Neutrophil exhaustion and impaired functionality in psoriatic arthritis patients

Abstract

Background: Neutrophils (polymorphonuclear leukocytes, PMNs) are the most abundant subtype of white blood cells and are among the main actors in the inflammatory response. Psoriatic arthritis (PsA) is a chronic inflammatory disease affecting both the axial and peripheral joints. Typically associated with psoriasis, PsA can also affect multiple systems and organs, including the nails and entheses. Despite the involvement of PMNs in PsA, their specific role in the disease remains poorly understood. This study aimed to characterize the biological functions of PMNs and neutrophil-related mediators in PsA patients.

Materials and methods: 31 PsA patients and 22 healthy controls (HCs) were prospectively recruited. PMNs were isolated from peripheral blood and subjected to in vitro stimulation with lipopolysaccharide (LPS), N-Formylmethionyl-leucyl-phenylalanine (fMLP), tumor necrosis factor (TNF), phorbol 12-myristate 13-acetate (PMA), or control medium. Highly purified peripheral blood PMNs (>99%) were evaluated for activation status, reactive oxygen species (ROS) production, phagocytic activity, granular enzyme and neutrophil extracellular traps (NETs) release. Serum levels of matrix metalloproteinase-9 (MMP-9), myeloperoxidase (MPO), TNF, interleukin 23 (IL-23), and interleukin 17 (IL-17) were measured by ELISA. Serum Citrullinated histone H3 (CitH3) was measured as a NET biomarker.

Results: Activated PMNs from PsA patients displayed reduced activation, decreased ROS production, and impaired phagocytic activity upon stimulation with TNF, compared to HCs. PMNs from PsA patients also displayed reduced granular enzyme (MPO) and NET release. Serum analyses revealed elevated levels of MMP-9, MPO, TNF, IL-23, IL-17, and CitH3 in PsA patients compared to HCs. Serum CitH3 levels positively correlated with MPO and TNF concentrations, and IL-17 concentrations were positively correlated with IL-23 levels in PsA patients. These findings indicate that PMNs from PsA patients show reduced in vitro activation and function, and an increased presence of neutrophil-derived mediators (MMP-9, MPO, TNF, IL-23, IL-17, and CitH3) in their serum.

Conclusions: Taken together, our findings suggest that PMNs from PsA patients exhibit an "exhausted" phenotype, highlighting their plasticity and multifaceted roles in PsA pathophysiology.

Keywords: inflammation; innate immunity; neutrophil extracellular traps; neutrophils; psoriatic arthritis.

Figure 1

PMNs from peripheral blood of PsA patients (PsA Pts, red borders) and HCs (HCs, black borders), freshly isolated (A–C) and/or stimulated with TNF (10 ng/mL) (D–F) for 60 min at +37 °C. Cells were stained for the neutrophil activation markers CD62L (A, D, J), CD66b (B, E, K), and CD11b (C, F, L), then subjected to cytofluorimetric analysis. Each data point in panels (A–F) represents one patient (PsA Pts) or one healthy control (HC). Mean fluorescence intensity (MFI) of CD62L, CD66b, and CD11b was calculated and normalized to non-stimulated cells (control medium). Results are presented as raw MFI (A–C) or percentage increase versus control (D–F) (mean ± SEM); *p < 0.05; ***p < 0.005, analyzed by Student’s-t-test or Mann-Whitney U test, depending on the distribution of the data. (G–I) Representative flow cytometric panels gated on live single cells, depicting forward (FSC) and side scatter (SSC) of EasySep-purified untouched PMNs (G, H). Cells positive for VioBlue include both dead cells and CCR3 cells (eosinophils), were excluded using a negative gate (I). Representative histograms illustrating MFI and cell count for CD62L (J), CD66b (K), and CD11b (L) on peripheral blood PMNs from HCs and PsA patients (PsA Pts), both non-stimulated (CTRL) and stimulated with TNF. MFI, Mean fluorescence intensity; FMO, Fluorescence minus one.

