Brief report: increased expression of a short splice variant of CTLA-4 exacerbates lupus in MRL/lpr mice.

OBJECTIVE: CTLA-4 is a negative regulator of the immune response expressed by regulatory T (Treg) cells and activated T cells. Polymorphisms in the CTLA4 gene have been associated with autoimmune diseases, including systemic lupus erythematosus. Disease-associated polymorphisms have been shown to affect the production of the different CTLA-4 variants through an effect on alternative splicing. This study was undertaken to evaluate the role of the 1/4 CTLA-4 isoform in lupus-prone mice. METHODS: We generated an MRL/lpr mouse strain that transgenically overexpresses a short isoform of CTLA-4 (1/4 CTLA-4) by backcrossing C57BL/6.1/4CTLA-4-transgenic mice to the MRL/lpr strain for 9 generations. A new antibody was generated to detect the expression of the 1/4 CTLA-4 isoform. Routine methods were used to evaluate kidney damage, humoral immunity, and cellular immunity. RESULTS: Expression of the 1/4 CTLA-4 isoform accelerated autoimmune disease. Transgenic mice died earlier, had more severe renal disease, and had higher titers of anti-double-stranded DNA antibodies than wild-type MRL/lpr mice. The acceleration of autoimmunity and disease pathology associated with the presence of the short (1/4) isoform of CTLA-4 was linked to increased numbers of activated T cells and B cells and heightened interferon-γ production, but not to altered expression of the full-length CTLA-4 molecule or Treg cell numbers. CONCLUSION: Our results indicate that the presence of the alternatively spliced 1/4 CTLA-4 isoform can further promote autoimmunity and autoimmune pathology in lupus-prone mice and suggest that altered splicing of CTLA4 contributes to the expression of autoimmune disease.

CREMα suppresses spleen tyrosine kinase expression in normal but not systemic lupus erythematosus T cells.

OBJECTIVE: T cells from patients with systemic lupus erythematosus (SLE) display increased amounts of spleen tyrosine kinase (Syk), which is involved in the aberrant CD3/T cell receptor-mediated signaling process, and increased amounts of CREMα, which suppresses the production of interleukin-2. Syk expression can be suppressed by CREMα. This study was undertaken to investigate why CREMα fails to suppress Syk expression in SLE T cells. METHODS: CREMα was overexpressed in healthy T cells by transfection with CREMα expression vector, and Syk expression and phosphorylation were measured. A newly identified cAMP response element (CRE) site on the SYK promoter was characterized by chromatin immunoprecipitation (ChIP) and electrophoretic mobility shift assay. The CREMα-mediated repression of Syk expression was further evaluated by analyzing SYK promoter activity. T cells from SLE patients and healthy individuals were subjected to ChIP to evaluate CREMα binding and histone H3 acetylation. RESULTS: Increased CREMα levels suppressed Syk expression by direct binding to a CRE site of the SYK promoter in T cells from healthy individuals but failed to do so in T cells from SLE patients. The failure of CREMα to suppress Syk expression in SLE T cells was due to weaker binding to the CRE site of the SYK promoter compared to healthy T cells because the promoter site is hypoacetylated in SLE T cells and therefore of limited access to transcription factors. CONCLUSION: Our findings indicate that epigenetic alteration of the SYK promoter in SLE T cells results in the inability of the transcriptional repressor CREMα to bind and suppress the expression of Syk, resulting in aberrant T cell signaling.

Promoter hypomethylation results in increased expression of protein phosphatase 2A in T cells from patients with systemic lupus erythematosus.

