Eduardo Bonilla

N‐acetylcysteine reduces disease activity by blocking mammalian target of rapamycin in T cells from systemic lupus erythematosus patients: A randomized, double‐blind, placebo‐controlled trial

OBJECTIVE Systemic lupus erythematosus (SLE) patients exhibit T cell dysfunction, which can be regulated through mitochondrial transmembrane potential (Δψm) and mammalian target of rapamycin (mTOR) by glutathione (GSH). This randomized, double-blind, placebo-controlled study was undertaken to examine the safety, tolerance, and efficacy of the GSH precursor N-acetylcysteine (NAC). METHODS A total of 36 SLE patients received either daily placebo or 1.2 gm, 2.4 gm, or 4.8 gm of NAC. Disease activity was evaluated monthly by the British Isles Lupus Assessment Group (BILAG) index, the SLE Disease Activity Index (SLEDAI), and the Fatigue Assessment Scale (FAS) before, during, and after a 3-month treatment period. Mitochondrial transmembrane potential and mTOR were assessed by flow cytometry. Forty-two healthy subjects matched to patients for age, sex, and ethnicity were studied as controls. RESULTS NAC up to 2.4 gm/day was tolerated by all patients, while 33% of those receiving 4.8 gm/day had reversible nausea. Placebo or NAC 1.2 gm/day did not influence disease activity. Considered together, 2.4 gm and 4.8 gm NAC reduced the SLEDAI score after 1 month (P = 0.0007), 2 months (P = 0.0009), 3 months (P = 0.0030), and 4 months (P = 0.0046); the BILAG score after 1 month (P = 0.029) and 3 months (P = 0.009); and the FAS score after 2 months (P = 0.0006) and 3 months (P = 0.005). NAC increased Δψm (P = 0.0001) in all T cells, profoundly reduced mTOR activity (P = 0.0009), enhanced apoptosis (P = 0.0004), reversed expansion of CD4-CD8- T cells (mean ± SEM 1.35 ± 0.12-fold change; P = 0.008), stimulated FoxP3 expression in CD4+CD25+ T cells (P = 0.045), and reduced anti-DNA production (P = 0.049). CONCLUSION This pilot study suggests that NAC safely improves lupus disease activity by blocking mTOR in T lymphocytes.

Activation of mammalian target of rapamycin controls the loss of TCRzeta in lupus T cells through HRES-1/Rab4-regulated lysosomal degradation.

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. Activation of mTOR was inducible by NO, a key trigger of MHP, which in turn enhanced the expression of HRES-1/Rab4, a small GTPase that regulates recycling of surface receptors through early endosomes. Expression of HRES-1/Rab4 was increased in CD4+ lupus T cells, and in accordance with its dominant impact on the endocytic recycling of CD4, it was inversely correlated with diminished CD4 expression. HRES-1/Rab4 overexpression was also inversely correlated with diminished TCRζ protein levels. Pull-down studies revealed a direct interaction of HRES-1/Rab4 with CD4 and TCRζ. Importantly, the deficiency of the TCRζ chain and of Lck and the compensatory up-regulation of FcεRIγ and Syk, which mediate enhanced calcium fluxing in lupus T cells, were reversed in patients treated with rapamcyin in vivo. Knockdown of HRES-1/Rab4 by small interfering RNA and inhibitors of lysosomal function augmented TCRζ protein levels in vitro. The results suggest that activation of mTOR causes the loss of TCRζ in lupus T cells through HRES-1/Rab4-dependent lysosomal degradation.

Haplotypes of the HRES-1 Endogenous Retrovirus Are Associated With Development and Disease Manifestations of Systemic Lupus Erythematosus

