Effects of Activin A on Cytokine Expression in Fibroblasts and Keratinocytes Under Atopic Dermatitis-Like Conditions

Abstract

Background

Activin A is involved in the inflammatory response, wound repair, and skin fibrosis. Serum activin A concentrations change during the menstrual cycle, and this can lead to periodic exacerbation of atopic dermatitis.

Objective

To determine the effect of activin A on the cytokine expression profiles of human fibroblasts and keratinocytes under atopic dermatitis-like conditions.

Methods

Cultured human fibroblasts and keratinocytes were treated with activin A in combination with interleukin (IL)-4 and IL-13 to determine the changes in cytokine concentrations.

Results

Activin A decreased the expression of IL-1β and IL-23 mRNA in fibroblasts. IL-4 and IL-13 increased the expression of IL-6 and IL-16 and decreased the expression of IL-1α, IL-1β, IL-8, IL-10, and IL-23 in fibroblasts. In keratinocytes, IL-4 and IL-13 increased the expression of IL-1α and IL-1β and decreased the expression of IL-8. Activin A treatment in combination with IL-4 and IL-13 significantly increased the expression of IL-1α, IL-6, IL-8, IL-10, IL-16, and IL-23 and decreased the expression of IL-1β in fibroblasts. In keratinocytes, only IL-8 expression was significantly increased following treatment with activin A, IL-4, and IL-13.

Conclusion

Activin A modulates cytokine expression in a cell type dependent manner under conditions mimicking atopic dermatitis, suggesting its potential involvement in cutaneous inflammatory regulation.

INTRODUCTION

Atopic dermatitis (AD) is a chronic inflammatory disorder of the skin caused by an abnormal immune response. Various inflammatory mediators interact in AD, and proinflammatory cytokines are key players in the regulation of inflammatory responses. AD lesions exhibit increased expression of Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13. By downregulating filaggrin, loricrin, and involucrin, IL-4 and IL-13 compromise the integrity of the skin barrier¹,²,³.

Activin is a cytokine that belongs to the transforming growth factor-β (TGF-β) superfamily. It possesses a dimeric structure linked by disulfide bonds and exists as three major isoforms, including activin A, activin B, and activin AB, depending on the subunits that comprise it. Of these, activin A that is composed of 2 βA subunits is the most abundantly synthesized and plays a major role in inflammatory situations. Activin is recognized by receptors located on the cell membrane and acts primarily through the SMAD2/3 signaling pathway⁴,⁵.

In skin tissue, activin A is primarily secreted by fibroblasts and keratinocytes. It has been reported to modulate the production of multiple inflammatory mediators, including tumor necrosis factor-α, IL-1β, IL-6, inducible nitric oxide synthase, and cyclooxygenase 2, and is primarily involved in wound healing and fibrotic responses⁴,⁵. Activin A expression was significantly increased in the skin of wounded rats from days 1 to 7 after injury⁵. Activin A is overexpressed in keloid lesion fibroblasts, and its inhibition has been proposed as a new approach for keloid treatment⁶. AD shares certain inflammatory and immune response pathways with the pathological mechanisms of keloids⁷.

Common exacerbating factors for AD include psychological stress, infectious episodes, and exposure to allergens⁸. In female patients, AD symptoms of AD are sometimes exacerbated during the menstrual cycle⁹,¹⁰. Estrogen and progesterone promote the activity of Th2 cells and regulatory T cells and this can exacerbate AD and cause itching¹¹. Activin A blood levels change cyclically with menstruation, and it is thought to be involved in the aggravation of dermatitis.

The interaction of activin A with various cells and its effects on cytokine expression have also been studied. Activin A is involved in migration and differentiation by promoting protease production in murine macrophages and mast cells, and it induces a Th2 immune response by promoting the differentiation of these cells into M2 macrophages¹²,¹³,¹⁴. It also weakly stimulates the production of TGF-β in human lung fibroblasts¹⁵. However, the effects of activin A on cytokine expression in human dermal fibroblasts and epidermal keratinocytes have not yet been studied in detail.

Therefore, in this study, we aimed to determine the role of activin A in skin tissue by identifying changes in cytokine profiles caused by the interaction of activin A with fibroblasts and keratinocytes under AD-like conditions.

MATERIALS AND METHODS

Cell culture

Human dermal fibroblasts (Gibco™ Life Technologies Corp., Waltham, MA, USA) and human epidermal keratinocytes (Gibco™ Life Technologies Corp.) were used. Human dermal fibroblasts were cultured in Medium 106 (Gibco™ Life Technologies Corp.) supplemented with Low Serum Growth Supplement containing antibiotics until passage 3. Human epidermal keratinocytes were cultured in EpiLife™ Medium (Gibco™ Life Technologies Corp.) supplemented with Human Keratinocyte Growth Supplement containing antibiotics until passage 3.

Cytokine and activin A treatment

Human dermal fibroblasts and epidermal keratinocytes were treated for 24 hours with serum-free medium containing IL-4 (50 ng/ml), IL-13 (50 ng/ml), and activin A (10 and 25 ng/ml).

