1,25-dihydroxyvitamin D3 induces LL-37 and HBD-2 production in keratinocytes from diabetic foot ulcers promoting wound healing: an in vitro model

• Published in: PloS one 2014-10, Vol.9 (10), p.e111355
• Main Authors: Gonzalez-Curiel, Irma, Trujillo, Valentin, Montoya-Rosales, Alejandra, Rincon, Kublai, Rivas-Calderon, Bruno, deHaro-Acosta, Jeny, Marin-Luevano, Paulina, Lozano-Lopez, Daniel, Enciso-Moreno, Jose A, Rivas-Santiago, Bruno
• Format: Article
• Language: English
• Subjects: Adult; Aged; Aged, 80 and over; Antibiotics; Antiinfectives and antibacterials; Antimicrobial activity; Antimicrobial Cationic Peptides - biosynthesis; Antimicrobial Cationic Peptides - genetics; Antimicrobial peptides; Bacterial infections; beta-Defensins - biosynthesis; beta-Defensins - genetics; Biology and Life Sciences; Biopsy; Bone surgery; Calcitriol; Case-Control Studies; Cathelicidins - biosynthesis; Cathelicidins - genetics; Cell culture; Cell growth; Cell Movement - drug effects; Cell Proliferation; Cells, Cultured; Conditioning; Culture Media, Conditioned; Cytometry; Defensins; Diabetes; Diabetes mellitus; Diabetic Foot - diagnosis; Diabetic Foot - genetics; Diabetic Foot - metabolism; E coli; Enzyme-linked immunosorbent assay; Epidermal growth factor; Escherichia coli; Feet; Female; Flow cytometry; Foot diseases; Gene expression; Gene Expression Regulation - drug effects; Humans; Infections; Isoleucine; Keratinocytes; Keratinocytes - drug effects; Keratinocytes - metabolism; Leg ulcers; Male; Medical research; Medicine and Health Sciences; Microorganisms; Middle Aged; Patients; Peptides; Phenotype; Phenotypes; Receptors, Calcitriol - genetics; Receptors, Calcitriol - metabolism; Skin; Ulcers; Vitamin D - analogs & derivatives; Vitamin D - pharmacology; Vitamin D receptors; Vitamin D3; Wound Healing
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0111355

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.