Abstract
BackgroundThe accumulation of M1-polarized macrophages and excessive inflammation are important in the pathogenesis of diabetic foot ulcer (DFU). However, the underlying mechanism of DFU pathogenesis and the crucial regulators of DFU are less well known. Our previous study reported that kallikrein-binding protein (KBP), an angiogenesis inhibitor, was significantly upregulated in diabetic patients compared to its levels in controls. The effects of KBP on monocyte chemotaxis and macrophage M1 polarization were elucidated in this study.MethodsPlasma KBP levels and monocyte counts were assessed by ELISA and flow cytometry. Wound closure rates in different groups were monitored daily. The phenotype and recruitment of macrophages were measured by real-time PCR, western blot and immunofluorescence assays. The expression of Notch and NF-κB signalling pathway members was determined by the methods mentioned above. ChIP and dual-luciferase reporter gene assays were employed to explore the binding and transcriptional regulation of Hes1 and iNOS.ResultsWe found that plasma KBP levels and circulating monocytes were elevated in diabetic patients compared to those in nondiabetic controls, and both were higher in diabetic patients with DFU than in diabetic patients without DFU. KBP delayed wound healing in normal mice; correspondingly, KBP-neutralizing antibody ameliorated delayed wound healing in diabetic mice. Circulating monocytes and macrophage infiltration in the wound were upregulated in KBP-TG mice compared to those in control mice. KBP promoted the recruitment and M1 polarization of macrophages. Mechanistically, KBP upregulated iNOS by activating the Notch1/RBP-Jκ/Hes1 signalling pathway. Hes1 downregulated CYLD, a negative regulator of NF-κB signalling, and then activated the IKK/IκBα/NF-κB signalling pathway.ConclusionsOur findings demonstrate that KBP is the key regulator of excessive inflammation in DFUs and provide a novel target for DFU therapy.
Highlights
The accumulation of M1-polarized macrophages and excessive inflammation are important in the pathogenesis of diabetic foot ulcer (DFU)
We found that the plasma level of kallikrein-binding protein (KBP) in the DM group (17.9 ± 13.8 μg/mL) was higher than that in the Nondiabetic control (NDM) group (6.4 ± 2.0 μg/mL), and the level in the DM Diabetic patients with diabetic foot ulcer (w/DFU) group (25.9 ± 14 μg/mL) was higher than that in the DM w/o DFU group (12.3 ± 10.6 μg/mL) (Fig. 1a)
There were more monocytes in the peripheral blood of the DM group (5.0 ± 2.1 × 109/L) than in the peripheral blood of the NDM group (3.8 ± 1.1 × 109/L), and the number of monocytes was much higher in the DM w/DFU group (6.2 ± 2.1 × 109/L) than in the DM w/o DFU (4.1 ± 1.6 × 109/L) and NDM groups (Fig. 1b)
Summary
The accumulation of M1-polarized macrophages and excessive inflammation are important in the pathogenesis of diabetic foot ulcer (DFU). Macrophages, which derive from monocytes and upstream progenitor cells, are involved in all the phases of wound healing [5]. Macrophages 2assume a spectrum of activation states ranging from pro-inflammatory M1 macrophages that induce an inflammatory response with the secretion of inflammatory factor [9] to anti-inflammatory M2 macrophages that promote the absorption of inflammation and wound healing [10,11,12]. Delayed diabetic healing is characterized by excessive inflammation with the prolonged accumulation of M1 macrophages and elevated pro-inflammatory cytokines. Anti-inflammatory factors and growth factors secreted by M2-polarized macrophages are downregulated [16]. The reason for this abnormal phenotypic transformation in M1/M2 macrophages in diabetic patients is not well defined
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