The Research Into Mechanism Of CCL2 Mediating Myelomonocytic Cell Differentiation And Immune Tolerance Establishment

Tumor is a kind of infinite hyperplasia lesion, which results from cells in a particular tissue that lost their ability on normal growth control under the effect of multiple carcinogenic factors. The occurrence and development of the tumor have close relationship with the status of tumor cells and the local environment. Tumor escapes from immune surveillance by gene augmentation and protein modification of tumor cells, which hides or discards the target that the immune system recognizes and attacks to. On the other side, by organizing a complex network, tumor cells establish a specific tumor microenvironment for the sustaining formation of angiogenesis, tumor invasion and distant metastasis. It has been well accepted that, tumor microenvironment provides fundamental external guarantee for the progress of tumor distant metastasis. More and more attention has been paid on the treatment strategy, which aims to intervention in tumor growth and progression by breaking the tumor microenvironment. Similar with tumor microenvironment, maternal-fetal interface is also an immune tolerance microenvironment during early pregnancy, which is advantageous to the embryo successfully implanted into the womb. Trophoblast cells, as a part of embryo, have similar characteristics with malignant tumor cells in significant proliferation and invasion ability. However, unlike in tumor cells, such abilities of trophoblast cells are highly restricted in space and time. Trophoblast cells will stop proliferation and invasion, once the embryo successfully implanted in the womb. According to this, many researches attempt to utilize the study of function and regulatory mechanism of trophoblast cells to explore new strategy for cancer treatment.Tumor microenvironment composed by cells components, including tumor cells, stromal cells, fibroblasts and immune cells, and non-cells components, such as a variety of inhibitory cytokines, chemokines, signaling molecules and extracellular matrix. As a heterogeneous cell population, immune cells in tumor microenvironment comprise tumor associated macrophages (TAM), myeloid-derived suppressor cells (MDSC) tumor associated neutrophils (TAN), tumor associated Th17 cells, regulatory T cells (Treg) and regulatory dendritic cells (DCreg), etc. Tumor cells crosstalk with recruitment host immune cells to induce and reeducate the aggressive immune cells, which is in favor of the establishment of immune tolerance in tumor microenvironment. Previously researches have shown that, as the largest population of immune cells in tumor microenvironment, proportion of the TAM in some tumor can be as much as half. These cells have characteristics of M2 type macrophages (alternatively activated macrophages), which can produce a variety of anti-inflammatory cytokines, including TGF-β, IL-4, IL-10 and PGE2. The number and sites of TAM infiltrating tumor tissue are closely related to the prognosis of the tumor and the outcome of clinical treatment. It suggests that, TAM plays an important role in establishment and maintaining of tumor microenvironment. Additionally, monocytic myeloid-derived suppressor cells (Mo-MDSC) is a kind of newfound immunosuppressive cells, which widely distributed in the in tumor tissue and peripheral blood of patients. On one hand, these cells promote tumor angiogenesis through the release of VEGF and bFGF and MMPs, on the other hand, inhibites antitumor immune response mediated by T cells, NK cells and macrophages via high expression of immunosuppressive molecules, such as COX2, ARG-1, IDO1, and IL-4Ra. It has been proved that, as the major contributors of maintaining immune tolerance in tumor microenvironment, both TAM and Mo-MDSC are differentiated from CD14+ myelomonocytic cells. The significantly increased TAM and Mo-MDSC in tumor tissue might be the result of recruitment, induction and differentiation of CD14+ myelomonocytic cells by tumor microenvironment. However, the specific mechanisms during this process are