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Nuclear factor (erythroid-derived 2)-like-2 pathway modulates substance P–induced human mast cell activation and degranulation in the hair follicle

2018; Elsevier BV; Volume: 142; Issue: 4 Linguagem: Inglês

10.1016/j.jaci.2018.04.039

ISSN

1097-6825

Autores

Laura Jadkauskaite, Rajia Bahri, Nilofer Farjo, Bessam Farjo, Gail Jenkins, Ranjit K. Bhogal, Iain S. Haslam, Silvia Bulfone‐Paus, Ralf Paus,

Tópico(s)

Mast cells and histamine

Resumo

Mast cells (MCs) are immune cells distributed throughout most tissues that respond to allergic and inflammatory reactions via inhibitory and activating receptors.1Bulfone-Paus S. Nilsson G. Draber P. Blank U. Levi-Schaffer F. Positive and negative signals in mast cell activation.Trends Immunol. 2017; 38: 657-667Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar The proinflammatory neuropeptide substance P (SP) not only causes MC degranulationE1Peters E.M.J. Liotiri S. Bodo E. Hagen E. Biro T. Arck P.C. et al.Probing the effects of stress mediators on the human hair follicle: substance P holds central position.Am J Pathol. 2007; 171: 1872-1886Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, E2Corrigan F. Vink R. Turner R.J. Inflammation in acute CNS injury: a focus on the role of substance P.Br J Pharmacol. 2016; 173: 703-715Crossref PubMed Scopus (47) Google Scholar, 2Liu N. Wang L.-H. Guo L.-L. Wang G.-Q. Zhou X.-P. Jiang Y. et al.Chronic restraint stress inhibits hair growth via substance P mediated by reactive oxygen species in mice.PloS One. 2013; 8: e61574Crossref PubMed Scopus (54) Google Scholar but also can generate damaging reactive oxygen species via activation of neurokinin-1 receptor and G-protein couple receptor MRGPRX2.1Bulfone-Paus S. Nilsson G. Draber P. Blank U. Levi-Schaffer F. Positive and negative signals in mast cell activation.Trends Immunol. 2017; 38: 657-667Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar SP-induced MC degranulation can thus result in the excessive accumulation of reactive oxygen species, which inhibits hair growth,2Liu N. Wang L.-H. Guo L.-L. Wang G.-Q. Zhou X.-P. Jiang Y. et al.Chronic restraint stress inhibits hair growth via substance P mediated by reactive oxygen species in mice.PloS One. 2013; 8: e61574Crossref PubMed Scopus (54) Google Scholar besides being hair growth–inhibitory in itself.E1Peters E.M.J. Liotiri S. Bodo E. Hagen E. Biro T. Arck P.C. et al.Probing the effects of stress mediators on the human hair follicle: substance P holds central position.Am J Pathol. 2007; 171: 1872-1886Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, E3Peters E.M.J. Handjiski B. Kuhlmei A. Hagen E. Bielas H. Braun A. et al.Neurogenic inflammation in stress-induced termination of murine hair growth is promoted by nerve growth factor.Am J Pathol. 2004; 165: 259-271Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar Importantly, IgE-induced MC degranulation in the RBL-2H3 cell line can be suppressed by activation of the nuclear factor (erythroid-derived 2)-like 2 (Nrf2) target gene heme-oxygenase 1 (HO-1).3Matsushima M. Takagi K. Ogawa M. Hirose E. Ota Y. Abe F. et al.Heme oxygenase-1 mediates the anti-allergic actions of quercetin in rodent mast cells.Inflamm Res. 2009; 58: 705-715Crossref PubMed Scopus (34) Google Scholar Therefore, we were interested in the role of Nrf2 in the SP-mediated activation of primary human MCs in situ and in vitro. Nrf2 is the “master regulator” of redox homeostasis, controlling cellular responses to oxidative stress by transcriptional regulation of antioxidant genes.E4Bai Y. Wang X. Zhao S. Ma C. Cui J. Zheng Y. Sulforaphane protects against cardiovascular disease via Nrf2 activation.Oxid Med Cell Longev. 