Epidermal Growth Factor Attenuated the Expression of Inflammatory Cytokines in Human Epidermal Keratinocyte Exposed to Propionibacterium acnes

Abstract

Background: While the beneficial effects of topical epidermal growth factor (EGF) on wound healing have been repeatedly reported, there are few reports about the effects of EGF on inflammatory skin diseases including acne.

Objective: To clarify the effects of EGF on acne, it was investigated whether recombinant human EGF (rhEGF) signalling can affect Propionibacterium acnes-induced cytokine expression in human epidermal keratinocytes.

Methods: The cultured normal human epidermal keratinocytes (NHK) were co-treated with P. acnes and rhEGF, and mRNA levels of interleukin (IL)-1α, IL-8 and tumor necrosis factor (TNF)-α; toll-like receptor 2 (TLR2); and nuclear factor kappa B (NF-κB) were determined. Specific enzyme-linked immunosorbent assay kits were used to measure the IL-1α, IL-8 and TLR2 expression as well as the NF-κB activation in P. acnes and rhEGF-treated NHK. After infecting the cultured NHK with live P. acnes, an increased expression of IL-1α, IL-8 and TNF-α was detected, which was prevented by rhEGF co-treatment.

Results: TLR2 and NF-κB activity increased after P. acnes treatment, and rhEGF treatment decreased TLR2 expression and NF-κB activity dose-dependently. The inhibition of EGF receptor by gefitinib attenuated the inhibitory effect of rhEGF on these increased expressions of proinflammatory cytokines and TLR2 and activity of NF-κB in NHK stimulated by P. acnes.

Conclusion: These results suggest that EGF attenuated P. acnes-induced inflammatory responses, at least in part, through the modulation of TLR2 signalling, and the topical application of rhEGF may be beneficial to relieve the inflammatory reactions of acne.

Keywords: Acne vulgaris; Epidermal growth factor; Propionibacterium acnes; Toll-like receptor 2.

Fig. 1. The effect of recombinant human epidermal growth factor (rhEGF) on interleukin (IL)-1α, IL-8 and tumor necrosis factor (TNF)-α expression in Propionibacterium acnes-stimulated normal human epidermal keratinocytes (NHK). NHK were stimulated with 10 multiplicity of infection (MOI) P. acnes for 6 and 48 hours in the presence of 0.1, 1 or 10 ng/ml rhEGF, or 5 µM nicotinamide. The messenger ribonucleic acid (mRNA) expression levels of IL-1α, IL-8 and TNF-α were analysed through real-time quantitative reverse transcription–polymerase chain reaction (RT-qPCR). The secretion of IL-1α, IL-8 and TNF-α was determined by using specific enzyme-linked immunosorbent assay (ELISA) kits. (A) The mRNA expression of IL-1α, IL-8 and TNF-α induced by P. acnes was inhibited by rhEGF in a concentration-dependent manner. 5 µM nicotinamide, which was used as a positive control, was also observed to reduce the mRNA levels of IL-1α, IL-8 and TNF-α. (B) Similar to the gene result, in a concentration-dependent manner, rhEGF downregulated the protein expression of IL-1α, IL-8 and TNF-α induced by P. acnes. Results are presented as mean±standard deviation (SD), representative of three separate experiments. Asterisks indicate statistically significant differences (^**p<0.01).

Fig. 2. The Propionibacterium acnes-induced expression of toll-like receptor 2 (TLR2) and nuclear factor kappa B (NF-κB) activity were reduced by recombinant human epidermal growth factor (rhEGF). Normal human epidermal keratinocytes (NHK) were stimulated with 10 multiplicity of infection (MOI) P. acnes for 6 and 48 hours in the presence of 0.1, 1, or 10 ng/ml rhEGF, or 5 µM nicotinamide. The messenger ribonucleic acid (mRNA) expression levels of TLR2, P38α and NF-κB were analysed through real-time quantitative reverse transcription–polymerase chain reaction (RT-qPCR). The protein expression of TLR2 and NF-κB activity were determined by using specific enzyme-linked immunosorbent assay (ELISA). (A, B) The gene and protein of TLR2 in P. acnes-stimulated NHK was inhibited by rhEGF and 5 µM nicotinamide. (C, D) In P. acnes-stimulated NHK, the mRNA expression levels of NF-κB were downregulated by rhEGF in a concentration-dependent manner, but not P38α. In contrast, 5 µM nicotinamide inhibited the mRNA expression levels of P38α in P. acnes-treated NHK, but not NF-κB. (E) Similar to the gene result, the P. acnes-induced activity of NF-κB was inhibited by rhEGF in a concentration-dependent manner. Results are presented as mean±standard deviation (SD), representative of three separate experiments. Asterisks indicate statistically significant differences (^*p<0.05, ^**p<0.01).

Fig. 3. The effects of the inhibition of interleukin (IL)-1α, IL-8 and tumor necrosis factor (TNF)-α expression on recombinant human epidermal growth factor (rhEGF) attenuated by gefitinib. Normal human epidermal keratinocytes (NHK) were treated with Propionibacterium acnes in the presence or absence of 1 or 10 ng/ml rhEGF for 48 hours, with pretreatment with 100 nM gefitinib, which was a selective epidermal growth factor (EGF) receptor inhibitor. Then, stimulation supernatants were collected, and the protein expressions of IL-1, IL-8 and TNF-α were determined by using specific enzyme-linked immunosorbent assay. Gefitinib of 100 nM attenuated the inhibitor effect of rhEGF on IL-1α (A), IL-8 (B) and TNF-α (C) in P. acnes-treated NHK. Results are presented as mean±standard deviation (SD), representative of three separate experiments. Asterisks indicate statistically significant differences (^*p<0.05, ^**p<0.01, ^***p<0.001).

Fig. 4. Gefitinib reduced the inhibition effect of recombinant human epidermal growth factor (rhEGF) on toll-like receptor 2 (TLR2) and nuclear factor kappa B (NF-κB) activity increased by Propionibacterium acnes. Normal human epidermal keratinocytes (NHK) were treated with P. acnes in the presence or absence of 1 or 10 ng/ml rhEGF for 6 and 48 hours, with pretreatment with 100 nM gefitinib. After treatment, the total ribonucleic acid (RNA) was prepared from the cells, and the relative gene expressions of TLR2, P38α and NF-κB were analysed through real-time quantitative reverse transcription–polymerase chain reaction (RT-qPCR); then, the total protein was collected from the cells, and the protein expression of TLR2 was detected by using enzyme-linked immunosorbent assay (ELISA). To assess NF-κB activity, nuclear protein was collected by using nuclear extraction kits, and a specific ELISA kit was used for measuring NF-κB activity. (A, B) TLR2 gene and protein expression was inhibited by 1 and 10 ng/ml rhEGF, and was reduced by 100 nM gefitinib in P. acnes-treated NHK. (C) Gefitinib and rhEGF did not affect the P38α mRNA expression level in P. acnes-stimulated NHK. The inhibition effect of rhEGF on gene expression (D) and NF-κB activity (E) was inhibited by 100 nM gefitinib. Results are presented as mean±standard deviation (SD), representative of three separate experiments. Asterisks indicate statistically significant differences (^*p<0.05, ^**p<0.01).

Fig. 5. Scheme of the pathway that mediates the epidermal growth factor (EGF)-induced inactivation of proinflammatory cytokines. TLR2: toll-like receptor 2, EGFR; EGF receptor, NF-κB: nuclear factor kappa B.