Mir-10b Regulated Klf4Mediated-Macrophage Polarization And Inhibited T_H1T_H17cell Response

Background and Objection:Macrophages have long been considered to be important immune effector cells.It plays an important role the in a broad spectrum of pathological and physiology procedure and respond to endogenous stimuli that are rapidly generated following injury or infection.which is first confirmed cell in the immune system. It is known that they are among the first cells to arrive at sites of wounding and/or infection and is characterize by a high capacity to present antigen,where they perform several functions. Clinical and experimental studies support an important role for the macrophage in a broad spectrum of acute (e.g., pathogen infection, sepsis) and chronic inflammatory conditions (e.g.insulin resistance, atherosclerosis, chronic wounds, and tumorigenesis).Circulating monocytes give rise to a variety of tissue resident macrophages throughout the body,as well as to specialized cells such as dendritic cells (DCs) and osteoclasts. With the stimulation of granulocyte-macrophage colony stimulating factor and multi-colony stimulating factor, bone marrow stem cell develops into Mononuclear cells.Monocytes are known to originate in the bone marrow from a common myeloid progenitor that is shared with neutrophils, and they are then released into the peripheral blood, where they circulate for several days before entering tissues and replenishing the tissue macrophage populations involved inflammation and host defense,,which are induced by pro-inflammatory and environmental signals.Immunologic effector cell exhibit remarkable plasticity that allows them to develop into different type of cell and serve distinct functions in the regulation of the inflammatory response. As more and more studies about the molecular mechanisms that govern Th cell polarization, the regulation of macrophage polarization is paid huge attention to.With a huge number of studies confirmed, it is well-accepted that macrophage is divided into M1macrophage (Classically activate) and M2macrophage (Alternatively activate). Macrophages stimulated with LPS or INF-γassume an M1pro-inflammatory phenotype characterized by a high expression level of iNOS, a high capacity to present antigen and production of pro-inflammatory cytokines such as TNF-a, IL-1β,monocyte chemoattractant protein-1(MCP-1). M1macrophages are critical effector cells that kill microorganisms and thus benefit the host. However, this pro-inflammatory activity must be carefully titrated, as sustained activation can predispose to chronic inflammatory states such as obesity and insulin resistance. M-CSF, IL-4, IL-13, IL-10, IL-4and TGF-βstimulation leads to M2macrophages.,which produce of anti-inflammatory cytokines such as IL-10、 CCL17,CCL18, CCL22和TGF-aand promote tissue repair and remodeling as well as tumor progression.Tumor-associated-macrophage is resembling M2macrophage,which is associated with cancer initiation and promotion. Accumulating evidence suggests that M2macrophages can protect against insulin resistance, eliminate parasites, and promote tissue remodeling and repair. Interestingly, it helps weight loss, known to improve insulin sensitivity and to induce remodeling of adipose tissue. MicroRNAs (miRNAs), a newly discovered class of non-protein-coding small RNA, have been shown to play a vital role in processes such as development, cellular proliferation, and more recently apoptosis.It combine mRNA of target gene and supress gene expression post-transcriptionally.Accumulating evidence suggests that miRNAs constitute an integrated part of the regulatory networks in innate immunity and adaptive. More than a dozen miRNA have been shown to play a critical role in regulating polarization of macrophage and Th-17cell. Studies found that miR-146a is rapidly upregulated in human monocytic cells stimulated with lipo-polysaccharide(LPS), a TLR4ligand, and acts as a negative feedback regulator of TLR signaling, presumably by targeting tumor necrosis factor receptor-associated factor6and interleukin(IL)-1receptor-associated kinase-1, miR-146a upregulation may be an essential component of endotoxin tolerance in innate immune response.miR-155is induced in macrophage in response to both bacterial and viral-derived antigens that activate TLR4,TLR2,TLR3or TLR9.It was estimated that miR-155plays both positive and negative regulator roles in immune responses. miR-21is induced in monocytic cells by LPS stimulation. Upregulated miR-21was found to dampen LPS-induced nuclear factor-κB activation and IL-6expression but enhance IL-10expression. miR-21can also trigger inflammatory responses by binding to TLR7and TLR8.In all of miRNAs, miR-10b is of great interest to us. Because it not only initiates invasion and metastasis in cancers, but also controls cancer cell growthe by regulating cell-cycle and apoptosis.Ma first concerned that miR-10b promote tumor metastasis, and miR-10b has been expressed by sever breast cancer metastasis,.Inhibition of miR-10b can reduce MDA-MB-231cell invasion ability,and over-expression miR-10b of SUM149cell which is unable to metastasis can metasta distantly in nude mouse. Clinical studies also prove that expression of miR-10b of metastasis breast cancer is higher than no metastasis.Gabriely estimated that inhibition fo miR-10b can restrain the growth of malignant glioma cells by inducing cell apoptosis. The malignant glioma survival rate of more expression miR-10b