Research ArticleSKIN INFLAMMATION

Interleukin-17 receptor D constitutes an alternative receptor for interleukin-17A important in psoriasis-like skin inflammation

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Science Immunology  07 Jun 2019:
Vol. 4, Issue 36, eaau9657
DOI: 10.1126/sciimmunol.aau9657
  • Fig. 1 Il17rc deficiency is insufficient to completely abolish IMQ-induced psoriasis-like skin inflammation.

    WT, Il17a and Il17f DKO, and Il17rc KO mice (n = 10) were subjected to IMQ-induced psoriasis-like skin inflammation and euthanized together with the untreated WT mice 5 days after disease induction. (A) Representative H&E staining of skin sections (10×) and statistical data. (B to D) The frequencies of leukocytes (B), neutrophils (C), and γδ T cells (D) in dermis. (E) The relative mRNA level of selected genes from the total skin. The results were repeated three times with consistent results. Data were shown as means ± SEM. P values were determined by unpaired t test or one-way ANOVA, followed by Tukey’s post hoc test. *P < 0.05 compared with indicated group.

  • Fig. 2 mIL-17RD binds to mIL-17A and forms a heterodimer with mIL-17RA.

    (A) The binding of His-tagged IL-17 family cytokines (IL-17A to IL-17F) to IL-17RD–expressing 293T cells. (B) The interaction between IL-17RA/IL-17RD and IL-17-RD/IL-17RD detected by BiFC. (C) The binding of IL-17A to IL-17RA/IL-17RD or IL-17-RD/IL-17RD–expressing 293T cells. FITC, fluorescein isothiocyanate. (D) The binding of IL-17 family cytokines to IL-17RA/IL-17RD or IL-17-RD/IL-17RD dimer. (E) The interaction between IL-17RA/IL-17RD and IL-17-RD/IL-17RD detected by immunoprecipitation. IP, immunoprecipitation; IB, immunoblot. (F) The interaction between mIL-17RA and mIL-17RD in mouse primary keratinocytes. (G) The colocalization of mIL-17RA and mIL-17RD in primary mouse keratinocytes. (A to G) Results shown are representative data of three independent experiments. DAPI, 4′,6-diamidino-2-phenylindole; PE, phycoerythrin. (H) The relative mRNA levels of selected genes from primary mouse keratinocytes stimulated with different cytokines (means ± SEM). All results were repeated three times with consistent results. P values were determined by unpaired t test. *P < 0.05 compared with indicated group.

  • Fig. 3 hIL-17RD binds to hIL-17A and forms a heterodimer with hIL-17RA.

    (A) The binding of biotin-labeled hIL-17 to hIL-17RD–expressing 293T cells. (B) The interaction between hIL-17RA and hIL-17RD in human keratinocytes cell line (HaCaT) after hIL-17A stimulation. (C) The interaction between hIL-17RA and hIL-17RD in primary human keratinocytes. (D) The colocalization of hIL-17RA and hIL-17RD in primary mouse keratinocytes. (E) The relative mRNA level of selected genes from WT or IL17RD knockdown (KD) primary human keratinocytes stimulated with hIL-17A (means ± SEM). (F) The mRNA level of IL-17RC and IL-17RD in patients with psoriasis (means ± SEM). (G) The protein level of IL-17RD in patients with psoriasis (means ± SEM). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (A to F) All results were repeated three times with consistent results. P values were determined by unpaired t test. (A to G) P < 0.05 compared with indicated group and ****P < 0.0001 compared with normal patients.

  • Fig. 4 IL-17RD is required for the induction of IMQ-induced psoriasis-like skin inflammation.

    WT and Il17rd KO mice were subjected to IMQ-induced psoriasis-like skin inflammation and euthanized on day 5 after disease induction together with the untreated mice (n = 10). (A) Representative H&E staining skin sections (10×) and statistical data. (B to C) The frequencies and absolute cell number of leukocytes (B) and neutrophils (C) in dermis. (D to E) The frequencies of γδ T cells (D) and ILC3s (E) in dermis. (F) The relative mRNA level of selected genes from the total skin. All results were repeated three times with consistent results. Data were shown as means ± SEM. P values were determined by unpaired t test or one-way ANOVA, followed by Tukey’s post hoc test. *P < 0.05 compared with WT and #P < 0.05 compared with untreated.

