Persistent mitochondrial hyperpolarization (MHP) and enhanced calcium fluxing underlie aberrant T cell activation and death pathway selection in systemic lupus erythematosus. Treatment with rapamycin, which effectively controls disease activity, normalizes CD3/CD28-induced calcium fluxing but fails to influence MHP, suggesting that altered calcium fluxing is downstream or independent of mitochondrial dysfunction. In this article, we show that activity of the mammalian target of rapamycin (mTOR), which is a sensor of the mitochondrial transmembrane potential, is increased in lupus T cells. S ystemic lupus erythematosus (SLE)3 is an autoimmune disease of unknown etiology characterized by T and B cell dysfunction and production of antinuclear Abs (1). Dysregulation of cell death is thought to play a key role in driving antinuclear Ab production, since the source of immunogenic nuclear material is necrotic or apoptotic cells in SLE (2). There is enhanced spontaneous apoptosis of circulating T cells in SLE, which has been linked to chronic lymphopenia (3) and compartmentalized release of autoantigens (4). Paradoxically, there is decreased activation-induced T cell death in SLE (5-7), which may contribute to persistence of autoreactive cells.The mitochondria play crucial roles in activation and death pathway selection in T lymphocytes (2). Lupus T cells exhibit mitochondrial dysfunction, which is characterized by the elevation of the mitochondrial transmembrane potential (⌬ m ) or persistent mitochondrial hyperpolarization (MHP) and consequential ATP depletion, resulting in decrease of activation-induced apoptosis and predisposition of T cells for necrosis (6). ATP depletion in lupus T cells was recently confirmed by Krishnan et al. (8). We proposed that increased release of necrotic materials from T cells could drive disease pathogenesis by activating macrophages and dendritic cells and enhancing their capacity to produce NO and IFN-␣ in SLE (2). Indeed, dendritic cells exposed to necrotic, but not apoptotic, cells induce lupus like-disease in MRL mice and accelerate the disease of MRL/lpr mice (9).Enhanced T cell activation-induced calcium fluxing has been identified as a central defect in abnormal activation and cytokine production by lupus T cells (10). Induction of MHP and mitochondrial biogenesis by NO augments cytoplasmic calcium levels and regenerates the enhanced rapid calcium signaling profile of lupus T cells (11). Dysregulation of signaling through the TCR has also been shown to be a critical determinant of abnormal calcium fluxing in SLE (12, 13). The TCR/CD3 -chain (TCR) expression is diminished in SLE T cells, and it is functionally replaced by the FcR type I ␥-chain (FcRI␥), a protein normally found in other cell types (14). TCR signaling through FcRI␥ and its adaptor protein Syk is associated with elevated calcium fluxing but only in the absence of TCR (12). It has been shown that forced expression of The costs of publication of this article were defrayed in part by the payment of page charges. This ...

