gms | German Medical Science

Introduction: Neutrophils are key protagonists in inflammatory arthritis, but their phenotypic and functional heterogeneity remains poorly understood.

Methods: We analyzed neutrophils from patients with inflammatory arthritis and healthy controls directly after isolation and after aging in vitro for two days with or without GM-CSF and IFN-γ. We used flow cytometry to quantify protein expression, reactive oxygen species production and phagocytosis. To quantify NETosis across different neutrophil phenotypes, we developed a new method based on imaging flow cytometry and deep learning. A cloud-based convolutional neural network was trained and validated, allowing us to differentiate between normal and pro-NETotic neutrophils.

Results: Neutrophil aging led to robust upregulation of CXCR4 and downregulation of CD62L on the surface of healthy and arthritic blood neutrophils, and CXCR4 upregulation was inhibited by concomitant activation with GM-CSF and IFN-γ.

On average, CXCR4+ neutrophils aged in vitro displayed lower expression of the activation and IFN-γ-driven markers HLA-DR, CD64, CD32, PD-L1 and ICAM-1 compared to CXCR4- cells of the same group, suggesting that aging and activation act as distinct but intertwined drivers of different neutrophil states.

Single cell analysis of CXCR4+ neutrophils aged in vitro illustrated expression gradients of the markers CD64, ICAM-1, HLA-DR, PD-L1, CD62L, CD16 and IFNgR. Concordantly, a small group of CXCR4+ neutrophils was characterized by high CD64 expression, indicating the combination of aging and activation in the same cell. This cell state appeared more frequently in the blood of patients with inflammatory arthritis compared to healthy controls, although the rarity of these cells impeded definitive conclusions.

Functionally, CXCR4+ neutrophils had a strongly reduced capacity for phagocytosis and ROS production, indicating exhaustion. This finding was robust to activation with different cytokines and correlated with CXCR4 expression abundance. Imaging flow cytometry with a custom deep learning-based classifier revealed that CXCR4+ neutrophils were in a pro-NETotic state characterized by nuclear decondensation.

Conclusion: CXCR4+ neutrophils represent a heterogenous population of neutrophils. In vitro, CXCR4+ neutrophils display an exhausted, pro-NETotic phenotype with reduced ROS production and impaired ability for phagocytosis. These findings provide new functional hints for a potentially pathogenic role of CXCR4+ pro-NETotic neutrophils in inflammatory arthritis.