Functional muscle recovery with nanoparticle-directed M2 macrophage polarization in mice

Abstract

Persistence of inflammation, and associated limits in tissue regeneration, are believed to be due in part to the imbalance of M1 over M2 macrophages. Here, we hypothesized that providing a sustained source of an antiinflammatory polarizing cytokine would shift the balance of macrophages at a site of tissue damage to improve functional regeneration. Specifically, IL-4-conjugated gold nanoparticles (PA4) were injected into injured murine skeletal muscle, resulting in improved histology and an ∼40% increase in muscle force compared with mice treated with vehicle only. Macrophages were the predominant infiltrating immune cell, and treatment with PA4 resulted in an approximately twofold increase in the percentage of macrophages expressing the M2a phenotype and an approximately twofold decrease in M1 macrophages, compared with mice treated with vehicle only. Intramuscular injection of soluble IL-4 did not shift macrophage polarization or result in functional muscle improvements. Depletion of monocytes/macrophages eliminated the therapeutic effects of PA4, suggesting that improvement in muscle function was the result of M2-shifted macrophage polarization. The ability of PA4 to direct macrophage polarization in vivo may be beneficial in the treatment of many injuries and inflammatory diseases.

Keywords: IL-4; macrophage polarization; muscle regeneration; nanomedicine; regenerative medicine.

Fig. 1.

Mcs/Mφs are required for regeneration of muscle function following ischemic injury. (A) Timeline showing clodronate treatment, injury, and injection to the ischemic TA. (B) Quantification of CD45⁺ cells in the TA, day 9, flow cytometry data. (C) Gating of CD45⁺ cells. (D–F) Number of Mφs (CD11c⁻/CD11b⁺/Ly6G⁻/Ly6C⁻/F4/80⁺) (D), Mcs (CD11c⁻/CD11b⁺/Ly6G⁻/Ly6C⁺/F4/80⁻) (E), and Mcs/Mφs (CD11c⁻/CD11b⁺/Ly6G⁻/Ly6C⁺/F4/80⁺) (F) in the TA. (G and H) Maximum TA contraction force and velocity of three tests. Force was normalized to TA mass. Data are means ± SEM, n = 7. *P < 0.05; ***P < 0.001; NS, not significant. Clod, clodronate; Cont, contralateral; Isch, ischemic.

Fig. 2.

AuNP Synthesis and human IL-4 conjugation. (A and B) Schematic showing partial PEGylation and subsequent IL-4 conjugation to AuNPs. (C–E) DLS size distribution of 30-nm (C), 60-nm (D), and 100-nm (E) AuNP core (black), AuNP-PEG (light color), and PA4 (dark color) particles. Data are from a representative synthesis. (F) PA4 DLS size distribution after IL-4 conjugation (dark pink) and after 1 d in RPMI + 10% HI-FBS at 37 °C (light pink). DLS distribution of RPMI + 10% HI-FBS (gray) is shown. Data are means ± SD, n = 3. (G) Thirty-nanometer-core PA4 were incubated at 37 °C, 5% CO₂ for 7 d, and the release of IL-4 into 1% BSA or RPMI + 10% HI-FBS was quantified with ELISA. Data are means ± SD, n = 3.

Fig. 3.

PA4 directed M2a Mφ polarization. (A and B) THP-1 derived Mφs were treated for 3 d with 20 ng/mL IL-4 as 30-nm PA4 or soluble IL-4 or 30-nm AuNP-PEG. Plots show expression of the M2a (CD206) or M2c (CD163) vs. the M1 marker (CCR7). (C) Percentage of M2a (CCR7⁻/CD163⁻/CD206⁺) Mφs. (D) CD206 median fluorescent intensity (MFI) within the CD206⁺ gate. (E and F) Mφs were treated for 1 d with 20 ng/mL IL-4 as 30-nm PA4 or soluble IL-4, AuNP-PEG, or basal RPMI. On day 5, flow cytometry was used to quantify M2a (CCR7⁻/CD206⁺) Mφs. (F) CD206 MFI within the CD206⁺/CCR7⁻ gate. Data are means ± SD, n = 4. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant. (G–I) Mφs were treated with 20 ng/mL IL-4 as soluble IL-4 or PA4 with varying degrees of IL-4 conjugation, 1×, 0.5×, 0.1×, and 0.01× (relative to the PA4s used throughout the work). (G) Flow cytometry was used to quantify M2a (CD206⁺/CCR7⁻) Mφs. (H) CD206 MFI within the CD206⁺ gate. (I) Percentage of M1 (CD206⁻/CCR7⁺) Mφs. Data are means ± SEM, n = 4. *P < 0.05; **P < 0.01; ***P < 0.001 (Dunnett’s comparison vs. basal media control).

