Skin wound recovery of diabetic mice is slow after injury
to evaluate the wound closure rate of control mice and diabetic mice, a 5mm full-thickness round cut on the back was made, which could be normally healed within 11 days. As shown in the images in Figure 1A and 1B, the wound closure of diabetic mice was continuously slower than that of control group. The wound area of DM was significantly larger than that of CON on days of 3 (P < 0.05), 5 (P < 0.01), 7 (P < 0.05), 9 (P < 0.05), and 11 (P < 0.01). On the 11th day of healing, the wound of CON was completely healed, while the DM still had obvious wounds. Then, the thicknesses of epidermal and dermal were assessed. The epidermal thickness of DM exhibited a significant reduction over 60% than CM wound (P < 0.01, Figure 1B, 1C, 1D). DM group displayed an ~35% reduction in dermal thickness compared with CON group (P < 0.05, Figure 1B, 1C, 1D). Moreover, we also calculated the number of endothelium blood vessels in DM and CON. Figure 1E showed that the number of endothelium blood vessels in DM was also significantly less than that of normal mice (P < 0.01) at 7-days after injury. Taken together, the DM exibited a slower recovery rate, thinner epidermis and dermis, and less regeneration of blood vessels than normal mice.
mTOR inhibitor INK128 promotes skin wound healing and angiogenesis in diabetic mice
The wound recovery rate of DM and DM+INK128 was assessed. As shown in Figure 2A that the wound healing rate in DM+INK128 did significantly increased than DM. The wound area of DM was significantly larger than that of DM+INK128 on days of 5 (P < 0.05), 7 (P < 0.01), 9 (P < 0.05), and 11 (P < 0.05). The epidermal thickness of DM was similar with that of DM+INK128 (P > 0.05, Figure 2B, 2D), whereas DM displayed a significant reduction in dermal thickness compared to DM+INK128 (P < 0.05, Figure 2B, 2C, 2D). Figure 2E showed that the number of endothelium blood vessels in DM was also significantly less than that of DM+INK128 (P < 0.01) at day 7, the proliferative phase after injury. These results suggested that INK128 could promote diabetic skin wound healing.
The percentage of MDSCs is increased in high glucose environment
The key for proper wound healing is whether the various reactions in the inflammatory can transition to proliferative phases appropriately (30). As reported that the MDSCs in peripheral blood mononuclear cell (PBMC) of type I diabetic patients are significantly accumulated than healthy human (15), we doubted that whether the slow recovery of wound was related to the accumulation of MDSCs. Thus, the amount of MDSCs at inflammatory (3 days after injury) and proliferative (7 days after injury) phases in BM, spleen, and PBMC were detected.
We found that the percentage of MDSCs in BM, PBMC and spleen were significantly higher in DM than that in control group both at inflammatory and proliferative phases after injury (Figure 3A-F; P < 0.05). In diabetic mice, the infiltration of MDSCs cells in the skin tissue around the wound increased on inflammatory and proliferative phases after injury comparing with control mice through immunofluorescence staining Gr-1 of skin tissue (Figure 3G, H). These results indicate the persistence of inflammation during wound healing process in diabetic mice.
To investigate whether the increased glucose concentration in DM contributed to the accumulation of MDSCs, the BM cells were isolated, treated with IL-6, GM-CSF and graded glucose of 5mM, 10mM, 20mM, 30mM, 60mM, and 120mM for 4 days to generate MDSCs. Under the gradient addition of glucose in BM cells, the percentage of MDSCs exhibited an increasing trend (Figure 3I). Then we chose 5mM and 30mM glucose concentration for further in vitro experiments corresponding to control mice and diabetic mice, for 25-35mM is commonly recognized range in hyperglycemic study. Taken together, the results showed that MDSCs were abnormally accumulated in DM and high glucose promoted the increase of MDSCs proportion.
High glucose promotes the activation of mTOR signaling in MDSCs
To confirm the role of mTOR signaling on accumulation of MDSCs in high glucose. The activation of mTOR pathway were assessed by evaluating the protein expression of downstream signaling molecules phosphorylated mammalian target of rapamycin (p-4EBP1), 4EBP1, phosphorylated ribosomal protein S6 (p-S6) and S6, with β-Tublin as the reference. Our results showed that p-4EBP1 and p-S6 were significantly highly expressed in MDSCs isolated from diabetic mice than control mice (Figure 4A). Moreover, the protein expression of p-4EBP1 and p-S6 in BM-derived MDSCs under the presence of 5mM and 30mM glucose were also evaluated. The results showed that p-4EBP1 and p-S6 were highly expressed in 30mM than that in 5mM (Figure 4B). These results indicated that mTOR signaling in MDSCs was activated in high glucose microenvironment.
mTOR inhibitor INK128 inhibist the accumulation of MDSCs in high glucose
To explore the role of mTOR on MDSCs expansion, gradient doses (0nM, 25nM, 50nM, and 100nM) of INK128 was added to BM in the process of generating MDSCs. Figure 5A showed that the percentage of MDSCs under addition of 25mM, 50mM and 100mM INK128 was significantly decreased compared to control, which demonstrated that INK128 could suppress BM cells differentiate into MDSCs. BM cells under treatments of 5mM glucose, 30mM glucose, and 30mM glucose + 50nM INK128 showed that high glucose promoted MDSCs expansion, which can be inhibited by INK128 (Figure 5B).