Objective. Systemic lupus erythematosus (SLE) is an autoimmune disease of unknown origin. Current treatment options are often ineffective or poorly tolerated. Recent observations have revealed mitochondrial hyperpolarization and enhanced Ca 2؉ fluxing in T cells from SLE patients. Rapamycin, a lipophilic macrolide antibiotic that regulates mitochondrial transmembrane potential and Ca 2؉ fluxing, has been used safely and effectively to treat renal transplant rejection since 1999. In addition, rapamycin has been shown to ameliorate T cell function and to prolong survival in lupus-prone MRL/lpr mice. We therefore undertook the present study to investigate whether rapamycin is beneficial in patients with SLE.Methods. Nine patients with clinically active SLE that had been treated unsuccessfully with other immunosuppressive medications began therapy with rapamycin, 2 mg/day orally. Disease activity was assessed with the British Isles Lupus Assessment Group (BILAG) score, SLE Disease Activity Index (SLEDAI), and requirement for prednisone therapy. Mitochondrial transmembrane potential and Ca 2؉ fluxing were assessed by flow cytometry.Results. In patients treated with rapamycin, the BILAG score was reduced by a mean ؎ SEM of 1.93 ؎ 0.9 (P ‫؍‬ 0.0218), the SLEDAI by 5.3 ؎ 0.8 (P ‫؍‬ 0.00002), and concurrent prednisone use by 26.4 ؎ 6.7 mg/day (P ‫؍‬ 0.0062) compared with pre-rapamycin treatment. While mitochondrial hyperpolarization persisted, pretreatment cytosolic and mitochondrial Ca 2؉ levels and T cell activation-induced rapid Ca 2؉ fluxing were normalized in rapamycin-treated patients.Conclusion. Rapamycin appears to be a safe and effective therapy for SLE that has been refractory to traditional medications. Mitochondrial dysfunction and Ca 2؉ fluxing could serve as biomarkers to guide decisions regarding future therapeutic interventions in SLE.Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown origin. It involves multiple organs including the joints, skin, kidneys, and central nervous system and is characterized by a waxing and waning course. We have previously discovered that mitochondrial dysfunction, characterized by elevation of the mitochondrial transmembrane potential and depletion of ATP, plays key roles in aberrant apoptosis and predisposition to necrosis, and likely significantly contributes to the inflammatory process in SLE (1). Recent observations have indicated that mitochondrial dysfunction is associated with enhanced Ca 2ϩ fluxing in T lymphocytes from patients with SLE (2,3), providing potential new therapeutic targets (4-6).Rapamycin (sirolimus) is a lipophilic macrolide isolated from a strain of Streptomyces hygroscopius. The intracellular rapamycin receptor is a small, 12-kd FK-506 binding protein (FKBP12). The rapamycin-FKBP12 complex interacts with mammalian target of rapamycin (mTOR), a protein kinase that controls T cell activation (7), by influencing gene transcription and multiple metabolic pathways, including Ca 2ϩ fluxing (8) and maintenance of the mitochondri...

Systemic sclerosis (SSc) is a multisystem life-threatening fibrosing disorder that lacks effective treatment. The link between the inflammation observed in organs such as the skin and profibrotic mechanisms is not well understood. The plasmacytoid dendritic cell (pDC) is a key cell type mediating Toll-like receptor (TLR)-induced inflammation in autoimmune disease patients, including lupus and skin diseases with interface dermatitis. However, the role of pDCs in fibrosis is less clear. We show that pDCs infiltrate the skin of SSc patients and are chronically activated, leading to secretion of interferon-α (IFN-α) and CXCL4, which are both hallmarks of the disease. We demonstrate that the secretion of CXCL4 is under the control of phosphatidylinositol 3-kinase δ and is due to the aberrant presence of TLR8 on pDCs of SSc patients, which is not seen in healthy donors or in lupus pDCs, and that CXCL4 primarily acts by potentiating TLR8- but also TLR9-induced IFN production by pDCs. Depleting pDCs prevented disease in a mouse model of scleroderma and could revert fibrosis in mice with established disease. In contrast, the disease was exacerbated in mice transgenic for TLR8 with recruitment of pDCs to the fibrotic skin, whereas TLR7 only partially contributed to the inflammatory response, indicating that TLR8 is the key RNA-sensing TLR involved in the establishment of fibrosis. We conclude that the pDC is an essential cell type involved in the pathogenesis of SSc and its removal using depleting antibodies or attenuating pDC function could be a novel approach to treat SSc patients.