The catalytic subunit α isoform of protein phosphatase 2A (PP2Acα) activity, protein, and mRNA have been found increased in systemic lupus erythematosus (SLE) T cells and to contribute to decreased IL-2 production. The PP2Acα promoter activity is controlled epigenetically through the methylation of a CpG within a cAMP response element (CRE) motif defined by its promoter. We considered that hypomethylation may account for the increased expression of PP2Acα in patients with SLE. Using bisulfite sequencing, we found that SLE T cells displayed decreased DNA methylation in the promoter region compared with normal T cells. More importantly, we found that the CRE-defined CpG, which binds p-CREB, is significantly less methylated in SLE compared with normal T cells, and the levels of methylation correlated with decreased amounts of DNA methyltransferase 1 transcripts. Methylation intensity correlated inversely with levels of PP2Acα mRNA and SLE disease activity. Chromatin immunoprecipitation assays revealed more binding of p-CREB to the CRE site in SLE T cells, resulting in increased expression of PP2Acα. We propose that PP2Acα represents a new methylation-sensitive gene that, like the previously reported CD70 and CD11a, contributes to the pathogenesis of SLE.

Cutting edge: Calcium/Calmodulin-dependent protein kinase type IV is essential for mesangial cell proliferation and lupus nephritis.

Renal involvement in systemic lupus erythematosus remains a major cause of morbidity and mortality. Although immune parameters that instigate renal damage have been characterized, their link to local processes, which execute tissue damage, is poorly understood. Using genetic-deletion and pharmacological-inhibition approaches, we demonstrated that calcium/calmodulin-dependent protein kinase type IV, which contributes to altered cytokine production in systemic lupus erythematosus patients, controls spontaneous and platelet-derived growth factor-stimulated mesangial cell proliferation and promotes IL-6 production through AP-1. Our studies identified calcium/calmodulin-dependent protein kinase type IV as a valuable treatment target for lupus nephritis and point out the importance of local kidney factors in the expression of tissue damage that, if properly targeted, should enhance clinical benefit and limit toxicity.

cAMP-responsive element modulator (CREM)α protein induces interleukin 17A expression and mediates epigenetic alterations at the interleukin-17A gene locus in patients with systemic lupus erythematosus.

IL-17A is a proinflammatory cytokine that is produced by specialized T helper cells and contributes to the development of several autoimmune diseases such as systemic lupus erythematosus (SLE). Transcription factor cAMP-responsive element modulator (CREM)α displays increased expression levels in T cells from SLE patients and has been described to account for aberrant T cell function in SLE pathogenesis. In this report, we provide evidence that CREMα physically binds to a cAMP-responsive element, CRE (-111/-104), within the proximal human IL17A promoter and increases its activity. Chromatin immunoprecipitation assays reveal that activated naïve CD4(+) T cells as well as T cells from SLE patients display increased CREMα binding to this site compared with T cells from healthy controls. The histone H3 modification pattern at the CRE site (-111/-104) and neighboring conserved noncoding sequences within the human IL17A gene locus suggests an accessible chromatin structure (H3K27 hypomethylation/H3K18 hyperacetylation) in activated naïve CD4(+) T cells and SLE T cells. H3K27 hypomethylation is accompanied by decreased cytosine phosphate guanosine (CpG)-DNA methylation in these regions in SLE T cells. Decreased recruitment of histone deacetylase (HDAC)1 and DNA methyltransferase (DNMT)3a to the CRE site (-111/-104) probably accounts for the observed epigenetic alterations. Reporter studies confirmed that DNA methylation of the IL17A promoter indeed abrogates its inducibility. Our findings demonstrate an extended role for CREMα in the immunopathogenesis of SLE because it contributes to increased expression of IL-17A.

Transcriptional activation of the cAMP-responsive modulator promoter in human T cells is regulated by protein phosphatase 2A-mediated dephosphorylation of SP-1 and reflects disease activity in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a complex autoimmune disease with numerous abnormalities recorded at the cellular, molecular, and genetic level. Expression of the basic leucine zipper transcription factor cAMP-responsive element modulator (CREM)α was reported to be abnormally increased in T cells from SLE patients. CREMα suppresses IL-2 and T cell receptor ζ chain gene transcription by direct binding to the respective promoters. Here, we show that increased CREM expression is the result of enhanced promoter activity. DNA binding analyses reveal direct binding of transcription factor specificity protein-1 (SP-1) to the CREM promoter resulting in enhanced transcriptional activity and increased CREM expression. Protein phosphatase 2A is known to activate SP-1 through dephosphorylation at its serine residue 59. Our results show that nuclei from SLE T cells contain lower levels of Ser(59)-phosphorylated SP-1 protein and a stronger SP-1 binding to the CREM promoter. We conclude that protein phosphatase 2A accounts for enhanced SP-1 dephosphorylation at Ser(59) in SLE T cells. More importantly, CREM promoter activity mirrors reliably disease activity in SLE patients, underscoring its potential role as a biomarker for the prediction of flares in SLE patients.

