Galectin-9 inhibition of the MIF-CD74/CD44 pathway suppresses chronic arthritis

Abstract

The destructive potential of rheumatoid arthritis (RA) lies in the aggressive behavior of fibroblast-like synoviocytes (FLSs), which actively contribute to the erosion of cartilage and bone and may persist even in the face of apparent clinical remission. Therapeutic approaches targeting RA-FLSs have been developed to treat RA; however, there are no clinically approved drugs available at present. Here, single-cell RNA sequencing of RA-FLSs identified a distinct macrophage migration inhibitory factor (MIF)^high subset with mitochondrial and endoplasmic reticulum dysfunction. MIF^high conditions led to increased survival, proliferation, and migration of FLSs, along with the upregulation of CD44 and the CD44v6 isoform expression. We next explored whether a stable, recombinant form of galectin-9 (sGal-9), which acts as a CD44 blockade, regulates the MIF-induced aggressive phenotype of RA-FLSs. We found that sGal-9 remarkably reduced the increased proliferation, migration, and invasion of RA-FLSs by inhibiting the MIF-CD44 pathway. Moreover, both local and systemic administration of sGal-9 substantially inhibited excessive cartilage and bone destruction by RA-FLSs in a xenotransplantation arthritis model and alleviated the severity of collagen-induced arthritis in mice, comparable to Enbrel and tofacitinib. Conclusively, these data suggest that sGal-9 is effective at repressing destructive phenotypes of RA-FLSs as a novel anti-MIF agent.

Keywords: CD44; CIA; ER; MIF; collagen-induced arthritis; endoplasmic reticulum; fibroblast-like synoviocytes; galectin-9; macrophage migration inhibitory factor; rheumatoid arthritis.

Graphical abstract

Figure 1

Expression and functional characterization of the MIF^high subset in RA-FLSs (A) t-SNE plot showing clusters analyzed by single-cell RNA seq using 10x Genomics in cultured RA-FLSs isolated from three patients. (B) Violin plot representing the expression levels of MIF across the nine clusters. (C) Heatmap depicting differentially upregulated genes in MIF^high FLS associated with mitochondria function, ER function, apoptosis, and actin filament organization across the nine clusters of RA-FLSs. Rows represent genes, and columns represent clusters. The color of each cell of the heatmap indicates the expression levels, where red signifies higher expression of genes associated with the gene set in the respective cluster compared to other clusters, while blue indicates lower expression. (D) Changes in ER stress and mitochondria-related proteins in RA-FLSs treated with recombinant MIF (100 ng/mL). The cells were stimulated with recombinant MIF for the indicated times (hours), and the expression of GRP78, CHOP, BAX, and BCL2 was assessed by western blot analysis. (E) Expression levels of BCL2 and GRP78 in RA-FLSs with lower (<25^th percentile) and higher (>75^th percentile) intracellular MIF expression, as determined by flow cytometry analysis. The data were quantified as the mean fluorescence intensity. Histograms are representative of experiments conducted at least three times. The bar graphs show the mean ± SD. Statistical differences were assessed using an unpaired two-tailed t test (E). ∗p < 0.05; ∗∗∗∗p < 0.0001.

Figure 2

Solid-phase ELISA for the interaction between CD44 and sGal-9 (A) Illustration of the plate-based binding assay. The plate was coated with 20 μM (100 μL) sGal-9, followed by incubation with CD44 Fc chimera protein (CD44-Fc) or human IgG1 Fc protein (IgG1-Fc) at different concentrations in the absence or presence of the anti-CD44 antibody (α-CD44 Ab; 3.5 μg/mL). Fc-Bio, biotinylated anti-human IgG Fc antibody; HRP, streptavidin-conjugated horseradish peroxidase. (B) Dose-dependent increase in sGal-9 binding to CD44-Fc. (C) Inhibition of the interaction between sGal-9 and CD44 by addition of (α-CD44 Ab). Data are presented as mean ± SD. Statistical differences were assessed using one-way ANOVA with Tukey’s multiple comparisons test (C). ∗∗∗∗p < 0.0001.

Figure 3

Suppression of MIF-stimulated survival, migration, and invasion of RA-FLSs by sGal-9 (A) Changes in MIF-induced expression of GRP78 and caspase-3 by sGal-9. RA-FLSs were treated with recombinant MIF (100 ng/mL) in the absence or presence of sGal-9 (1 nM) for 24 h. Levels of GRP78 and caspase-3 were determined by western blot analysis. (B) sGal-9-induced apoptosis in MIF-stimulated RA-FLSs. Apoptosis levels were assessed using the APOPercentage apoptosis assay, which utilizes an intense, pink-colored dye reagent that is taken up by apoptosis-committed cells. Apoptotic RA-FLSs, therefore, appeared pink. Scale bar, 400 μm. (C and D) Effect of sGal-9 on wound migration and invasion of RA-FLSs induced by MIF. Cells were incubated with recombinant MIF (100 ng/mL) in the absence or presence of sGal-9 (1 nM) for 16 h. The number of migrated cells in wound area (C) and invaded cells in Matrigel chamber (D) were manually counted. Scale bars, 1,000 μm for (C) and 100 μm for (D). (E and F) Reduction of MIF-induced CD44 and CD44v6 expression by sGal-9 as determined by real-time PCR (E) and western blot analysis (F). Data are representative of experiments conducted at least three times and presented as mean ± SD. Statistical differences were assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (A–F) and Kruskal-Wallis test with Dunn’s multiple comparisons test (E). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 4

