The circular RNA landscape in multiple sclerosis: Disease-specific associated variants and exon methylation shape circular RNA expression profile

BACKGROUND: Circular RNAs (circRNAs) are a class of non-coding RNAs increasingly emerging as crucial actors in the pathogenesis of human diseases, including autoimmune and neurological disorders as multiple sclerosis (MS). Despite several efforts, the mechanisms regulating circRNAs expression are still largely unknown and the circRNA profile and regulation in MS-relevant cell models has not been completely investigated. In this work, we aimed at exploring the global landscape of circRNA expression in MS patients, also evaluating a possible correlation with their genetic and epigenetic background. METHODS: We performed RNA-seq experiments on circRNA-enriched samples, derived from peripheral blood mononuclear cells (PBMCs) of 10 MS patients and 10 matched controls and performed differential circRNA expression. The genetic background was evaluated using array genotyping, and an expression quantitative trait loci (eQTL) analysis was carried out. RESULTS: Expression analysis revealed 166 differentially expressed circRNAs in MS patients, 125 of which are downregulated. One of the top dysregulated circRNAs, hsa_circ_0007990, derives from the PGAP3 gene, encoding a protein relevant for the control of autoimmune responses. The downregulation of this circRNA was confirmed in two independent replication cohorts, suggesting its implementation as a possible RNA-based biomarker. The eQTL analysis evidenced a significant association between 89 MS-associated loci and the expression of at least one circRNA, suggesting that MS-associated variants could impact on disease pathogenesis by altering circRNA profiles. Finally, we found a significant correlation between exon methylation and circRNA expression levels, supporting the hypothesis that epigenetic features may play an important role in the definition of the cell circRNA pool. CONCLUSION: We described the circRNA expression profile of PBMCs in MS patients, suggesting that MS-associated variants may tune the expression levels of circRNAs acting as circ-QTLs , and proposing a role for exon-based DNA methylation in regulating circRNA expression

5-HT reduces human CD4⁺ T cell migration towards CXCL12 in a dose-dependent manner.

<p>Human primary (n = 3, Panel A; n = 3, Panel B) or murine naive (n = 3, Panel A) CD4⁺ T cells were treated with the indicated concentrations of 5-HT for 30 min at RT and then allowed to migrate towards CXCL12. Number of migrated human CD4⁺ T cells resulted 1232±354 compared to 685±179 for untreated and 5-HT-treated cells, respectively (mean ± SEM; Panel A). Percentages of migrated cells for each condition were calculated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022482#s4" target="_blank">Materials and Methods</a>”. Data were statistically analyzed with either <i>t</i>-test (A) or Bonferroni's multiple comparison test (B); *<i>P</i><0.05, ***<i>P</i><0.001.</p

5-HT₃ receptor activation enhances T cell transmigration through CXCL12-coated endothelial cells.

<p>Human resting CD4⁺ T cells were treated with 2.5 µM 2M-5HT or 100 µM ondansetron for 30 min at RT. Then, CD4⁺ T cells were incubated (+) or not (−) with the CXCR4 antagonist AMD3100 (10 µM) and immediately used for a transendothelial migration assay towards CCL2 through CXCL12-coated endothelial cells. In absence of AMD3100, number of migrated control cells resulted 1524±270 compared to 1888±252 and 1234±398 for 5-HT and ondansetron treated cells, respectively (mean ± SEM). Percentages of migrated cells for each condition were calculated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022482#s4" target="_blank">Materials and Methods</a>”. n = 5, *<i>P</i><0.05, Bonferroni's multiple comparison test.</p

5-HT₃ receptor activity selectively modulates CXCL12-induced chemotaxis of human CD4⁺ T cells.

<p>Human resting CD4⁺ T cells were treated with 2.5 µM 2M-5HT (n = 3, Panel A) or 100 µM ondansetron (n = 3, Panel B) for 30 min at RT, and then allowed to migrate towards CXCL12, CCL2 or CCL5. Percentages of migrated cells for each condition were calculated as described in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022482#s4" target="_blank">Materials and Methods</a>”. **<i>P</i><0.01, ***<i>P</i><0.001, <i>t</i>-test.</p

5-HT is produced and released by human CD4⁺ T cells.

<p>(A) Cell-released 5-HT was quantified by EIA. Data are mean of triplicate ± SEM (n = 3). (B) Representative flowcytometric analysis of three experiments performed showing intracellular 5-HT in primary and activated human T cells (MFI, normalized to isotype control staining, was 70.5±3.5 and 3±1 for resting and activated T cells, respectively; n = 3, <i>P</i><0.01).</p

Resting and activated human CD4⁺ T cells express functional 5-HT₃ receptor.

