Jasn2011060543 1.10

Upregulation of miR-142-3p in Peripheral BloodMononuclear Cells of Operationally Tolerant Patientswith a Renal Transplant Richard Danger,*† Annaïck Pallier,* Magali Giral,*†‡ Marc Martínez-Llordella,§Juan José Lozano, Nicolas Degauque,* Alberto Sanchez-Fueyo,§ Jean-Paul Soulillou,*†‡and Sophie Brouard*‡ *Institut National de la Santé Et de la Recherche Médicale UMR643 and Institut de Transplantation Urologie,Néphrologie, Nantes, France; †Université de Nantes, Nantes, France; ‡Centre Hospitalier Universitaire Hôtel-Dieu,Nantes, France; §Liver Unit, Hospital Clinic Barcelona, CIBEREHD, Barcelona, Spain; and Bioinformatics Platform,CIBEREHD, Barcelona, Spain ABSTRACTAchieving drug-free tolerance or successfully using only small doses of immunosuppression is a major goalin organ transplantation. To investigate the potential mechanisms by which some kidney transplantrecipients can achieve operational tolerance, we compared the expression profiles of microRNA inperipheral blood mononuclear cells of operationally tolerant patients with those of stable patients treatedwith conventional immunosuppression. B cells from operationally tolerant patients overexpressed miR-142-3p. The expression of miR-142-3p was stable over time and was not modulated by immunosuppres-sion. In Raji B cells, overexpression of miR-142-3p modulated nearly 1000 genes related to the immuneresponse of B cells, including potential miR-142-3p targets and molecules previously identified in theblood of operationally tolerant patients. Furthermore, our results suggested that a negative feedbackloop involving TGF-b signaling and miR-142-3p expression in B cells may contribute to the maintenance oftolerance. In summary, miR-142-3p expression in peripheral blood mononuclear cells correlates with op-erational tolerance. Whether upregulation of miR-142-3p modulates inflammatory responses to promotetolerance or is a result of this tolerance state requires further study.
J Am Soc Nephrol 23: ccc–ccc, 2012. doi: 10.1681/ASN.2011060543 The use of minimal doses of immunosuppression or performing biopsies in such patients can be even achievement of drug-free tolerance is a major challenging in terms of ethical considerations and goal in organ transplantation.1,2 Although the kid- patient adherence. In these different studies, several ney is less susceptible to successful immunosup- key pathways were highlighted, such as a pathway pressive drug withdrawal than the liver, where implicating the TGF-b gene;10 in addition, several around 20% of transplant patients can be success- other genes have been highlighted as "key leader fully weaned off immunonsuppression,3,4 an in- genes," such as BANK-1 (B-cell scaffold protein creasing number of kidney transplant recipientswho continue to display good graft function in Received June 7, 2011. Accepted November 18, 2011.
the absence of immunosuppressive drugs havebeen described in the literature.5–8 We and others J.-P.S. and S.B. contributed equally to this work.
have looked at the gene expression profile in Published online ahead of print. Publication date available at PBMCs of such "operationally tolerant" kidney transplant recipients.7–12 The blood is a popular Correspondence: Dr. Sophie Brouard, INSERM UMR 643- 30 Bd choice for analysis because it provides a noninva- Jean Monnet, 44093 Nantes Cedex 93, France. Email: sive means for potential biomarker discovery, which is important in the case of tolerance; Copyright 2012 by the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012 ISSN : 1046-6673/2304-ccc with ankyrin repeats 1),11 a modulator of B-cell hyperactiva- in PBMCs from healthy volunteers before and 24 hours after tion through AKT upon CD40 activation.13 The implication of phytohemagglutinin A (PHA) and IL-2 stimulation B-cell–related genes correlated with a high number of B cells14 Fifty-two miRNAs displayed a fold change and to a gene signature enriched in B-cell–related genes in the (FC) superior to 2 (i.e., log2FC.1) and 59 others, an FC in- blood of operationally tolerant kidney recipients, which was sub- ferior to 2 (i.e., log2FC.-1), indicating a strong modulation of sequently confirmed in three different studies.7,8,12 The role of B expression after PHA/IL-2 stimulation cells has also been reported in experimental rodent models, in and Among the eight miRNAs dif- which transfer of B cells from tolerant rats prolonged graft ferentially expressed between operationally tolerant and STA survival when administered to untreated recipients.15 recipients, miR-450b-5p, miR-142-3p, and miR-324-5p were The mechanisms involved in the maintenance of this downregulated (FC=0.21, 0.29, and 0.42, respectively; Figure phenomenon remain elusive, and the identification of related 1B) and miR-876-3p was upregulated (FC=3.4; Figure 1B) biomarkers remains instrumental to achieving safe drug after PHA/IL-2 stimulation. We confirmed the downregula- minimization or complete weaning in clinical practice.
tion of miR-142-3p as early as 2 hours and at 24 hours after miRNA are small, endogenous, noncoding RNAs that can PHA/IL-2 stimulation, using individual Taqman microRNA regulate the expression of a variety of genes by directly destabi- assays (Figure 2A).
lizing their target mRNA.16,17 Furthermore, the incomplete Because miR-142-3p (1) appeared as the highest differen- pattern of target recognition allows a single miRNA to target tially expressed miRNA between operationally tolerant and hundreds of mRNAs, and conversely, a single mRNA to be STA recipients, (2) was underexpressed after PHA/IL-2 stim- targeted by multiple miRNAs, thus affecting a broad range of ulation (Figure 2A), (3) is specific to the hematopoietic line- gene networks.18 Numerous studies have reported on the dif- age,24–26 and (4) plays a role in lymphocyte functions,27 we ferential expression of miRNA in physiologic disorders or dis- focused on the potential role of this miRNA in our study. This eases,19,20 and miRNA has been found to be modulated in biopsy miRNA is overexpressed in operationally tolerant compared specimens from kidney transplant recipients.21–23 In this with STA patients according to TLDA assays (Figure 1C).
