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. 2017 Feb:117:32-43.
doi: 10.1016/j.biomaterials.2016.11.021. Epub 2016 Nov 16.

Selective recruitment of non-classical monocytes promotes skeletal muscle repair

Cheryl L San Emeterio  1 , Claire E Olingy  1 , Yihsuan Chu  1 , Edward A Botchwey  2
Affiliations

Selective recruitment of non-classical monocytes promotes skeletal muscle repair

Cheryl L San Emeterio et al. Biomaterials. 2017 Feb.

Abstract

Regeneration of traumatic defects in skeletal muscle requires the synchronized behavior of multiple cells that participate in repair. The inflammatory cascade that is rapidly initiated after injury serves as a powerful node at which to guide the progression of healing and influence tissue repair. Here, we examine the role that myeloid cells play in the healing of traumatic skeletal muscle injury, and leverage their pro-regenerative functions using local delivery of the immunomodulatory small molecule FTY720. We demonstrate that increasing the frequency of non-classical monocytes in inflamed muscle coincides with increased numbers of CD206+ alternatively activated macrophages. Animals treated with immunomodulatory materials had greater defect closure and more vascularization in the acute phases of injury. In the later stages of repair, during which parenchymal tissue growth occurs, we observed improved regeneration of muscle fibers and decreased fibrotic tissue following localization of pro-regenerative inflammation. These results highlight non-classical monocytes as a novel therapeutic target to improve the regenerative outcome after traumatic skeletal muscle injury.

Keywords: Inflammation; Innate immunity; Macrophages; Monocytes; Regenerative medicine; Skeletal muscle.

