Recombinant human proteoglycan-4 reduces phagocytosis of urate crystals and downstream nuclear factor kappa B and inflammasome activation and production of cytokines and chemokines in human and murine macrophages

Abstract

BackgroundGout is an inflammatory arthritis caused by monosodium urate monohydrate (MSU) crystals’ joint deposition. MSU phagocytosis by resident macrophages is a key step in gout pathogenesis. MSU phagocytosis triggers nuclear factor kappa B (NFκB) activation and production of cytokines and chemokines. Proteoglycan-4 (PRG4) is a glycoprotein produced by synovial fibroblasts and exerts an anti-inflammatory effect in the joint mediated by its interaction with cell surface receptor CD44. PRG4 also binds and antagonizes TLR2 and TLR4. The objective of this study is to evaluate the efficacy of recombinant human PRG4 (rhPRG4) in suppressing MSU-induced inflammation and mechanical allodynia in vitro and in vivo.MethodsTHP-1 macrophages were incubated with MSU crystals ± rhPRG4 or bovine submaxillary mucin (BSM), and crystal phagocytosis, cytokines and chemokines expression and production were determined. NFκB p65 subunit nuclear translocation, NLRP3 induction, caspase-1 activation and conversion of proIL-1β to mature IL-1β were studied. MSU phagocytosis by Prg4+/+ and Prg4−/− peritoneal macrophages was determined in the absence or presence of rhPRG4, BSM, anti-CD44, anti-TLR2, anti-TLR4 and isotype control antibodies. Rhodamine-labeled rhPRG4 was incubated with murine macrophages and receptor colocalization studies were performed. Lewis rats underwent intra-articular injection of MSU crystals followed by intra-articular treatment with PBS or rhPRG4. Weight bearing and SF myeloperoxidase activities were determined.ResultsrhPRG4 reduced MSU crystal phagocytosis at 4 h (p < 0.01) and IL-1β, TNF-α, IL-8 and MCP-1 expression and production at 6 h (p < 0.05). BSM did not alter MSU phagocytosis or IL-1β production in human and murine macrophages. rhPRG4 treatment reduced NFκB nuclear translocation, NLRP3 expression, caspase-1 activation and generation of mature IL-1β (p < 0.05). MSU-stimulated IL-1β production was higher in Prg4−/− macrophages compared to Prg4+/+ macrophages (p < 0.001). rhPRG4, anti-CD44, anti-TLR2 and anti-TLR4 antibody treatments reduced MSU phagocytosis and IL-1β production in murine macrophages (p < 0.05). rhPRG4 preferentially colocalized with CD44 on Prg4−/− peritoneal macrophages compared to TLR2 or TLR4 (p < 0.01). rhPRG4 normalized weight bearing and reduced SF myeloperoxidase activity compared to PBS in vivo.ConclusionrhPRG4 inhibits MSU crystal phagocytosis and exhibits an anti-inflammatory and anti-nociceptive activity in vitro and in vivo. rhPRG4’s anti-inflammatory mechanism may be due to targeting CD44 on macrophages.

Design and Biological Evaluation of Colchicine-CD44-Targeted Peptide Conjugate in an In Vitro Model of Crystal Induced Inflammation

“…Bone marrow-derived macrophages (BMDMs) from CD44 wild-type animals (Cd44 +/+ ) appeared to internalize MSU crystals to a greater extent when compared to the CD44 knockout (Cd44 −/− ) animals (white arrows point to MSU crystals in DAPI-stained BMDMs; Figure 1D). This observation is consistent with the phagocytic role of the CD44 receptor and validates our earlier observations that PRG4, a CD44 ligand, prevents MSU phagocytosis by macrophages [33]. Using flow cytometry to detect MSU phagocytosis via a change in macrophage cell side-scatter [33], we observed that the CD44 receptor-ligand, HA, significantly reduced MSU phagocytosis by THP-1 macrophages.…”

“…This observation is consistent with the phagocytic role of the CD44 receptor and validates our earlier observations that PRG4, a CD44 ligand, prevents MSU phagocytosis by macrophages [33]. Using flow cytometry to detect MSU phagocytosis via a change in macrophage cell side-scatter [33], we observed that the CD44 receptor-ligand, HA, significantly reduced MSU phagocytosis by THP-1 macrophages. Figure 1E depicts the representative flow cytometry plots of MSU-treated macrophages in the absence or presence of HA (100 µg/mL).…”

“…MSU treatment appeared to increase the number of cells in the P2 region while HA co-treatment reduced the number of cells in the P2 region. We previously validated this indirect phagocytosis assay against a direct method of assessing crystal uptake by cells using cell counting under the microscope [33]. The quantitative analysis of the percentage of macrophage cell population in the P2 region across different experimental groups is shown in Figure 1F.…”

“…Binding of HA and PRG4 to CD44 on the surface of macrophages was shown to result in ligand internalization [32,33]. Furthermore, we have previously shown that recombinant human PRG4 inhibits MSU phagocytosis by macrophages in a mechanism that may involve CD44 and/or TLR receptor interactions [33]. The utility of CD44 as a targeting receptor on macrophages was also reported by utilizing HA-coated nanoparticles to repolarize inflammatory macrophages to an anti-inflammatory phenotype [34,35].…”

“…A number of endogenous ligands to CD44 have been identified including high molecular weight hyaluronic acid (HA) and the glycoprotein, proteoglycan-4 (PRG4), two major components of joint synovial fluid (SF) [31]. Binding of HA and PRG4 to CD44 on the surface of macrophages was shown to result in ligand internalization [32,33]. Furthermore, we have previously shown that recombinant human PRG4 inhibits MSU phagocytosis by macrophages in a mechanism that may involve CD44 and/or TLR receptor interactions [33].…”