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IL-31 regulates differentiation and filaggrin expression in human organotypic skin models  Christian Cornelissen, MSc, Yvonne Marquardt, Katharina Czaja,

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Presentation on theme: "IL-31 regulates differentiation and filaggrin expression in human organotypic skin models  Christian Cornelissen, MSc, Yvonne Marquardt, Katharina Czaja,"— Presentation transcript:

1 IL-31 regulates differentiation and filaggrin expression in human organotypic skin models 
Christian Cornelissen, MSc, Yvonne Marquardt, Katharina Czaja, Jörg Wenzel, MD, Jorge Frank, MD, Juliane Lüscher-Firzlaff, MD, Bernhard Lüscher, PhD, Jens M. Baron, MD  Journal of Allergy and Clinical Immunology  Volume 129, Issue 2, Pages e8 (February 2012) DOI: /j.jaci Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 IL-31 disturbs differentiation of NHEKs in organotypic skin equivalents. A, 3D organotypic skin equivalents were stimulated with or without rhIL-31 (100 ng/mL; every second day), and histological sections were stained for the indicated markers at day 8 (1 of 6 models). B and C, Organotypic skin equivalents were harvested at the days indicated, and FLG (Fig 1, B) and KRT10 (Fig 1, C) expression was determined by using quantitative RT-PCR. Relative expression is displayed (compared with nonstimulated day 2, normalized to HPRT). One representative experiment of 3 is shown. D, NHEKs from 3 different donors were stimulated with rhIL-31 (100 ng/mL). The indicated genes were analyzed by using quantitative RT-PCR. HE, Hematoxylin-eosin. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 IL-31 induces IL20 and IL24 in HaCaT-IL31RA cells. A, IL-31RA protein expression was evaluated in clone 14 cells treated with or without dox (1 μg/mL) for 16 hours and visualized by using indirect confocal immunofluoresence microscopy (panels on the left) and fluorescence-activated cell sorting (panel on the right). B, Clone 14 cells were treated with or without dox (1 μg/mL, 16 hours), stimulated with rhIL-31 for 45 minutes, fixed, and subsequently stained for IL-31RA (green) and phospho-STAT3 (red). The DNA was stained with Hoechst C, The expression of IL20 and IL24 was analyzed by using quantitative RT-PCR in 2 individual clones in response to dox (16 hours), rhIL-31 (1 hour), or cotreatment. D, The cells were treated as in panel C. In addition, the indicated kinase inhibitors (JAK inhibitor I [100 nM]; c-Jun N-terminal kinase inhibitor II [20 μM]; SB202190, selective for p38 MAP kinases [20 μM]; U0126, selective for extracellular signal-regulated kinases [20 μM]; Wortmannin, selective for PI3K kinases [500 nM]) were added 1 hour prior to stimulation with IL-31. The expression of IL20 (left panel) and IL24 (right panel) was analyzed by using quantitative RT-PCR. Mean values and standard deviations of 3 independent experiments are displayed. ∗∗P < .01. dox, Doxycycline; n.d., not detectable. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 IL-31 inhibits proliferation in HaCaT-IL31RA cells. A, The proliferation rate of HaCaT-IL31RA clone 14 cells was measured by counting cells incubated in the presence or absence of dox (1 μg/mL) and IL-31 (100 ng/mL). The data are of 3 biological replicates with standard deviation. B, Cell cycle distribution of HaCaT-IL31RA clone 14 cells was determined in response to dox/IL-31 treatment after 48 hours. The percentage of cells in G1, S, and G2 phases was determined by using fluorescence-activated cell sorting. Displayed are mean values with standard deviation of 3 biological replicates. ∗∗P < .01. dox, Doxycycline. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 IL-31 disturbs the differentiation of HaCaT-IL31RA cells in organotypic 3D cocultures. A, Clone 14 cells organotypic skin equivalents were grown in the presence or absence of doxycycline and rhIL-31 (100 ng/mL). Histology (hematoxylin-eosin [HE]) and protein expression was analyzed at day 7 (identical to Fig E5, A). B, Epidermal thickness of HE-stained sections was measured. ∗P < .05. C, Epidermal constitution, the alignment of the stratum basale, and the development of the stratum granulosum of HE-stained sections were measured. ∗P < .05 (Mann-Whitney U test). D, The expression of KRT10 and FLG mRNA was determined from biopsies of the 3D recontructs shown in panel A and Fig E5. A representative experiment performed in duplicates is shown. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 IL-20 and IL-24 impair differentiation of HaCaT-IL31RA cells in organotypic 3D cocultures. A, HaCaT-IL31RA clone 14 cells were stimulated with doxycycline/IL-31 (1 μg/mL/100 ng/mL) and IL20 and IL24 mRNA measured by quantitative RT-PCR. B, Fluorescence-activated cell sorting analysis of fixed cells incubated with antibodies specific for IL-20Rα, IL-20Rβ, and IL-22Rα and secondary antibodies coupled to Alexa-Fluor 488. C, Clone 14 organotypic skin equivalents in the presence or absence of IL-20 (20 ng/mL) and IL-24 (20 ng/mL), harvested at day 7 and stained as indicated. D, Total RNA was isolated from the organotypic skin equivalents (panel C) and from comparable samples of clone 12 and of 3D equivalents of primary NHEKs that were grown in the presence or absence of IL-20 (20 ng/mL) and IL-24 (20 ng/mL). The expression of FLG and KRT10 was analyzed by using quantitative RT-PCR. HE, Hematoxylin-eosin. