| Mannan-binding lectin (MBL) is the member of type C serum lectin superfamily which is recognized as one of the most important molecules for innate immune system. This kind of highly conservative glycosylated protein is mainly synthesized by hepatocytes and subsequently released in peripheral blood. Generally, Serials oligomer of MBL based on the tripolymer composed by3homogeneous peptide chains circled in vivo. MBL is capable of activating2MBL associated serine proteases, as of MASP-1, MASP-2and verified to initiate activity of complement system by lectin pathway through recognizing wide spectrum of carbohydrate moieties ligand on the surface of variety of pathogenic microorganisms. The complex then conduct the cell lysis and opsonophagocytosis indirectly. Receptors for MBL known today is collectin receptor combined with which MBL conduct such modulatory function as direct opsonophagocytosis, inflammation regulation and process of cell apoptosis. Meanwhile, soluble Patteren Recognition Receptor (PRR), including MBL as well as C1q, Pulmonary surfatcant-associated protein A (SP-A) and SP-D are members of defense collagen super family whose ligands are so called Pathogen-Associated Molecule Pattern (PAMP) which are identical to one or even more than one pathogene populations and especially essential for their survival but not for the host. MBL is able to prompt rapid effectively protective immune responses by the combination between globular carboxyl structure and specific PAMP. Additionally, MBL possesses broad spectrum of binding site and structure such as immunoglobulin to conduct phagocytosis of apoptotic cells, MBL-dependent activation of Th cells and cell mediated cytotoxic effects as well. In recent years, it was found that, MBL from rat can enhance the phagocytosis of Escherichia coli and Staphylococcus aureus for Kupffer cells in liver. Artificial reconstructive MBL was also detected to prompt phagocytosis of THP-1towards Escherichia coli. On the other hand, cytokine profile and antigen presenting cell (APC) were believed to play vital roles in determing the type and intensity of immune responses after exposure to dangerous signals. Furthermore, it was reported that MBL possesses ability to bind to both monocytes (Mo) and dendritic cells (DC) and to regulate secretion of cytokine profile leading to inhibition of release of proinflamatory cytokine as TNF-a from LPS/CD14induced Mo and up-regulating expression of cytokines with inhibitory characteristics. T cells are also protected from DC-SIGN mediated infection. Data showed that, SP-A and SP-D were well illustrated to bind to sorts of immune cells, such as Mo, DC and T cells, developing functions as modulators in either innate immune or adaptive immune responses. A enhanced expression of surface receptors on macrophages and release of regulatory cytokines and free radicals as well as clearance of autoapoptotic cells were observed in research on these two members of collectin super family.DC, as the most potent professional cells for antigen-presenting, are responsible for induction of immune activity and immune modulation in vivo upon activation mainly via cytokines production and interaction with T cells and B cells. Initially, DCs was thought to be poorly phagocytic. Accumulating evidence has been shown on this point that DCs have a potent phagocytic activity which is mostly dedicated to inform and signal the adaptive immune system about peripheral tissue cues through their unique ability to sample tissue antigens. The capability of migration to the draning lymph nodes, presentation extracellular antigens and initiation tissue-specific T cell immunity are also highlighted simultaneously, the way in which adaptive immune responses are activated and regulated. Therefore, DCs were unique and of great importance for effective induction of immune responses. However, there are far less DCs living inside human body with much functional complexity, by which our understanding of DC differentiation in vitro, antigen presenting and regulation function etc. is greatly limited. The primary in vitro experiment suggested that, human immature DCs could induce the generation of T cells with allogeneic antigen reactivity. Later, Toll like receptor (TLR) agonists derived from invader pathogenes were identified to block conventional differentiation of CD14+Mo at early stage into immature DCs resulting in a deviated phenotype during infection. These TLR induced APCs were characterized by a retained expression of CD14and a lack of CD la, which attracted attention for their regulatory functions. Moreover, it is reported similarly that, culture with Yssel’s medium enabled a phenotypically and functionally novel Mo-derived DCs subset that lack IL-12synthesis but produce high levels of IL-10with altered inhibitory characteristic. The first24hours were regarded as the most critical period in determining DC differentiation, because DC is likely to differentiate into conventional phenotype with addition of stimulatory factors after then.It is worthy to focus on CD1family molecules that are mainly responsible for presenting of most lipid antigen during the process of antimicrobial immunity in vivo. They are normally of high expression at early differentiation of Mo-derived DCs and regarded as surface bio-marker in cell development. The expression of CD1a appears to be dependent on the functional state of the DCs, as CD la expression is high when cells are capturing antigen and is down-regulated when antigen presentation is occurring. Pathologically, the expression of CD la correlates with CD83positive DC