still poorly understood.Multiple chemokines and cytokines existing in the tumor microenvironment play important roles in recruitment and reeducating of immune cell, improving angiogenic ability of vascular endothelial cells, promoting invasion and adhesion ability of tumor cells. It has been reported, chemokine (C-C motif) ligand2 (CCL2) is remarkable increased in oral squamous cell carcinoma (OSCC), prostate cancer, lung cancer, ovarian cancer and rectal cancer. CCL2 is first confirmed by combining with its receptor CCR2 to recruit myelomonocytic cells and macrophages. Many studies have discovered that, expression level of CCL2 in tumor tissues has close relationship with clinical outcomes such as tumor size and whether metastasis or not. Further research indicates that, CCL2 can inhibit production of Thl type inflammatory cytokines to maintain oral immune tolerance microenvironment; induce T cells polarizes towards Th2 cells with anti-inflammatory ability; up-regulate the expression of signal transducer and activator of transcription-3 (STAT-3) in myelomonocytic cells, which also be considered as the necessary signals for amplification and activation of Mo-MDSC. That suggests:elevated CCL2 in the tumor microenvironment may play an important role in the process of recruitment, induction and differentiation of myelomonocytic cells.The rearrangement of the cytoskeleton is involved in the change of cell structure and function during proliferation, migration and invasion of tumor cells and reeducation of immune cells in tumor microenvironment. Kinesin superfamily (Kif) is a kind of microtubules-deepened molecular motor proteins, which participate in many cellular processes, including cytoskeleton rearrangement, mitosis, and intracellular transport and so on. It has been reported that, as a member of this super family, Kif4A is not only the key factor in many cellular processes, but also is an important trigger of tumor occurrence and development. According to the research on OSCC and long cancer, the expression level of Kif4A negatively correlated with prognosis of tumor. Notably, a report shows that, it is involved in T cell activation. These clues indicate that, Kif4A might have a potential regulatory function on the differentiation of immune cells, such as T cells and macrophages, in tumor microenvironment. Based on the phenomenon that elevated CCL2 in the tumor microenvironment, we employ an in vitro co-culture system to explore the function and related mechanism of CCL2 in mediating myelomonocytic cells differentiating into Mo-MDSC with strong immunosuppressive ability and M2 macrophages with a predominate expression of anti-inflammatory cytokines. Furthermore, we detected the roles of Kif4A in this process, in order to further explain the potential function and mechanism of CCL2 in establishment and maintain of immune tolerance microenvironment. Part I A research into the effects of CCL2 and Kif4A on macrophages recruitment and differentiation OBJECTIVE:to explore whether CCL2 or Kif4A have any effects on the recruitment and differentiation of macrophages. METHODS:1. Establish an in vitro co-culture system of tumor cells and macrophages.Human OSCC cells line Cal-27 and monocyte cell line THP-1 derived MO macrophages are employed to establish an in vitro co-culture system with 0.4 μm Transwell. 2. Analyze the change of Kif4A and CCL2/CCR2 in coculture system. We use western blotting to analyze the expression of Kif4A and CCL2/CCR2 in both Cal-27 and THP-1 derived macrophages, ELISA to detected CCL2 in supernatant of coculture system, and REQ-PCR to confirm the resource of CCL2. 