Vitamin D enhances production of soluble ST2, inhibiting the action of IL-33
Authors: Paul E. Pfeffer, Yin-Huai Chen, Grzegorz Woszczek, Nick C. Matthews, Elfy Chevretton, Atul Gupta, Sejal Saglani, Andrew Bush, Chris Corrigan, David J. Cousins, Catherine M. Hawrylowicz
Journal: Journal of Allergy and Clinical Immunology (2014)

Vitamin D insufficiency is an environmental factor that has been strongly associated with asthma and its severity.1Mann E.H. Chambers E.S. Pfeffer P.E. Hawrylowicz C.M. Immunoregulatory mechanisms of vitamin D relevant to respiratory health and asthma.Ann N Y Acad Sci. 2014; 1317: 57-69Crossref PubMed Scopus (50) Google Scholar The genes IL33 and IL1RL1 have been repeatedly identified as predisposing to asthma risk in genome-wide association studies.2Grotenboer N.S. Ketelaar M.E. Koppelman G.H. Nawijn M.C. Decoding asthma: translating genetic variation in IL33 and IL1RL1 into disease pathophysiology.J Allergy Clin Immunol. 2013; 131: 856-865Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar IL-33 is an alarmin cytokine that acts on multiple pulmonary cell types, including TH2 lymphocytes, mast cells, and innate lymphoid cells, to promote TH2-type cytokine secretion and airway inflammatory responses of the kind observed in asthmatic patients.3Garlanda C. Dinarello C.A. Mantovani A. The interleukin-1 family: back to the future.Immunity. 2013; 39: 1003-1018Abstract Full Text Full Text PDF PubMed Scopus (1227) Google Scholar, 4Liew F.Y. Pitman N.I. McInnes I.B. Disease-associated functions of IL-33: the new kid in the IL-1 family.Nat Rev Immunol. 2010; 10: 103-110Crossref PubMed Scopus (791) Google Scholar The receptor for IL-33 is encoded by IL1RL1; differential splicing of the gene can produce a functional membrane-bound receptor (ST2L) or a soluble decoy receptor (sST2).5Oboki K. Ohno T. Kajiwara N. Saito H. Nakae S. IL-33 and IL-33 receptors in host defense and diseases.Allergol Int. 2010; 59: 143-160Crossref PubMed Scopus (170) Google Scholar Therefore we investigated whether IL1RL1 is regulated by vitamin D in cells relevant to asthma. We did this first by measuring gene expression by means of quantitative real-time PCR with 2 different TaqMan probe sets: Hs01073300, which detects mRNA splice variants encoding both the membrane-bound and soluble receptors (the total mRNA for IL1RL1), and Hs00249389, which detects mRNA only for the splice variant encoding the membrane-bound receptor ST2L (Fig 1, A). Methods are provided in this article's Online Repository at www.jacionline.org. Both probe sets display similar efficiency of amplification (data not shown). Human primary bronchial epithelial cells (HBECs), CD4 lymphocytes, CD8 lymphocytes, eosinophils, and LUVA mast cells were cultured in the presence or absence of 1α,25-dihydroxyvitamin D3 (1,25[OH]D3), the active form of vitamin D (Fig 1, B-D, and see Fig E1, A and B, in this article's Online Repository at www.jacionline.org). Addition of 1,25(OH)D3 significantly increased the total number of IL1RL1 mRNA transcripts expressed by HBECs and CD4 and CD8 lymphocytes, as measured by using the Hs01073300 probe set. However, 1,25(OH)D3 did not significantly increase expression of IL1RL1 mRNA transcripts by primary eosinophils and LUVA mast cells, despite it significantly increasing expression of the gene cathelicidin antimicrobial peptide (CAMP), which is known to be induced by vitamin D (see Fig E1, A and B). Higher IL1RL1 expression levels were detected with the Hs01073300 probe set than with the Hs00249389 probe set in HBEC and lymphocyte cultures. Although 1,25(OH)D3 increased the total copies of IL1RL1 mRNA measured by using the Hs01073300 probe set in these cell