Vaishali R. Moulton

Abnormalities of T cell signaling in systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is an autoimmune disease resulting from a loss of tolerance to multiple self antigens, and characterized by autoantibody production and inflammatory cell infiltration in target organs, such as the kidneys and brain. T cells are critical players in SLE pathophysiology as they regulate B cell responses and also infiltrate target tissues, leading to tissue damage. Abnormal signaling events link to defective gene transcription and altered cytokine production, contributing to the aberrant phenotype of T cells in SLE. Study of signaling and gene transcription abnormalities in SLE T cells has led to the identification of novel targets for therapy.

T cell signaling abnormalities contribute to aberrant immune cell function and autoimmunity

Systemic lupus erythematosus (SLE) is a prototype systemic autoimmune disease that results from a break in immune tolerance to self-antigens, leading to multi-organ destruction. Autoantibody deposition and inflammatory cell infiltration in target organs such as kidneys and brain lead to complications of this disease. Dysregulation of cellular and humoral immune response elements, along with organ-defined molecular aberrations, form the basis of SLE pathogenesis. Aberrant T lymphocyte activation due to signaling abnormalities, linked to defective gene transcription and altered cytokine production, are important contributors to SLE pathophysiology. A better understanding of signaling and gene regulation defects in SLE T cells will lead to the identification of specific novel molecular targets and predictive biomarkers for therapy.

Alternative Splicing Factor/Splicing Factor 2 Regulates the Expression of the ζ Subunit of the Human T Cell Receptor-associated CD3 Complex

T cells from patients with systemic lupus erythematosus express decreased levels of the T cell receptor-associated CD3 ζ chain, a feature directly linked to their aberrant function. The decrease in CD3ζ protein expression is in part due to decreased levels of functional wild type isoform of the 3′-untranslated region (UTR) of CD3ζ mRNA with concomitant increased levels of an unstable alternatively spliced isoform. In order to identify factors involved in the post-transcriptional regulation of CD3ζ, we performed mass spectrometric analysis of Jurkat T cell nuclear proteins “pulled down” by a CD3ζ 3′-UTR oligonucleotide, which identified the splicing protein alternative splicing factor/splicing factor 2 (ASF/SF2). We show for the first time that ASF/SF2 binds specifically to the 3′-UTR of CD3ζ and regulates expression of CD3ζ protein by limiting the production of the alternatively spliced isoform. During activation of human T cells, an increase in the wild type CD3ζ mRNA is associated with increased expression of ASF/SF2. Finally, we show a significant correlation between ASF/SF2 and CD3ζ protein levels in T cells from systemic lupus erythematosus patients. Thus, our results identify ASF/SF2 as a novel factor in the regulation of alternative splicing of the 3′-UTR of CD3ζ and protein expression in human T cells.

Estrogen upregulates cyclic AMP response element modulator α expression and downregulates interleukin-2 production by human T lymphocytes.

Systemic lupus erythematosus (SLE) is an autoimmune disease with a complex multifactorial pathogenesis. T lymphocytes play a critical role in disease pathogenesis and display abnormal gene expression and poor interleukin (IL)-2 production. We previously showed that the expression of the transcriptional repressor cyclic AMP response element modulator α (CREMα) is increased in SLE T cells and contributes to reduced IL-2 production. Although estrogen is implicated in the onset and exacerbation of SLE, the precise nature of molecular events regulated by estrogen in immune cell function is not well understood. Here, we asked whether estrogen regulates the expression of CREMα in human T lymphocytes. We show that exposure of human T cells to 17-β-estradiol leads to a dose-dependent increase in CREMα mRNA expression, and this increase appears to be mediated through the estrogen receptors α and β. We show that the increased expression of CREMα is due to increased transcriptional activity of the CREM promoter and is mediated by increased expression and binding of the Sp1 transcriptional activator. We further show that estrogen treatment leads to a dose-dependent decrease in IL-2 mRNA and cytokine production by T cells. Finally, the effect of β-estradiol on CREMα is observed more frequently in T cells from women than from men. We conclude that estrogen can modulate the expression of CREMα and lead to IL-2 suppression in human T lymphocytes, thus revealing a molecular link between hormones and the immune system in SLE.

