Increased Neutrophil Secretion Induced by NLRP3 Mutation Links the Inflammasome to Azurophilic Granule Exocytosis

Abstract

Heterozygous mutations in the NLRP3 gene in patients with cryopyrin associated periodic syndrome (CAPS) lead to hyper-responsive inflammasome function. CAPS is a systemic auto-inflammatory syndrome characterized by the activation of the innate immune system induced by elevated pro-inflammatory cytokines, but the involvement of selective innate immune cells in this process is not fully understood. Neutrophil secretion and the toxic components of their granules are mediators of inflammation associated with several human diseases and inflammatory conditions. Here, using the Nlrp3A350V inducible mouse model (MWS CreT) that recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle-Wells sub-phenotype of CAPS, we studied the relationship between hyper-activation of the inflammasome and neutrophil exocytosis. Using a flow cytometry approach, we show that Nlrp3A350V (MWS) neutrophils express normal basal levels of CD11b at the plasma membrane and that the upregulation of CD11b from secretory vesicles in response to several plasma membrane or endocytic agonist including the bacterial-derived mimetic peptide formyl-Leu-Met-Phe (fMLF) and the unmethylated oligonucleotide CpG is normal in MWS neutrophils. Significant but modest CD11b upregulation in MWS neutrophils compared to wild type was only observed in response to GM-CSF and CpG. The same pattern was observed for the secretion of matrix metalloproteinase-9 (MMP-9) from gelatinase granules in that MMP-9 secretion in MWS neutrophils was not different from that observed in wild-type neutrophils except when stimulated with GM-CSF and CpG. In contrast, azurophilic granule secretion, whose cargoes constitute the most toxic secretory and pro-inflammatory factors of the neutrophil, was markedly dysregulated in MWS neutrophils under both basal and stimulated conditions. This could not be attributed to paracrine effects of secretory cytokines because IL-1β secretion by neutrophils was undetectable under these experimental conditions. The increased azurophilic granule exocytosis in MWS neutrophils was attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. In agreement with a possible neutrophil contribution to systemic inflammation in CAPS, the levels of neutrophil secretory proteins were significantly elevated in the plasma from Nlrp3A350V mice. Altogether, our data indicates an azurophilic granule-selective dysregulation of neutrophil exocytosis in CAPS.

Therapeutic targeting of neutrophil exocytosis

“…Neutrophil exocytosis inhibitors are also potentially useful in autoinflammatory diseases induced by genetic alterations. Neutrophils from the Nlrp3 A350V inducible mouse model ( MWS CreT ) recapitulates human patients with the A352V mutation in NLRP3 observed in the Muckle‐Wells sub‐phenotype (MWS) of cryopyrin‐associated periodic syndrome (CAPS) are characterized by normal gelatinase granule exocytosis but exacerbated azurophilic granule cargo secretion even under non‐stimulated conditions . The increased azurophilic granule exocytosis in MWS neutrophils is attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20.…”

“…Given before LPS injection, Nexinhib20 significantly prevents LPS-induced neutrophil exocytosis and decreases plasma levels of neutrophil secretory proteins. 205 Furthermore, Nexinhib20-treated mice showed decreased tissue infiltration by inflammatory neutrophils in kidney and liver, 208 222 The increased azurophilic granule exocytosis in MWS neutrophils is attenuated by treatment with the neutrophil exocytosis inhibitor Nexinhib20. Although in vivo validation is pending and this is a multifactorial syndrome, it is possible that decreasing neutrophil exocytosis may have beneficial effects alone or in combination with other available therapies.…”

NLRP3 Inflammasome Assembly in Neutrophils Is Supported by PAD4 and Promotes NETosis Under Sterile Conditions

“…In neutrophils the NLRP3 inflammasome was found to be activated after bacterial infection (20,21) or after lipopolysaccharide (LPS) pretreatment with subsequent ATP stimulation (22). In addition, an activating mutation (A352V) in NLRP3 leading to Muckle Wells syndrome is associated with excessive neutrophil granule exocytosis (23) and a gain-of-function mutation in NLRP3, which results in Familial Mediterranean Fever (FMF) is subsequently linked to augmented NETosis (24,25).…”

The role of neutrophils in thrombosis

“…131 Mechanisms involved in neutrophil exocytosis and inflammasome activation are also described. 132 The P2X7 receptor (P2X7R) is expressed on macrophages and dendritic cells, which upon activation by adenosine triphosphate (ATP), induces the NLRP3 inflammasome assembly and caspase-1 dependent processing, releasing the proinflammatory cytokines IL-1β and IL-18. 133 Expression of functional P2X7R has been described in other human and murine hematopoietic cells, however the role of these receptors in neutrophils is not entirely clear.…”