Although oxidative stress has been implicated in acute acetaminophen-induced liver failure and in chronic liver cirrhosis and hepatocellular carcinoma (HCC), no common underlying metabolic pathway has been identified. Recent case reports suggest a link between the pentose phosphate pathway (PPP) enzyme transaldolase (TAL; encoded by TALDO1) and liver failure in children. Here, we show that Taldo1 -/-and Taldo1 +/-mice spontaneously developed HCC, and Taldo1 -/-mice had increased susceptibility to acetaminophen-induced liver failure. Oxidative stress in Taldo1 -/-livers was characterized by the accumulation of sedoheptulose 7-phosphate, failure to recycle ribose 5-phosphate for the oxidative PPP, depleted NADPH and glutathione levels, and increased production of lipid hydroperoxides. Furthermore, we found evidence of hepatic mitochondrial dysfunction, as indicated by loss of transmembrane potential, diminished mitochondrial mass, and reduced ATP/ADP ratio. Reduced β-catenin phosphorylation and enhanced c-Jun expression in Taldo1 -/-livers reflected adaptation to oxidative stress. Taldo1 -/-hepatocytes were resistant to CD95/Fas-mediated apoptosis in vitro and in vivo. Remarkably, lifelong administration of the potent antioxidant N-acetylcysteine (NAC) prevented acetaminophen-induced liver failure, restored Fas-dependent hepatocyte apoptosis, and blocked hepatocarcinogenesis in Taldo1 -/-mice. These data reveal a protective role for the TAL-mediated branch of the PPP against hepatocarcinogenesis and identify NAC as a promising treatment for liver disease in TAL deficiency.

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.

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We have directly assessed the ability of interferon regulatory factor-1 (IRF-1) to act as a tumor suppressor gene in human breast cancer cells and explored whether this suppressor function is mechanistically conferred by affecting cell cycle transition, apoptosis and/or caspase activation. We have used a dual approach, measuring whether overexpression of wild-type IRF-1 or a dominant negative IRF-1 (dnIRF-1) produce opposing effects on breast cancer cell proliferation in vitro or tumorigenicity in athymic nude mice. Mechanistic studies determined the effects of blocking endogenous IRF-1 expression on cell cycle transition by flow cytometry, on apoptosis by Annexin V staining, and on caspase activation by fluorescent substrate cleavage. IRF-1 mRNA (P < or = 0.001) and protein (P < or = 0.001) are highly expressed in non-tumorigenic, normal, mammary epithelial cells, with intermediate expression in tumorigenic, but non-metastatic, cells and very low expression in metastatic cell lines. In MCF-7 cells transfected with a wild-type IRF-1 (MCF-7/IRF-1), IRF-1 mRNA expression inversely correlates with the rate of cell proliferation (r = -0.91; P = 0.002). Conversely, expression of dnIRF-1 in both MCF-7 (MCF-7/dnIRF-1; p53 wild-type) and T47D cells (T47D/dnIRF-1; p53 mutant) increases cell proliferation (P < or = 0.001). In athymic nude mice, the incidence of MCF-7/IRF-1 xenografts is reduced (P = 0.045), whereas MCF-7/dnIRF-1 xenografts exhibit a significantly higher tumor incidence (P < or = 0.001). Effects of IRF-1/dnIRF-1 are mediated through changes in the rates of apoptosis and not through cell cycle regulation. MCF-7/dnIRF-1 cells exhibit a 50% decrease in basal apoptosis (P = 0.007) and a significant reduction in caspase 8 activity (P = 0.03); similar effects occur in T47D/dnIRF-1 cells, where the effects on apoptosis appear to be mediated through inhibition of caspases 3/7 (P < 0.001) and caspase 8 (P = 0.03). These data establish a functional role for IRF-1 in the growth suppression of breast cancer cells and strongly implicate IRF-1 as a tumor suppressor gene in breast cancer that acts, independent of p53, to control apoptosis.

Nitric oxide (NO) has been shown to regulate T cell functions under physiological conditions, but overproduction of NO may contribute to T lymphocyte dysfunction. NO-dependent tissue injury has been implicated in a variety of rheumatic diseases, including systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA). Several studies reported increased endogenous NO synthesis in both SLE and RA, and recent evidence suggests that NO contributes to T cell dysfunction in both autoimmune diseases. The depletion of intracellular glutathione may be a key factor predisposing patients with SLE to mitochondrial dysfunction, characterized by mitochondrial hyperpolarization, ATP depletion and predisposition to death by necrosis. Thus, changes in glutathione metabolism may influence the effect of increased NO production in the pathogenesis of autoimmunity.