The Roles Of IL-27 In The Regulation Of CIITA And Class II MHC Expression

Interleukin-27(IL-27) is IL-12-related pleiotropic cytokine that has seemingly paradoxical functions in inflammation.IL-27 contains two subunits,p28 and EBI3 and signals through a heterodimeric receptor(IL-27R) consisting of the WSX-1 subunit(also called TCCR) and the gp130 subunit.Several cell types,including T cells,natural killer cells,B cells,monocytes,mast cells and neutrophils,express IL-27R.Ligation of IL-27R induces intracellular signaling involving Janus kinases and STAT transcription factors.Originally,IL-27 was thought to promote inflammation by inducing T helper type 1(TH1) differentiation early during immune responses.Several studies have shown that during the early stage of TH1 differentiation,IL-27 induces expression of the transcription factor T-bet,which triggers upregulation of theβ2-subunit of the interleukin 12 receptor(IL-12Rβ2) on naive T cells,thus conferring responsiveness to IL-12.An IL-27-induced T-bet-independent pathway of TH1 differentiation has also been reported.However,in infectious and autoimmune animal models,mice deficient in IL-27R develop excessive,pathological inflammation during both TH1 and TH2 responses.Regarding the anti-inflammatory effects of IL-27,it has been shown that IL-27 inhibits the proliferation of activated CD4+T cells by suppressing IL-2 production.Furthermore, IL-27 suppresses the proinflammatory immune responses of IL-17-producing T helper cells(TH-17 cells) in several animal models,including experimental autoimmune encephalomyelitis(EAE),an autoimmune inflammatory demyelinating disease of the central nervous system and a model of human multiple sclerosis.Recent works also identified a novel role of IL-27 in the induction of CD4+ and CD8+ effector T cells positive for IL-10,and demonstrated IL-10 was involved in the IL-27-mediated suppression of IL-17 but was dispensable for the IL-27-induced suppression of IL-2. IL-10 mediated the suppressive effect of IL-27 on encephalitogenic T cells in adoptively transferred EAE.The mechanisms that orchestrate the pro-inflammatory and anti-inflammatory effects of IL-27 are incompletely understood.Major histocompatibility complex classⅡ(MHCⅡ) proteins and their accessory molecules,such as the invariant chain(Ii) and the nonclassical MHCⅡproteins(DM and DO),are crucial for restricting immune reactivity to self versus foreign antigens during thymic education,and their expression level is also crucial for determining T-cell activation in the periphery.MHCⅡexpression is regulated by multiple cytokines,including interferon-gamma(IFN-γ),which can enhance the immune response by upregulating MHCⅡexpression in immune cells.MHCⅡgene transcription is regulated centrally by the master regulator of its activation,the classⅡtransactivator(CⅡTA).The gene encoding CⅡTA uses at least three distinct promoters (CⅡTApI,CⅡTApⅢand CⅡTApⅣ),and display tissue-restricted expression and differential responses to cytokines.Our study focused on the the mechanisms by which IL-27 mediated its pro-inflammatory effects.Firstly we explored the immunoregulatory effects of IL-27 on endothelial cells(EC).We reveal a role for IL-27 in the induction of MHC expression in primary human umbilical vein endothelial cells(HUVECs).Stimulation of HUVECs by IL-27 rapidly induces IFN regulatory factor-1(IRF-1),and dramatically increases the expression of CⅡTA isoformⅣ.Expression of CⅡTAⅣcorrelates with increased MHC classⅡgene expression.IL-27 also enhances expression of MHC classⅠmolecules.Furthermore,expression ofβ2-m and TAP-1 transcripts increases in response to IL-27.Microarray analysis demonstrates that IL-27 significantly up-regulates a panel of genes that correlate with immune regulation,including the chemokines CXCL9,CXCL10 and CX3CL1 in HUVECs. This first demonstration that both MHCⅡandⅠexpression are increased in EC after IL-27 stimulation suggests that IL-27 may be important in conferring immune function on vascular endothelium.We further examined the effects of IL-27 on THP-1 monocytic cells,a well characterized model of monocytes.Similarly,we revealed that IL-27 increased both CⅡTAⅢand CⅡTAⅣmRNA levels,leading to an enhanced suface expression of classⅡmolecules and elevated mRNA levels of HLA-DM and DO.In addition,IL-27 up-regulates beta-2m,TAP-1 and classⅠMHC expression.Furthermore IL-27 enhances co-stimulatory molecules CD80 and CD86 and adhesion molecule CD54 expression in THP-1 cells.These observations indicate a crucial role of IL-27 in regulating the antigen presentation function of monocytes/macrophages.To delineate the signaling mechanisms by which IL-27 regulate the expression of CⅡTA and classⅡMHC in THP-1 cells,we use ERK inhibitor U0126,p38MAPK inhibitor SB203580 and PI3K inhibitor LY294002 to examine the roles of various signaling pathway in IL-27 induced CⅡTA and classⅡMHC expression.Our results demonstrate that both p38MAPK and PI3K signalings are essential for optimal expression of classⅡMHC in THP-1 cells in response to IL-27,whereas ERK activation plays an inhibitory role in IL-27 induced classⅡMHC expression.PI3K is also required for maximal expression of classⅠMHC expression in THP-1 cells. These data suggest that PI3K signal playways are critically involved in IL-27-mediated effects,yet ERK act as negative regulator of classⅡexpression. Further work using TLR2 agonist Pam3CSK4 and TLR4 agonist LPS shows that these TLR signalings partially prevent IL-27 induced CⅡTA and MHC expression in THP-1 cells.The inhibitory effect of LPS on IL-27 effects is partially mediated through ERK and p38MAPK signaling.IL-27 has been reported to have potent anti-tumor activity against a varity of solid tumors through mechamis involving the induction of CD4+ and CD8+ T cells,and NK cells.IL-27 can also inhibit tumor angiogenesis through induction of chemokines IP-10 and MIG.Another work we have done is construction of IL-27 expression vectors to assess the anti-leukemic activity of IL-27 against murine leukemia models. We used overlap PCR to amplify the signal peptide and the single chain IL-27 gene, and inserted the gene into a variety of eukaryiotic expression vectors including pIRES2-EGFP,pcDNA3.1,p3×FLAG-CMV-9 and PCAGEN.Immunoprecipitation and western blot have been performed to confirm the expression and secrection of IL-27 from the supernatant of 293T cells transfected with IL-27 expressing vectors. Expression of IL-27 was also detected in the serum of mice treated with IL-27 expressing vectors by hydrodynamic gene transfer(HGT).The bioactivity of IL-27 was confirmed by the induction of CⅡTA expression in HUVECs.In conclusion,our work for the first time demonstrates that IL-27 increases CⅡTA and classⅡMHC expression in both monocytes and endothelial cells.IL-27 also up-regulates the expression classⅠMHC,co-stimulatory molecules and adhesion molecules.In addition,IL-27 could activate the critcal transcription factor IRF-1. ERK,p38MAPK and PI3K signaling pathways can be directly activated by IL-27,and they play cucial roles in modulating IL-27 mediated effects.Our results demonstrate novel roles of IL-27 in the regulation of antigen presentation and uncover a new mechanism by which IL-27 promote the activation of T cells and the generation of effective adaptive immune responses.