MiR-34a Promotes PDC And CDC Development By Targeting WNT1

Dendritic cells (DCs) derived from hematopoietic stem cells play crucial roles ininnate and adaptive immune response and in maintaining immune tolerance toself-tissues. DCs are sparsely distributed in the lymphoid tissues, non-lymphoid tissues,and circulation. They are a heterogeneous group of cells that have been divided intodifferent subsets with distinct patterns of cell-surface molecule expression, which alsohave distinct life span and immune functions. Two major subsets of DCs in mice andhumans are plasmacytoid DCs (pDCs) and conventional DCs (cDCs)； Although thegeneral pathway of DC development is obtained, the molecular regulations involved inthis pathway remain unclear.MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionallyregulate gene expression by binding to their target mRNAs, thereby inhibiting theirtranslation or affecting their stabilities. More than60%of all human coding genes arepredicted to contain miRNA binding sites in their3’ untranslated region. Therefore,miRNAs are key regulators in many biology processes. Many studies have alreadydemonstrated that miRNAs are involved in the modulation of hematopoietic lineagecommitment such as in T cells, B cells and NK cells commitment. Some miRNAs arealso functional in DC development and activation. Treatment of monocytes withmiR-21or miR-34a inhibitors partially stall MDDC differentiation, and the result isenhanced upon inhibition of both miRNAs. miR-221and miR-222are more highlyexpressed in cDCs than in pDCs. Inhibition of function of either of these two miRNAsin BM cells by using their agonist oligo increased pDC/cDC ratio, which suggests thatmir-221/mir222may promote pDC development. However, the manner by whichmicroRNAs regulate DC development remains largely unclear.To further assess the function of miR-34a on hematopoietic system, we initiallyconstructed the miR-34a overexpressing retrovirus plasmid pMDH-PGK-GFP-mir-34aand confirmed that this plasmid can functionally encode the miR-34a mature sequenceby real time PCR and Northern blot. Thus, we transduced mouse BM cells enriched for HSC/progenitor cells with viruses, onto which mouse miR-34a was integrated.Transduced cells were transplanted into lethally irradiated recipients. Flow cytometryanalysis of the BM and spleen in stably engrafted primary chimera mice showedincreasing numbers of cDCs and pDCs denoting chimerism eight weeks to12weeksafter transplantation, as well as reduced percentage of B cells in BM, which isconsistent with the findings of David Baltimore.To verify the phenotype of miR-34a chimera mice further, we established amiR-34a-TG mouse model by micro-injecting linearized pMDH-PGK-GFP-mir-34aplasmid into ES cells with C57/BL6background. Quantitative PCR indicated that theexpression of miR-34a was dramatically increased in miR-34a TG mice. FACS analysisof BM and spleen from miR-34a-TG mice showed similar phenotypes as those frommiR-34a chimera mice. These data suggest that miR-34a is involved in the developmentof pDCs and cDCs, as well as B cells. Thereafter, we focused on the function ofmiR-34a on cDC and pDC development.To explain why overexpression of miR-34a can increase the levels of cDCs andpDCs, we stained the BM and spleen cDCs and pDCs with annexin V to evaluate theapoptosis of cDC and pDC in mir-34a-TG mice. FACS analysis showed no significantlydifference on the apoptosis of cDC and pDC between wild-type (WT) and miR-34a-TGmice. We further investigated whether this increase can be attributed to the changedproliferation of cDCs and pDCs, we injected BrdU (1mg/mice) into mice through i.p.and stained BM and spleen cells with anti-BrdU antibody, followed by FACS analysis.The percentage of BrdU positive cells among cDCs or pDCs also exhibited nosignificant difference between WT and miR-34aTG, which suggests that miR-34a alsodid not affect the proliferation or self-renewal of both cDCs and pDC in vivo. Thesefindings demonstrate that the increase in cDCs and pDCs in miR-34a TG mice is notcaused by changes in apoptosis and proliferation.Overexpression of miR-34a does not affect cDC/pDC apoptosis and proliferation.Thus, we try to investigate whether this increase in cDC/pDCs can be attributed to thefact that miR-34a promotes DC development. First, we examined the percentages ofcells, including GMP, CMP, PreDC, and ProDC, at different checkpoints during DCdevelopment using FACS. The results also show that the percentages of these subsets,including GMP/CMP, ProDC and PreDC, of miR-34a TG mice were similar to those ofWT mice. This finding suggests that miR-34a was not involved in the development DC progenitors and may promote preDC development into cDC/pDC.To explore the molecular mechanism of DC development regulation by miR-34a,we first searched for the predicted targets of miR-34a by using online software. We thenselected some genes, such as E2F2and WNT1, which may contribute to DCdevelopment. To