Regulation And Mechanisms Of C-reactive Protein To The Immuno-functions Of The Dendritic Cells

Immuno-inflammation is a major patho-physiological mechanism in the development and progress of Coronary heart disease (CHD). C-reactive protein (CRP) is one of the most popular inflammatory marker and considered as a very important predictor for the risk of CHD consistently. However, as for the causal association between CRP and CHD, it has been a hot controversy issue internationally.Dendritic cells play a critical role in the inflammatory process of CHD. They initiate the immuno-inflammation and regulate them at the same time. In our previous studies, we have reported that OX-LDL and Advanced glycation end products activate DCs and initiate immuno-inflammtion in CHD, while some drugs or materials which inhibit the progress of CHD can regulate DCs negatively and alleviate the inflammatory response correspondingly.It is reported that DCs express CD32, the important receptor of CRP, indicating the regulation of CRP to the DCs functions. However, there are opposite reports as for the regulation of CRP to DCs currently. In addition, seldom results can been found for the molecular mechanisms of regulation of CRP to DCs.Based on the critical role of DCs in CHD, the widespread controversy about the causal relationship between CRP and CHD as well as the inflammatory regulation of CRP to DCs at present, we carried out this study to explore the immuno-regulations of CRP to DCs and the molecular mechanisms. The results of this study will provide evidence for the clarification of the causal relationship between CRP and CHD.Part â… The regulation of C-reactive protein to the immuno-functions of the dendritic cellsAims:To investigate the effects of CRP to the immuno-phenotypes, cytokines expression and the capacity to stimulate the proliferation of T cells of DCs.Methods:(1) Isolated CD14+monocyte, and differentiated them to the monocyte-derived dendritic cells (MoDC) by cytokines.(2) Groups:Control, LPS (100ng/mL) and CRP (5,15,30μg/mL). The cells and culture medium were taken at48hours.(3) The immuno-phenotype of MoDC was examined by FACS.(4) The expression of the inflammatory cytokines, including IL-12,IL-10,TNF-å'ŒIL-6, were examined by quantitative PCR and ELISA.(5) The CD4+CD45RA+T cells were isolated, and the DCs treated with CRP (30μg/mL) or LPS (100ng/mL) for48hours were miexed with the isolated T cells with the concentration ratio being1:5,1:10and1:20. The allogeneic mixed lymphocyte reactions were used to examine the effects of DCs to the proliferation of T cells.Results:(1) MoDC were successfully cultured after morphological identification;(2) The FACS results showed that CRP (15and30μg/mL) upregulated CD80and CD86(p<0.05), while no significant effects for CD83, CD40and MHC-â…¡(p>0.05);LPS upregulated the expression of CD80, CD86, CD83, CD40and CD1a(p<0.05);(3) Based on the PCR and ELISA results, IL-12expression was not significantly changed by CRP (30μg/mL)(p>0.05). For IL-10expression, it has decreased tendency (p>0.05). In addition, CRP decreased TNF-α mRNA expression, but has no effects to IL-6(p>0.05). On the other hand, LPS increased the expression of IL12and IL-10significantly (p<0.05), and LPS upregulated TNF-α and IL-6mRNA significantly also (p<0.05);(4) CRP didn’t activate the proliferation of T cells, but with an inhibitory tendency (p>0.05), While LPS promoted the T cells proliferation significantly (p<0.05), and with the strongest effect being DC:T=1:10.Conclusions:(1) With consistency to the previous reported results, LPS induces matured DCs and promotes T cells to proliferate.(2) CRP upregulates the DCs phenotypes partly, but doesn’t induce inflammatory cytokines expression, neither T cells proliferation.(3) It is reasonable to consider that CRP induces semi-maturation DCs, regulates the immuno-functions negatively, and thus maintains the homeostasis of DCs.Part â…¡The examination and analysis of the whole genome expression microarray of CRP and LPS to DCs independentlyAims:To explore the differences and similarities of the immune regulation and their molecular mechanisms between CRP and LPS to DCs.Methods:(1) Cultured MoDC (from3differently healthy male blood donors) were treated with medium, CRP (30μg/mL) or LPS (100ng/mL) respectively. The total RNA were extracted at48hours and the Affymetrix affymetrix u133plus2.0was used to examine the whole genome expression;(2) TwoClassDif was used to screen the differentially expressed genes;(3) The online software MAS2.0was used to analyze the gene functions (GO/Biological function) and signaling pathway (PATHWAY/KEGG);(4) The signaling transduction network (Signal-Net) was constructed based on the differentially expressed genes induced by