Charles F. Spurlock

Expression and functions of long noncoding RNAs during human T helper cell differentiation

Long noncoding RNAs (lncRNAs) regulate an array of biological processes in cells and organ systems. Less is known about their expression and function in lymphocyte lineages. Here we have identified >2000 lncRNAs expressed in human T cell cultures and those which display a TH lineage specific pattern of expression and are intragenic or adjacent to TH lineage specific genes encoding proteins with immunologic functions. One lncRNA cluster selectively expressed by the effector TH2 lineage consists of four alternatively spliced transcripts that regulate expression of TH2 cytokines, IL-4, IL-5 and IL-13. Genes encoding this lncRNA cluster in humans overlap the RAD50 gene and thus are contiguous with the previously described TH2 locus control region (LCR) in the mouse. Given its genomic synteny with the TH2 LCR, we refer to this lncRNA cluster as TH2-LCR lncRNA.

Methotrexate Inhibits NF‐κB Activity Via Long Intergenic (Noncoding) RNA–p21 Induction

To determine interrelationships between the expression of long intergenic (noncoding) RNA–p21 (lincRNA‐p21), NF‐κB activity, and responses to methotrexate (MTX) in rheumatoid arthritis (RA) by analyzing patient blood samples and cell culture models.

Methotrexate increases expression of cell cycle checkpoint genes via JNK activation

OBJECTIVE To assess defects in expression of critical cell cycle checkpoint genes and proteins in patients with rheumatoid arthritis (RA) relative to presence or absence of methotrexate (MTX) treatment, and to investigate the role of JNK in induction of these genes by MTX. METHODS Flow cytometric analysis was used to quantify changes in levels of intracellular proteins, measure reactive oxygen species (ROS), and determine apoptosis in different lymphoid populations. Quantitative reverse transcription-polymerase chain reaction was used to identify changes in cell cycle checkpoint target genes. RESULTS RA patients expressed reduced baseline levels of MAPK9, TP53, CDKN1A, CDKN1B, CHEK2, and RANGAP1 messenger RNA (mRNA) and JNK total protein. The reduction in expression of mRNA for MAPK9, TP53, CDKN1A, and CDKN1B was greater in patients not receiving MTX than in those receiving low-dose MTX, with no difference in expression levels of CHEK2 and RANGAP1 mRNA between MTX-treated and non-MTX-treated patients. Further, JNK levels were inversely correlated with C-reactive protein levels in RA patients. In tissue culture, MTX induced expression of both p53 and p21 by JNK-2- and JNK-1-dependent mechanisms, respectively, while CHEK2 and RANGAP1 were not induced by MTX. MTX also induced ROS production, JNK activation, and sensitivity to apoptosis in activated T cells. Supplementation with tetrahydrobiopterin blocked these MTX-mediated effects. CONCLUSION Our findings support the notion that MTX restores some, but not all, of the proteins contributing to cell cycle checkpoint deficiencies in RA T cells, via a JNK-dependent pathway.

Long non-coding RNAs in innate and adaptive immunity.

Long noncoding RNAs (lncRNAs) represent a newly discovered class of regulatory molecules that impact a variety of biological processes in cells and organ systems. In humans, it is estimated that there may be more than twice as many lncRNA genes than protein-coding genes. However, only a handful of lncRNAs have been analyzed in detail. In this review, we describe expression and functions of lncRNAs that have been demonstrated to impact innate and adaptive immunity. These emerging paradigms illustrate remarkably diverse mechanisms that lncRNAs utilize to impact the transcriptional programs of immune cells required to fight against pathogens and maintain normal health and homeostasis.

Methotrexate induces production of IL-1 and IL-6 in the monocytic cell line U937.

