Neutrophils are primed for enhanced inflammasome activation in skin microvasculature during adhesion via E-selectin

Summary: Abstract Body: Introduction: Neutrophils play a key role in psoriatic inflammation and recruitment into inflamed skin is initiated by tethering and rolling on E-selectin upregulated on endothelium in the microcirculation. We hypothesized that E-selectin ligation and clustering of L-selectin on the neutrophil membrane signals inflammasome activation that includes release of calprotectin (S100A8/A9), to promote progression of psoriatic plaque formation. Method: Neutrophils were isolated from the blood of healthy adult donors on an IRB from UC Davis. Neutrophils were shear mixed with polymer microspheres coated with recombinant human E-selectin (Eb) to simulate tethering and rolling adhesion. Cells were co-stimulated with LPS, S100A8/A9, or TNFα at 37°C. GMI-1687 is a small molecule antagonist that blocks E-selectin recognition of sLex on L-selectin. Neutrophil suspensions were incubated and shear mixed at 37°C for 20 minutes. FLICA is a fluorescent peptide that reports on Caspase-1 cleavage that initiates activation of the inflammasome and analyzed by flow cytometry. Results: Binding of Eb was a potent agonist of Caspase-1 activation as indicated by increased uptake of FLICA fluorescence. Eb binding to neutrophils increased FLICA by 100% and was blocked to baseline by pretreatment with GMI-1687. Superposition of Eb binding with addition of LPS, S100A8/A9, or TNFα was synergistic, increasing FLICA by 1-fold, 1.7-fold, and 1.5 fold respectively, over Eb alone. Conclusion: Psoriatic neutrophils in circulation are primed for enhanced inflammatory response by elevated b2-integrin binding affinity and calprotectin levels that increase with stimulation via TNFa. Here we report that rolling on inflamed endothelium via E-selectin/L-selectin can trigger inflammasome activation that is enhanced in the presence of calprotectin and TNFα, which may exacerbate the pathogenesis of psoriasis. Yoshiaki Matsushima^{1, 2}, Hideaki Uchida¹, Xuesong Wu¹, Samuel Hwang¹, Scott Simon³ 1. Dermatology, UC Davis Health, Sacramento, CA, United States. 2. Dermatology, Mie University, Graduate School of Medicine, Tsu, Mie Prefecture, Japan. 3. A-Chip LLC, Davis, CA, United States. Cell Communication Networks and Stromal Biology