Multiplex immunostaining and transcriptomic profiling identify novel immune cell markers for non-alcoholic steatohepatitis and primary sclerosing cholangitis

Background and aims: Adipose tissue dysfunction with infiltration of pro-inflammatory macrophages is a hallmark in obesity leading to an impairment of metabolic homeostasis.Non-alcoholic fatty liver disease (NAFLD) is intimately associated with obesity and it has been shown that pro-inflammatory adipose tissue macrophages (ATMs) induce hepatic inflammation although their role in fibrogenesis is still unknown.The aim of the present study is to assess the role of ATMs in hepatic fibrogenesis by modulating the pro-inflammatory phenotype of ATMs.Method: Dextran (500 kDa) nanocarriers conjugated with dexamethasone (dexa) were used to modulate ATMs' phenotype in a NAFLD models based on high-fat (HFD) and high fat high cholesterol (HFHC) diets.After feeding animals for 5 months, dextran conjugates were administered i.p. twice per week for 5 additional weeks.In order to study dextran uptake, i.p. administration of dextran-FITC was evaluated by flow cytometry and immunofluorescence.Hepatic and AT macrophages' phenotype switch was assessed by flow cytometry and gene expression analysis.The anti-inflammatory effect of dexaconjugates in liver and adipose tissue was evaluated by assessing key inflammatory markers expression by qPCR and immunostaining.Changes in hepatic fibrosis and NAS score were explored by histopathological analysis.In-vitro phenotypic modulation of human ATMs incubated with dextran conjugates were evaluated by qPCR.The effect of human ATMs on hepatic stellate cells (HSCs) activation was assessed by culturing HSCs with ATMs conditioned media.Results: Animals fed with HFHC diet displayed increased liver and adipose tissue macrophage infiltration with an enhanced proinflammatory phenotype compared to animals receiving HFD.Compared to HFD, liver tissue from HFHC model showed higher NASH score and fibrosis degree.Dextran-dexa conjugates were predominantly engulfed by macrophages from the AT.Conjugated nanocarriers administration significantly switched the pro-inflammatory phenotype of ATMs without significant differences in liver macrophages.Dextran-dexa treatment caused a significant reduction of hepatic inflammation, NAS score, and hepatic fibrosis in the HFHC model.Human ATMs treated with dextran-dexa attenuated their proinflammatory phenotype and reduced HSCs activation.Conclusion: Dextran-dexamethasone conjugates administrated i.p. are predominantly uptaken by adipose tissue macrophages inducing a switch of their pro-inflammatory phenotype which in turns attenuates liver inflammation and fibrosis.In vitro modulation of human ATMs by dextran nanocarriers reduce activated phenotype of HSCs.These results demonstrate that modulating the pro-inflammatory phenotype of ATMs might be a good strategy to reduce hepatic fibrosis and inflammation in NAFLD.