The interaction of lubricin/proteoglycan 4 (PRG4) with toll-like receptors 2 and 4: an anti-inflammatory role of PRG4 in synovial fluid

Abstract

Background: Lubricin/proteoglycan-4 (PRG4) is a mucinous glycoprotein secreted by synovial fibroblasts and superficial zone chondrocytes. PRG4 has a homeostatic multifaceted role in the joint. PRG4 intra-articular treatment retards progression of cartilage degeneration in pre-clinical posttraumatic osteoarthritis models. The objective of this study is to evaluate the binding of recombinant human PRG4 (rhPRG4) and native human PRG4 (nhPRG4) to toll-like receptors 2 and 4 (TLR2 and TLR4) and whether this interaction underpins a PRG4 anti-inflammatory role in synovial fluid (SF) from patients with osteoarthritis (OA) and rheumatoid arthritis (RA).

Methods: rhPRG4 and nhPRG4 binding to TLR2 and TLR4 was evaluated using a direct enzyme linked immunosorbent assay (ELISA). Association of rhPRG4 with TLR2 and TLR4 overexpressing human embryonic kidney (HEK) cells was studied by flow cytometry. Activation of TLR2 and TLR4 on HEK cells by agonists Pam3CSK4 and lipopolysaccharide (LPS) was studied in the absence or presence of nhPRG4 at 50, 100 and 150 μg/ml. Activation of TLR2 and TLR4 by OA SF and RA SF and the effect of nhPRG4 SF treatment on receptor activation was assessed. PRG4 was immunoprecipitated from pooled OA and RA SF. TLR2 and TLR4 activation by pooled OA and RA SF with or without PRG4 immunoprecipitation was compared.

Results: rhPRG4 and nhPRG4 exhibited concentration-dependent binding to TLR2 and TLR4. rhPRG4 associated with TLR2- and TLR4-HEK cells in a time-dependent manner. Co-incubation of nhPRG4 (50, 100 and 150 μg/ml) and Pam3CSK4 or LPS reduced TLR2 or TLR4 activation compared to Pam3CSK4 or LPS alone (p <0.05). OA SF and RA SF activated TLR2 and TLR4 and nhPRG4 treatment reduced SF-induced receptor activation (p <0.001). PRG4 depletion by immunoprecipitation significantly increased TLR2 activation by OA SF and RA SF (p <0.001).

Conclusion: PRG4 binds to TLR2 and TLR4 and this binding mediates a novel anti-inflammatory role for PRG4.

Fig. 1

Binding of recombinant human proteoglycan-4 (rhPRG4) and native human PRG4 (nhPRG4) to recombinant toll-like receptors 2 and 4 (TLR2 and TLR4) using a direct binding enzyme linked immunosorbent assay. PRG4 concentrations are reported in μg/mL and pmoles per well, based on a predicted molecular weight of 240 kDa. TLR2 and TLR4 are expressed as pmoles coated per well, based on predicted molecular weights of 66 and 70 kDa, respectively. The 450 nm absorbance values were normalized to non-specific binding. Data represent the mean ± SD from four independent experiments. Dashed line represents non-specific binding (background). a Concentration-dependent binding of rhPRG4 and nhPRG4 to immobilized TLR2. rhPRG4 exhibited a concentration-dependent binding at 50, 10 and 1 μg/mL. nhPRG4 exhibited a concentration-dependent binding at 50 and 10 μg/mL; *p <0.001. b Concentration-dependent binding of rhPRG4 and nhPRG4 to immobilized TLR4. rhPRG4 and nhPRG4 exhibited concentration-dependent binding at 50 and 10 μg/mL; *p <0.001

Fig. 2

Association of recombinant human proteoglycan-4 (rhPRG4) with toll-like receptor-2 and 4 (TLR2 and TLR4) expressing human embryonic kidney (TLR2-HEK and TLR4-HEK) cells. Data represent the mean ± SD from four independent experiments. a Representative flow cytometry scatter plots of rhodamine-labeled rhPRG4 (20 μg/mL) association with TLR2-HEK and TLR4-HEK cells following incubation for 12 and 24 h. A threshold was set at red fluorescence intensity = 10. Cell-associated fluorescence higher than 10 was considered positive. Following incubation of rhodamine-rhPRG4 with TLR2-HEK and TLR4-HEK cells for 12 or 24 h, cell association was observed. b Quantitative analysis of the percentage of positive TLR2-HEK and TLR4-HEK cells following incubation with rhodamine-rhPRG4 (20 μg/mL) for 12 and 24 h. rhPRG4 was significantly associated with TLR2-HEK and TLR4-HEK at 24 h compared to 12 h and controls. The 12-h association between rhPRG4 and TLR2-HEK cells was significantly higher than with TLR4-HEK cells and both were higher than control; *p <0.001, ** p <0.05