2015; 2015: 407580Crossref PubMed Scopus (120) Google Scholar, 4Haslam I.S. Jadkauskaite L. Szabó I.L. Staege S. Hesebeck-Brinckmann J. Jenkins G. et al.Oxidative damage control in a human (mini-) organ: Nrf2 activation protects against oxidative stress-induced hair growth inhibition.J Invest Dermatol. 2017; 137: 295-304Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar The connective tissue sheath (CTS) of human hair follicles (HFs) contains abundant MCs, which allows one to investigate primary human MC phenotype and activities under physiologically relevant conditions ex vivo.E1Peters E.M.J. Liotiri S. Bodo E. Hagen E. Biro T. Arck P.C. et al.Probing the effects of stress mediators on the human hair follicle: substance P holds central position.Am J Pathol. 2007; 171: 1872-1886Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, E5Ito N. Sugawara K. Bodó E. Takigawa M. van Beek N. Ito T. et al.Corticotropin-releasing hormone stimulates the in situ generation of mast cells from precursors in the human hair follicle mesenchyme.J Invest Dermatol. 2010; 130: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 5Sugawara K. Biro T. Tsuruta D. Toth B.I. Kromminga A. Zakany N. et al.Endocannabinoids limit excessive mast cell maturation and activation in human skin.J Allergy Clin Immunol. 2012; 129: 726-738Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Therefore, we investigated whether the Nrf2 transcriptional pathway can be activated within human CTS-MCs and whether this modulates SP-induced MC degranulation and secretory activity in vitro and in situ. Double immunostaining for Nrf2/MC tryptase (MCT) in the CTS was performed on human scalp HF sections.5Sugawara K. Biro T. Tsuruta D. Toth B.I. Kromminga A. Zakany N. et al.Endocannabinoids limit excessive mast cell maturation and activation in human skin.J Allergy Clin Immunol. 2012; 129: 726-738Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Overall, no significant difference in the number of Nrf2/MCT double-positive cells was found (Fig 1, A) and treatment with sulforaphane (SFN) did not increase the number of Nrf2/MCT double-positive cells (Fig 1, B). Recently, we demonstrated Nrf2-mediated upregulation of HO-1 in human HFs CTS by the well-established Nrf2 activator SFN.4Haslam I.S. Jadkauskaite L. Szabó I.L. Staege S. Hesebeck-Brinckmann J. Jenkins G. et al.Oxidative damage control in a human (mini-) organ: Nrf2 activation protects against oxidative stress-induced hair growth inhibition.J Invest Dermatol. 2017; 137: 295-304Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar Therefore, isolated human HFs were pretreated with SFN for 24 hours, followed by SP stimulation for 4 hours to activate native human CTS-MCs ex vivo. This significantly increased the number of HO-1/MCT double-positive MCs in the CTS (Fig 1, C and D), suggesting Nrf2 activation in these MCs. Next, Nrf2 activation by SFN was investigated in primary human peripheral blood-derived cultured MCs (PBDMCs).6Bahri R. Custovic A. Korosec P. Tsoumani M. Barron M. Wu J. et al.Mast cell activation test in the diagnosis of allergic disease and anaphylaxis.J Allergy Clin Immunol. 2018; 142: 485-496.e16Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar Increased Nrf2 phosphorylation is observed following Nrf2 activation, which is associated with increased nuclear Nrf2 protein translocation and transcriptional activity.E4Bai Y. Wang X. Zhao S. Ma C. Cui J. Zheng Y. Sulforaphane protects against cardiovascular disease via Nrf2 activation.Oxid Med Cell Longev. 2015; 2015: 407580Crossref PubMed Scopus (120) Google Scholar Using immunohistochemistry, we found that Nrf2 displayed both cytoplasmic and nuclear localizations under basal conditions, while SFN treatment increased nuclear Nrf2 accumulation (Fig 1, E and F). In Laboratory of Allergic Diseases 2 (LAD2) cells,E6Guhl S. Babina M. Neou A. Zuberbier T. Artuc M. Mast cell lines HMC-1 and LAD2 in comparison with mature human skin mast cells drastically reduced levels of tryptase and chymase in mast cell lines.Exp Dermatol. 