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. 2008 Dec 15;181(12):8504-12.
doi: 10.4049/jimmunol.181.12.8504.

IL-17A enhances vitamin D3-induced expression of cathelicidin antimicrobial peptide in human keratinocytes

Mark Peric  1 , Sarah Koglin, Song-Min Kim, Shin Morizane, Robert Besch, Jörg C Prinz, Thomas Ruzicka, Richard L Gallo, Jürgen Schauber
Affiliations

IL-17A enhances vitamin D3-induced expression of cathelicidin antimicrobial peptide in human keratinocytes

Mark Peric et al. J Immunol. .

Abstract

Cathelicidin is strongly expressed in lesional skin in psoriasis and may play an important role as both an antimicrobial peptide and as an autoinflammatory mediator in this chronic skin disease. The mechanism of increased cathelicidin in psoriatic keratinocytes is not known, but recent observations have found that psoriasis has abundant Th17 cells that produce IL-17A and IL-22. We found that human keratinocytes stimulated with supernatants from T cells isolated from lesional psoriatic skin increased expression of cathelicidin when stimulated in the presence of 1,25-dihydroxyvitamin D(3) (1,25D(3)). This increase was signaled through the IL-17RA. In vitro, IL-17A, but not IL-22, enhanced cathelicidin mRNA and peptide expression in keratinocytes dependent on the presence of 1,25D(3). At the same time, coincubation with 1,25D(3) blocked induction of human beta-defensin 2 (HBD2), IL-6, and IL-8, which are other target genes of IL-17A VSports手机版. Act1, an adaptor associated with IL-17RA and essential for IL-17A signaling, mediated cathelicidin induction, as its suppression by small interfering RNA inhibited HBD2 and cathelicidin. Both, 1,25D(3) and IL-17A signaled cathelicidin induction through MEK-ERK. These results suggest that increased IL-17A in psoriatic skin increases cathelicidin through a vitamin D(3)-, Act1-, and MEK-ERK-dependent mechanism. Therapy targeting this cathelicidin-regulating system might be beneficial in patients suffering from psoriasis. .

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Figures (VSports手机版)

