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Systemic lupus erythematosus (SLE) patients exhibit depletion of the intracellular antioxidant glutathione and downstream activation of the metabolic sensor, mechanistic target of rapamycin (mTOR). Since reversal of glutathione depletion by the amino acid precursor, N -acetylcysteine (NAC), is therapeutic in SLE, its mechanism of impact on the metabolome was examined within the context of a double-blind placebo-controlled trial. Quantitative metabolome profiling of peripheral blood lymphocytes (PBL) was performed in 36 SLE patients and 42 healthy controls matched for age, gender, and ethnicity of patients using mass spectrometry that covers all major metabolic pathways. mTOR activity was assessed by western blot and flow cytometry. Metabolome changes in lupus PBL affected 27 of 80 KEGG pathways at FDR p < 0.05 with most prominent impact on the pentose phosphate pathway (PPP). While cysteine was depleted, cystine, kynurenine, cytosine, and dCTP were the most increased metabolites. Area under the receiver operating characteristic curve (AUC) logistic regression approach identified kynurenine (AUC = 0.859), dCTP (AUC = 0.762), and methionine sulfoxide (AUC = 0.708), as top predictors of SLE. Kynurenine was the top predictor of NAC effect in SLE (AUC = 0.851). NAC treatment significantly reduced kynurenine levels relative to placebo in vivo (raw p = 2.8 × 10 −7 , FDR corrected p = 6.6 × 10 −5 ). Kynurenine stimulated mTOR activity in healthy control PBL in vitro. Metabolome changes in lupus PBL reveal a dominant impact on the PPP that reflect greater demand for nucleotides and oxidative stress. The PPP-connected and NAC-responsive accumulation of kynurenine and its stimulation of mTOR are identified as novel metabolic checkpoints in lupus pathogenesis.

Oxidative stress modulates carcinogenesis in the liver; however, direct evidence for metabolic control of oxidative stress during pathogenesis, particularly, of progression from cirrhosis to hepatocellular carcinoma (HCC), has been lacking. Deficiency of transaldolase (TAL), a rate-limiting enzyme of the non-oxidative branch of the pentose phosphate pathway (PPP), restricts growth and predisposes to cirrhosis and HCC in mice and humans. Here, we show that mitochondrial oxidative stress and progression from cirrhosis to HCC and acetaminophen-induced liver necrosis are critically dependent on NADPH depletion and polyol buildup by aldose reductase (AR), while this enzyme protects from carbon trapping in the PPP and growth restriction in TAL deficiency. Both TAL and AR are confined to the cytosol; however, their inactivation distorts mitochondrial redox homeostasis in opposite directions. The results suggest that AR acts as a rheostat of carbon recycling and NADPH output of the PPP with broad implications for disease progression from cirrhosis to HCC. In this study, Oaks and Patel et al. characterize the crosstalk between the pentose phosphate pathway and mitochondrial redox homeostasis in the context of aldose reductase and transaldolase deficiency and the contribution of pentose phosphate pathway mitochondria deregulation to the progression from cirrhosis to hepatocellular carcinoma.

Background Despite the high prevalence of liver dysfunction in systemic lupus erythematosus (SLE), the role of the liver in disease pathogenesis is still widely unknown. Recently, we showed that prior to disease onset, SLE splenocytes overexpress the small GTPase HRES-1/Rab4A and the mechanistic target of rapamycin (mTOR), which regulate endosomal recycling and mitophagy via Drp11. Furthermore, we showed that the inhibition of mTOR or Rab4A resulted in the improvement of SLE in the kidneys and spleen1. Objectives Here, we characterize the phenotype of liver mitochondria before the onset of SLE in MRL/lpr mice and the rescue of liver dysfunction by inhibition of HRES-1/Rab4A or mTOR with 3-PEHPC or rapamycin, respectively. Methods 4-week-old MRL/lpr, C57BL/6.lpr, and MRL/MpJ mice were sacrificed with age and gender matched C57BL/6 controls. O2 consumption was measured by a Clark electrode. Reactive oxygen intermediates, reactive nitrogen intermediates, and mitochondrial mass were measured by flow cytometry. Hepatocyte mitotic figures were counted in histopathologic sections of livers at 400x magnification. Protein expression was measured by western blot analysis. Additionally, we treated 4-week-old MRL/lpr mice with either 125μg/kg of the Rab geranylgeranyl transferase inhibitor, 3-PEHPC, or 1mg/kg rapamycin for 10 weeks. Results Both male and female MRL/lpr liver mitochondria had increased oxygen consumption at complex II of the electron transport chain (ETC) and reduced state 3/state 4 respiration ratios. Male, but not female, MRL/lpr mitochondria had increased mass, nitric oxide production, and peroxynitrite. We found increased mitotic figures in MRL/lpr livers. Expression of HRES-1/Rab4A was significantly increased in all lupus-prone mice. In turn, there was decreased Drp1 expression in MRL/lpr livers. Drp1 phosphorylation at Ser616, an activating modification, was reduced in MRL/lpr livers. MRL/lpr had increased S6K and AKT expression and phosphorylation. 3-PEHPC increased the ratio of phospho-4EBP1 to total 4EBP1. Rapamycin dramatically increased the total and phosphorylated levels of 4EBP1 and AKT while reducing total phospho-S6K. 3-PEHPC increased the expression of complex I while rapamycin increased expression of complex II and IV of the ETC. Rab4A levels were unchanged by 3-PEHPC and were increased by rapamycin compared to vehicle. This was accompanied by an increase in Drp1 expression in the rapamycin cohort. 3-PEHPC blocked the phosphorylation of Ser637, which when phosphorylated prevents mitochondrial fission. Conclusions Before the onset of SLE, there is mitochondrial dysfunction in the liver mitochondria of MRL/lpr mice. The result of mitochondrial dysfunction is increased oxidative stress that may initiate proliferation of hepatocytes. Rab4A overexpression occurred in the livers of all lupus-prone mice before SLE onset and may be the central regulator of mitochondrial dysfunction, mitophagy, and mTOR activation. We show that these changes are mitigated by inhibition of mTOR or geranylgeranylation of Rab4A in MRL/lpr. References Caza TN, Fernandez DR, Talaber G, et al. HRES-1/Rab4-mediated depletion of Drp1 impairs mitochondrial homeostasis and represents a target for treatment in SLE. Ann Rheum Dis 2013. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5891