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Figures

Figure 1
Figure 1. A novel volumetric muscle loss model in the murine spinotrapezius muscle enables study of cellular interactions during healing
(A, B) A 1mm circular volumetric defect made in the spinotrapezius of wildtype mice heals over 7 days, as quantified by (C) desmin immunostaining. (D) High-powered 3D images show granulation tissue under brightfield microscopy at days 3 and 7 post-injury, coinciding with accumulation of immunostained CD206+ cells. (E) Revascularization (shown by CD31 immunostaining) occurs along the defect edge at day 3 post-injury and among regenerating fibers at day 7. (F) Merged 3D images show the distribution of CD206+ cells among muscle fibers and revascularized muscle tissue. Data presented as mean ± S.E.M. Statistical analyses were performed using two-tailed t-tests. *p<0.05, n=4–5 animals per group.
Figure 2
Figure 2. Spinotrapezius volumetric muscle loss results in dynamic myeloid cell kinetics at both the systemic and tissue levels
(A) Following volumetric muscle injury in wildtype mice, blood neutrophils peak at day 3 post-injury and return to baseline levels by day 7 (blood neutrophils gated on CD11b+SSChiLy6Ghi). (B) Blood Ly6Chi monocytes increase over 7 days, while blood Ly6Clo monocytes gradually increase and plateau over days 3–7 (blood monocytes gated on CD11b+ SSCloCD115+). (C) Neutrophils in injured muscle tissue peak at day 3 and decrease by day 7 post-injury (tissue neutrophils gated on MerTK-CD64-CD11c-CD11b+SSChiLy6Ghi). (D) Ly6Chi monocytes in injured muscle tissue decrease from day 1 to day 7 post-injury, while Ly6Clo monocytes increase from day 1 to 3 and decrease by day 7 (tissue monocytes gated on CD11b+SSCloMerTK-CD64-CD11c-). (E) MerTK+CD64+ macrophages increase from day 1 to 7 post-injury. (F) CD206+ macrophages increase from day 1 to 7 post-injury. Data presented as mean ± S.E.M. Statistical analyses were performed using one-way ANOVA. †p<0.05 compared to Day 0 for Ly6Clo monocytes or *p<0.05 compared to Day 0 for all other cell types, n=4–12 animals per group.
Figure 3
Figure 3. Circulating latex bead-labeled non-classical monocytes are directly recruited to injured spinotrapezius muscle, where they give rise to alternatively activated macrophages
(A) Ly6Clo monocytes were selectively labeled by intravenous (i.v.) injection of latex (LX) beads 1 day prior to VML surgery in CX3CR1GFP/+ mice. (B) PLGA thin films were acutely implanted over spinotrapezius VML defects. (C) Both CX3CR1hi and CX3CR1lo cells, as well as LX+ cells, are recruited to the defect 3 days post-injury. (D) LX beads co-localize spatially with immunostained CD68+CD206+ macrophages 3 days post-injury. (E) Conditioned media from macrophages stimulated with IL-4 (M(IL-4)) promotes differentiation of C2C12 cells in vitro compared to conditioned media from macrophages stimulated with LPS and IFN-γ (M(LPS, IFN-γ)). Data presented as mean ± S.D.
Figure 4
Figure 4. On-site delivery of FTY720 increases Ly6Clo, CX3CR1hi monocytes in injured muscle
(A) Delivery of FTY720 from PLGA thin films to VML injury in wildtype mice increases the frequency of Ly6Clo monocytes, but not Ly6Chi monocytes 3 days post-injury compared to unloaded PLGA and no implant controls (tissue monocytes gated on CD11b+SSClo). (B) Few CX3CR1-GFP+ cells are seen in uninjured, contralateral spinotrapezius muscle in CX3CR1GFP/+ mice. (C) More CX3CR1hi cells are recruited to peri-defect tissue at day 3 in CX3CR1GFP/+ mice treated with FTY720. (D) LX+ cells cluster around defect in FTY720-treated CX3CR1GFP/+ mice at day 3 post-injury. Data presented as mean ± S.E.M. Statistical analyses were performed using two-way ANOVA. *p<0.05 compared to Ly6Clo monocytes in PLGA and no implant controls, n=3–4 animals per group.
Figure 5
Figure 5. FTY720 increases the frequency of CD68+CD206+ cells in injured muscle of CX3CR1GFP/+ mice
(A) FTY720-treated mice have a greater proportion of the defect region filled with infiltrating cells at 3 days post-injury, including LX+ and CX3CR1+ cells. (B) Quantification of cellular infiltration in CX3CR1/LX bead/lectin-labeled muscles shows a higher percentage of cellular infiltration in defects treated with FTY720. (C) FTY720-treated mice have a greater proportion of the defect region filled with CD68+CD206+ cells compared to PLGA controls 3 days post-injury. (D) Quantification of cellular infiltration in CD68/CD206/CD31 immunostained muscles shows a higher percentage of cellular infiltration in defects treated with FTY720. (E, F) Mice receiving FTY720 have increased density of CD68+CD206+ cells 3 days post-injury. (G) CD68+CD206+ cells are significantly closer to CD31+ blood vessels in the presence of FTY720 3 days post-injury. Data presented as mean ± S.E.M. Statistical analyses were performed using two-tailed t-tests. *p<0.05, n=3–4 animals per group.
Figure 6
Figure 6. On-site delivery of FTY720 promotes muscle healing 3 days post-injury in CX3CR1GFP/+ mice
(A) FTY720-treated mice have increased vascularization in the defect region, as detected by CD31 immunostaining and (B) quantification of CD31+ vessel density. (C) FTY720-treated mice have more collagen deposition within the defect region, as visualized by two-photon microscopy and (D) quantification of the non-collagenous void area. (E) Mice treated with FTY720 have a smaller defect area as measured by desmin immunostaining and (F) quantification of desmin-negative area (dotted red line indicates the mean value of wildtype mice undergoing VML and treated with no implant). Data presented as mean ± S.E.M. Statistical analyses were performed using two-tailed t-tests. *p<0.05, n=3–4 animals per group.
Figure 7
Figure 7. Local immunomodulation with FTY720 improves muscle repair 7 days post-injury in wildtype mice
(A) FTY720-treated mice have larger and more desmin+ muscle fibers (detected by immunostaining) that more closely recapitulates the structure of an uninjured contralateral control, as well as less dense and aligned collagen (detected by two-photon microscopy) compared to PLGA controls. (B) FTY720-treated mice have a reduced volume ratio of collagen to desmin. (C–D) FTY720-treated mice have regenerated muscle fibers with a larger diameter (dotted red line indicates the mean value of wildtype mice undergoing VML and treated with no implant). Data presented as mean ± S.E.M. Statistical analyses were performed using two-tailed t-tests. *p<0.05, n=3–4 animals per group.
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