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. 2024 May 17:19:4411-4427.
doi: 10.2147/IJN.S454445. eCollection 2024.

Administration of Liposomal-Based Pde3b Gene Therapy Protects Mice Against Collagen-Induced Rheumatoid Arthritis via Modulating Macrophage Polarization

Longmin Chen  1 , Qing Zhou  2 , Xun Fang  1 , Qianqian Xu  2 , Yuan Zou  2 , Jing Zhang  2
Affiliations

Administration of Liposomal-Based Pde3b Gene Therapy Protects Mice Against Collagen-Induced Rheumatoid Arthritis via Modulating Macrophage Polarization

Longmin Chen et al. Int J Nanomedicine. .

Abstract

Background: Rheumatoid arthritis (RA) is a chronic and systemic autoimmune disease characterized by synovial inflammation and joint destruction. Despite progress in RA therapy, it remains difficult to achieve long-term remission in RA patients. Phosphodiesterase 3B (Pde3b) is a member of the phosphohydrolyase family that are involved in many signal transduction pathways. However, its role in RA is yet to be fully addressed.

Methods: Studies were conducted in arthritic DBA/1 mice, a suitable mouse strain for collagen-induced rheumatoid arthritis (CIA), to dissect the role of Pde3b in RA pathogenesis. Next, RNAi-based therapy with Pde3b siRNA-loaded liposomes was assessed in a CIA model. To study the mechanism involved, we investigated the effect of Pde3b knockdown on macrophage polarization and related signaling pathway.

Results: We demonstrated that mice with CIA exhibited upregulated Pde3b expression in macrophages. Notably, intravenous administration of liposomes loaded with Pde3b siRNA promoted the macrophage anti-inflammatory program and alleviated CIA in mice, as indicated by the reduced inflammatory response, synoviocyte infiltration, and bone and cartilage erosion. Mechanistic study revealed that depletion of Pde3b increased cAMP levels, by which it enhanced PKA-CREB-C/EBPβ pathway to transcribe the expression of anti-inflammatory program-related genes.

Conclusion: Our results support that Pde3b is involved in the pathogenesis of RA, and Pde3b siRNA-loaded liposomes might serve as a promising therapeutic approach against RA.

