Expression of long non-coding RNAs in autoimmunity and linkage to enhancer function and autoimmune disease risk genetic variants.

Genome-wide association studies have identified numerous genetic variants conferring autoimmune disease risk. Most of these genetic variants lie outside protein-coding genes hampering mechanistic explorations. Numerous mRNAs are also differentially expressed in autoimmune disease but their regulation is also unclear. The majority of the human genome is transcribed yet its biologic significance is incompletely understood. We performed whole genome RNA-sequencing [RNA-seq] to categorize expression of mRNAs, known and novel long non-coding RNAs [lncRNAs] in leukocytes from subjects with autoimmune disease and identified annotated and novel lncRNAs differentially expressed across multiple disorders. We found that loci transcribing novel lncRNAs were not randomly distributed across the genome but co-localized with leukocyte transcriptional enhancers, especially super-enhancers, and near genetic variants associated with autoimmune disease risk. We propose that alterations in enhancer function, including lncRNA expression, produced by genetics and environment, change cellular phenotypes contributing to disease risk and pathogenesis and represent attractive therapeutic targets.

Reciprocal regulation of Rag expression in thymocytes by the zinc-finger proteins, Zfp608 and Zfp609.

Recombination-activating gene 1 (Rag1) and Rag2 enzymes are required for T cell receptor assembly and thymocyte development. The mechanisms underlying the transcriptional activation and repression of Rag1 and Rag2 are incompletely understood. The zinc-finger protein, Zfp608, represses Rag1 and Rag2 expression when expressed in thymocytes blocking T-cell maturation. Here we show that the related zinc-finger protein, Zfp609, is necessary for Rag1 and Rag2 expression in developing thymocytes. Zfp608 represses Rag1 and Rag2 expression indirectly by repressing the expression of Zfp609. Thus, the balance of Zfp608 and Zfp609 plays a critical role in regulating Rag1 and Rag2 expression, which may manifest itself not only during development of immature thymocytes into mature T cells but also in generation of the T-cell arm of the adaptive immune system, which does not fully develop until after birth.

Lineage-specific adjacent IFNG and IL26 genes share a common distal enhancer element.

Certain groups of physically linked genes remain linked over long periods of evolutionary time. The general view is that such evolutionary conservation confers 'fitness' to the species. Why gene order confers 'fitness' to the species is incompletely understood. For example, linkage of IL26 and IFNG is preserved over evolutionary time yet Th17 lineages express IL26 and Th1 lineages express IFNG. We considered the hypothesis that distal enhancer elements may be shared between adjacent genes, which would require linkage be maintained in evolution. We test this hypothesis using a bacterial artificial chromosome transgenic model with deletions of specific conserved non-coding sequences. We identify one enhancer element uniquely required for IL26 expression but not for IFNG expression. We identify a second enhancer element positioned between IL26 and IFNG required for both IL26 and IFNG expression. One function of this enhancer is to facilitate recruitment of RNA polymerase II to promoters of both genes. Thus, sharing of distal enhancers between adjacent genes may contribute to evolutionary preservation of gene order.

Gene-expression signatures: biomarkers toward diagnosing multiple sclerosis.

Identification of biomarkers contributing to disease diagnosis, classification or prognosis could be of considerable utility. For example, primary methods to diagnose multiple sclerosis (MS) include magnetic resonance imaging and detection of immunological abnormalities in cerebrospinal fluid. We determined whether gene-expression differences in blood discriminated MS subjects from comparator groups, and identified panels of ratios that performed with varying degrees of accuracy depending upon complexity of comparator groups. High levels of overall accuracy were achieved by comparing MS with homogeneous comparator groups. Overall accuracy was compromised when MS was compared with a heterogeneous comparator group. Results, validated in independent cohorts, indicate that gene-expression differences in blood accurately exclude or include a diagnosis of MS and suggest that these approaches may provide clinically useful prediction of MS.

Peripheral blood gene expression profiles in metabolic syndrome, coronary artery disease and type 2 diabetes.

