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. 2024 Jan 15:10:1324692.
doi: 10.3389/fmolb.2023.1324692. eCollection 2023.

Exosomes released by environmental pollutant-stimulated Keratinocytes/PBMCs can trigger psoriatic inflammation in recipient cells via the AhR signaling pathway

Hye Ran Kim  1 , So Yeon Lee  1 , Ga Eun You  2 , Chun Wook Park  1 , Hye One Kim  1 , Bo Young Chung  1
Affiliations

Exosomes released by environmental pollutant-stimulated Keratinocytes/PBMCs can trigger psoriatic inflammation in recipient cells via the AhR signaling pathway

Hye Ran Kim et al. Front Mol Biosci. .

Abstract

Introduction: Exosomes, pivotal in intercellular communication during skin disease pathogenesis, have garnered substantial attention. However, the impact of environmental pollutants, such as benzo[a]pyrene (BaP) and 2, 3, 7, 8-tetrachlorodibenzo-p-dioxin (TCDD), on exosome release amid inflammatory skin diseases remains unexplored. This study addresses this gap by examining the influence of BaP and TCDD on exosome function, specifically focusing on immune-related pathway alterations in normal recipient keratinocytes and peripheral blood mononuclear cells (PBMCs). Methods: HaCaT cells were treated with exosomes from BaP- or TCDD-treated keratinocytes. Proinflammatory cytokines and chemokines, including TNF-α, IL-1β, IL-6, IL-8, CXCL1, and CXCL5, were assessed. The involvement of the p65NF-κB/p38MAPK/ERK signaling pathway in recipient keratinocytes was investigated. Aryl hydrocarbon receptor (AhR) silencing was employed to elucidate its role in mediating the proinflammatory response induced by exosomes from BaP- or TCDD-treated keratinocytes. Results and discussion: Treatment with exosomes from BaP- or TCDD-treated keratinocytes induced a significant increase in proinflammatory cytokines and chemokines in HaCaT cells. The upregulation implicated the p65NF-κB/p38MAPK/ERK signaling pathway. AhR silencing attenuated this response, suggesting a role for AhR in mediating this response. In PBMCs from healthy controls, exosomes from BaP-stimulated PBMCs of psoriatic patients led to increased expression of proinflammatory cytokines and modulation of Th1/Th17 cell distribution via AhR activation. These findings unveil a novel dimension in the interplay between environmental xenobiotic agents (BaP and TCDD) and exosomal functions. The study establishes their influence on psoriatic inflammatory responses, shedding light on the underlying mechanisms mediated through the AhR signaling pathway in recipient keratinocytes and PBMCs.

Keywords: 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); aryl hydrocarbon receptor; benzo[a]pyrene; exosomes; psoriasis.

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Conflict of interest statement

Author GY was employed by the company Biosolution, Seoul Technopark. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

