Lymphoid Microrna Profiles During Allograft Rejection

Abstract

MicroRNAs (miRNAs) are highly conserved non-coding RNAs that regulate gene expression by translational repression or by mRNA degradation. MiRNAs as immune regulators may govern expression of genes relevant to allograft rejection, tolerance induction and post-transplant infection in recipients of organ transplants. We performed a broad analysis on miRNAs expressed by lymphocytes that may regulate immune functions post-transplant. Groups of syngeneic (C57BL/6 → C57BL/6) and allogeneic (BALB/c →C57BL/6) recipients (n=3-9) of heterotopic heart transplants were performed and spleen, PBMC and graft infiltrating cells (GILs) were obtained for miRNA microarray analysis using TaqMan microfluidic cards. Data were analyzed using SDS and RQ software to define miRNAs that are differentially expressed (P< 0.05). Of the 335 mature mouse miRNAs examined, forty-four miRNAs were significantly differentially expressed in allogeneic GILs (P< 0.05) as compared to syngeneic GILs. Eleven (10 increased, 1 decreased) miRNAs were significantly changed in the spleen while ten (6 increased, 4 decreased) showed significant differential expression in PBMC. Interestingly five miRNAs, miRs-146a, 337-5p, 342-3p, 410 and 449a shared differential expression in both the PBMC and GILs and two miRNAs, miR-7a and miR-182 were significantly increased in PBMC, GILs and spleen. Experiments were performed to examine the kinetics of miR-182 expression by T cells in response to alloantigen. T cell proliferation and miR-182 expression were analyzed on days 1-5 in MLR using C57BL/6 responder T cells and BALB/c stimulator splenocytes. Maximal expression of miR-182 coincided with maximal proliferation on day 5. Treatment with the calcineurin inhibitor CsA markedly decreased the expression of miR-182 in T cells, while the expression of the housekeeping gene snoRNA135 remained stable. These results suggest that IL-2 may induce miR-182 expression. CD4+T cells are the likely source of miR-182 as mice deficient in CD8+ T cells (B6.129S2-Tap1tm1Arp/J) expressed miR-182 as similar levels as wild-type mice when used as recipients of BALB/c allografts. Since miR-182 is differentially expressed in the spleen, GILs and PBMC during graft rejection, we analyzed corresponding plasma samples for miR-182. There was a specific and significant increase in miR-182 in the plasma of the recipients of allogeneic grafts as compared to recipients of syngeneic grafts suggesting the potential as a biomarker of graft status. Taken together, our results suggest that lymphocyte-associated miRNAs may be novel targets for therapeutics to improve graft outcomes.