Interleukin 37 limits monosodium urate crystal-induced innate immune responses in human and murine models of gout

Abstract

Background: Interleukin (IL)-37 has emerged as a fundamental inhibitor of innate immunity. Acute gout is a self-limiting inflammatory response to monosodium urate (MSU) crystals. In the current study, we assessed the preventive and therapeutic effect of recombinant human IL-37 (rhIL-37) in human and murine gout models.

Methods: We investigated the expression of IL-37 in patients with active and inactive gouty arthritis and assessed the effect of rhIL-37 in human and murine gout models: a human monocyte cell line (THP-1) and human synovial cells (containing macrophage-like and fibroblast-like synoviocytes) exposed to MSU crystals, a peritoneal murine model of gout and a murine gouty arthritis model. After inhibition of Mer receptor tyrosine kinase (Mertk), levels of IL-1β, IL-8 and chemokine (C-C motif) ligand 2 (CCL-2) were detected by ELISA and expression of mammalian homologs of the drosophila Mad gene 3 (Smad), suppressor of cytokine signaling 3 (SOCS3), NACHT-LRR-PYD-containing protein 3 (NLRP3), and IL-8R of THP-1 were assessed by qPCR and western blot to explore the molecular mechanisms.

Results: Our studies strongly indicated that rhIL-37 played a potent immunosuppressive role in the pathogenesis of experimental gout models both in vitro and in vivo, by downregulating proinflammatory cytokines and chemokines, markedly reducing neutrophil and monocyte recruitment, and mitigating pathological joint inflammation. In our studies, rhIL-37 suppressed MSU-induced innate immune responses by enhancing expression of Smad3 and IL-1R8 to trigger multiple intracellular switches to block inflammation, including inhibition of NLRP3 and activation of SOCS3. Mertk signaling participated in rhIL-37 inhibitory pathways in gout models. By inhibition of Mertk, the anti-inflammatory effect of rhIL-37 was partly abrogated, and IL-1R8, Smad3 and S​OCS3 expression were suppressed, whereas NLRP3 expression was reactivated.

Conclusions: Our studies reveal that IL-37 limits runaway inflammation initiated by MSU crystal-induced immune responses, partly in a Mertk-dependent fashion. Thus, rhIL-37 has both preventive and therapeutic effects in gouty arthritis.

Keywords: Gout; IL-1R8; IL-37; MSU; Mertk; NLRP3; Smad3; S​OCS3.

Fig. 1

Expression of IL-37 in patients with gouty arthritis. a H&E staining of synovial tissue from a person with active gouty arthritis. b Immunohistochemical staining of IL-37 in the same synovial tissue. c H&E staining of subcutaneous tophus from patients with chronic tophaceous gout. d Immunohistochemical staining of IL-37 (arrow) in the same subcutaneous tophus. e Concentration of IL-37 in serum from patients with acute gout (AG, n = 16) or non-acute gout (NAG, n = 15), and from healthy controls (HC, n = 18), and in synovial fliud (SF) from patients with acute gout (n = 5); *P < 0.05,**P < 0.01,***P < 0.001. f Expression of pro-IL-37 mRNA in the peripheral blood mononuclear cells (PBMCs) from patients with gout (AG, n = 10, NAG, n = 10); **P < 0.01

Fig. 2

IL-37 is inducible in the peripheral blood mononuclear cells (PBMCs) stimulated by monosodium urate (MSU). a Concentration of IL-37 protein in PBMC after MSU stimulation for 18 h; **P < 0.01,***P < 0.001. b The expression of pro-IL-37 mRNA in the PBMCs after MSU stimulation for 18 h; *P < 0.05,**P < 0.01,***P < 0.001

