Neutrophilic Activity Biomarkers (Plasma Neutrophil Extracellular Traps and Calprotectin) in Established Patients with Rheumatoid Arthritis Receiving Biological or JAK Inhibitors: A Clinical and Ultrasonographic Study

Abstract

Introduction: This study assesses the accuracy of neutrophil activation markers, including neutrophil extracellular traps (NETs) and calprotectin, as biomarkers of disease activity in patients with established rheumatoid arthritis (RA). We also analyse the relationship between NETs and various types of therapies as well as their association with autoimmunity.

Methods: Observational cross-sectional study of patients with RA receiving treatment with biological disease-modifying antirheumatic drugs or Janus kinase inhibitors (JAK-inhibitors) for at least 3 months. Plasma calprotectin levels were measured using an enzyme-linked immunosorbent assay test kit and NETs by measuring their remnants in plasma (neutrophil elastase-DNA and histone-DNA complexes). We also assessed clinical disease activity, joint ultrasound findings and autoantibody status [reumatoid factor (RF), anti-citrullinated peptide/protein antibodies (ACPAs) and anti-carbamylated protein (anti-CarP)]. Associations between neutrophilic biomarkers and clinical or ultrasound scores were sought using correlation analysis. The discriminatory capacity of both neutrophilic biomarkers to detect ultrasound synovitis was analysed through receiver-operating characteristic (ROC) curves.

Results: One hundred fourteen patients were included. Two control groups were included to compare NET levels. The active control group consisted of 15 patients. The second control group consisted of 30 healthy subjects. Plasma NET levels did not correlate with clinical disease status, regardless of the clinic index analysed or the biological therapy administered. No significant correlation was observed between NET remnants and ultrasound synovitis. There was no correlation between plasma NET and autoantibodies. In contrast, plasma calprotectin positively correlated with clinical parameters (swollen joint count [SJC] rho = 0.49; P < 0.001, Clinical Disease Activity Index [CDAI] rho = 0.30; P < 0.001) and ultrasound parameters (rho > 0.50; P < 0.001). Notably, this correlation was stronger than that observed with acute phase reactants.

Conclusion: While NET formation induced by neutrophils may play a role in RA pathogenesis, our study raises questions about the utility of NET remnants in peripheral circulation as a biomarker for inflammatory activity. In contrast, this study strongly supports the usefulness of calprotectin as a biomarker of inflammatory activity in patients with RA.

Keywords: Autoimmunity; Calprotectin; Joint ultrasound; NETs; Rheumatoid arthritis.

Fig. 1

ROC curves of plasma calprotectin, hsCRP and ESR vs ultrasound synovitis according to type of treatment. a Main group (n = 114); b anti-Il6r (n = 56); c JAKi (n = 28); d anti-TNF (n = 30). Anti-Il6r monoclonal antibodies against IL-6 receptors; JAKi JAK inhibitors; anti-TNF tumour necrosis factor inhibitors; hsCRP high sensitivity C-reactive protein; ESR erythrocyte sedimentation rate; ROC receiver-operating characteristic

Fig. 2

ROC curves of NE-DNA complexes, hsCRP and ESR vs ultrasound synovitis according to type of treatment. a Main group (n = 114); b anti-Il6r (n = 56); c JAKi (n = 28); d anti-TNF (n = 30). Anti-Il6r monoclonal antibodies against interleukin-6 receptors; JAKi JAK inhibitors; anti-TNF tumour necrosis factor inhibitors; NE-DNA complex neutrophil elastase-DNA complexes (NETs); hsCRP high sensitivity C-reactive protein; ESR erythrocyte sedimentation rate; ROC receiver-operating characteristic

Fig. 3

ROC curves of H3-DNA complexes, hsCRP and ESR vs ultrasound synovitis according to type of treatment. a Main group (n = 114); b anti-Il6r (n = 56); c JAKi (n = 28); d anti-TNF (n = 30). Anti-Il6r monoclonal antibodies against interleukin-6 receptors; JAKi Janus kinase inhibitors; anti-TNF tumour necrosis factor inhibitors; H3-DNA complex histone-DNA complexes (NETs); hsCRP high-sensitivity C-reactive protein; ESR erythrocyte sedimentation rate; ROC receiver-operating characteristic