Pathogenesis of Human Systemic Lupus Erythematosus: A Cellular Perspective

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease affecting multiple organs. A complex interaction of genetics, environment, and hormones leads to immune dysregulation and breakdown of tolerance to self-antigens, resulting in autoantibody production, inflammation, and destruction of end-organs. Emerging evidence on the role of these factors has increased our knowledge of this complex disease, guiding therapeutic strategies and identifying putative biomarkers. Recent findings include the characterization of genetic/epigenetic factors linked to SLE, as well as cellular effectors. Novel observations have provided an improved understanding of the contribution of tissue-specific factors and associated damage, T and B lymphocytes, as well as innate immune cell subsets and their corresponding abnormalities. The intricate web of involved factors and pathways dictates the adoption of tailored therapeutic approaches to conquer this disease.

Splicing factor SF2/ASF rescues IL-2 production in T cells from systemic lupus erythematosus patients by activating IL-2 transcription

T cells from patients with systemic lupus erythematosus (SLE) produce insufficient amounts of the vital cytokine IL-2. We previously showed that SLE T cells express decreased levels of the T-cell receptor–CD3ζ chain and forced expression of CD3ζ into SLE T cells restores IL-2 production. We recently showed that the serine arginine protein splicing factor 2/alternative splicing factor (SF2/ASF) enhances the expression of CD3ζ chain by limiting the production of an unstable splice variant. Here we demonstrate that SF2/ASF levels are decreased in patients with SLE and more so in those with active disease. More importantly, we reveal a function of SF2/ASF, independent of T-cell receptor/CD3 signaling, whereby it is recruited to the IL-2 promoter, increases transcriptional activity, and enhances IL-2 production in SLE T cells. Our results demonstrate that SF2/ASF regulates IL-2 production and that decreased SF2/ASF expression in SLE T cells contributes to deficient IL-2 production.

The RNA-stabilizing Protein HuR Regulates the Expression of ζ Chain of the Human T Cell Receptor-associated CD3 Complex

T cell dysfunction is crucial to the pathogenesis of systemic lupus erythematosus (SLE); however, the molecular mechanisms involved in the deficient expression of the T cell receptor-associated CD3ζ chain in SLE are not clear. SLE T cells express abnormally increased levels of an alternatively spliced isoform of CD3ζ that lacks a 562-bp region in its 3′-untranslated region (UTR). We showed previously that two adenosine/uridine-rich elements (ARE) in this splice-deleted region of CD3ζ transcript are critical for the mRNA stability and protein expression of CD3ζ. In this study we show for the first time that the mRNA-stabilizing protein HuR binds to these two ARE bearing regions of CD3ζ 3′-UTR. Knockdown of HuR resulted in decreased expression of the CD3ζ chain, whereas overexpression led to the increase of CD3ζ chain levels. Additionally, overexpression of HuR in human T cells resulted in increased mRNA stability of CD3ζ. Our results identify the 3′-UTR of CD3ζ as a novel target for the mRNA-stabilizing protein HuR. Thus, the absence of two critical AREs in the alternatively spliced CD3ζ 3′-UTR found in SLE T cells may result in decreased HuR binding, representing a possible molecular mechanism contributing to the reduced stability and expression of CD3ζ in SLE.

Ischemia-mediated aggregation of the actin cytoskeleton is one of the major initial events resulting in ischemia-reperfusion injury

Ischemia-reperfusion (IR) injury represents a major clinical challenge, which contributes to morbidity and mortality during surgery. The critical role of natural immunoglobulin M (IgM) and complement in tissue injury has been demonstrated. However, cellular mechanisms that result in the deposition of natural IgM and the activation of complement are still unclear. In this report, using a murine intestinal IR injury model, we demonstrated that the beta-actin protein in the small intestine was cleaved and actin filaments in the columnar epithelial cells were aggregated after a transient disruption during 30 min of ischemia. Ischemia also led to deposition of natural IgM and complement 3 (C3). A low dose of cytochalasin D, a depolymerization reagent of the actin cytoskeleton, attenuated this deposition and also attenuated intestinal tissue injury in a dose-dependent manner. In contrast, high doses of cytochalasin D failed to worsen the injury. These data indicate that ischemia-mediated aggregation of the actin cytoskeleton, rather than its disruption, results directly in the deposition of natural IgM and C3. We conclude that ischemia-mediated aggregation of the actin cytoskeleton leads to the deposition of natural IgM and the activation of complement, as well as tissue injury.