A novel intronic cAMP response element modulator (CREM) promoter is regulated by activator protein-1 (AP-1) and accounts for altered activation-induced CREM expression in T cells from patients with systemic lupus erythematosus.

The transcriptional repressor cAMP response element modulator (CREM) α has important roles in normal T cell physiology and contributes to aberrant T cell function in patients with systemic lupus erythematosus (SLE). Recently, we characterized a specificity protein-1-dependent promoter located upstream of the CREM gene that accounts for increased basal CREM expression in SLE T cells and reflects disease activity. Here, we identify a novel intronic CREM promoter (denoted P2) in front of the second exon of the CREM gene that harbors putative binding sites for TATA-binding proteins and the transcriptional activator AP-1. DNA binding studies, chromatin immunoprecipitation, and reporter assays confirmed the functional relevance of these sites, and T cell activation through CD3/CD28 stimulation or phorbol 12-myristate 13-acetate/ionomycin treatment enhances P2 promoter activity. Although the basal CREM levels are increased in T cells from SLE patients compared with healthy controls, there are remarkable differences in the regulation of CREM expression in response to T cell activation. Whereas T cells from healthy individuals display increased CREM expression after T cell activation, most likely through AP-1-dependent up-regulation of the P2 promoter, SLE T cells fail to further increase their basal CREM levels upon T cell activation due to a decreased content of the AP-1 family member c-Fos. Because CREM trans-represses c-fos transcription in SLE T cells, we propose an autoregulatory feedback mechanism between CREM and AP-1. Our findings extend the understanding of CREM gene regulation in the context of T cell activation and disclose another difference in the transcriptional machinery in SLE T cells.

Suppression of autoimmunity and organ pathology in lupus-prone mice upon inhibition of calcium/calmodulin-dependent protein kinase type IV.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a chronic inflammatory disease associated with aberrant immune cell function. Treatment involves the use of indiscriminate immunosuppression, which results in significant side effects. SLE T cells express high levels of calcium/calmodulin-dependent protein kinase type IV (CaMKIV), which translocates to the nucleus upon engagement of the T cell receptor-CD3 complex and accounts for abnormal T cell function. The purpose of this study was to determine whether inhibition of CaMKIV would improve disease pathology. METHODS: We treated MRL/lpr mice with KN-93, a CaMKIV inhibitor, starting at week 8 or week 12 of age and continuing through week 16 and evaluated skin lesions, proteinuria, kidney histopathology, proinflammatory cytokine production, and costimulatory molecule expression. We also determined the effect of silencing of CAMK4 on interferon-γ (IFNγ) expression by human SLE T cells. RESULTS: CaMKIV inhibition in MRL/lpr mice resulted in significant suppression of nephritis and skin disease, decreased expression of the costimulatory molecules CD86 and CD80 on B cells, and suppression of IFNγ and tumor necrosis factor α production. In human SLE T cells, silencing of CAMK4 resulted in suppression of IFNγ production. CONCLUSION: We conclude that suppression of CaMKIV mitigates disease development in lupus-prone mice by suppressing cytokine production and costimulatory molecule expression. Specific silencing of CAMK4 in human T cells results in similar suppression of IFNγ production. Our data justify the development of small-molecule CaMKIV inhibitors for the treatment of patients with SLE.

Methylation status of CpG islands flanking a cAMP response element motif on the protein phosphatase 2Ac alpha promoter determines CREB binding and activity.