study, we investigated whether miRNA is modulated in theblood of patients with operational tolerance compared with a Validation of miR-142-3p Overexpression and cohort of patients with stable graft function under classic Stability over Time in PBMCs from Operationally Tolerant RecipientsIndividual miR-142-3p Taqman quantitative PCR (qPCR)assays displays a good correlation (r=0.885; P,0.0001) (Figure 1D). Furthermore, we validated the overexpression of miR-142-3p in operationally tolerant recipients on 16 independent miRNA Profiling in PBMC from Kidney PBMC samples (6 operationally tolerant and 10 STA patients) Transplant Recipients (P=0.02; FC=1.58) (Figure 2B). The post-transplantation We first searched for a global miRNA profile in PBMCs from time was significantly higher in operationally tolerant than kidney transplant recipients using miRNA Taqman low- in STA patients (P=0.02; Table 1); however, we did not found density arrays (TLDAs). The expression of 381 miRNAs was any correlation between this time and miR-142-3p expression measured in PBMCs from 9 operationally tolerant patients and value (r=0.0342; P=0.845; . No signif- 10 STA recipients. A total of 266 miRNAs were expressed with a icant difference was observed between the subgroups of pa- quantification cycle (Cq) inferior to 35 in at least half of sam- tients for any of the clinical variables tested (age, sex, creatine ples from each group. We selected the eight top-ranked miRNAs level in blood, proteinuria, number of HLA mismatches). Fi- on the basis of Mann–Whitney tests between the two groups of nally, the expression of mir-142-3p was stable over time, as patients (Figure 1, A and B). According to the expression values tested in PBMCs collected from three operationally tolerant of these eight miRNAs, the clear separation of the two groups of recipients at two different time points (5.5, 11, and 13 months) patients by clinical status was further observed using principal (Figure 2C).
component analysis Among the eightdifferentially expressed miRNAs, four were overexpressed Overexpression of miR-142-3p in the Blood of (miR-450b-5p, miR142-3p, miR-876-3p, and miR-106b) and Operationally Tolerant Recipients Is Not Due four were underexpressed (miR508-3p, miR-148b, miR-324-5p, to the Absence of Immunosuppression and miR-98) in PBMCs from operationally tolerant compared Because operationally tolerant patients display stable graft with STA patients.
function but no longer receive immunosuppression andbecause healthy volunteers also displayed an increased expres- miRNA Profiling in PBMCs from Healthy Volunteers sion of miR-142-3p compared with STA patients (P=0.0038; after Polyclonal Activation FC=1.54) (data not shown), we hypothesized that immuno- To further analyze the basal expression and modulation of suppression treatment may modulate the blood expression of these eight specific miRNAs, we performed miRNA profiling miR-142-3p. We thus measured its expression in PBMCs from Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012

Figure 1. Differential miRNA in PBMCs from operationally tolerant compared with STA kidney transplant recipients. (A) The eight top-ranked miRNAs according to Mann–Whitney tests are represented in this heat map, in which blue bars represent STA patients (n=10)and green bars represent operationally tolerant (n=10) patients. The heat map represents normalized and color-coded relative ex-pression values (22DDCq), in which red values indicate overexpression and green values indicate underexpression. (B) The eight miRNAare classified according to their uncorrected P values, which were used only for ranking miRNA rather than for an absolute identificationof differential miRNA. FCs of miRNA after PHA/IL-2 activation of PBMCs from healthy volunteers are displayed in last column. (C)Relative expression values of miR-142-3p from TLDA assays are displayed in a scatter plot. (D) Correlation between TLDA assays andindividual Taqman qPCR assays for miR-142-3p (9 operationally tolerant and 10 STA patients).
tolerant liver transplant recipients before and after the patients compared with STA recipients (P=0.01; FC= 2.84) (Figure 3B), entered an immunosuppressive drug weaning protocol whereas no difference was observed compared with healthy (n=27), a population not available in the context of kidney volunteers (data not shown). Therefore, purified B cells from transplantation. No difference was found in the PBMC expres- healthy volunteers were cultured over 24 hours with physio- sion of miR-142-3p with or without immunosuppression regimen logic and high doses of cyclosporine A (40 and 400 ng/ml).
in these liver recipients (Figure 2D).
As shown in Figure 3D, no difference was observed for eitherdose of cyclosporine A used. These results are concordant with miR-142-3p Is Overexpressed in B Cells from the absence of modulation of miR-142-3p in blood from liver- Operationally Tolerant Patients tolerant recipients before and after withdrawal of the treatment We then analyzed the expression of miR-142-3p in purified and suggest that miR-142-3p is not affected by immunosup- blood leukocyte subpopulations (T and B lymphocytes, pressive drugs in total PBMC or purified B cells.
