Recombinant Human Proteoglycan 4 Regulates Phagocytic Activation of Monocytes and Reduces IL-1β Secretion by Urate Crystal Stimulated Gout PBMCs

Abstract

Objectives: To compare phagocytic activities of monocytes in peripheral blood mononuclear cells (PBMCs) from acute gout patients and normal subjects, examine monosodium urate monohydrate (MSU) crystal-induced IL-1β secretion ± recombinant human proteoglycan 4 (rhPRG4) or interleukin-1 receptor antagonist (IL-1RA), and study the anti-inflammatory mechanism of rhPRG4 in MSU stimulated monocytes.

Methods: Acute gout PBMCs were collected from patients in the Emergency Department and normal PBMCs were obtained from a commercial source. Monocytes in PBMCs were identified by flow cytometry. PBMCs were primed with Pam3CSK4 (1μg/mL) for 24h and phagocytic activation of monocytes was determined using fluorescently labeled latex beads. MSU (200μg/mL) stimulated IL-1β secretion was determined by ELISA. Reactive oxygen species (ROS) generation in monocytes was determined fluorometrically. PBMCs were incubated with IL-1RA (250ng/mL) or rhPRG4 (200μg/mL) and bead phagocytosis by monocytes was determined. THP-1 monocytes were treated with MSU crystals ± rhPRG4 and cellular levels of NLRP3 protein, pro-IL-1β, secreted IL-1β, and activities of caspase-1 and protein phosphatase-2A (PP2A) were quantified. The peritoneal influx of inflammatory and anti-inflammatory monocytes and neutrophils in Prg4 deficient mice was studied and the impact of rhPRG4 on immune cell trafficking was assessed.

Results: Enhanced phagocytic activation of gout monocytes under basal conditions (p<0.001) was associated with ROS generation and MSU stimulated IL-1β secretion (p<0.05). rhPRG4 reduced bead phagocytosis by normal and gout monocytes compared to IL-1RA and both treatments were efficacious in reducing IL-1β secretion (p<0.05). rhPRG4 reduced pro-IL-1β content, caspase-1 activity, conversion of pro-IL-1β to mature IL-1β and restored PP2A activity in monocytes (p<0.05). PP2A inhibition reversed rhPRG4's effects on pro-IL-1β and mature IL-1β in MSU stimulated monocytes. Neutrophils accumulated in peritoneal cavities of Prg4 deficient mice (p<0.01) and rhPRG4 treatment reduced neutrophil accumulation and enhanced anti-inflammatory monocyte influx (p<0.05).

Conclusions: MSU phagocytosis was higher in gout monocytes resulting in higher ROS and IL-1β secretion. rhPRG4 reduced monocyte phagocytic activation to a greater extent than IL-1RA and reduced IL-1β secretion. The anti-inflammatory activity of rhPRG4 in monocytes is partially mediated by PP2A, and in vivo, PRG4 plays a role in regulating the trafficking of immune cells into the site of a gout flare.

Keywords: PRG4; acute gout flare; interleukin-1 receptor antagonist (IL-1 ra); monocytes; urate crystals.

