L-Theanine Alleviates IMQ-Induced Psoriasis Like Skin Inflammation by Downregulating the Production of IL-23 and Chemokines



doi: 10.3389/fphar.2021.719842.


eCollection 2021.

Affiliations

Item in Clipboard

Yaohan Xu et al.


Front Pharmacol.


.

Abstract

Psoriasis, the most common skin inflammatory disease, is characterized by massive keratinocyte proliferation and immune cell infiltration into epidermis. L-Theanine (L-THE), a nonproteinogenic amino acid derived from green tea (Camellia sinensis), has been proved to possess the properties of anti-inflammatory, antidepressants and neuroprotective. However, whether L-THE has a therapeutic effect on psoriasis is still unknown. In this study, we found that the epidermal thickness and inflammatory response were significantly reduced in Imiquimod (IMQ)-induced psoriasis mice by applying with L-THE on mice skin. The expression of proliferation and inflammation associated genes such as keratin 17, IL-23 and CXCL1-3 was also downregulated by L-THE. Furthermore, L-THE inhibited the production of IL-23 in dendritic cells (DCs) after IMQ treatment, and decreased the levels of chemokines in keratinocytes treated with IL-17A by downregulating the expression of IL-17RA. RNA-seq and KEGG analysis revealed that L-THE significantly regulated the expression of IL-17A and NF-κB signaling pathway-associated genes. Metabolomics analysis displayed that L-THE promoted propanoate metabolism which has been reported to inhibit the activity of TH17 cells. Therefore, our results demonstrated that L-THE significantly decreases the levels of IL-23 and chemokines, and attenuates IMQ-induced psoriasis like skin inflammation by inhibiting the activation of NF-κB and IL-17A signaling pathways, and promoting the propanoate metabolism. Our findings suggest that topical applied L-THE can be used as a topical drug candidate for the treatment of psoriasis or as an adjuvant treatment of ustekinumab or secukinumab to prevent the relapse of psoriasis.


Keywords:

IL-23; L-Theanine; chemokines; dendritic cells; psoriasis.

Conflict of interest statement

The 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

L-THE decreases epidermal thickness in mice with IMQ-induced psoriasis like skin inflammation. (A) C57BL/6 mice (n = 6 per group) were subjected to a daily topical dose of IMQ cream on the shaved back or ear for five consecutive days, and were topical applied with 10 mM or 100 mM L-THE on the skin for twice per day. (B) Mice ear thickness was measured relative to the contralateral ear from IMQ-induced psoriasis mice treated with L-THE or H2O. (C) H&E staining ear skin sections obtained from IMQ-induced psoriasis mice treated with L-THE or H2O for 5 days. (D) The ear epidermal thickness was measured in (C) by the software ImageJ. (E) H&E staining back skin sections obtained from IMQ-induced psoriasis mice treated with L-THE or H2O for 5 days. (F) The ear epidermal thickness was measured in (D) by the software ImageJ. Data is representative of three independent experiments. p values are determined by two-way ANOVA. **p < 0.01, ***p < 0.001.


FIGURE 2



FIGURE 2

L-THE regulates the expression of genes related to the proliferation and differentiation of keratinocytes. (A,B) Heatmap (A) and Volcano plot. (B) showed the expression of KRT genes based on RNA-seq data from IMQ-induced psoriasis mice treated with 100 mM L-THE or H2O for 5 days. (C) RT-PCR analysis of the mRNA levels of KRT10, KRT1, KRT6A, and KRT17 in IMQ-induced psoriasis mice (n = 6 per group) treated with 100 mM L-THE or H2O for 5 days. (D,F) IHC staining of KRT10, KRT6A and Ki67 in ear or back skin sections obtained from IMQ-induced psoriasis mice (n = 6 per group) treated with 100 mM L-THE or H2O for 5 days. Data is representative of three independent experiments. p values are determined by two-way ANOVA. ***p < 0.001.


