Ram Raj Singh

A role for sex chromosome complement in the female bias in autoimmune disease

Most autoimmune diseases are more common in women than in men. This may be caused by differences in sex hormones, sex chromosomes, or both. In this study, we determined if there was a contribution of sex chromosomes to sex differences in susceptibility to two immunologically distinct disease models, experimental autoimmune encephalomyelitis (EAE) and pristane-induced lupus. Transgenic SJL mice were created to permit a comparison between XX and XY within a common gonadal type. Mice of the XX sex chromosome complement, as compared with XY, demonstrated greater susceptibility to both EAE and lupus. This is the first evidence that the XX sex chromosome complement, as compared with XY, confers greater susceptibility to autoimmune disease.

Differential Contribution of IL-4 and STAT6 vs STAT4 to the Development of Lupus Nephritis

Mechanisms that initiate lupus nephritis and cause progression to end-stage renal disease remain poorly understood. In this study, we show that lupus-prone New Zealand Mixed 2410 mice that develop a severe glomerulosclerosis and rapidly progressive renal disease overexpress IL-4 in vivo. In these mice, STAT6 deficiency or anti-IL-4 Ab treatment decreases type 2 cytokine responses and ameliorates kidney disease, particularly glomerulosclerosis, despite the presence of high levels of IgG anti-dsDNA Abs. STAT4 deficiency, however, decreases type 1 and increases type 2 cytokine responses, and accelerates nephritis, in the absence of high levels of IgG anti-dsDNA Abs. Thus, STAT6 and IL-4 may selectively contribute to the development of glomerulosclerosis, whereas STAT4 may play a role in autoantibody production.

Repeated α-Galactosylceramide Administration Results in Expansion of NK T Cells and Alleviates Inflammatory Dermatitis in MRL-lpr/lpr Mice

NK T (NKT) cells expressing the invariant Vα14-Jα18 TCR α-chain recognize glycolipid Ags such as α-galactosylceramide (α-GalCer) presented by the MHC class I-like molecule CD1d. Upon activation by α-GalCer, invariant NKT cells secrete multiple cytokines and confer protection in certain immune-mediated disorders. Here we have investigated the role of NKT cells in the development of inflammatory dermatitis in MRL-lpr/lpr mice, which shares features with lupus in humans. Our results show that the numbers Sand functions of NKT (TCRβ+CD1d/α-GalCer tetramer+) cells, particularly of the NK1.1− subset, are reduced in MRL-lpr/lpr mice compared with MRL-fas/fas and/or nonautoimmune C3H/Hej and BALB/c mice. Repeated treatments with α-GalCer result in the expansion of NKT cells and alleviate dermatitis in MRL-lpr/lpr mice. Our results indicate that NKT cell deficiency can be corrected by repeated α-GalCer treatment and that NKT cells may play a protective role in inflammatory dermatitis of lupus-prone mice.

Treatment with a consensus peptide based on amino acid sequences in autoantibodies prevents T cell activation by autoantigens and delays disease onset in murine lupus.

OBJECTIVE To test the hypothesis that an artificial peptide, based on an algorithm describing T cell stimulatory sequences from the VH regions of murine IgG antibodies to DNA, is an effective tolerogen in vivo in the (NZB/NZW)F1 (BWF1) mouse model of systemic lupus erythematosus. METHODS Using proliferative T cell responses to 439 Ig peptides, an algorithm was constructed that describes the amino acid sequences likely to stimulate BWF1 T cells. Stimulatory (pConsensus [pCONS]) or nonstimulatory (pNegative [pNEG]) peptides were synthesized. Groups of 10-week-old (healthy) or 20-week-old (diseased) BWF1 mice received monthly intravenous injections of 1,000 microg of peptide or saline. Ex vivo splenic T cell responses and in vivo clinical effects were measured. RESULTS Tolerance was induced by pCONS, but not by pNEG, with respect to ex vivo T cell proliferation and T cell help for antibodies to DNA. T cell help for IgG anti-DNA was impaired not only after T cell stimulation by pCONS but also after stimulation by some peptides from nucleosomal and Ro antigens. Treatment with pCONS significantly delayed the onset of nephritis and inhibited increases in the plasma levels of total IgG, IgG antibodies to DNA, nucleosome, cardiolipin, interferon-gamma, and interleukin-4. In contrast, antibody responses to an exogenous antigen were not impaired. Survival was significantly prolonged in both healthy and diseased mice treated with pCONS. CONCLUSION Induction of immune tolerance in response to treatment with pCONS in autoreactive T cell helper populations is highly effective in delaying the appearance of multiple autoantibodies, cytokine increases, and nephritis in BWF1 mice, and dramatically prolongs survival. A striking effect is the ability of the peptide to tolerize T cell help for anti-DNA that is induced by multiple, structurally unrelated self antigens.