  • Fig. 5 Il17rd deficiency in nonhemopoietic cells causes resistance to IMQ-induced psoriasis-like skin inflammation.

    (A) IL-17RD expression in the skin tissue of WT mice (n = 4). Left: Relative mRNA level of Il17rd in total skin, and different types of cells from the skin at steady state. N/A, not applicable. Right: Relative mRNA level of Il17rd from the total skin, CD45 cells and CD45+ cells of the skin of WT mice treated with or without IMQ for 5 days (n = 5). *P < 0.05 compared with total skin and #P < 0.05 compared with indicated group. (B to D) The frequencies of leukocytes [(B), top], neutrophils [(B), bottom], γδ T cells (C), and ILC3s (D) in the dermis of chimeric mice (n = 6). n.s., not significant. (E) The relative mRNA level of selected genes from the total skin of chimeric mice. The results were repeated three times with consistent results. Data were shown as means ± SEM. P values were determined by unpaired t test or one-way ANOVA, followed by Tukey’s post hoc test. *P < 0.05 compared with indicated group.

  • Fig. 6 Il17rd deficiency reduces IL-17A downstream gene expression.

    (A) The percentages of IL-17RD–related genes among all IL-17A regulated genes. (B) KEGG pathway analysis of IL-17RD–related pathways (marked by “*”) among IL-17A up- and down-regulated pathways. (C) Verify the mRNA level of selected genes from WT and Il17rd KO primary mouse keratinocytes by qPCR (means ± SEM). PPAR, peroxisome proliferator–activated receptor; AGE-RAGE, advanced glycation end-products and their receptor. P values were determined by one-way ANOVA, followed by Tukey’s post hoc test. #P < 0.05 compared with untreated WT and *P < 0.05 compared with IL-17A–treated WT.

  • Fig. 7 IL-17RC and IL-17RD differentially regulates IL-17A downstream events in keratinocytes.

    (A) The saturation binding of IL-17RC–ecd and IL-17RD–ecd to IL-17A (means ± SEM). CBS, citrate-buffered saline; OD, optical density. (B) Phosphorylation of p38, IκB, Erk, and JNK in primary mouse keratinocytes cultured with IL-17A for different time points. Results shown are representative data of three independent experiments. (C) Heatmap of normalized RPKM data from WT, Il17rc KO, and Il17rd KO keratinocytes treated with IL-17A. The top enriched pathways of each cluster were listed aside. HTLV-I, human T cell leukemia virus I. (D) Venn diagram showing the numbers and overlap of the differentially expressed genes (comparing to IL-17A–treated WT) among the four comparisons. (E) The pathways that are commonly down-regulated by Il17rc and Il17rd KO.

  • Fig. 8 IL-17A–dependent IL-23 expression in keratinocytes.

    The primary mouse keratinocytes were cultured and stimulated with IL-17A (A) or indicated cytokines (B) in vitro. The relative mRNA level of Il23a, Il12a, and Il12b was detected by qPCR. *P < 0.05 compared with untreated WT and #P < 0.05 compared with IL-17A–treated WT. (C) Immunofluorescence staining of IL-23 in the skin sections of WT, Il17rd KO, Il17rc KO, and Il17a and Il17f DKO mice treated with IMQ. MFI, median fluorescence intensity. (D to F) Representative H&E staining of skin sections (D), frequency of γδ T cells (E), and the relative mRNA level of Il23 from the total skin (F) in Il23 chimeric mice. *P < 0.05 compared with indicated group. EM, extracellular matrix. (G and H) The relative Il23 mRNA level (G) and the absolute number (H) of keratinocytes, Langerhans cells, dermal DCs, and macrophages in the skin of WT and Il17rd KO mice. (I) The relative mRNA level of Il23a from the total skin. The results were repeated three times with consistent results. Data were shown as means ± SEM. P values were determined by unpaired t test or one-way ANOVA, followed by Tukey’s post hoc test. *P < 0.05 compared with WT and #P < 0.05 compared with untreated.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/4/36/eaau9657/DC1

    Material and Methods

    Fig. S1. Test of knockout and knockdown efficiency in Il17rc KO mice and IL17RD knockdown human keratinocytes.