2010; 19: 845-847Crossref PubMed Scopus (78) Google Scholar, E7Kirshenbaum A.S. Akin C. Wu Y. Rottem M. Goff J.P. Beaven M.A. et al.Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia: activation following aggregation of FcepsilonRI or FcgammaRI.Leuk Res. 2003; 27: 677-682Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar instead, Nrf2 localization was only cytoplasmic in vehicle- and SFN-treated conditions (see Fig E1 in this article's Online Repository at www.jacionline.org). Furthermore, we asked whether the expression of known Nrf2 target genes and proinflammatory MC genes is modulated by SFN stimulation for 24 hours or SP stimulation for 6 hours in PBDMCs. SFN treatment significantly increased transcription of the Nrf2 target genes HO-1 and NADP(H) dehydrogenase-quinone 1 (Fig 1, G and H). Treatment with SP significantly upregulated mRNA expression of the proinflammatory cytokine IL-1β, whereas pretreatment with SFN before SP significantly reduced IL-1β levels (Fig 1, I). However, these transcriptional effects of SP and SFN were not observed for the MC activation markers CD69E8Diaz-Agustín B. Escribano L. Bravo P. Herrero S. Nuñez R. Navalón R. et al.The CD69 early activation molecule is overexpressed in human bone marrow mast cells from adults with indolent systemic mast cell disease.Br J Haematol. 1999; 106: 400-405Crossref PubMed Scopus (36) Google Scholar or TNF-α (see Fig E2, A and B, in this article's Online Repository at www.jacionline.org). In LAD2, a similar effect was detected (see Fig E3 in this article's Online Repository at www.jacionline.org). Furthermore, we investigated whether Nrf2 activation could modulate SP-induced MC degranulation. In LAD2 cells, SP increased surface expression of MC degranulation markers CD107a and CD63 by nearly 70%, whereas SFN alone or given before SP had no effect (see Fig E4, A and B, in this article's Online Repository at www.jacionline.org), which was also the case when β-hexosaminidase release was measured (Fig E4, C). Therefore, PBDMCs were investigated next. Although their surface expression of CD63 and CD107a after SP stimulation was approximately 30%, pretreatment of PBDMCs with SFN for 1 hour before SP treatment significantly reduced the SP-induced upregulation of both markers (Fig 2, A-C). Furthermore, SP induced β-hexosaminidase release in PBDMCs, whereas SFN pretreatment reduced β-hexosaminidase levels (Fig 2, D). Pretreatment with SFN for 24 hours did not show this protective effect (see Fig E5, A and B, in this article's Online Repository at www.jacionline.org). Importantly, the inhibitory effect of SFN varied in intensity between PBDMC cultures. Another Nrf2 activator, quercetin, reportedly induces rapid HO-1 upregulation in RBL-2H3 cells and suppresses their degranulation.3Matsushima M. Takagi K. Ogawa M. Hirose E. Ota Y. Abe F. et al.Heme oxygenase-1 mediates the anti-allergic actions of quercetin in rodent mast cells.Inflamm Res. 2009; 58: 705-715Crossref PubMed Scopus (34) Google Scholar Thus, it is possible that the 1-hour stimulation with SFN used in this study stimulates a similarly rapid increase in HO-1 expression and enzymatic activity to suppress SP-induced degranulation.3Matsushima M. Takagi K. Ogawa M. Hirose E. Ota Y. Abe F. et al.Heme oxygenase-1 mediates the anti-allergic actions of quercetin in rodent mast cells.Inflamm Res. 2009; 58: 705-715Crossref PubMed Scopus (34) Google Scholar MCs are a major source of prostaglandin E2 (PGE2) and prostaglandin D2 (PGD2) production,7Gaudenzio N. Sibilano R. Marichal T. Starkl P. Reber L.L. Cenac N. et al.Different activation signals induce distinct mast cell degranulation strategies.J Clin Invest. 