Supplementary Material for: Markers Involved in Innate Immunity and Neutrophil Activation are Elevated during Acute Human Anaphylaxis: Validation of a Microarray Study

<b><i>Background:</i></b> We have previously identified the upregulation of the innate immune response, neutrophil activation, and apoptosis during anaphylaxis using a microarray approach. This study aimed to validate the differential gene expression and investigate protein concentrations of “hub genes” and upstream regulators during anaphylaxis. <b><i>Methods:</i></b> Samples were collected from patients with anaphylaxis on their arrival at the emergency department, and after 1 and 3 h. mRNA levels of 11 genes (<i>interleukin-6 [IL-6], IL-10, oncostatin M [OSM], S100A8, S100A9, matrix metalloproteinase 9 [MMP9], FASL, toll-like receptor 4 [TLR4], MYD88, triggering receptor expressed on myeloid cells 1 [TREM1]</i>, and <i>cluster of differentiation 64 [CD64]</i>) were measured in peripheral blood leucocytes using qPCR. Serum protein concentrations were measured by ELISA or cytometric bead array for 6 of these candidates. <b><i>Results:</i></b> Of 69 anaphylaxis patients enrolled, 36 (52%) had severe reactions, and 38 (55%) were female. Increases in both mRNA and protein of IL-10, S100A9, MMP9, and TREM1 were observed. <i>OSM</i>, <i>S100A8</i>, <i>TLR4</i>, and <i>CD64</i> were upregulated and IL-6 protein concentrations were increased during anaphylaxis. Both <i>FASL</i> and soluble Fas ligand decreased during anaphylaxis. <b><i>Conclusion:</i></b> These results provide evidence for the involvement of innate immune pathways and myeloid cells during human anaphylaxis, validating previous microarray findings. Elevated <i>S100A8</i>, <i>S100A9</i>, <i>TLR4</i>, and <i>TREM1</i> expression, and increased S100A9 and soluble TREM1 protein concentrations strongly suggest that neutrophils are activated during acute anaphylaxis.

<b><i>背景：</i></b> 我们此前通过基因芯片（microarray）技术鉴定出，过敏性反应过程中存在先天免疫应答（innate immune response）上调、中性粒细胞（neutrophil）活化与细胞凋亡（apoptosis）现象。本研究旨在验证过敏性反应中的差异基因表达情况，并探究核心基因（hub genes）与上游调控因子的蛋白浓度水平。<b><i>方法：</i></b> 研究样本采集自急诊就诊时的严重过敏反应患者，分别于就诊即刻、1小时及3小时后采集。针对11个基因，即白细胞介素-6（interleukin-6, IL-6）、IL-10、制瘤素M（oncostatin M, OSM）、S100A8、S100A9、基质金属蛋白酶9（matrix metalloproteinase 9, MMP9）、FASL、Toll样受体4（toll-like receptor 4, TLR4）、MYD88、髓系细胞触发受体1（triggering receptor expressed on myeloid cells 1, TREM1）以及分化簇64（cluster of differentiation 64, CD64），我们采用实时定量PCR（qPCR）对外周血白细胞中的mRNA水平进行检测。此外，针对其中6个候选基因，采用酶联免疫吸附试验（ELISA）或流式微球阵列（cytometric bead array）检测其血清蛋白浓度。<b><i>结果：</i></b> 本研究共纳入69名严重过敏反应患者，其中36例（52%）为重度过敏反应，38例（55%）为女性。研究观察到IL-10、S100A9、MMP9及TREM1的mRNA与蛋白水平均出现上调。OSM、S100A8、TLR4及CD64的表达水平升高，且IL-6的蛋白浓度在过敏反应过程中有所增加。FASL与可溶性FAS配体（soluble Fas ligand）的水平则在过敏反应期间出现下降。<b><i>结论：</i></b> 本研究结果证实，先天免疫通路与髓系细胞参与人类严重过敏反应过程，验证了此前基因芯片的研究发现。S100A8、S100A9、TLR4及TREM1的表达上调，以及S100A9与可溶性TREM1的蛋白浓度升高，均强烈提示中性粒细胞在急性严重过敏反应期间发生活化。