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Effects of cold atmospheric plasma (CAP) on ß-defensins, inflammatory cytokines, and apoptosis-related molecules in keratinocytes in vitro and in vivo.

Arndt S, Landthaler M, Zimmermann JL, Unger P, Wacker E, Shimizu T, Li YF, Morfill GE, Bosserhoff AK, Karrer S - PLoS ONE (2015)

Bottom Line: In summary, our results suggest that a 2 min application of CAP induces gene expression of key regulators important for inflammation and wound healing without causing proliferation, migration or cell death in keratinocytes.The induction of ß-defensins in keratinocytes describes an absolutely new plasma strategy.Activation of antimicrobial peptides supports the well-known antibacterial effect of CAP treatment, whereas the mechanism of ß-defensin activation by CAP is not investigated so far.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Regensburg, D-93042 Regensburg, Germany.

ABSTRACT
Cold atmospheric plasma (CAP) has been gaining increasing interest as a new approach for the treatment of skin diseases or wounds. Although this approach has demonstrated promising antibacterial activity, its exact mechanism of action remains unclear. This study explored in vitro and in vivo whether CAP influences gene expression and molecular mechanisms in keratinocytes. Our results revealed that a 2 min CAP treatment using the MicroPlaSter ß in analogy to the performed clinical studies for wound treatment induces expression of IL-8, TGF-ß1, and TGF-ß2. In vitro and in vivo assays indicated that keratinocyte proliferation, migration, and apoptotic mechanisms were not affected by the CAP treatment under the applied conditions. Further, we observed that antimicrobial peptides of the ß-defensin family are upregulated after CAP treatment. In summary, our results suggest that a 2 min application of CAP induces gene expression of key regulators important for inflammation and wound healing without causing proliferation, migration or cell death in keratinocytes. The induction of ß-defensins in keratinocytes describes an absolutely new plasma strategy. Activation of antimicrobial peptides supports the well-known antibacterial effect of CAP treatment, whereas the mechanism of ß-defensin activation by CAP is not investigated so far.

No MeSH data available.


Related in: MedlinePlus

CAP effects on keratinocyte proliferation and migration.(a) Cell proliferation was determined using a XTT proliferation assay 24 h, 48 h and 72 h after the CAP exposure for 2 min and compared to control (ctr.). (b) Representative examples of immunohistochemical stains of formalin-fixed paraffin-embedded skin sections using Ki67, a proliferation marker that stains the nuclei of cells in G1, S, G2 and early mitosis. Highly proliferative, Ki67-positive keratinocytes were observed within the basal layer of the epidermis (I) and the bulge region of the hair follicle (II), whereas no difference between 5x placebo or 5x CAP treatment for 2 min was observed. Magnification as indicated. (c, d) Cell migration was determined using a wound healing assay. The migration rate was calculated 7 h and 12 h after culture insert was removed and displayed as the percentage relative to untreated control (ctr. set 100%). Representative images are shown immediately after culture insert was removed (0 h) and 7 h and 12 h later.
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pone.0120041.g003: CAP effects on keratinocyte proliferation and migration.(a) Cell proliferation was determined using a XTT proliferation assay 24 h, 48 h and 72 h after the CAP exposure for 2 min and compared to control (ctr.). (b) Representative examples of immunohistochemical stains of formalin-fixed paraffin-embedded skin sections using Ki67, a proliferation marker that stains the nuclei of cells in G1, S, G2 and early mitosis. Highly proliferative, Ki67-positive keratinocytes were observed within the basal layer of the epidermis (I) and the bulge region of the hair follicle (II), whereas no difference between 5x placebo or 5x CAP treatment for 2 min was observed. Magnification as indicated. (c, d) Cell migration was determined using a wound healing assay. The migration rate was calculated 7 h and 12 h after culture insert was removed and displayed as the percentage relative to untreated control (ctr. set 100%). Representative images are shown immediately after culture insert was removed (0 h) and 7 h and 12 h later.

Mentions: Proliferation of keratinocytes in vitro was not significantly changed after CAP treatment for 2 min analyzed 24 h, 48 h, and 72 h after exposure (Fig. 3a). Ki67 staining of skin sections confirmed our in vitro results, showing that the proliferation of cells was limited to keratinocytes of the basal layer of the epidermis (I) and the bulge region of the outer root sheath of the hair follicle (II) and did not differ between CAP and placebo control after 5x exposure for 2 min (Fig. 3b). Furthermore, we wanted to know whether migration is affected by the CAP treatment in keratinocytes because in our previous study we could show that a 30 sec treatment with CAP significantly activates fibroblast migration [20]. The treatment time for this assay has to be reduced because it is known that CAP treatment temporarily results in a detachment of the cells and inferential leads to a delayed migration ability [18]. In contrast to our results obtained for fibroblasts [20], we observed that migration was not significantly modified in keratinocytes treated with CAP for 30 sec (Fig. 3c, d).