A one-year, phase I/IIa, open-label pilot trial of imatinib mesylate in the treatment of systemic sclerosis-associated active interstitial lung disease

OBJECTIVE Transforming growth factor β (TGFβ) and platelet-derived growth factor (PDGF) may play a critical role in systemic sclerosis (SSc)-related interstitial lung disease (ILD), and imatinib is a potent inhibitor of TGFβ and PDGF production. In this 1-year, phase I/IIa open-label pilot study of imatinib in patients with SSc-related active ILD, our primary aim was to assess the safety of imatinib; we also explored its efficacy. METHODS We recruited 20 SSc patients with a forced vital capacity (FVC) of <85% predicted, dyspnea on exertion, and presence of a ground-glass appearance on high-resolution computed tomography. Patients received oral therapy with imatinib (up to 600 mg/day) for a period of 1 year. Adverse events were recorded, pulmonary function was tested, and the modified Rodnan skin thickness score (MRSS) was assessed every 3 months. The course of changes in lung function, the Health Assessment Questionnaire (HAQ) disability index (DI), and the MRSS were modeled over the period of study to explore treatment efficacy. RESULTS The majority of patients were female (65%), Caucasian (75%), and had diffuse cutaneous SSc (70%). At baseline, the mean ± SD FVC % predicted was 65.2 ± 14.0 and the mean ± SD MRSS was 18.7 ± 10.1. The mean ± SD dosage of imatinib was 445 ± 125 mg/day. Of the 20 SSc patients, 12 completed the study, 7 discontinued because of adverse events (AEs), and 1 patient was lost to followup. Common AEs (≥20%) included fatigue, facial/lower extremity edema, nausea and vomiting, diarrhea, generalized rash, and new-onset proteinuria. Treatment with imatinib showed a trend toward improvement in the FVC % predicted (1.74%; P not significant) and the MRSS (3.9 units; P < 0.001). CONCLUSION Use of high-dose daily therapy with imatinib (600 mg/day) in SSc patients with ILD was associated with a large number of AEs. Our experience with AEs suggests that dosages of imatinib lower than 600 mg/day may be appropriate and that further dose ranging analysis is needed in order to understand the therapeutic index of imatinib in SSc.

Immune tolerance to autoantibody-derived peptides delays development of autoimmunity in murine lupus.

Mechanisms that initiate and maintain autoantibody (autoAb) production in individuals with autoimmune diseases like SLE are poorly understood. Inadequate suppression of autoreactive T cells and/or unusual activation of T and B cells may underlie the persistence of pathogenic autoAbs in lupus. Here, we examine the possibility that in mice with lupus, autoAb molecules may be upregulating their own production by activating self-reactive T cells via their own processed peptides; downregulation of this circuit may decrease autoAb production and delay the development of lupus. We found that before the onset of clinical disease, lupus-prone (NZB/NZW) F1 [BWF1] (but not MHC-matched nonautoimmune mice) developed spontaneous T cell autoimmunity to peptides from variable regions of heavy chains (VH) of syngeneic anti-DNA mAbs but not to peptides from the VH region of an mAb to an exogenous antigen. Tolerizing young BWF1 mice with intravenous injections of autoAb-derived determinants substantially delayed development of anti-DNA antibodies and nephritis and prolonged survival. Thus, in such an autoAb-mediated disease, the presence of autoreactive T cells against VH region determinants of autoAbs may represent an important mechanism involved in the regulation of autoimmunity. Our findings show that tolerizing such autoreactive T cells can postpone the development of an autoimmune disease like SLE.

