Radhika Raheja

Comprehensive evaluation of serum microRNAs as biomarkers in multiple sclerosis

Objective: To identify circulating microRNAs (miRNAs) linked to disease stage and disability in multiple sclerosis (MS). Methods: Sera from 296 participants including patients with MS, other neurologic diseases (Alzheimer disease and amyotrophic lateral sclerosis), and inflammatory diseases (rheumatoid arthritis and asthma) and healthy controls (HCs) were tested. miRNA profiles were determined using LNA (locked nucleic acid)-based quantitative PCR. Patients with MS were categorized according to disease stage and disability. In the discovery phase, 652 miRNAs were measured in sera from 26 patients with MS and 20 HCs. Following this, significant miRNAs (p < 0.05) from the discovery set were validated using quantitative PCR in 58 patients with MS, 30 HCs, and in 74 samples from other disease controls (Alzheimer disease, amyotrophic lateral sclerosis, asthma, and rheumatoid arthritis). Results: We validated 7 miRNAs that differentiate patients with MS from HCs (p < 0.05 in both the discovery and validation phase); miR-320a upregulation was the most significantly changing serum miRNA in patients with MS. We also identified 2 miRNAs linked to disease progression, with miR-27a-3p being the most significant. Ten miRNAs correlated with the Expanded Disability Status Scale of which miR.199a.5p had the strongest correlation with disability. Of the 15 unique miRNAs we identified in the different group comparisons, 12 have previously been reported to be associated with MS but not in serum. Conclusions: Our findings identify circulating serum miRNAs as potential biomarkers to diagnose and monitor disease status in MS. Classification of evidence: This study provides Class III evidence that circulating serum miRNAs can be used as biomarker for MS.

Identification of a novel mechanism of action of fingolimod (FTY720) on human effector T cell function through TCF-1 upregulation

BackgroundFingolimod (FTY720), the first oral treatment for multiple sclerosis (MS), blocks immune cell trafficking and prevents disease relapses by downregulation of sphingosine-1-phosphate receptor. We determined the effect of FTY720 on human T cell activation and effector function.MethodsT cells from MS patients and healthy controls were isolated to measure gene expression profiles in the presence or absence of FTY720 using nanostring and quantitative real-time polymerase chain reaction (qPCR). Cytokine protein expression was measured using luminex assay and flow cytometry analysis. Lentivirus vector carrying short hairpin RNA (shRNA) was used to knock down the expression of specific genes in CD4+ T cells. Chromatin immunoprecipitation was performed to assess T cell factor 1 (TCF-1) binding to promoter regions. Luciferase assays were performed to test the direct regulation of interferon gamma (IFN-γ) and granzyme B (GZMB) by TCF-1. Western blot analysis was used to assess the phosphorylation status of Akt and GSK3β.ResultsWe showed that FTY720 treatment not only affects T cell trafficking but also T cell activation. Patients treated with FTY720 showed a significant reduction in circulating CD4 T cells. Activation of T cells in presence of FTY720 showed a less inflammatory phenotype with reduced production of IFN-γ and GZMB. This decreased effector phenotype of FTY720-treated T cells was dependent on the upregulation of TCF-1. FTY720-induced TCF-1 downregulated the pathogenic cytokines IFN-γ and GZMB by binding to their promoter/enhancer regions and mediating epigenetic modifications. Furthermore, we observed that TCF-1 expression was lower in T cells from multiple sclerosis patients than in those from healthy individuals, and FTY720 treatment increased TCF-1 expression in multiple sclerosis patients.ConclusionsThese results reveal a previously unknown mechanism of the effect of FTY720 on human CD4+ T cell modulation in multiple sclerosis and demonstrate the role of TCF-1 in human T cell activation and effector function.

Association Between Serum MicroRNAs and Magnetic Resonance Imaging Measures of Multiple Sclerosis Severity

