The Role of Macrophage Migration Inhibitory Factor (MIF) Family Proteins in Wound Repair and Obesity

肥胖是一种与慢性脂肪组织炎症（CAI）相关的疾病，是包括2型糖尿病（T2DM）在内的几种疾病的危险因素。重要的是，肥胖患者往往表现出伤口修复延迟和伤口愈合障碍的风险增加，这仍然是一个未解决的问题，特别是在整形和重建手术。大量证据表明，趋化因子样炎症细胞因子巨噬细胞迁移抑制因子（MIF）促进肥胖、T2DM、CAI，同时抑制伤口修复。相比之下，在这种情况下，对最近发现的mif同源物d -多巴胺互变酶（DDT）知之甚少。申请人在此提案之前的初步工作已经为急性伤口愈合障碍患者脂肪组织中MIF和DDT的相互调节提供了证据。在小鼠LPS注射模型中观察到炎症脂肪组织中MIF和DDT的相互调节，脂肪细胞被确定为MIF和DDT差异表达的细胞来源。有趣的是，MIF和DDT的同步上调和下调分别被认为对体外伤口修复有不利影响。此外，只有MIF通过其受体CXCR2和CXCR4调节单核细胞向脂多糖注射脂肪组织的迁移，而DDT似乎没有表现出促进迁移的作用。本研究旨在通过小鼠体内模型、患者组织标本和机制实验，全面研究MIF和DDT在CAI、胰岛素抵抗和肥胖伤口修复中的作用。首先，将野生型小鼠和Ddt基因全基因敲除的小鼠进行高脂肪饮食（HFD），以研究迄今未知的Ddt对肥胖、胰岛素抵抗和CAI的影响。接下来，脂肪组织特异性敲除Mif和Ddt的小鼠（或对照组的野生型）被喂食HFD，将进行切口和切除损伤，以表征脂肪组织来源的Mif和Ddt在肥胖条件下伤口修复中的确切作用。我们预测，在我们的初步实验中观察到的MIF和DDT的反向作用也将适用于我们的体内模型。为了建立潜在的治疗策略，将在体内模型中进一步研究给予重组MIF/DDT蛋白和/或MIF/DDT中和抗体的可行性。在第三部分，体内实验研究将通过评估肥胖和瘦弱个体的原始人类脂肪组织样本来补充。为此，将使用脂肪组织样本和人成纤维细胞/角化细胞的体外共培养试验来检查MIF和ddt依赖的伤口愈合过程。最后，为了确定有助于MIF和DDT相互作用的受体，将进行机制研究，以解决CD74、CD44、CXCR2和/或CXCR4以及相关信号通路的作用。

Obesity is a condition associated with chronic adipose tissue inflammation (CAI) and represents a risk factor for several diseases including type 2 diabetes mellitus (T2DM). Importantly, obese patients often show delayed wound repair and an increased risk of wound healing disorders which remains an unsolved issue, particularly in Plastic and Reconstructive Surgery. A strong body of evidence suggests that the chemokine-like inflammatory cytokine macrophage migration inhibitory factor (MIF) promotes obesity, T2DM, CAI, while inhibiting wound repair. In contrast, little is known about MIFs recently discovered homolog D-dopachrome tautomerase (DDT) in this context. Preliminary work by the applicants leading up to this proposal has provided evidence for a reciprocal regulation of MIF and DDT in adipose tissue of patients with acute wound healing disorders. Reciprocal MIF and DDT regulation in inflamed adipose tissue was observed in a murine LPS injection model and adipocytes were identified as the cellular source for differential MIF and DDT expression. Interestingly, the coincident up-regulation versus down-regulation of MIF and DDT, respectively, was seen to have a detrimental effect on wound repair in vitro. Furthermore, Only MIF regulated monocyte migration into LPS-injected adipose tissue via its receptors CXCR2 and CXCR4 while DDT did not appear to exhibit pro-migratory effects.Here we aim to comprehensively study the role of MIF and DDT in the context of CAI, insulin resistance and wound repair under obesity, applying murine in vivo models, patient tissue specimens, and mechanistic experiments. First, wildtype mice and mice with a global Ddt knockout will undergo a high fat diet (HFD) to investigate the hitherto unknown effect of DDT on obesity, insulin resistance and CAI. Next, mice with an adipose tissue-specific knockout of Mif and Ddt (or wildtype in the control) that are fed a HFD will undergo incisional and excisional wounding to characterize the precise role of adipose tissue-derived MIF and DDT on wound repair under obese conditions. We predict that the inverse role of MIF and DDT that was observed in our preliminary experiments also will apply to our in vivo models. To establish potential therapeutic strategies, the feasibility of administering recombinant MIF/DDT protein and/or MIF/DDT neutralizing antibodies will additionally be studied in the in vivo models. In a third part, the experimental in vivo studies will be complemented by evaluation of primary human adipose tissue samples of obese and lean individuals. To this end, MIF- and DDT-dependent wound healing processes will be examined using in vitro co-culture assays of the adipose tissue samples and human fibroblasts/keratinocytes. Lastly, to identify the receptors contributing to MIF and DDT interactions, mechanistic studies will be performed to address the role of CD74, CD44, CXCR2, and/or CXCR4 as well as the relevant signaling pathways.