Effects of cold atmospheric plasma (CAP) on ß-defensins, inflammatory cytokines, and apoptosis-related molecules in keratinocytes in vitro and in vivo.

Arndt S, Landthaler M, Zimmermann JL, Unger P, Wacker E, Shimizu T, Li YF, Morfill GE, Bosserhoff AK, Karrer S - PLoS ONE (2015)

CAP effects on keratinocyte proliferation and migration.(a) Cell proliferation was determined using a XTT proliferation assay 24 h, 48 h and 72 h after the CAP exposure for 2 min and compared to control (ctr.). (b) Representative examples of immunohistochemical stains of formalin-fixed paraffin-embedded skin sections using Ki67, a proliferation marker that stains the nuclei of cells in G1, S, G2 and early mitosis. Highly proliferative, Ki67-positive keratinocytes were observed within the basal layer of the epidermis (I) and the bulge region of the hair follicle (II), whereas no difference between 5x placebo or 5x CAP treatment for 2 min was observed. Magnification as indicated. (c, d) Cell migration was determined using a wound healing assay. The migration rate was calculated 7 h and 12 h after culture insert was removed and displayed as the percentage relative to untreated control (ctr. set 100%). Representative images are shown immediately after culture insert was removed (0 h) and 7 h and 12 h later.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4359157&req=5

pone.0120041.g003: CAP effects on keratinocyte proliferation and migration.(a) Cell proliferation was determined using a XTT proliferation assay 24 h, 48 h and 72 h after the CAP exposure for 2 min and compared to control (ctr.). (b) Representative examples of immunohistochemical stains of formalin-fixed paraffin-embedded skin sections using Ki67, a proliferation marker that stains the nuclei of cells in G1, S, G2 and early mitosis. Highly proliferative, Ki67-positive keratinocytes were observed within the basal layer of the epidermis (I) and the bulge region of the hair follicle (II), whereas no difference between 5x placebo or 5x CAP treatment for 2 min was observed. Magnification as indicated. (c, d) Cell migration was determined using a wound healing assay. The migration rate was calculated 7 h and 12 h after culture insert was removed and displayed as the percentage relative to untreated control (ctr. set 100%). Representative images are shown immediately after culture insert was removed (0 h) and 7 h and 12 h later.
Mentions: Proliferation of keratinocytes in vitro was not significantly changed after CAP treatment for 2 min analyzed 24 h, 48 h, and 72 h after exposure (Fig. 3a). Ki67 staining of skin sections confirmed our in vitro results, showing that the proliferation of cells was limited to keratinocytes of the basal layer of the epidermis (I) and the bulge region of the outer root sheath of the hair follicle (II) and did not differ between CAP and placebo control after 5x exposure for 2 min (Fig. 3b). Furthermore, we wanted to know whether migration is affected by the CAP treatment in keratinocytes because in our previous study we could show that a 30 sec treatment with CAP significantly activates fibroblast migration [20]. The treatment time for this assay has to be reduced because it is known that CAP treatment temporarily results in a detachment of the cells and inferential leads to a delayed migration ability [18]. In contrast to our results obtained for fibroblasts [20], we observed that migration was not significantly modified in keratinocytes treated with CAP for 30 sec (Fig. 3c, d).

Bottom Line: In summary, our results suggest that a 2 min application of CAP induces gene expression of key regulators important for inflammation and wound healing without causing proliferation, migration or cell death in keratinocytes.The induction of ß-defensins in keratinocytes describes an absolutely new plasma strategy.Activation of antimicrobial peptides supports the well-known antibacterial effect of CAP treatment, whereas the mechanism of ß-defensin activation by CAP is not investigated so far.

View Article: PubMed Central - PubMed

Affiliation: Institute of Pathology, University Regensburg, D-93042 Regensburg, Germany.

ABSTRACT
Cold atmospheric plasma (CAP) has been gaining increasing interest as a new approach for the treatment of skin diseases or wounds. Although this approach has demonstrated promising antibacterial activity, its exact mechanism of action remains unclear. This study explored in vitro and in vivo whether CAP influences gene expression and molecular mechanisms in keratinocytes. Our results revealed that a 2 min CAP treatment using the MicroPlaSter ß in analogy to the performed clinical studies for wound treatment induces expression of IL-8, TGF-ß1, and TGF-ß2. In vitro and in vivo assays indicated that keratinocyte proliferation, migration, and apoptotic mechanisms were not affected by the CAP treatment under the applied conditions. Further, we observed that antimicrobial peptides of the ß-defensin family are upregulated after CAP treatment. In summary, our results suggest that a 2 min application of CAP induces gene expression of key regulators important for inflammation and wound healing without causing proliferation, migration or cell death in keratinocytes. The induction of ß-defensins in keratinocytes describes an absolutely new plasma strategy. Activation of antimicrobial peptides supports the well-known antibacterial effect of CAP treatment, whereas the mechanism of ß-defensin activation by CAP is not investigated so far.

No MeSH data available.


Related in: MedlinePlus