RNA isolation and quantitative reverse transcription polymerase chain reaction (PCR) analysis

RNA was extracted from cultured cells using an RNeasy Mini Kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. The cDNA was transcribed from 1 μg of RNA using the RevertAid™ First Strand cDNA synthesis kit and Oligo (dT)₁₈ primers (Thermo Fisher Scientific Inc., Vilnius, Lithuania). The primers used in this study are listed in Table 1.

Table 1. Primers used in quantitative reverse transcription polymerase chain reaction.

Genes	Primer sequences	Product size (bp)	Annealing temperature (℃)
IL-1α	5'-TGTATGTGACTGCCCAAGATGAAG-3'	96	60
	5'-AGAGGAGGTTGGTCTCACTACC-3'
IL-1R1	5'-ATTTCTGGGCTTCTAGTCTGGTGTTTC-3'	80	60
	5'-AACGTGCCAGTGTGGAGTGA-3'
IL-1β	5'-TGATGGCTTATTACAGTGGCAATG-3'	140	60
	5'-GTAGTGGTGGTCGGAGATTCG-3'
IL-6	5'-GTGTTGCCTGCTGCTGCCTTC-3'	194	60
	5'-AGTGCCTCTTTGCTGCTTTC-3'
IL-8	5'-GACATCTCCAAACCTTTCCAC-3'	160	60
	5'-CTTCTCCACAACCCTCTGC-3'
IL-10	5'-GAACCAAGACCCAGACATC-3'	137	60
	5'-CATTCTTCACCTGCTCCAC-3'
IL-16	5'-TTGGACACAGGGTTCTCGCTCA-3'	128	60
	5'-AGCAGGGGAGATAACGGACTGA-3'
IL-23	5'-GAGCCTTCTCTGCTCCCTGATA-3'	121	60
	5'-GACTGAGGCTTGGAATCTGCTG-3'
β-actin	5'-GCGGAGAAGATGACCCAGATC-3'	77	60
	5'-GGATAGCACAGCCTGGATAG-3'

IL: interleukin, IL-1R1: interleukin-1 receptor 1.

Products were denatured at 95°C for 10 minutes, and this was followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minutes using the StepOnePlus™ real-time PCR system and Power SYBR™ Green PCR Master Mix (Applied Biosystems™, Foster, CA, USA). The crossing point of the target gene and β-actin was quantified by calculating the following formula: 2^{−(target gene–ß actin)}.

Statistical analysis

All statistical analyses were performed using SPSS Statistics version 25.0 (IBM Corp., Armonk, NY, USA). Comparisons were made using one-way analysis of variance followed by Tukey’s post hoc test, with p-values of less than 0.05 considered significant.

Ethics statement

This study was conducted using commercially available cell lines. As no patients or human subjects were involved, Institutional Review Board approval was not required.

RESULTS

Effects of activin A on cytokine profiles of human fibroblasts and human keratinocytes

Human fibroblasts were treated with 25 ng/ml of activin A for 24 hours, and cytokine mRNA expression was compared to that of the control. The results revealed 0.25- and 0.78-fold decreases in IL-1β and IL-23 mRNA expression, respectively. No statistically significant differences were observed in the number of human keratinocytes. The results are presented in Fig. 1.

Fig. 1. Effects of activin A on cytokine mRNA profiles of (A) human fibroblasts and (B) human keratinocytes.

IL: interleukin.

^*p<0.05.

Effects of AD environment on cytokine profiles of human fibroblasts and human keratinocytes

To create an AD-like environment, fibroblasts and keratinocytes were treated with 50 ng/ml IL-4 and IL-13 for 24 hours. Comparing the expression of cytokines to that of the untreated control, the expression of IL-6 and IL-16 was increased in fibroblasts (2.78- and 1.25-fold, respectively), and the expression of IL-1α, IL-1β, IL-8, IL-10, and IL-23 was significantly decreased (0.65-, 0.55-, 0.27-, 0.70-, and 0.54-fold, respectively). In keratinocytes, the expression of IL-1α and IL-1β increased (2.47- and 2.51-fold, respectively), and the expression of IL-8 decreased by 0.84-fold. The results are presented in Fig. 2.

Fig. 2. Cytokine mRNA profiles of (A) human fibroblasts and (B) human keratinocytes under atopic dermatitis conditions with activin A modulation.

IL: interleukin.

^*p<0.05.

Effects of activin A on cytokine profiles of human fibroblasts and human keratinocytes in the environment of AD

Cultures supplemented with IL-4 and IL-13 were treated with activin A concentrations of 10 ng/ml and 25 ng/ml, respectively, for 24 hours and compared to control cultures supplemented with IL-4 and IL-13 without activin A. In fibroblasts, the expression of IL-1α, IL-6, IL-8, IL-10, IL-16, and IL-23 was significantly increased by activin A (3.78-, 1.33-, 3.38-, 1.37-, 1.41-, and 1.66-fold, respectively) at an activin A concentration of 25 ng/ml. In contrast, IL-1β expression was decreased (0.70-fold at an activin A concentration of 25 ng/ml). In keratinocytes, the overall cytokine expression increased, but only IL-8 expression exhibited a significant change (1.29-fold at 25 ng/ml activin A concentration). The results are presented in Fig. 2.