types, it did not increase the number of transcripts encoding ST2L detected with Hs00249389, indicating that vitamin D selectively upregulates the expression of mRNA for the soluble decoy receptor sST2. sST2 concentrations in culture supernatants were measured by means of ELISA to confirm the findings of gene expression studies at the protein level. sST2 concentrations were significantly increased by 1,25(OH)D3 in both HBEC and CD4 lymphocyte cultures (Fig 1, E and F). In contrast, CD8 lymphocytes produced less than 200 pg/mL sST2 in all conditions, and there was no evidence of upregulation by 1,25(OH)D3 (data not shown). Vitamin D circulates primarily as the inactive precursor 25-hydroxyvitamin D3 (25[OH]D3); however, epithelial cells constitutively express CYP27B1, the enzyme that converts 25(OH)D3 to 1,25(OH)D3.6Hansdottir S. Monick M.M. Hinde S.L. Lovan N. Look D.C. Hunninghake G.W. Respiratory epithelial cells convert inactive vitamin D to its active form: potential effects on host defence.J Immunol. 2008; 181: 7090-7099Crossref PubMed Scopus (436) Google Scholar HBECs cultured with 100 nmol/L 25(OH)D3, a physiologic concentration, increased expression of IL1RL1 (see Fig E1, C) and produced significantly greater amounts of sST2 (Fig 1, G). Primary human nasal epithelial cells (HNECs) cultured with a similar concentration range of 25(OH)D3 also showed a clear concentration-dependent increase in sST2 production (Fig 1, H). CD4 lymphocytes did not respond to 25(OH)D3, which is consistent with their lower expression of CYP27B1 (see Fig E1, D). An IL-33 sensitive bioassay was developed using the LUVA mast cell line to test the biological activity of sST2. Twenty-four hours of exposure of LUVA cell cultures to IL-33 resulted in marked homotypic aggregation, which was associated with increased expression of CD54 (intercellular adhesion molecule 1; see Fig E2, A and B, in this article's Online Repository at www.jacionline.org). IL-33 increased CD54 expression on LUVA cells in a concentration-dependent manner (Fig 2, A). Other stimuli can also induce mast cell CD54 expression, and a similar concentration-dependent induction of CD54 expression was achieved with a combination of IFN-γ and IL-4 (Fig 2, A). Treatment of LUVA cells with a recombinant sST2-Fc chimera significantly inhibited IL-33–induced CD54 expression (P = .0076; Fig 2, B, and see Fig E2, C) but not IFN-γ plus IL-4–induced CD54 expression (see Fig E2, D). Addition of 1,25(OH)D3 itself to LUVA cell cultures did not significantly alter constitutive or induced CD54 expression (P > .05; see Fig E2, E), as previously reported for another mast cell line.7Babina M. Krautheim M. Grutzkau A. Henz B.M. Human leukemic (HMC-1) mast cells are responsive to 1alpha, 25-dihydroxyvitamin D(3): selective promotion of ICAM-3 expression and constitutive presence of vitamin D(3) receptor.Biochem Biophys Res Commun. 2000; 273: 1104-1110Crossref PubMed Scopus (19) Google Scholar To examine the effect of vitamin D–enhanced epithelial sST2 production on the IL-33 bioassay, HNECs were cultured in the presence or absence of 25(OH)D3 for 48 hours, and then the supernatants were added to cultures of LUVA cells stimulated with concentrations of IL-33 or IFN-γ plus IL-4 that induced comparable CD54 expression. Conditioned medium from vitamin D–treated epithelial cells significantly reduced IL-33–induced CD54 expression on LUVA cells compared with conditioned medium from matched epithelial cell cultures not treated with vitamin D (P = .015; Fig 2, C and E). In contrast, there was no significant difference in IFN-γ plus IL-4–induced CD54 expression between LUVA cells cultured with the different epithelial