Objective Accumulation of mitochondria underlies T-cell dysfunction in systemic lupus erythematosus (SLE). Mitochondrial turnover involves endosomal traffic regulated by HRES-1/Rab4, a small GTPase that is overexpressed in lupus T cells. Therefore, we investigated whether (1) HRES-1/Rab4 impacts mitochondrial homeostasis and (2) Rab geranylgeranyl transferase inhibitor 3-PEHPC blocks mitochondrial accumulation in T cells, autoimmunity and disease development in lupus-prone mice. Methods Mitochondria were evaluated in peripheral blood lymphocytes (PBL) of 38 SLE patients and 21 healthy controls and mouse models by flow cytometry, microscopy and western blot. MRL/lpr mice were treated with 125 μg/kg 3-PEHPC or 1 mg/kg rapamycin for 10 weeks, from 4 weeks of age. Disease was monitored by antinuclear antibody (ANA) production, proteinuria, and renal histology. Results Overexpression of HRES-1/Rab4 increased the mitochondrial mass of PBL (1.4-fold; p=0.019) and Jurkat cells (2-fold; p=0.000016) and depleted the mitophagy initiator protein Drp1 both in human (−49%; p=0.01) and mouse lymphocytes (−41%; p=0.03). Drp1 protein levels were profoundly diminished in PBL of SLE patients (−86±3%; p=0.012). T cells of 4-week-old MRL/lpr mice exhibited 4.7-fold over-expression of Rab4A (p=0.0002), the murine homologue of HRES-1/Rab4, and depletion of Drp1 that preceded the accumulation of mitochondria, ANA production and nephritis. 3-PEHPC increased Drp1 (p=0.03) and reduced mitochondrial mass in T cells (p=0.02) and diminished ANA production (p=0.021), proteinuria (p=0.00004), and nephritis scores of lupus-prone mice (p<0.001). Conclusions These data reveal a pathogenic role for HRES-1/Rab4-mediated Drp1 depletion and identify endocytic control of mitophagy as a treatment target in SLE.

The mechanistic target of rapamycin (mTOR) is a central regulator in cell growth, activation, proliferation, and survival. Activation of the mTOR pathway underlies the pathogenesis of systemic lupus erythematosus (SLE). While mTOR activation and its therapeutic reversal were originally discovered in T cells, recent investigations have also uncovered roles in other cell subsets including B cells, macrophages, and “non-immune” organs such as the liver and the kidney. Activation of mTOR complex 1 (mTORC1) precedes the onset of SLE and associated co-morbidities, such as anti-phospholipid syndrome (APS), and may act as an early marker of disease pathogenesis. Six case reports have now been published that document the development of SLE in patients with genetic activation of mTORC1. Targeting mTORC1 over-activation with N-acetylcysteine, rapamycin, and rapalogs provides an opportunity to supplant current therapies with severe side effect profiles such as prednisone or cyclophosphamide. In the present review, we will discuss the recent explosion of findings in support for a central role for mTOR activation in SLE.