To further confirm INK128 inhibit the accumulation of glusoce-induced MDSCs expansion in vivo, STZ-induced diabetic mice were treated with vehicle and 1mg/kg INK128 for 45 days. The percentage of MDSCs in BM, PBMC and spleen was significantly lower in DM+INK128 group than that in DM group both at inflammatory and proliferative phases after injury (Figure 5C-I; P < 0.05). In INK128 treated diabetic mice, the infiltration of MDSCs in the skin tissue around the wound significantly decreased at inflammatory and proliferative phases after injury through immunofluorescence staining Gr-1 of skin tissue (Figure 5J, K). Taken together, these results suggested high glucose caused the accumulation of MDSCs on mTOR-dependent manner and INK128 inhibited the expansion of MDSCs in vitro and in vivo.
INK128 suppresses functional gene expression of high glucose-induced MDSCs
To examine whether the function of MDSC cells was changed in diabetic mice, the MDSCs were isolated from spleens and the expression of several functional molecules including p47phox, gp91phox, arginase-1 (Arg-1), and inducible nitric oxide synthase (iNOS) were detected. The results showed that the expression levels of them were significantly higher in diabetic mice than that in control mice (P < 0.05; Figure 6A-C). Moreover, the effect of INK128 on MDSCs function was also evaluated in vitro. The gene expression levels of Arg-1, iNOS, and IL-6 were assessed in MDSCs supplemented with glucose of 5mM, 30mM, and 30mM+INK128. These genes presented a higher expression level in 30mM glucose than that of 5mM and INK128 suppressed the elevation of gene expression in 30mM group (P < 0.05; Figure 6D-F). Together, the results demonstrated that the MDSCs function were disordered in DM and high glucose environments, which could explain for the slow wound healing. Moreover, INK128 could help retrieve their function, thus promote wound healing.
INK128 inhibits high glucose-induced differentiation of MDSCs into macrophages
Macrophages are considered as the primary effector cells in regulating wound healing, unregulated macrophage activation represent a source of excessive inflammation, leading to aberrant wound healing (8, 31, 32). MDSCs have the potential to differentiate to macrophages in chronic inflammation (33). Diabetes presents a systemic inflammatory state. It is unclear whether high glucose promotes macrophage development and mTOR signaling is involved. Therefore, the amount of macrophage in BM and spleen of CON, DM, DM+INK128 were detected. Figure 7A-D showed that the percentage of CD11b+F4/80+ macrophages increased in diabetic mice compared with control group and INK128 reduced macrophages in DM (P < 0.05). Moreover, in the skin tissue around the wound of diabetic mice showed massive macrophage infiltration which was mitigated by INK128 treatment (Figure 7E, F).
To explore whether mTOR signaling was involved in differentiation of MDSC cells into macrophages in high glucose in vitro, BM cells were incubated with IL-6, GM-CSF as well as 5mM glucose, 30mM glucose, and 30mM glucose + INK128 for 4 days. The MDSCs treated with 30mM glucose displayed a higher macrophage amount than that of 5mM glucose and 30mM glucose + INK128 (Figure 7G), which demonstrated that high glucose could promote MDSCs differentiated into macrophage, which was on a mTOR-dependent manner. Some studies have demonstrated that S100A8 and S100A9 proteins are directly involved in inhibiting MDSCs maturation (33). Our result showed that INK128 significantly increased the expression levels of S100A8 and S100A9 (P < 0.05; Figure 7H). Taken together, high glucose promoted MDSCs to differentiate into macrophage, and INK128 suppressed the differentiation.
INK128 reduces M-MDSCs differentiated into pro-inflammatory macrophages induced by high glucose
The phenotype of mice MDSCs is CD11b+Gr-1+, which can be further divided into two subtypes, including G-MDSCs and M-MDSCs. It was reported that M-MDSCs is the subtype which can differentiate into macrophages, therefore, we detected the percentage of M-MDSCs. Figure 8A-D demonstrated that the percentage of M-MDSCs in DM was significantly higher than that in CON and INK128 reduces the M-MDSC in DM (P < 0.05). In vitro, the percentage of M-MDSCs decreased within the increace of INK128 (Figure 8E). Moreover, the percentage of M-MDSCs in 30mM glucose group was significantly higher than that in 5mM glucose and 30mM glucose + INK128 (P < 0.01; Figure 8F). In summary, the results suggested the percentage of M-MDSCs increased in diabetes and high glucose environment, which can be inhibited by INK128.
Macrophages can be divided into pro-inflammatory (M1) and anti-inflammatory (M2) types. In the early stage of wound formation, M1 macrophages infiltrate the periwound tissue, swallow pathogens and necrotic tissue, and play a cleaning role. In the later stage, M2 macrophages cells perform repair functions. The continuous presence of M1 type causes persistence of inflammatory state poor wound healing.(34) In our study, inflammatory cell model BMDM and RAW264.7 were co-cultured with MDSCs pretreated with glucose 5mM, 30mM, and 30mM+INK128 (Figure 8G). Then, the expression of M1 macrophage markers, i.e., IL-6, and iNOS in BMDM cells, IL-6 and IL-1β in RAW264.7 cells were detected. The expression of M2 macrophage markers, i.e., CD206, IL-10 in BMDM cells, CD206 and IGF-1 in RAW264.7 cells were detected. The result showed that the relative expression levels of CD206 and IL-10 were significantly lower, and IL-6 and iNOS were significantly higher under 30mM glucose treatment in BMDM than that under 5mM glucose and 30mM glucose+INK128 (Figure 8H). The relative expression levels of CD206 and IGF-1 were significantly lower, and IL-6 and IL-1β were significantly higher under 30mM glucose treatment in RAW164.7 than that under 5mM glucose and 30mM glucose+INK128 (Figure 8I). These results indicated that MDSCs from high glucose environment promoted macrophages differentiate towards M1 type and INK128 suppressed the effect of high glucose. Taken together, these findings demonstrated that high glucose caused M-MDSCs to differentiate into M1 type which can be inhibited by INK128.