cell–conditioned media (P > .05; Fig 2, D and E). Although mediators in the epithelial cell–conditioned medium other than sST2 might conceivably have affected LUVA cell CD54 expression, the selective effect of vitamin D treatment on IL-33–induced CD54 expression suggests that the sST2 in the vitamin D–treated epithelial cell supernatants is biologically active. The capacity of IL-33 to induce production of TH2-type proinflammatory cytokines by multiple cell types likely underpins the reported genetic associations of IL33 and IL1RL1 with asthma. Here we report the novel finding that 100 nmol/L vitamin D is able to augment expression by epithelial cells and lymphocytes of the soluble decoy receptor sST2, which in turn inhibits the actions of IL-33. Importantly, this effect occurs at physiologic concentrations (vitamin D sufficiency is defined as a serum 25[OH]D level of 75 to 150 nmol/L), and similar concentrations of 1,25(OH)D3 have been shown to be able to be generated from 25(OH)D3 in culture.1Mann E.H. Chambers E.S. Pfeffer P.E. Hawrylowicz C.M. Immunoregulatory mechanisms of vitamin D relevant to respiratory health and asthma.Ann N Y Acad Sci. 2014; 1317: 57-69Crossref PubMed Scopus (50) Google Scholar We hypothesize that the capacity of vitamin D to augment the synthesis of an inhibitor of IL-33 in situ in the airways mucosa is of potential benefit in the limitation of asthmatic mucosal inflammation. Furthermore, this might in part account for the paradox that epidemiologic studies have repeatedly revealed associations between vitamin D insufficiency and both the risk of more severe asthma1Mann E.H. Chambers E.S. Pfeffer P.E. Hawrylowicz C.M. Immunoregulatory mechanisms of vitamin D relevant to respiratory health and asthma.Ann N Y Acad Sci. 2014; 1317: 57-69Crossref PubMed Scopus (50) Google Scholar and increased serum IgE concentrations,8Sharief S. Jariwala S. Kumar J. Muntner P. Melamed M.L. Vitamin D levels and food and environmental allergies in the United States: results from the National Health and Nutrition Examination Survey 2005-2006.J Allergy Clin Immunol. 2011; 127: 1195-1202Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar whereas other studies have reported that in culture vitamin D can act directly on TH2 lymphocytes to promote TH2 cytokine secretion (eg, Boonstra et al9Boonstra A. Barrat F.J. Crain C. Heath V.L. Savelkoul H.F.J. O'Garra A. 1alpha,25-Dihydroxyvitamin D3 has a direct effect on naive CD4(+) T cells to enhance the development of Th2 cells.J Immunol. 2001; 167: 4974-4980Crossref PubMed Scopus (881) Google Scholar). The apparent beneficial association of vitamin D in asthma in vivo, which in many patients is a TH2-type cytokine pathology, suggests that the direct action of vitamin D in promoting TH2 lymphocyte responses is less important in vivo than other vitamin D–mediated mechanisms (eg, vitamin D upregulated production of sST2 and the induction of regulatory mechanisms) in inhibiting TH2-type cytokine responses. Because the enhancement by vitamin D of sST2 production is concentration dependent, this supports therapeutic strategies to boost pulmonary vitamin D levels to reduce asthmatic inflammation. Ultrahigh-purity 1,25(OH)D3 and 25(OH)D3 (Enzo Life Sciences, Exeter, United Kingdom) were placed in aliquots dissolved in dimethyl sulfoxide (Sigma-Aldrich, Gillingham, United Kingdom). Recombinant proteins were obtained as follows: IL-33, IFN-γ, IL-4, human sST2-Fc chimera, and isotype were all from R&D System (Abingdon, United Kingdom). Primary HBECs were acquired from Lonza (Basel, Switzerland) and locally from endobronchial brushings obtained at fiberoptic bronchoscopy from adult volunteers (Guy's Research