To determine if individuals with metabolic disorders possess unique gene expression profiles, we compared transcript levels in peripheral blood from patients with coronary artery disease (CAD), type 2 diabetes (T2D) and their precursor state, metabolic syndrome to those of control (CTRL) subjects and subjects with rheumatoid arthritis (RA). The gene expression profile of each metabolic state was distinguishable from CTRLs and correlated with other metabolic states more than with RA. Of note, subjects in the metabolic cohorts overexpressed gene sets that participate in the innate immune response. Genes involved in activation of the pro-inflammatory transcription factor, NF-κB, were overexpressed in CAD whereas genes differentially expressed in T2D have key roles in T-cell activation and signaling. Reverse transcriptase PCR validation confirmed microarray results. Furthermore, several genes differentially expressed in human metabolic disorders have been previously shown to participate in inflammatory responses in murine models of obesity and T2D. Taken together, these data demonstrate that peripheral blood from individuals with metabolic disorders display overlapping and non-overlapping patterns of gene expression indicative of unique, underlying immune processes.

Selective matrix attachment regions in T helper cell subsets support loop conformation in the Ifng gene.

Cytokine genes undergo progressive changes in chromatin organization when naïve CD4+ T helper (Th) cells differentiate into committed Th1 and Th2 lineages. Here, we analyzed nuclear matrix attachment regions (MARs) in the Ifng gene by DNA array technique in unactivated and activated CD4+ Th cells. This approach was combined with analysis of spatial organization of the Ifng gene by chromosome conformation capture approach to assess the relationship between the gene conformation and matrix attachment organization in functionally different cell subsets. We report that the Ifng gene in unactivated cells displays a linear conformation, but in T-cell receptor-activated cells, it adopts a loop conformation. The selective MARs support the spatial gene organization and characteristically define the Ifng gene in functionally different cell subsets. The pattern of interaction of the Ifng gene with the nuclear matrix dynamically changes in a lineage-specific manner in parallel with the changes in Ifng gene conformation. The data suggest that such structural dynamics provide the means for transcriptional regulation of the Ifng gene in the course of activation and differentiation of CD4+Th cells.

Deregulated stress system in non-obese diabetic lymphocyte.

Lymphopenia-induced homeostatic expansion in non-obese diabetic (NOD) mice may lead to autoimmunity. We demonstrated that NOD lymphocytes are more susceptible to apoptosis than those of non-diabetic C57BL/6 or NOD.H2(h4) mice in vivo and in vitro, which may be an underlying mechanism causing lymphopenia in NOD mice. Gene expression profiling identified a set of genes that are differentially expressed between NOD and B6 mice. Identity of these genes suggested that NOD T cells have a deregulated stress response system, especially heat-shock protein family, making them overly sensitive to apoptosis. Thus, we hypothesize that this strain-specific gene expression profile may confer a liability upon NOD T cells making them more susceptible to apoptosis that may lead to lymphopenia in NOD mice and contribute to development of autoimmunity.

Highly conserved gene expression profiles in humans with allergic rhinitis altered by immunotherapy.

BACKGROUND: Atopic diseases, resulting from hypersensitivity to a wide variety of allergens, affect 10-20% of the population. Immunotherapy is an effective treatment for atopic diseases, but its mechanisms are not fully understood. OBJECTIVE: We studied gene expression profiles in the peripheral blood mononuclear cells (PBMC) and examined whether the individuals with allergic rhinitis (AR) have a unique gene expression profile and how the immunotherapy affect the gene expression profiles. METHODS: We used cDNA microarray and 'expression analysis systemic explorer' to examine the gene expression profiles in the PBMC of atopic subjects and other groups. RESULTS: We identified a highly conserved gene expression profile in atopic subjects that permitted their accurate segregation from control or autoimmune subjects. A major feature of this profile was the under-expression of a variety of genes that encode proteins required for apoptosis and over-expression of genes that encode proteins critical for stress responses and signal transduction. We also identified 563 genes that can segregate individuals with AR based upon receipt of immunotherapy. CONCLUSION: There is a highly conserved gene expression profile in the PBMC of individuals with AR. This profile can be used to identify individuals with AR and to evaluate responses to immunotherapy. Quantitative endpoints, such as gene expression, may assist clinicians faced with clinical decisions in the diagnosis of patients and the evaluation of response to therapy. The knowledge of the possible genetic basis for immunotherapy efficacy may also lead to novel therapeutic approaches for atopic diseases.

