The Phenotype Of Kupffer Cells In The Process Of Development And Resolution Of Liver Fibrosis

Kupffer cells （KCs）, settled in the liver, are non-parenchymal cells within the hepaticsinusoids, belong to the members of the monocyte-macrophage cell lines of the body. KCsaccount for80%-90%of the total number of monocyte-macrophage cell lines, although foronly15%of the total number of liver cells. KCs are located in the lobular portal vein area,play an important defensive role in liver. More and more researchers pay attention to theexperimental studies about KCs, owing to their important functions both under physiologicaland pathological conditions. KCs not only phagocytose and clear non-specifically bacteriaand exogenous antigenic substances in the blood, but play a role in specific immune response,anti-tumor immune response, endotoxin detoxification, anti-infective, regulatingmicrocirculation and metabolism, etc. Under pathological conditions, the KCs can beactivated by endotoxin and tumor necrosis factor（TNF）, then release TNF, transforminggrowth factor（TGF）, interferon（IFN）, interleukin-1（IL-1）, interleukin-6（IL-6）, oxygenfreeradical（OFR）, nitric oxide（NO） and other inflammatory mediators, which are involved in theoccurrence and development of liver injury. The KCs are a class of very strong heterogeneitymacrophages, with different phenotypes in different microenvironmental conditions, that is tosay the KCs can be divided into several subpopulations with different functions. At present,the accepted macrophage subpopulations are mainly two categories of M1and M2. Themacrophages are polarized into classically activated macrophages （CAMφ, M1-typemacrophages） by Th1-type cytokines （gamma-IFN, TNF-α） and polarized into alternativelyactivated macrophages （AAMφ, M2-type macrophages） by Th2cytokines （IL4/IL13）. Themajor functions of M1macrophages include elimination of extracellular pathogens andpromoting inflammation response by synthesizing and secreting pro-inflammatory cytokines,including TNF-α, IL-1, IL-6, etc. While the main functions of M2macrophages are inhibitionof inflammatory reaction by synthetizing and secreting anti-inflammatory cytokines IL-10,participation in tissue repair. In previous studies M2can be divided into M2a, M2b, and M2csubpopulations. However, the classification of KCs subpopulations is still unclear in in vivoanimal models of liver fibrosis.Liver fibrosis, remains with the early stages of cirrhosis, is caused by persistent damage-repair response, which leads to abnormal extracellular matrix deposition and furtherdevelops to a pathological process of liver structure and liver function abnormalities. A largenumber of studies have shown that liver fibrosis can be reversed after removing the injury secreting a variety of collagen fiber and extracellular matrix, which has been considered to bethe central link of the liver fibrosis development. However, the researchers have found thatmacrophages are central regulators of hepatic fibrogenesis and fibrosis resolution, couldactivate the quiescent hepatic stellate cells to promote hepatic fibrosis in the process of liverfibrosis. While during the hepatic fibrosis reversal process, the macrophages promoteapoptosis of activated hepatic stellate cell and degrade fibrillar collagen, to facilitate thereversal of liver fibrosis. Studies have shown that the bi-directional regulation ofmacrophages in the development and reversal of liver fibrosis is carried out by differentmacrophage subpopulations. However, the classification of the macrophage subpopulations inanimal models is still unclear, the regulation of liver macrophages （KCs） in liver fibrosis isgetting more attention. In this study, we aim to find which phenotype of KCs plays a keyregulatory role in development and reversal process of liver fibrosis respectively. And we alsotry to discover molecular mechanisms of KCs in hepatic fibrosis, which may give valuableclues for prevention and mitigation of liver fibrosis.We established flow cytometry sorting to obtain the KCs subpopulations at the sametime through a variety of cell surface markers. After flow cytometry sorting, the yield of KCs（F4/80+） was （1.26±0.24）×106per mice and the purity of KCs was （97.6±0.7）%. As forcontamination, hepatic sinus endothelial cells （CD146+） was （0.5±0.3）%and T lymphocyte（CD3+） was less than0.5%. The purity, activity and yield of KCs can satisfy the follow-upfunctional validation studies in vitro and further proteomic analysis. Then we established themouse model of liver fibrosis induced by CCl₄, and separated KCs from liver tissue in acuteinflammation phase, the development and reversal phase of liver fibrosis. We analyzed thedifference of surface markers, the differential expression of inflammatory cytokines andfibrosis-related cytokines to confirm the phenotype of KCs in different phase of liver fibrosis.After first injection CCl₄, the serum levels of ALT and AST significantly increased andreached to a maximum value in48h. HE and Masson staining showed that a large number ofinflammatory cells infiltrated into the liver. Damage to liver cells is serious, but nohyperplasia of fiber collagen. The results suggested that there were serious acute injuryresponse in liver after first injection CCl₄, leading to necrosis of a large number ofhepatocytes. With continuous stimulation of CCl₄, serum levels of aminotransferase, thecontent of fiber collagen and the expression of α-SMA increased gradually in liver tissue, andaccompanied with chronic inflammatory response, which suggested successful establishmentof CCl₄-induced liver fibrosis in mice. After stopping injection, with the extension of recovertime, the serum levels of ALT and AST gradually decreased to normal level and the expression of α-SMA in liver tissue markly declined. Although collagen fiber elimination isslow, the indicators are close to normal at6weeks of natural recovery （R6W）.Then we detected the changes of numbers of KCs and the expression of cytokines ofKCs, which confirm that KCs were participated in the process of development and reversal ofliver fibrosis. The mount of KCs in liver significantly increased during the development ofliver fibrosis while gradually decreased during the reversal process of liver fibrosis andtrended to normal level. KCs exert bi-directional regulation by secreting several differentcytokines in the development and reversal process of liver fibrosis. The experimental resultsshowed that during the acute inflammation, the main functions of intrahepatic inherent KCs（CD45+F4/80hi） were recruitment of monocytes into the liver and expression of inflammatorycytokines, promoting inflammation response and leading to liver damage. And the mainfunctions of KCs（CD45+F4/80low）were to promote hepatic stellate cell activation and expressfibrosis-related cytokines, facilitatingthe development of liver fibrosis. The phenotype ofnormal KCs is M2b type. In short, there are three different cell subpopulations playing animportant role in process of liver fibrosis, namely M1KCs in the period of acuteinflammation while M2c KCs in the development phase and M2a KCs in the reversal stage.Hence, phenotype switching among KCs subpopulations occurred during fibrogenesis,development and reversal process of liver fibrosis. The determination of phenotype of KCsprovides theoretical basis for molecular mechanisms of phenotypic changes of KCs. Thesedatas also provide clues for anti-fibrotic therapy of fibrosis in clinic application. However,this study only describes the phenomenon that KCs playing different biological functions arewith different phenotypes at different stage of liver fibrosis, there is no further investigationof its mechanism.