Protein phosphatase 2A (PP2A) is a major serine/threonine protein phosphatase in eukaryotic cells and is involved in many essential aspects of cell function. The catalytic subunit of the enzyme (PP2Ac), a part of the core enzyme, has two isoforms, alpha (PP2Ac alpha) and beta (PP2Ac beta), of which PP2Ac alpha is the major form expressed in vivo. Deregulation of PP2A expression has been linked to several diseases, but the mechanisms that control the expression of this enzyme are still unclear. We conducted experiments to decipher molecular mechanisms involved in the regulation of the PP2Ac alpha promoter in human primary T cells. After preparing serially truncated PP2Ac alpha promoter luciferase constructs, we found that the region stretching around 240 bases upstream from the translation initiation site was of functional significance and included a cAMP response element motif flanked by three GC boxes. Shift assays revealed that CREB/phosphorylated CREB and stable protein 1 could bind to the region. Furthermore, we demonstrated that methylation of deoxycytosine in the CpG islands limited binding of phosphorylated CREB and the activity of the PP2Ac alpha promoter. In contrast, the binding of stable protein 1 to a GC box within the core promoter region was not affected by DNA methylation. Primary T cells treated with 5-azacitidine, a DNA methyltransferase inhibitor, showed increased expression of PP2Ac alpha mRNA. We propose that conditions associated with hypomethylation of CpG islands, such as drug-induced lupus, permit increased PP2Ac expression.

Differential expression and molecular associations of Syk in systemic lupus erythematosus T cells.

Diminished expression of TCR zeta and reciprocal up-regulation and association of FcRgamma with the TCR/CD3 complex is a hallmark of systemic lupus erythematosus (SLE) T cells. In this study we explored whether differential molecular associations of the spleen tyrosine kinase Syk that preferentially binds to FcRgamma contribute to pathological amplification of signals downstream of this "rewired TCR" in SLE. We detected higher amounts of Syk expression and activity in SLE compared with normal T cells. Selective inhibition of the activity of Syk reduced the strength of TCR-induced calcium responses and slowed the rapid kinetics of actin polymerization exclusively in SLE T cells. Syk and ZAP-70 also associated differently with key molecules involved in cytoskeletal and calcium signaling in SLE T cells. Thus, while Vav-1 and LAT preferentially bound to Syk, phospholipase C-gamma1 bound to both Syk and ZAP-70. Our results show that differential associations of Syk family kinases contribute to the enhanced TCR-induced signaling responses in SLE T cells. Thus, we propose molecular targeting of Syk as a measure to control abnormal T cell responses in SLE.

Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys.

Double negative (DN) T cells are expanded in patients with systemic lupus erythematosus (SLE) and stimulate autoantibody production as efficiently as CD4(+) T cells. In this study, we demonstrate that DN T cells from patients with SLE produce significant amounts of IL-17 and IFN-gamma, and expand when stimulated in vitro with an anti-CD3 Ab in the presence of accessory cells. Furthermore, IL-17(+) and DN T cells are found in kidney biopsies of patients with lupus nephritis. Our findings establish that DN T cells produce the inflammatory cytokines IL-17 and IFN-gamma, and suggest that they contribute to the pathogenesis of kidney damage in patients with SLE.

PP2A dephosphorylates Elf-1 and determines the expression of CD3zeta and FcRgamma in human systemic lupus erythematosus T cells.

T cells from patients with systemic lupus erythematosus are characterized by decreased expression of CD3zeta-chain and increased expression of FcRgamma-chain, which becomes part of the CD3 complex and contributes to aberrant signaling. Elf-1 enhances the expression of CD3zeta, whereas it suppresses the expression of FcRgamma gene and lupus T cells have decreased amounts of DNA-binding 98 kDa form of Elf-1. We show that the aberrantly increased PP2A in lupus T cells dephosphorylates Elf-1 at Thr-231. Dephosphorylation results in limited expression and binding of the 98 kDa Elf-1 form to the CD3zeta and FcRgamma promoters. Suppression of the expression of the PP2A leads to increased expression of CD3zeta and decreased expression of FcRgamma genes and correction of the early signaling response. Therefore, PP2A serves as a central determinant of abnormal T cell function in human lupus and may represent an appropriate treatment target.