monocytes, and NK cells) from healthy volunteers (n=3). Pu-rification was typically greater than 95%. Figure 3A shows that miR-142-3p Transfection in the Raji B-Cell Line miR-142-3p is expressed in all blood cell subsets, confirming Because miRNA regulate mRNA levels,16,17 we overexpressed previous reports.24–26 Because miR-142-3p was highly ex- mir-142-3p in the Raji B-cell line using synthetic mimics and pressed in T and B lymphocytes from healthy volunteers, we performed gene expression profiling using microarrays next analyzed its expression in these two populations in trans- 24 hours after transfection. A total of 22,332 spots were filtered plant recipients. No difference was observed in the expression and the overexpression of miR-142-3p was found to induce of miR-142-3p in the T lymphocyte subset (Figure 3C). In the up- and downregulation of 492 and 489 transcripts, re- contrast, the expression of miR-142-3p was significantly spectively. To provide a more comprehensive biologic inter- higher in total B cells purified from operationally tolerant pretation of our finding, GOminer software was used to J Am Soc Nephrol 23: ccc–ccc, 2012 miRNA in Tolerant Recipients network related to "Inflammation re-sponse, antimicrobial response and cell de-velopment" with 49 of these genes wasfound (Withinthis gene network, we identified a subgroupof TGF-b–related genes in which the TGF-breceptor 1 gene (TGFBR1), a possible target ofmiR-142-3p, plays a central role (Figure 4A),together with a subgroup of IFN-g–relatedgenes (Figure 4B), and a subgroup of genesrelated to B cells (Figure 4C).
The analysis of the detailed B-cell–related gene network after miR-142-3p transfectionhighlighted the upregulation of genes previ-ously identified in PBMCs from operationallytolerant recipients, such as the key moleculesM S 4 A 1 ( C D 2 0 ) , C R 2 , C D 3 8 , 1 0 a n dBANK-1.11,12 We next validated the array expression patterns of a few select targets and miRNAexpression. The upregulation of miR-142-3p,MS4A1, and BANK1 in Raji cells transfected Figure 2. miR-142-3p expression in PBMCs. (A) miR-142-3p was downregulated in with miR-142-3p mimics was confirmed PBMCs from healthy volunteers after PHA/IL-2 stimulation as early as 2 hours and using individual Taqman assays (Figure 5A).
remained low for up to 24 hours. Mean6 SEM of miR-142-3p relative expression(22DDCq relative to RNU6) in total PBMCs from three healthy volunteers stimulated Implication of miR-142-3p in TGF-b with PHA (2 mg/ml) and IL-2 (150 U/ml) are represented. (B) miR-142-3p was signifi- cantly overexpressed in PBMCs from operationally tolerant (Op-Tol) kidney transplant Using individual qPCR, we also confirmed the recipients compared with STA patients. qPCR measurements were performed using significant downregulation of TGFBR1 gene individual Taqman qPCR assays with independent PBMC samples from TLDA assays transcripts subsequent to miR-142-3p trans- (10 STA and 6 operationally tolerant patients). Means 6 SEM of miR-142-3p relative fection in the Raji cell line compared with con- expression (22DDCq relative to miR-374b) are represented. (C) miR-142-3p expression did not differ in PBMCs from liver transplant recipients before (n=11) and after (n=16) P=0.03; FC=0.47; Figure 5A). We then immunosuppression weaning. Means 6 SEM of miR-142-3p relative expression noted that the addition of TGF-b to the Raji (22DDCq relative to miR-374b) are represented. (D) miR-142-3p expression was stable culture media (5 ng/ml over 24 hours) in- between the two time points (5.5, 11, and 13 months between blood collections) for duced an increase in miR-142-3p expression, three operationally tolerant patients. Means 6 SEM of miR-142-3p relative expression whereas PHA stimulation did not (Figure 5B).
(22DDCq relative to miR-374b) are represented. *P,0.05.
Similarly, purified B cells from healthy volun-teers were activated with independent and de- identify the over-represented gene ontology (GO) categories pendent B-cell receptor signaling for 24 hours (Figure 3D). As based on the differential gene lists compared with all other observed after PHA stimulation in Raji cells (Figure 5B), there is expressed genes in the microarray.28 Among the 492 over- only nonsignificant and marginal regulation of miR-142-3p. This expressed genes, the GO categories "immune response" finding reinforces the fact that miR-142-3p needs specific stimu- (GO:0006955) and "B-cell activation" (GO:0042113) were lation, such as TGF-b, for its modulation (Figure 5B).
identified with a total of 25 genes. Among the 489 underex- We then measured TGF-b1 and TGFBR1 transcript levels in pressed genes, GO categories related to cell communication purified B cells from operationally tolerant and STA patients. We (GO:0007154), "vesicle-mediated transport" (GO:0016192), found that TGF-b1 expression is increased in B cells from opera- and "small GTPase [guanosine triphosphatase] mediated sig- tionally tolerant patients (FC=1.4 compared with STA; P=0.04) nal transduction" (GO:0007264) were identified. We then (Figure 5C), whereas the level of TGFBR1 did not significantly identified 66 potential miR-142-3p targets downregulated differ between operationally tolerant and STA patients (Figure 5D).
subsequent to the miR-142-3p overexpression among the242 genes predicted to be miR-142-3p targets (among at least4 of 11 established miRNA target prediction databases com- puted by miRecords software).29 Mixing these 66 potentialtarget genes and the 25 upregulated immune-related genes The achievement of long-term drug-free tolerance in solid and using Ingenuity pathways analysis software, a gene organ transplantation is thought to be possible on the basis of Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012 Table 1. Summary of clinical data for the 35 kidney recipients used for measurement of miR-142-3p expression Proteinuria Donor/Living Immunosuppression Mismatches 121.1632.5 0.2360.17 Operationally tolerant 110.1622.1 0.2560.34 Mean, minimum (min), and maximum (max) values apply only to recipient age, recipient gender, time between graft and analysis, creatine level, and proteinuria.