subset. There is evidence suggesting that, CD1a is required for induction of potent cell-mediated immunity to Mycobacterium and can be considered clinically to assess the course of leprosy. CD1+CD83+Mo-derived DCs were highly efficient APCs for CD1-restricted T cells activation and for the clinical treatment with further instructional significance on process monitoring.After isolation and purification of human peripheral blood Monocyte (PBMC), we explored the effect of MBL during differentiation to DCs among different groups by observing cellular morphology on early differentiation, immature stage and mature stage consecutively. Surface marker molecules, cellular function and corresponding mechanism were examined respectively. Here we reported the inhibitory induction of peripheral blood mononuclear cells derived monocytes to conventional dendritic cells when administrated with MBL at very beginning of DC cultivation in vitro and explored the corresponding molecular mechanisms. An alternative phenotype with a reduced expression of CD1a and a retained expression of CD14is presented. It has been observed with up-regulated release of IL-10and IL-6, and functioned as an inhibitor for T-cell proliferation. IL-12was detected at a lower level relatively, but no differences were found in comparative analysis with samples treated with no mannan-banding lectin. Regarding that granulocyte macrophage colony stimulating factor dependent JAK2/STAT5was known as a pivotal transducer during the monocyte-derived dentritic cells development, we therefore examined this pathway and findings showed an attenuated activity of both STAT5and JNK but a stimulated STAT3accumulation in the dentritic cell precursors. Furthermore, phosphorylation of extracellular signal-regulated kinase (ERK) was inhibited as well. Taken together, data indicated that, encounter of Mo with MBL results in an inhibitory differentiation into immature conventional dentritic cells from CD14+monocytes in vitro leading to a deviated phenotype of dentritic cells, which enables mannan-binding lectin to balance the intensity of innate and adaptive immune response indirectly through impeding the eventual generation of monocytes derived dentritic cells. We revealed the possible mechanism that how MBL exert effects on adaptive immunity and elaborated that MBL can probably regulate differentiation of Mo, prompting generation of CD14+CD1alow regulatory DC subset with altered phenotype and function in each stage. Therefore, MBL is conceivable to be regarded as a bridge to integrate innate and adaptive immune responses with novel dual effects both on defense against pathogenes infection and regulation of immune responses intensity.CHAPER1MBL induces differentiation of Mo into regulatory DCs The molecule of CD1a is highly homologous with MHCI and MHCII and has similar capability of recognization and presenting of antigen peptide. Most reported shown that, CD la can be only expressed by Langerhans cells (LC) and some DC resided in skin and thymocytes. Besides, the expression of CD1a correlates tightly with maturation of DCs, as immature DCs are more capable on antigen capture than mature DCs with relatively higher expression of CD la. DCs mature rapidly under stimulation of pathogens resulting in increased capability of antigen presenting and down-regulated ability of antigen capture with a reduced expression of CD1a.We isolated PBMC from fresh heparinized blood of selected healthy donors using Ficoll-Hypaque gradient centrifugation and MACs. High purified Mo were obtained and a large number of Mo-derived DCs were induced using GM-CSF and IL-4simultaneously. At early stage, purified MBL and unrelated protein HSA were added where indicated. Cells were havested at day2and day5respectively aftef routine culture. We examined the expression of surface molecules as markers during Mo development including CD14and CD1a which are regarded as important instructor of Mo differentiation towards DCs by Flow cytometre (FCM). Meanwhile, expression of costimulatory molecules such as CD80, CD40, CD86, CD1a, CD83and class Ⅱ MHC HLA-DR were tested as well to determine maturation of DCs. PCR and ELISA were performed to examine secretion of cytokine profile on protein and gene level respectively.Data showed that, compared with control culture with GI medium alone, experimental group was observed with a less quantity for cell counting ultimately and poor proliferation stimulated by GM-CSF which was thought to have great importance for maintaining survival and activity of CD14+monocytes. MBL did not induce apoptosis in treated cells. Additionally, expression of co-stimulatory molecules such as CD80, CD40and of HLA-DR decreased as well, whereas cells did not differ in expression of CD86between groups with or without stimulation of MBL. Moreover, there was only a weak expression of CD83among all cases examined. The group added by MBL produced undetectable levels of IL-α, IL-1b, TNF-a and IL-12in gene expression relatively, but were observed with high production of IL-10in contrast to group cultured in GI medium. ELISA was performed for further confirmation. Findings were identical to the observation of genetic test. Besides, the expression of IL-6was stimulated as well by MBL, suggesting that it is probably to obtain an altered DC subset with deviated phenotype and function from conventional DCs with early encounter of MBLThis part of work enables a steady and sufficient supply of MBL protein with high purity and natural biological activity after experimental identification. High purified Monocytes were obtained from MACs isolation and