DataSheet1_Haptenation of Macrophage Migration Inhibitory Factor: A Potential Biomarker for Contact Hypersensitivity.docx

The immunological response in contact hypersensitivity is incited by small electrophilic compounds, known as haptens, that react with endogenous proteins after skin absorption. However, the identity of hapten-modified proteins seen as immunogenic remains as yet largely unknown. In a recent study, we have for the first time identified a hapten-modified protein in the local lymph nodes of mice treated topically with the model hapten tetramethylrhodamine isothiocyanate (TRITC). The TRITC modification was located on the N-terminal proline of the protein macrophage migration inhibitory factor (MIF). The focus of the current study was to investigate the presence of the same hapten-protein conjugate in blood samples from mice treated topically with TRITC. Furthermore, TRITC modifications of the two major blood proteins, namely hemoglobin (Hb) and albumin (Alb), as well as TRITC modifications of MIF other than the N-terminal proline, were examined. Following incubation with different molar ratios of TRITC, a proteomic approach was applied to characterize conjugate formation of the three aforementioned proteins, using high resolution mass spectrometry (HRMS). The targeted screening of the TRITC-treated mice blood and lymph node samples for these sites led to the identification of only the same TRITC-MIF conjugate previously detected in the lymph nodes. No Hb and Alb conjugates were detected. Quantification of both the TRITC-modified and unmodified N-terminal peptide of MIF in blood and lymph node samples gave interesting insights of MIF’s role in murine contact hypersensitivity. Incubation of MIF with four different haptens encompassing different reactivity mechanisms and potencies, showed adduct formation at different amino acid residues, suggesting that MIF can be the preferred target for a wide variety of haptens. The present study provides essential progress toward understanding of hapten-protein conjugate formation in contact hypersensitivity and identifies hapten-modified MIF as a potential biomarker for this condition. Further investigation of MIF as a target protein can be a next step to determine if MIF is a biomarker that can be used to develop better diagnostic tools and targeted therapeutics for individuals with allergic contact dermatitis.

接触性超敏反应的免疫应答由被称为半抗原（hapten）的小型亲电化合物引发，这类物质经皮肤吸收后可与内源性蛋白质结合。然而，被识别为具有免疫原性的半抗原修饰蛋白的具体种类，迄今仍鲜为人知。在前期一项研究中，本团队首次在使用模式半抗原四甲基罗丹明异硫氰酸酯（TRITC）外用处理的小鼠局部淋巴结中，鉴定出一种半抗原修饰蛋白，其TRITC修饰位点位于巨噬细胞移动抑制因子（MIF）的N端脯氨酸残基处。本研究的核心目标，是探究经TRITC外用处理的小鼠血液样本中是否存在同款半抗原-蛋白结合物。此外，本研究还检测了两种主要血液蛋白质——血红蛋白（Hb）与白蛋白（Alb）——的TRITC修饰情况，以及MIF除N端脯氨酸残基外其他位点的TRITC修饰。在与不同摩尔比的TRITC共孵育后，本研究采用蛋白质组学方法结合高分辨质谱（HRMS），对上述三种蛋白质的结合物形成情况进行了表征。针对TRITC处理的小鼠血液与淋巴结样本的上述位点进行靶向筛选后，仅检测到此前在淋巴结中发现的同款TRITC-MIF结合物，未检出Hb与Alb的结合物。对血液与淋巴结样本中TRITC修饰及未修饰的MIF N端肽段进行定量分析，为阐明MIF在小鼠接触性超敏反应中的作用提供了重要见解。将MIF与四种具备不同反应机制与反应强度的半抗原共孵育后，可在不同氨基酸残基处检测到加合物形成，这表明MIF可能是多种半抗原的优先结合靶点。本研究为阐明接触性超敏反应中的半抗原-蛋白结合物形成机制取得了重要进展，并将半抗原修饰的MIF鉴定为该疾病的潜在生物标志物。后续可将MIF作为靶蛋白开展进一步研究，以明确其是否可作为生物标志物，用于为变应性接触性皮炎患者开发更优质的诊断工具与靶向治疗手段。