Exosome-mediated delivery of super-repressor IκBα alleviates inflammation and joint damages in rheumatoid arthritis

Abstract

Background: This study aims to investigate the potential anti-inflammatory effects of exosomes engineered to carry super-repressor IκB (Exo-srIκB), an exosome-based NF-κB inhibitor, in the context of RA.

Methods: Peripheral blood mononuclear cells (PBMCs) and synovial fluid mononuclear cells (SFMCs) were collected from patients diagnosed with RA and treated with Exo-srIκB to test the therapeutic potential. Flow cytometry analysis was performed to assess the production of inflammatory cytokines (IL-17A and GM-CSF) by the cells. ELISA was utilized to measure the levels of TNF-α, IL-17A, IL-6, and GM-CSF. Arthritis was induced in SKG mice by intraperitoneal injection of curdlan. DBA/1 J mice were used in collagen-induced arthritis (CIA) experiments. After the development of arthritis, mice were injected with either Exo-Naïve (control exosome) or Exo-srIκB. Arthritis scores were recorded biweekly, and histological observations of the ankle joint were conducted using H&E and safranin-O staining. Additionally, bone erosion was evaluated using micro-CT imaging.

Results: In the ex vivo study involving human PBMCs and SFMCs, treatment with Exo-srIκB demonstrated a notable reduction in inflammatory cytokines. Furthermore, in both the SKG and CIA models, Exo-srIκB treatment exhibited significant reductions in inflammation, cartilage destruction, and bone erosion within the joint tissues when compared to the Exo-Naive control group. Additionally, the radiographic score assessed through microCT showed a significant decrease compared to the Exo-Naive control group.

Conclusion: Overall, these findings suggest that Exo-srIκB possesses anti-inflammatory properties in human RA cells and animal models, making it a promising therapeutic candidate for the treatment of RA.

Keywords: Exosome; Inflammation; NF-κB; Rheumatoid arthritis; Treatment.

Fig. 1

Production and characterization of Exo-srIκB. A DNA constructs and blue-light-mediated fusion of recombinant proteins were used to produce Exo-srIκB, as shown in schematic diagrams. B Representative images for transmission electron microscopy of Exo-srIκB morphology. C Representative panels for concentration and size distribution of Exo-srIκB were determined by a NanoSight (NS300) instrument. D Immunoblotting of Expi293F cells (producing cells) and Expi293F cell-derived exosomes to analyze the expression of recombinant proteins (srIκB, CRY2, and CD9) and exosome markers (CD9, CD81, TSG101, Alix, and GAPDH) and cell organelle markers (GM130, lamin B1, prohibitin, and calnexin). Exo-Naïve (non-engineered cell and exosome) were used as negative controls of Exo-srIκB

Fig. 2

Assessment of cell viability with Exo-srIκB. A The cell viability of PBMCs and SFMCs was assessed using the MTS assay, taking into account the duration of Exo-srIκB treatment. Statistical significance was determined using the Kruskal–Wallis test with Dunn’s multiple comparisons. The presented values represent the mean ± SEM. B A representative gating strategy for flow cytometry was employed to evaluate the survival rate. C Viability dyes were used to stain and measure PBMC and SFMC viability. Statistical significance was determined using a T-test. The presented values represent the mean ± SEM, and symbols represent individual sample. NS, not significant

Fig. 3

Exo-srIκB suppresses inflammatory cytokines in both PBMCs and SFMCs obtained from patients with rheumatoid arthritis. A, B Flow cytometry analysis was performed to assess the percentages of IL-17A and GM-CSF-positive cells in PBMCs and SFMCs. C, D ELISA was conducted to measure TNF-α, IL-17A, IL-6, and GM-CSF levels in ex vivo supernatants from PBMCs and SFMCs. Statistical significance was determined using the Wilcoxon matched-pairs signed-rank test. The symbols represent individual sample. NS, not significant. *P < 0.05

Fig. 4

Exo-srIκB demonstrates the ability to suppress arthritis in the SKG mouse model. A The animal study protocol is depicted, where 11-week-old female SKG mice were treated with either Exo-Naïve or Exo-srIκB starting from the 3rd week after arthritis induction, which was achieved by injecting curdlan. Arthritis scores were assessed based on the clinical severity of arthritis in each group, with a total of nine mice per group. Two-way analysis of variance (ANOVA) was performed to determine statistical significance for the clinical score. B A representative tissue stain of the ankle joint at the end of the experiment is displayed in the right panel, along with the analysis of histological scores for inflammation. Kruskal–Wallis test with Dunn’s multiple comparisons was performed to determine statistical significance. C The number of cells exhibiting co-expression of TNF-α or IL-17A among CD4-positive T cells per high-power field (HPF) was counted. The prevalence of CD4-positive T cells co-expressing TNF-α or IL-17A was significantly reduced in the ankle joints of mice treated with Exo-srIκB compared to those receiving Exo-Naïve treatment. Over 150 cells in each field were selected, ensuring the exclusion of nonspecific signals. A Mann–Whitney U-test was performed to determine statistical significance. D Analysis of the frequencies of IFN-γ, IL-17A, and TNF-α-producing cells was conducted on SKG splenocytes. Mann–Whitney U-test was performed to determine statistical significance. The values presented are the mean ± SEM, and the symbols represent individual sample. *P < 0.05, **P < 0.01

Fig. 5

Exo-srIκB exhibits the ability to suppress arthritis and reduce radiographic scores in CIA mouse model. A The upper panel shows the study protocol schematic. Arthritis scores were evaluated according to the clinical severity in each group, with five mice per group. Two-way analysis of variance (ANOVA) was performed to determine statistical significance for the clinical score. B In the upper panel, a representative tissue stain of the ankle joint at the end of the experiment is displayed. The lower panel presents the analysis of histological scores, including synovial hyperplasia, cartilage destruction, pannus formation, and bone erosion. C A representative microCT scan was shown. Radiographic scores were evaluated and analyzed for each group. Mann–Whitney U-test or Kruskal–Wallis test with Dunn’s multiple comparisons was performed to determine statistical significance for histologic and radiographic scores. Values are the mean ± SEM. Symbols represent the individual sample. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001