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E1 IL-31 disturbs differentiation of hair follicle keratinocytes of a patient with AD. 3D organotypic skin equivalents of hair follicle keratinocytes obtained from an atopic patient with elevated total IgE serum levels, allergic rhinitis, and moderate AD were stimulated with or without rhIL-31 (100 ng/mL; every second day), harvested at day 5, and histological sections stained for the indicated proteins. HE, Hematoxylin-eosin. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E2 IL-31 regulated genes. A, NHEKs were stimulated with rhIL-31 for 1 hour and mRNA was isolated and analyzed on Affymetrix Exon arrays. Displayed are genes that are regulated more than 2-fold compared with a nonstimulated control in 2 independent experiments. B, Schematic representation of the IL10 gene cluster located on chromosome 1 that spans a region of about 140 kb. The IL19 gene is transcribed from 2 different promoters. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E3 HaCaT cells express IL-31RA in response to IFN-γ and subsequently respond to IL-31. A, HaCaT cells were stimulated with IFN-γ (20 ng/mL) for 24 hours. Then, IL31RA and OSMRβ expression was analyzed by using quantitative RT-PCR. B, The expression of IL-31RA at the cell surface was evaluated in cells treated as described in panel A using FACS. C, HaCaT cells, either pretreated or not with IFN-γ for 24 hours, were stimulated with rhIL-31 (100 ng/mL). The expression of IL20 and IL24 was analyzed by using quantitative RT-PCR. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E4 Characterization of HaCaT cell clones carrying an inducible IL31RA transgene. A, HaCaT cells were infected with pSLIK-IL31RA and individual clones selected with G418. The clones were stimulated with or without doxycycline (1 μg/mL) for 16 hours and the expression of IL31RA mRNA was analyzed by using quantitative RT-PCR. B, IL-31RA protein expression was analyzed in lysates of cells treated with or without doxycycline (1 μg/mL, 16 hours) by western blotting. Tubulin served as loading control. C, The expression of IL20 and IL24 was analyzed by using quantitative RT-PCR in response to 16 hour dox/1 hour IL-31 stimulation. The IL20 expression in individual clones is displayed as CT values because the basal expression values for IL20 in the different clones were either not detectable or very low. The IL24 expression is shown as relative expression compared with cells without dox and IL-31 treatment. HPRT expression was used for normalization. D, HaCaT-IL31RA cells were stimulated with or without dox (1 μg/mL) and IL-31 (100 ng/mL) as indicated. The cells were then stained for AnnexinV and analyzed by using fluorescence-activated cell sorting. Displayed are mean values with standard deviation of 3 experiments. The differences were not statistically significant. dox, Doxycycline. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E5 IL-31 interferes with HaCaT-IL31RA cell differentiation. A and C, Clone 14 (Fig E5, A) and clone 12 (Fig E5, C) cells and primary dermal fibroblasts were grown as organotypic skin equivalents in the presence or absence of dox and/or rhIL-31 (100 ng/mL). Dox and IL-31 were added every second day. The skin equivalents were harvested after 7 and 14 days and histological sections were stained with hematoxylin-eosin (HE). B, Clone 12 cells and primary dermal fibroblasts were grown as organotypic skin equivalents in the presence or absence of dox and rhIL-31 (100 ng/mL). Dox and IL-31 were added every second day. The skin equivalents were harvested after 7 days and analyzed histologically (HE) or stained for the indicated marker proteins. These results are from the same experiment as shown in panel C. D, Clone 14 cells and primary dermal fibroblasts were grown as organotypic skin equivalents in the presence or absence of dox. rhIL-31 (100 ng/mL) was added for the indicated times, and the skin equivalents were harvested after 7 days and processed as described in panels A to C. dox, Doxycycline. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E6 IL-31 reduces lipid envelope formation. A, 3D organotypic skin equivalents were stimulated with or without rhIL-31 (100 ng/mL; every second day) and histological sections were stained with Nile red. B, Organotypic skin equivalents from HaCaT-IL31RA cells and NHEKs were harvested at day 7 (HaCaT) or days 5, 8, and 14 (NHEKs) and the expression of spingomyelinase (SMPD1), sphingomyelin synthase (SGMS1), steroid sulfatase (STS), and phospholipase A2 (PLA2G4A) was measured by using quantitative RT-PCR. C, Sections of 3D organotypic skin equivalents (as in panel A) were stained for ceramid. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2011 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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