Figure 1

Analysis of the phagocytic activity of monocytes in peripheral blood mononuclear cells (PBMCs) of normal subjects (n=3) and gout patients (n=7) and its relationship to interleukin-1 beta (IL-1β) release in response to priming with Pam3CSK4, a toll-like receptor 2 ligand, and treatment with monosodium urate monohydrate (MSU) crystals. To assess phagocytic activation, PBMCs were treated with Pam3CSK4 (1μg/mL) for 24h followed by co-incubation with FITC-labeled rabbit IgG-coated latex beads and determination of percent bead-positive monocytes. Alternatively, PBMCs were incubated with MSU crystals (200μg/mL) and secreted IL-1β levels were determined by ELISA at 6h. Analysis of FITC-bead positive monocytes was performed using 2-way ANOVA followed by Tukey’s post-hoc test, and analysis of IL-1β levels was performed by multiple t-tests followed by a post-hoc false discovery rate analysis using two stage step-up approach. ns, non-significant; *p < 0.05; ***p < 0.001. (A) Flow cytometry gating strategy for monocytes in PBMCs. Identification of monocytes was conducted using the expected forward and side scatter (FSC-A and SSC-A) ranges and single cells were gated using FSC-A and FSC-H. Viable cells were identified using Zombie Violet viability dye and monocytes were confirmed using CD14 and CD45 surface markers. Subsequently, bead positive monocytes were gated based on thresholds established using fluorescence minus one control. (B) Representative flow cytometry histograms depicting a higher percentage of bead-positive monocytes in a gout patient compared to a normal subject. (C) Monocytes of gout patients had higher basal phagocytic activities compared to normal subjects. TLR2 ligand priming increased phagocytic activities of normal subjects’ monocytes but not gout patients and phagocytic activities in primed samples were not different between normal and gout subjects. (D) A combination of TLR2 ligand priming and MSU crystal challenge resulted in higher secreted IL-1β levels from gout patients’ PBMCs.

Figure 2

Dose-response of interleukin-1 receptor antagonist (IL-1RA) and recombinant human proteoglycan 4 (rhPRG4) in Pam3CSK4 (P), a toll-like receptor 2 ligand, and monosodium urate monohydrate (M) crystal-stimulated peripheral blood mononuclear cells (PBMCs) of normal subjects and efficacy of IL-1RA and rhPRG4 in modulating phagocytic activation of monocytes of normal subjects (n=3) and gout patients (n=7 to 12) and secretion of mature interleukin-1 beta (IL-1β) by urate crystal-stimulated gout PBMCs. To assess phagocytic activation, PBMCs were treated with Pam3CSK4 (1μg/mL) for 24h followed by co-incubation with FITC-labeled rabbit IgG-coated latex beads and determination of percent bead-positive monocytes, using the gating strategy shown in Figure 1A . Alternatively, PBMCs were incubated with urate crystals (200μg/mL) and secreted IL-1β levels were determined by ELISA. Gout PBMCs were categorized, based on urate crystal-induced IL-1β secretion, as either high IL-1β secreting (IL-1β concentrations > 500pg/mL) (n=6) or low IL-1β secreting (IL-1β concentrations < 500pg/mL) (n=6) and the differential efficacies of IL-1RA and rhPRG4 on both populations was investigated. Statistical analyses included one and two-way ANOVAs followed by Tukey’s post-hoc test. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (A) IL-1RA (250ng/mL and higher) reduced IL-1β secretion by PBMCs of normal subjects. (B) rhPRG4 (100&200μg/mL) reduced IL-1β secretion by PBMCs of normal subjects. (C) IL-1RA (250ng/mL) and rhPRG4 (200μg/mL) reduced phagocytic activation of monocytes in PBMCs of normal subjects. rhPRG4 treatment reduced bead phagocytosis by normal monocytes compared to IL-1RA. (D) rhPRG4 (200μg/mL) reduced bead phagocytosis by gout monocytes, while IL-1RA (250ng/mL) did not alter fluorescent bead uptake by the same monocytes. (E) IL-1RA (250ng/mL) and rhPRG4 (200μg/mL) reduced IL-1β secretion by gout PBMCs by similar magnitudes. PBMCs from patients receiving colchicine (n=5) are highlighted in red. 4 out of 5 samples are classified as low-IL-1β secreting gout PBMCs. (F) IL-1RA (250ng/mL) and rhPRG4 (200μg/mL) reduced IL-1β secretion from high-IL-1β secreting gout PBMCs but not from low-IL-1β secreting PBMCs.

