Lsectin Dc On Expression Of Helper T Cells Subgroup Effects And Its Mechanism

Dendritic cells (DCs), the most important antigen-presenting cells (APCs),instruct the activation and differentiation of naive CD4~+T cells and regulate theimmune responses. When organism is infected by pathogen, pattern-recognitionreceptors (PRRs) expressed on DC binds the pathogen-associated molecular patterns(PAMPs) on the microbial pathogen, which induces the maturation and activation ofDC. The activated DCs present antigen to CD4~+T cells to instruct its activation anddifferentiation. According to the pattern of specific cytokines and function, helperCD4~+T cells can be divided into Th1, Th2and Th17cell subsets. Th1cellspredominantly produce IFN-γ and protect organism from infection of intracellularpathogens, whereas Th2cells secrete mainly IL-4, IL-5, and IL-13and regulatehumoral and allergic responses. Th17cells produce IL-17as its signature cytokines.They mediate defence against extracellular bacterial and involve in the pathogenesisof autoimmune diseases. Thus, differentiated helper CD4~+T cells play important rolesin fighting foreign pathogen infection. Currently, it is well described that Tcell-intrinsic signal pathway and transcription factors regulate the differentiation ofhelper CD4~+T cells, but the molecular mechanism by which DC participates in thedifferentiation of CD4~+T cells is unclear.C-type lectin receptors (CLRs) comprise a large family of receptors that containcarbohydrate recognition domain in its extracellular domain and predominantlyexpress on myeloid cells. They play important roles in regulating immune responses.After binding the fungi-derived carbohydrate, CLRs directly trigger intracellularsignaling of DC or affect TLRs-meidated signaling. Recent studies indicate thatCLRs-mediated signaling mainly induces Th17responses.LSECtin, a C-type lectin receptor, is colned by our experiment from liver andspecially expressed on liver sinusoidal endothelial cells and Kupffer cells. Ourprevious studies indicated that LSECtin binds activated T cells and surpresses T cellactivation in vitro. In vivo, LSECtin expressed in the liver limited the ability ofhepatic T-cell immunity to protect the liver from immunohyperresponsiveness. Liveris an immune-privileged organ. Its immune responses are prone to tolerance, which isdifferent from immune responses induced by blood and bone marrow derived system.DC derived from bone marrow play important roles in immune responses and bridgethe innate and adaptive immunity.In this paper, we first demonstrated that LSECtin expressed on bonemarrow-derived DC (BMDC). BMDCs were stimulated with LPS, IFN-γ, IL-4andTGF-β for different times. We found that TGF-β significantly decreased the expression of LSECtin, whereas LPS, IFN-γ and IL-4did not affect its expression.Therefore, LSECtin is significantly down-regulated by TGF-β in BMDC.To study the role of LSECtin on DC, we cultured LSECtin~+/~+or LSECtin-/-BMDCwith DO11.10na ve CD4~+T cells in the presence of different concentration ofOVA323-339peptide. The proliferation of na ve CD4~+T cells activated by LSECtin-/-BMDC is not altered. In contrast to LSECtin~+/~+BMDC, LSECtin-/-BMDC inhibitedthe production of IFN-γ (Th1signature cytokine) and IL-17(Th17signature cytokine)and promoted the production of IL-13(Th2signature cytokine) from na ve CD4~+T.The transcription factors T-bet, RORγ/RORα and GATA3in CD4~+T cells control theexpression of IFN-γ, IL-17and IL-13respectively. We found that CD4~+T cellsactivated by LSECtin-/-BMDC have less expression of genes encoding Th1and Th17master genes T-bet and Rora/Rorc than did those activated by LSECtin~+/~+BMDC. Onthe contrary, the expression of genes encoding Th2master gene (Gata3) isup-regulated in CD4~+T cells activated by LSECtin-/-BMDC. Cytokines IL-12p70andIL-6/IL-23are critical for the differentiation of Th1and Th17cells respectively, sotheir corresponding receptors IL-12R and IL-6R/IL-23R play important roles indriving Th1and Th17differnentiation. However, the expression of IL-12r andIl-6r/Il23r was unaltered between T cells activated with LSECtin~+/~+and LSECtin-/-BMDC. These results indicated that LSECtin-deficient DC affect the Th1and Th17cells development via impairing the expression of T cell-intrinsic transcription factors,but not cytokine receptors.IL-12p70and IL-6/IL-23secreted from DC are critical in Th1and Th17differentiation respectively. We observed that IL-12p70, IL-6and IL-23cytokines aresignificantly decreased when LSECtin-/-BMDCs were cultured with na ve CD4~+Tcells in the presence of OVA323-339peptide, compared with LSECtin~+/~+BMDC. Toassess the functional importance of