IL-17 producing CD4+ T cells mediate accelerated ischemia/reperfusion-induced injury in autoimmunity-prone mice.

Elements of the innate and adaptive immune response have been implicated in the development of tissue damage after ischemic reperfusion (I/R). Here we demonstrate that T cells infiltrate the intestine of C57BL/6 mice subjected to intestinal I/R during the first hour of reperfusion. The intensity of the T cell infiltration was higher in B6.MRL/lpr mice subjected to intestinal I/R and reflected more severe tissue damage than that observed in control mice. Depletion of T cells limited I/R damage in B6.MRL/lpr mice, whereas repletion of B6.MRL/lpr lymph node-derived T cells into the I/R-resistant Rag-1(-/-) mouse reconstituted tissue injury. The tissue-infiltrating T cells were found to produce IL-17. Finally, IL-23 deficient mice, which are known not to produce IL-17, displayed significantly less intestinal damage when subjected to I/R. Our data assign T cells a major role in intestinal I/R damage by virtue of producing the pro-inflammatory cytokine IL-17.

Protein phosphatase 2A is a negative regulator of IL-2 production in patients with systemic lupus erythematosus.

Decreased IL-2 production in systemic lupus erythematosus (SLE) represents a central component of the disease immunopathology. We report that the message, protein, and enzymatic activity of the catalytic subunit of protein phosphatase 2A (PP2Ac), but not PP1, are increased in patients with SLE regardless of disease activity and treatment and in a disease-specific manner. Treatment of SLE T cells with PP2Ac-siRNA decreased the protein levels and activity of PP2Ac in a specific manner and increased the levels of phosphorylated cAMP response element-binding protein and its binding to the IL2 and c-fos promoters, as well as increased activator protein 1 activity, causing normalization of IL-2 production. Our data document increased activity of PP2A as a novel SLE disease-specific abnormality and define a distinct mechanism whereby it represses IL-2 production. We propose the use of PP2Ac-siRNA as a novel tool to correct T cell IL-2 production in SLE patients.

Systemic lupus erythematosus serum IgG increases CREM binding to the IL-2 promoter and suppresses IL-2 production through CaMKIV.

Systemic lupus erythematosus (SLE) T cells express high levels of cAMP response element modulator (CREM) that binds to the IL-2 promoter and represses the transcription of the IL-2 gene. This study was designed to identify pathways that lead to increased binding of CREM to the IL-2 promoter in SLE T cells. Ca(2+)/calmodulin-dependent kinase IV (CaMKIV) was found to be increased in the nucleus of SLE T cells and to be involved in the overexpression of CREM and its binding to the IL-2 promoter. Treatment of normal T cells with SLE serum resulted in increased expression of CREM protein, increased binding of CREM to the IL-2 promoter, and decreased IL-2 promoter activity and IL-2 production. This process was abolished when a dominant inactive form of CaMKIV was expressed in normal T cells. The effect of SLE serum resided within the IgG fraction and was specifically attributed to anti-TCR/CD3 autoantibodies. This study identifies CaMKIV as being responsible for the increased expression of CREM and the decreased production of IL-2 in SLE T cells and demonstrates that anti-TCR/CD3 antibodies present in SLE sera can account for the increased expression of CREM and the suppression of IL-2 production.

Increased expression of STAT3 in SLE T cells contributes to enhanced chemokine-mediated cell migration.

Exposure of T cells to inflammatory cytokines leads to phosphorylation-dependent activation of signal transducer and activator of transcription (STAT) 3. T cells from patients with systemic lupus erythematosus (SLE) display increased levels of total and phosphorylated STAT3 which resides primarily in the nucleus. Increased STAT3 is associated with increased expression of target genes. Silencing of STAT3 expression using a small interfering RNA approach resulted in decreased chemokine-provoked SLE T cell migration. Our data suggest that inhibition of STAT3 expression may reverse the signaling aberrations involved in SLE T cell migration.

Dissecting the molecular mechanisms of TCR zeta chain downregulation and T cell signaling abnormalities in human systemic lupus erythematosus.

