Authors L. DEVISSCHER (1), C. SCOTT (2), S. LEFERE (1), S. RAEVENS (1), E. BOGAERTS (1), A. PARIDAENS (1), X. VERHELST (1), A. GEERTS (1), M. GUILLIAMS (2), H. VAN VLIERBERGHE (1) / [1] Ghent University, Ghent, Belgium, Gastro-enterology and Hepatology, [2] Ghent University, Ghent, Belgium, Department of Biomedical Molecular Biology
Introduction Kupffer cells (KCs) and liver infiltrating bone-marrow (BM) monocyte-derived macrophages (mo-Mf) have been denoted as key players in the pathogenesis of non-alcoholic steatohepatitis (NASH). Despite this, to date it has not been possible to accurately discriminate between these two populations due to the lack of specific markers. Additionally, KCs were believed to be derived solely from embryonic progenitors, which are maintained by self-renewal, however, it has recently been demonstrated that BM monocytes can differentiate into bona fide KCs (mo-KCs) when required. To date, it is also unclear if mo-KCs are present during NASH. Understanding which of the distinct macrophage populations are present and the roles they play in NASH is crucial to furthering our understanding of NASH pathogenesis and the development of novel therapies.
Aim
By using newly defined specific markers including Clec4F and Tim4 alongside BM chimeras, we aimed at accurately characterize the dynamic changes and origins of the distinct liver macrophage subsets in experimental-induced NASH and recovery.
Methods Immunohistopathology and flow cytometry were used to determine the level of steatosis, steatohepatitis and recovery in methionine and choline deficient (MCD) diet fed mice. Flow cytometric analysis including the specific markers Clec4F and Tim4 and BM chimeras were applied to identify the distinct liver macrophage subsets and their origins.
Results Mice fed the MCD diet for 8 weeks gradually developed severe steatohepatitis while replacement of MCD diet by normal chow resulted in full recovery after 4 weeks. Ly6CloClec4F-Tim4- infiltrated mo-Mf were observed from week 2 of MCD feeding, further increased during MCD feeding and returned to baseline during recovery. The absolute number of KCs, characterized as Ly6CloClec4F+Tim4+ cells, did not differ significantly between mice fed either MCD or the control diet (CD) over the duration of feeding or during recovery. However, an increased proportion of Ki-67+ proliferating KCs were observed in mice fed MCD diet compared with mice fed control diet. In line with this, we observed the development of a new population of Ly6CloClec4F+Tim4- KCs, only typically present in minor numbers in steady state, previously identified as mo-KCs. Mo-KCs developed from week 4 on the MCD diet and remained present during recovery. As lack of Tim4 expression is only a temporal marker of mo-KCs, with mo-KCs gradually gaining Tim4 expression after their differentiation, we utilised BM chimeras to both validate the presence of mo-KCs and determine their longevity. Interestingly, while these cells do develop from monocytes during MCD feeding and peak during initial recovery, they do not have the capacity to self-renew as their numbers are reduced by week 4 recovery.
Conclusions Our findings demonstrate that during NASH pathogenesis and recovery the KC pool is maintained by proliferation and the differentiation of short-lived mo-KCs in the MCD diet model.