OBJECTIVE Endogenous retroviral sequences represent a link between viral and genetic factors that may influence the development of systemic lupus erythematosus (SLE). The HRES-1 human endogenous retroviral sequence is centrally located at the 1q42 chromosomal region relative to microsatellites previously associated with SLE. We therefore undertook the present study to determine the haplotypes of the HRES-1 locus and their linkage to SLE. METHODS One hundred six patients with SLE, 82 unrelated healthy Caucasian individuals, and 70 healthy members of 34 lupus families were examined. HRES-1 was amplified by polymerase chain reaction (PCR) and analyzed by sequencing and restriction enzyme mapping. Microsatellites were analyzed by PCR. Haplotype construction and transmission disequilibrium testing (TDT) were performed in lupus families. RESULTS Based on 4 single-nucleotide polymorphisms (SNPs) within a 935-base interval, we detected 6 HRES-1 haplotypes that were differentially segregated in unrelated Caucasian patients and control subjects (chi(2) = 16.86, P = 0.0048) and were in linkage disequilibrium (LD) with the D1S225 microsatellite (P = 0.0002). The microsatellites D1S225, D1S235, and D1S2785 (but not D1S229) were linked to SLE by TDT. Interestingly, LD between HRES-1 SNPs at bases 653 and 1259 was reduced in patients with SLE (P = 0.048). The HRES-1 653C/1259C-harboring alleles were associated with the presence of renal disease (P = 0.0021) and with the absence of lung disease (P = 0.0323), while the 956A allele was associated with the antiphospholipid syndrome in patients with SLE (P = 0.0036). CONCLUSION The HRES-1 locus represents a recombination hot spot at the 1q42 chromosomal region that influences the development and disease manifestations of SLE.

Association of common mitochondrial DNA variants with multiple sclerosis and systemic lupus erythematosus

Mitochondrial dysfunction has been implicated in the pathogenesis of multiple sclerosis (MS) and systemic lupus erythematosus (SLE). This study re-investigates the roles of previously suggested candidate genes of energy metabolism (Complex I genes located in the nucleus and in the mitochondria) in patients with MS relative to ethnically matched SLE patients and healthy controls. After stringent correction for multiple testing, we reproduce the association of the mitochondrial (mt)DNA haplotype K* with MS, but reject the importance of previously suggested borderline associations with nuclear genes of Complex I. In addition, we detect the association of common variants of the mitochondrial ND2 and ATP6 genes with both MS and SLE, which raises the possibility of a shared mitochondrial genetic background of these two autoimmune diseases.

Molecular mimicry and immunomodulation by the HRES-1 endogenous retrovirus in SLE

Genetic and environmental factors are believed to influence development of systemic lupus erythematosus (SLE). Endogenous retroviruses (ERV) correspond to the integrated proviral form of infectious retroviruses, which are trapped within the genome due to mutations. ERV represent a key molecular link between the host genome and infectious viral particles. ERV-encoded proteins are recognized by antiviral immune responses and become targets of autoreactivity. Alternatively, ERV protein may influence cellular processes and the life cycle of infectious viruses. As examples, the HRES-1 human ERV encodes a 28-kDa nuclear autoantigen and a 24-kDa small GTP-ase, termed HRES-1/Rab4. HRES-1/p28 is a nuclear autoantigen recognized by cross-reactive antiviral antibodies, while HRES-1/Rab4 regulates surface expression of CD4 and the transferrin receptor (TFR) through endosome recycling. Expression of HRES-1/Rab4 is induced by the tat gene of HIV-1, which in turn down-regulates expression of CD4 and susceptibility to re-infection by HIV-1. CD4 and the TFR play essential roles in formation of the immunological synapse (IS) during normal T-cell activation by a cognate MHC class II peptide complex. The key intracellular transducer of T-cell activation, Lck, is brought to the IS via binding to CD4. T-cell receptorζ (TCRζ) chain binds to the TFR. Abnormal T-cell responses in SLE have been associated with reduced lck and TCRζ chain levels. HRES-1 is centrally located on chromosome 1 at q42 relative to lupus-linked microsatellite markers and polymorphic HRES-1 alleles have been linked to the development of SLE. 1q42 is one of the three most common fragile sites in the human genome, and is inducible by DNA demethylation, a known mechanism of retroviral gene activation. Molecular mimicry and immunomodulation by a ERV, such as HRES-1, may contribute to self-reactivity and abnormal T and B-cell functions in SLE.

Signaling Abnormalities in Systemic Lupus Erythematosus as Potential Drug Targets

