10.6084/m9.figshare.11323454.v1 Tsai C.-M. Tsai C.-M. Riestra A.M. Riestra A.M. Ali S.R. Ali S.R. Fong J.J. Fong J.J. Liu J.Z. Liu J.Z. Hughes G. Hughes G. Varki A. Varki A. Nizet V. Nizet V. Supplementary Material for: Siglec-14 Enhances NLRP3-Inflammasome Activation in Macrophages Karger Publishers 2019 Siglec Interleukin-1β Caspase-1 Inflammasome Macrophages Innate immunity Vimentin 2019-12-05 15:18:07 Dataset 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.