Supplementary Material for: MIP-3α Expression in Macrophages Is NOD Dependent

<i>Background:</i> The first identified susceptibility gene for Crohn’s disease, <i>NOD2</i>, acts as a sensor for the bacterial-wall peptidoglycan fragment muramyl dipeptide (MDP) and activates the transcription factor nuclear factor-ĸB (NF-ĸB). Upon NF-ĸB activation, intestinal macrophages (IMACs) induce expression of macrophage inflammatory protein (MIP)-3α to attract memory T lymphocytes. We therefore investigated the influence of NOD2 ligation of IMAC differentiation and functional MIP-3α induction. <i>Methods:</i> Human embryonal kidney HEK293 cells were transfected with NOD2 wild-type (NOD2^WT) and the NOD2 SNP13 variant (NOD2^L1007fsinsC) and stimulated with MDP. Recruitment of CD45R0⁺ and Th17 cells was determined by immunohistochemistry. <i>Results:</i> Endogenous NOD2 stimulation was followed by a dose-dependent increase in MIP-3α secretion in MONO-MAC-6 (MM6) cells. MIP-3α mRNA was also significantly (* p &lt; 0.05) induced in HEK293 transfected with NOD2^WT via MDP ligation. In vivo cell-cell contacts between IMACs and CD45R0⁺ memory T cells as well as recruitment of Th17 cells in patients of NOD2 variants were unchanged as compared to wild-type patients. <i>Conclusion:</i> Our data demonstrate a dose-dependent increase in MIP-3α secretion in the human myeloid cell line MM6 upon MDP. However, MIP-3α-driven recruitment of Th17 cells or CD45R0⁺ memory T lymphocytes is not affected in patients carrying heterozygous NOD2 variants.

背景：首个被鉴定的克罗恩病（Crohn’s disease）易感基因NOD2，可作为细菌细胞壁肽聚糖片段胞壁酰二肽（muramyl dipeptide, MDP）的感受器，并激活转录因子核因子κB（nuclear factor-κB, NF-κB）。核因子κB激活后，肠巨噬细胞（intestinal macrophages, IMACs）会诱导巨噬细胞炎性蛋白（macrophage inflammatory protein, MIP）-3α的表达，以招募记忆T淋巴细胞。因此本研究探讨了NOD2配体对IMAC分化及功能性MIP-3α诱导的影响。
方法：将野生型NOD2（NOD2^WT）及NOD2 SNP13变异体（NOD2^L1007fsinsC）转染至人类胚胎肾HEK293细胞，并用MDP进行刺激。通过免疫组化法检测CD45R0⁺细胞及Th17细胞的招募情况。
结果：内源性NOD2受刺激后，MONO-MAC-6（MM6）细胞中MIP-3α的分泌呈剂量依赖性升高。在经MDP配体处理并转染了NOD2^WT的HEK293细胞中，MIP-3α的mRNA水平也显著升高（*p < 0.05）。与野生型患者相比，携带NOD2变异体的患者中，IMACs与CD45R0⁺记忆T细胞之间的体内细胞-细胞接触，以及Th17细胞的招募情况均无明显变化。
结论：本研究数据证实，在经MDP处理后，人类髓系细胞系MM6中MIP-3α的分泌呈剂量依赖性升高。但携带杂合型NOD2变异体的患者，其MIP-3α介导的Th17细胞或CD45R0⁺记忆T淋巴细胞招募过程并未受到影响。