is significant reduction. Compared with placebo group, tumor growth dropped significantly of glioma model in mice when injecting inhibition of miR-10b. All this studies concerns miR-10b promote tumor growth.Kruppel-like factor4(Klf4; also called gut-enriched Kriippel-like factor) is a zinc finger-containing transcription factor, plays a role in cell cycle regulation, apoptosis, and differentiation. In accordance with these studies, Klf4expression is frequently lost in several human tumors, including esophageal, stomach, colorectal and bladder cancers, which indicates its tumor suppressor role. It has been reported to inhibit metastasis of several cancers, such as colorectal, esophageal, and pancreatic cancer cells. Moreover, Klf4inhibits cell proliferation by inducing cell cycle arrest in CRC; and regulates esophageal carcinogenesis by affecting proliferation, apoptosis, and cell invasion. Klf4is acting as cancer suppressor genes during tumor proliferation and metastasis,which is on the contrary of miR-10b. Recently studier estimated that miR-10b regulate colon cancer and promote tumor proliferation and metastasis by targeting Klf4.Members of KLFs gene family have been shown to play important roles in a diverse array of cellular processes including hematopoiesis.It has been reported that Klf4,KLF2,KLF3is associated with macrophage macrophage polarization and activation.Huge studies have reported that Klf4is expressed in a stage-specific pattern during myelopoiesis and function to promote monocyte differentiation.In Klf4-/-mice,mononuclear cells differentiation is severely affected, the mononuclear cells from the bone marrow and spleen experience a substantial reduction and mononuclear cell of circulation almost disappear. It is identified that Klf4as an essential regulator of macrophage M1/M2polarization and attendant functions.Since1889, some scholars put forward the "seeds" and "soil" hypothesis, tumor microenvironment began to be taken seriously, inflammatory microenvironment has been listed as one of the top ten characteristics of tumor. Tumor has a closely relation with inflammation. During tumor initiation, they create an inflammatory environment that is mutagenic and promotes growth.It is identified that Klf4as an essential regulator of macrophage M1/M2polarization and attendant functions. Meanwhile Klf4is target gene of miR-10b,which regulate tumor progression and metastases.Base on that,we put a forward the hypothesis that wether miR-10b could regulate macrophage polarization by modulating Klf4. To improve the hypothesis, wo do reacher as follows:to determine wether different macrophage polarization could change the expression of miR-10b in macrophage, we evaluated the expression of miR-10Ob in M1and M2macrophage;then we transfect miR-10b inhibitor and miR-10b mimics to answer wether over-expression and inhibition of miR-10b could regulate macrophage polarization. One of remarkable ability of macrophage is presenting antigen to accelerate T helper cell polarization.We next analyze wether inhibition miR-10b could weak Thl cell and Th17cell polarization response and reduce the sensitive of endotoxin in mice and the ability of lear the bacterial infection in mice. At last,we produce the CD4+CD45RBhiT cells colitis mice model,which is resembling human inflammatory brown disease, to explore inhibition miR-10b could release the T cell induced colitis.Methods and materialsTo explore wether miR-10b modulate M1macrophage polarization and macrophage polarization is consistent with miR-10b expression.Human monocytes were obtained from the periphery blood5-10ml from healthy donors by density gradient centrifugation(Ficoll centrifugation). The monocytes was cultured7days with stimulation of GM-CSF(10ng/mL), then co-stimulated by LPS (100ng/mL)and IFN-γ(20ng/mL) to polarize M1macrophage.RT-PCR analyze of miR-10b mRNA in M1macrophage at0h,12h,24h.We also evaluated expression of miR-10b in miR-10b.The monocytes from healthy donors was co-stimulated by IL-4(10ng/mL)and L13(10ng/mL),RT-PCR analyze of miR-10b mRNA in Ml macrophage at0h,12h,24h. Next we speculated that wether varying expression of miR-10b has influence on macrophage polarization.The monocytes was cultured7days with stimulation of GM-CSF(10ng/mL), then co-stimulated by LPS (100ng/mL)and IFN-γ(20ng/mL) to polarize M1macrophage for24h to form M1macrophage, then was removed the culture medium,and was washed by PBS for3time, which eliminated the influence of M1polarization factor. The cell was co-stimulated by IL-4(10ng/mL)and L13(10ng/mL)for24h to polarize M2macrophage.Then they were removed the influence of M2polarization factor as the same way and simulated into M1macrophage. RT-PCR analyze of miR-10b mRNA in M1,M2.The monocytes was stimulated into Ml macrophage as mentioned above, than transfected miR-10b inhibitor and miR-10b mimic for48h,then was simulated with GM-CSF(20ng/mL)for4h to polarize M1macrophage.RT-PCR analyze of highly specific macrophage marker expression of M1,M2,which iNOS is marker of M1macrophage.and Arg-1,Klf4and IL-4R are marker of M2macrophage.To explore wether inhibition expression of miRlOb in M1macrophage could reduce TH1cell response. The monocytes was isolated from C57BL/6mice and was cultured with simulation GM-CSF(10ng/mL)for5days,then was transfected miR-10b inhibitor for48h.Then the cells were co-cultured with CD4+T cell which was screening by magnetic cell sorting;magnetic activated cell sorting. RT-PCR analyzed