A Tissue-Tended Mycophenolate-Modified Nanoparticle Alleviates Systemic Lupus Erythematosus in MRL/ Lpr Mouse Model Mainly by Promoting Local M2-Like Macrophagocytes Polarization

Abstract

Background: Mycophenolate mofetil (MMF), for which the bioactive metabolite is mycophenolic acid (MPA), is a frequently used immunosuppressant for systemic lupus erythematosus (SLE). However, its short half-life and poor biodistribution into cells and tissues hinder its clinical efficacy. Our dextran mycophenolate-based nanoparticles (MPA@Dex-MPA NPs) have greatly improved the pharmacokinetics of MMF/MPA. We here tested the therapeutic efficacy of MPA@Dex-MPA NPs against SLE and investigated the underlying mechanism.

Methods: The tissue and immune cell biodistributions of MPA@Dex-MPA NPs were traced using live fluorescence imaging system and flow cytometry, respectively. Serological proinflammatory mediators and kidney damage were detected to assess the efficacy of MPA@Dex-MPA NPs treatments of MRL/lpr lupus-prone mice. Immune cell changes in the kidney and spleen were further analyzed post-treatment via flow cytometry. Bone marrow-derived macrophages were used to investigate the potential mechanism.

Results: MPA@Dex-MPA NPs exhibited superior therapeutic efficacy and safety in the MRL/lpr mice using significantly lower administration dosage (one-fifth) and frequency (once/3 days) compared to MMF/MPA used in ordinary practice. The overall prognosis of the mice was improved as they showed lower levels of serological proinflammatory mediators. Moreover, kidney injury was alleviated with reduced pathological signs and decreased urine protein-creatinine ratio. Further investigations of the underlying mechanism revealed a preferential penetration and persistent retention of MPA@Dex-MPA NPs in the spleen and kidney, where they were mostly phagocytosed by macrophages. The macrophages were found to be polarized towards a CD206⁺ M2-like phenotype, with a downregulation of surface CD80 and CD40, and reduced TNF-α production in the spleen and kidney and in vitro. The expansion of T cells was also significantly inhibited in these two organs.

Conclusion: Our research improved the efficacy of MPA for MRL/lpr mice through synthesizing MPA@Dex-MPA NPs to enhance its tissue biodistribution and explored the possible mechanism, providing a promising strategy for SLE therapy.

Keywords: macrophage polarization; mycophenolate; nanoparticles; systemic lupus erythematosus; tissue damage.

Graphical abstract

Figure 1

Biodistribution of MPA@Dex-MPA NPs among the tissues and immune cells of MRL/lpr mice. (A) Preparation procedure for MPA@Dex-MPA NPs. (B) Fluorescence microscopy images of the liver, spleen, kidney, lung and heart of MRL/lpr mice at 0, 0.5, 1, 2, 6, 24, 48 and 72 h post intraperitoneal injection of Cy7-labeled MPA@Dex-MPA NPs. (C) Cellular internalization of Cy5-labeled MPA@Dex-MPA NPs in the spleen and kidney. The proportions of T cells, B cells, DCs and macrophages that internalized the Cy5-labeled MPA@Dex-MPA NPs were analyzed at 6 h post intraperitoneal injection through flow cytometry analysis (n = 5/group). Error bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2

MPA@Dex-MPA NPs treatment improve the survival and reduce the levels of serological proinflammatory mediators in MRL/lpr mice. (A) Schema for the animal experiment (n = 8/group). (B) Survival curves for the MRL/lpr mice during the experiment (n = 8/group). (C) Serum levels of anti-dsDNA IgG in the MRL/lpr mice from age of 12 to 24 weeks (n = 4–8/group). (D and E) Serum concentrations of anti-dsDNA IgG (D) and cytokines (IL-6, IL-17A, TNF-α, IFN-γ) (E) at the age of 24 weeks (n = 4–6/group). Error bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 3

MPA@Dex-MPA NPs improve renal function and alleviate the pathological signs in the kidneys of the MRL/lpr mice. (A) The UPCR was obtained by measuring the urine protein and creatinine concentrations during the animal experiment (n = 4–8/group). (B) The UPCR and the BUN level was determined at the end of animal experiment (n = 4–6/group). (C) The representative micrographs of glomeruli stained by PAS. (D) The glomerulonephritis score was assessed. (E) The representative immunofluorescence micrographs of the deposition of IgG and complement 3 in the glomeruli (green, IgG; red, C3; blue, nucleus; n = 4–6/group). The fluorescence intensity of (F) IgG and (G) C3 was assessed. Scale bar, 20 μm. Error bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Changes of the immune cells in the kidney and spleen of MRL/lpr mouse after MPA@Dex-MPA NPs therapy. Counts were taken of infiltrating cells per kidney including the following: (A) CD45⁺ immune cells; (B) T cells; (C) CD11b⁺ myeloid cells; and (D) F4/80⁺ macrophages. These calculations were made using precision count beads and flow cytometry. (E and F) The percentages of CD80⁺ macrophages (E) and CD206⁺ M2-like macrophages (F) among the renal macrophages were also calculated. (G and H) The expressions of CD40 (G) and CD206 (H) among the splenic F4/80⁺ macrophages were assayed using flow cytometry. (I) Flow cytometric analysis of the percentage of splenic T cells among the total immune cell population. (J) Proportions of CD4⁺ T cells, CD8⁺ T cells and CD4⁻CD8⁻ DNT cells among the total T cell population. (K) Flow cytometric analysis of the percentage of splenic CD138⁺ plasma cells among the CD45⁺ immune cells. Error bars represent the mean ±SEM, n = 4–6/group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

MPA@Dex-MPA NPs inhibit the viability and inflammatory function of BMDMs in vitro. (A) Representative confocal microscope images of the BMDM uptake of Nile Red-MPA@Dex-MPA NPs at 2 h post-treatment. Blue, nucleus; red, NPs. Scale bar, 20 μm. (B) CCK-8 analysis of BMDM viability after treatment with dextran, fMPA or MPA@Dex-MPA NPs for 24, 48 or 72 h, relative to the PBS control group. (C) Apoptotic response of BMDMs measured at 72 h by flow cytometry analysis. (D–F) Flow cytometry analysis of the percentages of CD40-, CD80-, and MHC-II- (D), or CD86-positive (E) BMDMs, and also of CD206⁺ M2-like BMDMs (F) after 24 h-pretreatment of the cells and further stimulation with lipopolysaccharide and IFN-γ for 12 h. (G) Supernatant level of TNF-α. Error bars represent the mean ± SEM, n = 5/group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

MPA@Dex-MPA NPs treatments exhibit an excellent long-term safety profile in C57BL/6 mice. (A) Body weight gaining curves for the mice during the 4 weeks’ treatment period. (B) Serum levels of ALT and AST detected by biochemical analyzer (n = 5/group). (C) BUN and serum creatinine levels, also detected by biochemical analyzer (n = 5/group). (D–F) Hemoglobin level (D), platelets count (E), and total white blood cell and lymphocyte counts (F) were determined using a hematology analyzer (n = 5/group). Error bars represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

H&E staining of the major organs in the C57BL/6 mice indicated no obvious pathological manifestations after a 4 week’s MPA@Dex-MPA NPs treatment regimen. Representative H&E staining images are shown of the heart, liver, spleen, lung and kidney (n = 5/group). Scale bar, 50 μm.