Periodic Reporting for period 1 - MAFLDCs (Elucidating Dendritic Cell Heterogeneity and Functions in Metabolic Associated Fatty Liver Disease)
Okres sprawozdawczy: 2021-04-01 do 2023-03-31
The key objective of this project was to identify the role of liver dendritic cells (DCs) in metabolic (dysfunction) associated liver disease (MAFLD). The rise in obesity and insulin resistance and therefore the accumulation of triglycerides and free fatty acids in the liver has resulted in a growing epidemic of NAFLD. This disease consists of a largely asymptomatic spectrum, whereby most patients have simple steatosis, a disease characterised by the retention of fat in the liver. The majority of patients do not progress through the disease spectrum, but for approximately twenty percent of patients, they will develop the more progressive form of disease, non-alcoholic steatohepatitis (NASH). This can lead to fibrosis, cirrhosis and in some instances, liver cancer (Hepatocellular Carcinoma; HCC). With the increasing prevalence of disease and the current lack of therapies, MAFLD is predicted to become the leading cause of liver transplantation in the Western world by 2030. However, despite its prevalence the mechanisms that drive disease progression in MAFLD remain incompletely understood. As key cytokine-producing antigen presenting cells, liver dendritic cells (DCs) unquestionably play important roles in the induction and regulation of hepatic inflammation, particularly in the context of T cell responses. However, due to difficulties in targeting them and potential heterogeneity within the total DC pool, studies extrapolating their functional characterisation, especially in the context of MAFLD, are limited. In addition, the existing research pertaining to their contribution in MAFLD progression is conflicting. Whilst some have observed a protective role of DCs in MAFLD, others have suggested they contribute to MAFLD development. The lack of effective therapies targeting MAFLD combined with the conflicting studies on DC role in MAFLD highlighted a significant gap in the literature that had the potential to provide information to guide therapeutic advances. The overall objectives of this project were to: characterise the heterogeneity of hepatic DCs in both steady state and during the development of NAFLD; identify the T cell responses induced by distinct cDC subsets; determine the consequences of manipulation cDC1 populations.
The beginning stages of the project were designed to extensively profile liver DCs, utilising data from the host labs previous CITE-Seq experiments to guide the development of flow cytometry panels. Having identified an expansion of hepatic cDCs with a mature, migratory phenotype we confirmed this by flow cytometry and sought locate these cells. Using flow cytometry we observed increased mature cDC1s and cDC2s in mice with MAFLD. Moreover, using a combination of confocal microscopy and RNAScope we observed localisation of cDCs in close proximity to the hepatic portal vein in the steady state which changes to form small clusters of cells including cDCs, macrophages and T cells in the peri-portal space in models of MAFLD. The changes in cDC subsets we observed during MAFLD, occurred directly prior to increases in cytotoxic T cells, suggesting they were driving the T cell responses. However, when we assessed the impact hepatic cDCs from mice with MAFLD had on T cells in vitro we observed no significant differences on their ability to induce a T cell response. To determine whether this also occurred in vivo we transferred naïve T cells from OTI/OTII mice into mice with MAFLD. We observed that the naïve T cells proliferated and developed a cytotoxic profile when adopted into mice with MAFLD. Despite this, depletion of cDC1s had little effect on MAFLD-induced liver damage and work is ongoing to delineate which cDC subsets can be manipulated to reduce progression of MAFLD. Most of these data have already been presented at the European Fatty Liver Conference in June 2022 (oral) and at the International Dendritic Cell Symposium in October 2022 (poster). These results will also be published in a high impact journal following the completion of some final experiments.
This MSCA has pushed the frontiers of hepatic dendritic cell research forward in numerous ways. As recently discussed in the 16th International Symposium on Dendritic Cells 2022, subsets of DCs are incompletely characterised and as a result confusion in their nomenclature is rampant. Moreover, the ability to characterise hepatic dendritic cells effectively has been surprisingly lacking in the literature to date, this fellowship utilised single cell technologies to identify subsets of liver DCs in both steady state and during MAFLD. This in itself will enable substantial progress in MAFLD research. Without specific markers previous studies have drawn conclusions regarding liver DCs using CD11c-CRE mice, a marker that is now appreciated to be vastly prolific, expressed on natural killer cells, some innate lymphoid cells as well as some macrophages. This has resulted in a plethora of conflicting studies showing both a protective and damaging role of DCs in MAFLD. Using the results from this MSCA, markers for mature liver cDCs, that are upregulated in MAFLD, have been identified and can therefore be targeted as potential contributors in disease progression. This research has the potential to identify novel therapeutic strategies which are so urgently required by society given the increasing prevalence of MAFLD, the lack of effective treatments and the increasing demand for liver transplantation.