observations in both liver4 and kidney3,5transplantation. We and others have reportedon an increased number of peripheral B cellsand have identified modifications in bloodgene expression in operationally tolerantkidney transplant recipients that involveTGF-b signaling and B-cell–related path-ways.7,8,10,12,14 However, a clear mechanismor biologic process of peripheral regulationhas yet to be identified in these patients.
miRNAs are small RNA molecules with im-portant roles in immune modulation, homeo-stasis, the development of immune diseasesand the regulation of physiologic pro-cesses.19 Deregulated miRNA expressionhas been shown to be involved in severalhuman immune-related diseases, such asmultiple sclerosis,30,31 cancer,32,33 and rheu-matoid arthritis,34 but their function andregulation processes are still far from beingtotally understood. In renal transplantation,two studies reported on the identification of Figure 3. miR-142-3p expression in purified blood cell populations. (A) miR-142-3p miRNA profile signatures in biopsy samples expression in isolated subpopulations from PBMCs of three healthy volunteers (except from kidney transplant patients with acute NK cells; n=2). B ly, B lymphocytes; mono, mononuclear cells; T ly, T lymphocytes. (B) rejection episodes,21,23 suggesting that miR-142-3p expression exhibited increased expression in total B lymphocytes isolated miRNA expression profiling may be used from kidney operationally tolerant (Op-Tol) recipients (n=5) compared with STA pa- to monitor allograft status. To our knowl- tients (n=12) (P=0.0098; FC= 2.84). (C) miR-142-3p expression was similar in total T edge, and particularly in operationally toler- lymphocytes isolated from kidney operationally tolerant recipients (n=5) compared ant kidney transplant recipients, no miRNA with STA patients (n=8). (D) miR-142-3p expression was not significantly modulated in expression analyses have been performed in purified B lymphocytes from healthy volunteers after B-cell–independent and B-cell–dependent stimulation and after cyclosporine A incubation during 24 hours of culture (n=4). Means 6 SEM of miR-142-3p relative expression (22DDCq relative to RNU6) are In this study, we report on the modulation represented. BCR, B-cell receptor. *P,0.05.
of expression of eight miRNAs in PBMCs J Am Soc Nephrol 23: ccc–ccc, 2012 miRNA in Tolerant Recipients

Figure 4. Three subgroups of genes with modulated function after miR-142-3p overexpression in Raji cells. (A–C) These three sub-groups of genes were extracted from the gene network created using IPA software . Genes in red are upregulatedand genes in green are downregulated in Raji transfected by miR-142-3p compared with control mimic. PP, protein–protein binding;PD, protein–DNA binding; MB, group/complex membership; TR, translocation.
from kidney graft recipients with drug-free operational toler- described for other miRNAs, such as miR-125b, miR-16b, ance compared with patients with stable graft function under or miR-148b.35–37 miR-142-3p expression has been reported immunosuppression. Our choice to compare operationally as playing a role in CD4+CD25+Treg function.27 Although tolerant patients with patients who have stable graft function miR-142-3p was highly expressed in T lymphocytes, we did under immunosuppression was based on the fact that the latter not observe any differential expression in T lymphocytes be- population is the most likely to benefit from immunosuppres- tween operationally tolerant and STA recipients (Figure 3C).
sion minimization, whereas patients with chronic rejection or In contrast, we found a significant overexpression of miR- healthy volunteers would not. Unsupervised hierarchical clus- 142-3p in the B-lymphocyte subset of operationally tolerant tering analysis based on the expression of these eight miRNAs compared with STA patients (P=0.0098; FC=2.84) (Figure only led to the clustering of operationally tolerant patients 3B) and also compared with patients with signs of chronic together (Figure 1A). Among these eight miRNA, miR-142-3p antibody-mediated rejection (data not shown). We also found was highly expressed in PBMCs from operationally tolerant that its expression was not modulated by immunosuppressive patients (Figure 1C and Figure 2B). This miRNA has been treatment in tolerant liver transplant recipients (Figure 2D) described as a hematopoietic-restricted lineage miRNA.24–27 or when purified B cells from human volunteers were cultured We found that miR-142-3p was decreased after PHA/IL-2 with cyclosporine A in vitro (Figure 3D), indicating that its activation, further favoring a regulatory loop, as already overexpression in operationally tolerant kidney transplant Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012 between miR-142-3p expression and post-transplantation time and did find miR-142-3p expression to be stable over time (Figure2C); these results indicate that miR-142-3p isindependent of post-transplantation time.
Recently, Guo and colleagues demon- strated that miRNA decreased protein pro-duction mostly by lowering mRNA levels.17Thus, we used gene microarrays to measurethe effect of miR-142-3p overexpression inB cells, looking at the effect of experimentalmiR-142-3p transfection in a stable RajiB-cell line. Whereas the overexpression ofthis single miRNA repressed many genes, weobserved the paradoxical biologic effect ofmiR-142-3p, which also induced gene up-regulation, as already described for othermiRNAs.39 A large number of genes relatedto B cells, IFN-g, and TGF-b signaling wereupregulated after overexpression of miR-142-3p in the Raji B-cell line. Of note, inprevious studies we had already observedsome of these genes, such as MS4A1 Figure 5. miR-142-3p expression in Raji cells. (A) Confirmation of the overexpression of (CD20); this gene was part of the 49-gene miR-142-3p, BANK1, and CD20 and the underexpression of TGFBR1 in Raji cells signature that correctly classified kidney transfected with miR-142-3p mimic compared with a control mimic (n=4). (B) miR-142- operationally tolerant patients10 and was 3p expression was increased by TGF-b in Raji cells, whereas PHA had marginal effects part of the best-classifier genes in the blood in Raji cells, compared with control (PBS), after 24 hours of culture. Means 6 SEM of and urine of operationally tolerant patients miR-142-3p FC (control/stimulation) are represented. (C) TGF-b expression was sig- in the study by Newell and colleagues.7 nificantly increased (P=0.04) in purified B cells from operationally tolerant (Op-Tol) Similarly, miR-142-3p overexpression led (n=4) and STA (n=6) patients, whereas TGFBR1 was not. (D) Means 6 SEM of gene to the upregulation of BANK1 transcripts, relative expression (22DDCq relative to RNU6 for miR-142-3p or relative to HPRT1 for one of the key leader genes upregulated in other genes) are represented. *P,0.05.