    Fig. S2. IL-17RD functions as a receptor for IL-17A but not IL-17A/F in mouse keratinocytes.

    Fig. S3. IL-17RC and IL-17RD are both required for the full development of IMQ-induced psoriasis-like skin inflammation.

    Fig. S4. IL-17RD expression in keratinocytes is important for IMQ-induced psoriasis-like skin inflammation.

    Fig. S5. Densitometry analysis of the phosphorylation of p38, IκB, Erk, and JNK in primary mouse keratinocytes.

    Fig. S6. The expression of psoriasis-related genes in WT, Il17rd KO, Il17rc KO, and Il17rc and l17rd DKO keratinocytes stimulated with IL-17A.

    Fig. S7. Secretion of CXCL1 and CCL20 from IL-17A–stimulated WT, Il17rd KO, Il17rc KO, and Il17rc and l17rd DKO keratinocytes.

    Fig. S8. The expression of psoriasis-related genes in WT, Il17rd KO, and Il17rc KO keratinocytes stimulated with IL-17F or IL-17A/F.

    Fig. S9. IL-17A–induced IL-23 expression was diminished by Il17rc KO, Il17rd KO, Act1 KO, and NF-κB or ERK inhibition.

    Fig. S10. Reduced IL-23 level is associated with reduced IL-17 level in the skin of Il17rd KO mice.

    Fig. S11. IL-17RD deficiency did not significantly reduce lipopolysaccharide and TNF-α signaling in mouse primary keratinocytes.

    Fig. S12. Gating strategy for granulocytes, γδ T cells, and ILC3s.

    Table S1. List of primer sequences used for RT-qPCR analysis.

    Table S2. List of antibodies used in this study.

    Data file S1. Complete list of differential regulated genes in Il17rd KO keratinocytes compared with WT upon IL-17A stimulation.

    Data file S2. Raw data.

  • Supplementary Materials

    The PDF file includes:

    • Material and Methods
    • Fig. S1. Test of knockout and knockdown efficiency in Il17rc KO mice and IL17RD knockdown human keratinocytes.
    • Fig. S2. IL-17RD functions as a receptor for IL-17A but not IL-17A/F in mouse keratinocytes.
    • Fig. S3. IL-17RC and IL-17RD are both required for the full development of IMQ-induced psoriasis-like skin inflammation.
    • Fig. S4. IL-17RD expression in keratinocytes is important for IMQ-induced psoriasis-like skin inflammation.
    • Fig. S5. Densitometry analysis of the phosphorylation of p38, IκB, Erk, and JNK in primary mouse keratinocytes.
    • Fig. S6. The expression of psoriasis-related genes in WT, Il17rd KO, Il17rc KO, and Il17rc and l17rd DKO keratinocytes stimulated with IL-17A.
    • Fig. S7. Secretion of CXCL1 and CCL20 from IL-17A–stimulated WT, Il17rd KO, Il17rc KO, and Il17rc and l17rd DKO keratinocytes.
    • Fig. S8. The expression of psoriasis-related genes in WT, Il17rd KO, and Il17rc KO keratinocytes stimulated with IL-17F or IL-17A/F.
    • Fig. S9. IL-17A–induced IL-23 expression was diminished by Il17rc KO, Il17rd KO, Act1 KO, and NF-κB or ERK inhibition.
    • Fig. S10. Reduced IL-23 level is associated with reduced IL-17 level in the skin of Il17rd KO mice.
    • Fig. S11. IL-17RD deficiency did not significantly reduce lipopolysaccharide and TNF-α signaling in mouse primary keratinocytes.
    • Fig. S12. Gating strategy for granulocytes, γδ T cells, and ILC3s.
    • Table S1. List of primer sequences used for RT-qPCR analysis.
    • Table S2. List of antibodies used in this study.

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    Other Supplementary Material for this manuscript includes the following:

    • Data file S1 (Microsoft Excel format). Complete list of differential regulated genes in Il17rd KO keratinocytes compared with WT upon IL-17A stimulation.
    • Data file S2 (Microsoft Excel format). Raw data.

    Files in this Data Supplement:

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