A High-dimensional Atlas Of Mouse Liver Immune Cell Reveals Immunomodulatory Effects Of Mesenchymal Stem Cell In Acute Liver Injury

BackgroundAcute liver injury（ALI）induced by chemicals or viruses can progress rapidly to acute liver failure,which carries a high mortality.Numerous studies reported that ALI provokes rapid recruitment of immune cells to liver and triggers immune response,progression or restriction of ALI depend on the ultimate outcome of the liver immune response.MSCs are multipotent stromal cells that have multidirectional differentiation capacities.It has been reported that transplantation of MSCs ameliorates liver injury by regulated the liver immune response.Due to flow cytometry use fluorophores tagged antibodies and thus is confounded by overlapping emission,they do not increase the number of parameters that can be detected.Studies of the immunomodulatory effect of MSCs have focused on limited immune cell subsets as well as a few parameters.However,the immune system is a complex and mobile network of various cells,and therefore,systematic analyses of the composition and immune cell profiles in the liver from homeostasis to ALI,systematic assessment the interaction between the immune system and MSCs in ALI are needed.Tanner and his colleagues firstly described mass cytometry in 2009.Mass cytometry uses stable metal isotopes instead of fluorophores conjugated antibodies and adapt inductively coupled plasma mass spectrometry（ICP-MS）as detection system,enables the simultaneous measurement of>40 markers per single cell.In order to systematically analyze the immunomodulatory effects of MSCs in ALI using mass cytometry,the following four experiments were performed.Firstly,the mass cytometry antibody panel was designed to identify hepatic immune cell,using 43 antibodies including hepatic immune cell subset marker,immunomodulatory molecules related markers,markers for cytokine receptors and chemokine receptors.Then each antibody was conjugated to a metal isotope.An appropriate unsupervised computational approach was used to analysis and visualization of high-dimensional mass cytometry data.Secondly,we used mass cytometry of 43 antibodies performed on normal mice liver immune cells.Liver immune landscape of normal mice was profiled.Based on above experiments,immune landscape and dynamics in ALI mice liver was mapped to identify the specific cell subsets related to liver injury and repair.At last,the composition,frequency,dynamics and specific immune cells subsets correlated with MSCs-treated ALI were identified,revealing the immunomodulatory effects of MSCs in ALI systematically,which will facilitate the clinical applications of MSC-based treatments for ALI.MethodsWe first assembled a panel of metal isotope tagged antibodies to define cell surface markers of liver innate and adaptive immune cells.For the conjunction of the indicated isotope to a given antibody,Max PAR×8 Antibody Conjugation Kit was used.We optimized the antibody staining concentration,validated the reliability of mass cytometry data by flow cytometry and the accuracy of unsupervised computational approaches by manual gate analysis.Liver immune cells were prepared and characterized their CD45 expression and cell viability by flow cytometry.Liver immune cell suspensions from normal mice were collected and reacted with the metal-isotope-conjugated antibody cocktail for further mass cytometry analysis.Mass cytometry data were processed using the t-Distribution Stochastic Neighbor Embedding（t-SNE）and X shift.Then the liver immune landscape of normal mice was profiled.ALI models of C57BL/6 were established by were intraperitoneally administered Carbon Tetrachloride（CCl₄）,at day 1,2,3 and 7,Liver immune cell suspensions from ALI mice were collected for further mass cytometry analysis.The liver immune landscape of ALI mice was mapped.Mouse MSCs were isolated and characterized by analyzing the marker expression and inducing osteogenic and adipogenic differentiation.Six hours after CCl₄administration,the ALI mice were divided into two groups:MSC-treated group（5×10⁵MSCs were administrated through tail vein）and placebo-treated group（PBS was administrated through tail vein）.To determine the efficacy of MSCs against ALI,liver tissue and serum were collected for histological and biochemical analyses.Mortality was recorded every 24 h until.Liver immune cell suspensions from ALI treated with or without MSCs mice were collected for further mass cytometry analysis.The liver immune cell subsets related to MSC treatment in ALI mice was identified.ResultsWe designed a panel of metal isotope tagged antibodies to identify liver innate and adaptive immune cells.Each antibody was conjugated with indicated isotope.The relative frequencies of each cell population obtained by mass cytometry were similar with flow cytometry,confirming the reliability of the mass cytometry.The relative frequencies of each cell population obtained by manual analysis were similar with unsupervised computational approach analysis,confirming the accuracy of the computational approach analysis.Liver immune cells expressed high level of CD45（>96%）and were almost alive（>98%）.Then we profiled the liver immune landscape of normal mice by mass cytometry.The data were analyzed by t-SNE and X shift,up to 25 clusters were identified,includingγδT cell,Ly6C^hiCD103^lowCD8⁺T cell,Ly6C^lowCD103^hiCD8⁺T cell,na?ve CD4⁺T cell,effector CD4⁺T cell,Ig M⁺Ig D⁺B cell,Ig M⁺Ig D^-B cell,liver conventional nature killer cell（c NK）,resident NK cell,monocyte,monocyte-derived macrophage,Plasmacytoid dendritic cell（p DC）,CD103⁺DC,CD11b⁺DC,CD11b⁺CD11c⁺MHCII^-DC,monocyte-derived DC,neutrophil and eosnophils.The liver immune landscape of ALI mice was mapped.ALI provokes rapid recruitment of immune cells to liver and triggers immune cell activation and proliferation.The number of immune cells in ALI livers peaked at day 2 and gradually decreased with injured liver repaired.But the phenotypic of immune cell subsets in repaired livers were different from normal livers.Mouse MSCs expressed high levels of CD44,SCA-1 and CD29,but rare expressed CD31,CD86,CD11b,CD45 and MCH II-1a.MSCs can also differentiate into osteoblasts and adipocytes.MSCs treatment significantly alleviated CCl₄-induced ALI.During the injured phase,MSCs inhibited a systemic response by reducing the numbers of Ly6C^lowCD103^hiCD8⁺T cells,conventional NK cells,and Ig M⁺Ig D⁺B cells;suppressing the activation of Ly6C^hiCD103^lowCD8⁺T cells;downregulating MHC II and Ig M expression inIg M⁺Ig D⁺B cells;and increasing the number of immunosuppressive monocyte-derived macrophages.During the recovery phase,MSCs promoted the retention of Ly6C^lowCD103^hiCD8⁺T cells and maintained the immunosuppressive activity of monocyte-derived macrophages.The response to MSC treatment differed between the injured and recovery phases,emphasizing the benefit of dynamic assessment of the immunomodulatory effects of MSCs.ConclusionLiver immune landscape of normal mice,ALI mice treated with MSC or not was profiled.Our data identified the specific cell subsets related to liver injury and repair,evaluated the distribution and phenotype of liver immune cell subsets with liver injury and repair.We also mapped the MSCs treatment induced alterations of hepatic immune system,which will facilitate the clinical applications of MSC-based treatments for ALI.