the blood of kidney operationally tolerantrecipients,11 independent of immunosup- recipients was not just a consequence of the absence of im- pressive treatment.12 BANK1 is an inhibitory adaptor protein highly expressed in peripheral B cells and is a modulator of Interestingly, these data are in accordance with an increased hyperactive B-cell responses by inhibiting AKT activation number of peripheral B cells14 and with the specific B-cell– upon CD40 signaling.13 enriched gene profile that we and others previously reported Finally, the TGF-b signaling pathway was also affected after in the blood of these patients.7,8,10,12 Although the increased miR-142-3p overexpression in the Raji cell line and in B cells expression of miR-142-3p in PBMCs from operationally tol- from operationally tolerant patients. Interestingly, miR-106b, erant kidney recipients is probably in part due to the increased one of the eight differentially overexpressed miRNA in PBMCs number of blood B cells in these patients, because miR-142-3p from operationally tolerant kidney transplant recipients, also expression in PBMCs is correlated with B cell number (data not affects downstream effector molecules of TGF-b signaling.40,41 shown), we report that purified B cells from operationally tol- In a previous report, we showed that among the specific and erant also expressed more miR-142-3p. This miRNA has already unique blood signature of 49 genes associated with tolerance, been reported to be expressed specifically in hematopoietic tis- 27% of the genes modulated in blood from operationally tol- sues and particularly in B cells, but a precise role in the B-cell erant patients could be regulated by the TGF-b even though compartment has yet to be attributed.24–27,38 TGF-b was not significantly increased in total PBMCs from miR-142-3p expression has also been associated with tubular operationally tolerant patients (increased from 30% only).10 atrophy and interstitial fibrosis of renal transplants.22 Moreover, TGF-b is involved in various animal models of tolerance,42–44 miR-142-3p was overexpressed in B lymphocytes from the blood plays a role in immune regulation and homeostasis of Treg of operationally tolerant patients, and this analysis performed in cells,45 and is known for its intrinsic suppressive properties.46 peripheral blood does not exclude a different expression profile We also report here that TGF-b stimulation of Raji cells within the graft itself. In addition, we did not find any correlation induced an increased expression of miR-142-3p and that J Am Soc Nephrol 23: ccc–ccc, 2012 miRNA in Tolerant Recipients TGF-b1 expression is increased in B cells from operationally protocol (Agilent Technologies Inc., Palo Alto, CA). A total of tolerant patients, whereas TGFBR1 is not; this finding 22,332 spots were filtered. GOminer software and Ingenuity Pathway suggests a negative feedback loop between this cytokine and Analysis 6.5 software (Ingenuity Systems Inc.) were used to assess miR-142-3p in B cells. Such a process of regulation, already biologic significance of genes selected with Mann–Whitney tests. Raw described for different miRNAs,40 suggests that miRNAs act as microarray data were deposited in the Gene Expression Ominbus key gene switches and as fine-tuning molecules, depending on (GEO) database (accession number GSE28456).
the compartment and the specific biologic context.20 There-fore, our data suggest that mir-142-3p is also implicated in the Statistical Analyses TGF-b pathway. However, because the modulation of one The nonparametric Mann–Whitney test, Kruskal Wallis test, or miRNA may affect multiple mRNAs that are also regulated paired Wilcoxon test was used for group comparisons using Graph by several other miRNAs, a direct link between these two mol- PadPrism software, version 4. Differences were defined as statistically ecules cannot be predicted at this stage.
significant with P,0.05 and highly significant with P,0.01.
Our findings show that overexpression of miR-142-3p in B Additional details can be found in cells correlates to the state of operational tolerance in kidney transplant recipients. They also point toward a possiblenegative feedback loop between TGF-b and miR-142-3p inB cells. The mechanism driving and maintaining spontaneoustolerance in which TGF-b could be implicated remains un- clear. Further investigations are now needed to find outwhether this overexpression of miR-142-3p in B cells contrib- We thank all the patients who participated in this study and the utes to controlling inflammatory responses and tolerance physicians who helped us recruit patients: J.F. Subra, F. Villemain, maintenance or is only a consequence of this tolerance state.
C. Legendre, E. Thervet, F.J. Bemelman, G. Roussey, G. Orlando,A. Garnier, H. Jambon, H. Le Monies De Sagazan, L. Braun, C. Noël,E. Pillebout, M.C. Moal, C. Cantarell, A. Hoitsma, M. Ranbant, A.
Testa. We thank the transcriptome core facility of Nantes for technicalassistance with gene expression microarrays. We also thank Yohann Foucher for critical review of the manuscript.
