Role of the annexin A1 anti-inflammatory protein in the regulation of NLRP3 inflammassome in isolated neutrophils

Abstract

Inflammasomes are cytoplasmic platforms that have in common a nucleotide-binding oligomerization domain (NOD) in their structure and are activated by several pathogens, as well as DAMPs (damage-associated molecular patterns) and environmental irritants. NLRP3 is the most studied member of the NLRP receptor family and forms a multiprotein inflammasome complex with the adaptor protein ASC and procaspase-1. This multiprotein is able to recognize several microorganisms through activation of cell surface receptors, such as TLR-2 and TLR-4, lectin (dectin-1) and NOD-type receptors, triggering a pro-inflammatory response via nuclear factor kappa B (NFºB) and/or via NLRP3, with production of cytokines (IL-1±, IL-1² and TNF-±) and chemokines. The release of pro-inflammatory mediators induces the influx of immune cells and consequent inflammatory response, which may be protective or deleterious, depending on the degree of immune response and the substances involved. In this context, the annexin A1 (ANXA1) protein plays a significant role in the control of the inflammatory cascade, especially related to the regulation of IL-1², but its relationship with the NLRP3 pathway has not been established. Aim: To evaluate in in vitro assays the role of the ANXA1 anti-inflammatory protein in the regulation of the NLRP3 inflammasome. Methods: C57BL/6 wild-type (WT) and ANXA1 knockout mice (ANXA1-/-) will receive i.p. of 0.3% carrageenan and, after 3 hours, the peritoneal lavage (neutrophil population > 95%) will be collected. WT and ANXA1-/- neutrophils will be stimulated with lipopolysaccharide (LPS), followed by NLRP3 agonists, nigericin or ATP. In addition, to check the exogenous effect of ANXA1, neutrophils will be pretreated with the mimetic peptide of ANXA1, Ac2-26, and then given the NLRP3 agonists. The following analyzes will be performed: western blotting and Elisa to measure the release of IL-1² and caspase-1; immunofluorescence to detect NLRP3, ASC and ANXA1 co-localization; release of lactate dehydrogenase (LDH) to detect cell viability. The results will contribute to a better understanding of the role of ANXA1 in the inflammatory response, as well as its relationship with the NLRP3 pathway, indicating possible therapeutic targets.