A gene expression signature for recent onset rheumatoid arthritis in peripheral blood mononuclear cells.

BACKGROUND: In previous studies the presence of a distinct gene expression pattern has been shown in peripheral blood cells from patients with autoimmune disease. OBJECTIVE: To determine whether other specific signatures might be used to identify subsets of these autoimmune diseases and whether gene expression patterns in early disease might identify pathogenetic factors. METHODS: Peripheral blood mononuclear cells were acquired from patients with rheumatoid arthritis (RA) and analysed by microarrays containing over 4300 named human genes. Patients with RA for <2 years were compared with subjects with longstanding RA (average duration 10 years) and with patients with other immune or autoimmune diagnoses. RESULTS: Cluster analyses permitted separation of the patients with early RA (ERA) from those with longstanding disease. Comparison with other patient groups suggested that the ERA signature showed some overlap with that seen in the normal immune response to viral antigen as well as with a subset of patients with systemic lupus erythematosus. CONCLUSIONS: The ERA signature may reflect, in part, a response to an unknown infectious agent. Furthermore, shared features with some lupus patients suggest that common aetiological factors and pathogenetic pathways may be involved in these two autoimmune disorders.

Transcriptional reprogramming during T helper cell differentiation.

Our laboratory employs reporter transgenic mice as model systems to study the transcriptional reprogramming that accompanies T helper cell differentiation. These studies demonstrate that changes in the activity of simple transcriptional elements associated with the IFN-gamma gene can recapitulate alterations in gene expression. In addition, our studies have revealed a key role for the transcription factor, CAMP response element binding protein (CREB), in the protection of differentiating T cells from apoptosis. Together, these findings further our understanding of the logic employed by T cells to alter gene expression profiles in response to differentiation signals.

Defective Th function induced by a dominant-negative cAMP response element binding protein mutation is reversed by Bcl-2.

cAMP response element binding protein (CREB) is a critical regulator of diverse stimulus-dependent transcriptional events. Following TCR stimulation, CREB is rapidly induced in CD4+ Th cell precursors, but not in effector Th cells. However, its role in mature T cell function is incompletely defined. Transgenic mice expressing a CREB dominant-negative (dn) mutation in the T cell lineage exhibited normal T cell development in the thymus, normal T cell homeostasis in the periphery, and normal T cell clonal expansion following Ag challenge. However, this mutation caused selective inhibition of Th cell function in vitro and in vivo, and increased susceptibility of Th cells to activation-induced cell death. Th cells expressing the CREB-dn mutation contained reduced levels of the inhibitor of programmed cell death, BCL-2; overexpression of BCL-2 in transgenic mice reversed both susceptibility to activation-induced cell death in CREB-dn T cells and the defect in effector cytokine production. Thus, CREB plays a critical role in Th cell function and development of Th cell-mediated adaptive immune responses, at least in part, by inhibiting stimulus-dependent cell death.

TCR and IL-12 receptor signals cooperate to activate an individual response element in the IFN-gamma promoter in effector Th cells.

IFN-gamma is a key regulatory cytokine of the immune system. Reporter transgenic mice expressing the luciferase gene under the control of separate TCR-response elements (TCR-RE) from the IFN-gamma promoter or expressing the green fluorescent protein gene under the control of an IFN-gamma "minigene" were employed to explore the basis for IL-12 regulation of IFN-gamma gene transcription. In the absence of TCR stimulation, IL-12 did not activate the TCR-REs but did induce green fluorescent protein expression. TCR plus IL-12R stimulation of effector Th cells resulted in: 1) enhanced activation of the proximal, but not the distal, TCR-RE, and 2) increased induction of cJun-proximal TCR-RE complexes and c-Jun protein expression. Overexpression of cJun, but not cFos, increased activity of the proximal TCR-RE in T cells. These results suggest that IL-12R signaling affects IFN-gamma gene transcription by at least two separate mechanisms; IL-12R signaling without TCR signaling targets promoter regions outside of the approximately 100-bp IFN-gamma TCR-RE, and IL-12R signaling also stimulates TCR-induced activity of the proximal TCR-RE.

