the benzene metabolite 1 4 benzoquinone reduces t reg function a potential mechanism for tobacco smoke associated atopic dermatitis

Accepted Manuscript The benzene metabolite 1,4-benzoquinone reduces Treg function – a potential mechanism for tobacco smoke-associated atopic dermatitis Marcus Winter, MSc, Loreen Thürmann, MSc, Zuguang Gu, PhD, Gerrit Schüürmann, PhD, Gunda Herberth, PhD, Denise Hinz, PhD, Martin von Bergen, PhD, Hauke Harms, PhD, Sven Olek, PhD, Stefan Röder, PhD, Michael Borte, MD, Roland Eils, PhD, Irina Lehmann, PhD, Saskia Trump, PhD PII: S0091-6749(17)30339-1 DOI: 10.1016/j.jaci.2017.01.034 Reference: YMAI 12677 To appear in: Journal of Allergy and Clinical Immunology Received Date: November 2016 Revised Date: 22 December 2016 Accepted Date: 23 January 2017 Please cite this article as: Winter M, Thürmann L, Gu Z, Schüürmann G, Herberth G, Hinz D, von Bergen M, Harms H, Olek S, Röder S, Borte M, Eils R, Lehmann I, Trump S, The benzene metabolite 1,4-benzoquinone reduces Treg function – a potential mechanism for tobacco smoke-associated atopic dermatitis, Journal of Allergy and Clinical Immunology (2017), doi: 10.1016/j.jaci.2017.01.034 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain Winter et al ACCEPTED MANUSCRIPT Letter to the Editor The benzene metabolite 1,4-benzoquinone reduces Treg function – a potential mechanism for tobacco smoke-associated atopic dermatitis Marcus Winter, MSca, Loreen Thürmann, MSca, Zuguang Gu, PhDb,c, Gerrit Schüürmann, PhDd,e, Gunda Herberth, PhDa, Denise Hinz, PhDa,f, Martin von Bergen, PhDg,h, Hauke Harms, PhDi, Sven Olek, PhDj, Stefan Röder, PhDa, Michael Bortek, MD, Roland Eilsb,c,l,m, PhD, Irina Lehmann, PhDa, Saskia Trump, PhDa SC 11 Research – UFZ, Permoserstraße 15, 04318 Leipzig, Germany b 13 Neuenheimer Feld 267, 69120 Heidelberg, Germany c 15 16 69120 Heidelberg, Germany d 17 18 e 22 23 24 Germany f 27 La Jolla Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA g Department of Molecular Systems Biology, UFZ, Helmholtz-Centre for Environmental Research, Permoserstraße 15, 04318 Leipzig, Germany h 25 26 Technical University Bergakademie Freiberg, Leipziger Straße29, 09596 Freiberg, AC C 21 Department of Ecological Chemistry, Helmholtz Centre for Environmental Research – UFZ, Permoserstraße 15, 04318 Leipzig, Germany 19 20 Heidelberg Center for Personalized Oncology (DKFZ-HIPO), Im Neuenheimer Feld 280, D 14 Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im TE 12 Department of Environmental Immunology, Helmholtz Centre for Environmental M AN U a EP 10 RI PT Department of Biochemistry, Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany i Department of Environmental Microbiology, Helmholtz Centre for Environmental Research, Leipzig, Permoserstraße 15, 04318 Leipzig, Germany Winter et al ACCEPTED MANUSCRIPT 28 j 29 k 30 32 33 34 Municipal Hospital “St Georg” Children’s Hospital, Delitzscher Straße 141, 04129 Leipzig, Germany l Institute of Pharmacy and Molecular Biotechnology (IPMB), and BioQuant Center, University of Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany m Translational RI PT 31 Epiontis GmbH, Rudower Chaussee 29, 12489 Berlin, Germany Lung Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), University of Heidelberg, Germany 35 Corresponding author 37 Dr Saskia Trump, Helmholtz-Centre for Environmental Research – UFZ, Department of 38 Environmental Immunology, Permoserstraße 15, 04318 Leipzig, Germany, 39 phone: +49 341 235 1550, fax: +49 341 235 1787, mail: saskia.trump@ufz.de M AN U SC 36 40 Sources of funding 42 Marcus Winter was supported by the “Helmholtz Impulse and Networking Fund” 43 through the “Helmholtz Interdisciplinary Graduate School