Figure 3

Analysis of monosodium urate monohydrate (MSU) crystal phagocytosis and its relationship to reactive oxygen species (ROS) generation in monocytes of peripheral blood mononuclear cells (PBMCs) of normal subjects (n=3) and gout patients (n=7) and secretion of interleukin-1 beta (IL-1β). PBMCs were primed with Pam3CSK4 (1μg/mL) for 24h followed by MSU crystals (200μg/mL) and MSU crystal phagocytosis was determined by assessing the change in monocytes’ side scatter (SSC-A) profile and mature IL-1β levels were determined by ELISA at 2h. ROS generation in monocytes was determined using DCFDA/H2DCFDA probe, and geometric means of fluorescence intensities (FI) of Alexa Fluor 488-positive monocytes were calculated and compared across groups. To further delineate the role of ROS in IL-1β secretion, PBMCs of normal subjects (n=4) were primed with Pam3CSK4 (1μg/mL) for 24h followed by MSU crystals (200μg/mL) ± N-acetylcysteine (NAC) (20 mM). Statistical analyses included Student’s t-test and one-way ANOVA followed by Tukey’s post-hoc test. ns, non-significant; *p < 0.05; ****p < 0.0001. (A) Representative flow cytometry histograms showing increased monocytes with SSC-A values above a pre-determined threshold indicative of MSU crystal phagocytosis. (B) Phagocytosis of MSU crystals by gout monocytes was higher than normal monocytes. (C) IL-1β secretion from MSU-challenged gout PBMCs was higher than normal PBMCs. (D) Representative flow cytometry histograms of DCFDA/H2DCFDA stained normal monocytes at baseline and following MSU crystal incubation. Monocytes with Alexa Fluor 488 FI values above 1.0 x 10³ were considered positive and the FI geometric mean of this population was determined. Gating of monocytes was performed as shown in Figure 1A . (E) ROS generation was higher in gout monocytes compared to normal monocytes. (F) Representative flow cytometry histogram showing an increase in ROS generation in monocytes as a result of MSU crystal exposure compared to control, which was attenuated with NAC treatment. (G) NAC treatment reduced ROS generation in monocytes of MSU crystal challenged PBMCs. (H) NAC treatment reduced IL-1β release by MSU challenged PBMCs.

Figure 4

Impact of recombinant human proteoglycan-4 (rhPRG4) or interleukin-1 receptor antagonist (IL-1RA) treatments on phagocytic activation of human THP-1 monocytes, intracellular pro interleukin-1 beta (pro-IL-1β), secreted mature IL-1β and activation of NLRP3 in response to monosodium urate monohydrate (MSU) crystal challenge and role of protein phosphatase-2A (PP2A) in mediating rhPRG4’s effect. Reactive oxygen species (ROS) generation in THP-1 monocytes ± rhPRG4 was determined using the DCFDA/H2DCFDA probe, and geometric means of fluorescence intensities (FI) of Alexa Fluor 488-positive cells were calculated and compared across groups at 2h post treatments. THP-1 monocytes were primed with Pam3CSK4 (1μg/mL) for 24h ± rhPRG4 (200μg/mL) or IL-1RA (250ng/mL) followed by co-incubation with FITC-beads for 2h and THP-1 phagocytic activation was determined as shown in Figure 1A . Alternatively, THP-1 monocytes were challenged with MSU crystals (200μg/mL) for 6h ± rhPRG4 (200μg/mL) or IL-1RA (250ng/mL) followed by analysis of IL-1β gene expression, intracellular pro-IL-1β and secreted mature IL-1β by ELISA and PP2A activity following PP2A immunoprecipitation. Intracellular pro-IL-1β and PP2A activity were normalized to total isolated protein. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (A) Representative flow cytometry histograms showing enhanced phagocytosis of FITC labeled beads by TLR2 ligand primed THP-1 monocytes and rhPRG4 treatment appeared to reduce phagocytosis of FITC-labeled beads (shown by arrows). (B) rhPRG4 treatment reduced FITC-labeled beads’ phagocytosis by THP-1 monocytes. (C) Representative flow cytometry histograms showing reduced ROS fluorescence intensity with rhPRG4 treatment (shown by an arrow). (D) rhPRG4 treatment reduced ROS generation in THP-1 monocytes. (E) rhPRG4 reduced IL-1β gene expression in THP-1 monocytes. (F) rhPRG4 reduced pro-IL-1β levels in THP-1 monocytes. (G) rhPRG4 reduced mature IL-1β secreted by THP-1 monocytes. (H) rhPRG4 treatment increased PP2A activity in MSU challenged THP-1 monocytes.