LSECtin-dependent IL-12p70and IL-6expression,exogenous IL-12p70or IL-6was added to the culture. We found that exogenousIL-12p70and IL-6restored the IFN-γ and IL-17-secreting capacity of CD4~+T cellsactivated by LSECtin-/-BMDC.Costimulatory molecules expressed on BMDCs play important roles in thedifferentiation of helper T subsets, such as CD11c, CD40, CD80and MHC-II（I-A/I-E）, so we detected the expression of these costimulatory molecules onLSECtin~+/~+and LSECtin-/-BMDC. Our results showed that the expression of CD11c,CD40, CD80and MHC-II（I-A/I-E）was not different between LSECtin~+/~+andLSECtin-/-BMDC. In addition, the cytokine production capacity of LSECtin~+/~+andLSECtin-/-BMDC was not different after stimulating with OVA323-339peptide.Compared with DCs and T cells co-culture system, BMDCs treated with OVA323-339 peptide produced very low level cytokines. Thus, our data showed thatLSECtin-dependent IL-12p70and IL-6/IL-23expression are dependent on thepresence of CD4~+T cells in the co-culture system. In addition, our previous resultsdemonstrated that CD4~+T cells expressed LSECtin unidentified ligand. Collectively,these results strongly suggested that LSECtin on DC promote the production ofcytokines via interaction with its endogenous ligand on T cells and thereby drive thedifferentiation of Th1and Th17.To verify the hypothesis, we test a panel of monoclonal antibody includingCCA023, CFD051and CCB059to LSECtin as an agonist to trigger LSECtinsignaling for their induction of changes in cytokine production after crosslinking.CCA023, CFD051and CCB059recognized transmembrane, neck and CRD domainof LSECtin respectively. We treated DCs with immobilized three LSECtin mAbs,including CCA023, CFD051and CCB059, and isotype control mIgG1. Theproduction of TNF-α were significantly increased by DCs after treatment withCFD051for24h, compared with CCA023, CCB059and isotype control mIgG1,suggesting that only CFD051-LSECtin signaling promotes cytokine production. Tofurther investigate the function of LSECtin-mediated signaling on DC, we treatedDCs with CFD051or isotype control mIgG1. MDDCs treated with CFD051for12hformed many more dendrites (a sign of DC maturation) than did those stimulated withcontrol mIgG1(morphology level). And costimulatory molecules expression ofHLA-DR, CD80, CD83and CD86on DC is also increased by LSECtin-mediatedsignaling. Thus, we demonstrated that CFD051-LSECtin signaling increased thematuration status of MDDCs.More important, we need to prove that LSECtin-mediated signaling promote thecytokine production from DCs, such as IL-6. DCs were treated with differentconcentration CFD051or isotype control mIgG1for24h and IL-6and IL-10in thesupernatant were detected by ELISA. We found that CFD051-LSECtin signalingpromoted the production of IL-6and IL-10in a dose dependent manner. Likewise,DCs were treated with the same concentration CFD051or isotype control mIgG1fordifferent time points. We found that CFD051-LSECtin signaling induced rapid buttransient expression of cytokine IL-6, IL-10and TNF-α mRNA, which peakedapproximately3h after LSECtin ligation. Taken together, we demonstrated thatCFD051-LSECtin signaling promoted IL-6, TNF-α and IL-10production.Next, we want to know the mechanism by which LSECtin mediated intracellularsignaling. Analyzing the amino acid sequence of LSECtin, we found that there is noimmunoreceptor tyrosine-based activatory motif (ITAM) in its intracellular domain.However, there is a peptide sequence WXYWXYWXYY close to the transmembrane domain, where X is a small amino acid and Y is a positively charged residue.ITAM-containing adapter proteins DAP12and FceRIg transduce activating signals.The DAP12and FceRIg-associated receptors usually have a positively charged in orclose to their transmembrane regions for pairing with DAP12and FceRIg.Co-Immunoprecipitation and Immunoblot analysis showed that LSECtin wasassociated selectively with DAP12but not with FceRIg. And the interaction ofLSECtin and DAP12was mediated through the transmembrane region of LSECtin. Inaddition, the Jurkat cells were stably co-transfected with LSECtin and DAP12. Wefound that DAP12stablized the surface expression of LSECtin, preliminarily provingthat the interaction between LSECtin and DAP12is functional. Thus, our resultssuggested that LSECtin-mediated intracellular signaling is dependent on theexpression of DAP12.Taken together, our results indicate that LSECtin on DC recognizes itsunidentified ligand on T cells and triggers the intracellular signaling via its interactionwith DAP12. LSECtin-mediated signaling promotes the production of IL-12p70andIL-6/IL-23from DC and thereby drives the differentiation of Th1and Th17.