Supplementary Material for: Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages Tsai C.-M. Riestra A.M. Ali S.R. Fong J.J. Liu J.Z. Hughes G. Varki A. Nizet V. 10.6084/m9.figshare.11323454.v1 https://karger.figshare.com/articles/dataset/Supplementary_Material_for_Siglec-14_Enhances_NLRP3-Inflammasome_Activation_in_Macrophages/11323454 Pathogenic microorganisms are sensed by the inflammasome, resulting in the release of the pro-immune and proinflammatory cytokine interleukin-1β (IL-1β). In humans, the paired sialic acid-binding Ig-like lectin receptors Siglec-5 (inhibitory) and Siglec-14 (activating) have been shown to have reciprocal roles in regulating macrophage immune responses, but their interaction with IL-1β signaling and the inflammasome has not been characterized. Here we show that in response to known inflammasome activators (ATP, nigericin) or the sialic acid-expressing human bacterial pathogen group B <i>Streptococcus</i> (GBS), the presence of Siglec-14 enhances, whereas Siglec-5 reduces, inflammasome activation and macrophage IL-1β release. Human THP-1 macrophages stably transfected with Siglec-14 exhibited increased caspase-1 activation, IL-1β release and pyroptosis after GBS infection, in a manner blocked by a specific inhibitor of nucleotide-binding domain leucine-rich repeat protein 3 (NLRP3), a protein involved in inflammasome assembly. Another leading pathogen, <i>Streptococcus pneumoniae</i>, lacks sialic acid but rather prominently expresses a sialidase, which cleaves sialic acid from macrophages, eliminating <i>cis</i>- interactions with the lectin receptor, thus attenuating Siglec-14 induced IL-1β secretion. Vimentin, a cytoskeletal protein released during macrophage inflammatory activation is known to induce the inflammasome. We found that vimentin has increased interaction with Siglec-14 compared to Siglec-5, and this interaction heightened IL-1β production by Siglec-14-expressing cells. Siglec-14 is absent from some humans because of a <i>SIGLEC5/14</i> fusion polymorphism, and we found increased IL-1β expression in primary macrophages from <i>SIGLEC14</i><sup>+/+</sup> individuals compared to those with the <i>SIGLEC14</i><sup>–/+</sup> and <i>SIGLEC14</i><sup>–/–</sup> genotypes. Collectively, our results identify a new immunoregulatory role of Siglec-14 as a positive regulator of NLRP3 inflammasome activation. 2019-12-05 15:18:07 Siglec Interleukin-1β Caspase-1 Inflammasome Macrophages Innate immunity Vimentin