**3. Discussion**

In the current study, IFN-γKO mice exhibited significant attenuation in wound closure, wound breaking strength, and myofibroblast differentiation in the proliferation phase compared with WT mice through prolonged neutrophil accumulation and enhanced MMP-2 activation.

IFN-γ contributes to macrophage activation [14], neutrophil recruitment, and cell clearance by apoptosis [15]. Yet the question of how IFN-γ contributes to wound healing, especially in the proliferative phase, remains controversial and poorly understood. Regarding the role of IFN-γ in skin wounds, we previously reported that IFN-γ plays a key role in the early phase of the wound healing process in a study on mice deficient in invariant natural killer T (*i*NKT) cells, which are major IFN-γ-producing cells [21]. In addition, IFN-γ-treated LEPCs, which initiate blood vessel regeneration [22], or TNF-<sup>α</sup>- and IFN-γ-treated monocytes/macrophages [18] have been reported to be involved in the promotion of wound healing. In the current study, IFN-γKO mice exhibited significant attenuation in wound closure on Day 10 in association with prolonged neutrophil accumulation. In contrast to this, Ishida and Kondo et al. [14] demonstrated that IFN-γ deficiency accelerated the wound healing process in association with an early-phase reduction in the infiltration of myeloperoxidase (MPO)+ neutrophils, F4/80<sup>+</sup> macrophages, and CD3+ T cells.

Our murine wound model was at low risk for microbial infection as we used a clean procedure for the wounding and occlusive dressings for the wounds (closed wounds) until tissue collection. The model used by Ishida and Kondo et al. [14], in contrast, analyzed open wounds, i.e., wounds that had not been covered with occlusive dressings. This difference may have affected the different results of our two studies as a variation in environmental moisture and the microbial load at the wound site may have affected the findings. This possibility is strengthened by our previous finding that, compared with WT mice, IL-17AKO mice exhibited accelerated wound healing under closed-wound conditions but delayed wound healing under open-wound conditions [23].

In the present study, IFN-γKO mice exhibited diminished wound breaking strength, reduced myofibroblast differentiation, and low levels of *COL1A1* and *COL3A1* expression. Although several reports have demonstrated that IFN-γ can inhibit collagen synthesis by fibroblasts in vitro [12,13], in wound sites, IFN-γ can contribute to collagen deposition [21]. Previously, Hata et al. reported that TGF-β1 induces myofibroblast differentiation and collagen synthesis in the proliferation phase [24]. In this study, TGF-β1 expression was decreased in IFN-γKO mice compared with WT mice. Thus, our results are likely to be related to a delay in collagen synthesis.

In the current study, IFN-γKO mice exhibited delayed wound repair in the proliferative phase along with an increased neutrophil count, suggesting that accumulated neutrophils may suppress the healing process. In normal acute wounds, neutrophils are infiltrated immediately after skin injury and initially play a key role in antimicrobial activity; later, these cells undergo apoptosis and are engulfed by macrophages [25]. In non-healing wounds, however, prolonged neutrophil accumulation often leads to persistent inflammation through the production of proteases such as MMPs [26]. The functions of *MMP-2* [6], *MMP-8* [5], and *MMP-9* [27] have been studied with regard to the wound healing process. MMP-2 is not considered to play a critical role in normal acute murine wounds [6]. In non-healing wounds in humans, however, high levels of *MMP-2* activity have been detected [28]. In the current study, IFN-γKO mice exhibited delayed wound healing associated with a significant increase in *MMP-2* expression on recruited neutrophils at the wound sites. We also confirmed that pro-*MMP-2* activity levels in peritoneal neutrophils were significantly suppressed by IFN-γ stimulation. In fact, our current results demonstrate that delayed wound healing in IFN-γKO mice can be recovered under neutropenic conditions induced by treatment with the anti-Gr-1 monoclonal antibody, and that this recovery is associated with low levels of MMP-2. Previously, Qin et al. [29] reported the transcriptional suppression of *MMP-2* gene expression in human astroglioma cells by IFN-γ administration. Our skin wound model likewise suggests that IFN-γ could be involved in *MMP-2* expression.

MMP-2 has been reported to involve tissue remodeling by degrading extracellular matrix components such as type III collagen, type IV collagen, fibronectin and elastin [30,31]. At wound sites, upregulation of *MMP-2* expression has been observed in both granulation and scar tissues after skin injury [32]: during normal wound repair, *MMP-2* expression reached peak levels on Day 3 after wound creation and declined thereafter to the baseline level [33]. In this study, we showed that IFN-γKO mice exhibited decreased wound breaking strength along with upregulated *MMP-2* activity, suggesting that MMP-2 in the proliferative phase may reduce the strength of wounded skin. Thus, MMP-2 is likely to contribute positively in the early phase of wound healing, and negatively from the proliferative phase onward.

In this study, our in vitro gelatin zymography experiment detected pro-*MMP-2* activity but not MMP-2 itself in peritoneal neutrophils. It has previously been reported that MMP-2 is secreted as a zymogen (pro-MMP-2), and that membrane-bound MMP-14 (MT-1-MMP), which is mainly expressed on fibroblasts and cancer cells, activates secreted MMP-2 by cleaving its pro-domain [34]. Thus, the absence of MMP-14-expressing cells such as fibroblasts may be related to the absence of detectable MMP-2 in our in vitro analysis.

In conclusion, the present study demonstrated that IFN-γ plays an important role in the proliferation phase of skin wound healing and in the neutrophilic inflammatory response at the wound site. To date, little was known about IFN-γ's function in the proliferation phase; here, we have shown that it contributes significantly to wound strength and the suppression of inflammation. Inflammation is deeply involved in wound healing [35], but little is known about therapy for inflammatory responses at the wound sites. IFN-γ therapy has already been used as a treatment for pulmonary fibrosis [36] and may also be useful in a novel approach to the treatment of augmented fibrosis in the skin, such as hypertrophic scarring, keloid scarring and scleroderma. However, we did not confirm the effects of IFN-γ administration in this study. Further investigation is necessary to clarify the effects of IFN-γ therapy on augmented fibrosis in skin as well as its optimal dose and route.