Costimulation reverses the defect in IL-2 but not effector cytokine production by T cells with impaired IkappaBalpha degradation.

Although the transcriptional basis for states of unresponsiveness in primary T cells is unclear, tolerant B lymphocytes exhibit inhibition of both c-Jun N-terminal kinase induction and IkappaBalpha (inhibitor of NF-kappaBalpha) degradation, leading to lower levels of both nuclear AP-1 and NF-kappaB. Expression of an IkappaBalpha mutant resistant to signal-induced degradation in transgenic T cells caused markedly deficient effector cytokine (IL-4, IFN-gamma) production after primary TCR stimulation despite a detectable level of nuclear NF-kappaB. A TCR response element from the IFN-gamma promoter, despite lacking detectable NF-kappaB/Rel sites, was also unresponsive to TCR ligation. Nuclear induction of AP-1 proteins in response to T cell activation was diminished in transgenic T cells. Costimulation induced by anti-CD28 mAb increased IL-2 production, but failed to reverse the defects in effector cytokine production. Taken together, these data indicate that impaired NF-kappaB/Rel signaling in T cells interferes with the signal transduction pathways required for efficient induction of effector cytokine production.

Regulation of the activity of IFN-gamma promoter elements during Th cell differentiation.

Before they can deliver their effector functions, CD4+ Th cells must differentiate into Th1 or Th2 subsets. We have prepared reporter transgenic mice that express the luciferase gene under the control of proximal (prox.IFN-gamma) and distal (dist.IFN-gamma) regulatory elements from the IFN-gamma promoter to permit investigation of mechanisms that regulate IFN-gamma gene transcription during Th cell differentiation. Precursor Th cells (pTh) contain high levels of cAMP response element binding protein-activation transcription factor-1 (CREB-ATF1) proteins that bind these promoter elements from the IFN-gamma gene, and these cells fail to express promoter activity. Restimulated effector Th (eTh) cells have reduced levels of CREB-ATF1 proteins, their nuclear extracts exhibit reduced CREB-ATF1 binding and greater Jun and Jun-ATF2 binding to dist.IFN-gamma), and eTh cells express promoter activity. CREB directly competes with effector T cell nuclear proteins for dist.IFN-gamma binding, and overexpression of CREB inhibits both prox.IFN-gamma- and dist.IFN-gamma-directed transcription in Jurkat T cells. IL-12-stimulated Thl differentiation increases dist.IFN-gamma activity in restimulated eTh1 cells; eTh1 nuclear extracts form increased levels of Jun-ATF2-dist.IFN-gamma complexes. Taken together, these data suggest that both de-repression and trans-activation contribute to the induction of IFN-gamma gene transcription during Th1 differentiation.

Differential expression of transcription directed by a discrete NF-AT binding element from the IL-4 promoter in naive and effector CD4 T cells.

Acquiring the ability to selectively produce IFN-gamma or IL-4 is a fundamental property of the immune system and enables T cell subsets (Th1, Th2) to deliver their effector functions. To create an experimental system to examine regulation of critical promoter elements within the IL-4 gene, we have prepared and analyzed transgenic mice expressing the luciferase gene under the control of the distal NF-AT binding site from the IL-4 promoter. This site is immediately adjacent to an AP-1 site, and this NF-AT/AP-1 composite site is termed either P1 or PubB. The distal NF-AT site we have used to prepare these reporter transgenic mice lacks the AP-1 binding site but contains the NF-AT binding site (P1(NF-AT)) These transgenic mice were also intercrossed with transgenic mice expressing a single MHC class II-restricted TCR. Expression of transcriptional activity under the control of P1(NF-AT) was observed only in effector T cells, and not naive T cells, after stimulation with Ag or polyclonal stimuli. By contrast, gel mobility shift assays showed that nuclear extracts from both naive and effector T cells contained NF-AT, which could effectively bind to the P1(NF-AT) element. IL-4-stimulated Th2 differentiation did not increase the TCR responsiveness of the P1(NF-AT) element by more than 2-fold but increased production of IL-4 protein by more than 10-fold. These data suggest that factors interacting with the P1(NF-AT) element regulate transcriptional activity in a naive/effector T cell-specific manner but not in a Th1/Th2-specific manner. Th1/Th2-specific regulation of the composite P1 element may result from regulation of transacting factors that bind to the AP-1 portion of this element.