3. Block Kif4A and neutralize CCL2 with neutralizing antibody. We block the expression of Kif4A in cells of coculture system with siRNA and neutralize CCL2 in supernatant with neutralizing antibody.4. Cell migration assay. Cell migration assay was carried out using 24-well transwell inserts (8 μm pore size membrane). In brief, cells (1×105cells/well) were placed into the upper chamber of transwell inserts with 100 μL of serum-free medium. The other type of cells (1×105 cells/well) in 600 μL RPMI 1640 medium containing 10% FBS were added into the lower chambers of the transwell plate. Cells were incubated at 37℃ in 5% CO2 for 24h. The migrated cells on the lower side of the membrane were fixed by 10% formalin and stained with eosin. Five random fields of each well were photographed and counted.5. Analyze the expression of anti/pro-inflammatory cytokines and suppressive molecular. Amplification of cytokines such as TNF-α, IL-1β,L-6, IL-12p40, TGF-β, IL-4 and IL-10; and suppressive molecular such as ARG-1, COX2, IDO1, IL-4R and NOS2 were carried out by RTQ-PCR. Relative gene expression was normalized to the expression of GAPDH mRNA and expressed as fold change for each relative gene reaction among the samples.RESULTS:1. Both Kif4A and CCR2 are increased in THP-1 derived macrophages via co-culturing with OSCC. Western blotting analyzes expression of Kif4A in Cal-27 and THP-1-derived macrophages in co-culture system after 24 hours culturing. Compared with the monoculture group, coculturing with Cal-27 cells significantly increased Kif4A expression in THP-1-derived macrophages (p< 0.01). However, there was no obviously change of Kif4A that can be detected in Cal-27 between co-culture system and monocultures group.2. Blockade of Kif4A ablates the significant expression of CCL2 in co-culture system. Supernatant CCL2 concentration was analyzed by ELISA, secreted levels of CCL2 were remarkable elevated in both Cal-27 cells and THP-1-derived macrophage when co-cultured with each other compared with cells monoculture groups (p<0.01). Treated Cal-27 cells or THP-1-derived macrophage with siKif4A, it dramatically prevented the up-regulation of CCL2, especially in the Cal-27 chambers of co-culture system (p<0.01).3. Cal-27 cells enhanced migration of the macrophage via Kif4A-CCL2 induction. Cell migration assay shows that, after 24 hours, remarkable increased numbers of THP-1-derived macrophages migrated toward Cal-27 cells in the lower chamber, as compared with the lower chamber containing medium alone (p<0.01). Both anti-CCL2 neutralizing mAb and siKif4A significantly prevented the up-regulation of macrophages migration(p<0.01).4. Kif4A-CCL2 modulated THP-1-derived macrophages towards M2-type cytokine profile and high expression of suppressive molecules. RTQ-PCR analysis suggested, Kif4A involve in regulating macrophages towards M2-type. In addition, it also significantly improved expression of suppressive molecules such as ARG-1, COX2, IDO1 and IL-4Ra in THP-1-derived macrophages. Both these processes were achieved by regulate expression of CCL2.CONCLUSIONS:The results provided experimental evidences that a remarkable interaction may occur between tumor cells and macrophages, which gave rise to increasing expression of Kif4A in both two types of cells and elevated CCL2 level in supernatant. In addition, the up-regulated expression of CCR2 on macrophages and high concentration of CCL2, which, in turn, promoted recruitment and polarization of macrophages, could be modulated by Kif4A.Part ⅡA research on mechanism of CCL2 regulating myelomonocytic cell differentiation into MDSCOBJECTIVE:to explore the regulatory role of CCL2 in myelomonocytic cell differentiation into Mo-MDSC. To further expose mechanism of CCL2 in establishing of immune tolerance microenvironment.METHODS:1. Examine the ratio of Mo-MDSC in peripheral blood of early pregnant women. Flow cytometry analysis Mo-MDSC (CD14+HLA-DR-/low) in peripheral blood of early pregnant women and non-pregnant women.2. To obtain CD14+myelomonocytic cells and CD4+T cells. PBMC were isolated from the freshly obtained peripheral blood of healthy non-pregnant female donors by density gradient centrifugation. For isolation of CD 14+ myelomonocytic cells, PBMC were resuspended in MACS with CD 14 MicroBeads in it. For isolation of CD4+T cells, PBMC were purified using CD4 Microbeads and MS separation column. The purity of the CD14+ myelomonocytic cells and CD4+ T cells was>95% as assessed by flow cytometry.3. Coculture system studies. Trophoblast cell line HTR8/SVneo and CD14+ myelomonocytic