FIGURE 3



FIGURE 3

L-THE downregulates the expression of inflammatory genes. (A,B) Volcano plot. (A) showed the expression of inflammatory response associated genes, and heatmap. (B) showed the significantly differentiated genes about interleukin, CXCL, and CCL family genes based on RNA-seq data from IMQ-induced psoriasis mice treated with 100 mM L-THE or H2O for 5 days. (C) RT-PCR analysis of the mRNA levels of IL-23p19, TNF-α, CXCL2, and S100A8 in IMQ-induced psoriasis mice (n = 6 per group) treated with 100 mM L-THE or H2O for 0 or 5 days. (D) ELISA analysis of IL-23A in IMQ-induced psoriasis mice (n = 6 per group) treated with 100 mM L-THE or H2O for 0 or 5 days. Data is representative of three independent experiments. p values are determined by two-way ANOVA. ***p < 0.001.


FIGURE 4



FIGURE 4

L-THE regulates the gene expression profiles of extracellular space, cell surface and extracellular region. (A) Scatter plot for GO analysis based on RNA-seq data from IMQ-induced psoriasis mice treated with 100 mM L-THE or H2O for 5 days. (B–E) Volcano plot showed the expression of extracellular space (B), cell surface (C), extracellular region (D), and immune system process. (E) associated genes.


FIGURE 5



FIGURE 5

L-THE treatment regulates the expression of cytokine-cytokine receptor interaction, chemokine signaling pathway, and IL-17A signaling pathway associated genes. (A) Scatter plot for KEGG analysis based on RNA-seq data from IMQ-induced psoriasis mice treated with 100 mM L-THE or H2O for 5 days. (B–F) Volcano plot showed the expression of cytokine-cytokine receptor interaction (B), chemokine signaling pathway (C), TNF signaling pathway (D), IL-17A signaling pathway (E), and NF-κB signaling pathway associated genes.


FIGURE 6



FIGURE 6

L-THE upregulates the propanoate metabolism by increasing the expression of propanoate metabolism associated genes. (A) Scatter plot for KEGG analysis based on untargeted metabolism data from IMQ-induced psoriasis mice treated with 100 mM L-THE or H2O for 5 days. (B) Heatmap showed the significantly changed production of phenylalanine and propanoate metabolism based on untargeted metabolism data from IMQ-induced psoriasis mice treated with 100 mM L-THE or H2O for 5 days. (C,D) Volcano plot. (C) showed the expression of propanoate metabolism associated genes, and heatmap. (D) showed the significantly differentiated propanoate metabolism genes based on RNA-seq data from IMQ-induced psoriasis mice treated with 100 mM L-THE or H2O for 5 days. (E) The FPKM Value of Acss2, Echdc1, Ldhb, and Acacb in (D).


FIGURE 7



FIGURE 7

L-THE inhibites the expression of IL-23 and chemokines in vitro. (A) Q-PCR analysis of IL-23p19 and TNF-α in mRNA in BMDCs were treated with 4 μg/ml IMQ for indicated time points after pretreatment with L-THE for 12 h. (B) ELISA analysis of IL-23 protein in BMDCs were treated with 4 μg/ml IMQ for indicated time points after pretreatment with L-THE for 12 h. (C) Q-PCR analysis of CXCL1-3, and CCL2 mRNA in mouse primary keratinocytes were treated with 100 ng/ml IL-17A for indicated time points after pretreatment with L-THE for 12 h. (D) ELISA analysis of CXCL1 protein in mouse primary keratinocytes were treated with 100 ng/ml IL-17A for indicated time points after pretreatment with L-THE for 12 h. (E) Q-PCR analysis of IL-17RA mRNA in mouse primary keratinocytes were treated with 100 ng/ml IL-17A for indicated time points after pretreatment with L-THE for 12 h. Data is representative of three independent experiments. p values are determined by two-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

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