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.