Ann Dermatol.  2016 Oct;28(5):579-585. 10.5021/ad.2016.28.5.579.

Induction of Interleukin-22 (IL-22) production in CD4+ T Cells by IL-17A Secreted from CpG-Stimulated Keratinocytes

• Li ZJ
• Choi DK
• Sohn KC
• Lim SK
• Im M
• Lee Y
• Seo YJ
• Kim CD
• Lee JH

• KMID: 2382882
• DOI: http://doi.org/10.5021/ad.2016.28.5.579

Abstract

BACKGROUND
Interleukin-17A (IL-17A) is mainly secreted from Th17 cells that are activated by various stimuli including CpG oligodeoxynucleotide, a Toll-like receptor 9 (TLR9) ligand. Recently, it has been demonstrated that keratinocytes play an important role in the pathogenesis of psoriasis.
OBJECTIVE
To investigate the potential role of keratinocytes, we examined whether TLR9 ligand CpG induces IL-17A expression in keratinocytes.
METHODS
We used HaCaT keratinocytes as a model system, and determined CpG-induced IL-17A using enzyme-linked immunosorbent assay and Western blot.
RESULTS
When HaCaT keratinocytes were treated with CpG, the expression of several cytokines including IL-17A, tumor necrosis factor-α and CCL20 was markedly increased. Treatment with nuclear factor (NF)-κB inhibitor significantly blocked the CpG-induced IL-17A production, indicating that CpG induced IL-17A expression through the NF-κB signaling pathway. In addition, IL-17A secreted from keratinocytes stimulated the CD4⁺ T cells, resulting in strong induction of IL-22 production.
CONCLUSION
Since IL-22 is an important mediator for psoriatic inflammation, our data suggest that keratinocytes can participate in the pathogenesis of psoriasis via the TLR9-dependent IL-17A production.

Keyword

CpG; Interleukin-17A; Keratinocytes; NF-κB; Psoriasis

MeSH Terms

Blotting, Western
Cytokines
Enzyme-Linked Immunosorbent Assay
Inflammation
Interleukin-17*
Keratinocytes*
Necrosis
Psoriasis
T-Lymphocytes*
Th17 Cells
Toll-Like Receptor 9
Cytokines
Interleukin-17
Toll-Like Receptor 9

Figure

• 

  Fig. 1 CpG induces interleukin-17A (IL-17A) expression in HaCaT keratinocytes. (A) Cells were treated with 10 µM CpG for the indicated time points, and cytokine expression was assessed by quantitative real-time polymerase chain reaction. (B) After treatment with CpG, medium was collected and IL-17A was determined by ELISA. The mean values±standard deviation are averages of triplicate measurements. *p<0.05 versus time 0 hour. (C) Expression of IL-17A was detected in normal and psoriatic lesional skin tissues by immunohistochemistry. It clearly shows that expression of IL-17A is increased in psoriatic lesional skin compared to normal skin. TNF-α: tumor necrosis factor-α.

• 

  Fig. 2 CpG induces interleukin-17A (IL-17A) expression via nuclear factor (NF)-κB-dependent way. (A) HaCaT keratinocytes were transduced with 1 multiplicity of infection of NF-κB reporter adenovirus for overnight. Cells were replenished with fresh medium, and then treated with 10 µM CpG for 24 hours. Luciferase assay was performed and data are expressed as fold induction. Data represent the mean±standard deviation (SD) (n=3). *p<0.05. (B) Cells were treated with 10 µM CpG for the indicated time points, then phosphorylated-IκBα (p-IκBα) and CIAP2 were analyzed by Western blot. (C) Cells were pretreated with NF-κB inhibitor Bay-11 (10 ng/ml) or CpG antagonist G-ODN (4 µM) for 30 minutes, then treated with 10 µM CpG for 2 days. Secreted IL-17A was determined by ELISA. The mean values±SD are averages of triplicate measurements. *p<0.05.

• 

  Fig. 3 Interleukin-17A (IL-17A) secreted from keratinocytes affects IL-22 production in CD4+ T cells. (A) CD4+ T cells were isolated from human blood. After a first round of stimulation with CD3/CD28, CD4+ T cells were treated with conditioned medium obtained from CpG-treated HaCaT keratinocytes (CpG-CM) and/or conditioned medium obtained from CpG-non-treated HaCaT keratinocytes (CTL-CM). For preparation of conditioned medium, HaCaT keratinocytes were treated with 10 µM CpG for 24 hours, then cells were replenished with fresh medium and incubated for a further 24 hours. After collecting the medium, cell debris was discarded by centrifugation and supernatant was used as the conditioned medium. (B) Conditioned medium was neutralized with preincubation with anti-IL-17A antibody. Normal IgG was used as a negative control. Secreted IL-22 was determined by ELISA. The mean values±standard deviation are averages of triplicate measurements. *p<0.05.

• 

  Fig. 4 Proposed model for the crosstalk between keratinocytes and CD4+ T cells. Keratinocytes recognize the Toll-like receptor 9 (TLR9) agonist and then release interleukin-17A (IL-17A) in a nuclear factor (NF)-κB-dependent way. Secreted IL-17A activates CD4+ T cells to produce IL-22, which in turn affects keratinocytes pathogenically.

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