available for DC induction in vitro with high cytoactivity. Being relatively shout-lived, DCThis method applied were completely conceivable and repeatable for subsequent DC research quantitatively. At the same time, we identified a probable regulatory DC subset treated by MBL at early stage of Mo differentiation and finished primarily the characterization on phenotypic analysis and cytokines expression profile of of these DCs.CHAPER2Primary explorations on mechanism in generation of MBL-induced regulatory DCResearch of recent years showed that, there is low IL-12secretion detected from plasma derived DC which directs Th2differentiation in both human and murine system, whereas, Mo derived DC favors differentiation of Thl cells by producing high levels of IL-12. Because these DC subsets were derived from different cell populations, it remained to be determinded whether the same precursor has the potential to differentiate into DC with different cytokine production profiles. It is discovered by Yssel etc. that, when Mo was induced by Yssel’s medium which is IMDM enriched with human transferrin, insulin, linoleic acid, oleic acid and palmitic acid, it will differentiate to CD14+DC. These DCs remains CD1a-and lacks IL-12production even upon stimulation of LPS and IFN-γ, which were designated as mDC2, mDC2is characterized by both lack of IL-12and CD1a negative expression. They are identified by regulating differentiation of T cells and enhancing production of Th0/Th2cells. Myeloid dendritic cell differntiation depends on the activity of GM-CSF, a cytokine that signals through Janus kinases (JAKs) and signal transducers and activators of transcription (STATs), especially JAK2and STAT5.Based on this, we co-cultured these MBL-induced DCs together with T cells and found that addition of MBL exerted effects on early development of immature DC leading to the inhibition of either proliferation or activation of T cells and their secretion of IFN-γ as well. The lack of IL-12and CD1a expression by these MBL-induced DCs attenuated their capacity of antigen presenting but did not affect their capability of antigen uptake. Meanwhile, significant decreased expression of STAT5and JNK were observed through Westernblot signal pathway analysis. We also examined the extracellular regulatory protein kinases (ERK), finding an decreased activity of phosphorylation and stimulated expression of STAT3, by which way MBL probably regulated Mo differentiation phenotypically and cytokine production profile targeting the pathway of GM-CSF dependent STAT5/JAK2. Moreover, these mDC are verified here that they can be matured into CD83+cells.CHAPER3Effects on maturation of monocyte-derived dendritic cells by mannan-binding lectin in vitroBased on the DC research in our lab previously, we explored the further effects of MBL on Mo-derived DC maturation and corresponding mechanism in this part. DC were generated by routine induction culture and harvested at day5. Immature DC cells then can be obtained from this culture system with repeatable classical cellular phenotype. After that, cells were designated into different groups for another2days, among which MBL was added in experimental case and HSA group was considered as control test with unrelated proteins. Mature cells were harvested as DC development at day7and supernatant was collected of each group. FCM and ELISA were performed to analyze effects on maturation of Mo-derived DC of MBL both on cellular and molecular levels. Western Blot was ran to determine the targeting signal pathways controlled by stimulatory factors at protein level. Data showed that, under the condition of existence of innate recognition molecule of MBL, up-regulated changes occurred on such surface markers as CD83, CD86, CD80, CD40and HLA-DR as a molecule of MHCⅡ, resulting in enhancement of proliferation for non antigen specific homologous naive T cells. Contrast to the role in the process of development of DC precursors to immature DCs, MBL stimulated expression of STAT5but attenuated activity of both STAT3and SOCS. Production of IL-12and were increased while nither TNF-a nor IL-6secretion was recorded, which suggested that, MBL possessed the ability to prompt the mature process of immature DC including functional facilitation in each stage of APC development, from which the intensity of innate immune reponses were regulated. Eventually adaptive immune response system was directed through these altered APCs and effecter immune cells.In conclusion, in this study we focus on the additional capability of MBL besides its conventional role in innate immune response system as a vital recognizer and defense molecule responsible for invading pathogens clearance, confirming that MBL exerts effects on human adaptive immune responses by regulating both development process and function of APCs, especially influent the differentiation towards conventional DC of Mo at early stage. Inhibitory DCs with deviated phenotype were generated as a probable regulatory DC subset at specific stage of development. These CD14+CD1alow cells conducted negative regulation through interaction with naive T cells and characterized by lack of IL-12synthesis, producing medium levels of both IL-10and IL-6. Most co-stimulatory surface molecules were down-regulated. Furthermore, the regulatory mechanisms were revealed primarily on MBL mediated balance between intensity of innate immune and adaptive immune response in the pathological state such as infection by presenting analysis of molecular mechanism and the signal transduction pathways. |
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