Ethics Committee, South London REC Office 3, REC approval no. 09/H0804/108, March 16, 2010). HNECs were obtained from resected nasal turbinates from adult patients undergoing nasal turbinectomy for clinical indications (City Road & Hampstead NRES Committee London, REC approval no. 12/LO/1931, January 14, 2013). Excised tissue was dissected and then treated with Liberase TL (Roche Diagnostics, Indianapolis, Ind) before isolation of epithelial cells with CD326 magnetic microbeads (magnetic cell sorting [MACS]; Miltenyi Biotech, Bisley, United Kingdom). Cells were expanded in flasks of Bronchial Epithelial Cell Growth Medium, which was constituted by supplementing Bronchial Epithelial Basal Medium (Lonza) with SingleQuot Supplements (Lonza). Epithelial cells were used for experiments at passages 3 to 5. Cell purity after passage checked by means of flow cytometry with an allophycocyanin-labeled anti-human CD326 antibody (Miltenyi Biotech) and appropriate isotype control was greater than 99%. Monolayers for cell-culture experiments were inspected daily by using light microscopy, and once approaching confluence, medium was changed to Bronchial Epithelial Cell Growth Medium containing all SingleQuot Supplements except bovine pituitary extract, retinoic acid, and hydrocortisone. Cell-culture experiments with HBECs were conducted with triplicate wells for each condition in each experiment. For conditioned medium, culture supernatant was collected and centrifuged at 250g for 7 minutes before aliquots of the supernatant were used as conditioned medium. PBMCs were obtained by means of density centrifugation with Lymphoprep density gradient medium (Axis-Shield, Dundee, United Kingdom). CD4+ and CD8+ lymphocytes were isolated by using Dynabeads (Invitrogen, Life Technologies, Paisley, United Kingdom). Cell purity was assessed by means of flow cytometry with appropriate isotype controls and was 95% to 98% for all experiments. Lymphocytes were cultured at 1 × 106 cells/mL in RPMI 1640 (Gibco, Life Technologies, Paisley, United Kingdom) supplemented with 50 U/mL IL-2 (Eurocetus, Harefield, United Kingdom), 10% FBS (PAA, Yeovil, United Kingdom), 2 mmol/L l-glutamine (Gibco, Life Technologies), and 0.1% 50 mg/mL gentamicin (Gibco, Life Technologies) on cell-culture plates coated with anti-CD3 (1 μg/mL; OKT-3) for 7 days. PBMCs were obtained, as described previously. Eosinophils were isolated with a MACS negative selection kit (MACS, Miltenyi Biotech), according to an adapted manufacturer's protocol. Cell purity was 96% to 99% for all experiments. Eosinophils were cultured at 1 × 106 cells/mL in RPMI 1640 supplemented with 10% FBS, 2 mmol/L l-glutamine, and 0.1% 50 mg/mL gentamicin for 20 hours. Epithelial cell monolayers were lysed with Qiazol reagent (Qiagen, Manchester, United Kingdom) before storage at −80°C pending extraction of mRNA with an miRNeasy Mini Kit (Qiagen), according to an adapted manufacturer's protocol with an off-column DNA digest with TurboDNase (Ambion, Life Technologies, Paisley, United Kingdom). mRNA was extracted from lymphocyte culture cell pellets by using an RNeasy Mini Kit (Qiagen), according to the manufacturer's protocol. mRNA was quantified with a NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, Mass) and converted to cDNA by using RevertAid Reverse Transcriptase and complementary reagents (Fermentas, Thermo Scientific). Relative quantification of IL1RL1 relative to the 18S housekeeping gene was conducted in triplicate by means of real-time quantitative PCR with the TaqMan primers Hs01073300 and Hs00249389 (Applied Biosystems, Life Technologies, Paisley, United Kingdom). Concentrations of sST2 protein in cell-culture supernatants were quantified by using a Human ST2 ELISA DuoSet (R&D Systems), according to the manufacturer's protocol. The LUVA mast cell line was a kind gift from Dr J. Steinke, University of Virginia.E1Laidlaw T.M. Steinke J.W. Tinana A.M. Feng C. Xing W. Lam B.K. et al.Characterization of a novel human mast cell line that responds to stem cell factor and expresses functional FcεRI.J Allergy Clin Immunol. 