2016; 126: 3981-3998Crossref PubMed Scopus (215) Google Scholar and release of these prostaglandins can occur very quickly upon stimulation.E9Moon T.C. Campos-Alberto E. Yoshimura T. Bredo G. Rieger A.M. Puttagunta L. et al.Expression of DP2 (CRTh2), a prostaglandin D2 receptor, in human mast cells.PloS One. 2014; 9: e108595Crossref PubMed Scopus (42) Google Scholar Therefore, we also investigated by ELISA how Nrf2 activation by SFN for 1 hour (Fig 2, E and F) or 24 hours (see Fig E6, A and B, in this article's Online Repository at www.jacionline.org) impacted on the SP-induced (1-hour) secretion of these PGs by PBDMCs into the supernatant. This revealed a significant increase in PGD2 secretion following SP treatment, whereas pretreatment with SFN before SP tendentially reduced this, although the level of significance was not reached (Fig 2, E). PGE2 release was largely unaffected in this setting (Fig 2, F). SFN and SP treatment did not alter the levels of prostaglandin D2 synthase (PTGDs) and prostaglandin E2 synthase (PTGEs) transcription (Fig E6, C and D). Finally, we asked whether the SP-induced degranulation of native human skin MCs can be suppressed by SFN ex vivo, using toluidine blue histochemistry. As expected,E1Peters E.M.J. Liotiri S. Bodo E. Hagen E. Biro T. Arck P.C. et al.Probing the effects of stress mediators on the human hair follicle: substance P holds central position.Am J Pathol. 2007; 171: 1872-1886Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar SP stimulated CTS-MC degranulation in situ. Yet, there was no significant SP effect on perifollicular MC number in comparison to control. However, SFN pretreatment reduced SP-stimulated anaphylactic MC degranulation and overall MC number (Fig 2, G-I). Our results provide the first evidence that primary human MCs exhibit substantial Nrf2 activity, which can effectively suppress MC activation and degranulation induced by SP. Interestingly, PGD2 levels are increased in balding scalp HFs, whereas PGE2 levels are decreased,E10Larson A.R. Zhan Q. Johnson E. Fragoso A.C. Wan M. Murphy G.F. A prostaglandin D-synthase-positive mast cell gradient characterizes scalp patterning.J Cutan Pathol. 2014; 41: 364-369Crossref PubMed Scopus (4) Google Scholar, E11Garza L.A. Liu Y. Yang Z. Alagesan B. Lawson J.A. Norberg S.M. et al.Prostaglandin D2 inhibits hair growth and is elevated in bald scalp of men with androgenetic alopecia.Sci Transl Med. 2012; 4: 126ra34Crossref PubMed Scopus (183) Google Scholar and the intrafollicular balance between PGD2 and PGE2 appears to control optimal hair growth.E12Bernard B.A. Michelet J.F. Colombe L. Prostanoid receptors in anagen human hair follicles.Exp Dermatol. 2008; 17: 63-72PubMed Google Scholar, 8Nieves A. Garza L.A. Does prostaglandin D2 hold the cure to male pattern baldness?.Exp Dermatol. 2014; 23: 224-227Crossref PubMed Scopus (48) Google Scholar Increased MC numbers/degranulation have been reported in androgenic alopecia,E13Jaworsky C. Kligman A.M. Murphy G.F. Characterization of inflammatory infiltrates in male pattern alopecia: implications for pathogenesis.Br J Dermatol. 1992; 127: 239-246Crossref PubMed Scopus (174) Google Scholar whereas the number, degranulation, and proliferation of CTS-MCs is increased in the most common inflammatory hair growth disorder, alopecia areata.E13Jaworsky C. Kligman A.M. Murphy G.F. Characterization of inflammatory infiltrates in male pattern alopecia: implications for pathogenesis.Br J Dermatol. 1992; 127: 239-246Crossref PubMed Scopus (174) Google Scholar, E14Cetin E.D. Savk E. Uslu M. Eskin M. Karul A. Investigation of the inflammatory mechanisms in alopecia areata.Am J Dermatopathol. 