Abnormal expression of key signaling molecules and defective function of T lymphocytes play a significant role in the pathogenesis of systemic lupus erythematosus (SLE). Probing on altered expression of genes that may predispose to SLE revealed that the expression of TCR zeta chain is defective in the majority of SLE patients. Current research has been directed towards understanding the molecular basis of TCR zeta chain deficiency and dissecting the T cell signalling abnormalities in SLE T cells. Latest developments suggest that interplay of abnormal transcriptional factor expression, aberrant mRNA processing/editing, unbiquitination, proteolysis, and the effects of oxidative stress as well as changes in chromatin structure invariably contribute to TCR zeta chain deficiency in SLE T cells. On the other hand, multiple factors, including altered receptor structure, modulation of membrane clustering, lipid-raft distribution of signaling molecules, and defective signal silencing mechanisms, play a key role in delivering the increased TCR/CD3-mediated intracellular calcium response in SLE T cells.

Activation of the Ets transcription factor Elf-1 requires phosphorylation and glycosylation: defective expression of activated Elf-1 is involved in the decreased TCR zeta chain gene expression in patients with systemic lupus erythematosus.

Elf-1, a member of the Ets transcription factor family with an estimated molecular mass of 68 kDa, is involved in the transcriptional regulation of several hematopoietic cell genes. It is shown that following O-GlcNAc glycosylation and phosphorylation by PKC theta, the cytoplasm-located, 80-kDa Elf-1 translocates to the nucleus as a 98-kDa protein. In the nucleus, Elf-1 binds to the promoter of the TCR zeta gene and promotes its transcription in Jurkat and fresh human T cells. It is also shown that in the majority of patients with systemic lupus erythematosus (SLE), who are known to express decreased levels of T cell receptor (TCR) zeta chain and mRNA, the 80-kDa Elf-1 protein does not undergo proper post-transcriptional modification, which results in low levels of the 98-kDa protein, lack of Elf-binding to the TCR zeta promoter, and decreased gene transcription. Therefore, a novel activation pathway for a member of the Ets family of transcription factors, which is defective in patients with systemic autoimmunity, has been revealed.

Geldanamycin treatment inhibits hemorrhage-induced increases in KLF6 and iNOS expression in unresuscitated mouse organs: role of inducible HSP70.

The aim of this study was to determine whether hemorrhage affects the levels of a variety of stress-related proteins and whether changes can be inhibited by drugs reported to provide protection from ischemia and reperfusion injury. Male Swiss Webster mice were subjected to a 40% hemorrhage without resuscitation. Western blot analysis indicated that c-Jun (an AP-1 protein), Kruppel-like factor 6 (KFL6), and inducible nitric oxide synthase (iNOS) were upregulated sequentially in that order. Pretreatment of mice with geldanamycin (GA) 16 h before hemorrhage effectively inhibited the expression of the proteins KLF6 and iNOS, whereas caffeic acid phenethyl ester did not. GA pretreatment increased inducible heat shock protein (HSP) 70 but not HSP90 in both sham and hemorrhagic tissues. The overexpressed inducible HSP70 formed complexes with KLF6 and iNOS. These results suggest that GA may be therapeutically useful for reducing hemorrhage-induced injury when used as a presurgical treatment or when added to resuscitation fluids.

Pathogenesis of human systemic lupus erythematosus: recent advances.

Systemic lupus erythematosus (SLE) is an autoimmune disease with manifestations derived from the involvement of multiple organs including the kidneys, joints, nervous system and hematopoietic organs. Immune system aberrations, as well as heritable, hormonal and environmental factors interplay in the expression of organ damage. Recent contributions from different fields have developed our understanding of SLE and reshaped current pathogenic models. Here, we review recent findings that deal with (i) genes associated with disease expression; (ii) immune cell molecular abnormalities that lead to autoimmune pathology; (iii) the role of hormones and sex chromosomes in the development of disease; and (iv) environmental and epigenetic factors thought to contribute to the expression of SLE. Finally, we highlight molecular defects intimately associated with the disease process of SLE that might represent ideal therapeutic targets and disease biomarkers.