Human keratinocytes' response to injury upregulates CCL20 and other genes linking innate and adaptive immunity

Abstract

In the early stages of wound healing, keratinocytes (KCs) become "activated" and release inflammatory molecules such as IL-1 and IL-8, which are linked to innate immune responses and neutrophil recruitment. It is unclear, however, whether KCs release molecules linked to adaptive immune responses, e.g., CCL20, in their early state of activation without signals from infiltrating T cells. This study aims to isolate the immediate alterations in protective and inflammatory gene expression that occur in epidermal KCs, with a particular focus on molecules associated with cell-mediated immunity. We used dispase-separated epidermis, followed by intercellular disassociation by trypsinization, as a model for epidermal injury. We obtained a pure population of KCs using flow cytometry. As a control for uninjured epidermis, we performed laser capture microdissection on normal human skin. Sorted KCs had an early burst of upregulated gene expression, which included CCL20, IL-15, IL-23A, IFN-κ, and several antimicrobial peptides. Our results provide insight into the potential role of KCs as contributors to cell-mediated inflammation, and expand knowledge about gene modulation that occurs during early wound healing. Our findings may be relevant to cutaneous diseases such as psoriasis, where micro-injury can trigger the formation of psoriatic plaques at the site of trauma.

Figure 1. Isolation of a pure population of KCs by FACS and normal epidermis by laser capture microdissection

(a) Sorted KCs were isolated from epidermal single-cell suspensions with flow cytometry. Using antibodies for CD207 and c-kit, we identified three cell populations: CD207 ⁺ c-kit ⁻ cells (Langerhans cells), CD207 ⁻ c-kit ⁺ (melanocytes), and CD207 ⁻ c-kit ⁻ cells. We collected CD207 ⁻ c-kit ⁻ cells as KCs. (b) Principal components analysis of microarray data for sorted KCs (KC sorted), cultured KCs (KC in vitro), and tissue isolated by LCM, including normal epidermis (KC LCM), normal dermis, and bulk (dermis + epidermis). PC1 accounts for 39% of the variance. (c) Heat map of top 100 differentially expressed genes for sorted KCs (KC sorted) and normal epidermis (KC LCM).

Figure 2. Differentially expressed genes in microarray for KC sorted vs. KC LCM are validated by quantitative RT-PCR

We performed quantitative RT-PCR using mRNA from sorted KCs (n=6) and KC LCM (n=6). All data was normalized to hARP (human acidic ribosomal protein) housekeeping gene. A two-tailed student’s t-test confirmed the differential expression of all genes tested (* p < 0.05, ** p < 0.01). Error bar denotes mean +/− SD.

Figure 3. TNF regulates expression of CCL20 and IL23A by KCs

(a) Cell lines of NHK (n=3) were treated with 10 ng ml⁻¹ TNF and/or Etanercept 10 μg ml⁻¹. As measured by RT-PCR, CCL20 and IL-23A mRNA at 24 hours were increased compared to control (p=0.005, p=0.0038) and Etanercept neutralized TNF-induced expression of CCL20 and IL-23A (p=0.0026, p=0.0037) (b) Disassociated epidermal cells (n=3) were cultured for 16 hours +/− Etanercept. CCL20 mRNA expression was increased for both conditions compared to control. (c) Compared to control, CCL20 protein in supernatants of disassociated epidermal cells was increased in cells cultured +/− Etanercept (p=0.0001, p=0.0002). Etanercept inhibited CCL20 protein compared to cells cultured in media only (p=0.0414). Mean +/− SD. Statistical significance determined by two-tailed t-test *p<0.05, **p<0.01, ***p<0.001

Figure 4. CCL20 mRNA and protein are increased in normal adult skin irradiated with ultraviolet B light

Archived material was used from a study in which we irradiated the skin of normal adults (n=10) with 2MED narrowband UVB light (312 nm). Biopsies were before irradiation and 24 hours later. (a) CCL20 mRNA expression, as measured by quantitative RT-PCR, was upregulated in 10/10 patients after UVB irradiation, with a mean 34-fold increase (p = 0.002). Lines denote the mean normalized gene expression. (b) Immunohistochemistry staining for CCL20 shows increased protein expression after UVB irradiation. The insert shows CD3 antibody staining as a control for non-specific staining of damaged epithelial cells. Scale bar = 1 μm