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

Gonzalez-Curiel I, Trujillo V, Montoya-Rosales A, Rincon K, Rivas-Calderon B, deHaro-Acosta J, Marin-Luevano P, Lozano-Lopez D, Enciso-Moreno JA, Rivas-Santiago B - PLoS ONE (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, Zacatecas, Mexico; Department of Immunology, Faculty of Medicine, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico.

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.

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KCM antimicrobial activity in E. coli.KCMs from DFUs that showed higher LL-37 and HBD-2 concentrations were tested for antimicrobial activity against S. aureus, P. aeruginosa and E. coli in a modified radial diffusion assay. Eight KCMs from DFUs cell cultures showed clear inhibition areas (arrow heads) only against E. coli (A and B) comparable with the solid-phase synthesized LL-37 (SP). mprf gene expression in the different clinical isolates (C). Each bar represents the mean ± SD of five independent experiments.
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pone-0111355-g005: KCM antimicrobial activity in E. coli.KCMs from DFUs that showed higher LL-37 and HBD-2 concentrations were tested for antimicrobial activity against S. aureus, P. aeruginosa and E. coli in a modified radial diffusion assay. Eight KCMs from DFUs cell cultures showed clear inhibition areas (arrow heads) only against E. coli (A and B) comparable with the solid-phase synthesized LL-37 (SP). mprf gene expression in the different clinical isolates (C). Each bar represents the mean ± SD of five independent experiments.

Mentions: We collected eight (KCM) from vitamin D- treated DFUs cell cultures, which responded favorably to the treatment reflected by the LL-37 production and we aimed to determine the antimicrobial activity of these KCMs against most common strains. Thus, we isolated and characterized P. aeruginosa and S. aureus strains from ulcerative exudates. Screening KCM for their antibacterial activity was conducted using radial diffusion assays. The results showed that KCM did not result in larger zones of inhibition against clinical isolates neither inhibition areas were observed with synthetic peptides (Figure S1), suggesting that both strains have some biological component that confers its resistance. Therefore, we used E. coli as reference strain to verify the above observation. The results demonstrated that these supernatants showed a clear inhibition area against E. coli (1.033±0.1614 cm diameter) (Figure 5A–B). Since not all KCM showed antimicrobial activity against clinical isolates, we wondered whether clinical isolates had some resistance gene for AMPs antimicrobial activity. Interestingly, we found multiple peptide resistance factor (mprf) gene in both clinical isolates, S. aureus strain has a higher copy number than P. aeruginosa (Figure 5C).


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

Gonzalez-Curiel I, Trujillo V, Montoya-Rosales A, Rincon K, Rivas-Calderon B, deHaro-Acosta J, Marin-Luevano P, Lozano-Lopez D, Enciso-Moreno JA, Rivas-Santiago B - PLoS ONE (2014)

KCM antimicrobial activity in E. coli.KCMs from DFUs that showed higher LL-37 and HBD-2 concentrations were tested for antimicrobial activity against S. aureus, P. aeruginosa and E. coli in a modified radial diffusion assay. Eight KCMs from DFUs cell cultures showed clear inhibition areas (arrow heads) only against E. coli (A and B) comparable with the solid-phase synthesized LL-37 (SP). mprf gene expression in the different clinical isolates (C). Each bar represents the mean ± SD of five independent experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4206472&req=5

pone-0111355-g005: KCM antimicrobial activity in E. coli.KCMs from DFUs that showed higher LL-37 and HBD-2 concentrations were tested for antimicrobial activity against S. aureus, P. aeruginosa and E. coli in a modified radial diffusion assay. Eight KCMs from DFUs cell cultures showed clear inhibition areas (arrow heads) only against E. coli (A and B) comparable with the solid-phase synthesized LL-37 (SP). mprf gene expression in the different clinical isolates (C). Each bar represents the mean ± SD of five independent experiments.
Mentions: We collected eight (KCM) from vitamin D- treated DFUs cell cultures, which responded favorably to the treatment reflected by the LL-37 production and we aimed to determine the antimicrobial activity of these KCMs against most common strains. Thus, we isolated and characterized P. aeruginosa and S. aureus strains from ulcerative exudates. Screening KCM for their antibacterial activity was conducted using radial diffusion assays. The results showed that KCM did not result in larger zones of inhibition against clinical isolates neither inhibition areas were observed with synthetic peptides (Figure S1), suggesting that both strains have some biological component that confers its resistance. Therefore, we used E. coli as reference strain to verify the above observation. The results demonstrated that these supernatants showed a clear inhibition area against E. coli (1.033±0.1614 cm diameter) (Figure 5A–B). Since not all KCM showed antimicrobial activity against clinical isolates, we wondered whether clinical isolates had some resistance gene for AMPs antimicrobial activity. Interestingly, we found multiple peptide resistance factor (mprf) gene in both clinical isolates, S. aureus strain has a higher copy number than P. aeruginosa (Figure 5C).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Unit Zacatecas, Mexican Institute of Social Security-IMSS, Zacatecas, Mexico; Department of Immunology, Faculty of Medicine, Autonomous University of San Luis Potosi, San Luis Potosi, Mexico.

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.

Show MeSH
Related in: MedlinePlus