Regulation of macrophage Function by ADAR1 in metabolic disease

Macrophages exhibit remarkable functional plasticity in response to environmental cues, and emerging evidence indicates that post-transcriptional regulation plays a pivotal role in orchestrating these dynamic phenotypic states. ADAR1, the enzyme responsible for adenosine-to-inosine (A-to-I) RNA editing, is well established as a suppressor of aberrant type I interferon responses. However, its role in regulating human macrophage biology under steady-state conditions and in metabolic disease remains poorly defined. The overarching aim of this thesis was to elucidate how ADAR1 influences macrophage function in health and disease. In Paper I, we systematically profiled ADAR1 expression across human immune cell populations and identified a pronounced enrichment of ADAR1 protein in macrophages. This enrichment was driven by monocyte-to-macrophage differentiation upon tissue entry and was mediated by usage of an alternative transcription start site (TSS) and a regulatory element upstream of the canonical TSS, rather than increased mRNA abundance. Using loss-of-function approaches in primary human macrophages combined with unbiased profiling of the A-to-I RNA editome, we demonstrate that ADAR1 orchestrates prototypical macrophages programs associated with efferocytosis, endocytosis, lysosomal function, and lipid metabolism. Notably, these programs substantially overlap with those defining lipid-associated macrophages (LAM), a population implicated in metabolic dysfunction. Consistent with this link, analysis of human adipose tissue and matched peripheral blood samples from a longitudinal weight-loss intervention revealed that ADAR1 expression in LAMs is dynamically regulated in a cell-specific manner following weight loss. Mechanistically, we attributed this induction to tissue-derived free fatty acids, suggesting that ADAR1 upregulation represents a buffering response of the lipolytic and lipotoxic environment characteristic of early weight-loss stages. Collectively, these findings extend the functional scope of ADAR1 beyond its established role in autoimmunity, providing novel insights into how post- transcriptional regulation governs human macrophage plasticity in steady and in metabolic disease contexts. By leveraging clinical cohorts, computational analyses, and mechanistic studies in primary human cells, this work bridges fundamental molecular mechanisms with translational relevance. A secondary aim of this thesis was to develop more physiologically relevant human experimental models of liver disease. In Paper II, we established a three- dimensional human hepatic spheroid model composed of hepatocytes and key non-parenchymal cells (NPC), including hepatic stellate cells and liver sinusoidal endothelial cells. We demonstrated the robustness of this model over time and its ability to recapitulate key features of fatty liver disease under metabolic stress, including induction of CYP2E1 and COL1A1 expression. In Paper III, we extended this hepatic spheroid model by incorporating liver macrophages to functionally validate a predicted protective macrophage subpopulation. Single-cell RNA sequencing of lean and obese human livers identified a macrophage population (liver macrophage 2; LM2) characterized by high expression of secreted antioxidant enzyme PRDX2, which was markedly depleted in individuals with obesity. To functionally assess the antioxidant buffering capacity of LM2 macrophages, we generated hepatic spheroids containing all NPCs or NPCs lacking LM2 under obesogenic conditions. Absence of LM2 macrophages resulted in reduced PRDX2 secretion, accompanied by significantly increased in reactive oxygen species and lipid peroxidation products. Together, these findings provide functional validation of computational predictions and demonstrate that LM2 macrophages exert a protective antioxidant role in the liver under metabolic stress. Taken together, Papers II and III highlight how physiologically relevant human spheroid models can advance our understanding of liver disease mechanisms and support the development of more effective and translatable therapeutic strategies. List of scientific papers I. ADAR1 controls macrophage scavenging and lipid-buffering programs in metabolic tissues. Fardellas A*, Barreby E, Brice M, Nock S, Russick J, Vankova A, Edberg C, Robertsen I, Hertel J K, Stål P, Mellgren G, Karlsson C, Hjelmesaeth J S, Hagström H, Näslund E, Lauschke V M, Fernø J, Chen P, Morgantini C, Aouadi M, Björkström N K *. [Manuscript] *Corresponding authors II. Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis. Hurrell T*, Kastrinou-Lampou V*, Fardellas A, Hendriks DFG, Nordling Å, Johansson I, Baze A, Parmentier C, Richert L, Ingelman- Sundberg M. Cells. 9, 964 (2020). doi: https://doi.org/10.3390/cells9040964 *TH and VKL contributed equally III. Human resident liver myeloid cells protect against metabolic stress in obesity. Barreby E, Strunz B, Nock S, Naudet L, Shen JX, Johansson H, Sönnerborg I, Ma J, Urgard E, Pallett LJ, Hu Y, Fardellas A, Azzimato V, Vankova A, Levi L, Morgantini M, Maini MK, Stål P, Rosshart SP, Coquet JM, Nowak G, Näslund E, Lauschke VM, Ellis E, Björkström NK, Chen P, Aouadi A. Nat Metab 5, 1188-1203 (2023). doi: https://doi.org/10.1038/s42255-023-00834-7