Systemic lupus erythematosus (SLE) is an autoimmune inflammatory disease characterized by T-cell, B-cell, and dendritic cell dysfunction and antinuclear autoantibody production. Much of the knowledge that has been gained about SLE in recent years is related to molecular signaling abnormalities present in the disease. Signaling through the T-cell receptor (TCR) is affected in SLE by alterations in the localization, amount, and activity of numerous protein kinases. TCR stimulation releases calcium from intracellular stores, which triggers an influx of extracellular calcium and activates the transcription of many genes, including interleukin-2. Short-term calcium fluxing is exaggerated in SLE, but long-term calcium fluxing is diminished and may account for sub-optimal interleukin-2 production. SLE T-cells have persistently hyperpolarized mitochondria associated with increased mitochondrial mass, high levels of reactive oxygen species (ROS) and low levels of ATP, which decrease activation-induced apoptosis and instead predispose T cells for necrosis, thus stimulating inflammation in SLE. The pentose phosphate pathway impacts the mitochondrial potential and represents a target for possible intervention. Nitric oxide (NO) is a potential link to tie together the signaling and mitochondrial abnormalities in SLE. NO-induced mitochondrial biogenesis recapitulates the TCR-stimulated calcium fluxing abnormalities of SLE T-cells. Since mitochondria can store calcium, the increase in mitochondrial mass may be implicated in the aberrant calcium fluxing in SLE T cells. The mammalian target of rapamycin senses the mitochondrial potential and regulates calcium release, serving as a novel target of treatment of SLE.

Liver injury correlates with biomarkers of autoimmunity and disease activity and represents an organ system involvement in patients with systemic lupus erythematosus

Liver disease (LD), defined as ≥ 2-fold elevation of aspartate aminotransferase (AST) or alanine aminotransferase (ALT), was examined in a longitudinal study of systemic lupus erythematosus (SLE) patients. Among 435 patients, 90 (20.7%) had LD with a greater prevalence in males (15/39; 38.5%) than females (75/396; 18.9%; p = 0.01). SLE disease activity index (SLEDAI) was greater in LD patients (7.8 ± 0.7) relative to those without (5.8 ± 0.3; p = 0.0025). Anti-smooth muscle antibodies, anti-DNA antibodies, hypocomplementemia, proteinuria, leucopenia, thrombocytopenia, and anti-phospholipid syndrome were increased in LD. An absence of LD was noted in patients receiving rapamycin relative to azathioprine, cyclosporine A, or cyclophosphamide. An absence of LD was also noted in patients treated with N-acetylcysteine. LFTs were normalized and SLEDAI was diminished with increased prednisone use in 76/90 LD patients over 12.1 ± 2.6 months. Thus, LD is attributed to autoimmunity and disease activity, it responds to prednisone, and it is potentially preventable by rapamycin or N-acetylcysteine treatment.

CCL genes in multiple sclerosis and systemic lupus erythematosus

This follow up study aims to refine the roles of previously suggested candidate genes (CC chemokine ligands or CCLs) in multiple sclerosis (MS), and to test these markers in another autoimmune disorder, systemic lupus erythematosus (SLE). After stringent correction for multiple testing, we reject the importance of previously suggested borderline associations with CCLs in MS. A new finding is the differential distribution of CCL8 marker alleles and a haplotype in extreme severity subgroups of MS. In SLE, this study reveals strong associations with a marker and a haplotype encompassing the CCL14 gene, which suggests that a lupus relevant variant may lie within or in the proximity of this haplotype.

Association of psoriasis and psoriatic arthritis with systemic lupus erythematosus

Objective: The prevalence of psoriasis (Ps) and psoriatic arthritis (PsA) in patients with systemic lupus erythematosus (SLE) is largely unknown, which was investigated in this study. Methods: The prevalence of Ps and PsA was examined in 445 patients with SLE. The diagnosis of psoriasis was made clinically by a dermatologist or rheumatologist, while the diagnosis of PsA was made on the basis of CASPAR criteria. The diagnosis of SLE was based on the American College of Rheumatology (ACR) classification criteria. Clinical and laboratory variables were compared in SLE patients with and without Ps (SLE/Ps+ and Ps-) as well as in those with and without PsA (SLE/PsA+ and PsA-). Statistical analyses were performed with chi-square test using Graphpad Prism software. Results: Among 445 patients with SLE, 23 (5.1%) had Ps, out of which 20 (4.5%) had PsA which is greater than what is observed in the general population (p < 0.0001). The prevalence of malar rash, discoid rash, photosensitivity, and arthritis were increased in SLE patients with Ps and PsA. Antiphospholipid antibodies (APLAs) were less common in SLE patient with concurrent Ps and PsA. Conclusions: The prevalence of PsA is increased and it may represent a distinct clinical entity in patients with SLE. Correspondence to: Andras Perl, Division of Rheumatology, Department of Medicine, Suny Upstate Medical University, 750 East Adams Street, 8310 Weiskotten Hall, Syracuse, 13210, USA, Tel: 315 464-4194, E-mail: perla@upstate.edu