A total of 86 individuals were enrolled in this study: 15 operationally The Institut de Transplantation Urologie belongs to the Fondation tolerant patients, 34 STA patients, 10 healthy volunteers, 11liver recipients Centaure, which supports a French research network in trans- with stable graft function, and 16 drug-free liver recipients from the plantation. R.D. was supported by the Fondation Centaure and by a Nantes hospital in France and the Barcelona hospital in Spain. The two grant from the Fondation pour la Recherche Médicale.
local ethics committees approved all aspects of the study, and all patientsgave informed consent. The clinical information is described in the , clinical data are summarized in Table 1, and detailed clinical data are provided in .
miRNA ProfilingmiRNA profiling was performed using TLDA microRNACards pool Aset, version 2.0 (Applied Biosystems, Foster City, CA), in accordance with the manufacturer's recommendations. Normalization was per-formed by subtracting the mean Cq of the measured miRNA.47 After 1. Danger R, Giral M, Soulillou JP, Brouard S: Rationale and criteria of eligibility for calcineurin inhibitor interruption following kidney trans- normalization, miRNA were ranked using P values from nonparametric plantation. Curr Opin Organ Transplant 13: 609–613, 2008 Mann–Whitney tests, which do not require normal assumptions or 2. Ashton-Chess J, Giral M, Brouard S, Soulillou JP: Spontaneous opera- asymptotic conditions. These uncorrected P values for multiple testing tional tolerance after immunosuppressive drug withdrawal in clinical were used only for ranking miRNA and are not an absolute identifi- renal allotransplantation. Transplantation 84: 1215–1219, 2007 cation of differential miRNA.
3. Orlando G, Soker S, Wood K: Operational tolerance after liver trans- plantation. J Hepatol 50: 1247–1257, 2009 4. Martínez-Llordella M, Puig-Pey I, Orlando G, Ramoni M, Tisone G, miRNA Individual Assays Rimola A, Lerut J, Latinne D, Margarit C, Bilbao I, Brouard S, Individual miRNA expression was measured with Taqman miRNA Hernández-Fuentes M, Soulillou JP, Sánchez-Fueyo A: Multiparameter assays (Applied Biosystems) using probes for miR-142-3p (assay ID: immune profiling of operational tolerance in liver transplantation. Am J 000464), RNU6 (assay ID: 001973), and miR-374b (assay ID: 001319), Transplant 7: 309–319, 2007 starting with 10 ng of total RNA, on an ABI Prism 7900 HT.
5. Roussey-Kesler G, Giral M, Moreau A, Subra JF, Legendre C, Noël C, Pillebout E, Brouard S, Soulillou JP: Clinical operational tolerance afterkidney transplantation. Am J Transplant 6: 736–746, 2006 Gene Expression Microarray Analysis 6. Kawai T, Cosimi AB, Spitzer TR, Tolkoff-Rubin N, Suthanthiran M, Gene expression was measured using whole human genome 4344K Saidman SL, Shaffer J, Preffer FI, Ding R, Sharma V, Fishman JA, Dey B, Agilent microarrays, following the manufacturer's two-color Ko DS, Hertl M, Goes NB, Wong W, Williams WW Jr, Colvin RB, Sykes Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012 M, Sachs DH: HLA-mismatched renal transplantation without mainte- 23. Sui W, Dai Y, Huang Y, Lan H, Yan Q, Huang H: Microarray analysis of nance immunosuppression. N Engl J Med 358: 353–361, 2008 MicroRNA expression in acute rejection after renal transplantation.
7. Newell KA, Asare A, Kirk AD, Gisler TD, Bourcier K, Suthanthiran M, Transpl Immunol 19: 81–85, 2008 Burlingham WJ, Marks WH, Sanz I, Lechler RI, Hernandez-Fuentes MP, 24. Chen CZ, Li L, Lodish HF, Bartel DP: MicroRNAs modulate hemato- Turka LA, Seyfert-Margolis VL Immune Tolerance Network ST507 Study poietic lineage differentiation. Science 303: 83–86, 2004 Group: Identification of a B cell signature associated with renal trans- 25. Landgraf P, Rusu M, Sheridan R, Sewer A, Iovino N, Aravin A, Pfeffer S, plant tolerance in humans. J Clin Invest 120: 1836–1847, 2010 Rice A, Kamphorst AO, Landthaler M, Lin C, Socci ND, Hermida L, Fulci 8. Sagoo P, Perucha E, Sawitzki B, Tomiuk S, Stephens DA, Miqueu P, V, Chiaretti S, Foà R, Schliwka J, Fuchs U, Novosel A, Müller RU, Chapman S, Craciun L, Sergeant R, Brouard S, Rovis F, Jimenez E, Schermer B, Bissels U, Inman J, Phan Q, Chien M, Weir DB, Choksi R, De Ballow A, Giral M, Rebollo-Mesa I, Le Moine A, Braudeau C, Hilton R, Vita G, Frezzetti D, Trompeter HI, Hornung V, Teng G, Hartmann G, Gerstmayer B, Bourcier K, Sharif A, Krajewska M, Lord GM, Roberts I, Palkovits M, Di Lauro R, Wernet P, Macino G, Rogler CE, Nagle JW, Ju J, Goldman M, Wood KJ, Newell K, Seyfert-Margolis V, Warrens AN, Papavasiliou FN, Benzing T, Lichter P, Tam W, Brownstein MJ, Bosio A, Janssen U, Volk HD, Soulillou JP, Hernandez-Fuentes MP, Lechler RI: Borkhardt A, Russo JJ, Sander C, Zavolan M, Tuschl T: A mammalian Development of a cross-platform biomarker signature to detect renal microRNA expression atlas based on small RNA library sequencing.
