Elucidating the role of iron metabolizing macrophages in adipose tissue

描述(申请人提供)：在过去的十年里，肥胖患者的脂肪组织(AT)炎症及其与胰岛素敏感性的联系日益受到重视。胰岛素抵抗在我们人群中的流行使得我们必须更好地了解免疫细胞在维持健康AT中的作用。巨噬细胞是AT中的主要免疫细胞，已知在肥胖时，巨噬细胞会改变为炎性表型，以应对不健康的脂肪堆积。瘦肉AT含有一群抗炎的“M2样”AT巨噬细胞(M2ATM)，其在维持健康瘦肉状态中的作用尚不清楚。我们的实验室最近发现了一个M2ATM亚群，细胞内铁含量增加了两倍，参与铁摄取、储存和释放的基因(如CD163)的表达也增加了。在瘦肉型AT中，这一群体占M2 ATM的25%，在肥胖的小鼠和人类AT中也存在。以前的研究表明，脂肪细胞需要铁来进行脂肪生成，但过量的铁具有细胞毒性，并导致胰岛素敏感性的全身性变化。因此，健康的脂肪细胞必须保持细胞内铁浓度的狭窄范围。目前尚不清楚MFeHI自动取款机是否有助于脂肪细胞铁稳态。我们假设MFeHI ATM调节脂肪细胞对铁的供应，从而影响瘦肉型和肥胖型AT的胰岛素敏感性。为了确定MFeHIATM和脂肪细胞之间可能的铁交换，我们将在铁负载处理后与铁超载的遗传模型共同培养这些细胞。在最初的研究中，我们成功地量化了用柠檬酸亚铁铵和螯合剂8-羟基氯喹处理后巨噬细胞内铁的增加。我们还建议在小鼠体内使用CD163靶向的含有氯屈膦酸盐的脂质体来耗尽MFeHI ATM。耗竭将使我们能够确定MFeHI自动取款机对全身代谢状态的影响。我们对腹膜内钙黄绿素荧光脂质体的初步研究表明，CD163靶向脂质体是一种特异性靶向ATM的新技术。最后，对MFeHI和MFelo ATM的比较代谢组学分析将作为一种公正的假说生成方法来表征MFeHI的表型。根据我们目前对铁在细胞功能中的理解，我们希望在MFeHI人群中发现线粒体和/或脂质氧化途径的变化。这项建议中的研究将使我们能够确定MFeHI人群在维持健康的脂肪细胞铁浓度方面所起的作用，从而确定胰岛素敏感性。

DESCRIPTION (provided by applicant): In the last decade, there has been growing appreciation of adipose tissue (AT) inflammation in obesity, and its link to insulin sensitivity. Te prevalence of insulin resistance in our population makes it imperative that we come to a better understanding of the role of immune cells in maintaining healthy AT. Macrophages are the predominant immune cell in AT and are known to change to an inflammatory phenotype in response to unhealthy lipid accumulation in obesity. Lean AT contains a resident population of anti-inflammatory "M2-like" AT macrophages (M2 ATMs), whose role in the maintenance of a healthy lean state is not well understood. Our lab has recently identified a "MFehi" sub-population of M2 ATMs, with a two-fold increase in intracellular iron content and increased expression of genes involved in iron uptake (e.g. CD163), storage and release. This population composes 25% of M2 ATMs in lean AT and also exists in obese mouse and human AT. Previous studies have demonstrated that adipocytes require iron for adipogenesis, but excess iron is cytotoxic and leads to systemic changes in insulin sensitivity. Therefore, healthy adipocytes must maintain a narrow range of intracellular iron concentrations. It remains unknown whether MFehi ATMs contribute to adipocyte iron homeostasis. We hypothesize that MFehi ATMs modulate iron availability to adipocytes, and thereby impact insulin sensitivity of lean and obese AT. To identify possible iron exchange between MFehi ATMs and adipocytes, we will co-culture these cells after iron-loading treatment and from genetic models of iron overload. In initial studies, we have successfully quantified increased intracellular iron in macrophages after treatment with ferrous ammonium citrate and the chelator 8-hydroxychloroquine. We also propose to deplete MFehi ATMs in vivo in mice using CD163-targeted clodronate-containing liposomes. Depletion will allow us to determine the impact of MFehi ATMs on the systemic metabolic state. Our preliminary studies with fluorescent calcein-loaded liposomes in the peritoneum have revealed CD163-targeted liposomes as a novel technique to specifically target ATMs. Lastly, comparative metabolomics analysis of MFehi and MFelo ATMs will serve as an unbiased hypothesis-generating approach to characterizing the MFehi phenotype. From our current understanding of iron in cell function, we expect to find changes in mitochondrial and/or lipid oxidation pathways in the MFehi population. The studies in this proposal will allow us to define the role of the MFehi population in maintaining healthy adipocyte iron concentrations and, thereby, insulin sensitivity.