Role of Annexin A1 in NLRP3 Inflammasome Activation in Murine Neutrophils.

José Marcos Sanches,Gustavo H B Duarte,Rebeca D Correia-Silva,Cristiane D Gil,Vanessa Moreira,Patrícia O Carvalho,Anna Maria A P Fernandes,Sonia M Oliani,Salvador Sánchez-Vinces,Karina R Bortoluci

doi:10.3390/cells10010121

Abstract

This study evaluated the role of endogenous and exogenous annexin A1 (AnxA1) in the activation of the NLRP3 inflammasome in isolated peritoneal neutrophils. C57BL/6 wild-type (WT) and AnxA1 knockout mice (AnxA1-/-) received 0.3% carrageenan intraperitoneally and, after 3 h, the peritoneal exudate was collected. WT and AnxA1-/- neutrophils were then stimulated with lipopolysaccharide, followed by the NLRP3 agonists nigericin or ATP. To determine the exogenous effect of AnxA1, the neutrophils were pretreated with the AnxA1-derived peptide Ac2-26 followed by the NLRP3 agonists. Ac2-26 administration reduced NLRP3-derived IL-1β production by WT neutrophils after nigericin and ATP stimulation. However, IL-1β release was impaired in AnxA1-/- neutrophils stimulated by both agonists, and there was no further impairment in IL-1β release with Ac2-26 treatment before stimulation. Despite this, ATP- and nigericin-stimulated AnxA1-/- neutrophils had increased levels of cleaved caspase-1. The lipidomics of supernatants from nigericin-stimulated WT and AnxA1-/- neutrophils showed potential lipid biomarkers of cell stress and activation, including specific sphingolipids and glycerophospholipids. AnxA1 peptidomimetic treatment also increased the concentration of phosphatidylserines and oxidized phosphocholines, which are lipid biomarkers related to the inflammatory resolution pathway. Together, our results indicate that exogenous AnxA1 negatively regulates NLRP3-derived IL-1β production by neutrophils, while endogenous AnxA1 is required for the activation of the NLRP3 machinery.

Highlights

NLRP3 Inflammasome-Derived IL-1β Production is Impaired in annexin A1 (AnxA1)-/- Neutrophils
NLRP3 inflammasome activation by nigericin and adenosine triphosphate (ATP) caused a significant increase in the release of IL-1β by WT neutrophils, an effect that was reduced by the addition of Ac2-26 (Figure 1B)
These findings were corroborated by the immunoblot analysis of cell extracts, showing decreased levels of pro-IL-1β in Ac2-26 -treated WT cells compared to non-treated cells (Figure 1D,F)

Summary

Introduction

NLRP3 (NOD-like receptor pyrin domain-containing protein 3, cryopyrin, or NALP3). Is the most studied member of the intracellular pattern recognition NOD (nuclear-binding and oligomerization domain)-like receptors [1,2]. The NLRP3 inflammasome is activated by a broad range of pathogen- and damage-associated molecular patterns, such as adenosine triphosphate (ATP), uric acid crystals and β-amyloid plaques, as well as environmental irritants [2]. Inflammasome activation, which consists of a sensor (NLRP3), an adaptor (apoptosis-associated spec-like protein, ASC; known as PYCARD), and an effector (caspase 1), culminates in the release of mature IL-1β and IL-18 forms through membrane pore formation by gasdermin D cleavage [1,2]. Investigations of novel signaling components that regulate inflammasome activation are crucial to prevent or treat human inflammatory diseases

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Conclusion