for Environmental Research 44 (HIGRADE)” Further support for Saskia Trump came from the Helmholtz Initiative for 45 Personalized Medicine (iMed) EP TE D 41 46 Capsule summary 48 A direct inhibitory effect of the benzene metabolite 1,4-benzoquinone on Treg functional 49 activity was observed, which seems to contribute to the benzene-/tobacco smoke 50 exposure-mediated risk increase of early childhood atopic dermatitis by reducing Treg 51 activity AC C 47 52 53 54 Winter et al ACCEPTED MANUSCRIPT To the Editor: 56 Exposure to environmental chemicals can contribute to the development or 57 exacerbation of atopic dermatitis (AD) Benzene is one of the chemicals previously been 58 implicated in the etiology of AD1 Albeit a ubiquitous environmental chemical, non- 59 occupational exposure to benzene mainly results from active or passive tobacco smoke 60 exposure in homes or public places Exposure to benzene has been shown to exert 61 diverse effects on blood cells in particular on lymphocytes While chronic exposure to 62 high levels of benzene may cause leukemia and probably other lymphohematopoietic 63 cancers, already low levels of benzene have been described to decrease the number of 64 peripheral lymphocytes2 in humans including circulating CD4+ T cells, CD4+/CD8+ ratios, 65 B cells, neutrophils, and platelets3 66 Although benzene itself is not regarded as toxic its metabolization products are believed 67 to be the main source of benzene toxicity Benzene degradation in humans preferentially 68 occurs via the formation of phenol from the initial oxidation product benzene oxide4 69 Phenol is then further degraded to the highly reactive electrophile 1,4-benzoquinone 70 (1,4-BQ, see Online Repository, Figure E1), which can readily react with cellular 71 nucleophiles including proteins or DNA There is a nonlinear relationship between 72 benzene exposure and metabolite formation causing a favored production of reactive 73 quinones in case of low dose benzene exposure (< ppm)5 74 We have previously reported that prenatal benzene exposure is associated with 75 decreased cord blood regulatory T cells (Treg) numbers and a subsequent increased risk 76 for the development of atopic dermatitis in early childhood1 This reduction of 77 immunocompetent cells is likely a contributing factor to the increased risk of atopic 78 dermatitis described in relation to benzene/environmental tobacco smoke exposure To 79 further elucidate the processes underlying the benzene-associated Treg deficiency, we 80 used an in vitro model based on expanded human Treg cells (CD4+CD25hiCD45RA+, for 81 details see Online Repository, Figure E2) derived from healthy blood donors As public AC C EP TE D M AN U SC RI PT 55 Winter et al ACCEPTED MANUSCRIPT available RNASeq data revealed that neither resting nor activated Treg cells express the 83 necessary enzymes for the degradation of benzene via phenol (see Online Repository, 84 Table E1) we directly exposed in vitro expanded Treg to the benzene metabolite 1,4-BQ 85 While Treg exposure (independent donors, n = 5) to 50 nM 1,4-BQ for 24h, corresponding 86 to concentrations previously observed in smokers6, had no effect on FOXP3 expression 87 as monitored by FACS (see Online Repository, Figure E4), it led to a significant decrease 88 in IL-10 production (Table E2, signed rank sum test, p = 0.005) - determined by CBA 89 assays in the cell culture supernatants (CBA assays performed at least in triplicates, see 90 Online Repository) We hypothesize that this decrease in IL-10 production, a key anti- 91 inflammatory cytokine that is critical for efficient Treg function contributed to the 92 impairment in Treg function subsequently observed in in