Mult Scler. 2014 Sep;20(10):1363-70.
MiR-126: a novel route for natalizumab action?
Maria Meira, Claudia Sievers, Francine Hoffmann, Tobias Derfuss, Jens Kuhle, Ludwig Kappos, and Raija LP Lindberg.
Clinical Neuroimmunology, Departments of Biomedicine and Neurology, University Hospital Basel, Switzerland.
MicroRNAs (miRNAs) have emerged as a family of post-transcriptional regulators of gene expression that mediate diverse aspects of immunity. MiRNA dysregulation has been found in multiple sclerosis (MS), reflecting the growing need to identify disease-specific miRNA expression signatures. Our previous low-density array studies reveal differential miR-126 expression in the CD4(+)T cells of untreated relapsing-remitting MS (RRMS) patients. Here, we investigated miR-126 expression in natalizumab-treated patients.
We isolated CD4(+) T cells from untreated (n = 12) and natalizumab-treated MS patients (n = 24), and from healthy volunteers (n = 12). We analyzed the expression of miRNAs and potential targets by real time reverse transcription polymerase chain reaction (RT-PCR). We assessed specific inhibition of miR-126, in vitro.
MiR-126 was down-regulated in cells of patients under natalizumab treatment and up-regulated during relapse, supporting a regulatory role in MS immunopathogenesis. MiR-126 expression correlated with the expression of POU2AF1, a regulator of Spi-B that binds to the promoter/enhancer sequences of JC virus (JCV), the pathogen of progressive multifocal leukoencephalopathy (PML), a rare complication of natalizumab treatment. The same trend was found for Spi-B. Strong up-regulation of both genes appeared to be treatment duration-dependent. Specific inhibition experiments supported the link between the expression of miR-126 and POU2AF1/Spi-B.
Our findings provided deeper insight into the mode of action of natalizumab, with possible implications for understanding both the effects of natalizumab on MS activity and its specific adverse event profile.
Multiple sclerosis (MS) is a chronic inflammatory and neurodegenerative disorder of the central nervous system characterized by immune-mediated demyelination and axonal injury. The profound heterogeneity in clinical course and response to therapy adds to the complexity of this disabling disease of still unknown etiology. MicroRNAs (miRNAs), a class of small non-coding RNA molecules that mediate post-transcriptional gene silencing, have recently emerged as important factors to consider. In fact, deregulation of miRNAs has been widely documented in a variety of human diseases including cancer and MS, giving an incentive for the development of their use as diagnostic, prognostic and predictive biomarkers.
We have extensively been studying the impact of MS therapies on gene expression and miRNA expression profiles. In 2008, our longitudinal analysis of gene expression in peripheral blood of MS patients treated with natalizumab, (Tysabri, Biogen Idec and Elan Pharmaceuticals), an approved disease-modifying therapy for RRMS, revealed a number of altered genes related to immune response, signal transduction, adhesion and metabolism . More recently, our study of B cells from patients treated with natalizumab revealed a distinct pattern of ten differentially expressed miRNAs in natalizumab treated compared to untreated patients .
In the present study, we hypothesized that natalizumab might also impact on the previously uncovered differential miRNA expression profile in CD4+T cells from RRMS patients . We particularly focused on expression of miR-126, and confirmed its up-regulation in RRMS compared to healthy volunteers (HVs) (Figure 1). We then showed that natalizumab “normalized” miR-126 expression back to the level of HVs, therefore suggesting that natalizumab could mediate some effects through miRNA regulation.
Figure 1. Differential expression of miR-126 in CD4+T cells of HVs and RRMS patients. Transcriptional expression of miR-126 was analyzed with real-time RT-PCR in CD4+T cells from HVs, RRMS patients whom were untreated or natalizumab-treated for either less than 24 months or more than 24 months (n = 12 in each group). c: p < 0.001; b: p < 0.01; a: p < 0.05.
We further examined the affected pathways downstream of miR-126 expression. Of interest, miR-126 blockade has been reported to result in augmented expression of the transcriptional co-activator POU2AF1 . Here, in accordance with the expression of miR-126, POU2AF1 relative expression was significantly down regulated in CD4+T cells from RRMS compared to HVs, and reversely up-regulated in cells from natalizumab treated compared to untreated patients (Figure 2A). Notably, significantly higher expression of POU2AF1 was found in patients treated longer than 2 years with natalizumab.
Regarding the importance of natalizumab therapy on the development of progressive multifocal leukoencephalopathy PML , we further examined expression of Spi-B, a transcription factor regulated by POU2AF1 that binds unique sequences of the promoter/enhancer of the JC virus (JCV) PML pathogen . Interestingly, we uncovered significant higher expression of Spi-B mRNA in CD4+T cells from natalizumab patients ex vivo, with higher levels of expression in patients treated longer than 2 years with natalizumab (Figure 2B).
Figure 2. Differential expression of POU2AF1 and Spi-B mRNA. Transcriptional expression of POU2AF1 (A) and Spi-B (B) were analyzed with real-time RT-PCR in CD4+T cells from HVs, RRMS patients, who were untreated or natalizumab treated for either less than 24 months or more than 24 months (n = 12 in each group). c: p < 0.001; b: p < 0.01; a: p < 0.05
Knowing the involvement of Spi-B in early JC virus gene expression and its critical role in JC virus activity, its increased expression in conjunction to natalizumab treatment duration, an independent important risk factor for PML in MS, suggested a more than coincidental relation of this natalizumab effect on Spi-B mRNA expression in CD4+T cells.
The mechanism by which CD4+T cells could contribute to the process of JCV activation in PML remains unknown, however, we propose here a model where the initial up-regulation of Spi-B mRNA through miR-126-mediated POU2AF1 regulation would occur in activated CD4+T cells, which would then be transferred by exchange of genetic material to infected carriers cells such as B cells.
Importance of the study: Our present data provide new insights into the role of miRNAs in MS pathogenesis and add to our understanding of the molecular mechanism of action of natalizumab that impact on the therapeutic effects but also on its potential adverse events.
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Acknowledgements: This study was supported by the Swiss National Science Foundation (SNSF; grant no: 310030_132644) and the Swiss Multiple Sclerosis Society (SMSG).
Prof Raija LP Lindberg, Ph.D.
Clinical Neuroimmunology, Departments of Biomedicine and Neurology, University Hospital Basel, Hebelstrasse 20, Basel, CH-4031, Switzerland.