Populations of Oriental White-backed Vulture Gyps bengalensis and Long-billed Vulture G. indicus declined in India between the mid 1980s and late 1990s. Regional reports from India described declines of 95–100% across a wide area. This study was conducted to investigate the breeding success and pattern of mortality in two vulture colonies (Dholewala and Changa Manga) within Punjab Province, Pakistan between December 2000 and June 2001. Breeding success was found to be 62% in Dholewala and 59% in Changa Manga. A total of 668 sick and dead vultures were collected of which 591 were less than one month post mortem. No significant variation was found in the weekly mortality rate of adult and subadult vultures during the study period spanning winter through summer. A peak in mortality rate was observed during late April and early May that corresponded to mortality of newly fledged juveniles. Minimum annual mortality rate in the adult breeding population was calculated to be 11.4% and 18.6% in Dholewala and Changa Manga respectively. In a subsample of dead vultures (n = 185) visceral gout was found in 80% of adults, 63% of subadults, 19% of juveniles and 13% of nestlings. These mortality rates were consistent with a rapid population decline. Results imply that the mortality factor responsible for the decline in Gyps vultures described in India is also present in Pakistan and will potentially lead to a population decline of a comparable magnitude.

The Oriental white-backed vulture (OWBV; Gyps bengalensis ) was once one of the most common raptors in the Indian subcontinent 1 . A population decline of >95%, starting in the 1990s, was first noted at Keoladeo National Park, India 2 . Since then, catastrophic declines, also involving Gyps indicus and Gyps tenuirostris , have continued to be reported across the subcontinent 3 . Consequently these vultures are now listed as critically endangered by BirdLife International 4 . In 2000, the Peregrine Fund initiated its Asian Vulture Crisis Project with the Ornithological Society of Pakistan, establishing study sites at 16 OWBV colonies in the Kasur, Khanewal and Muzaffargarh–Layyah Districts of Pakistan to measure mortality at over 2,400 active nest sites 5 . Between 2000 and 2003, high annual adult and subadult mortality (5–86%) and resulting population declines (34–95%) (ref. 5 and M.G., manuscript in preparation) were associated with renal failure and visceral gout. Here, we provide results that directly correlate residues of the anti-inflammatory drug diclofenac with renal failure. Diclofenac residues and renal disease were reproduced experimentally in OWBVs by direct oral exposure and through feeding vultures diclofenac-treated livestock. We propose that residues of veterinary diclofenac are responsible for the OWBV decline.

The population declines affecting Asian Gyps vultures are among the most rapid and geographically widespread recorded for any species. This paper describes the rates and patterns of mortality and population change over 4 years at three Oriental white-backed vulture Gyps bengalensis colonies in Pakistan: Dholewala (initially 421 pairs), Toawala (initially 445 pairs) and Changa Manga (initially 758 pairs). Vulture mortality led to the extirpation of two of these colonies (Changa Manga and Dholewala) in 3 years, and a decline of 54.3% in the third. Visceral gout, indicative of diclofenac poisoning, was the largest single cause of death in vultures examined. Annual adult mortality from diclofenac poisoning was significantly positively correlated with annual population declines at each colony indicating a direct causal relationship. Visceral gout occurred in temporal and spatial clusters suggesting multiple point sources of diclofenac exposure. The spatial and temporal distribution of dead vultures and approximate time since death were used to estimate minimum rates at which colonies encountered carcasses with sufficient diclofenac to cause mortality of 1.26–1.88 carcasses per colony per month. By estimating total carcass consumption at each colony, the percentage of carcasses contaminated with diclofenac was calculated as 1.41–3.02%, exceeding the minimum required to have caused the observed population decline. With populations declining by approximately 50% annually, the long term survival of Gyps vultures in South Asia will require the removal of diclofenac from vulture food and establishment of captive populations for future restoration once the environment is free from contamination.

Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is important for downregulation of T-cell activation, and CTLA-4 gene polymorphisms have been implicated as risk factors for rheumatoid arthritis (RA). Previous studies of the association between the +49 polymorphism of the CTLA-4 gene in RA have provided conflicting results. In order to determine association of the CTLA-4 gene with RA in Chinese Han population, we used denaturing gradient gel electrophoresis (DGGE) to genotype polymorphisms of four SNPs (MH30, +49, CT60 and JO31) of the CTLA-4 gene in 326 RA patients and 250 healthy controls. Furthermore, meta-analysis of all available studies relating +49 polymorphism to the risk of RA was performed to confirm the disease association. Among the SNPs examined, the genotype frequencies of CTLA-4 +49 and CT60 in RA patients differed significantly from controls ( P =0.028 and 0.007). In addition, the distribution of four haplotypes constructed by these two SNPs was significantly different between patients and controls ( χ 2 =10.58, d.f. =3, P =0.014). The meta-analysis also revealed that in both European and Asian populations, the CLTA-4 +49 G allele was associated with the risk of RA. These results suggested that the CTLA-4 gene might be involved in the susceptibility to RA in the Chinese Han population and both +49 and CT60 of CTLA-4 gene might be the causal variants in RA disease.