transplant tolerance in humans. J Clin Invest 120: 1848–1861, 2010 Cell 129: 1401–1414, 2007 9. Braud C, Baeten D, Giral M, Pallier A, Ashton-Chess J, Braudeau C, 26. Merkerova M, Belickova M, Bruchova H: Differential expression of Chevalier C, Lebars A, Léger J, Moreau A, Pechkova E, Nicolini C, microRNAs in hematopoietic cell lineages. Eur J Haematol 81: 304–310, Soulillou JP, Brouard S: Immunosuppressive drug-free operational im- mune tolerance in human kidney transplant recipients: Part I. Blood gene 27. Huang B, Zhao J, Lei Z, Shen S, Li D, Shen GX, Zhang GM, Feng ZH: expression statistical analysis. J Cell Biochem 103: 1681–1692, 2008 miR-142-3p restricts cAMP production in CD4+CD25- T cells and 10. Brouard S, Mansfield E, Braud C, Li L, Giral M, Hsieh SC, Baeten D, CD4+CD25+ TREG cells by targeting AC9 mRNA. EMBO Rep 10: 180– Zhang M, Ashton-Chess J, Braudeau C, Hsieh F, Dupont A, Pallier A, Moreau A, Louis S, Ruiz C, Salvatierra O, Soulillou JP, Sarwal M: Iden- 28. Zeeberg BR, Feng W, Wang G, Wang MD, Fojo AT, Sunshine M, tification of a peripheral blood transcriptional biomarker panel associ- Narasimhan S, Kane DW, Reinhold WC, Lababidi S, Bussey KJ, Riss J, ated with operational renal allograft tolerance. Proc Natl Acad Sci USA Barrett JC, Weinstein JN: GoMiner: A resource for biological interpre- 104: 15448–15453, 2007 tation of genomic and proteomic data. Genome Biol 4: R28–R28, 2003 11. Sivozhelezov V, Braud C, Giacomelli L, Pechkova E, Giral M, Soulillou JP, 29. Xiao F, Zuo Z, Cai G, Kang S, Gao X, Li T: miRecords: An integrated Brouard S, Nicolini C: Immunosuppressive drug-free operational immune resource for microRNA-target interactions. Nucleic Acids Res tolerance in human kidney transplants recipients. Part II. Non-statistical 37[Database issue]: D105–D110, 2009 gene microarray analysis. J Cell Biochem 103: 1693–1706, 2008 30. Otaegui D, Baranzini SE, Armañanzas R, Calvo B, Muñoz-Culla M, 12. Pallier A, Hillion S, Danger R, Giral M, Racapé M, Degauque N, Dugast E, Khankhanian P, Inza I, Lozano JA, Castillo-Triviño T, Asensio A, Olaskoaga Ashton-Chess J, Pettré S, Lozano JJ, Bataille R, Devys A, Cesbron-Gautier J, López de Munain A: Differential micro RNA expression in PBMC from A, Braudeau C, Larrose C, Soulillou JP, Brouard S: Patients with drug-free multiple sclerosis patients. PLoS ONE 4: e6309, 2009 long-term graft function display increased numbers of peripheral B cells 31. Junker A, Krumbholz M, Eisele S, Mohan H, Augstein F, Bittner R, with a memory and inhibitory phenotype. Kidney Int 78: 503–513, 2010 Lassmann H, Wekerle H, Hohlfeld R, Meinl E: MicroRNA profiling of 13. Aiba Y, Yamazaki T, Okada T, Gotoh K, Sanjo H, Ogata M, Kurosaki T: multiple sclerosis lesions identifies modulators of the regulatory pro- BANK negatively regulates Akt activation and subsequent B cell re- tein CD47. Brain 132: 3342–3352, 2009 sponses. Immunity 24: 259–268, 2006 32. Farazi TA, Spitzer JI, Morozov P, Tuschl T: miRNAs in human cancer.
14. Louis S, Braudeau C, Giral M, Dupont A, Moizant F, Robillard N, Moreau J Pathol 223: 102–115, 2011 A, Soulillou JP, Brouard S: Contrasting CD25hiCD4+T cells/FOXP3 33. Marcucci G, Mrózek K, Radmacher MD, Garzon R, Bloomfield CD: The patterns in chronic rejection and operational drug-free tolerance.
prognostic and functional role of microRNAs in acute myeloid leuke- Transplantation 81: 398–407, 2006 mia. Blood 117: 1121–1129, 2011 15. Le Texier L, Thebault P, Lavault A, Usal C, Merieau E, Quillard T, 34. Pauley KM, Satoh M, Chan AL, Bubb MR, Reeves WH, Chan EK: Up- Charreau B, Soulillou JP, Cuturi MC, Brouard S, Chiffoleau E: Long-term regulated miR-146a expression in peripheral blood mononuclear cells allograft tolerance is characterized by the accumulation of B cells ex- from rheumatoid arthritis patients. Arthritis Res Ther 10: R101, 2008 hibiting an inhibited profile. Am J Transplant 11: 429–438, 2011 35. Tili E, Michaille JJ, Cimino A, Costinean S, Dumitru CD, Adair B, Fabbri 16. Baek D, Villén J, Shin C, Camargo FD, Gygi SP, Bartel DP: The impact of M, Alder H, Liu CG, Calin GA, Croce CM: Modulation of miR-155 and microRNAs on protein output. Nature 455: 64–71, 2008 miR-125b levels following lipopolysaccharide/TNF-alpha stimulation 17. Guo H, Ingolia NT, Weissman JS, Bartel DP: Mammalian microRNAs pre- and their possible roles in regulating the response to endotoxin shock.
dominantly act to decrease target mRNA levels. Nature 466: 835–840, 2010 J Immunol 179: 5082–5089, 2007 18. Peter ME: Targeting of mRNAs by multiple miRNAs: The next step.