cells are coculture at 1:1 for 40 hours..4. Cytokine and Chemokine Analysis. The amount of human cytokine and chemokine, including CCL2, TGF-β, IL-4, IL-6, IL-8 and IL-10 in the coculture system and control supernatant was measured using the cytokine-specific enzyme-linked immunosorbent assay (ELISA) kit.5. Sort different proportion of CD14+ myelomonocytic cells from coculture system. CD14+ myelomonocytic cells were sorted into CD14+HLA-DR-/low cells and CD14+HLA-DR+cells and the purity of the cells after sorting was>98%.6. T-cell suppression assay. To determine the suppressive capacity of MDSC, CD14+ cells, CD14+HLA-DR-/low cells or CD14+HLA-DR+ cells sorted from coculture system were cultured with allogeneic CD4+ T cells at 1:1 ratio for 3,5, 7days. CD4+T cells are stained with CFSE before culture with other cells.7. Analyze the expression of suppressive molecular. Amplification of IDO1, ARG-1, IL-4Ra, NOS2 and COX2 from CD14+myelomonocytic cells, CD14+HLA-DR-/low cells or CD14+HLA-DR+ cells which sorted from coculture system were carried out by RTQ-PCR.8. Neutralize CCL2. The coculture system added 10 μg/mL anti-CCL2 neutralizing mAb for 40 hr after, flow cytometry analysis the portion of Mo-MDSC in CD14+ myelomonocytic cells, suppression level of T-cell. RTQ-PCR tests the amplification of suppressive molecular.9. STAT-3 expression of Mo-MDSC and CD14+ myelomonocytic cells in coculture system. The protein level of STAT-3 and pSTAT-3 are analyzed by western blotting.RESULTS:1. Mo-MDSC is increased in peripheral blood of early pregnant women and can be induced by trophoblast cells in vitro. There is a significant increased in the percentage of Mo-MDSC in PB CD14+ myelomonocytic cells of early pregnant women as compared to non-pregnant donors (p<0.01). After coculture with trophoblast cell line HTR8/SVneo cells for 40 hours, there is a significant increase in the percentage of CD14+HLA-DR-/low cells (p<0.01).2. Mo-MDSC from coculture system inhibits autologous T-cell proliferation. Mo-MDSC isolated from coculture system inhibited proliferation of CFSE-labeled autologous CD4+T cells efficiently (p<0.01). In addition, sorted CD14+HLA-DR+ myelomonocytic cells from coculture system were used as controls, they failed to suppress proliferation of the responding autologous3. Trophoblast cells modulate CD14+ myelomonocytic cells’ CCL2 production. CCL2 level in coculture system is tested by ELISA. CCL2 level incease> 100-fold upon coculturing these cells for 40 hours. RTQ-PCR results show that, the main source of CCL2 is CD14+ myelomonocytic cells, and trophoblast cells promote this progress via a cell-cell contact manner. Furthermore, we also find the secreted level of TGF-β was slightly increased in the supernatants of coculture system, but no major changes were found in cytokines such as IL-4, IL-8, IL-6 and IL-10.4. HTR8/SVneo cells expand CD14+HLA-DR-/low MDSCs via CCL2. When anti-CCL2 is added to the media of coculture system, there is a significant decrease in the percentage of CD14+ HLA-DR-/low MDSC(p<0.01). The inhibitory ability of CD14+myelomonocytic cells on CD4+T cell proliferation was significant decreased when CCL2 was neutralized(p<0.01). Furthermore, ARG-1 has significant decreased when treated with anti-CCL2(p<0.01).5. CD14+HLA-DR-/low MDSCs express STAT3 and pSTAT3, and this expression correlates with CCL2. The expression of STAT3 is detected by western blotting. There is a statistically higher expression of STAT3 protein in CD14+ myelomonocytic cells from coculture system (p<0.01). we find a significant lower level of expression of STAT3 in coculture system without functional CCL2 (p<0.01).CONCLUSIONS:Trophoblast cells, as a part of embryo, have similar characteristics with malignant tumor cells, can significantly up-regulate the expression of CCL2 in CD14+ myelomonocytic cells. Elevated CCL2 can promote myelomonocytic cell differentiation into Mo-MDSC via STAT-3 signaling.This study further confirmed that CCL2 play an important role in promoting myelomonocytic cell differentiation, which provides a new perspective for tumor immunotherapy.