expression of T-bet mRNA; Elisa and Flow Injection Analysis analyzed IFN-y level.To explore wether inhibition expression of miR10b in M1macrophage could reduce TJ17cell response. The monocytes was isolated from C57BL/6mice and was cultured with simulation GM-CSF(10ng/mL)for5days,then was transfected miR-10b inhibitor for48h.Then the cells were co-cultured with CD4+T cell which was screening by magnetic cell sorting; magnetic activated cell sorting. RT-PCR analyzed expression of RORyt mRNA;Elisa and Flow Injection Analysis analyzed IL-17level.We monitored the sensitive of endotoxin in mice to discuss wether inhibition miR-10b could restrain M1macrophage polarization in vitro experiment. Rinsed the mice abdominal with5%broth medium, peritoneal macrophage were collected and were planted on the60mm culture dish, after2h were removed suspension cell. The adherent cells were planted into6holes culture plate,2×106per hole and were transfected with miR-10b inhibitor.10mouse were divided into2group.The cells were injected into mice enterocoelia for48h.The experimental group was injected LPS via enterocoelia. Two groups of Survival rate was observed,blood serum was collected after6h for Elisa analyzing IL-6, TNF-a level.To explain inhibition of miR-10b could reduce the ability of lear the bacterial infection in mice, we performed injection miR-10b antagomir via tail vein in20mouse(2x104CFU per one),after48h, injection Listeria bacteria via tail vein of10mouse in experimental group.At24h,48h,every5mouse was put to execution respectively and collected blood serum for Elisa of IL-6, TNF-alevel.Liver and spleen were collected to homogenate, daub on the LB agarose petri dishes, observe the bacteria colony formation.At last, we verify whether inhibition of miR-10b could reduce CD4+CD45RBhiT cell induced colitis,which is resembling human inflammatory brown disease. miR-10b antagomir is injected into mice via tail vein,twice a week for3weeks. Screening CD4+CD45RBhiT cells by flow cytom and MACS,5x105cells each mice were injected via enterocoelia in Rag-/-mice,miR-10b antagomir were injected twice a week,for6weeks.Observe two groups of mice’s body weight,color, motion and change in stool, colonic inflammation histological pathology.To explore wether miR-10b could regulate Klf4to modulate M1/M2polarization, we isolate monocyte from mice bone marrow, Stimulate with GM-CSF(10ng/mL) for5day,then transfect miR-10b mimic for48h,then Stimulate with rIL-4/IL-13for4h and24respectively. Detection protein and mRNA of Klf4. The cells were transiently cotransfected in96-well plates with luciferase reporter vector containing3’-UTR variants and either control or miR-10b inhibitor. After48h, luciferase activity was measured with Dual-Glo Luciferase Assay System (Promega, WI), and Renilla luciferase activity was normalized to Firefly luciferase activity.ResultMacrophage polarization is affected by many factors such as microenvironment. This article is discussed that miR-10b can regulate M1/M2polarization by targeting Klf4for first time. Expression of miR-lOb is elevated in M1macrophage and that is not found in M2macrophage. We also found that expression of miR-10b varied with macrophage polarization. The highest expression of miR-10b in M1macrophage,which was dramatic declined in M2macrophage,but it recovered high expression again after stimulation of influence factor of M1macrophage. The result concerns that macrophage polarization can affect expression of miR-10b and on the contrary the expression of miR-10b can also influence the polarization of macrophage. The remarkable factor of Ml macrophage such as iNOS is reduce as inhibition of miR-10b but that of M2macrophage is elevated. It means that changing expression of miR-10b can affect polarization of macrophage. M1macrophage induced TH1and TH17cell response and M2macrophage induce TH2cell response. Inhibition of expression of miR-10b can reduce level of T-bet mRNA, ROR t mRNA expression and IFN-γ,IL-17level.In vitro experiment, survival rate experimental group is significantly higher than control group,and IL-6,TNF-aof blood serum is lower than cntrol group. We also find experimental group has more the bacteria colony formation than control group. The result emphasize on the inhibition of miR-10b can restrain M1macrophage polarization and reduce THland TH17cell response by vitro experiment. TH1and TH17cell play a crucial role on human inflammatory brown disease, we find inhibition of miR-10b can release T cell induced colitis which is resembling IBD. It estimate that inhibition of miR-10b can restrain M1macrophage polarization and reduce TH1and TH17cell response. We also found elevated miR-10b can reduce mRNA and protein level of Klf4in M2macrophage. Klf4is the direct target of miR-10b, and the expression of Klf4is regulated by miR-10b.ConclusionThis article first report expression of miR-10b in M1and m2macrophage,which indicate that expression of miR-10b not only take an adoptive change with macrophage polarization,but also it could modulate polarization of macrophage.Inhibition miR-10b can reduce Thl and Th2cell response.In vitrvo experiment, it estimate that inhibition miR-10b can restrain M1polarization. In vivo experimentation, inhibition miR-10b can reduce the sensitive of endotoxin in mice and the ability of lear the bacterial infection in mice. It also release T cell induce colitis. All the data estimate the hypothesis that miR-10b could target Klf4to regulate M1/M2polarization.