Dicer Insufficiency and MicroRNA-155 Overexpression in Lupus Regulatory T Cells: An Apparent Paradox in the Setting of an Inflammatory Milieu

Systemic lupus erythematosus is a chronic autoimmune disease characterized by loss of tolerance to self-Ags and activation of autoreactive T cells. Regulatory T (Treg) cells play a critical role in controlling the activation of autoreactive T cells. In this study, we investigated mechanisms of potential Treg cell defects in systemic lupus erythematosus using MRL-Faslpr/lpr (MRL/lpr) and MRL-Fas+/+ mouse models. We found a significant increase in CD4+CD25+Foxp3+ Treg cells, albeit with an altered phenotype (CD62L−CD69+) and with a reduced suppressive capacity, in the lymphoid organs of MRL strains compared with non-autoimmune C3H/HeOuj mice. A search for mechanisms underlying the altered Treg cell phenotype in MRL/lpr mice led us to find a profound reduction in Dicer expression and an altered microRNA (miRNA, miR) profile in MRL/lpr Treg cells. Despite having a reduced level of Dicer, MRL/lpr Treg cells exhibited a significant overexpression of several miRNAs, including let-7a, let-7f, miR-16, miR-23a, miR-23b, miR-27a, and miR-155. Using computational approaches, we identified one of the upregulated miRNAs, miR-155, that can target CD62L and may thus confer the altered Treg cell phenotype in MRL/lpr mice. In fact, the induced overexpression of miR-155 in otherwise normal (C3H/HeOuj) Treg cells reduced their CD62L expression, which mimics the altered Treg cell phenotype in MRL/lpr mice. These data suggest a role of Dicer and miR-155 in regulating Treg cell phenotype. Furthermore, simultaneous appearance of Dicer insufficiency and miR-155 overexpression in diseased mice suggests a Dicer-independent alternative mechanism of miRNA regulation under inflammatory conditions.

Immunoregulatory Role of CD1d in the Hydrocarbon Oil-Induced Model of Lupus Nephritis

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that is accompanied by the emergence of autoreactive T cells and a reduction in regulatory T cells. Humans and mice with SLE have reduced numbers of CD1d-restricted NK T cells, suggesting a role for these cells in the regulation of SLE. In this study, we show that CD1d deficiency exacerbates lupus nephritis induced by the hydrocarbon oil pristane. This exacerbation in disease is associated with: 1) reduced TNF-α and IL-4 production by T cells, especially during the disease induction phase; and 2) expansion of marginal zone B cells. Strikingly, inoculation of pristane in wild-type mice resulted in reduced numbers and/or functions of NK T cells and CD1d-expressing dendritic cells. These findings suggest that CD1d may play an immunoregulatory role in the development of lupus in the pristane-induced model.

Prevalence of rheumatoid arthritis in the adult Indian population.

SummaryThe prevalence of rheumatoid arthritis was studied in the adult Indian population. As the first step, a house-to-house survey of a rural population near Delhi was conducted by two trained health workers. The target population comprised 44 551 adults (over 16 years old). The health workers identified the possible cases of rheumatoid arthritis (RA) using a questionnaire. These cases were then further evaluated by the authors using the 1987 revised ARA criteria for the diagnosis of RA. A response rate of 89.5% was obtained and 3393 persons were listed as possible cases of RA by the health workers. Of these, 299 satisfied the revised ARA criteria for the diagnosis of RA, giving a prevalence of 0.75%. Projected to the whole population, this would give a total of about seven million patients in India. The prevalence of RA in India is quite similar to that reported from the developed countries. It is higher than that reported from China, Indonesia, Philippines and rural Africa. These findings are in keeping with the fact that the north Indian population is genetically closer to the Caucasians than to other ethnic groups.

The natural killer T cell ligand α-galactosylceramide prevents or promotes pristane-induced lupus in mice

Systemic lupus erythematosus is a systemic autoimmune disease characterized by inflammation in organs such as kidneys and presence of autoantibodies against nuclear antigens. We have previously shown that CD1d deficiency in BALB/c mice exacerbates lupus nephritis and autoantibody production induced by the hydrocarbon oil pristane. Here, we have tested the impact of activating CD1d‐restricted natural killer T (NKT) cells on pristane‐induced lupus‐like autoimmunity in BALB/c and SJL mice. Repeated in vivo treatment of pristane‐injected BALB/c mice with the NKT cell ligand α‐galactosylceramide (α‐GalCer) prior to the onset of florid disease suppressed proteinuria, in a manner that was dependent on CD1d and IL‐4 expression. In sharp contrast, however, similar treatment of pristane‐injected SJL mice with α‐GalCer resulted in increased proteinuria. Consistent with these dichotomous effects of NKT cell activation on the development of lupus‐like autoimmunity, NKT cells in BALB/c and SJL/J mice exhibited a mixed Th1/Th2 and a Th1‐biased cytokine production profile, respectively. These findings demonstrate that NKT cell activation with α‐GalCer suppresses or promotes pristane‐induced lupus‐like autoimmunity in mice, in a strain‐dependent manner.