FIGURE 1
FIGURE 1
Characterization of exosomes derived from HaCaT cells and PBMCs and exosome uptake analysis. (A) Size distribution of exosomes by nanoparticle tracking analysis (n = 4). (B) The expression of CD63, CD9, HSP70, Alix and TSG101 in exosomes and the corresponding total cell lysates. The relative expression was normalized to GAPDH (n = 3). The densitometric value of each independent band was compared with the total cell lysate control. (C) The representative immunofluorescence images of exosomes derived from BaP-treated HaCaT cells-treated recipient HaCaT cells or BaP-treated PS PBMC-treated recipient HC PBMCs. Nuclei were counterstained with DAPI (blue). Scale bar, 75 μm. exo, exosomes, BaP, benzo [a]pyrene. PS, psoriasis patients, HC, healthy controls, PBMC, peripheral blood mononuclear cell.
FIGURE 2
FIGURE 2
The effects of exosomes derived from BaP- or TCDD-treated HaCaT cells on proinflammatory cytokine/chemokine mRNA or protein expression in recipient HaCaT cells. (A) The mRNA expression of TNF-α, IL-1β, IL-6, IL-8, CXCL1 and CXCL5. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. Data represent the mean ± S.D. of 3 independent experiments. **p < 0.01 and ***p < 0.001. (B) Immunoblotting of TNF-α, IL-1β, IL-6, IL-8, CXCL1 and CXCL5. The relative expression was normalized to GAPDH (n = 3). The relative intensities of western blots were compared with the control and measured by ImageJ software. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. Data represent the mean ± S.D. of 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001.
FIGURE 3
FIGURE 3
The effects of exosomes derived from BaP- or TCDD-treated HaCaT cells on the p65/NF-κB, p38/MAPK and ERK/MAPK signaling pathways in recipient HaCaT cells. (A) P-P65, P-P38 and P-ERK expression treated with exosomes derived from BaP- or TCDD-treated HaCaT cells by Western blotting. The relative expression was normalized to GAPDH (n = 3). The relative intensity of each band was quantified by densitometric scan. (B) TNF-α, IL-1β, IL-6, IL-8, CXCL1, and CXCL5 expression with the exosome-derived from BaP- or TCDD-treated HaCaT cell in the absence or presence of inhibitors of P65 (PDTC, 10 µM), P38 (SB203580, 10 µM), or ERK (PD980599, 5 µM) by quantitative PCR. Statistics: mean ± S.D. (n = 3). Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. **p < 0.01 and ***p < 0.001. PDTC, pyrrolidine dithiocarbamate, exo, exosome, BaP, benzo [a]pyrene.
FIGURE 4
FIGURE 4
The effects of exosomes derived from BaP- or TCDD-treated HaCaT cells on the AhR and CYP1A1 expression in recipient HaCaT cells. (A) The treatment of recipient HaCaT cells with exosomes derived from BaP- or TCDD-treated HaCaT cells increased AhR and CYP1A1 mRNA expression. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. Data represent the mean ± SD of 3 independent experiments. **p < 0.01 and ***p < 0.001. (B) The protein expression of AhR and CYP1A1. The relative expression was normalized to that of GAPDH (n = 3). The relative intensities were compared with the control.
FIGURE 5
FIGURE 5
The Upregulation of Cytokine and Chemokine Expression in Recipient HaCaT Cells Treated with BaP- or TCDD-Stimulated HaCaT Cell-Derived Exosomes was dependent on AhR signaling pathway. (A) A 77% reduction in AhR mRNA was shown in the AhR siRNA-transfected cells compared to the nontransfected cells. HaCaT cells were transfected with AhR siRNA for 24 h. (B) Cell viability of AhR siRNA transfected HaCaT cells by MTT assay. Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. Data represent the mean ± SD of 3 independent experiments. (C) AhR silencing attenuated the upregulation induced by exosomes from TCDD-treated HaCaT cells or BaP-treated HaCaT cells on TNF-α, IL-1β, IL-6, IL-8, CXCL1, and CXCL5 in recipient HaCaT cells. Data represent the mean ± S.D. (n = 3). Statistical analysis was performed by one-way ANOVA followed by Tukey’s multiple comparison test. **p < 0.01 and ***p < 0.001. exo, exosome, BaP, benzo [a]pyrene.
FIGURE 6
FIGURE 6
The effects of exosomes derived from BaP-treated PS PBMCs on AhR and CYP1A1 expression in recipient HC PBMCs. (A) Representative images of western blots for AhR and CYP1A1. The relative expression was normalized to GAPDH. The results are representative of three independent experiments. The relative intensities of western blots were measured by ImageJ. (B) mRNA expression of AhR and CYP1A1. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. Data represent the mean ± S.D. of 3 independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001.
FIGURE 7
FIGURE 7
The effects of exosomes derived from BaP-treated PS PBMCs on proinflammatory cytokine expression, and the distribution of IL-17A-positive and IFN-γ-positive CD4+ T cells in recipient HC PBMCs. (A) The expressions of IL-6, IL-17A, IL-22, IL-23, and IFN-γ by immunofluorescence. Results are a representation of one sample of each group (PS = 3, HC = 3). Scale bar = 75 µm. The fluorescence intensity was semi-quantitatively analyzed and the results are presented as the mean optical density with standard deviation based on three different digital images. Statistical significance was determined by one-way ANOVA followed by Tukey’s multiple comparison test. ***p < 0.001 (B) Representative dot plots and the percentages of IL-17A-positive and IFN-γ-positive CD4+ T cells. The percentage of IL-17A-positive and IFN-γ-positive CD4+ T cells by flow cytometric analysis. The percentage of dead cells in IL-17 and CD4 was 1.86% for HC, 1.09% for HC/PS-exo, and 0.21% for HC/PS Bap-exo. For INF-y and CD4, the percentages were 0.38% for both HC and HC/PS-exo, and 0.11% for HC/PS Bap-exo and 0.57%. Results are a representation of one sample of each group (PS = 3, HC = 3). Statistics: mean ± S.D. **p < 0.05 and ***p < 0.001. exo, exosome, BaP, benzo [a]pyrene, PS, psoriasis patient, HC, healthy control.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This study was supported by the National Research Foundation of Korea (NRF-2021R1F1A1059510), Hallym University Research Fund, Hallym University Medical Center Research Fund, and Hallym University Research Fund 2023 (HURF-2023-32). The funders had no involvement in the study design, data collection, analysis, interpretation, writing, or submission decisions.
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