IL-33/ST2 Activation Is involved in Ro60-Regulated Photosensitivity in Cutaneous Lupus Erythematosus

Abstract

Interleukin- (IL-) 33 contributes to various inflammatory processes. IL-33/ST2 activation participates in systemic lupus erythematous via binding to the receptor of Suppression of Tumorigenicity 2 protein (ST2). However, whether IL-33/ST2 interferes with the nosogenesis of cutaneous lupus erythematosus (CLE) has not been reported so far. Herein, we proposed to disclose the impacts on IL-33/ST2 activation and Ro60 on CLE and their potential implications in the photosensitization of CLE cells. IL-33, ST2, and Ro60 in CLE patients' skin lesions were detected. Murine keratinocytes stimulated with or without IL-33 were irradiated by ultraviolet B (UVB), and the levels of Ro60 and inflammation markers were determined. Keratinocytes were cocultured with J774.2 macrophages and stimulated with IL-33 for analysis of chemostasis. The results identified that IL-33, ST2, and downstream inflammation markers were significantly upregulated in CLE lesions with Ro60 overexpression. Additionally, IL-33 treatment promoted the upregulation of Ro60 induced by UVB treatment in murine keratinocytes. Moreover, IL-33 stimulates keratinocytes to induce macrophage migration via enhancing the generation of the chemokine (C-C motif) ligands 17 and 22. Meanwhile, the silencing of ST2 or nuclear factor-kappa B (NF-κB) suppression abolished IL-33-induced upregulation of Ro60 in keratinocytes. Similarly, the inhibition of SOX17 expression was followed by downregulation of Ro60 in keratinocytes following IL-33 stimulation. In addition, UVB irradiation upregulated SOX17 in keratinocytes. Conclusively, the IL-33/ST2 axis interferes with Ro60-regulated photosensitization via activating the NF-κB- and PI3K/Akt- and SOX17-related pathways.

Figure 1

IL-33 and ST2 are upregulated in CLE patients' cutaneous lesions. (a) Immunofluorescence analysis of IL-33 in paraffin sections. (b) Numerical quantification of the positivity percentage per square millimeter of IL-33-stained sections. (c) Immunofluorescence analysis of ST2 in paraffin sections. (d) Numerical quantification of the positivity percentage per square millimeter of ST2-stained sections. (e) qRT-PCR analysis of IL-33 and ST2 mRNA expression in fresh skin tissues. (f) qRT-PCR analysis of RANTES, MCP-1, and IP-10 mRNA in fresh skin tissues. (g, h) Immunofluorescence analysis of Ro60 colocalization and ST2 expression in lesional skin tissues. Normal samples (n = 10), nonlesional samples (n = 15), and lesional samples (n = 15). Data points and error bars represent mean ± SEM. Representative images are shown. Bar = 50 μm. ^∗p < 0.05, ^∗∗p < 0.01. CLE: cutaneous lupus erythematosus.

Figure 2

IL-33 promotes proinflammatory cytokine secretion and Ro60 expression in murine KCs upon UVB irradiation. In vitro, the PAM212 KCs were subjected to UVB irradiation, either alone or combined with IL-33 and bovine serum albumin (BSA) stimulation. (a) qRT-PCR determination of ST2, SOX17, CCL2, RANTES, ICAM-1, MCP-1, and IP-10 mRNA expression in KCs. (b) qRT-PCR detection of Ro60 mRNA in KCs. (c) Western blotting detection of Ro60 protein in cell lysates and supernatants of KCs. (d) Densitometry analysis of Western blotting bands of supernatant Ro60. (e) Densitometry analysis of Western blotting bands of cell lysate Ro60. (f) Western blotting analysis of Ro60 in cells transfected with ST2 siRNA or control siRNA and subjected to UVB and IL-33 treatments. (g) Densitometry analysis of Western blotting bands of Ro60 in ST2 siRNA- or control siRNA-transfected cells with UVB and IL-33 treatments. (h, i) Immunofluorescence analysis of ST2 and Ro60 in KCs subjected to different treatments. Data were obtained from three experiments conducted independently. Data points and error bars denote mean ± SEM. Bar = 5 μm. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. KC: keratinocyte.

Figure 3

Inhibition of the PI3K/Akt and NF-κB Axis affects the regulation of Ro60 and proinflammatory cytokines by IL-33/ST2 in KCs. PAM212 cells cultured in vitro were treated with ultraviolet B irradiation and IL-33 stimulation. (a) qRT-PCR determination of Ro60 mRNA expression in different treatment groups. (b) Western blotting determination of Ro60 protein expression in supernatants and cell lysates in different treatment groups. (c) Densitometry analysis of Western blot bands. (d) qRT-PCR detection of RANTES, MCP-1, and IP-10 mRNA levels in KCs. Data were obtained from three experiments conducted independently. Data points and error bars denote mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01. KC: keratinocyte.

Figure 4

Macrophage chemoattraction is induced by IL-33/ST2 activation in KCs. (a) Transwell detection of the migration of macrophages in different treatment groups and numerical representation. (b) Detection of ICAM-1 and CCL22 mRNA by qRT-PCR in IL-33-stimulated KCs. (c) Western blot analysis of ICAM-1 and CCL22 expression levels in the supernatants collected from IL-33-stimulated KCs. Data were obtained from three experiments conducted independently. Data points and error bars denote mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01. KC: keratinocyte.

Figure 5

Ultraviolet B (UVB) irradiation enhances SOX17 expression in murine KCs post-UVB irradiation. (a) qRT-PCR determination of SOX17 mRNA expression in KCs following UVB irradiation. (b) Immunofluorescence analysis of SOX17 expression in KCs following UVB irradiation. (c) FCM detection of SOX17 expression in KCs. Data were obtained from three experiments run independently. Data points and error bars denote mean ± SEM. ^∗∗p < 0.01. KC: keratinocyte.

Figure 6

SOX17 is pivotal in IL-33's effect on KCs. (a) Detection of SOX17, NF-κB, PI3K, AKT, and Ro60 mRNA expression in KCs following IL-33 treatment by qRT-PCR. (b) Western blot determination of SOX17, NF-κB, NF-KBP65, PI3K, p-PI3K AKT, p-AKT, and Ro60 expression in KCs following IL-33 irradiation. (c) qRT-PCR determination of SOX17 expression in KCs after SOx17 siRNA transfection. (d) Western blot determination of SOX17 expression in KCs after SOX17 siRNA transfection. (e) qRT-PCR determination of SOX17, CCL2, and ICAM-1 mRNA expression in KCs following IL-33 stimulation. (f) Western blot determination of SOX17, NF-κB, NF-KBP65, PI3K, p-PI3K AKT, p-AKT, and Ro60 expression in KCs following SOX17 siRNA transfection and IL-33 treatment. Data were obtained from three experiments conducted independently. Data points and error bars denote mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01. KC: keratinocyte.