IntroductionMethotrexate (MTX) has been for decades a standard treatment in a wide range of conditions, from malignancies to rheumatoid arthritis (RA). Despite this long experience, the mechanisms of action of MTX remain incompletely understood. Reported immunologic effects of MTX include induction of increased production of some cytokines, an effect that seems to be at odds with the generally anti-inflammatory effects of this drug in diseases like RA. To further elucidate these immune activities, we examined effects of MTX on the human monocytic cell line U937.MethodsThe U937 cell line was treated in vitro with pharmacologic-range concentrations of MTX and effects on production of interleukin (IL)-1, IL-6 and TNF alpha were measured. Changes in gene expression for IL-1 and IL-6 and specificities in the Jun-N-terminal kinase (JNK) signaling pathway including JNK 1, JNK2, JUN and FOS were also determined. The contribution of NF-kB, folate and adenosine pathways to the observed effects was determined by adding appropriate inhibitors to the MTX cultures.ResultsMTX mediated a dose-dependent increase in IL-1 and IL-6 in U937 cells, as measured by secreted proteins and levels of gene expression. The increased cytokine expression was inhibited by addition of parthenolide and folinic acid, but not by caffeine and theophylline, suggesting that NF-kB and folates, but not adenosine, were involved in mediating the observed effects. When U937 cells were cultured with MTX, upregulated expression of JUN and FOS, but not JNK 1 or 2, also was observed.ConclusionsMTX induces expression of proinflammatory cytokines in U937 monocytic cells. These effects might mediate the known toxicities of MTX including pneumonitis, mucositis and decreased bone mineral density.

Expression of IL-33 and its epigenetic regulation in Multiple Sclerosis.

We examined the expression of IL‐33 as an indicator of an innate immune response in relapsing remitting MS (RRMS) and controls. We proposed a link between the expression of IL‐33 and IL‐33 regulated genes to histone deacetylase (HDAC) activity and in particular HDAC3, an enzyme that plays a role in the epigenetic regulation of a number genes including those which regulate inflammation.

Long noncoding RNAs in T lymphocytes

Long noncoding RNAs are recently discovered regulatory RNA molecules that do not code for proteins but influence a vast array of biologic processes. In vertebrates, the number of long noncoding RNA genes is thought to greatly exceed the number of protein‐coding genes. It is also thought that long noncoding RNAs drive the biologic complexity observed in vertebrates compared with that in invertebrates. Evidence of this complexity has been found in the T‐lymphocyte compartment of the adaptive immune system. In the present review, we describe our current level of understanding of the expression of specific long or large intergenic or intervening long noncoding RNAs during T‐lymphocyte development in the thymus and differentiation in the periphery and highlight the mechanisms of action that specific long noncoding RNAs employ to regulate T‐lymphocyte function, both in vitro and in vivo.

Cutting Edge: Chronic NF-κB Activation in CD4+ T Cells in Rheumatoid Arthritis Is Genetically Determined by HLA Risk Alleles

Of identified genetic variants, HLA polymorphisms confer the greatest risk for developing autoimmune diseases, including rheumatoid arthritis (HLA-DRB1*04). There are strong influences of HLA polymorphisms on cell type–specific gene expression in B cells and monocytes. Their influence on gene expression in CD4+ T cells is not known. We determined transcript and proteins levels of target genes in lymphocyte/monocyte subsets in healthy controls and rheumatoid arthritis subjects as a function of HLA-DRB1*04 haplotype. We identified gene expression dependent on HLA-DRB1*04 genotype in CD4+ T cells. NF-κB activity in CD4+ T cells was also dependent on HLA-DRB1*04 genotype, and blocking HLA-DR inhibited NF-κB activity in CD4+ T cells and normalized gene expression, as did pharmacologic inhibition of NF-κB. We conclude that interactions between TCR and MHC class II encoded by HLA-DRB1*04 create a proinflammatory “hum” altering CD4+ T cell phenotype.

Biogenesis and Transcriptional Regulation of Long Noncoding RNAs in the Human Immune System

The central dogma of molecular biology states that DNA makes RNA makes protein. Discoveries over the last quarter of a century found that the process of DNA transcription into RNA gives rise to a diverse array of functional RNA species, including genes that code for protein and noncoding RNAs. For decades, the focus has been on understanding how protein-coding genes are regulated to influence protein expression. However, with the completion of the Human Genome Project and follow-up ENCODE data, it is now appreciated that only 2–3% of the genome codes for protein-coding gene exons and that the bulk of the transcribed genome, apart from ribosomal RNAs, is at the level of noncoding RNA genes. In this article, we focus on the biogenesis and regulation of a distinct class of noncoding RNA molecules termed long, noncoding RNAs in the context of the immune system.

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.