CRP.Results:(1) The quality of RNA was qualified for chip examination;(2) The number of the differentially expressed genes for CRP is815. Among them, the number of the specific genes for CRP is613, with432upregulated and181down-regulated. The number of the differentially expressed genes for LPS is1477, and among them, the specific ones for LPS are1303(804upregulated and499down-regulated). Additionally, a small number of genes are differentially expressed by both CRP and LPS;(3) Among the CRP specifically down-regulated genes, significant GO for immuno-functions of DCs include inflammatory response and chemotaxis, and significant PATHWAY is involved in Cytokine-cytokine receptor interaction and Toll-like receptor signaling pathway;(4) Among the LPS specifically up-regulated genes, significant GO for DCs immuno-functions include, majorly, positive regulation of I-kappaB kinase/NF-kappaB cascade, positive regulation of IL-2biosynthesis, response to interferon-gamma, positive regulation of interleukin-4biosynthesis, interleukin-6-mediated signaling pathway and positive regulation of IL-10biosynthesis, etc. Some significant PATHWAY included Toll-like receptor signaling pathway, Cytokine-cytokine receptor interaction, B cell receptor signaling pathway and T cell receptor signaling pathway, etc.(5) Among differentially expressed genes by CRP, some GO and PATHWAY were closely related to lipid metabolism, such as GO:lipoprotein catabolism and GO:cholesterol biosynthesis; PATHWAY:short-chain fatty acid metabolism, Adipocytokine signaling pathway and PPAR signaling pathway.(6) There were two classes of genes majorly in the Signal-Net map, one type is chemokine, chemokine receptor and transcription factor, including CCL2(MCP-1), CCL3(MIP-1α),CCL4(MIP-1B), CCL7(MCP-3), CXCL1-CXCL3, CX3CR1and NFATC2. All these genes were down-regulated. Another type was cell-cycle and apoptosis related genes, such as CDKN1B, CCND1, CCND2, CCNE2, PTEN and FOXO1.Conclusions:The high-throughput screening data demonstrates that,(1) Contrary to LPS induced DCs maturation and initiation of T cell proliferation, CRP down-regulates the immune related genes of DCs, including inflammatory cytokines, chemokines and their receptor;(2) CRP is involved in lipid metabolism by regulating DCs, which is different from LPS;(3) CRP might change the expression of the apoptotic genes, and thus regulates the DCs functions.Part â…¢The prelimilary study of the molecular mechanisms of immuno-homeostasis by CRP regulated DCsAims:To validate the expression of the apoptotic genes in the Signal-Net including PTEN, FOXO1and CDKN1B (p27.kip1) and to explore the apoptosis and mechanisms of DCs by CRP.Methods:(1) The cultured MoDC was treated and grouped as the second part;(2) Quantitative PCR was used to validate the expression of PTEN, FOXO1and CDKN1B (p27.kip1);(3) Western Blot was used to examine the protein expression of the above3genes. And the protein Caspase-7and PARP were tested also;(4) The apoptosis of MoDC was examined by FACS.Results:(1) By PCR and Western Blot, CRP induced the upregulation of PTEN and CDKN1B (p27.kip1) significantly, with FOXO1being upregulated in some degree; LPS upregulated the expression of FOXO1significantly, with the upregulation tendency of CDKN1B (p27.kip1) also;(2) Both CRP and LPS increased and activated the expression of Caspase-7and degraded PARP;(3) CRP and LPS induced the apoptosis of DCs.Conclusions:(1) With consistency to the microarray data, CRP upregulates the expression of the tumor suppressor genes including PTEN, FOXO1and CDKN1B (p27.kip1);(2) By upregulating the above3genes, CRP activates Caspase-7and degrades PARP;(3) CRP induces the apoptosis of DCs, and thus maintains the homeostasis of immuno-inflammation of DCs. Summary of the whole study1. Contrary to LPS induced immuno-inflammation, CRP regulates the immuno-functions of DCs negatively by inducing smDCs and down-regulating the expression of the CC and CXC chemokines.2. CRP induces the apoptosis of DCs by upregulating the expression of the tumor suppressor genes including PTEN, FOXO1and CDKN1B (p27.kip1), activating Caspase-7, and degrading PARP, and thus maintains the immune homeostasis of DCs.Novelty and Value1. We demonstrated that CRP regulates DCs negatively by stimulating smDC formation and by decreasing the expression of CC and CXC chemokines.2. We revealed that CRP induces DCs apoptosis and maintains immuno-homeostasis by upregulating the expression of the tumor suppressor genes including PTEN, FOXO1and CDKN1B (p27.kip1), and then activating Caspase-7and degrading PARP.3. The results of this study enriches the immune regulatory mechanisms of DCs, and paves the way for further exploration of the negative regulation of DCs as well as their functions in CHD.