Background/Purpose: Neutrophil extracellular traps (NETs) contain numerous bactericidal proteins and are an important defense mechanism against microorganisms.  Clearance of NETs is impaired in a subset of patients with systemic lupus erythematosus (SLE), which may contribute to disease pathogenesis.  Additionally, we recently described that NETs are vigorously and spontaneously released from low density granulocytes (LDGs) isolated from lupus patients and that these lattices are toxic to the endothelium, expose immunostimulatory molecules and may participate in organ damage through incompletely characterized pathways.   We propose that in antigen presenting cells, NETs may act as strong activators of inflammatory pathways, such as the inflammasome, contributing to organ damage.  We have examined the role of NETs in inflammasome activation and have specifically characterized the ability of one NET-associated antibacterial peptide, LL37, in stimulating this process.

Methods: Primary human macrophages were derived from monocytes isolated from control and lupus patients.  Murine macrophages were cultured from bone marrow.  M1 and M2 differentiation were promoted by growth in GM-CSF or M-CSF, respectively and confirmed by FACs.  Spontaneously-induced NETs from LDGs were isolated by micrococcal nuclease incubation to allow their release into the supernatant.  Macrophages were primed for 4 hours with 100 ng/ml LPS, prior to stimulation with LL37 peptide or NETs for 2 hours.  Active IL-1beta and IL-18 release was determined by ELISA.  Quantification of caspase-1 activation was determined by incubating macrophages with a specific fluorogenic marker, followed by fluorescent microscopy.

Results: Stimulation of human macrophages with NETs resulted in activation of caspase-1 and in IL-1beta and IL-18 activation and secretion. Incubation of macrophages with the NET-associated peptide LL37 resulted in robust activation of caspase-1 and release of IL-1beta and IL-18; this activation was enhanced in M1 versus M2 macrophages.  Caspase-1 activation required the NLRP3 inflammasome, as murine macrophages deficient in NLRP3, ASC or caspase-1 did not respond to the murine LL37 analog mCRAMP, whereas wild-type macrophages demonstrated caspase-1 activation and IL-1beta release.  Comparison of control versus lupus macrophage dose responses to LL37 revealed a lower threshold for inflammasome activation in lupus macrophages, suggesting that exposure to this molecule in vivo may trigger enhanced inflammatory responses.  Exposure of lupus macrophages to NETs also led to a significant increase in caspase-1 activation when compared to control macrophages. 

Conclusion:  Macrophages isolated from lupus patients have enhanced activation of the inflammasome in response to NETs or to the NET-associated peptide LL37.  As NETs are increased in SLE, these results suggest that these structures could be an important trigger for inflammasome activation and the resultant downstream inflammatory pathways in SLE patients.  

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/neutrophil-extracellular-trap-associated-protein-activation-of-the-inflammasome-is-enhanced-in-lupus-m1-macrophages/