miR-466a Targeting of TGF-β2 Contributes to FoxP3+ Regulatory T Cell Differentiation in a Murine Model of Allogeneic Transplantation

Abstract

The promise of inducing immunological tolerance through regulatory T cell (Treg) control of effector T cell function is crucial for developing future therapeutic strategies to treat allograft rejection as well as inflammatory autoimmune diseases. In the current study, we used murine allograft rejection as a model to identify microRNA (miRNA) regulation of Treg differentiation from naïve CD4 cells. We performed miRNA expression array in CD4+ T cells in the draining lymph node (dLN) of mice which received syngeneic or allogeneic grafts to determine the molecular mechanisms that hinder the expansion of Tregs. We identified an increase in miRNA cluster 297-669 (C2MC) after allogeneic transplantation, in CD4+ T cells, such that 10 of the 27 upregulated miRNAs were all from this cluster, with one of its members, mmu-miR-466a-3p (miR-466a-3p), targeting transforming growth factor beta 2 (TGF-β2), as identified through reporter luciferase assay. Transfection of miR-466a-3p in CD4+ T cells led to a decreased inducible FoxP3+ Treg generation while inhibiting miR-466a-3p expression through locked nucleic acid resulting in increased Tregs and a reduction in effector T cells. Furthermore, in vivo inhibition of miR-466a-3p in an allogeneic skin-graft model attenuated T cell response against the graft through an increase in TGF-β2. TGF-β2 was as effective as TGF-β1 at both inducing Tregs and through adoptive transfer, mitigating host effector T cell response against the allograft. Together, the current study demonstrates for the first time a new role for miRNA-466a-3p and TGF-β2 in the regulation of Treg differentiation and thus offers novel avenues to control inflammatory disorders.

Immune and microRNA responses toHelicobacter muridaruminfection and indole-3-carbinol during colitis

“…U6, SnorD96, Snor68, Snor234, and Snor202 were evaluated for stability among groups and SnorD96 was chosen for normalization. SnorD96 has also been used by others[ 39 , 40 ]. Primers were purchased from Qiagen, Maryland.…”

Cannabinoid Receptor 1 Blockade Attenuates Obesity and Adipose Tissue Type 1 Inflammation Through miR-30e-5p Regulation of Delta-Like-4 in Macrophages and Consequently Downregulation of Th1 Cells

“…Bone marrow derived macrophages (BMDM) were generated by isolating bone marrow cells from the tibia and femur of 6–8 week old naïve female C57Bl6/J mice and culturing in complete DMEM/F12 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, and 1U/mL macrophage-colony stimulating factor (M-CSF, Biolegend 576406) for 7 days as previously described (21). Lymphocytes from mesenteric, inguinal, axillary, and cervical lymph nodes were isolated from naïve 6–8 week old female C57Bl6/J mice as previously described (34). Day 7 BMDM were re-plated in complete RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES buffer, and 0.0002% β-mercaptoethanol.…”

The Role of MicroRNAs in Regulatory T Cells

“…Micro466a-3p. Becker et al analysed the microRNA expression profile of CD4 + T cells from the draining lymph node of a murine model of allogenic transplantation and found that micro466a-3p expression was increased after allogeneic transplantation [71]. To further research the role of micro466a-3p in Treg, micro466a-3p was transfected into CD4 + T cells, which could reduce iTregs, while inhibiting micro446-3p expression in CD4 + T cells could increase Tregs and reduce effector cells.…”