36. Liu X, Zhan Z, Xu L, Ma F, Li D, Guo Z, Li N, Cao X: MicroRNA-148/152 Oncogene 29: 2161–2164, 2010 impair innate response and antigen presentation of TLR-triggered 19. O'Connell RM, Rao DS, Chaudhuri AA, Baltimore D: Physiological dendritic cells by targeting CaMKIIa. J Immunol 185: 7244–7251, 2010 and pathological roles for microRNAs in the immune system. Nat Rev 37. De Santis G, Ferracin M, Biondani A, Caniatti L, Rosaria Tola M, Immunol 10: 111–122, 2010 Castellazzi M, Zagatti B, Battistini L, Borsellino G, Fainardi E, Gavioli R, 20. Schott J, Stoecklin G: Networks controlling mRNA decay in the immune Negrini M, Furlan R, Granieri E: Altered miRNA expression in T regu- system. Wiley Interdiscip Rev RNA 1: 432–456, 2010 latory cells in course of multiple sclerosis. J Neuroimmunol 226: 165– 21. Anglicheau D, Sharma VK, Ding R, Hummel A, Snopkowski C, Dadhania D, Seshan SV, Suthanthiran M: MicroRNA expression profiles predictive 38. Malumbres R, Sarosiek KA, Cubedo E, Ruiz JW, Jiang X, Gascoyne RD, of human renal allograft status. Proc Natl Acad Sci USA 106:5330–5335 Tibshirani R, Lossos IS: Differentiation stage-specific expression of 22. Scian MJ, Maluf DG, Archer KJ, Suh JL, Massey D, Fassnacht RC, microRNAs in B lymphocytes and diffuse large B-cell lymphomas.
Whitehill B, Sharma A, King A, Gehr T, Cotterell A, Posner MP, Mas V: Blood 113: 3754–3764, 2009 Gene expression changes are associated with loss of kidney graft 39. Srikantan S, Marasa BS, Becker KG, Gorospe M, Abdelmohsen K: Par- function and interstitial fibrosis and tubular atrophy: Diagnosis versus adoxical microRNAs: individual gene repressors, global translation prediction. Transplantation 91: 657–665, 2011 enhancers. Cell Cycle 10: 751–759, 2011 J Am Soc Nephrol 23: ccc–ccc, 2012 miRNA in Tolerant Recipients 40. Petrocca F, Visone R, Onelli MR, Shah MH, Nicoloso MS, de Martino I, 44. Josien R, Douillard P, Guillot C, Müschen M, Anegon I, Chetritt J, Iliopoulos D, Pilozzi E, Liu CG, Negrini M, Cavazzini L, Volinia S, Alder H, Menoret S, Vignes C, Soulillou JP, Cuturi MC: A critical role for trans- Ruco LP, Baldassarre G, Croce CM, Vecchione A: E2F1-regulated forming growth factor-beta in donor transfusion-induced allograft tol- microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis erance. J Clin Invest 102: 1920–1926, 1998 in gastric cancer. Cancer Cell 13: 272–286, 2008 45. Bommireddy R, Doetschman T: TGFbeta1 and Treg cells: Alliance for 41. Wang H, Liu J, Zong Y, Xu Y, Deng W, Zhu H, Liu Y, Ma C, Huang L, tolerance. Trends Mol Med 13: 492–501, 2007 Zhang L, Qin C: miR-106b aberrantly expressed in a double transgenic 46. Wan YY, Flavell RA: ‘Yin-Yang' functions of transforming growth factor- mouse model for Alzheimer's disease targets TGF-b type II receptor.
beta and T regulatory cells in immune regulation. Immunol Rev 220: Brain Res 1357: 166–174, 2010 42. Chiffoleau E, Bériou G, Dutartre P, Usal C, Soulillou JP, Cuturi MC: Role 47. Mestdagh P, Van Vlierberghe P, De Weer A, Muth D, Westermann for thymic and splenic regulatory CD4+ T cells induced by donor F, Speleman F, Vandesompele J: A novel and universal method for dendritic cells in allograft tolerance by LF15-0195 treatment. J Immunol microRNA RT-qPCR data normalization. Genome Biol 10: R64, 168: 5058–5069, 2002 43. Gagne K, Brouard S, Guillet M, Cuturi MC, Soulillou JP: TGF-beta1 and donor dendritic cells are common key components in donor-specificblood transfusion and anti-class II heart graft enhancement, whereastolerance induction also required inflammatory cytokines down-regulation.
This article contains supplemental material online at Eur J Immunol 31: 3111–3120, 2001 Journal of the American Society of Nephrology J Am Soc Nephrol 23: ccc–ccc, 2012

Source: http://servidor-ciberehd.upc.es/pubs/Danger_etal_2012.pdf

Microsoft word - life sciences p2 nov 2011 memo eng version 1.doc

NATIONAL LIFE SCIENCES P2 VERSION 1 (NEW CONTENT) FOR FULL-TIME CANDIDATES NOVEMBER 2011 MARKS: 150 This memorandum consists of 11 pages. Copyright reserved Please turn over Life Sciences/P2 (Version 1) (Full-time) DBE/November 2011 NSC – Memorandum PRINCIPLES RELATED TO MARKING LIFE SCIENCES 2011