FIGURE 1
FIGURE 1
1,25D3 enables supernatants from T cells isolated from psoriatic plaques to increase cathelicidin expression in human keratinocytes. A, Keratinocytes in lesional skin strongly express IL-17RA and cathelicidin peptide as demonstrated by staining of paraffin sections of skin from patients with psoriasis with an IL-17RA and hCAP18/LL-37 Ab, respectively. B, Human keratinocytes (HaCaT) were treated with supernatants from T cells isolated from psoriatic plaques in the presence or absence of 1,25D3 (10−11 M). To block IL-17A signaling, an IL-17RA blocking Ab was added 30 min before stimulation. Cells were harvested after 24 h and cathelicidin transcript levels were analyzed by quantitative real-time PCR. C, In a control experiment, HaCaT keratinocytes were treated for 24 h with 1,25D3 (10−8 M), IL-17A (10 ng/ml), or the combination. Cathelicidin transcript abundance was analyzed by quantitative real-time PCR. Data are mean ± SD of a single experiment performed in triplicate and are representative of three independent experiments *, p < 0.05, determined by Student’s t test.
FIGURE 2
FIGURE 2
IL-17A enhances the effect of 1,25D3 on cathelicidin mRNA and cathelicidin peptide hCAP18 in primary human keratinocytes. A, NHEK were treated with IL-17A (10 ng/ml) in the presence of increasing concentrations of 1,25D3 (10 −11–10−8 M) for 24 h. Cathelicidin transcript abundance was analyzed by quantitative real-time PCR. B, NHEK were treated for 24 h with 1,25D3 (10−8 M), and increasing concentrations of IL-17A (1–100 ng/ml). Cathelicidin was measured by quantitative real-time PCR. C, Keratinocytes were stimulated with IL-17A (10 ng/ml) in the presence of 1,25D3 (10−8 M). Cathelicidin induction as measured by quantitative real-time PCR at the indicated time points was calculated relative to the induction by 1,25D3 alone. Data are mean ±SD of a single experiment performed in triplicate and are representative of three independent experiments.*, p < 0.05 and **, p < 0.01, determined by Student’s t test. D, To evaluate cathelicidin peptide induction, NHEK were treated for 24 h with 1,25D3 (10−8 M), IL-17A (100 ng/ml), or the combination. Cathelicidin hCAP18/LL-37 protein expression was analyzed in NHEK lysates by Western blot using an Ab that detects hCAP18 and LL-37. Synthetic LL-37 peptide was used as a positive control (far right lane).
FIGURE 3
FIGURE 3
NHEK express IL-17RA while 1,25D3 differentially affects IL-17A-induced gene expression in keratinocytes. A, Expression of the IL-17RA was confirmed in primary NHEK by immunofluorescence staining and TissueFAXS analyses. Cell nuclei were detected by DAPI. B, NHEK were stimulated with IL-17A (10 ng/ml) and 1,25D3 (10−8 M) for 24 h, and cathelicidin transcript abundance was measured by quantitative real-time PCR. In addition, cells were preincubated 2 h with an IL-17RA blocking Ab before stimulation. As an additional control, an IL-17A neutralizing Ab was added to culture medium containing IL-17A 2 h before the medium was applied to the cells. The 1,25D3 stimulation enabled IL-17A to increase cathelicidin, which was inhibited by the IL-17RA blocking Ab or the IL-17A neutralizing Ab. To evaluate the expression of other IL-17A regulated genes, NHEK were stimulated with IL-17A (10 ng/ml) and 1,25D3 (10−8 M) for 24 h and HBD2 (C) and IL-6 (D) transcript abundance was measured by quantitative real-time PCR. IL-8 expression (D) in cell culture supernatants was analyzed by ELISA. Cells were incubated with an IL-17RA blocking Ab 2 h before stimulation. HBD2 expression was induced by IL-17A alone, and induction was attenuated in the presence of 1,25D3. C, Further inhibition of IL-17A signaling inhibited HBD2 induction. D, Similar to HBD2 expression, IL-17A induced IL-6 (left) and IL-8 (right) expression that was decreased when 1,25D3 was present. Data are mean ± SD of a single experiment performed in triplicate and are representative of three independent experiments.*, p < 0.05 and **, p < 0.01, determined by Student’s t test.
FIGURE 4
FIGURE 4
Th17 cytokines IL-17A and IL-22 differentially affect antimicrobial peptide expression. To compare the effects of the Th17 cytokines, IL-17A (top) and IL-22 (bottom) keratinocytes were treated with IL-17A (10 ng/ml) or IL-22 (10 ng/ml) in the presence of 1,25D3 (10−8 M). Cells were harvested 24 h after stimulation and mRNA levels for cathelicidin and HBD2 were analyzed by quantitative real-time PCR. A, 1,25D3 enabled IL-17A to increase cathelicidin, whereas IL-22 had no effect. Both, IL-17A and IL-22 induced HBD2 in keratinocytes as measured by quantitative real-time PCR and again 1,25D3 attenuated HBD2 induction. B, The combination of IL-17A and IL-22 showed the same effect on cathelicidin transcript abundance as IL-17A alone. C, To evaluate the influence of cell differentiation on cathelicidin induction, NHEK were grown in medium containing low (0.06 mM) or high (1.7 mM) concentrations of calcium for 24 h. Cells were then stimulated with IL-17A (10 ng/ml) in the presence or absence of 1,25D3 (10−8 M) for another 24 h. Cathelicidin transcript abundance was analyzed by quantitative real-time PCR. Induction of cathelicidin by 1,25D3 did not change with increasing calcium concentration, but the effect of IL-17A was significantly stronger in keratinocytes treated with 1.7 mM calcium. Data are mean ± SD of a single experiment performed in triplicate and are representative of three independent experiments.*, p < 0.05; **, p < 0.01; and ***, p < 0.001, determined using Student’s t test.
FIGURE 5
FIGURE 5
Act1 mediates the effect of IL-17A on cathelicidin expression in keratinocytes. Act1 is a recently identified adaptor protein involved in IL-17RA signaling. To investigate the role of Act1 in IL-17A increased cathelicidin NHEK were transfected with siRNA to decrease Act1 expression before stimulation with IL-17A (10 ng/ml) and 1,25D3 (10−8 M). Silencing of Act1 was confirmed by quantitative real-time PCR. A, The siRNA suppression of Act1 blocked enhancement of cathelicidin by IL-17A in the presence of 1,25D3. B, 1,25D3-induced cathelicidin was not affected. C, NHEK were transfected with siRNA oligonucleotides for Act1 and stimulated with 1,25D3 (10−8 M), IL-17A (10 ng/ml), or the combination. Again, silencing of Act1 blocked induction of HBD2 (top) and IL-6 (bottom). Data are mean ± SD of a single experiment performed in triplicate and are representative of three independent experiments. **, p <0.01, determined by Student’s t test. D, The corresponding cathelicidin peptide hCAP18 expression levels are displayed as observed by Western blot. Similar to cathelicidin mRNA expression, silencing of Act1 blocked IL-17A-enhanced cathelicidin peptide.
FIGURE 6
FIGURE 6
IL-17A increases cathelicidin through activation of a transcriptional mechanism. To investigate the mechanism of IL-17A-enhanced cathelicidin, NHEK were stimulated with IL-17A (10 ng/ml) alone or in combination with 1,25D3 (10−8 M). ActD (5 μg/ml) was added to inhibit active mRNA transcription and cathelicidin was measured by quantitative real-time PCR. A, ActD completely blocked the effect of IL-17A on cathelicidin. To evaluate the effect of IL-17A on cathelicidin mRNA stability, 5 cells were treated with 1,25D3 and the combination of IL-17A with 1,25D3. ActD was added 24 h after stimulation and cells were harvested at different time points. Cathelicidin expression was analyzed by quantitative real-time PCR and calculated relative to the time of ActD addition. Data are mean ± SD of a single experiment performed in triplicate and are representative of three independent experiments. **, p < 0.01, determined by Student’s t test.
FIGURE 7
FIGURE 7
IL-17A and 1,25D3 increase cathelicidin by activation of MEK-ERK. To investigate the role of MEK-ERK signaling in IL-17A-induced cathelicidin, NHEK were treated with the specific MEK inhibitor PD98059 (20 μM) before stimulation with IL-17A or 1,25D3. Inhibition of MEK-ERK blocked increased cathelicidin by IL-17A in the presence of 1,25D3. A, The 1,25D3-induced cathelicidin was also diminished. To investigate whether MEK-ERK was involved in induction of other innate immune genes by IL-17A, NHEK were treated with PD98059 and subsequently stimulated with 1,25D3 (10−8 M), IL-17A (10 ng/ml), or the combination. B, Again, inhibition of MEK-ERK blocked induction of HBD2. Data are mean ± SD of a single experiment performed in triplicate and are representative of three independent experiments.*, p < 0.05 and **, p < 0.01, determined by Student’s t test. C, The inhibitory effect of PD98059 on MEK-ERK was confirmed by Western blot analyses of phospho-p44/p42. Similar to cathelicidin mRNA expression, inhibition of MEK-ERK signaling blocked IL-17A with 1,25D3-enhanced cathelicidin peptide hCAP18.
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