Myeloid cell-specific sirtuin 6 deficiency delays wound healing in mice by modulating inflammation and macrophage phenotypes

Abstract

We recently reported that myeloid cell-expressed sirtuin 6 (Sirt6) plays a crucial role in M1 macrophage polarization and chemotaxis. Given the prominent role of macrophages during wound repair and macrophage heterogeneity, we hypothesized that a Sirt6 deficiency in myeloid cells would delay skin wound closure by affecting the phenotypes of macrophages in wounds. To address this question, a full-thickness excisional lesion was made in the dorsal skin of myeloid cell-specific Sirt6 knockout (KO) and wild-type mice. Wound closure was delayed in the KO mice, which exhibited less collagen deposition, suppressed angiogenesis, and reduced expression of wound healing-related genes compared to the wild-type mice. Using immunohistochemical, flow cytometric, and gene-expression analyses of macrophage subpopulations from wound tissue, we identified increased infiltration of M1 macrophages with a concomitant decrease in M2 macrophage numbers in the KO mice compared to the wild-type mice. Consistent with the in vivo wound closure defects observed in the KO mice, keratinocytes and fibroblasts treated with KO macrophage-derived conditioned medium migrated slower than those treated with wild-type macrophage-derived conditioned medium. An analysis of downstream signaling pathways indicated that impaired Akt signaling underlies the decreased M2 phenotypic switching in KO mice. These results suggest that a macrophage phenotypic switch induced by Sirt6 deficiency contributes to impaired wound healing in mice.

Fig. 1. Delayed wound healing in mS6KO mice.

a The process used to generate myeloid cell-specific Sirt6 knockout (mS6KO) mice is shown. b Excisional wounds were made by 6-mm-diameter punches. The wound sites were photographed at the indicated time. A day 0 picture was taken immediately after injury. Representative results from five individual mice from each group are shown. c Wound photographs were analyzed at the indicated times to determine wound closure in WT and KO mice. Values represent the mean ± SEM (n = 8). ^*p < 0.05 and ^**p < 0.01 vs. WT. d Histological sections of mouse skin wounds were examined by H&E staining. Bars = 250 μm

Fig. 2. Decreases in collagen deposition and angiogenesis in mS6KO mice.

a Representative photomicrographs of wounds stained with Masson’s trichrome or immunostained with an anti-vWF antibody at 0 and 14 days after injury are shown (original magnification, 200×). Bars = 50 μm. b The mRNA levels of extracellular matrix-related genes in wounds were determined by real-time RT-PCR (n = 8). c Skin wounds of WT and KO mice were double stained for vimentin and α-SMA expression to identify myofibroblasts. The vimentin and α-SMA-positive cells were counted in sections of the wounds harvested on day 14 (n = 6). Values represent the mean ± SEM. ^*p < 0.05 and ^**p < 0.01 vs. WT

Fig. 3. Increase in macrophage infiltration into wound sites in mS6KO mice.

a Immunohistochemistry was used to identify infiltrating macrophages (F4/80⁺ cells) at 0, 3, and 7 days after injury. Bars = 50 μm. b F4/80-positive macrophages were counted in wound sections. c The mRNA level of F4/80 (Adgre1) in the wound site was determined by real-time RT-PCR (n = 8). d, e Single-cell suspensions were prepared by enzymatically digesting and gently dissociating skin wounds. After excluding dead cells, the cells were analyzed by flow cytometry to identify all macrophages (F4/80^hiLy6g^loCD11b^hi) and M2 macrophages (F4/80^hiLy6c^loMHCII^hi) (n = 5–6). Values represent the mean ± SEM. ^*p < 0.05 and ^**p < 0.01 vs. WT

Fig. 4. Decrease in the number of M2 macrophages in wound sites in mS6KO mice.

The skin wounds of WT and KO mice were double stained for F4/80 and iNOS to identify M1 macrophages (a) or F4/80 and MRC1 to identify M2 macrophages (b). Digitally merged signals are shown in the right panels. Bars = 50 μm. Representative results from three independent experiments are shown

Fig. 5. Temporal changes in the expression of markers for M1 and M2 macrophages in wound sites.

a, b The mRNA levels of M1 and M2 marker genes in the wound site were determined by real-time RT-PCR (n = 8). c, d The levels of M1 and M2 cytokines in the tissue were measured using ELISA (n = 6). Values represent the mean ± SEM. ^*p < 0.05 and ^**p < 0.01 vs. WT

Fig. 6. Delayed migration of skin keratinocytes and fibroblasts in wound scratch assays.

Bone marrow macrophages (BMMs, 2 × 10⁶) isolated from WT or KO mice were treated with TNF-α (10 ng/ml), IL-1β (10 ng/ml), and IL-6 (10 ng/ml) for 3 h, and then conditioned medium (CM) was harvested. Scratch wounds were made in monolayers of HaCaT (a) and MDFB cells (b). For rescue experiments, KO BMMs were transduced with AdSirt6. Representative images show the progression of wound closure at the indicated times. The scratch area is indicated by a dotted line. The rate of wound closure was calculated by measuring the scratch area under the same magnification at different times. Values represent the mean ± SEM (n = 8) ^**p < 0.01 vs. WT; ^#p < 0.05; and ^##p < 0.01 vs. KO

Fig. 7. Regulation of M2 polarization by Sirt6.

a BMMs were treated with 10 ng/ml IL-4 and 10 ng/ml MCSF for 6 h, and the expression patterns of M2 marker genes were compared by RT-PCR. b BMMs were treated with 10 ng/ml IL-4 for the indicated periods, and then whole-cell extracts were generated and analyzed by Western blotting. c, d BMMs from mS6KO mice were transduced with an adenovirus expressing β-galactosidase (AdLacZ), Sirt6 (AdSirt6), or CA-Akt (AdAkt) at a multiplicity of infection (MOI) of 100 and treated with IL-4 for the indicated times. Protein and mRNA levels were analyzed. e, f BMMs from WT mice were treated with 10 ng/ml IL-4 and 10 ng/ml MCSF for 6 h with or without Akt inhibitors (8 μM MK2206 and 10 μM Akti), and then the protein levels in the whole cell extract and mRNA levels of M2 marker genes were determined. Values represent the mean ± SEM (n = 3). ^*p < 0.05 and ^**p < 0.01 vs. AdLacZ or Veh; ^#p < 0.05 vs. IL-4. Veh Vehicle, MK MK2206, i Akti