vitro suppression assays 93 (Figure 1A) Exposed Treg were cocultured at different ratios with 5,6-carboxyfluorescein 94 diacetate succinimidyl ester (CFDA-SE) labeled responder T cells (CD4+CD25- as Tconv) of 95 the same donor for five days (see Online Repository for details) 1,4-BQ exposed Treg 96 showed a significant decrease in suppressive function compared to vehicle control 97 treated Treg of the same donor (Figure 1A, two-way ANOVA, p-value=0.002) Evaluation 98 of cytokine secretion during the duration of the suppression assay revealed a significant 99 increase in IL-13 and granzyme B (GrzB, Figure 1B and Online Repository, Table E3) 100 Elevated GrzB concentrations were most likely related to enhanced production by 101 responder T cells (Tconv) since they seem to be the main source of GrzB, secreting about 102 25-fold higher amounts of GrzB compared to Treg (see Online Repository Table E4) This 103 increase of GrzB as an important mediator of cell death and regulatory T cell activity 104 might point to an enhanced apoptosis rate in Treg cells, which - in addition to their 105 diminished IL-10 secretion - could account for the relative decrease in Treg suppression 106 by 1,4-BQ7 107 To evaluate whether these in vitro observations can be related to the in vivo situation, 108 we additionally studied the effect of benzene exposure in our prospective mother-child AC C EP TE D M AN U SC RI PT 82 Winter et al ACCEPTED MANUSCRIPT cohort LINA (see Online Repository, Table E5) As already earlier shown for cord blood 110 Treg1 we can provide a link between indoor benzene concentrations – a good proxy for 111 active or passive tobacco smoke exposure - and peripheral Treg numbers in mothers and 112 one year old children Benzene exposure during pregnancy was associated with a 113 decrease in maternal peripheral Treg numbers (Figure 1C, adj MR=0.89, 95% CI: 081- 114 0.97, n=561, for confounders see Online Repository) This inverse relationship between 115 perinatal benzene exposure, considering benzene measurements during pregnancy and 116 one year after birth, and Treg numbers was also observed in one year old children 117 (Figure 1C, MR=0.94, 95% CI: 0.88-0.99, n=480, for confounders see Online Repository) 118 Similar to the increase in IL-13, which we observed in our in vitro coculture assays (see 119 Online Repository, Table E3), a positive association was found between indoor benzene 120 concentrations and phytohaemagglutinin (PHA)-induced IL-13 concentrations in 121 maternal or infantile blood samples respectively (Figure 1C, mothers: MR=1.10, 95% CI: 122 1.01-1.20, n=570; children: MR=1.39, 95% CI: 1.15-1.68, n=489) 123 In addition, GrzB concentrations measured in PHA-stimulated whole blood samples 124 were positively associated with benzene concentrations (see Online Repository, 125 Figure E5, adj MR=1.20, 95% CI: 1.00-1.44, n=264) and negatively associated with the 126 number of Treg cells in these children (see Online Repository, Figure E5, adj MR=0.94, 127 95% CI: 0.89-0.99, n=261), further corroborating our in vitro findings In children the 128 high concentrations of PHA-stimulated IL-13 were associated with an increased risk for 129 these children to develop symptoms according to Hanifin and Rajka8 and/or physician- 130 diagnosed AD up to the age of six (adj OR: 1.31, 95% CI: 1.11-1.55, per ln(1 pg/ml) 131 increase in PHA-IL13, n=490, see Online Repository and Table E6) 132 Our results support the notion that even low levels of the reactive benzene metabolite 133 1,4-BQ can significantly impair the suppressive function of Treg by reducing their 134 potential to secrete the anti-inflammatory cytokine IL-10 This functional impairment 135 most