLE. Paradoxically, a loss of NADPH oxidase (NOX) activity, due to deficiency of NOX2 isozyme, is common genetic cause of chronic granulomatous disease (CGD) that can predispose to lupus in such patients as well as MRL/lpr mice. Given that antioxidants, such as NAC show overall benefit both in patients and mice, and other antioxidant treatments also improved lupus disease activity, at least in mice, a better understanding of redox signaling is relevant for overall disease pathogenesis, mechanisms of flares, and identification of biomarkers and targets for treatment. Therefore, this chapter critically evaluates the causes and consequences of oxidative stress in SLE.

Accumulation of dysfunctional mitochondria is the source of oxidative stress in T cells

In most nucleated cells, mitochondria are the main source of ROI. The transfer of electrons to molecular oxygen (O ₂ ) during ETC activity generates ROI, primarily superoxide anion, O ₂ ⁻ . This is a by-product of ETC activity which generates energy, stored as ΔΨm. In turn, this electrical energy is transformed into chemical energy in the form of ATP by the terminal ETC complex, complex V or F _O F ₁ ATPase. ETC activity is increased lupus T cells, primarily at complex I, which has been proposed as a potential source of ROI production in mammalian mitochondria. ETC complex I activity is elevated in “untouched” T cells of SLE patients over 2-fold relative to healthy and non-lupus psoriatic and rheumatoid arthritis (RA) disease controls studied in parallel. O ₂ consumption and ETC activity are also increased relative to greater mitochondrial mass and MHP in lupus T cells. In a state of mitochondrial hyperpolarization, H ⁺ ions are extruded from the mitochondrial matrix and cytochromes within the ETC become more reduced, promoting the transfer of electrons onto O ₂ , and thus generating more O ₂ ⁻ and oxidative stress . Among the ROI, the most toxic moiety is the hydroxyl radical (OH ⁻ ), which cannot be eliminated without causing oxidative damage. OH ⁻ is generated from O ₂ ⁻ and H ₂ O ₂ in the presence of metal ion through the Fenton reaction or UV light. The generation of OH ⁻ is primarily controlled by prevention via fully functional, endogenous antioxidant mechanisms, as described below, that is, converting O ₂ ⁻ into relatively stable, non-radical hydrogen peroxide (H ₂ O ₂ ) by superoxide dismutases and then into water by catalase. Besides ROI, redox signaling involves reactive nitrogen intermediates (RNI), such as NO and peroxynitrite (ONOO ⁻ ), the latter of which is generated by the reaction of NO with O ₂ ⁻ .

The accumulation of mitochondria contributes to oxidative stress in lupus T cells. This is caused in part by NO-induced MHP and mitochondrial biogenesis. As more recently uncovered, NO also induces the expression of the lupus susceptibility gene, HRES-1/Rab4 which, in turn, inhibits mitophagy. Thus, the accumulation of mitochondria is favored by both increased biogenesis and diminished turnover in lupus T cells. These mechanisms of altered mitochondrial homeostasis are detectable in patients with SLE as well as in lupus-prone NZB/WF1, Sle1.Sle2.Sle3 triple congenic, MRL, and MRL/lpr mice. In accordance with operation of metabolic genes outside the immune system, accumulation of mitochondria and oxidative stress also occur in the largest metabolic organ, the liver, of lupus-prone mice.

Genetic data also support mitochondrial dysfunction in SLE. Sle1c2 (a sub-locus of the major lupus susceptibility locus Sle1 ) has been identified as estrogen-related receptor γ (ESRRG). Sle1c2 congenic mice exhibit increased mitochondrial mass, as evidenced by elevated voltage-dependent anion channel (VDAC) protein levels, CD4 T cell hyperreactivity and cGVHD susceptibility. These findings are consistent with a role of ESRRG as a transcription factor involved in NO-dependent mitochondrial biogenesis. Genetic polymorphisms in human mitochondrial DNA have been also associated with SLE. The nt4917 and nt9055 mtDNA polymorphisms cause amino acid substitutions—(1) D→N in the ND2 subunit of ETC complex I and (2) A→T in the ATP6 subunit of complex V. The functional consequences of these amino acid changes are yet to be determined. Moreover, inactive alleles of mitochondrial uncoupling protein 2 (UCP2), a protein that reduces oxidative stress, are associated with susceptibility to several autoimmune diseases, including SLE, multiple sclerosis, rheumatoid arthritis, granulomatosis with polyangiitis, Crohn's disease, and ulcerative colitis.

Extramitochondrial generation of oxidative stress

In addition to mitochondria, phagocytic cells and endothelial cells generate ROI by NADPH oxidase (NOX) enzymes, mainly NOX2. Macrophages and granulocytes express NOX2, which produces ROI for the killing of ingested bacteria. Oxidative stress contributes to the destruction of organisms in phagocytic cells—ROI, generated by the respiratory burst with the involvement of NOX2, help in elimination of bacteria by neutrophils. Leftover DNA of persistent bacteria is thought to chronically stimulate the innate immune system and trigger SLE. Genetic mutations of NOX2 lead to chronic granulomatous disease (CGD), which is characterized by recurrent bacterial infections due to NOX2 deficiency in phagocytic cells. Discoid lupus was significantly associated with the X chromosome-linked recessive form of CGD relative to autosomal chromosome-linked recessive CGD. NOX2 deficiency exacerbated lupus disease activity in MRL/lpr mice, thus supporting a role for defective ROI generation in neutrophils. Paradoxically , the formation of neutrophil extracellular traps (NET) or NETosis was reduced in NOX2-deficient MRL/lpr mice. This is in contrast with increased NETosis in patients with SLE. Polymorphism in CYBB , which encodes the NOX2 subunit cytochrome b-245 heavy chain, has been associated with SLE in a Chinese population. Along these lines, polymorphism of NCF1 and NCF2 , encoding a 67-kilodalton activating cytosolic subunit of NOX2, has been associated with SLE in another Chinese cohort and in the US patients.