IL-17A expression is localised to both mononuclear and polymorphonuclear synovial cell infiltrates

Abstract

Introduction: This study examines the expression of IL-17A-secreting cells within the inflamed synovium and the relationship to in vivo joint hypoxia measurements.

Methods: IL-17A expression was quantified in synovial tissue (ST), serum and synovial fluid (SF) by immunohistochemistry and MSD-plex assays. IL-6 SF and serum levels were measured by MSD-plex assays. Dual immunofluorescence for IL-17A was quantified in ST CD15+ cells (neutrophils), Tryptase+ (mast cells) and CD4+ (T cells). Synovial tissue oxygen (tpO(2)) levels were measured under direct visualisation at arthroscopy. Synovial infiltration was assessed using immunohistochemistry for cell specific markers. Peripheral blood mononuclear and polymorphonuclear cells were isolated and exposed to normoxic or 3% hypoxic conditions. IL-17A and IL-6 were quantified as above in culture supernatants.

Results: IL-17A expression was localised to mononuclear and polymorphonuclear (PMN) cells in inflamed ST. Dual immunoflourescent staining co-localised IL-17A expression with CD15+ neutrophils Tryptase+ mast cells and CD4+T cells. % IL-17A positivity was highest on CD15+ neutrophils, followed by mast cells and then CD4+T-cells. The number of IL-17A-secreting PMN cells significantly correlated with sublining CD68 expression (r = 0.618, p<0.01). IL-17A SF levels correlated with IL-6 SF levels (r = 0.675, p<0.01). Patients categorized according to tp0(2)< or >20 mmHg, showed those with low tp0(2)<20 mmHg had significantly higher IL-17A+ mononuclear cells with no difference observed for PMNs. Exposure of mononuclear and polymorphonuclear cells to 3% hypoxia, significantly induced IL-6 in mononuclear cells, but had no effect on IL-17A expression in mononuclear and polymorphonuclear cells.

Conclusion: This study demonstrates IL-17A expression is localised to several immune cell subtypes within the inflamed synovial tissue, further supporting the concept that IL-17A is a key mediator in inflammatory arthritis. The association of hypoxia with Il-17A expression appears to be indirect, probably through hypoxia-induced pro-inflammatory pathways and leukocyte influx within the joint microenvironment.

Figure 1. IL-17A expression is localized to the inflamed joint.

IL-17A protein levels were measured by MSD Assay in paired serum/synovial fluid samples (n = 20) (A). Synovial fluid levels were significantly higher than serum levels. Values expressed as median ± range, *p<0.01, significance level. Immunohistochemistry was performed in synovial tissue sections from patients with inflammatory arthritis (n = 19). (B).The number of mononuclear cells (white bars) staining for IL-17A was higher than the number of IL-17A positive polymorphonuclear cells (grey bars). Results are expressed as the number of IL-17A positive cells per high powered field (HPF). (C) Representative images of IL-17A expression in RA (i) vs. PsA (ii) and mononuclear IL-17A expression (iii) and polymorphonuclear IL-17A expression (iv).

Figure 2. Localisation of IL-17A to neutrophils and mast cells within the inflamed synovium.

Representative images of RA synovial tissue section stained with antibodies against tryptase, CD15 and IL-17A. Merged images indicating co-localisation – yellow.

Figure 3. Increased systemic expression of IL-17A at low pO2.

(A) Patient synovial fluid samples (n = 22) were assessed by MSD assay for the expression of IL-17A. Cytokine levels were then grouped according to patient tpO₂ levels or >20mmHg. No significant difference in synovial fluid levels was observed between the two groups. (B) Synovial tissue pO₂ levels (n = 18) were also examined in relation to the expression of IL-17A positive mononuclear (white bars) and polymorphonuclear cells (grey bars). Patients with tpO₂ levels <20mmHg (n = 9) had significantly more IL-17A positive mononuclear cells than those with tpO₂ levels >20mmHg (n = 9) (p<0.05). Patients with tpO₂ levels <20mmHg (n = 9) also had a higher number of IL-17A positive polymorphonuclear cells than those with tpO₂ levels >20mmHg (n = 9). This difference was not statistically different. (C) Representative images of IL-17A expression on mononuclear cells from a patient with high tpO2 levels vs a patient with low tpO2 levels are shown.

Figure 4. The effect of 3% hypoxia on IL-17A and IL-6 in vitro.

Peripheral blood mononuclear cells (n = 12) and neutrophils (n = 6) were cultured for 24hrs under 3% hypoxic conditions and normoxia. Hypoxia had no effect on IL-17A expression (A), but significantly induced IL-6 expression in mononuclear cells (B). Hypoxia had no effect on IL-17A(C) or IL-6 (D) in neutrophils. Data expressed as the mean ± SEM. P<0.05 significantly different from normoxic conditions.