Importance MicroRNAs (miRNAs) are promising multiple sclerosis (MS) biomarkers. Establishing the association between miRNAs and magnetic resonance imaging (MRI) measures of disease severity will help define their significance and potential impact. Objective To correlate circulating miRNAs in the serum of patients with MS to brain and spinal MRI. Design, Setting, and Participants A cross-sectional study comparing serum miRNA samples with MRI metrics was conducted at a tertiary MS referral center. Two independent cohorts (41 and 79 patients) were retrospectively identified from the Comprehensive Longitudinal Investigation of Multiple Sclerosis at the Brigham and Womens Hospital. Expression of miRNA was determined by locked nucleic acid–based quantitative real-time polymerase chain reaction. Spearman correlation coefficients were used to test the association between miRNA and brain lesions (T2 hyperintense lesion volume [T2LV]), the ratio of T1 hypointense lesion volume [T1LV] to T2LV [T1:T2]), brain atrophy (whole brain and gray matter), and cervical spinal cord lesions (T2LV) and atrophy. The study was conducted from December 2013 to April 2016. Main Outcomes and Measures miRNA expression. Results Of the 120 patients included in the study, cohort 1 included 41 participants (7 [17.1%] men), with mean (SD) age of 47.7 (9.5) years; cohort 2 had 79 participants (26 [32.9%] men) with a mean (SD) age of 43.0 (7.5) years. Associations between miRNAs and MRIs were both protective and pathogenic. Regarding miRNA signatures, a topographic specificity differed for the brain vs the spinal cord, and the signature differed between T2LV and atrophy/destructive measures. Four miRNAs showed similar significant protective correlations with T1:T2 in both cohorts, with the highest for hsa.miR.143.3p (cohort 1: Spearman correlation coefficient rs = −0.452, P = .003; cohort 2: rs = −0.225, P = .046); the others included hsa.miR.142.5p (cohort 1: rs = −0.424, P = .006; cohort 2: rs = −0.226, P = .045), hsa.miR.181c.3p (cohort 1: rs = −0.383, P = .01; cohort 2: rs = −0.222, P = .049), and hsa.miR.181c.5p (cohort 1: rs = −0.433, P = .005; cohort 2: rs = −0.231, P = .04). In the 2 cohorts, hsa.miR.486.5p (cohort 1: rs = 0.348, P = .03; cohort 2: rs = 0.254, P = .02) and hsa.miR.92a.3p (cohort 1: rs = 0.392, P = .01; cohort 2: rs = 0.222, P = .049) showed similar significant pathogenic correlations with T1:T2; hsa.miR.375 (cohort 1: rs = −0.345, P = .03; cohort 2: rs = −0.257, P = .022) and hsa.miR.629.5p (cohort 1: rs = −0.350, P = .03; cohort 2: rs = −0.269, P = .02) showed significant pathogenic correlations with brain atrophy. Although we found several miRNAs associated with MRI outcomes, none of these associations remained significant when correcting for multiple comparisons, suggesting that further validation of our findings is needed. Conclusions and Relevance Serum miRNAs may serve as MS biomarkers for monitoring disease progression and act as surrogate markers to identify underlying disease processes.

Correlating serum micrornas and clinical parameters in amyotrophic lateral sclerosis: miRNA in ALS

Introduction: Amyotrophic lateral sclerosis (ALS) is a debilitating neurologic disorder with poor survival rates and no clear biomarkers for disease diagnosis and prognosis. Methods: We compared serum microRNA (miRNA) expression from patients with ALS with healthy controls and patients with multiple sclerosis and Alzheimer disease. We also correlated miRNA expression in cross‐sectional and longitudinal cohorts of ALS patients with clinical parameters. Results: We identified 7 miRNAs (miR‐192‐5p, miR‐192‐3p, miR‐1, miR‐133a‐3p, miR‐133b, miR‐144‐5p, miR‐19a‐3p) that were upregulated and 6 miRNAs (miR‐320c, miR‐320a, let‐7d‐3p, miR‐425‐5p, miR‐320b, miR‐139‐5p) that were downregulated in patients with ALS compared with healthy controls, patients with Alzheimer disease, and patients with multiple sclerosis. Changes in 4 miRNAs (miR‐136‐3p, miR‐30b‐5p, miR‐331‐3p, miR‐496) correlated positively and change in 1 miRNA (miR‐2110) correlated negatively with changes in clinical parameters in longitudinal analysis. Discussion: Our findings identified serum miRNAs that can serve as biomarkers for ALS diagnosis and progression. Muscle Nerve 58: 261–269, 2018

Monomethyl fumarate treatment impairs maturation of human myeloid dendritic cells and their ability to activate T cells

Background: Dimethyl fumarate (DMF) and its active metabolite monomethyl fumarate (MMF) effectively lead to reduction in disease relapses and active magnetic resonance imaging (MRI) lesions. DMF and MMF are known to be effective in modulating T- and B-cell responses; however, their effect on the phenotype and function of human myeloid dendritic cells (mDCs) is not fully understood. Objective: To investigate the role of MMF on human mDCs maturation and function. Methods: mDCs from healthy controls were isolated and cultured in vitro with MMF. The effect of MMF on mDC gene expression was determined by polymerase chain reaction (PCR) array after in vitro MMF treatment. The ability of mDCs to activate T cells was assessed by in vitro co-culture system. mDCs from DMF-treated multiple sclerosis (MS) patients were analyzed by flow cytometry and PCR. Results: MMF treatment induced a less mature phenotype of mDCs with reduced expression of major histocompatibility complex class II (MHC-II), co-stimulatory molecules CD86, CD40, CD83, and expression of nuclear factor κB (NF-κB) subunits RELA and RELB. mDCs from DMF-treated MS patients also showed the same immature phenotype. T cells co-cultured with MMF-treated mDCs showed reduced proliferation with decreased production of interferon gamma (IFN-γ), interleukin-17 (IL-17), and granulocyte-macrophage colony-stimulating factor (GM-CSF) compared to untreated cells. Conclusion: We report that MMF can modulate immune response by affecting human mDC function.

