Neutrophils in Rheumatoid Arthritis Synovium: Implications on Disease Activity and Inflammation State

Abstract

Background: Rheumatoid arthritis (RA) is characterized by chronic synovial inflammation driven by immune cell infiltration. While neutrophils have traditionally been associated with acute inflammation, emerging evidence suggests their significant role in chronic RA synovitis. Synovial pathology reports from our center reveal lymphocyte-predominant infiltration in most RA cases, with synovial neutrophils (SNs) observed in only 30% of patients. This finding suggests that neutrophil involvement in RA pathogenesis is not universal but subtype-specific, potentially linked to distinct clinical phenotypes.

Methods: We performed a retrospective analysis of synovial pathology and clinical data from 55 RA patients collected during 2023. Using both Hematoxylin-Eosin (H&E) staining and single-cell RNA sequencing, we analyzed the synovial tissue samples. Based on neutrophil counts, patients were classified into two groups: neutrophil-absent (<10 neutrophils) and neutrophil-present (≥10 neutrophils).

Results: In this cohort of 55 RA patients, the synovial neutrophil (SN) group demonstrated significantly elevated disease activity markers, including Disease Activity Score in 28 joints based on C-reactive protein (DAS28-CRP), swollen joint count (SJC28), Visual Analog Scale (VAS) pain scores, and tender joint count (TJC28) (p < 0.05 for all parameters). Synovial inflammatory infiltration and neovascularization were markedly increased in the SNs group (P < 0.05). Patients with SNs maintained higher disease activity and showed poorer therapeutic responses despite treatment with methotrexate and targeted biologics (TNF inhibitors, IL-6 inhibitors, or JAK inhibitors). Analysis revealed a positive correlation between lymphocyte and neutrophil counts, while multivariate analysis identified DAS28-CRP, synovial inflammation, and CD3+/CD68+ cell counts as predictors of SN infiltration. Single-cell RNA sequencing confirmed their significant presence in synovial tissue, supporting neutrophils' role in refractory disease.

Conclusion: Elevated neutrophil presence in RA synovium correlates with heightened clinical disease activity and an exacerbated inflammatory state. These findings underscore the potential significance of SNs in the pathology of RA.

Keywords: disease activity; immune cell infiltration; rheumatoid arthritis; synovial biopsy; synovial neutrophils.

Figure 1

Synovial biopsy device and procedure. (A) Key components: outer cannula (1.7mm diameter, 50mm length), sampling needle, puncture needle, and stylet. (B) Procedural steps: insertion of combined cannula-puncture needle into joint cavity; removal of puncture needle followed by sampling needle insertion; activation of firing mechanism; connection of sampling needle to negative pressure system facilitating synovial tissue acquisition.

Figure 2

Synovial pathology scoring in RA patients. X-axis represents patient distribution across scoring grades. Four panels display neovascularization, inflammatory infiltration, stromal activity, and synovial hyperplasia (scored 0–3).

Figure 3

Comparison of synovial immune cell infiltration in RA. Box plots illustrate lymphocyte (p=0.007), macrophage (p=0.010), and plasma cell (p=0.219) counts between neutrophil-negative and neutrophil-positive groups.

Figure 4

Correlation heatmap of clinical and synovial features in rheumatoid arthritis. Color intensity reflects both the strength and direction of relationships, with positive correlations depicted in red and negative correlations in blue. Analysis revealed significant positive correlations between synovial neutrophils (SNs) and multiple factors, including VAS pain scores, DAS28-CRP disease activity, synovial inflammatory infiltration, neovascularization, sublining lymphocytes, and macrophages. Synovial inflammatory infiltration demonstrated strong positive associations with disease activity indicators (VAS and DAS28-CRP), neovascularization, and inflammatory cell populations (lymphocytes and macrophages), indicating that elevated synovial inflammation corresponds with heightened disease activity and inflammatory response.

Figure 5

Relationship between lymphocyte count and neutrophil infiltration probability. Graph demonstrates nonlinear increase in neutrophil infiltration odds with rising lymphocyte counts (nonlinearity test p<0.05). Using 200 lymphocytes as reference point, counts exceeding 900 significantly increase neutrophil infiltration likelihood. Red line indicates mean odds ratio with pink area showing 95% confidence interval.

Figure 6

(A) The UMAP plot shows the cell types present in the tissue. (B) The violin plot displays the marker genes used for identifying these types. (C) The bar plot illustrates the proportion of cell numbers for each cell type in ACPA-positive samples.

Figure 7

(A) The UMAP plot shows two neutrophil subpopulations (Neu_CXCR2 and Neu_IDO1) in the tissue. (B) The dot plot displays differential gene expression profiles characterizing these neutrophil populations. (C) The bar plot illustrates the proportion of Neu_CXCR2 and Neu_IDO1 in Acpa-P positive samples.