OP0017 Single-cell immune profiling of neutrophils from patients with anti-neutrophil cytoplasmic antibody-associated vasculitis

Abstract

Background:

Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) manifests as a heterogeneous autoimmune pathology characterized by small vessel injury and subsequent multisystem organ involvement [1]. While the aberrant activation of neutrophils is recognized as a pivotal element in the pathogenesis of AAV, the heterogeneity and functional states of neutrophils remain poorly understood, limiting the development of therapies specifically targeting neutrophils. Additionally, there is a pressing need for reliable biomarkers capable of predicting therapeutic responses and monitoring disease progression.

Objectives:

This study aimed to characterize the transcriptomic profiles of neutrophils and delineate their functional perturbations at the single-cell level in patients with AAV. Additionally, it sought to identify specific neutrophil subgroups as potential biomarkers for AAV through in vitro analyses.

Methods:

Peripheral blood samples were collected from three patients with active granulomatosis with polyangiitis and three healthy controls, followed by single-cell RNA sequencing (scRNA-seq) to investigate the heterogeneity and functional characteristics of neutrophils. In a subsequent validation cohort comprising eighteen patients with AAV and eighteen healthy donors, flow cytometry and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to confirm the presence of specific neutrophil subtypes and to explore their potential clinical significance.

Results:

The entirety of the whole blood cells was divided into several principal subsets, including neutrophils, T/NK cells, B cells, myeloid cells and basophils. Neutrophils were further subdivided into seven distinct populations (Figure 1A), each exhibiting marked differences in gene expression profiles and functional characteristics (Figures 1C and 1D). Among these, Neu_CXCL8 subtypes were the predominant neutrophils in healthy individuals, specific expressing immunosuppression-related molecules, including ZFP36, NFKBIA, GABARAP and DUSP6. Neu_IFIT1 subtypes were primarily involved in interferon responses, whereas Neu_MMP8 subtypes played a key role in antimicrobial activity. Pseudotime analysis revealed that the PLA2G7_Neu subtypes were predominantly located at the terminal branches of the trajectory tree, indicative of a mature state (Figure 1B). Consistent with findings from single-cell analyses, flow cytometric assays and qRT-PCR experiments confirmed an elevated proportion of PLA2G7+ neutrophils in the peripheral blood of AAV patients compared to healthy controls (Figures 2A, 2D and 2E). The relative RNA levels of PLA2G7 in neutrophils were associated with disease exacerbation or relapse (Figure 2B), as well as renal involvement (Figure 2C). Further correlational analysis demonstrated a significant association between PLA2G7+ neutrophil subsets and both Birmingham Vasculitis Activity Score and C-reactive protein levels (Figure 2F), underscoring their potential clinical relevance as a biomarker in AAV. Figure 1Heterogeneity of neutrophils in AAV patients and healthy controls. (A) UMAP plot showing seven clusters of neutrophils. (B) Pseudotime trajectory of the indicated neutrophil clusters, with each point corresponding to a single cell. (C) Bubble heatmap showing the expression levels of selected signature genes in neutrophil subtypes. (D) Bubble heatmap showing gene enrichment analysis based on highly expressed genes in neutrophil subtypes. Figure 2(A) qRT-PCR analysis of PLA2G7 levels in neutrophils from the peripheral blood of AAV patients and HCs, and (B and C) grouping analysis. (D) Representative flow cytometry plot and (E) summary of the proportions of PLA2G7_Neu subtypes in the periphery from AAV patients and HCs. (F) The correlational analysis between PLA2G7 levels in neutrophils and both BVAS and CRP levels. *P < 0.05; **P < 0.01; ***P < 0.001.

Conclusion:

We initially employed scRNA-seq to characterize the heterogeneity of neutrophils and evaluate their potential as prognostic indicators of disease progression in AAV patients. These novel findings enhance our understanding of the dynamic functional roles of neutrophils in AAV and may facilitate the development of therapeutic strategies aimed at selectively targeting disease-specific neutrophil subsets.

REFERENCES:

[1] Kitching AR, Anders HJ, Basu N, Brouwer E, Gordon J, Jayne DR, et al. ANCA-associated vasculitis. Nature reviews Disease primers. 2020 Aug 27; 6(1):71.

Acknowledgements:

NIL.

Disclosure of Interests:

None declared. © The Authors 2025. This abstract is an open access article published in Annals of Rheumatic Diseases under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). Neither EULAR nor the publisher make any representation as to the accuracy of the content. The authors are solely responsible for the content in their abstract including accuracy of the facts, statements, results, conclusion, citing resources etc.