Anti-FLS effects of sGal-9 under non-MIF stimulatory conditions in vitro (A–D) sGal-9 suppresses the survival, migration, and invasion of RA-FLSs stimulated with media alone (without MIF). (A) Dose-dependent inhibition of FLS viability by sGal-9 in vitro. Cell viability was assessed using the MTT assay. (B) Dose-dependent reduction of lamellipodium formation by sGal-9. RA-FLSs were stained with Alexa Fluor 488-conjugated phalloidin (green) for visualization of F-actin, and nuclei were stained with DAPI (blue). Arrowheads indicate filopodia and lamellipodia. (C) Inhibition of wound migration of RA-FLSs by sGal-9. Cells were incubated in DMEM containing sGal-9 for 16 h. The migrated cells beyond the reference line were then quantified. Scale bar, 1,000 μm. (D) Reduction of RA-FLS invasion by sGal-9. RA-FLSs were incubated with sGal-9 for 16 h, and invaded cells were stained using violet solution. Scale bar, 100 μm. (E and F) sGal-9 suppression of IL-1β or TGF-β-induced migration and invasion of RA-FLSs. Wound migration (E) and invasion (F) of RA-FLSs treated with recombinant IL-1β (1 ng/mL) or TGF-β (10 ng/mL) in the presence or absence of sGal-9 (1 nM) for 16 h. Scale bar, 1,000 μm for (E) and 100 μm for (F). “None” images in Figures 3D and 4F are derived from the same experiment. Data are representative of more than three independent experiments and presented as mean ± SD. Statistical differences were assessed using two-way ANOVA with Tukey’s multiple comparisons test (A, E, and F) and Friedman test with Dunn’s multiple comparisons test (B–D). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 5

sGal-9 inhibition of RA-FLS migration and invasion in a humanized synovitis model (A) Human cartilage implants in left flanks (primary) were co-implanted with sGal-9-treated RA-FLSs for 60 days in SCID mice. Cartilage implants of the same size were placed in the right flank (contralateral) without RA-FLSs (n = 2). Scale bars, 200 μm (overview) and 50 μm (magnified image). Bar graphs on the right depict the severity of cartilage invasion and degradation. (B) Human cartilage was co-transplanted into left flanks of SCID mice along with RA-FLSs, while equivalent-sized human cartilage was transplanted into the right flank. sGal-9 (2 mg/kg) was subsequently administered intraperitoneally twice per week for 60 days (n = 4). Afterward, the transplanted tissues were retrieved and analyzed. The implants underwent H&E staining. The invaded regions with perichondrocytic degradation are marked by black arrowheads. Scale bars, 200 μm (overview) and 50 μm (magnified image). Bar graphs on the right depict the severity of cartilage invasion and degradation. Data are presented as mean ± SD. Statistical differences were assessed using two-way ANOVA with Tukey’s multiple comparisons test (A and B). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Figure 6

Amelioration of collagen-induced arthritis by sGal-9 (A) From 3 weeks after the primary immunization, mice with collagen-induced arthritis (CIA) were subcutaneously administered with sGal-9 (2 mg/kg, n = 6) once per week for 3 weeks. Arthritis severity was assessed at the indicated time point. Enbrel (10 mg/kg, 3 times per week, n = 6), serving as a positive control, was injected subcutaneously, while tofacitinib (6.2 mg/kg, once daily, n = 6) was fed orally. (B) Histology score for the synovial hyperplasia, cartilage destruction, pannus formation, and bone erosion in mice treated with vehicle alone versus with sGal-9, Enbrel, or tofacitinib. (C) Toluidine blue staining for assessment of cartilage damage. Scale bar, 100 μm. (D and E) Infiltration of CD3⁺ and F4/80⁺ cells in the affected joints of CIA mice treated with vehicle alone, sGal-9, Enbrel, and tofacitinib, as determined by immunostaining. Scale bar, 50 μm. (F) Cell proliferation determined by immunostaining for Ki-67⁺ cells. Scale bar, 50 μm. (G) Immunostaining for MIF expression in the joint tissues of CIA mice treated with either vehicle alone or sGal-9. Tissue sections were stained with an anti-MIF antibody, and the number of MIF-expressing FLSs was manually counted. Data are representative of more than three independent experiments and presented as mean ± SD. Statistical differences were analyzed using two-way ANOVA with Tukey’s multiple comparisons test (A), Kruskal-Wallis test with Dunn’s multiple comparisons test (B–D and F), one-way ANOVA with Tukey’s multiple comparisons test (E), and Mann-Whitney U test (G). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.