School of Medicine and Health Sciences Poster Presentations

Title

Cytokine production varies between hidradenitis suppurativa, chronic wounds and normal keratinocytes in an in-vitro wound closure model

Document Type

Poster

Keywords

hidradenitis; keratinocytes; cytokines; wounds; FACS

Publication Date

Spring 2017

Abstract

Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory disease of the apocrine glands. This disease affects approximately 1% of the population and there is currently no known cure. In order to develop therapies that target the molecular drivers of this disease, the molecular mechanisms of pathogenesis must be elucidated. Defective keratinocyte function has been implicated in the pathogenesis of HS. The purpose of this study was to compare keratinocyte function in HS, chronic wound (CW), and normal (N) skin samples.

Methods

Human epidermal keratinocytes were cultured to reach 80% confluence in 6 well plates. A scratch assay was performed using a 1 ml sterile tip of about 100um in diameter. Culture supernatants were collected at 0, 24, 48, 72, and 96 hours after scratch.

Supernatants were analyzed using Luminex immunoassays. A panel of cytokines previously demonstrated to be correlated with HS pathogenesis were measured at each time point.

Apoptosis was assessed by flow cytometry for each group at 96-hour time-point after initial scratch using a FITC-annexinV and Propidium Iodide (PI). Apoptotic cells stained annexinV positive only, whereas necrotic cells stained double positive for annexinV and PI. Cell viability was measured 96 hours post scratch by fluorescence microscopy with a live/dead vital dye staining method.

Results

Cell viability was similar at 96 hours post scratch in the normal and HS keratinocytes. However, cell viability was significantly lower in chronic wound keratinocytes at 96 hours (p=0.0138).

Flow cytometry for AnnexinV and PI demonstrated that CW keratinocytes had a significantly higher rate of apoptosis (annexinV positive) and necrosis (annexinV and PI double positive cells) than normal (p=0.0075) and HS (p=0.028) keratinocytes.

Significantly higher levels of IL-1α and VEGF were observed in the normal keratinocyte culture supernatant compared to CW. In contrast IL-22 levels were significantly lower in the HS culture supernatant than both CW and normal keratinocytes (p=0.0008). This finding indicates that IL-22 and its downstream pathways merit further investigation as molecules of interest in HS pathogenesis.

Conclusion

Using a keratinocyte wound healing model, we were able to show that keratinocytes harvested from HS, CW, and normal skin exhibit distinct behaviors in response to in-vitro wounding. Results indicate that keratinocyte viability and function are associated with, and may be influenced by, specific cytokines and growth factors such as IL-22. Inherent biologic mechanisms at the level of the keratinocyte may contribute to delayed healing in chronic wounds and the pathogenesis of hidradenitis suppurativa.

Creative Commons License

Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

Open Access

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Comments

Poster to be presented at GW Annual Research Days 2017.

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Cytokine production varies between hidradenitis suppurativa, chronic wounds and normal keratinocytes in an in-vitro wound closure model

Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory disease of the apocrine glands. This disease affects approximately 1% of the population and there is currently no known cure. In order to develop therapies that target the molecular drivers of this disease, the molecular mechanisms of pathogenesis must be elucidated. Defective keratinocyte function has been implicated in the pathogenesis of HS. The purpose of this study was to compare keratinocyte function in HS, chronic wound (CW), and normal (N) skin samples.

Methods

Human epidermal keratinocytes were cultured to reach 80% confluence in 6 well plates. A scratch assay was performed using a 1 ml sterile tip of about 100um in diameter. Culture supernatants were collected at 0, 24, 48, 72, and 96 hours after scratch.

Supernatants were analyzed using Luminex immunoassays. A panel of cytokines previously demonstrated to be correlated with HS pathogenesis were measured at each time point.

Apoptosis was assessed by flow cytometry for each group at 96-hour time-point after initial scratch using a FITC-annexinV and Propidium Iodide (PI). Apoptotic cells stained annexinV positive only, whereas necrotic cells stained double positive for annexinV and PI. Cell viability was measured 96 hours post scratch by fluorescence microscopy with a live/dead vital dye staining method.

Results

Cell viability was similar at 96 hours post scratch in the normal and HS keratinocytes. However, cell viability was significantly lower in chronic wound keratinocytes at 96 hours (p=0.0138).

Flow cytometry for AnnexinV and PI demonstrated that CW keratinocytes had a significantly higher rate of apoptosis (annexinV positive) and necrosis (annexinV and PI double positive cells) than normal (p=0.0075) and HS (p=0.028) keratinocytes.

Significantly higher levels of IL-1α and VEGF were observed in the normal keratinocyte culture supernatant compared to CW. In contrast IL-22 levels were significantly lower in the HS culture supernatant than both CW and normal keratinocytes (p=0.0008). This finding indicates that IL-22 and its downstream pathways merit further investigation as molecules of interest in HS pathogenesis.

Conclusion

Using a keratinocyte wound healing model, we were able to show that keratinocytes harvested from HS, CW, and normal skin exhibit distinct behaviors in response to in-vitro wounding. Results indicate that keratinocyte viability and function are associated with, and may be influenced by, specific cytokines and growth factors such as IL-22. Inherent biologic mechanisms at the level of the keratinocyte may contribute to delayed healing in chronic wounds and the pathogenesis of hidradenitis suppurativa.