2009; 31: 53-60Crossref PubMed Scopus (53) Google Scholar, 9Bertolini M. Zilio F. Rossi A. Kleditzsch P. Emelianov V.E. Gilhar A. et al.Abnormal interactions between perifollicular mast cells and CD8+ T-cells may contribute to the pathogenesis of alopecia areata.PloS One. 2014; 9: e94260Crossref PubMed Scopus (86) Google Scholar Therefore, therapeutic upregulation of Nrf2 activity in perifollicular MCs may be of particular interest in the future management of both androgenetic alopecia and alopecia areata. We thank Jiakai Wu for his help with primary MCs culture and generation, and Drs Arnold Kirshenbaum and Dean Metcalfe for kindly providing LAD2 cells. Occipital scalp HFs from male hair transplant surgery were delivered from the Farjo Medical Centre (Manchester, UK). Male patients who had undergone hair transplant surgery were suffering from androgenic alopecia, but the occipital scalp HFs are androgen-insensitive and not affected by androgenic alopecia; therefore, they are suitable for experimental procedures. The tissue was collected after informed patient consent according to the “Declaration of Helsinki Principles” together with institutional approval ethics from the University of Manchester. Isolated full-length scalp HFs in anagen VI were cultured in William's E media (Gibco, Leicestershire, UK) supplemented with 2 mM of l-glutamine (Invitrogen, Paisley, UK), 10 ng/mL hydrocortisone (Sigma-Aldrich, Gillingham, UK), 1 μg penicilin/streptomycin antibiotic mixture (Gibco), and insulin-transferin-selenium (Life Technologies, Paisley, UK) at 37°C 5% CO2 incubator.E15Philpott M.P. Green M.R. Kealey T. Human hair growth in vitro.J Cell Sci. 1990; 97: 463-471PubMed Google Scholar, E16Langan E.A. Philpott M.P. Kloepper J.E. Paus R. Human hair follicle organ culture: theory, application and perspectives.Exp Dermatol. 2015; 24: 903-911Crossref PubMed Scopus (107) Google Scholar Nrf2 activation was performed using 20 μM of SFN (Sigma-Aldrich) for 2, 4, and 24 hours and 2 μM of SP (Millipore, Nottingham, UK) for 4 hours. Double staining of Nrf2/MCT was performed using PerkinElmer TSA plus Kit (PerkinElmer, Beaconsfield, UK). Cryosections (6 μm) were fixed in 4% paraformaldehyde followed by permeabelization in 0.1% Triton X. The blocking was performed using 3% H2O2 (Sigma-Aldrich) followed by washes with TNT buffer. Additional blocking was done using TNB, and primary Nrf2 antibody (Abcam 31163, Cambridge, UK) was left overnight at 4°C before incubation with horseradish peroxidase goat-antirabbit (Life Technologies). Additional blocking was done using Bloxall (Vector, Peterborough, UK) before addition of secondary MCT antibody (Abcam 2378). PBDMCs were dried at room temperature after stimulation with SFN (5 μM) for 2 hours and stained for Nrf2 phospho-S40 (Abcam76026). Washes were done using TBS + 0.1% Tween 20. The blocking was performed using normal goat serum 10% and primary antibody was diluted 1:100 followed by overnight incubation. Subsequently, secondary antibody AF594 (goat anti-rabbit) (Life Technologies) 1:200 was added for 1 hour, followed by washes and counterstain with 4′-6-diamidino-2-phenylindole, dihydrochloride. MCT/HO-1 double immunostaining was performed using Vector Immpress kit (Vector, Peterborough, UK). Slides were blocked using Bloxall followed by incubation with 2.5% normal horse serum and primary HO-1 antibody (Sigma HPA000635, Sigma-Aldrich). Washes were performed using PBS, and secondary MCT antibody (Abcam 2378) was used. Toluidine blue histochemistry (Tol) was performed by fixing slides in acetone, followed by washes in water before staining in Tol blue working solution for 3 minutes. The analysis of immunostaining was performed using a Biozero-8000 microscope (Keyence, Milton Keynes, UK) and staining analysis was quantified by ImageJ software (NIH, Bethesda, Md). Degranulated MCs were identified by quantifying 5 or more extracellular metachromatic granules in the direct vicinity of clearly identifiable perifollicular MCs.E5Ito N. Sugawara K. Bodó E. Takigawa M. van Beek N. Ito T. et al.Corticotropin-releasing hormone stimulates the in situ generation of mast cells from precursors in the human hair follicle mesenchyme.J Invest Dermatol. 