Protective role of OPN-High macrophages in NASH

非酒精性脂肪性肝炎(NASH)正在成为儿童和成人肝脏疾病的主要全球原因。最近的研究表明，在NASH过程中，巨噬细胞(MFS)在肝脏中的表型具有很大的异质性。募集的MFS以两个亚群的形式存在，具有不同的激活状态：(1)与内环境平衡Kupffer细胞(KCs)非常相似的MFS，或(2)脂质相关MFS(LAM)。2020年，在NASH的肝脏中发现了一群高表达骨桥蛋白(OPN)的LAM。值得注意的是，与常驻和单核细胞来源的KCs相比，LAM具有不同的激活方式，并且具有独特的脂类代谢能力。然而，它们是否也与邻近细胞串扰，以防止NASH中的脂肪变性和纤维化，目前尚不清楚。虽然MFS在肝脏中的募集和随后的激活被认为是促炎和促纤维化事件，但目前一个重要的知识缺口是缺乏对OPNHigh MFS在NASH中是有害的还是具有保护作用的了解。初步数据显示，Spp1KI Mye是受保护的，而Spp1ΔMye的NASH活性得分比WT小鼠更差。因此，我们的结果表明了一种范式的转变，因为OPNHigh MFS可能会保护NASH。然而，OPNHigh MFS的细胞间通讯以及预防和治疗潜力仍有待确定。我们的主要假设是OPNHigh MFS通过向肝细胞(HEPs)和肝星状细胞(HSCs)传递信号，减少肝脏脂肪变性和纤维化，保护肝脏免受NASH的侵袭。目标1是确定OPNHigh MFS如何预防脂肪变性。我们假设OPNHigh MFS信号使HEPs上调精氨酸酶-2(ARG2)，从而增加线粒体生物能量和脂肪酸氧化(FAO)，减少亚硝化应激，防止脂肪变性。为了测试这一点，首先，我们将鉴定向HEPs发送信号上调ARG2的OPNHigh MF‘分泌体’蛋白质；其次，我们将确定已识别的OPNHigh MF分泌体蛋白如何反式激活HEPS中的ARG2启动子；以及第三，我们将阐明ARG2如何增加HEPS中的线粒体生物能量学和FAO，并减少亚硝化应激。目的2是剖析OPNHigh MFS如何预防纤维化。我们假设OPNHigh MFS通过分泌组蛋白发出信号，降低I型胶原蛋白，和/或改变NASH的“基底体”格局，以防止纤维化，并调节细胞行为。为了证明这一点，首先，我们将鉴定向HSCs发送信号以降低胶原-I沉积和/或增加其降解的OPNHigh MF分泌体蛋白；其次，我们将分析OPNHigh MFS是否改变了基质组格局，并建立OPNHigh MF-ECM相关网络；第三，我们将进行肝脏scRNA-seq，并通过计算确定OPNHigh MF-ECM网络如何调控NASH中的细胞行为。

Non-alcoholic steatohepatitis (NASH) is emerging as a major worldwide cause of liver disease in children and adults. Recent studies demonstrate substantial heterogeneity in the phenotype of macrophages (MFs) infiltrating the liver during NASH. Recruited MFs exist as two subsets, with distinct activation states: (1) those closely resembling homeostatic Kupffer cells (KCs), or (2) lipid-associated MFs (LAMs). In 2020, a cluster of LAMs with high expression of osteopontin (OPN) was identified in livers with NASH. Notably, LAMs are differentially activated, compared to resident- and monocyte-derived KCs, and with a distinct ability to metabolize lipids. However, whether they also crosstalk with neighboring cells, to prevent steatosis and fibrosis in NASH, remains unknown. While recruitment of MFs into the liver, and subsequent activation, are considered proinflammatory and profibrotic events, currently a significant knowledge gap is a lack of understanding of whether OPNHigh MFs, are detrimental or protective in NASH. Preliminary data demonstrates that Spp1KI Mye are protected, whereas Spp1ΔMye have worse NASH activity scores than WT mice. Thus, our results suggest a paradigm shift, in that OPNHigh MFs may protect from NASH. Yet, the intercellular communication, and preventive and therapeutic potential of OPNHigh MFs, remain to be determined. Our overarching hypothesis is that OPNHigh MFs, by signaling to hepatocytes (HEPs) and hepatic stellate cells (HSCs), reduce liver steatosis and fibrosis, and protect from NASH. Aim 1 is to identify how OPNHigh MFs protect from steatosis. We hypothesize that OPNHigh MFs signal HEPs to upregulate arginase-2 (ARG2), which increases mitochondrial bioenergetics and fatty acid oxidation (FAO), reduces nitrosative stress, and protects from steatosis. To test this, first, we will identify the OPNHigh MF ‘secretome’ proteins that signal HEPs to upregulate ARG2; second, we will determine how the identified OPNHigh MF secretome proteins transactivate the ARG2 promoter, in HEPs; and third, we will elucidate how ARG2 increases mitochondrial bioenergetics and FAO, and reduces nitrosative stress, in HEPs. Aim 2 is to dissect how OPNHigh MFs protect from fibrosis. We hypothesize that OPNHigh MFs signal through secretome proteins to lower collagen-I, and/or modify the ‘matrisome’ landscape, to prevent fibrosis, and regulate cell behavior, in NASH. To prove this, first, we will identify the OPNHigh MF secretome proteins that signal HSCs to lower collagen-I deposition and/or increase its degradation; second, we will analyze whether OPNHigh MFs modify the matrisome landscape, and establish the OPNHigh MF-ECM correlation network; and third, we will perform liver scRNA-seq, and computationally identify how the OPNHigh MF-ECM network regulates cell behavior in NASH.