**2. Results**

#### *2.1. Delayed Wound Healing in IFN-*γ*-Deficient Mice in the Proliferative Phase*

To examine the possible contribution of IFN-γ to wound healing, the rate of wound closure in IFN-γKO mice was compared with that in WT mice. Wound closure on Day 10 was significantly delayed in IFN-γKO mice compared with WT mice (Figure 1A,B). To confirm this e ffect, wound breaking strength was examined. Wound breaking strength on Day 14 was significantly delayed in IFN-γKO mice compared with WT mice (Figure 1C). As an alternate indicator of wound healing, we also evaluated α-SMA, which indicates myofibroblast di fferentiation. As shown in Figure 1D, the number of α-SMA<sup>+</sup> cells was significantly decreased in IFN-γKO mice. In addition, IFN-γKO mice exhibited lower *COL1A1*, *COL3A1*, and *TGF-*β*1* expression compared with WT mice on Day 14 (Figure 1E).

**Figure 1.** IFN-γ deficiency leads to impaired wound healing in skin. Wounds were created on the backs of WT or IFN-γKO mice. (**A**) Wound photographs in WT or IFN-γKO mice. (**B**) Percentage of wound closure was evaluated on Days 5, 7, and 10. (**C**) Wound breaking strength was measured on day 14. (**D**) The number of myofibroblasts stained with anti-α-SMA antibody on Day 10. The myofibroblast density/mm2 was determined by counting the positive cells within six visual fields (*n* = 6). Arrows indicate the re-epithelialized leading edges. (**E**) Real-time PCR was performed to detect *COL1A1*, *COL3A1*, and *TGF-*β mRNA isolated from the wound. Each column represents the mean ± SD. \* *p* < 0.05.

#### *2.2. Prolonged Accumulation of Neutrophils in IFN-*γ*KO Mice*

To define the role of inflammatory leukocytes during the wound healing process in IFN-γKO mice, wounded skin tissues were histologically examined in IFN-γKO and WT mice. As shown in Figure 2A, the former genotype exhibited prolonged accumulation of inflammatory leukocytes at the wound sites on Day 7. In the WT mice, in contrast, mainly fibroblasts were accumulated at the wound sites. Next, Ly6G, a marker specific to neutrophils, given that accumulated macrophages and eosinophils at the wound sites did not express Ly6G [19], was evaluated histologically. As shown in Figure 2B, the number of Ly6G+ cells on Day 7 was significantly greater in IFN-γKO mice. Consistent with these results, *CXCL1* (KC) and *CXCL2* (MIP-2) expression levels were also significantly higher in IFN-γKO mice than in WT mice on Day 7 (Figure 2C).

**Figure 2.** Prolonged accumulation of neutrophils in IFN-γ-KO mice. (**A**) Representative histological views of skin wounds on Day 7 are shown. (**B**) The number of neutrophils stained with anti-Ly6G antibody on Day 7. The Ly6G+ cell density/mm2 was determined by counting the positive cells in six visual fields (*n* = 6). (**C**) Real-time PCR was performed to detect *CXCL1* (KC) and *CXCL2* (MIP-2) mRNA isolated from the wound. Each column represents the mean ± SD. \* *p* < 0.05.

#### *2.3. Inhibited MMP-2 Activation by IFN-*γ

To define the mechanisms underlying IFN-γ-associated reductions in breaking strength and in *COL1A1* and *COL3A1* expression as well as IFN-γ-associated prolonged neutrophil accumulation, we examined mRNA expression levels of the collagen degradation-associated factors *MMP-2* and *MMP-9* in the wounded tissue. *MMP-2* mRNA expression on Day 14 was significantly increased in IFN-γKO mice compared with WT mice; with regard to *MMP-9* expression, in contrast, there was no significant difference between WT and IFN-γKO mice (Figure 3A). As shown in Figure 3B, from a morphological perspective, *MMP-2* is mainly expressed in neutrophils in IFN-γKO mice in contrast to WT mice. Next, because *MMP-2* expression was significantly increased in IFN-γKO mice, we examined the involvement of IFN-γ in the activity of neutrophil-derived MMP-2 and pro-MMP-2 activity by gelatin zymography. As shown in Figure 3C,D, pro-MMP-2 activity level was significantly suppressed by IFN-γ in a concentration-dependent manner, while MMP-2 activity, in contrast, was not detected in any experimental groups.

**Figure 3.** IFN-γ leads to inhibited MMP-2 activation. (**A**) Real-time PCR was performed to detect *MMP-2* and *MMP-9* mRNA isolated from the wound. (**B**) Representative histological views of wounded skin stained with MMP-2 antibody on Day 7. Red indicates MMP-2 positive cells. (**C**) Thioglycolate-elicited peritoneal neutrophils were treated with IFN-γ and lipopolysaccharide (LPS) for 24 h. The conditioned medium samples were analyzed for pro-MMP-2 activation by gelatin zymography. (**D**) The levels of pro-MMP-2 activation in (C) were analyzed using Image J image analysis software. Each column represents the mean ± SD. \* *p* < 0.05. M—marker.

#### *2.4. Wound Healing and MMP-2 Expression after Neutrophil Depletion Induced by Anti-Gr-1 Monoclonal Antibody in IFN-*γ*KO Mice*

As histological findings have revealed, MMP-2 derived mainly from neutrophils is involved in the delayed wound healing in IFN-γKO mice, as described above. Accordingly, we examined the effect of neutropenia induced by means of the anti-Gr-1 monoclonal antibody on wound closure and *MMP-2* expression. As shown in our recent study [20], the neutrophils in peripheral blood are completely depleted by this treatment. Wound closure on Day 10 was significantly accelerated in anti-Gr-1 antibody-treated mice compared with control IgG-treated mice (Figure 4A). As shown in Figure 4B,C, the accumulation of Ly6G+ neutrophils had almost completely disappeared on Day 10 after anti-Gr-1 antibody administration at the wound sites. In addition, *MMP-2* expression was significantly decreased in anti-Gr-1 antibody-treated mice (Figure 4D). In the control group, interestingly, *MMP-2* was mainly detected in infiltrating leukocytes, whereas *MMP-2*-expressing fibroblasts were frequently detected in the anti-Gr-1 antibody-treated group (Figure 4E).

**Figure 4.** Neutrophil depletion by means of anti-Gr-1 monoclonal antibody leads to decreased MMP-2. (**A**) IFN-γKO mice were injected intraperitoneally with anti-Gr-1 monoclonal antibody or control rat IgG 5 and 7 days after wound creation. Percentage of wound closure was evaluated on Day 10. (**B**) Representative histological views of skin wounds on Day 10 are shown. (**C**) The number of neutrophils stained with anti-Ly6G antibody on Day 10. The Ly6G+ cell density/mm<sup>2</sup> was determined by counting the positive cells in six visual fields (*n* = 6). (**D**) Real-time PCR was performed to detect *MMP-2* mRNA isolated from the wound. (**E**) Representative histological views of wounded skin stained with MMP-2 antibody on Day 10. Each column represents the mean ± SD. \* *p* < 0.05.