2010; 130: 995-1004Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar, 5Sugawara K. Biro T. Tsuruta D. Toth B.I. Kromminga A. Zakany N. et al.Endocannabinoids limit excessive mast cell maturation and activation in human skin.J Allergy Clin Immunol. 2012; 129: 726-738Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar Human peripheral blood was obtained from a blood bank in Manchester. Mononuclear cells were obtained and CD117+ progenitor cells were isolated by positive selection of FcRI block/CD117+ (Miltenyi Biotec GmbH, Surrey, UK) by magnetic cell sorting.E17Jensen B.M. Swindle E.J. Iwaki S. Gilfillan A.M. Generation, isolation, and maintenance of rodent mast cells and mast cell lines.Curr Protoc Immunol. 2006; (Chapter 3:Unit 3.23)Crossref PubMed Scopus (67) Google Scholar For the first 4 weeks, cells were cultured in StemSpam medium supplement with 1% penicilin/streptomycin (Invitrogen, Paisley, UK), 50 ng/mL IL-6 (Peprotech, London, UK), 10 ng/mL IL-3 (Peprotech), 100 ng/mL Stem Cell Factor (Peprotech), and 10 μg/mL low density lipoprotein (StemCell Technologies, Cambridge, UK). After 4 weeks, cells were cultured in Iscove's Modified Dulbecco's Medium (Thermofisher, Paisley, UK) supplemented with 50 μM 2β-mercaptoethanol (Sigma-Aldrich), 0.5% BSA (Life Technologies), 1% insulin-transferrin-selenium (Life Technologies), 1% penicilin/steptomycin (Invitrogen), 50 ng/mL IL-6 (Peprotech), and 100 ng/mL stem cell factor (rhSCF) (Peprotech). Cell viability and maturity after 8 weeks was measured using fluorochrome-conjugated antibodies FcεRI (Biolegend, London, UK) and CD117 (Biolegend) by FlowCytometry. LAD2 cells (kindly supplied by the National Institute of Allergy and Infectious Diseases: Arnold S. Kirshenbaum and Dean D. Metcalfe) were cultured in StemPro-34 serum-free medium (Invitrogen) supplemented with 100 U/mL penicilin/streptomycin (Invitrogen), 100 U/mL glutamine (Invitrogen), and 100 ng/mL rhSCF (Peprotech).E7Kirshenbaum A.S. Akin C. Wu Y. Rottem M. Goff J.P. Beaven M.A. et al.Characterization of novel stem cell factor responsive human mast cell lines LAD 1 and 2 established from a patient with mast cell sarcoma/leukemia: activation following aggregation of FcepsilonRI or FcgammaRI.Leuk Res. 2003; 27: 677-682Abstract Full Text Full Text PDF PubMed Scopus (414) Google Scholar MC activity and degranulation was measured using flow cytometry. Briefly, LAD2 cells and PBDMCs (1.5 × 105) were seeded in 96-well plate and stimulated with 5 μM SFN (Sigma-Aldrich) for 1 hour or 24 hours followed by stimulation with 5 μM SP (Millipore) for 1 hour. Subsequently, cells were resuspended in FACS-buffer (2% newborn calf serum, 0.1% NaN3, 2 mM EDTA in PBS) and stained with fluorochrome-conjugated antibodies CD63 and CD69 (Biolegend) for 30 minutes at 4°C followed wash and a Live/Dead Fixable Blue dead cell stain kit for UV excitation (Life Technologies). Samples were analyzed with LSR II or Fortessa (BD Biosciences, Oxford, UK). LAD2 cells or PBDMCs (1.5 × 105) were treated with 5 μM SFN for 24 hours followed by stimulation with 5 μM SP for 6 hours. Total RNA was extracted with an RNeasy Micro kit (Qiagen, Manchester, UK) according to the manufacturer's instructions. Complementary DNA was reverse transcribed using Tetro complementary DNA synthesis kit (Bioline, Wokingham, UK). Quantitative PCR was