FXR1: Linking cellular quiescence, immune genes and cancer

Cellular quiescence has been considered a homogeneous passive state wherein cells, in response to certain physiological stimuli, remain dormant until they are signaled to re-enter the cell cycle. However, in recent years, studies have indicated that quiescence is a heterogeneous active state with distinct gene expression profiles that might dictate the functional state of these cells and govern their transition into subsequent cell fates. Therefore, understanding these gene signatures and their underlying regulatory mechanisms is critical to explain aberrations under disease conditions. A comprehensive analysis of gene regulation in quiescence by Vasudevan et al led to the identification of the RNA binding protein FXR1 (Fragile X mental retardation syndrome related protein 1), a critical regulator of post-transcriptional gene expression in differentiation, development, and immunity. In a monocytic cell line, THP1 cultured under serum-starved conditions (>24 h), FXR1 associates with a modified microRNP complex (microRNA-protein) comprising of a target-specific miRNA (microRNA) and AGO2 (Argonaute-2 protein). This complex recruited onto the AU-rich element (ARE) of a target mRNA such as TNFa (Tumor necrosis factor alpha) resulted in enhanced translation. The mechanism of non-canonical translation in such a quiescent state is dependent on the ability of FXR1 to form FXR1/AGO2/microRNA complex that associates with a specialized translation factor, DAP5/p97 (Fig. 1). Interestingly, FXR1 is also associated with poor clinical outcomes in several cancers including NSCLC (non small cell lung cancer), ovarian, breast and HNSC (head and neck squamous cell carcinoma). This effect was attributed to its ability to regulate the expression of 2 oncogenes, protein kinase C and iota and epithelial cell transforming 2. In response to certain stimuli, cells enter a transition state where they can undergo quiescence, re-enter the cell cycle or terminally differentiate. While the role of FXR1 in quiescence has been studied, whether it can regulate mRNA levels of specific genes in this transition state and consequently alter cell function is not clear. In the current volume of Cell Cycle, Le Tonqueze O, et al analyzed the effect of FXR1 depletion on mRNA levels in THP1 cells cultured either with serum or under 24 h early serum-starved conditions representing the transition state. They showed that FXR1 regulates mRNA levels of proinflammatory (IL-1b and TNFa) and migration (CCL2 and CCL3) specific genes in the early serum starved conditions. Depletion of FXR1 in THP1 cells led to a reduction in CCL2 mRNA levels and conversely, CCL2 mRNA expression was increased upon overexpression of FXR1 in both serum-grown and early serum starved conditions. Additionally, mRNA and protein levels of IL-1b and TNFa were significantly reduced in the absence of FXR1 in early serum-starved cells (Fig. 1). IL-1b and TNFa could not be detected in serum-grown THP1 cells. The mechanism by which FXR1 increases RNA levels is not clear. Furthermore, FXR1 depletion significantly reduced cell migration of serum-starved THP1 monocytes. Thus, this paper highlights a novel function of FXR1 in regulating pro-inflammatory genes, chemokine expression, and cell migration. The specific miRNAs associated with this translational activation of proinflammatory and chemokine genes and the role of FXR1 in regulating gene expression in other cell types remains to be determined. However, studies by this group have revealed a hitherto unknown regulatory pathway that has major implications. Firstly, it suggests an unconventional role for miRNAs in upregulating gene expression under specific conditions. Since miRNAs are extensively studied as potential biomarkers and therapeutic targets, it is imperative to understand the cellular state during such analyses and its mechanism of action. Second, cancer cells are a heterogeneous population consisting of quiescent cancer stem cells (CSC) that contribute to the reactivation and recurrence of tumors. The regulation of genes that enable quiescence maintenance, resistance to therapy and re-entry into proliferation is not clearly understood. The overexpression of FXR1 in tumors and its role in maintaining translational activation in quiescent cells necessitates further exploration of this molecule in CSCs. Lastly, circulating monocytes are typically precursors of macrophages and are implicated in immune surveillance where they are recruited in response to microenvironment stimuli and elicit an inflammatory response. In this context, the role of FXR1 in dictating downstream functions needs to be elucidated. Overall, these studies lay precedent to unravel the physiological relevance of quiescence-associated networks in diseases.