validate whether miR-34a directly recognizes the3’ UTR of thesetranscripts, we cloned oligonucleotides representing the presumed target sites into the3’UTR of the luciferase gene. We also constructed the corresponding reporter vectorscontaining mutated binding sites. Luciferase activity was compared in293T cellsco-transfected with one of these constructs, and vector expressing miR-34a and TKplasmid. The overexpression of miR-34a had no effect on the oligonucleotide constructrepresenting the E2F23’UTR, which indicates that E2F2is not a miR-34a target. Bycontrast, miR-34a reduced the luciferase activity of the reporter construct containingmiR-34a binding site in WNT13’UTR, whereas no effect was observed for constructcontaining a mutated WNT1seed site. These findings suggest that WNT1is the targetof miR-34a. We also found that the amount of WNT1mRNA decreased based onRT-PCR assay, and the expression of WNT1protein was also blocked in CD11c+cellspurified from spleen of miR-34a-TG mice by Western blot. These observations suggestthat miR-34a downregulated the translation and transcription of WNT1.To examine whether WNT1is the functional target of miR-34a in DCs, wepreserved the expression of WINT1in BM cells from miR-34a-TG mice enriched forHSC/progenitor cells with mouse WINT1or empty control MSCV-basedGFP-expressing retrovirus, the expression of WNT1coded by pMSCM-wnt1in293Tcells was confirmed by western-blot. Transduced cells were transplanted into lethallyirradiated recipients to establish a bone marrow chimera model. One to four monthsafter the establishment of the model, we detected the percentage of DCs in the GFP+population that overexpressed WNT1and GFP-WT population. Percentages of bothcDCs and pDCs were rescued in WNT1overexpressing cells compared with controlcells. These data strongly suggest that WNT1is the functional target of miR-34a inDCs.Given that DCs serve a critical function in T cell response, we further explorewhether miR-34a overexpression in DCs can affect DC function on T cell response.First, we stimulated WT cDCs with OVA overnight in culture and then checkedmiR-34a expression using RT-PCR. miR-34a expression was downregulated in OVA-treated cDCs compared with untreated cDCs, which suggests that miR-34a mayserve a critical function in antigen-presentation or maturation of DCs. To test whethermiR-34a indeed affects the antigen-presentation of DCs, we checked the uptake ofFITC-labeled OVA protein by cDCs from WT or miR-34a TG mice at2,4, and6husing FACS. We found that the intensity of FITC in DCs from two groups of mice werenearly identical, which suggests that miR-34a overexpression did not affect theantigen-uptake of DCs. We also detected the expression levels of activation markers ofDCs, such as CD80and CD86, and we failed to find any difference in the expressions ofthese cDC activation markers from WT and miR-34a TG mice. These findings suggestthat miR-34a did not affect the activation of cDCs. However, when we co-culturedcDCs purified from WT or miR-34a-TG mice with OVA-immunized CD4+T cells, wefound that the response of CD4+T cells was significantly inhibited in the miR-34a-TGgroup, which suggests that miR-34a overexpression in cDCs can impair the response ofCD4+T cells. To further explore how miR-34a affects T cell response via DCs, we firstmeasured the expression profile of cytokines in cDCs by using RT-PCR. Surprisingly,IL-17a was significantly highly expressed in cDCs from miR-34a TG mice. Intracellularstaining also demonstrated that IL-17a was highly expressed in miR-34a overexpressingcDCs. Furthermore, the addition of IL-17a into co-culture system of cDCs and T cellscan also inhibit the proliferation of CD4+T cells. However, after neutralization ofIL-17a with its antibody, this inhibition disappeared. These results strongly suggest thatmiR-34a overexpression in cDCs can inhibit CD4+T cell response, which may bemediated by IL-17a.IL-17a may affect the response of CD4+T cells directly or indirectly using DCs asa mediator. When we stimulated CD4+T cells with anti-CD3/CD28antibodies in theabsence of DCs, we did not observe any inhibition after the addition of different dosesof IL-17a. These findings suggest that IL-17a may directly act on cDCs and exert aninhibition function on CD4+T cell activation via DCs. Some evidence has alreadyshown that WNT1can modulate TCF1to regulate RORγT expression, thus regulatingIL-17a transcription. We thus detected the expression of TCF1and RORγT in cDC frommiR-34a-TG mice. Consistent with our expectations, Q-PCR results showed that TCF1expression decreased, but that of RORγT significantly increased in cDCs frommiR-34a-TG mice compared with that in their WT counterpart.Taken togethor, miR-34a overexpression in DCs downregulates the WNT1 expression, thus resulting in the downregulation of TCF1, upregulation of RORγT andconsequent upregulation of IL-17a, which affects cDCs in turn, as well as the inhibitionof CD4+T cell activation via DCs.