Fig. 3

Recombinant human IL-37 (rhIL-37) treatment in THP-1 macrophages and human synoviocytes. a–c Concentration of secreted IL-1β, IL-8, chemokine (C-C motif) ligand 2 (CCL2) and transforming growth factor β (TGF-β) in synoviocytes or THP-1 left untreated or treated with rhIL-37 for 3 h, and then incubation with monosodium urate (MSU), ATP or lipopolysaccharide (LPS) for a further 6 h or 18 h. Mean ± s.e.m. *P < 0.05 for rhIL-37 treatment compared to non-rh-IL-37 treatment

Fig. 4

Recombinant human 1 L-37 (rhIL-37) reduces inflammatory cell recruitment in mice with monosodium (MSU) crystals induced peritonitis. a–c Neutrophil, monocyte and macrophage determination by flow cytometry: neutrophils are defined as Gr-1^high7/4⁺ (gate R1); monocytes are defined as Gr-1^inter7/4⁺ (gate R2) expressing low levels of F4/80 (gate R3); macrophages with high expression of F4/80 (gate R4) are thought to be resident macrophages. d Total and separate proportions of neutrophils and monocytes in all peritoneal exudate cells; **P < 0.01,***P < 0.001. e Concentration of neutrophil chemoattractant in peritoneal lavage fluid; ***P < 0.001. f Concentration of monocyte chemoattractant in peritoneal lavage fluid; **P < 0.01. g Different dosage of rhIL-37 was given preventively or therapeutically in mice with gouty arthritis, and foot thickness was evaluated; **P < 0.01, ***P < 0.001. h, i Histopathological analysis of joint from non-rhIL-37 treatment group by H&E staining (×100 original magnification (h) and × 200 original magnification (i)); arrow abundant inflammation in soft tissue and joint space. j Histopathological analysis of joint from rhIL-37 preventive group by H&E staining (×100 original magnification); arrow less inflammation in soft tissue and joint space. k, l Histopathological analysis of joint from rhIL-37 therapeutic group by H&E staining (×100 original magnification (k) and × 200 original magnification (l); arrow almost no inflammation in soft tissue and joint space

Fig. 5

Underlying mechanism involved in recombinant human IL-37 (rhIL-37) inhibitory properties. a Heat map of the mRNA qPCR-array analysis. Color intensity is proportional to the fold-regulation, which represents fold-change results in a biologically meaningful way. Fold-regulation values greater than 1.5 indicate positive or up-regulation (red), whereas values less than -1.5 indicate negative or downregulation (blue). With fold-change values greater than one the fold-regulation is equal to the fold-change.) Fold-change values less than one indicate negative or downregulation, and the fold-regulation is the negative inverse of the fold-change. Fold-change is expressed as a ratio by 2^-ΔΔct in the rhIL-37 intervention group/2^-ΔΔct in the non-rhIL-37 intervention group. b–d Fold-regulation in different rhIL-37 intervention groups vs the non-rhIL-37 intervention group, respectively; *P < 0.05

Fig. 6

Contribution of the Mertk inhibitor to the IL-37-mediated anti-inflammatory effect in monosodium urate (MSU)-induced models in vitro and in vivo. a–c Concentration of secreted IL-1β, IL-8 and CCL2 in THP-1 macrophages treated with or without recombinant human IL-37 (rhIL-37) for 3 h, followed by incubation for 1 h with or without Mertk inhibitor and then incubated with lipopolysaccharide (LPS) or MSU separately for a further 18 h; *P < 0.05. d Different dosage of rhIL-37 was given preventively or therapeutically with or without Mertk inhibitor intervention in mice with gouty arthritis, and foot thickness was evaluated; *P < 0.05. e, f Histopathological analysis by H&E staining in a joint from the group treated with rhIL-37 treatment and Mertk inhibitor intervention (×100 original magnification (e) and × 200 original magnification (f); arrow inflammation in soft tissue and joint space. g–k The protein level of Smad3, IL-1R8, S​OCS3 and NLRP3 was verified by western blotting in the IL-37 treatment groups with or without Mertk inhibitor intervention. Protein levels in different groups were expressed as a ratio to that of corresponding glyceraldehyde-3-phosphate dehydrogenase (GAPDH); *P < 0.05 **P < 0.01