Mechanism Of Myeloid-derived Suppressor Cell In Mice Liver Fibrogenesis

Liver fibrosis is the same outcome of all kinds of chronic liver diseases, and is the commonpathological basis of all chronic liver diseases. Although it could be reversed at early stage bycontrolling potential factors, the only treatment for severe cirrhosis nowadays is livertransplantation. Even if liver fibrosis has a high incidence, the pathogenic mechanism is stillunclear. In addition, currently there is no treatable-radically drug. Now hepatic stellate cell(HSC) is being considered as a new treatment target, but the anti-fibrosis mechanism is stillunclear.Under physical condition, HSC is in quiescent state. Under diversity liver injury factors,HSC can be activated into myofibroblasts which has properties of proliferation, fibrogenesisand contractility. Under the effect of virus, abnormal metal metabolism and autoimmunologicaldiseases, HSC can excrete several cytokines, which bind with the receptors locating at themembrane of HSC, then transmit the cell signal through the same or different intracellularsignal transduction pathways. With the help of some transcriptional factors, transductionsignals are transmitted into nuclear, then promots DNA replication, transcription and translationprocess. Finally HSC could be activated into a proliferation state, and excrete extracellularmatrix. In the end the liver becomes fibrosis.Myeloid-derived suppressor cells (MDSC) are identified as a heterogeneous cellpopulation in mice and humans. Under physiological conditions, these cells are generated in thebone marrow and differentiated into mature macrophages, dendritic cells (DCs), andgranulocytes, and are present to a lesser extent in the spleen.. The different progenitor cells thatform this population demonstrate a broad range of morphology and functional capacity. Incontrast, in pathological conditions, there is a dramatic expansion of cells with the samephenotype and immunosuppressive activity in various tissues. These immature cell populationswith potent immunosuppressive activity are important as negative regulators of immune responses. During chronic inflammation and cancer, immature cell populations significantlyexpand owing to a partial block in their differentiation into mature myeloid cells. MDSCssuppress proliferation and cytokine secretion in both T lymphocytes and Natural Killer (NK)cells, as well as induce apoptosis in T cell subsets. MDSCs inhibit immune responses bymediating other immune cells, such as T cells, macrophages, NK cells, dendritic cells, andTregs. In mice, MDSCs are generally characterized as Gr-1+CD11b+cells. Gr-1antigen isconsidered to be a marker of granulocytic differentiation, and the antibodies bind to twoepitopes, Ly6G and Ly6C. The use of these epitope-specific antibodies has led to theidentification of two MDSC subsets, monocytic, and granulocytic subsets, which have differentmorphological and functional features. As both Ly6C and Ly6G epitopes are bound byanti-Gr-1antibodies, the relative intensity of Gr-1staining is indicative of monocytic (low orintermediate) and granulocytic (high) subsets. In both humans and mice, MDSCs are markedby high expression of CD11b (a common myeloid cell surface marker).Macrophages, on the other hand, display a remarkable plasticity and can differentiate intofunctionally diverse subtypes, e.g.’classically activated’ M1and ’alternatively activated’ M2macrophages. Experimental animal models indicate that macrophages are not only critical forfibrosis progression, but also for fibrosis regression, because macrophages can also degradeextracellular matrix proteins and exert anti-inflammatory actions. M1macrophage can expresshigh level of iNOS and TNF-α, which mediates acute inflammation, and M2macrophagesexpress Arg-1and IL-10, which plays an important role in transfering acute inflammation intochronic inflammation and promoting liver fibrosis. Similarly, in tumor microenvironment, M2macrophages promote tumor progression, metastasis and angiogenesis by inhibiting T cellmediation anti-tumor response, On the contrary, M1macrophage has an effect of inhibitingtumor growth.Liver belongs to human immunological organ. Many researchers suggest that liver fibrosisrelates to immunological abnormal. Although chronic liver disease has many etiologies,including chronic viral hepatitis, alcohol abuse, metabolic syndrome, and autoimmune disorders, the cellular and pathological mechanisms leading to hepatic