Fig. 4.

PA4 enhanced muscle fiber regeneration and contraction force following ischemic injury. (A) Timeline showing surgery, treatment, and TA analysis. (B) Representative image showing the purple hue of AuNPs throughout the TA on day 6. (C) Representative H&E of ischemic TAs on day 15 hematoxylin (Center) and eosin (Right) channels. (Scale bars, 1.5 mm.) (D–F) Percentage of the TA cross-section associated with cell nuclei, muscle fibers, or empty area. Data are means ± SD, n = 3. *P < 0.05; **P < 0.01. Due to large variations in empty area on day 6, these data were excluded from statistics. (G and H) Maximum contraction force (25 V, 250 Hz) and velocity. Force was normalized to TA mass. Data are means ± SEM. Day 6, n = 5; day 15, n = 16 for PA4 and PBS, n = 5 for AuNP-PEG and bolus IL-4. *P < 0.05 (Bonferroni planned comparison).

Fig. 5.

Immune cells are cleared more rapidly following PA4 treatment. (A) Plots show the percentage of TA cells that were CD45⁺. (B–D) CD45⁺, Mcs (CD45⁺/CD11b⁺/CD3⁻/F4/80⁻/Gr1^{lo or-}), and Mφs (CD45⁺/CD11b⁺/CD3⁻/F4/80⁺) data normalized to TA mass (milligrams). (E) Distribution of CD45⁺ cells in the TA: Mφs (pink; CD11b⁺/CD3⁻/F4/80⁺), inflammatory Mcs (black; Ly6C^Hi/Gr1^Lo), Mcs (black above gray border; Ly6C^Hi/Gr1⁻), patrolling Mcs (gray; Ly6C^{Lo or-}/Gr1⁻), neutrophils (blue; Ly6C^Lo/Gr1^Hi), T cells (gray above the blue; CD3⁺), and CD11b⁻/CD3⁻ (white). (F) Gating of myeloid (CD45⁺/CD11b⁺/CD3⁻/F4/80⁻) cells on day 6. Plots show diminishing inflammatory Mcs (Ly6C^Hi/Gr1^Lo) with PA4 treatment. (G) Inflammatory Mcs (CD45⁺/CD11b⁺/CD3⁻/F4/80⁻/Gr1^Lo/Ly6C^Hi) in TA normalized by mass (milligrams). Data are means ± SEM, n = 5–10. *P < 0.05; **P < 0.01; ***P < 0.001. Asterisks above columns indicate significance vs. contralateral. Cont, contralateral; Isch, ischemic.

Fig. 6.

PA4 enhanced muscle regeneration by directing M2a polarization. (A) Plots show the percentage of Mφs (F4/80⁺/CD11b⁺) from the ischemic TAs expressing CD206 (M2a) or CD80 (M1). (B and D) Percentage of Mφs expressing the M2 (CD80⁻/CD86⁻/CD163⁺/CD206⁺) or M1 (CD80⁺/CD163⁻/CD206⁻) phenotype. (C and E) CD206 (M2a) or CD86 (M1) MFI. Data are means ± SEM, n = 3–5. *P < 0.05; **P < 0.01. (F and G) Mice were treated with either clodronate or PBS-liposomes and PA4. Maximum TA contraction force and velocity of three tests on day 9 are shown. Force was normalized to TA mass. Data are means ± SEM, n = 5. *P < 0.05. Isch, ischemic.