<p>Human CD4⁺ T cells were stimulated with anti-CD3 and anti-CD28 Abs, PHA or PMA plus ionomycin for 72 h. (A) Gene expression for 5-HT_3A receptor subunits was examined by real-time PCR. The graph represents the quantitative analysis of the experiments. Values are expressed as fold increase over resting T cells. Data are mean of triplicates ± SD of the 2^−ΔΔCt (n = 3); *<i>P</i><0.05, **<i>P</i><0.01, <i>t</i>-test. (B) T cell membranes were isolated by ultracentrifugation from total cell lysates and 20 µg/sample were separated on SDS-PAGE gel. A representative immunoblot of three experiments performed showing the expression of 5-HT_3A subunit is reported. Asterisk indicates the band corresponding to 5-HT_3A monomer. (C–G) Freshly isolated CD4⁺ T cells were subjected to electrophysiological patch clamp experiments in the whole-cell configuration. Cells were held at resting potential (−50 mV) and voltage pulses of 400 ms duration were applied in 20 mV steps ranging from −130 to −10 mV. Currents were recorded before (C, left panel) and after (C, right panel) addition of 3 µM 5-HT to the bath solution. Addition of 5-HT induced an inward current at hyperpolarizing voltages. Activity of Kv1.3 resulting in outward potassium current was seen at depolarizing voltages −30 and −10 mV. Pipette: Kgluconate containing solutions; bath: Nagluconate containing solutions. In the same experimental conditions, with the exception that cesium was used in the pipette solution and NaCl in the bath solution (D–G), currents were also recorded before (D, left panel) and after (D, right panel) addition of 3 µM of specific 5-HT₃ agonist 2M-5HT to the bath solution. Cesium blocked outward potassium currents and Kv1.3 activity was avoided by applying voltage pulses only from −130 to −50 mV in 20 mV steps. Addition of 3 µM 2M-5HT to a cell held at −90 mV holding potential in the whole-cell configuration caused a significant inward current within few seconds (E). Currents were recorded at −70 mV holding potential in outside-out patch after addition of 3 µM 2M-5HT (F, upper trace) and after addition of 100 µM amiloride, in the presence of 2M-5HT, to the same patch (F, lower trace). Amiloride did not reduce the current. Whole-cell currents were recorded in presence of ondansetron (100 µM) (G, left panel) and after addition of 3 µM 2M-5HT in the presence of 100 µM ondansetron (G, right panel). Applied voltages ranged from −130 to −10 mV. Data in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0022482#pone-0022482-g003" target="_blank">Figures 3C–G</a> are representative of at least five experiments in the same conditions giving similar results.</p

5-HT₃ receptor activity does not modify CXCR4 expression, CXCL12 binding and CXCL12-induced actin polymerization.

<p>(A) After treatment of resting human CD4⁺ T cells with 5-HT, the 5-HT₃ receptor agonist 2M-5HT or the 5-HT₃ receptor antagonist ondansetron for 0, 30 and 150 min, CXCR4 expression was analyzed by flow cytometry. Data refer to percentage of a representative experiment of three performed. (B) Binding of CXCL12-AF647 on human CD4⁺ T cells. The cells were treated for 30 min at RT with 5-HT, 2M-5HT, ondansetron or the specific CXCR4 antagonist AMD3100 (ref. 20). The cells were then incubated with increasing concentrations of fluorescent CXCL12, in the presence of the indicated chemicals. Data are expressed as MFI ± SEM vs. CXCL12-AF647 concentrations (n = 4; ***<i>P</i><0.001, Bonferroni's multiple comparison test). (C) Human CD4⁺ T cells were treated with 2M-5HT or ondansetron for 30 min at RT and then stimulated with CXCL12. Kinetics of CXCL12-induced F-actin formation were analyzed by phalloidin staining and flow cytometric analysis. Data represent MFI ± SEM vs. time (n = 3).</p

Role of CCR5, CCR2 and SDF-1 gene polymorphisms in a population of HIV-1 infected individuals

The finding that in addition to CD4 molecule HIV-1 uses, CCR5 or CXCR4 receptors to enter target cells prompted the research to identify polymorphisms in coreceptor genes affecting disease progression. In this study we analyzed the prevalence of CCR5-delta32, CCR2-641 and SDF1-3'A alleles in a highly selected group of 42 Long-Term Nonprogressors (LTNPs) compared to 112 subjects with a typical course of HIV-1 infection (TPs) and 117 healthy controls (HCs). In addition, we correlated CCR5, CCR2 and SDF-1 genotypes with molecular indexes of HIV-1 replication, cell-free RNA and both unspliced (US) and multiply spliced (MS) intracellular transcripts, to investigate the role of the mutant alleles in determining a long-term nonprogressive course of HIV-1 disease. Our results indicate a significantly higher prevalence of CCR5-delta32 allele in LTNPs compared to TPs (p=0.0434), while the proportions of CCR2-64I and SDF1-3'A alleles were comparable between the two groups. However, SDF-1 wild type LTNP subjects showed significantly lower levels of HIV-1 genomic RNA, US and MS transcripts than SDF1-3'A heterozygous ones (p=0.0021, 0.016, 0.0031, respectively), whereas both CCR5 and CCR2 wild type individuals had similar rates of viral replication compared to CCR5-delta32 and CCR2-64I heterozygous ones. CCR5, CCR2 and SDF-1 combined genotypes were also studied and this analysis did not identify a specific protective cluster of alleles in LTNPs. Taken together, our results indicate that genetic background involving CCR5, CCR2 and SDF-1 alleles may play a limited role in the natural history of HIV-1 infection