fibrosis, and as anend-stage cirrhosis are relatively common and uniform. Liver fibrosis is characterized by anaccumulation of extracellular matrix proteins and activated HSC. Portal fibroblasts andmyofibroblasts have been identified as major collagen-producing cells in the injured liver.Experimental models of liver fibrosis highlight the importance of hepatic macrophages,so-called Kupffer cells, for perpetuating an inflammatory phase resulting in the massive releaseof proinflammatory cytokines and chemokines as well as activation of HSC. Recent studiesdemonstrate that these actions are only partially conducted by liver-resident macrophages, butlargely depend on recruitment of monocytes into the liver, namely of the inflammatory Gr1+(Ly6C+) monocyte subset as precursors of tissue macrophages. As a result, we want toinvestigate if there is cross-talk between HSC and MDSC through mice models and co-culturetwo cells. Finally a new treatment target could be found to control liver fibrosis and liver cancer.Our main results are as follows:1.Liver fibrosis animal model was established successfully, and identified withimmunohistochemistry staining.2. HSC was isolated from liver fibrosis tissues, and cultured to get different activationstatus with different morphology and function.3. We analysed MDSC and Tregs in liver tissues, bone marrow and spleen. It was shownthat in two weeks model, liver histology presented that a majority of hepatocytes becamenecrosis. MDSC in liver tissue increased dramatically, and numbers of M1and M2in CCl4group were higher than those of oil groups, and the difference was significant statistically. Thepercentage of MDSC from bone marrow and Tregs from spleen in two groups both had nodifference statistically. This suggested that M1macrophage induced acute inflammation ofliver, and M2macrophage simultaneously increased, which suggested that M2macrophageswanted to transfer acute inflammation into chronic inflammation. In addition, in four weeksmodel, the percentage of M2macrophages in CCl4group was much higher than that of oilgroup，but the percentage of M1macrophages in CCl4group decreased. And the expression of surface and intracellular marker of M2macrophages, such as Arginase-1, IL-10and CD206,were up-regulated. All this indicated that M2macrophages plays an important role in liverfibrosis reversion.4. HCC animal model by transgenic method was established successfully. From the fourthweek after injection the tumor could be detected by ultrasound from animal imagingdepartment. The tumor grew with a diffuse manner. The mice were divided into two groups bydiet: low fat diet group(LFD) and high fat diet group(HFD). Finally we found that the survivalrate of HFD group was lower than that of LFD group. This indicated that HFD can promotetumorigenesis. Moreover, the average weight of tumors in HFD group was higher than that ofLFD.5. MDSC in liver cancer mice was analysed by flow cytometry. In liver cancer tissue, thenumber of CD11b+Ly6C+Ly6G+MDSC was much higher than that of control group. Thissuggested that MDSC regulated the tumorigenesis and development of liver cancer. In addition,the percentage of M2macrophage increased, which demonstrated that M2macrophages playsan crucial importance in tumor formation. Similarly, the percentage of M2macrophages inbone marrow of CCl4group increased as well. Moreover, CD4+CD25+FoxP3+Treg in spleenalso increased compared with control group, which suggested that MDSC presentedimmunological inhibitory effect through propagation of Tregs.6. MDSC could inhibit HSC proliferation after co-culture of HSC and MDSC，and HSCcould induce MDSC to differentiate into M2macrophages. Especially MDSC from humancould inhibit HSC proliferation by90%. This is the possible mechanism to resolute or reverseliver fibrosis in the future.ConclusionIn conclusion, we find firstly that MDSC plays an important role in promoting liverfibrogenesis and liver tumorigenesis. Meantime, at acute inflammation stage MDSC andMDSC-derived M1and M2macrophages both increase. Moreover, M2macrophage is verycrucial in fibrosis reversion and tumorigenesis. And MDSC promotes liver tumorigenesis through inducing propagation of Tregs. In addition, high fat diet is very important for tumordevelopment. Moreover, HSC proliferation could be inhibited by MDSC dramatically afterco-culture24hours, which suggests a possible target to inhibit fibrogenesis, and MDSC candifferentiate into M2macrophages after co-culture probably.