DISCUSSION

To date, various roles for activin A in the context of skin tissue have been identified. It is also involved in wound regeneration and scar formation. Activin A is primarily secreted by keratinocytes and fibroblasts and promotes their proliferation¹⁶. Overexpression of activin A promotes granulation tissue formation and re-epithelialization but results in excessive scarring. It is also involved in tissue repair processes in other organs such as the liver, lungs, pancreas, and kidneys. Additionally, activin A is regarded as an important regulator of inflammation. Prior studies have shown that its levels rise in blood and tissues under inflammatory conditions, and that it stimulates the production of multiple inflammatory mediators in cachexia⁴.

To understand the role of activin A in fibroblasts and keratinocytes, we examined cytokine expression in each cell type. Treatment of both cell types with activin A decreased the expression of IL-1β and IL-23 mRNA and increased the expression of IL-10 mRNA in fibroblasts. In vitro experiments using human monocytes indicated that activin A inhibited the conversion of IL-1β to its active form¹⁷. Similarly, in normal skin tissue, activin A regulates inflammation rather than induces inflammation.

In a previous study, treatment of epidermal skin models with IL-4, IL-13, and IL-31 induced features of AD¹⁸. IL-4 and IL-13 induce the activation of signal transducer and activator of transcription (STAT)6 and STAT3 through their receptors on keratinocytes and the JAK-STAT signaling pathway, and this, in turn, represses the transcription of the epidermal differentiation complex. This then inhibits the expression of filaggrin, loricrin, and involucrin, leading to skin barrier dysfunction³.

In the present study, we assessed the expression of inflammatory cytokines after treatment with IL-4 and IL-13 to create an AD environment and observed that the expression of inflammatory cytokines was significantly altered. In particular, the expression levels of IL-1α and IL-1β were significantly increased in keratinocytes, while the expression of IL-1α and IL-1β was decreased in fibroblasts. The expression of IL-6 was also significantly increased, indicating differences in response depending on the cell type. In a previous study, IL-23 expression was reduced in fibroblasts isolated from the spinous process entheses by IL-4 and IL-13¹⁹. Similarly, IL-23 mRNA expression was reduced by IL-4 and IL-13 in dermal fibroblasts.

Additional treatment with activin A in the AD environment resulted in a greater increase in the expression of proinflammatory cytokines compared to that in response to no treatment, particularly for IL-1α and IL-8 in fibroblasts.

IL-1α promotes the transcription of genes involved in the inflammatory response through the NF-kB pathway in keratinocytes, fibroblasts, and vascular endothelial cells²⁰. IL-1α is increased in the stratum corneum of mice with a loss-of-function mutation in filaggrin²¹. IL-8 has been demonstrated to be significantly reduced in the stratum corneum of the epidermis during the treatment of patients with AD in response to improvements in skin symptoms²². It also promotes wound healing by increasing the migration rate and intercellular adhesion of keratinocytes, limiting their proliferation, and reducing adenosine 5'-triphosphate-induced fibroblast contraction²³,²⁴. Thus, in the setting of AD, activin A may alter the cytokine expression profile, affecting the proliferation and inflammatory response of skin cells (Fig. 3).

Fig. 3. Effect of activin on cytokine profile changes in normal/atopic fibroblasts and keratinocytes.

IL: interleukin, AD: atopic dermatitis.

In women, types of inflammatory dermatitis such as AD and psoriasis may exhibit short-term acute exacerbations immediately before or during menstruation⁹. Worsening AD symptoms of AD during pregnancy have also been reported¹⁰. Blood activin A levels change with the menstrual cycle and gestational age, with the highest increases occurring after the mid-luteal phase and during the third trimester²⁵. Activin A is believed to be involved in the mechanisms that worsen dermatitis during menstruation or pregnancy.

One limitation of this study is that the effects of cell-to-cell interactions were excluded. In dermatitis, the epidermis and dermis interact²⁶. For example, the inflammatory response of fibroblasts is mediated by IL-1 derived from keratinocytes²⁷,²⁸. There are 3-dimensional (3D) models that simulate human skin tissue and allow for histological analysis and the elucidation of interactions between different cells. A reconstructed human epidermal model treated with an inflammatory cocktail to create an AD environment exhibited morphological changes and altered transcriptome profiles²⁹. Recent studies have demonstrated that activin A secreted by mesenchymal stem cells influences epidermal differentiation and regeneration through a paracrine effect³⁰. Therefore, further studies are necessary to examine the role of activin A using 3D models that simulate the AD environment and skin immune systems.

Second, cytokine regulation in this study was evaluated only at the mRNA level. Although immunoblot analyses were performed for related signaling and barrier proteins, these experiments did not directly quantify cytokine proteins, partly due to insufficient protein yield. Future studies using high-sensitivity protein assays will be required to clarify how activin A modulates cytokine production under AD conditions.

In summary, activin A modulates cytokine expression in a cell type dependent manner under conditions that resemble AD. These results suggest a potential role for activin A in inflammatory amplification within inflamed skin.