NORMATIVAS Y REGULACIONES URBANAS

and miR-590-5p to target the predicted seed region in the 3

′

was tested with a dual-luciferase reporter constructs containing the wild-type or target site-mutated 3’-UTRs. Specific inhibition was calculated as decrease in luciferase activity in wild-type 3’-UTR compared with target region-mutated 3’-UTR. Each individual experiment was internally normalized against an negative control miRNA (C. elegans cel-miR-67). Bars represent mean±s.e.m. Each experiment was performed three times in duplicate. *P<0.05; **P<0.01; ***P<0.001.

miR- 23b miR-98 FAS miR-98 miR-590-5p FASL miR-98 0 20 40 60 80 TRAIL Specific inhibition (%) miR-23b miR-590-5p TRAIL-R2 * *** *** ** miR- 23b miR- 98 FAS * miR- 98 miR- 590-5 p FASLG nsmi R miR- 98 60 70 80 90 100 110 120 TRAIL

Normalized expression versus nsmiR (%)

** miR- 23b miR- 590-5 p TRAIL-R2

Figure 5. Repression of extrinisic pathway apoptosis genes by miRNA in primary cytotoxic T lymphocytes. For each gene, values were normalized against treatment with negative control miRNA cel-miR-67. Relative mRNA expression was calculated versus housekeeping gene OAZ1 for all experiments. Bars represent mean±s.e.m. Each experiment was performed at least three times in triplicate. *P<0.01; **P<0.001.

by displaying increased proliferative capacity upon repeated antigen exposure with concomitant reduced expression of the pro-apoptotic FAS, FASLG, TRAIL and TRAIL-R2 at gene and protein level and increased the expression of miRNAs regulating these factors, that is, miR-23b, miR-98 and miR-590-5p.

One of the hallmarks of autoimmune disorders, and T1D in particular, is the acquisition of defects in the regulatory circuits that control cell proliferation and immune homeostasis. Two well-known pathways involved in immune homeostasis are the Fas/FasL18 _{and TRAIL/}

TRAIL-R pathways,19 _{however, their role in T1D appears to be two-fold.}20,21 _{On one hand, Fas}

and TRAIL expression by PBMCs is required for control of autoreactive cells,22 _{whereas on}

the other hand, expression of Fas or TRAIL on PBMCs may facilitate beta-cell lysis through increased death receptor:ligand interactions in pancreatic islets.23,24 _{It is conceivable that}

both mechanisms occur simultaneously during autoimmune disease progression. Yet, the immunoregulatory properties of Fas/FasL and TRAIL/TRAIL-R appear to hold greater clinical relevance.25 _{This is demonstrated for Fas by increased resistance to Fas-induced}

apoptosis by PBMC of T1D patients, which would lead to decreased immune regulation and thus increased susceptibility to autoimmunity.26 _{Indeed, a favorable response to treatment}

with high-dose immune suppression followed by autologous hematopoietic stem cell transplantation, correlated with an increase of Fas and Fas ligand expression to levels seen in healthy individuals.27 _{Although the role of TRAIL in T1D has not been studied as}

extensively as Fas, interference with TRAIL receptor signaling correlated with aggravation of autoimmune disease28 _{and administration of TRAIL protected against,}29 _{or alleviated}

severity30 _{of disease in murine models for T1D. These findings suggest that restoration of}

the apoptosis balance by increasing the expression of Fas and TRAIL pathway members would be beneficial despite possible negative effects on organ damage.

In this study, we focused on the differential expression of the Fas and TRAIL pathway between autoreactive cells from T1D patients and non-affected individuals. Although these two pathways are known regulators of T-cell death, it is likely that other factors contribute to the increased survival capacity of the T cells observed in this study. Indeed, our mRNA profiling experiments point to several other apoptosis regulators that appear differently expressed between autoreactive T cells in health versus disease. For instance, the pro-apoptotic BAX was expressed at lower levels in patient-derived autoreactive T cells, whereas survivin, a negative regulator of both Fas- and BAX-mediated apoptosis,31

was expressed higher in these cells. Further, several members of the caspase family were differentially expressed in patient-derived T cells, among which are the pro-inflammatory caspases CASP1, CASP4 and CASP5, as well as pro-apoptotic CASP7. Although the individual roles of each of these apoptosis regulators remain to be clarified, these collective findings point to overall anti-apoptotic mRNA expression profile in autoreactive T cells in T1D.

The autoreactive T cells used in this study present a rare opportunity to compare autoreactivity in health and disease. However, as these cells are expanded in vitro we cannot exclude culture artifacts from our analyses, despite the reproducibility of

3

the observed phenotypes. Significant effort was undertaken to analyze the transcriptome of polyclonal IGRP265-273T cells directly ex vivo, but unfortunately their low frequencies in peripheral blood prevented us to do so in a sufficiently robust and reproducible manner. Further, the fragile nature of autoreactive T cells derived from healthy individuals precluded miRNA overexpression studies, as the procedure of nucleofection resulted in an unacceptable rate of cell death. Therefore, a CMV-specific T-cell clone that expressed FAS and TRAIL at levels comparable to the healthy individual-derived autoreactive T cells were selected to determine the effect of miR-23b, miR-98 and miR-590-5p overexpression in primary T lymphocytes.