Figure 5

Role of protein phosphatase-2A (PP2A) in mediating rhPRG4’s anti-inflammatory effect in monosodium urate monohydrate (MSU) crystals challenged THP-1 human monocytes. THP-1 monocytes were primed with Pam3CSK4 (1μg/mL) for 24h followed by MSU (200μg/mL) crystals ± rhPRG4 (200μg/mL) ± okadaic acid (OKA) (5nM) for 6h and intracellular pro-interleukin-1 beta (pro-IL-1β), secreted mature IL-1β, NLRP3 protein, and caspase-1 activity were quantified. Intracellular pro-IL-1β, NLRP3 and caspase-1 activity were normalized to total isolated protein. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post-hoc test. ns, non-significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. (A) OKA co-treatment increased pro-IL-1β content in rhPRG4-treated THP-1 monocytes. (B) OKA co-treatment increased secreted IL-1β levels in rhPRG4-treated THP-1 monocytes. (C) NLRP3 protein content did not change as a result of rhPRG4 ± OKA treatments. (D) OKA co-treatment increased caspase-1 activity in rhPRG4-treated THP-1 monocytes.

Figure 6

Recruitment of inflammatory classical monocytes (CMs), anti-inflammatory nonclassical monocytes (NCMs) and neutrophils in monosodium urate (MSU) crystal induced peritoneal inflammation model in proteoglycan 4 (Prg4) gene-trap (Prg4^GT/GT ) and Prg4 competent (Prg4^+/+ ) mice. Peritoneal lavages (PLs) were collected at 6h and 24h. CMs were identified as CD11b+ Ly6C^hi CCR2+ and NCMs were identified as CD11b+ Ly6C^lo CD43^hi CX3CR1+. Neutrophils were identified using Ly6G and Ly6B.2 surface markers. PL cell populations of interest were determined using Precision Counting Beads. PL IL-1β and CXCL1 levels were determined by ELISAs. Recombinant human PRG4 (rhPRG4) (50μL; 1 mg/mL) or PBS (50μL) were administered intra-peritoneally at 6h following MSU crystal injection in the Prg4^GT/GT mice with PLs collected at 24h. We utilized 4 animals in each group at each time point balanced between males and females with an age range of 2-3 months. Statistical analyses included Student’s t-test and two-way ANOVA followed by Tukey’s post-hoc test. ns, non-significant; *p < 0.05; **p < 0.01. (A) Flow cytometry gating strategy to identify inflammatory CMs and anti-inflammatory NCMs. Singlets were initially identified followed by gating for viable cells using Zombie Violet dye as shown in Figure 1A . CMs were identified as CD11b+ Ly6C^hi CCR2+ and NCMs were identified as CD11b+ Ly6C^lo CD43^hi CX3CR1+. (B) Flow cytometry gating strategy to identify neutrophils. Singlets were initially identified followed by gating for viable cells using Zombie Violet dye as shown in Figure 1A . Neutrophils were identified as Ly6G+ Ly6B.2+. (C) CMs in Prg4^GT/GT and Prg4^+/+ PLs were higher at 6h. rhPRG4 treatment did not alter CMs in Prg4^GT/GT PLs at 24h. (D) NCMs in Prg4^GT/GT and Prg4^+/+ PLs were higher at 24h. rhPRG4 treatment increased NCMs in Prg4^GT/GT PLs. (E) Neutrophils in Prg4^GT/GT PLs were higher at 24h. rhPRG4 treatment reduced neutrophils in Prg4^GT/GT PLs. (F) PL IL-1β levels in Prg4^GT/GT animals at 24h were higher than Prg4^+/+ animals and rhPRG4 did not significantly modify IL-1β levels in Prg4^GT/GT animals. (G) PL CXCL1 levels in Prg4^GT/GT animals were not different from Prg4^+/+ animals at 6h and 24h. rhPRG4 treatment reduced CXCL1 levels in Prg4^GT/GT animals at 24h.