A post-transcriptional program of chemoresistance by AU-rich elements/TTP in cancer quiescence

Background Quiescence (G0) is a transient, cell cycle-arrested state. By entering G0, cancer cells survive unfavorable conditions such as chemotherapy and cause relapse. While G0 cells have been studied at the transcriptome level, how post-transcriptional regulation contributes to their chemoresistance remains unknown. Results We induced chemoresistant and quiescent (G0) leukemic cells by serum-starvation or chemotherapy treatment. To study post-transcriptional regulation in G0 leukemic cells, we systematically analyzed their transcriptome, translatome, and proteome. We find that our resistant G0 cells recapitulate gene expression profiles of in vivo chemoresistant leukemic and G0 models. In G0 cells, canonical translation initiation is inhibited; yet we find that inflammatory genes are highly translated, indicating alternative post-transcriptional regulation. Importantly, AU-rich elements (AREs) are significantly enriched in the up-regulated G0 translatome and transcriptome. Mechanistically, we find the stress-responsive p38 MAPK-MK2 signaling pathway stabilizes ARE mRNAs by phosphorylation and inactivation of mRNA decay factor, tristetraprolin (TTP) in G0. This permits expression of ARE-bearing TNFα and DUSP1 that promote chemoresistance. Conversely, inhibition of TTP phophorylation by p38 MAPK inhibitors and non-phosphorylatable TTP mutant decreases ARE mRNAs and sensitizes leukemic cells to chemotherapy. Furthermore, co-inhibiting p38 MAPK and TNFα—prior to or along with chemotherapy—substantially reduced chemoresistance in primary leukemic cells ex vivo and in vivo. Conclusions These studies uncover post-transcriptional regulation underlying chemoresistance in leukemia. Our data reveal the p38 MAPK-MK2-TTP axis as a key regulator of expression of ARE bearing mRNAs that promote chemoresistance. By disrupting this pathway, we developed an effective combination therapy against chemosurvival.

Identification of MS-specific serum miRNAs in an international multicenter study

Objective To identify circulating microRNAs (miRNAs) linked to disease, disease stage, and disability in MS across cohorts. Methods Samples were obtained from the Comprehensive Longitudinal Investigation of Multiple Sclerosis (CLIMB, Boston, MA), EPIC (San Francisco, CA), AMIR (Beirut, Lebanon) as part of the SUMMIT consortium, and Stockholm Prospective Assessment of Multiple Sclerosis (Stockholm, Sweden) cohorts. Serum miRNA expression was measured using locked nucleic acid–based quantitative PCR. Four groups were compared: (1) MS vs healthy control (HC), (2) relapsing-remitting (RR) vs HC, (3) secondary progressive (SP) vs HC, and (4) RR vs SP. A Wilcoxon rank-sum test was used for the comparisons. The association between each miRNA and the Expanded Disability Status Scale (EDSS) score was assessed using the Spearman correlation coefficient. For each comparison, the p values were corrected for multiple comparisons using the approach of Benjamini and Hochberg to control the false discovery rate. Results In the CLIMB cohort, 5 miRNAs (hsa-miR-484, hsa-miR-140-5p, hsa-miR-320a, hsa-miR-486-5p, and hsa-miR-320c) showed a significant difference between patients with MS and healthy individuals; among these, miR-484 remained significant after accounting for multiple comparisons (p = 0.01). When comparing RRMS with HCs, hsa-miR-484 showed a significant difference (p = 0.004) between the groups after accounting for multiple group comparisons. When SP and HC were compared, 6 miRNAs (hsa-miR-484, hsa-miR-140-5p, hsa-miR-142-5p, hsa-miR-320a, hsa-miR-320b, and hsa-miR-320c) remained significantly different after accounting for multiple comparisons. Disability correlation analysis with miRNA provided 4 miRNAs (hsa-miR-320a, hsa-miR-337-3p, hsa-miR-199a-5p, and hsa-miR-142-5p) that correlated with the EDSS during the internal reproducibility phase. Among these, hsa-miR-337-3p was the most statistically significant miRNA that negatively correlated with the EDSS in three of the MS cohorts tested. Conclusions These findings further confirm the use of circulating serum miRNAs as biomarkers to diagnose and monitor disease status in MS. Classification of evidence This study provides Class III evidence that levels of circulating miRNAs identify patients with MS.