Introduction: Critical roles for the recruitment of inflammatory cells have been described for a broad spectrum of therapeutic applications, including skeletal muscle repair and limb regeneration, as well as generic host responses to biomedical implants. Biphasic recruitment kinetics of Ly6Chi (“pro-inflammatory”) monocytes from the blood is a hallmark of acute inflammation and is followed temporally by recruitment of Ly6Clo (“anti-inflammatory”) monocytes that facilitate inflammation resolution and re-vascularization of damaged tissue. While macrophages have been widely appreciated for their functions in mediating host response to implanted materials, a role for their monocyte precursors has remained largely unexplored. Using the murine dorsal skinfold window chamber model to monitor inflammatory processes in situ, we designed a series of experiments to explore the role that circulating monocytes play in wound healing. We hypothesized that enhancing recruitment of Ly6Clo monocytes from blood would increase the generation of pro-regenerative “M2” macrophages and improve vascular remodeling.
Methods and Materials: To monitor monocyte recruitment and vascular remodeling in real-time, we performed dorsal skinfold window chambers in mice, which entails split thickness skin wounding. Poly(lactic-co-glycolic acid) (PLGA) thin films were made by using a solvent-casting technique with or without the small molecule FTY720 and subsequently implanted in window chambers. Selective labeling of Ly6Clo monocytes was performed by intravenous injection of fluorescently-labeled latex beads 1 day prior to surgery. In separate studies, labeling of Ly6Chi monocytes was performed by intravenous administration of clodronate liposomes (ClodLip) 2 days prior to surgery, followed by latex beads 16 hours later. Intravital microscopy and whole mount immunohistochemistry was used to investigate longitudinal changes to the vasculature and monocyte/macrophage-vessel interactions, respectively. Monocyte subsets were immunophenotyped and quantified using flow cytometry analysis of blood and collagenase-digested tissue.
Results and Discussion: Circulating Ly6Clo monocytes labeled with latex beads were seen in inflamed tissue surround unloaded PLGA implants by 3 days post-surgery (Fig. 1A) and preferentially became CD206+ M2-like macrophages (Fig. 1B). Conversely, Ly6Chi monocytes labeled via sequential administration of ClodLip and latex beads similarly homed to inflamed tissue (Fig. 1C), but displayed no preference for differentiating into CD206+ macrophages (Fig. 1D). Selective depletion of blood Ly6Clo monocytes with ClodLip (Fig. 1E) severely impaired the in situ generation of CD206+ macrophages (Fig. 1F).
Consequently, blood-derived Ly6Clo monocytes are biased progenitors of CD206+ wound macrophages that efficiently undergo differentiation post-extravasation. On site delivery of the immunomodulatory small molecule FTY720 increased the frequency of recruited Ly6Clo monocytes (data not shown) and CD206+F4/80+ macrophages 3 days post-surgery (Fig. 2A). In loss-of-function studies employing ClodLip, we observed that FTY720 no longer increased the frequency of M2 macrophages (Fig. 2B), indicating that circulating Ly6Clo monocytes are required. To explore the function of CD206+ macrophages, we probed the distribution of CD68+CD206+ cells in explanted whole tissue samples. FTY720 increased the frequency of CD68+CD206+ macrophages and positioned these cells around remodeling blood vessels (Fig. 2C). Moreover, FTY720-mediated regulation of monocyte fate corresponded with expansion of arterioles and angiogenic sprouting (Fig. 2D).
Conclusions: We have demonstrated that circulating Ly6Clo monocytes are robustly recruited to inflamed peri-implant tissue following injury and efficiently undergo differentiation into pro-regenerative macrophages. Recruitment of Ly6Clo monocytes by biomaterial-released FTY720 is a promising strategy for tuning the innate immune response to improve vascularization and wound healing.
National Institutes of Health grants R01AR056445-01A2 and R01DE019935-01 to Dr. Botchwey; National Science Foundation Graduate Research Fellowship to Claire Segar under Grant No. No. DGE-1148903; American Heart Association Pre-doctoral Fellowship 15PRE25090024 to Claire Segar