Keywords: Pde3b; liposomes; macrophages; rheumatoid arthritis.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Arthritic mice exhibit increased Pde3b expression in synovial macrophages. Representative immunofluorescence staining results for F4/80 and Pde3b in joint sections from controls and arthritic mice. Scale bars: 100 μm. Original magnification: ×400. St, synovial tissue.
Figure 2
Figure 2
Pde3b knockdown orchestrates alternative activation of macrophages. (a and b) Verification of the interference efficiency of Pde3b siRNAs in BMDMs at the mRNA (a) and protein (b) level. (c) Volcano plot of the differentially expressed genes (DEGs) between scramble and Pde3b siRNA-transfected BMDMs, as determined by RNA-seq. Four biological replicates were included for each group. (d) Heatmap showing the top 10 significantly upregulated genes in Pde3b siRNA-transfected BMDMs. (e) RT-qPCR analysis of Arg1 and Retnla mRNA expression in scramble and Pde3b siRNA-transfected BMDMs, either unstimulated or treated with IL-4 for 6 h. (f) Western blot analysis of Arg1 expression in BMDMs before and after IL-4 stimulation. (g) Representative FACS plots and quantitative data of CD206 expression in BMDMs with or without IL-4 stimulation for 24 h. (h) Flow cytometry analysis of CD86 expression in scramble and Pde3b siRNA-transfected BMDMs with or without LPS treatment for 24 h. (i) RT-qPCR analysis of relative mRNA levels of Tnf, Il6 and Il1b in BMDMs treated with or without LPS. (j) Gene set enrichment of genes describing for TCA cycle in Pde3b siRNA-transfected BMDMs, relative to scramble siRNA-transfected BMDMs. (k) OCR of scramble and Pde3b siRNA-transfected BMDMs with or without IL-4 stimulation for 24 h, which was assessed before and after sequential addition of oligomycin, FCCP, and antimycin A/rotenone. (l) Accordingly, basal OCR and spare respiratory capacity (SRC) were shown. Data were collected from three (a, b, e, g-i, k, and l) or four (f) independent experiments. Values are presented as mean ± SEM. Significance was determined by one-way ANOVA in (a and b) and by unpaired Student’s t test in other figure parts. *p < 0.05; **p < 0.01; ***p < 0.001. Arg1, Arginase 1; Unsti, unstimulated.
Figure 3
Figure 3
Characterization and biodistribution of the siRNA-loaded liposomes. (a) Schematic diagram showing generation of the Pde3b siRNA-loaded liposomes. (b) Hydrodynamic diameter, polymer dispersity index (PDI), and zeta-potential of blank and siRNA-loaded liposomes measured by dynamic light scattering (DLS). siRNA entrapment efficiency was evaluated by Ribogreen assay. (c) Representative transmission electron microscopy (TEM) image of siRNA-loaded liposomes. Scale bars: 300 nm. (d) Hydrodynamic diameter distribution of siRNA-loaded liposomes. (e) Colloid stability of siRNA-loaded liposomes in PBS solution. (f) The biosafety of siRNA-loaded liposomes in vitro determined by flow cytometry. (g) Ex vivo DiR fluorescence images showing the biodistribution of siRNA-loaded liposomes in arthritic mice at 24 h post injection. (h) Flow cytometry analysis of the DiO-labeled liposome distribution in the ankle joints of arthritic mice. (i) Temporal expression of Pde3b in synovial tissues after administration of the scramble siRNA- or Pde3b siRNA-loaded liposomes. Data are expressed as mean ± SEM. Statistical difference in (f) was analyzed using unpaired Student’s t test.
Figure 4
Figure 4
Therapeutic efficacy of the Pde3b siRNA-loaded liposomes in mice with collagen-induced arthritis (CIA). (a) Schematic outline of the experimental design and time line of liposome treatment. (b) Representative image of the hindlimbs at the endpoint of the experiment. (c) Paw thickness of CIA mice recorded during the experimental process. (d) Graph summarizing clinical score. Normal mice, n = 4; CIA mice treated with blank liposome and Pde3b siRNA, n = 7 per group; CIA mice treated with siRNA-loaded liposomes, n = 10 in each study group (c, d). (e) Representative images for H&E (top), toluidine blue (middle), and safranin O (bottom) staining of ankle joints at the endpoint of the experiment. Scale bars: 200 μm. Original magnification: ×200. (f-h) Analysis of plasma IL-1β (f), TNF-α (g), and IL-17A (h) levels in normal mice (n = 4), CIA mice treated with blank liposome (n = 4), Pde3b siRNA (n = 4), and siRNA-loaded liposomes (n = 5 per group). Values are expressed as mean ± SEM, and one-way ANOVA was employed for data analysis. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5
Figure 5
Administration of Pde3b siRNA-loaded liposomes prevents bone erosion in arthritic mice. (a) Representative 3D-reconstructed images of hind paws by micro-CT analysis. (b) Representative micro-CT images of the reconstructed trabecular structure in bone. (c-g) Quantitative micro-CT analysis of BMD (c), BS/BV (d), Tb.N (e), Tb.Th (f), and Tb.Sp (g) of the ankle joints at the endpoint of the experiment. Normal mice, n = 3; CIA mice treated with blank liposome and Pde3b siRNA, n = 4 for each group; CIA mice treated with siRNA-loaded liposomes, n = 5 for each group. Values are presented as mean ± SEM. Statistical significance was accessed by one-way ANOVA. *p < 0.05; **p < 0.01. BMD bone mineral density; BS/BV, bone surface vs bone volume; Tb.N, trabecular number; Tb.Th, trabecular bone thickness; Tb.Sp trabecular separation.
Figure 6
Figure 6
Silencing Pde3b promotes anti-inflammatory macrophage polarization in vivo. (a-f) Single-cell suspension was prepared from mouse synovial tissues and popliteal lymph nodes at the endpoint of the experiment and subject to flow cytometry analysis. (a) Representative FACS plots and percentages of F4/80+CD11b+ macrophages in the synovial tissues from normal mice and CIA mice treated with blank liposome, Pde3b siRNA, or siRNA-loaded liposomes. (b and c) Flow cytometry analysis of CD86 (b) and CD206 (c) expression in synovial macrophages. (d) Representative FACS plots and percentages of F4/80+CD11b+ macrophages in the popliteal lymph nodes from normal mice and CIA mice treated with blank liposome, Pde3b siRNA, or siRNA-loaded liposomes. (e and f) Flow cytometry analysis of CD86 (e) and CD206 (f) in macrophages from the popliteal lymph nodes. (g and h) RT-qPCR analysis of the mRNA abundance for the indicated genes in synovial tissues. n = 4 in each study group. Data are presented as mean ± SEM. Statistical difference was determined by one-way ANOVA. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 7
Figure 7
Pde3b knockdown enhances macrophage anti-inflammatory program by regulating PKA-CREB-C/EBPβ signaling. (a) ELISA analysis of cAMP in the culture supernatants of BMDMs transfected with scramble or Pde3b siRNA. (b) Western blot analysis of (p-)CREB expression in BMDMs before and after IL-4 stimulation. (c) Results for C/EBPβ expression in BMDMs with or without IL-4 stimulation for 24 h. (d) ChIP-qPCR was performed for C/EBPβ in the Arg1 promoter in scramble or Pde3b siRNA-transfected BMDMs following IL-4 stimulation. (e) Western blot analysis of C/EBPβ and Arg1 levels in scramble or Pde3b siRNA-transfected BMDMs under indicated culture conditions. Data were collected from three independent experiments (a-e). Values are expressed as mean ± SEM. Statistical significance was analyzed by unpaired Student’s t test. *p < 0.05; **p < 0.01. Unsti, unstimulated.
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Grants and funding

Our study was supported by the National Natural Science Foundation of China (82300929 and 82100892), Department of Science and Technology of Hubei Province Program Project (2022CFB739), the Intramural Research Program of the Central Hospital of Wuhan (21YJ01, 23YJ14), and Wuhan Talent Project.
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