Figure 2

PMNs from peripheral blood of PsA patients (red lines) and HCs (black lines) were incubated with 2’,7’-dichlorodihydrofluorescein diacetate (H₂DCFDA, 10 µM), washed, then stimulated with TNF (10 ng/mL) or control medium. Immediately following stimulation, PMNs were analyzed with a multimode microplate reader (EnSpire Multimode Plate reader, PerkinElmer), and DCF fluorescence was measured for 30 min at 2 min intervals. The results are expressed as Relative Fluorescence Unit (RFU) and percentage increase versus time 0 (mean ± SEM); *p < 0.05; **p < 0.01, analyzed by two-way ANOVA and Bonferroni post-test (A). PMNs from peripheral blood of PsA patients (PsA Pts, red border) and HCs (HCs, black border) were stimulated with TNF (10 ng/mL) for 60 min at +37 °C, then incubated with pHrodo^TM Green E. coli BioParticles® conjugate (100 µg/mL) for 60 min at +37 °C, with or without cytochalasin B (as inhibitor of phagocytosis, 10 µM), followed by flow cytometric analysis. Each point represents one patient (PsA Pts) or one healthy control (HCs). MFI of cells positive for pHrodo^TM was calculated and normalized to the unstimulated cells (control medium). Results were expressed as percentage increase versus control (mean ± SEM); *p < 0.05, analyzed by Student’s t-test (B). Representative histograms illustrating MFI and cell count for pHrodo assay data of peripheral blood PMNs from HCs and PsA patients (PsA Pts), in the presence or absence of cytochalasin B, after stimulation with TNF (C). MFI, Mean fluorescence intensity; FMO, Fluorescence minus one.

Figure 3

PMNs from peripheral blood of PsA patients (PsA Pts, red borders) and HCs (HCs, black borders) were cultured for 60 min at +37 °C in the presence or absence of TNF (10 ng/mL). The extracellular levels of dsDNA (A), MPO-DNA complexes (B), MPO (C), and MMP-9 (D) were measured by Quant-iT^TM PicoGreen^TM dsDNA Assay Kit or by ELISA, respectively. Each point in graphs (A–D) represents one patient (PsA Pts) or one healthy control (HCs). The extracellular levels of dsDNA, MPO-DNA complexes, MPO, and MMP-9 were calculated and normalized to unstimulated cells (control medium; mean ± SEM); *p < 0.05; **p < 0.01; p for MMP-9 = 0.06, analyzed by Student’s-t-test or Mann-Whitney U test, depending on the distribution of the data.

Figure 4

Serum concentrations of MMP-9 (A), MPO (B), TNF (C), IL-23 (D), and IL-17 (E) in PsA patients (PsA PTs, red borders) and HCs (HCs, black borders) were measured by ELISA. Each point in graphs (A–E) represents one patient (PsA Pts) or one healthy control (HCs). Results were expressed as mean ± SEM; *p < 0.05; **p < 0.01; **** p < 0.001. Student’s t-test or Mann-Whitney U test was employed according to the distribution of the data.

Figure 5

Serum concentrations of CitH3 in PsA patients (PsA PTs, red borders) and HCs (HCs, black borders) were measured by Citrullinated Histone H3 (clone11D3) ELISA. Each point represents one patient (PsA Pts) or one healthy control (HCs). Results were expressed as mean ± SEM; *** p < 0.005, analyzed by Student’s t-test or Mann-Whitney U test, depending on the distribution of the data (A). Multiple linear regression analysis between serum concentrations of CitH3, MPO (red line), and TNF (green line) in PsA patients, with CitH3 as the dependent variable. r² = 0.46; versus MPO p = 0.0001; versus TNF p = 0.0007 (B). ROC curve analysis of serum concentrations of CitH3 to evaluate the accuracy of CitH3 as a diagnostic biomarker for PsA. Area Under the Curve (AUC) = 0.78; Cut-Off = 1.65; Sensitivity = 0.90; Specificity = 0.64; CI = 2.51–7.07 (C).