Clearance of Dead Hepatocytes by Liver Macrophages in NASH

Non-alcoholic steatohepatitis (NASH) is a major cause of liver disease. However, there are no FDA-approved drugs to treat NASH, due in large part to an incomplete understanding of NASH mechanisms, particularly those involved in NASH-induced liver fibrosis. A key feature of NASH is the accumulation of dead hepatocytes (HCs), particularly apoptotic HCs (apHCs) and necroptotic HCs (necHCs). In other diseases, the impact of dead cells is largely determined by their fate, i.e., clearance by macrophages (Mφs), but surprisingly little is known about this in NASH. This proposal aims to address this fundamental gap and thereby gain new insights into potentially druggable and human-relevant NASH mechanisms, particularly those involving cellular cross-talk. A key concept is that the Mφ clearance of apoptotic cells ("efferocytosis") vs. necroptotic cells are distinct in terms of mecha- nisms and consequences, and I have shown the Mφ clearance of both apHC and necHCs is impaired in human and mouse NASH. Based on extensive preliminary data, my proposal addresses specific hypotheses on apHC efferocytosis in Aim 1 (K99) and necHC clearance in Aim 2 (R00). In Aim 1, I will test the hypothesis that loss of the efferocytosis receptor TIM4 in NASH Mφs causes defective apHC clearance and contributes to NASH pro- gression. I will ask if deletion of Mφ-TIM4 in NASH mice accelerates the progression to impaired efferocytosis and liver fibrosis in early NASH. Conversely, I will use an inducible model to restore Mφ-TIM4 during NASH. I have shown this restores apHC efferocytosis, and I will test the prediction that it will decrease HSC activation and liver fibrosis. I will next explore a mechanism-based therapeutic idea to restore Mφ-TIM4 in NASH, asking if this drug can improve efferocytosis and dampen NASH progression. Finally, I will conduct scRNA-seq analyses of the livers of the Mφ-TIM4-restoration model to probe the hypothesis that apHC efferocytosis leads to the secretion Mφ molecules that promote a pro-resolving Mφ phenotype and maintain HSCs in a quiescent state. In Aim 2, I will study a new pathway in NASH in which upregulation of the CD47-SIRPα axis impairs necHC clear- ance. I propose that Mφ necHC uptake induces Mφs to secrete protein(s) that down-regulate HC-TAZ, thereby suppressing the TAZ-IHH-HSC pathway involved in fibrotic NASH. I will seek proof for this idea in complementary models of NASH by testing the effect of combining HC-TAZ-silencing and SIRPα blockade. I will then use a Mφ- specific "secretome" mouse model and proteomics to identify the Mφ protein(s) induced by necHC uptake that lower HC-TAZ. Finally, I will address 2 fundamental questions: (1) How is SIRPα up-regulated in NASH Mφs?; and (2) Is Mφ uptake of necHCs in the setting of CD47/SIRPα-axis blockade receptor-mediated? My preliminary data in these areas have highlighted new roles for two Mφ proteins in NASH, TREM2 and LRP1. Through these Aims, I hope to gain new insights into NASH mechanisms and therapeutic concepts. My K99 project will allow me to receive invaluable knowledge-based and technical mentoring and career advice as I transition to inde- pendence, and the R00 encompasses an exciting research program that will help launch my independent career.