performed using StepOne real-time PCR system (Applied Biosystems, Warrington, UK) using Taqman fast advance master mix and probes (Applied Biosystems). The following probes from Applied Biosystems were used: HMOX1 (Hs01110250_m1), NADP(H) dehydrogenase-quinone 1 (Hs02512143_s1), CD69 (Hs00934033_m1), and most important proinflammatory mediator in skin disorders IL-1β (Hs00174097_m1). In addition, prostaglandin D synthase (Hs00168748_m1) and prostaglandin E synthase (Hs00610420_m1) were used. Samples were run using the StepOne Plus Real-Time PCR system and associated software (Applied Biosystems); relative expression was quantified against the housekeeping gene PPIA (Hs04194521_s1). PBDMCs (1 × 105) were cultured with 5 μM SFN for 1 hour and 24 hours followed by stimulation with 5 μM SP for 1 hour. Secretion of PGD2 and PGE2 was measured according to the manufacturer's instructions (CaymanChemical, Cambridge, UK). A total of 50 μL (25,000 cells) from the LAD2 cell or PBDMC cultures was taken and centrifuged to separate the supernatant and cell pellet. Cell pellets were lysed in 50 μL media culture 1% Triton X-100. β-Hexosaminidase was measured in supernatant as well as in the cell pellet by adding 100 μL β-hexosaminidase substrate, 10 mM p-nitrophenyl N-acetyl-beta-d-glucosamine (Sigma-Aldrich) in 0.1 mol Na2HPO4 buffer (pH 4.5) for 2 hours at 37°C, 5% CO2. The reaction was stopped by adding 100 μL of 0.2 mol glycine buffer (pH 10). Optical density was measured at 405 nm. hMC degranulation was assessed as % release of total β-hexosaminidase.Fig E2Expression of MC target genes in PBDMCs. A, Expression of CD69 mRNA in PBDMCs did not change after treatment with SFN or SP. B, The mRNA levels of TNF-α were increased after pretreatment with SFN before SP (n = 8 donors; data are mean ± SEM; 1-way ANOVA; significant indicate by *P < .05).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3The expression of Nrf2 and MCs target genes in human LAD2. HO-1 (A), NQO1 (B), CD69 (C), and IL-1β (D) in LAD2 were measured by quantitative RT-PCR and data reported as fold changes in normalized expression (n = 9 repeats; data are mean ± SEM; 1-way ANOVA; significance indicated by *P < .05; **P < .01; ***P < .001). NQO1, NADP(H) dehydrogenase-quinone 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E4LAD2 degranulation after treatment with SP and SFN (1 hour). A, SP alone increased CD63 expression on LAD2 surface. Pretreatment with SFN did not reduce SP-induced activity. B, Treatment with SP induced CD107a expression on LAD2, but SFN pretreatment did not have an effect against SP. C, Pretreatment with SFN before SP exposure did not reduce release of β-hexosaminidase (n = 3-4 repeats; data are mean ± SEM).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E5PBDMCs degranulation after treatment with SFN (24 hours) and SP (1 hour). The expression of both MC degranulation markers (A), CD63 (B), and CD107a (C) was not reduced after pretreatment with SFN for 24 hours (n = 4 donors; data are mean ± SEM). FSC-A, Forward scatter-area.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E6Measurement of secretion of PGD2 and PGE2 and mRNA levels of PTGDS and PTGES in PBDMCs. A, Pretreatment with SFN for 24 hours reduced SP-induced PGD2 release. B, Stimulation with SFN for 24 hours had a minimal suppression effect of PGE2 release during SP exposure. (n = 4 donors; data are mean ± SEM; 1-way ANOVA; significance indicated by **P < .01). C, The expression of PTGD mRNA was not affected by SFN and SP treatment. D, The PTGE mRNA levels were variable between 2 donors and no effect of SP and SFN was observed. (n = 2-5 donors; data are mean ± SD).View Large Image Figure ViewerDownload Hi-res image Download (PPT)

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