Ubiquitination Regulates Expression of the Serine/Arginine-rich Splicing Factor 1 (SRSF1) in Normal and Systemic Lupus Erythematosus (SLE) T Cells

Background: Mechanisms that control expression of the splicing factor SRSF1 in human T cells are unknown. Results: Ubiquitination and proteasome degradation of SRSF1 occur during T cell activation and in T cells from patients with systemic lupus erythematosus (SLE). Conclusion: Ubiquitin-proteasome degradation regulates SRSF1 expression in human T cells. Significance: Understanding how SRSF1 expression is regulated in SLE may enable new therapeutic approaches. T cells from patients with systemic lupus erythematosus (SLE) exhibit reduced expression of the critical T cell receptor (TCR)-associated CD3ζ signaling chain and are poor producers of the vital cytokine IL-2. By oligonucleotide pulldown and mass spectrometry discovery approaches, we identified the splicing regulator serine/arginine-rich splicing factor (SRSF) 1 or splicing factor 2/alternative splicing factor (SF2/ASF) to be important in the expression of CD3ζ chain. Importantly, increases in the expression of SRSF1 rescued IL-2 production in T cells from patients with SLE. In this study, we investigated the regulation of SRSF1 expression in resting and activated human T cells. We found that T cell stimulation induced a rapid and significant increase in mRNA expression of SRSF1; however, protein expression levels did not correlate with this increase. Co-engagement of CD28 induced a similar mRNA induction and reduction in protein levels. Proteasomal but not lysosomal degradation was involved in this down-regulation as evidenced by blocking with specific inhibitors MG132 and bafilomycin, respectively. Immunoprecipitation studies showed increased ubiquitination of SRSF1 in activated T cells. Interestingly, T cells from patients with SLE showed increased ubiquitination of SRSF1 when compared with those from healthy individuals. Our results demonstrate a novel mechanism of regulation of the splicing factor SRSF1 in human T cells and a potential molecular mechanism that controls its expression in SLE.

Why do women get lupus

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease which affects predominantly women, causing significant morbidity and mortality. A multi-system disease, SLE is characterized by the production and deposition of autoantibodies, and inflammatory cell infiltration leading to damage of target organs such as the skin, kidneys and brain. Both innate and adaptive arms of the immune system are dysregulated and contribute to disease pathogenesis. Nine out of ten patients afflicted with SLE are women, thus gender plays an important role in disease development. The gender bias in SLE reflects the role of sex chromosomes as well as sex hormones. The importance of the X chromosome was shown in a pristane-induced model of lupus — the XX sex chromosome complement conferred increased susceptibility to disease over the XY−mice [1]. The preponderance of disease is seen in the reproductive years such that before puberty, the female tomale prevalence is 3:1—which increases to 9:1 after the onset of puberty, strongly suggesting the role of hormones in disease pathogenesis. Numerous studies have implicated hormones especially estrogen in lupus pathogenesis. While estrogen levels per se are not found to be altered in the serum from lupus patients, the metabolism of this hormone is abnormally high in patients. There is increased oxidation of dehydroepiandrosterone (DHEA) to 16-hydroxyestrone and estriol resulting in increased levels of these metabolites in patients with SLE. Lower levels of androgens specifically DHEA are found in lupus patients [2]. Early studies in animal models of lupus (both NZB/NZW and MRL/lpr) have shown that lupusprone female mice succumbed to disease sooner than male mice. Studies from castrated mice both male and female have shown the important role of hormones in disease pathogenesis. Oopherectomized female mice have prolonged survival whereas castrated male mice that lost their androgen source die earlier. When either female ormale castratedmicewere given androgen supplements, disease improved; whereas estrogen administration worsened disease [2,3]. SLE is characterized by a Th2 cytokine environment, and high doses of estrogen are shown to promote a Th2 cytokine (IL4, IL5, IL6, IL10, TGFβ) profile. On the other hand, studies allude to the suppression of the Th1 (IL2, TNFα, IFNγ) cytokines by estrogen [4,5]. Mice treated with synthetic estrogen were susceptible to Listeria monocytogenes infection and their