GM-CSF Increases Uptake of Charcot-Leyden Crystals in Neutrophils and Can Induce NETopathic Airway Inflammation

Charcot-Leyden crystals (CLCs) are present during eosinophilic inflammation and involved in the pathogenesis of various airway diseases. Our laboratory has previously shown that CLC induces neutrophil recruitment and formation of extracellular neutrophil traps (NETs). NETs serve as a platform for complement activation, and activated complement proteins such as C5a stimulates NET formation, thereby sustaining increased inflammation. GM-CSF can trigger viable neutrophils to generate NETs, but data on the effects of GM-CSF on complement activation and CLC are lacking. We investigated whether GM-CSF affects NETosis via neutrophil uptake of CLC and whether the absence of C5ar1 and CSFRb2 could prevent CLC-induced inflammation. To understand how GM-CSF contributes to CLC uptake, we stimulated bone marrow-derived neutrophils (BMDN) with CLCs in the presence or absence of GM-CSF. GM-CSF significantly increased CLC uptake into BMDNs, resulting in increased NET formation. Intracellular complement (C3) and complement receptor (C5aR1) were increased in BMDN after crystal uptake and C5a complement was produced by these cells. WT mice showed CLC uptake and neutrophilia, and increased production of cytokines (CCL-2, IL-1β IL-5, IL-6, and TNF-α). CLCs were not taken up by BMDN from CSFRb2−/− or C5ar1−/− mice, and in vivo crystals administered to C5ar1−/− mice showed less crystal uptake by neutrophils. Based on our data, GM-CSF contributes to CLC uptake and neutrophil activation to generate complement factors and NETs. Our data suggest reconsidering treatment options for unresponsive asthmatic patients with NETopathic airway inflammation.