A Conserved Pathway Of Transdifferentiation In Murine Kupffer Cells

Background and objective:KCs are long lived resident macrophages in the liver.During chronic liver injury,KCs are activated and secrete pro-inflammatory and pro fibrotic cytokines,which act on the transdifferentiation of HSCs to myofibroblasts,and finally produce collagen.It is widely recognized that HSCs are the main source of collagen deposition in the liver and the core of liver fibrosis.KCs can assist the activation of HSCs to participate in fibrosis.However,the transdifferentiation of KCs has not been clarified.The transdifferentiation of HSCs into myofibroblasts is a key of liver fibrosis.In addition to the transdifferentiation of HSCs,there are abundent instance of transdifferentiation in the liver.For example,experimental studies have proved the transdifferentiation between hepatocytes and bile duct cells in animal models.Macrophages in extrahepatic tissues,such as skin,myocardium and kidney,can further differentiate into fibroblasts and secrete collagen,which is closely related to wound healing and repair.Through the above analogy,it is proposed that KCs can undergo transdifferentiation.After transdifferentiation into fibroblasts,KCs may produce collagen deposition,and may even participate in the repair of liver tissue injury and even liver fibrosis.The transdifferentiation of KCs provides a different perspective.The process of liver cirrhosis is regarded as a river,the transdifferentiation of HSCs can be regarded as a main road,while KCs can be used as a tributary,which provides new ideas and targets for the treatment of liver fibrosis in the future,and provides a new source for fibroblasts,improved the understanding of liver fibrosis.Materialsand methods: Firstly,parallel experiments were carried out in two different in vitro experimental models:liver non parenchymal cell culture and precision liver slice culture.The specific steps are as follows:(1)An animal model tracking of KCs lineage was built,using Emr1 cre + /-ribotag+ /-mice tracked KCs lineage.(2)KCs were isolated and cultured in vitro.(3)Precision liver slice culture was performed by precision liver slice culture technique.(4)The proteins expressed in cultured KCs were analyzed by immunofluorescence imaging analysis.(5)KCs was enriched by polymer immunocoprecipitation,RNA was extracted and c DNA was synthesized.(6)Real time quantitative PCR(fluigm).Secondly,collagen deposition was studied in mouse liver slices.KCs were depleted by chlorophosphonate liposomes.Precision liver slices were cultured.Masson trichrome staining and RT-PCR gene expression analysis were performed with liver slices.Finally,IL-1r knockout mice and NLRP3 knockout mice were used,the gene expression was analyzed by real-time quantitative PCR using precision liver slice culture technology,and the cell death in liver slices was quantitatively analyzed by LDH detection kit.Results:(1)In non parenchymal cell culture and liver slice cultures,Kupffer cell-associated genes expression is down-regulated and so is its transcription factors over time.(2)In non parenchymal cell culture and liver slice cultures,firbosis associated genes expression is up regulated and so is its transcription factors over time.(3)Collagen deposition in liver slices decreased after KCs depeltion.(4)There is no significant difference in the expression of cell death related genes in liver slices of IL-1R knockout mice and the control group.(5)There is no significant difference in the expression of cell death related genes in liver slices of NLRP3 knockout mice and the control group.Conclusion: KCs transdifferentiation occurs in both non parenchymal cell culture in vitro and precision liver slice culture.The transdifferentiation of KCs is relatively conservative in vitro.KCs transdifferentiate into fibroblast like cells,secreting collagen deposition related proteins.