2011; 127: 815-822Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar LUVA cells were expanded in flasks in StemPro-34 serum free medium with associated nutrient supplement (Gibco, Life Technologies), l-glutamine (Gibco, Life Technologies), and 1% penicillin-streptomycin (10,000 U/mL, Gibco, Life Technologies). For the IL-33 bioassay, LUVA cells were cultured for 24 hours at 0.5 million cells/mL in RPMI 1640 supplemented with 10% FBS, 2 mmol/L l-glutamine, and gentamicin. The cell cultures were stimulated with a concentration series of recombinant IL-33 or IFN-γ plus IL-4 in the presence/absence of recombinant sST2-Fc chimera, isotype-Fc chimera, 100 nmol/L 1,25(OH)D3, or 40% HNEC conditioned medium. Cell-surface expression of CD54 (intercellular adhesion molecule 1) was measured by means of flow cytometry with a phycoerythrin-labeled anti-human CD54 antibody (BioLegend, London, United Kingdom) and appropriate isotype control on a FACSCalibur flow cytometer (BD Biosciences, San Jose, Calif) for initial experiments (Figs 2, B, and E2, B) and an Attune flow cytometer (Life Technologies, Paisley, United Kingdom) for later experiments (Fig 2, A and C-E, and Fig E2, C-E). Surface expression of CD54 was quantified as the geometric mean fluorescence intensity (GeoMFI). For stimulated LUVA cell cultures, induction of CD54 was calculated as the CD54 GeoMFI for unstimulated cell cultures subtracted from the GeoMFI for stimulated cell cultures in that experiment. The percentage reduction in stimulus-induced CD54 expression on LUVA cells cultured with vitamin D–treated epithelial cell–conditioned medium compared with induction of CD54 with control conditioned medium for each stimulus concentration was calculated to analyze the effect of vitamin D–treated epithelial cell–conditioned medium on stimulus-induced CD54 expression by LUVA cells. Data were analyzed with GraphPad Prism 6 software (GraphPad Software, La Jolla, Calif). For analysis of parametric data, t tests, repeated-measures ANOVA, and 2-way repeated-measures ANOVA were used with posttest-corrected multiple comparisons where the ANOVA was significant. For nonparametric data, Wilcoxon matched-pairs signed-rank tests or Friedman tests (with Dunn multiple comparison tests for comparison with control conditions) were used. Unless otherwise stated, parametric data are shown as means ± SEMs, and nonparametric data are shown as box and whisker plots with medians, 25th to 75th percentiles, and minimum to maximum ranges.Fig E2Stimulation with IL-33 induces LUVA cell expression of CD54. A and B, IL-33 causes homotypic aggregation of LUVA cells (×10 magnification; Fig E2, A) associated with increased expression of CD54 (filled gray histogram, unstained; dashed line, isotype control; solid line, anti-CD54; Fig E2, B). C, Inhibition of IL-33–induced LUVA cell CD54 expression by the recombinant sST2-Fc chimera is not observed with an isotype Fc chimera (n = 3). D, IFN-γ plus IL-4–induced LUVA cell CD54 expression is not inhibited by addition of recombinant sST2-Fc chimera (n = 4). E, Effect of IL-33 on LUVA cell CD54 expression is not affected by addition of active vitamin D (100 nmol/L 1,25[OH]D3) directly to the LUVA cells (n = 4).View Large Image Figure ViewerDownload Hi-res image Download (PPT)