Differential transcription directed by discrete gamma interferon promoter elements in naive and memory (effector) CD4 T cells and CD8 T cells.

Acquisition of the ability to produce gamma interferon (IFN-gamma) is a fundamental property of memory T cells and enables one subset (T helper 1 [TH1]) to deliver its effector functions. To examine regulation of IFN-gamma gene expression in a model system which recapitulates TH1 differentiation, we prepared reporter transgenic mice which express the luciferase gene under the control of proximal and distal regulatory elements (prox.IFN gamma and dist.IFN gamma) from the IFN-gamma promoter. Memory T cells, but not naive T cells, secreted IFN-gamma and expressed both prox.IFN gamma and dist.IFN gamma transcriptional activities. Naive T cells required priming to become producers of IFN-gamma and to direct transcription by these elements. While both CD4+ and CD8+ T cells produced IFN-gamma, only CD4+ T cells expressed prox.IFN gamma transcriptional activity. Induction of transcriptional activity was inhibited by known antagonists of effector T-cell populations. Cyclosporin A inhibited transcriptional activity directed by both elements in effector T cells. Elevated cyclic AMP inhibited transcriptional activity directed by prox.IFN gamma in primed CD4+ T cells but enhanced transcriptional activity directed by dist.IFN gamma in primed CD8+ T cells. Taken together, these data show that prox.IFN gamma and dist.IFN gamma transcriptional activities mirror IFN-gamma gene expression in naive and memory CD4+ T cells but suggest that differences exist in regulation of IFN-gamma gene expression in CD4+ and CD8+ T-cell subsets.

Inhibitors of serotonin synthesis and antagonists of serotonin 1A receptors inhibit T lymphocyte function in vitro and cell-mediated immunity in vivo.

The serotonin (5HT1A) receptor subtype is one member of the 5HT1 receptor family and is constitutively expressed on Jurkat cells and is elevated on human T lymphocytes after mitogenic activation. Published reports show that human T lymphocytes and monocytes also release 5HT after stimulation with PHA or IFN-gamma. In lymphocytes and the central nervous system, the 5HT1A receptor is coupled to regulation of adenylate cyclase. The 5HT1A receptor agonists inhibit activation of adenylate cyclase. The purpose of the experiments reported here was to investigate further the role 5HT and the 5HT1A receptor may play in the regulation of human and murine T cell activity. For this purpose, human PBMC or murine spleen cells were used for experimental purposes rather than Jurkat cells. The results show that inhibition of 5HT synthesis inhibits IL-2-stimulated human T cell proliferation and that addition of 5-hydroxytryptophan, a precursor of 5HT, reverses inhibition of T cell proliferation. The 5HT1 receptor antagonist, metitepine, and the 5HT1A selective antagonist, pindobind-5HT1A, also block T cell proliferation. Inhibition by metitepine is reversed by 5HT and by the selective 5HT1A receptor agonist, 8-hydroxy-2-(di-n-propylamino) (8-OH-DPAT). Selective 5HT1A receptor antagonists cause elevation of cAMP in human T cells. In a murine model, selective 5HT1A receptor antagonists inhibit contact sensitivity responses but not Ab responses to oxazalone in vivo. Inhibition is reversed by 8-OH-DPAT. In addition, production of Th1 cytokines, such as IL-2 and IFN-gamma, by Ag-stimulated, immune murine spleen cells is inhibited by 5HT1A receptor antagonists in vitro but not by 5HT1C/2 receptor antagonists.

Elevation of intracellular cAMP in human T lymphocytes by an anti-CD44 mAb.

