Significance Chronic inflammation contributes to the progression of many diseases, including 7 of the 10 leading causes of death. Macrophages play a central role in regulating inflammation because they adopt proinflammatory (M1) and proregenerative (M2) phenotypes. While an initial M1 response is critical, the prolonged presence of M1 macrophages, or the imbalance of M1 over M2 macrophages, can cause tissue damage and inhibit regeneration. We demonstrate that gold nanoparticles can be used to deliver a cytokine to direct M2 macrophage polarization following muscle injury in vivo. The polarization shift promoted regeneration and increased muscle strength. The ability to direct macrophage polarization in an inflammatory microenvironment may be useful in the treatment of many injuries and inflammatory diseases.

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.

Acute inflammation is a protective response that kills invading pathogens, should be self-limiting, and leads to healing. However, uncontrolled activation of immune cells, and failure of the acute inflammatory response to be self-limiting, leads to chronic inflammation, resulting in tissue damage (1). Following tissue injury or infection, monocytes (Mcs) are recruited from circulation and differentiate to M1 macrophages (Mφs), which promote inflammation by the release of inflammatory cytokines, reactive oxygen species, proteases, and antimicrobial peptides (2). Subsequently, Mφs adopt potent antiinflammatory and proregenerative activity, broadly referred to as M2 Mφs. Beyond antagonizing M1 responses, M1-to-M2 phenotype switching is important in promoting tissue regeneration and restoring homeostasis (2⇓–4). M1–Mφ-dominated aberrant inflammation contributes to the pathogenesis of many chronic inflammatory conditions, including atherosclerosis, inflammatory bowel disease, asthma, rheumatoid arthritis, osteoarthritis, multiple sclerosis, and chronic venous leg ulcers (5⇓⇓⇓⇓–10). Hence, development of therapeutics that can dampen acute inflammation and promote M2 polarization are of considerable interest.

Mφ polarization plays a central role in directing skeletal muscle regeneration following injury (11, 12). Satellite cells, muscle resident stem cells, become activated to proliferate, migrate to the injury, fuse, and differentiate to form new myofibers (13, 14). Mφs directly control satellite-cell activation and maturation and are crucial to muscle regeneration (15, 16). M1 Mφs promote the proliferation of satellite and myogenic precursor cells, in vitro and in vivo, following human muscle injury, while M2 Mφs promote their differentiation (15). Imbalanced Mφ polarization, specifically skewing toward M1 phenotypes, has been shown to inhibit skeletal muscle repair (3, 16, 17). Transition to M2 phenotypes is critical to ultimately yield functional muscle (18).

The hypothesis underlying this study is that IL-4–conjugated gold nanoparticles (AuNPs) can direct M2a Mφ polarization, thereby enhancing regeneration of functional skeletal muscle following ischemic injury. IL-4 is an antiinflammatory cytokine that can induce the polarization of M1 Mφs toward the M2a state. Exogenous IL-4 is sufficient to drive accumulation of M2 Mφs through self-renewal, suggesting that IL-4 delivery can induce the expansion of therapeutic M2 Mφs without necessitating further recruitment of destructive M1 Mφs (19). IL-4 has been widely explored as a potential therapeutic in various inflammatory disease models, including autoimmune demyelinating disease, arthritis, and chronic skin inflammation (20⇓–22). However, its use has required repeated infusions due to its short half-life in vivo. Resultant high-dose requirements and systemic side effects have limited the use of IL-4 treatments (23). Here, we utilize nanoparticles (NPs) for IL-4 delivery, as they allow distribution throughout the targeted tissue and can extend retention time compared with bolus delivery. AuNPs were specifically used because they can be synthesized with monodisperse size over a clinically relevant range (24), can be injected, and show minimal toxic or immunogenic activity in humans (25, 26). Formulations of gold (Au) are Food and Drug Administration-approved for the treatment of arthritis, a chronic inflammatory condition (27).

Materials and Methods For additional methods, see SI Appendix. Surgery. Animal work was in compliance with National Institutes of Health and the Harvard Institutional Animal Care and Use Committee. Hindlimb ischemia was induced in female C57BL/6J mice (6–8 wk; Jackson Laboratories) by left unilateral femoral artery and vein ligation (40). On day 3, ischemic TAs were injected with 2 μg of IL-4 as PA4 or soluble IL-4, AuNP-PEG, or PBS; two 10-μL injections were given to each TA. Mφ Depletion. Clodronate or PBS liposomes (Liposoma) were administered (SI Appendix, Fig. S1A). Flow Cytometry. Cells from TAs and in vitro experiments were stained for surface markers of immune polarization. TA Force. TAs were mounted ex vivo between electrodes (45). Three tetanic contractions were evoked. Contraction force, the difference between maximum force and baseline, was normalized to TA mass. Contraction velocity was the slope of the force curve at stimulation (SI Appendix, Fig. S2). AuNP Synthesis. AuNPs were synthesized by hydroquinone reduction of Au onto citrate-stabilized seed particles (24). PEG and IL-4 Conjugation. AuNP concentration was calculated with the Beer–Lambert Law. Partial PEGylation of AuNPs (5-kDa PEG-SH; Laysan Bio Inc) occurred overnight. Subsequently, recombinant human or murine IL-4 (Peprotech) was conjugated in the presence of trehalose (Sigma catalog no. T0167). Subtractive analysis was used to quantify bound IL-4. Histology. Images were tiled across the TA cross-section. To assess the percentage of the cross-section composed of muscle fibers (eosin) or nuclei (hematoxylin), ImageJ color deconvolution was performed. Statistics. All analyses were performed on GraphPad Prism5. For experiments that involved more than one comparison, ANOVA with Tukey or Bonferroni post hoc test was used. Where noted, Dunnett’s comparison vs. a control condition was used. For assessment of muscle function, a power analysis was performed on G*Power3.1 (46). PA4 and PBS groups were performed with n = 16 (per the analysis), and Bonferroni planned comparison was used.

Acknowledgments This work was supported by National Institutes of Health Grant DP3DK108224; the Wyss Institute for Biologically Inspired Engineering at Harvard University; and the National Science Foundation Graduate Research Fellowship Program (T.M.R.).

Footnotes Author contributions: T.M.R. and D.J.M. designed research; T.M.R. performed research; T.M.R. analyzed data; and T.M.R. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

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