Non-coding Double-stranded RNA and Antimicrobial Peptide LL-37 Induce Growth Factor Expression from Keratinocytes and Endothelial Cells

Abstract

A critical function for skin is that when damaged it must simultaneously identify the nature of the injury, repair barrier function, and limit the intrusion of pathogenic organisms. These needs are carried out through the detection of damage-associated molecular patterns (DAMPs) and a response that includes secretion of cytokines, chemokines, growth factors, and antimicrobial peptides (AMPs). In this study, we analyzed how non-coding double-stranded RNA (dsRNAs) act as a DAMP in the skin and how the human cathelicidin AMP LL-37 might influence growth factor production in response to this DAMP. dsRNA alone significantly increased the expression of multiple growth factors in keratinocytes, endothelial cells, and fibroblasts. Furthermore, RNA sequencing transcriptome analysis found that multiple growth factors increase when cells are exposed to both LL-37 and dsRNA, a condition that mimics normal wounding. Quantitative PCR and/or ELISA validated that growth factors expressed by keratinocytes in these conditions included, but were not limited to, basic fibroblast growth factor (FGF2), heparin-binding EGF-like growth factor (HBEGF), vascular endothelial growth factor C (VEGFC), betacellulin (BTC), EGF, epiregulin (EREG), and other members of the transforming growth factor β superfamily. These results identify a novel role for DAMPs and AMPs in the stimulation of repair and highlight the complex interactions involved in the wound environment.

Keywords: antimicrobial peptide (AMP); betacellulin; cathelicidin; double-stranded RNA (dsRNA); fibroblast growth factor (FGF); insulin-like growth factor (IGF); keratinocyte; vascular endothelial growth factor (VEGF).

FIGURE 1.

NHEKs have a growth factor response to UVB poly(I:C), or LL-37 stimulation. A, analysis of RNA-seq gene profiling for genes in NHEKs after a 24-h exposure to 0.1 μg/ml poly(I:C) or 4 μm LL-37. Up-regulated genes with a greater than 2-fold change and a significant p value (p < 0.05) are represented. B and C, qPCR measurements of FGF2 and HBEGF cDNA created from 1 μg of RNA isolated from NHEKs at the indicated time points after the addition of 0.1 μg/ml of poly(I:C) (P(I:C)). D and E, ELISA of supernatant of NHEKs treated with 0.1 μg/ml poly(I:C). p values were calculated by a two-tailed Student's t test. The time course qPCR and the ELISA data are mean ± S.D. of biological replicates, n = 3, and are representative data from at least three independent experiments. Samples not detected by ELISA are indicated by the abbreviation N.D. Error bars represent S.D.

FIGURE 2.

UVB irradiation leads to increased expression of FGF2 in basal keratinocytes. A and B, qPCR of FGF2 and HBEGF from whole skin biopsies after a 24-h incubation in EpiLife medium following either no UVB irradiation (Control) or a 5 kJ/m² dose of UVB irradiation (UV). C–F, replica human punch biopsies from A and B were visualized via immunofluorescence of either non-irradiated human skin punch biopsies with IgG control (IgG), or a 5 kJ/m² UV dose (IgG + UV) showed background staining in the stratum corneum but not in basal keratinocytes. Anti-FGF2 staining detected low levels of expression in non-irradiated basal keratinocytes (FGF2) and increased FGF2 expression after a 5 kJ/m² UV dose (FGF2 + UV) (indicated by arrows). p values were calculated by a two-tailed Student's t test. Human biopsy qPCR data were from technical triplicates from n = 4 patients. The dotted line denotes the boundary between epidermal basal layer keratinocytes and the dermis. The scale bar represents 50 μm. Error bars represent S.E.

FIGURE 3.

