1,25-Dihydroxyvitamin D3 Induces LL-37 and HBD-2 Production in Keratinocytes from Diabetic Foot Ulcers Promoting Wound Healing: An In Vitro Model

Abstract

Diabetic foot ulcers (DFU) are one of the most common diabetes-related cause of hospitalization and often lead to severe infections and poor healing. It has been recently reported that patients with DFU have lower levels of antimicrobial peptides (AMPs) at the lesion area, which contributes with the impairment of wound healing. The aim of this study was to determine whether 1,25-dihydroxyvitamin D3 (1,25 (OH)2 D3) and L-isoleucine induced HBD-2 and LL-37 in primary cultures from DFU. We developed primary cell cultures from skin biopsies from 15 patients with DFU and 15 from healthy donors. Cultures were treated with 1,25 (OH)2D3 or L-isoleucine for 18 h. Keratinocytes phenotype was identified by western blot and flow cytometry. Real time qPCR for DEFB4, CAMP and VDR gene expression was performed as well as an ELISA to measure HBD-2 and LL-37 in supernatant. Antimicrobial activity, in vitro, wound healing and proliferation assays were performed with conditioned supernatant. The results show that primary culture from DFU treated with 1,25(OH)2D3, increased DEFB4 and CAMP gene expression and increased the production of HBD-2 and LL-37 in the culture supernatant. These supernatants had antimicrobial activity over E. coli and induced remarkable keratinocyte migration. In conclusion the 1,25(OH)2D3 restored the production of AMPs in primary cell from DFU which were capable to improve the in vitro wound healing assays, suggesting their potential therapeutic use on the treatment of DFU.

Unique profile of antimicrobial peptide expression in polymorphic light eruption lesions compared to healthy skin, atopic dermatitis, and psoriasis

“…Our observation of increased LL‐37 expression in lesional skin of psoriasis and decreased expression in AD patients is consistent with previous findings . LL‐37 is induced by UVB, 1,25‐dihydroxyvitamin D 3, components of bacterial infections and stress, cytokines such as IFN‐γ, TNF‐α, IL‐6, and activated TLRs . Rather low systemic levels of vitamin D have been reported in PLE and other photodermatoses; hence, LL‐37 similar as Tregs could be induced in PLE by other, nonvitamin D‐dependent factors .…”

“…It is known to be upregulated in the skin by LPS, TNF‐α, IL‐1β, IL‐1α and bacterial infections and also 1,25‐dihydroxyvitamin D 3. We found HBD‐2 to be highly expressed in PLE, especially in infiltrating cells in the dermis. In this regard, it is already known in PLE that there is a lack of neutrophils and TNF‐α, but an increase in IL‐1β production which could induce the expression of HBD‐2 .…”

Vitamin D and L-Isoleucine Promote Antimicrobial Peptide hBD-2 Production in Peripheral Blood Mononuclear Cells from Elderly Individuals

“…For stimulation, cells were treated with 50 µg / ml of L-isoleucine (Sigma-Aldrich, St. Louis, USA) for 24 hours as reported previously [15]. Similarly, cells were stimulated with 10 -7 M of active form vitamin D3 (1,25(OH)2D3) (Sigma-Aldrich, St. Louis, USA) or an equal amount of DMSO (Sigma-Aldrich, St. Louis, USA) (0.5% v / v, such as vehicle control) for 24 hours similar to other models reported elsewhere [16]. After incubation, the obtained supernatants were supplemented with protease inhibitor cocktails, divided into aliquots and stored at -70 ºC until use.…”

Multifunctional Activity of the β-Defensin-2 during Respiratory Infections

“…The scarce expression of HBD2 is seen during the chronic disease. The authors supposed that high glucose levels inhibit the expression of HBD2 in human keratinocytes [92].…”