Regulation of lymphocyte responses to activation of the CD3/TCR complex by other cell surface proteins expressed on lymphocytes is a well established phenomenon. CD44 is an example of such a cell surface protein. Anti-CD44 mAb have been identified which either stimulate or inhibit lymphocyte function. Certain anti-CD44 mAb augment proliferation and IL-2 production by T cells stimulated through the CD2 or CD3/TCR pathways. An anti-CD44 mAb with opposing properties has also been identified. This mAb inhibits activation of human T cells by preventing the rise in [Ca2+]i stimulated by OKT3. The purpose of experiments reported here was to further characterize this phenomenon. The results show that the anti-CD44 mAb, 212.3, does not inhibit inositol phosphate turnover stimulated by OKT3 but does inhibit elevation of intracellular [Ca2+]i in these same cells. Addition of 212.3 to purified human T cells results in a rapid increase in intracellular levels of cAMP. Elevation of cAMP by 212.3 is time- and concentration-dependent. Activation of adenylate cyclase by forskolin also results in elevation of intracellular cAMP and inhibition of the increase in [Ca2+]i stimulated by OKT3. Taken together, these data suggest that CD44 may be positively coupled to adenylate cyclase and that activation of adenylate cyclase by the anti-CD44 mAb, 212.3, may mediate the inhibition of the OKT3-stimulated elevation of [Ca2+]i.

Cell contact between T cells and synovial fibroblasts causes induction of adhesion molecules and cytokines.

Human activated T cells adhere to synovial fibroblast-like cells in vitro. The present study was conducted to investigate the consequences of T cell-synovial fibroblast interactions with regard to induction of adhesion molecules and proinflammatory cytokines. A sensitive Western blot technique, polymerase chain reaction (PCR) amplification, and fluorescence-activated cell sorter (FACS) analysis were used to analyze the induction of VCAM-1 and ICAM-1 expression in T cell-synovial fibroblast cocultures. VCAM-1 and ICAM-1 expression could be induced in synovial fibroblast-like cells by 2 h. PCR amplification showed that both forms of VCAM-1 mRNA are found after the interaction of synovial fibroblasts with T cells. Up-regulation of VCAM-1 and ICAM-1 was confined to synovial fibroblasts; T cells did not express VCAM-1 or increased ICAM-1. In contrast to the T cell-synoviocyte interaction, the interaction between T cells and dermal fibroblasts resulted in the up-regulation of ICAM-1 but not VCAM-1, suggesting tissue-specific regulation of VCAM-1. The T cell-synovial fibroblast interaction also resulted in increased levels of tumor necrosis factor (TNF), interferon-gamma, and interleukin-6 in coculture supernatant. Of the neutralizing antibodies used against these cytokines, only anti-TNF could significantly inhibit VCAM-1 and ICAM-1 expression. When T cells were separated from synoviocytes by a chamber that allowed medium exchange but no cell contact, VCAM-1 and ICAM-1 failed to be up-regulated and cytokine accumulation in cocultures was drastically reduced. Our results demonstrate mutual cell activation of T cells and synoviocytes upon cell contact as shown by the release of T cell- and synoviocyte-specific cytokines and suggest a cell contact-mediated and T cell-initiated mechanism for the chronic accumulation and retention of mononuclear cells via VCAM-1/ICAM-1 by synovial fibroblasts in the rheumatoid synovium.

Expression of 5HT1a receptors on activated human T cells. Regulation of cyclic AMP levels and T cell proliferation by 5-hydroxytryptamine.

The neurotransmitter, serotonin (5-hydroxytryptamine, 5HT), has been shown to affect function of cells of the immune system. More recently, specific 5HT receptors have been identified and partially characterized on Jurkat cells. Results presented here characterize the receptor on Jurkat cells as the 5HT1a receptor subtype and show that mitogen-activated but not resting human T cells also express the 5HT1a receptor subtype. Analysis of mRNA in Jurkat cells and activated and resting T cells by PCR or by Northern analysis revealed the presence of 5HT1a receptor. Pharmacologic analysis of this receptor demonstrated that the receptors on Jurkat cells and activated T cells are similar to each other and that they resemble the 5HT1a receptor found in the brain. Analysis of the second messenger pathways activated by the 5HT1a receptor show that ligand-binding to the Jurkat cell 5HT1a receptor results in elevation of intracellular inositol phosphates and Ca2+ and that ligand binding to the 5HT1a receptor on activated T cells modulated intracellular levels of cAMP.