Omentin-1 ameliorates the progress of osteoarthritis by promoting IL-4-dependent anti-inflammatory responses and M2 macrophage polarization

Abstract

Osteoarthritis (OA) is a prevalent joint disease commonly associated with aging and obesity, which can lead to pain, stiffness, joint dysfunction, and disability. Omentin-1 (also called intelectin-1) is a newly discovered adipokine, which plays a protective role in suppressing the secretion of pro-inflammatory cytokines. Based on data from the Gene Expression Omnibus (GEO) dataset and clinical samples obtained at our institution revealed, determined that omentin-1 and IL-4 (an anti-inflammatory cytokine) levels were significantly lower in OA patients than in normal controls. Omentin-1 was shown to induce IL-4-depedent anti-inflammatory responses and M2 macrophage polarization in OA synovial fibroblasts via the PI3K, ERK, and AMPK pathways. Administering omentin-1 was shown to block cartilage degradation and bone erosion resulting from anterior cruciate ligament transection by inhibiting the production of pro-inflammatory cytokines and promoting M2 macrophage polarization in vivo. Our findings indicate omentin-1 as a promising therapeutic avenue for the treatment for OA.

Keywords: Anti-inflammation; IL-4; M2 macrophage; Omentin-1; Osteoarthritis.

Figure 1

Omentin-1 and IL-4 expression levels are negatively correlated with OA progression. (A) Omentin-1 levels and (C) IL-4 levels in normal and OA synovial tissue samples retrieved from the GEO dataset; (B) Heatmap showing differentially expressed IL family genes from the GSE82107 dataset; (D) Positive correlation between omentin-1 and IL-4 based on the GSE29746 dataset; (E-H) IHC staining showing that the levels of omentin-1 and IL-4 were higher in human OA synovial tissue than in healthy tissue. Scale bar = 100 µm. (I) qPCR analysis showing that treating OASFs with omentin-1 increased IL family gene expression. n=6 for each group. * p<0.05 vs controls.

Figure 2

Omentin-1 inhibited the production of pro-inflammatory cytokines by upregulating IL-4 expression. (A and B) IHC staining showing that IL-1β, IL-6, IL-8, and TNF-α were higher in human OA synovial tissue than in healthy tissue. Scale bar = 100 µm. (C) Gene expression levels in normal and OA synovial tissue samples retrieved from the GEO dataset. (D and E) qPCR analysis showing the gene expression of OASFs treated with omentin-1 or transfected with IL-4 siRNA prior to stimulation using omentin-1, n=6 for each group. * p<0.05 vs controls. # p<0.05 vs omentin-1-treated group.

Figure 3

Omentin-1 promoted IL-4 production in OASFs via the PI3K, ERK, and AMPK pathways. (A and B) Ingenuity Pathway Analysis (IPA) pathway enrichment figure showing pathways that were significantly upregulated in the GSE82107 database; (C) Western blot analysis showing the phosphorylation of PI3K, ERK, and AMPK in OASFs treated with omentin-1; (D-F) Western blot quantified P85, ERK and AMPK phosphorylation and total protein. (G and H) qPCR analysis showing IL-4 expression in OASFs treated with PI3K inhibitors (Ly294002 and wortmannin), ERK inhibitor (ERK II), and AMPK inhibitor (Compound C) or transfected with indicated siRNA prior to stimulation using omentin-1. * p<0.05 vs controls. # p<0.05 vs omentin-1-treated group.

Figure 4

Omentin-1 enhanced IL-4-dependent polarization of the M2 macrophage. (A) Immunofluorescence staining for CD68, CD86, and CD163 in synovium tissue samples from normal controls and OA patients. Scale bar = 100 µm. (B) TissueFaxs analysis of fluorescence intensity for macrophage polarization in the ACLT model. (C-E) Flow cytometry analysis and qPCR of THP-1 cells incubated with PMA for 24 h prior to stimulation using LPS+IFN-γ or IL-4+IL-13 or Conditioned Medium (CM) from OASF with or without omentin-1 treatment and IL-4 siRNA transfection for 24 h. * p<0.05 vs OASFs group. # p<0.05 vs omentin-1-treated OASFs group.

Figure 5

Omentin-1 antagonized ACLT-induced breakdown of cartilage and bone. (A) Experiment flow involving ACLT induction and omentin-1 injection; (B) Weight-bearing asymmetry analysis; (C) Photomicrographs showing coronal and axial views of micro-CT images. Red arrow indicates the bone loss; (D-J) Graphic illustrations of BMD, BMC, bone volume, bone surface, trabecular numbers, trabecular bone thickness, and trabecular separation in the indicated groups; (K-M) Histological sections from knees stained with H&E and Safranin-O with corresponding OARSI and synovium scores. Black arrows indicate the cartilage damage or synovial hyperplasia. Scale bar =500 µm. Abbreviations: ST, synovial tissue; SB, subchondral bone; CA, cartilage. * p<0.05 vs control group. # p<0.05 vs ACLT group.

Figure 6

Omentin-1 increased IL-4 production and reduced the expression of pro-inflammatory cytokines in ACLT model. (A) Immunohistochemistry (IHC) staining for IL-4, IL-1β, IL-6, IL-8, and TNF-α and (B-F) quantification of IHC scores. Scale bar =100 µm. n=6 for each group. * p<0.05 vs control group. # p<0.05 vs ACLT group.

Figure 7

Omentin-1 treatment increased the number of M2 macrophages in ACLT model. (A) Immunofluorescence staining for CD68, CD86, and CD163; and (B) TissueFaxs analysis of fluorescence intensity of macrophage polarization in ACLT model. Scale bar =100 µm. * p<0.05 vs control group. # p<0.05 vs ACLT group.

Figure 8

Schematic diagram illustrating the mechanisms of omentin-1 function in OA. The schematic diagram summarizes the mechanisms underlying the omentin-1-induced increase in the production of IL-4 in human OASFs via the PI3K, ERK, and AMPK pathways, which inhibited the expression of pro-inflammatory cytokines, while facilitating M2 macrophage polarization and antagonizing OA progression.