Recently, miR-98 has been reported to regulate Fas expression, as well as Fas- mediated apoptosis in a dose-dependent manner in HeLa cells.15 _{Furthermore, miR-98 was}

implicated as mediator of the anti-inflammatory effects of glucocorticoids by suppressing Fas and Fas ligand, among other factors.32 _{miR-23b was recently shown to mediate}

neuronal apoptosis in hypoxia-induced brain damage33 _{and overexpression of miR-23b}

lead to reduced apoptosis in lymphoma cells, whereas inhibition of miR-23b increased cell death.34 _{These findings further underscore the general anti-apoptotic potential of}

these particular miRNAs. Thus far, little is known on the role of miR-590-5p in apoptosis regulation, although miR-590-5p is implicated in acute myeloid leukemia and cervical cancer.35,36 _{Interestingly, the seed region of miR-590-5p is identical to the seed region}

of miR-21, a miRNA overexpressed in a wide variety of malignancies,37-40 _{leading to its}

classification as an ‘oncomir’. Overexpression of miR-21 has many consequences, of which the increased resistance to apoptosis is predominant. It can be argued that cancer and autoimmune disease are on opposite sides of the immunological spectrum. Treatment of cancer focuses on activation of adaptive immunity, whereas the therapeutic strategies in autoimmunity target immune regulation. Yet, the finding that two independent but functionally related miRNAs are overexpressed in both cancer and autoimmune disease, with similar phenotypic consequences in both conditions, creates an interesting parallel between autoimmunity and neoplastic disease in which the elucidation of miRNA expression may provide novel therapeutic targets for both conditions.

miRNAs are known essential negative regulators of gene expression during T-cell development and differentiation.41 _{A striking example of this is given by miR-155,}

a miRNA extensively studied for its role in hematopoiesis and lymphocyte functioning. In mice miR-155 deficiency leads to immunodeficiency, likely through dysfunctioning dendritic cells, B and T cells. In contrast, increased expression of miR-155 is linked to tumor development,42-44 _{whereas aberrant expression of miR-155 has been observed}

in several autoimmune diseases including rheumatoid arthritis, multiple sclerosis and systemic lupus erythematosus.45 _{The fact that different levels of expression of a single}

miRNA can lead to both immune deficiency, autoimmune disease and cancer indicates the potential therapeutic value of understanding the regulatory mechanisms governing miRNA expression. Yet, over the past years insight into miRNA biogenesis has revealed its complexity and to date the exact transcriptional cues that regulate miRNA expression

remain unclear for the majority of miRNAs.27, 28 _{miRNA gene control involves various}

regulatory mechanisms, among which are transactivation and transrepression by nuclear transcription factors. Expression of miRNA can be influenced by extracellular cues and shows tissue- and developmental stage-specific expression.46 _{Expression of miR-23b is}

influenced by extracellular stimuli and inflammatory cues, among which are estrogen, TSH, interleukin (IL)-17 and type I interferons.47 _{miR-590-5p expression appears to be}

affected by IL-3, macrophage colony-stimulating factor and granulocyte-macrophage colony-stimulating factor and is under the control of the lysine methyl transferase SEDT1A (a regulator of cell cycle progression).35,48 _{To date, little is known about the molecular}

pathways regulating mir-98 expression, although v-myc avian myelocytomatosis viral oncogene homolog is implicated in the regulation of the let-7 family, to which miR-98 belongs.49 _{Although there is still much to be learned about the spatiotemporal expression}

of miRNAs, further elucidation of miRNA regulation may aid in identifying a potential common event in the etiogenesis of autoreactive T cells.

miRNAs are inherently pleiotropic and potentially target a wide range of mRNAs. Therefore, functional validation of in silico predicted target binding is essential and the biological relevance of miRNAs for complex biological processes such as apoptosis should ideally be tested only in relevant cell types.50 _{This is underscored by the discrepancy in}

miRNA efficacy observed in our study between dual-luciferase reporters assays and primary T cells. Limited availability of autoreactive T cells and technical difficulties manipulating these cells, such as low transfection efficacy and low tolerance for manipulation, impair functional miRNA studies for the most relevant cell types in autoimmunity. Here we used a top-down approach to identify disparities between autoreactive T-cell clones from T1D patients and healthy individuals and identified differential expression of two canonical extrinsic apoptosis pathways and miRNAs regulating them, leading to increased proliferative potential of diabetogenic autoreactive T cells.

Previously, great effort was put into understanding the roles of protein-coding genes in T1D. Our finding that miRNAs can reduce the expression of several key apoptotic molecules in cytotoxic T lymphocytes implicate them as modifiers of disease susceptibility in T1D. Increased expression of miRNAs may act as a biomarker, distinguishing autoreactive T cells in patients from those in healthy subjects. Understanding the mechanisms by which autoreactive T cells overexpress miRNAs leading to increased survival and proliferation upon self-recognition may offer novel targets for therapeutic intervention.

MATERIALS AND METHODS