Fig. 3

Concentration-dependent effect of native human proteoglycan 4 (nhPRG4) on agonist-induced activation of toll-like receptors 2 and 4 (TLR2 and TLR4) expressing human embryonic kidney (TLR2-HEK and TLR4-HEK) cells. Absorbance values across groups were normalized to untreated cells (control). Data represents the mean ± SD from four independent experiments. a Inhibition of Pam3CSK4-induced activation of TLR2 by nhPRG4 treatment. Pam3CSK4 significantly activated TLR2. nhPRG4 (50, 100 and 150 μg/mL) co-incubation significantly reduced Pam3CSK4-induced TLR2 activation. nhPRG4 (100 and 150 μg/mL) treatments were more efficacious in reducing TLR2 activation than nhPRG4 (50 μg/mL). nhPRG4 alone did not stimulate TLR2; *p <0.001. b Inhibition of lipopolysaccharide (LPS)-induced activation of TLR4 by nhPRG4 treatment. LPS significantly activated TLR4. nhPRG4 (50, 100 and 150 μg/mL) co-incubation significantly reduced LPS-induced TLR4 activation. nhPRG4 (100 and 150 μg/mL) treatments were more efficacious in reducing TLR4 activation than nhPRG4 (50 μg/mL). nhPRG4 (150 μg/mL) treatment was more efficacious in reducing TLR4 activation than nhPRG4 (100 μg/mL). nhPRG4 alone did not stimulate TLR4; *p <0.001, **p <0.01, ***p <0.05

Fig. 4

Activation of toll-like receptors 2 and 4 (TLR2 and TLR4) expressing human embryonic kidney (TLR2-HEK and TLR4-HEK) cells by synovial fluids (SF) from subjects with no joint arthropathy (normal; n = 3), patients with osteoarthritis (OA; n = 8) and patients with rheumatoid arthritis (RA; n = 5) and effect of native human proteoglycan-4 (nhPRG4) treatment. SF samples were incubated with TLR2- or TLR4-HEK cells (3.75 % dilution) at 37 °C for 48 h. Absorbance values across groups were normalized to untreated cells (control). Data represent the mean SD from three independent experiments. a Activation of TLR2 and TLR4 by normal, OA and RA SF. Normal SF did not significantly activate TLR2 or TLR4 compared to control. OA SF and RA SF significantly activated TLR2 and TLR4 compared to normal SF and control. RA SF significantly activated TLR2 compared to OA SF; *p <0.001. b Impact of nhPRG4 treatment (100 μg/mL) on Pam3CSK4, OA SF and RA SF-induced activation of TLR2. nhPRG4 treatment inhibited Pam3CSK4, OA SF and RA SF-induced TLR2 activation; *p <0.001. c Impact of nhPRG4 treatment (150 μg/mL) on lipopolysaccharide (LPS), OA SF and RA SF-induced activation of TLR4. nhPRG4 treatment inhibited LPS, OA SF and RA SF-induced TLR4 activation; *p <0.001

Fig. 5

Impact of proteoglycan-4 (PRG4) immunoprecipitation on activation of toll-like receptors 2 and 4 (TLR2 and TLR4) expressing human embryonic kidney (TLR2-HEK and TLR4-HEK) cells by pooled osteoarthritis (OA) (n = 5) and pooled rheumatoid arthritis (RA) (n = 5) synovial fluid (SF). OA or RA SF with or without PRG4 immunoprecipitation were incubated with TLR2-HEK or TLR4-HEK cells (0.5, 3.75, 5.0 and 10.0 % dilution) at 37 °C for 24 h. The 630 nm absorbance values across experimental groups were normalized to untreated controls. Data represent the mean ± SD from three independent experiments. a Impact of PRG4 immunoprecipitation on TLR2 activation by OA SF. TLR2 activation was significantly higher in PRG4-immunoprecipitated OA SF (OA SF (-PRG4)) compared to OA SF at 3.75, 5.0 and 10.0 %; *p <0.001. b Impact of PRG4 immunoprecipitation on TLR2 activation by RA SF. TLR2 activation was significantly higher in PRG4 immunoprecipitated RA SF (RA SF (-PRG4)) compared to RA SF at 5 and 10 %; *p <0.001. c Impact of PRG4 immunoprecipitation on TLR4 activation by OA SF and RA SF. There was no significant difference in TLR4 activation between OA SF and OA SF following PRG4 immunoprecipitation (OA SF (-PRG4)) across any SF dilutions. There was no significant difference in TLR4 activation between RA SF and RA SF following PRG4 immunoprecipitation (RA SF (-PRG4)) across any SF dilutions

Fig. 6

Impact of native human proteoglycan-4 (nhPRG4) treatment (100, 200 and 300 μg/mL) on toll-like receptor 2 (TLR2) activation by pooled osteoarthritis (OA) synovial fluid (SF) and pooled rheumatoid arthritis (RA) SF following proteoglycan-4 (PRG4) immunoprecipitation. TLR2-expressing human embryonic kidney (TLR2-HEK) cells were incubated with SF (5 % dilution for 24 h) in the absence or presence of nhPRG4. The 630 nm absorbance values across experimental groups were normalized to untreated controls. Data represent the mean ± SD from three independent experiments. a Impact of nhPRG4 on TLR2 activation of PRG4-immunoprecipitated OA SF (OA SF (-PRG4)). nhPRG4 treatments (200, 300 μg/mL) significantly reduced TLR2 activation by OA SF (-PRG4). nhPRG4 (300 μg/mL) treatment was more efficacious in reducing TLR2 activation than nhPRG4 (100 μg/mL); *p <0.001; **p <0.05. b Impact of nhPRG4 on TLR2 activation of PRG4-immunoprecipitated RA SF (RA SF (-PRG4)). nhPRG4 treatments (200, 300 μg/mL) significantly reduced TLR2 activation by RA SF (-PRG4). nhPRG4 (200 and 300 μg/mL) treatment was more efficacious in reducing TLR2 activation than nhPRG4 (100 μg/mlL); *p <0.001; **p <0.01