Session Title: Innate Immunity and Rheumatic Disease
Session Type: Abstract Submissions (ACR)
Background/Purpose Previously, we reported that a novel variant, p.Ser707Tyr, in phospholipase Cγ2 (PLCγ2) is the cause of a dominantly inherited autoinflammatory disease, APLAID (autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation). The APLAID patients suffered from early onset recurrent blistering skin lesions, pulmonary disease, arthralgia, inflammatory eye and bowel disease, and mild immunodeficiency. The hypermorphic mutation enhances the PLCγ2 activity and causes an increase in intracellular Ca2+ release from ER stores. As increased intracellular Ca2+signaling has been associated with NLRP3 inflammasome activation, we studied the role of the NLRP3 inflammasome in the pathogenesis of this disease.
Methods Human peripheral blood mononuclear cells (PBMCs) were isolated from healthy controls and two affected patients. Inflammasome activation was analyzed by Western blotting of secreted interleukin-1β (IL-1β). Intracellular Ca2+levels were measured with the FLIPR Calcium 4 assay kit.
Results First, we confirmed that PLC-inositol trisphosphate (InsP3)-mediated Ca2+ release can trigger the activation of the NLRP3 inflammasome in human PBMCs in much the same way as was previously shown in the mouse. Since the S707Y APLAID mutation disrupts the autoinhibition of PLCg2, which leads enhanced PLCγ2 activity, patients’ leukocytes had elevated basal levels of intracellular Ca2+. Upon stimulation with extracellular CaCl2, an activator of the NLRP3 inflammasome, patients’ cells release significantly higher amounts of Ca2+ into the cytosol than the cells of healthy controls. Consistent with that the increase of cytoplasmic Ca2+ mediates the activation of the NLRP3 inflammasome, in the absence of inflammasome activators, PBMCs from patients with APLAID secreted IL-1β whereas control PBMCs secreted IL-1β only following the stimulation with CaCl2. The IL-1β secretion from LPS-primed patients’ PBMCs was attenuated by use of a PLC inhibitor and intracellular Ca2+blockers. Finally, we found that the constitutive IL-1β secretion from patients PBMCs was substantially reduced by the treatment with NKH477, the water-soluble analog of forskolin, which is a potent activator of adenylyl cyclase. These results suggest cAMP as a potential target for therapy of APLAID and other NLRP3 mediated diseases.
Conclusion Our findings suggest that the inflammation in patients with APLAID is partially driven by the activation of the NLRP3 inflammasome. These data link two seemingly distinct molecular pathways and provide new insights in the pathogenesis of APLAID and autoinflammation.
J. J. Chae,
Y. H. Park,
I. Y. Hwang,
D. L. Kastner,
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ACR Meeting Abstracts - https://acrabstracts.org/abstract/connecting-two-pathways-through-ca2-signaling-nlrp3-inflammasome-activation-induced-by-a-hypermorphic-plcg2-mutation/