Growth factor response to poly(I:C) is enhanced by LL-37. A, analysis of RNA-seq gene profiling for genes in NHEKs after a 24-h exposure to dsRNA mimic 0.1 μg/ml poly(I:C) (P(I:C)), 4 μm LL-37 (LL), or 0.1 μg/ml poly(I:C) and 4 μm LL-37 (LL/P(I:C)) co-stimulation. Genes represented follow the guidelines established in Fig. 1. B, heat map generated from selected cytokines and growth factors from A. C–I, qPCR measurement of IL-6, VEGFC, BTC, EGF, FGF2, and HBEGF from NHEKs following protocols described in Fig. 1. J and K, ELISA of supernatant of NHEKs from H and I, respectively. p values were calculated by one-way analysis of variance. qPCR data are mean ± S.D. of biological replicates, n = 3, and are representative data from four independent experiments. Samples not detected by ELISA are indicated by the abbreviation N.D. Error bars represent S.D.

FIGURE 4.

Poly(I:C) and LL-37 can stimulate FGF2 expression in basal layer keratinocytes. A–D, immunofluorescence staining of human skin punch biopsies with IgG control (IgG) antibodies indicated no background staining in basal keratinocytes. Anti-FGF2 antibodies detected low levels of FGF2 expression in basal keratinocytes of untreated control tissue (Ctrl). Tissue incubated with LL-37 (LL) or poly(I:C) (P(I:C)) had increased FGF2 expression in basal keratinocytes. Tissue co-incubated with poly(I:C) and LL-37 (LL/P(I:C)) had increased FGF2 expression compared with individual treatments. E and F, qPCR measurement of FGF2 and HBEGF from HDMECs using the treatment protocol described in Fig. 1. G and H, qPCR measurement of FGF2 and HBEGF from fibroblasts protocols described in E and F. Staining was performed on replicate punch biopsies of human skin from Fig. 2. The dotted line denotes the boundary between epidermal basal layer keratinocytes and the dermis. The scale bar represents 50 μm. p values were calculated by one-way analysis of variance. Data are mean ± S.D. of biological replicates, n = 3, and are representative data from at least three independent experiments. Error bars represent S.D.

FIGURE 5.

Small ncRNAs induce growth factor expression. A, C, and E, qPCR measurement of FGF2, HBEGF, and VEGFC from cDNA created from 500 ng of RNA isolated from NHEK cells 24 h after the addition of 1.0 μg/ml ncRNA U1 (U1), 1.75 μm LL-37 (LL), or co-treatment with 1.0 μg/ml ncRNA U1 and 1.75 μm LL-37 (LL/U1). B, D, and E, ELISA of supernatant of fibroblasts treated in E and F. p values were calculated by one-way analysis of variance. Data are mean ± S.D. of biological replicates, n = 3, and are representative data from at least two independent experiments (ELISAs) or from four independent experiments (qPCR). Samples not detected by ELISA are indicated by the abbreviation N.D. Error bars represent S.D.

FIGURE 6.

LL-37 affects multiple signal pathways and has synergy with poly(I:C) in stimulating the NF-κB pathway. A, keratinocytes were pretreated with LL-37 followed by stimulation with poly(I:C) for 2 h before cells were harvested for immunoblotting analyses using antibodies against phosphorylated and total forms of the following proteins: p38, AKT, IKK, NF-κB, and ERK. B–E, keratinocytes were preincubated with either p38, IKK, EGFR, or an endosomal inhibitor for 30 min prior to stimulation with 4 μm LL-37 (LL) and 0.1 μg/ml poly(I:C) (P(I:C)) for 16 h before RNA was isolated and FGF2 mRNA was quantified via qPCR. F–I, siRNA knockdown was performed on keratinocytes 72 h before stimulation with 4 μm LL-37 and 0.1 μg/ml poly(I:C) for 16 h before cells were collected, RNA was isolated, and qPCR measurements were performed. p values were calculated by two-way analysis of variance. Data are mean ± S.D. of biological replicates, n = 3, and are representative data from at least two or more independent experiments. Error bars represent S.D.