NLRP3 inflammasome activity and pyroptosis are involved in CD206+ macrophage activation by MPO anti-neutrophil cytoplasmic antibodies

Abstract

Macrophages are key players in the pathology of anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). Existing studies and our previous studies have documented the role of CD206-positive M2 macrophages in the inflammatory process of AAV. Inflammasome activation is a critical pathway through which macrophages release inflammatory factors. In this study, we investigate the role of the inflammasome in macrophages in AAV and explore the role of CD206 in this process. We recruit newly diagnosed AAV patients and disease controls from our department. The expression and localization of the NOD-like receptor family, pyrin domain containing 3 (NLRP3) and CD206 in the kidney are determined via immunofluorescence experiments. Myeloperoxidase (MPO)-ANCA immunoglobulin G (MPO-ANCA IgG) is purified from new-onset AAV patients with MPO-ANCA and used to treat lipopolysaccharide (LPS)-primed macrophages in vitro. Our findings reveal that NLRP3 expression is significantly elevated in the kidneys of active AAV patients, accompanied by increased cleaved caspase-1 and N-terminal gasdermin-D (GSDMD) levels in peripheral blood mononuclear cells (PBMCs). In vitro, MPO-ANCA IgG induces NLRP3 inflammasome activation and interleukin (IL)-1β production in macrophages, which is associated with increased MPO expression and JNK signaling pathway activation. Immunofluorescence analysis demonstrates partial colocalization of CD206 and NLRP3 in AAV kidneys. Furthermore, silencing of MRC1 gene, which encodes CD206, reduces inflammasome activation induced by MPO-ANCA IgG. In conclusion, our study provides evidence that MPO-ANCA IgG contributes to NLRP3 inflammasome activation and macrophage pyroptosis, with CD206 playing a critical role in this process. These findings elucidate the mechanisms underlying inflammation in AAV and suggest potential therapeutic targets.

1. Introduction

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a group of life-threatening systemic autoimmune diseases with multiorgan involvement characterized by necrotizing small vessel vasculitis associated with myeloperoxidase (MPO) or proteinase 3 (PR3) ANCA [1]. Among the clinical manifestations of AAV, pauci-immune glomerulonephritis is the most common and severe manifestation of the disease and has a poor prognosis [2–5].

Although the outcome of AAV has improved with standard therapy, which includes high-dose glucocorticoids and immunosuppressants, the mortality rate among AAV patients remains high [6,7]. Emerging evidence suggests that monocyte and macrophage activation occur during AAV onset, contributing significantly to acute inflammatory injury and a mid- to long-term fibrotic response in affected organs [8]. Animal studies have shown that monocyte depletion can partially alleviate AAV-related kidney damage [9], but this approach has significant limitations in human applications. The development of more precise therapeutic strategies targeting monocytes or macrophages requires a deeper understanding of their alterations and mechanisms during the progression of AAV. In our previous study [10], we demonstrated that the peripheral blood mononuclear cells (PBMCs) and kidney-infiltrated macrophages in AAV predominantly exhibit activation and polarization toward CD206-positive cells and that this phenotype shift is correlated with disease activity and is implicated in kidney injury. In addition, ANCA IgG promotes CD206 expression in mouse bone marrow-derived macrophages (BMDMs) by increasing the protein level of MPO. However, the role of this class of cells in AAV disease development requires further investigation.

Inflammation is the principal contributor to tissue damage in AAV [11]. The inflammasome is a multiprotein complex that recognizes pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) [12]. Recent studies have revealed that inflammasomes, especially the NOD-like receptor (NLR) family pyrin domain-containing protein 3 (NLRP3) inflammasome, are central instigators of the inflammatory response in innate immune cells, including macrophages and their subsets. However, their role in AAV remains to be fully elucidated.