likely reduces Treg suppressive capacity resulting in higher GrzB production by AC C EP TE D M AN U SC RI PT 109 Winter et al ACCEPTED MANUSCRIPT effector cells and a subsequent further loss in Treg numbers presumably due to higher 137 apoptosis rates induced by GrzB In addition to the earlier reported induction of 138 miR-223 under benzene exposure leading to reduced Treg numbers1, in an indirect 139 manner we here demonstrate an additional direct inhibitory effect of the benzene 140 metabolite 1,4-BQ on Treg functional activity providing further mechanistic insides into 141 how benzene/tobacco smoke exposure can contribute to early childhood atopic 142 dermatitis by reducing Treg activity 145 Loreen Thürmann, MSca, 146 Zuguang Gu, PhDb,c, 147 Gerrit Schüürmann, PhDd,e, 148 Gunda Herberth, PhDa, 149 Denise Hinz, PhDa,f, 150 Martin von Bergen, PhDg,h, 151 Hauke Harms, PhDi, 152 Sven Olek, PhDj, 153 Stefan Röder, PhDa, 154 Michael Borte, MDk, 155 Roland Eils, PhDb,c,l,m, 156 Irina Lehmann, PhDa, 157 Saskia Trump, PhDa M AN U Marcus Winter, MSca, 158 AC C EP TE D 144 SC 143 RI PT 136 159 Affiliations: (a) Department of Environmental Immunology, Helmholtz Centre for 160 Environmental Research – UFZ, Permoserstraße 15, 04318 Leipzig, Germany, (b) 161 Division of Theoretical Bioinformatics, German Cancer Research Center (DKFZ), Im 162 Neuenheimer Feld 267, 69120 Heidelberg, Germany, (c) Heidelberg Center for Winter et al ACCEPTED MANUSCRIPT Personalized Oncology (DKFZ-HIPO), Im Neuenheimer Feld 280, 69120 Heidelberg, 164 Germany, (d) Department of Ecological Chemistry, Helmholtz Centre for Environmental 165 Research – UFZ, Permoserstraße 15, 04318 Leipzig, Germany, (e) Technical University 166 Bergakademie Freiberg, Leipziger Straße29, 09596 Freiberg, Germany, (f) La Jolla 167 Institute for Allergy and Immunology, 9420 Athena Circle, La Jolla, CA 92037, USA, (g) 168 Department of Molecular Systems Biology, UFZ, Helmholtz-Centre for Environmental 169 Research, Permoserstraße 15, 04318 Leipzig, Germany, (h) Department of Biochemistry, 170 Faculty of Biosciences, Pharmacy and Psychology, University of Leipzig, Brüderstrasse 171 34, 04103 Leipzig, Germany, (i) Department of Environmental Microbiology, Helmholtz 172 Centre for Environmental Research, Permoserstraße 15, 04318 Leipzig, Germany, (j) 173 Epiontis GmbH, Rudower Chaussee 29, 12489 Berlin, Germany, (k) Municipal Hospital 174 “St Georg” Children’s Hospital, Delitzscher Straße 141, 04129 Leipzig, (l) Institute of 175 Pharmacy and Molecular Biotechnology (IPMB), and BioQuant Center, University of 176 Heidelberg, Im Neuenheimer Feld 267, 69120 Heidelberg, (m) Translational Lung 177 Research Center Heidelberg (TLRC), German Center for Lung Research (DZL), University 178 of Heidelberg, Germany 181 182 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 TE References Herberth G, Bauer M, Gasch M, Hinz D, Roder S, Olek S, et al Maternal and cord blood miR-223 expression associates with prenatal tobacco smoke exposure and low regulatory T-cell numbers J Allergy Clin Immunol 2014; 133:543-50 Casale T, Sacco C, Ricci S, Loreti B, Pacchiarotti A, Cupelli V, et al Workers exposed to low levels of benzene present in urban air: Assessment of peripheral blood count variations Chemosphere 2016; 152:392-8 Lan Q, Zhang L, Li G, Vermeulen R, Weinberg RS, Dosemeci M, et al Hematotoxicity in Workers Exposed to Low Levels of Benzene Science 2004; 306:1774-6 Snyder R, Hedli CC An overview of benzene metabolism Environmental Health Perspectives 1996; 104:1165-71 Kirkeleit J, Riise T, Gjertsen BT, Moen BE, Bratveit M, Bruserud O Effects of Benzene on Human Hematopoiesis The Open Hematology Journal 2008; 2:87102 Yeowell-O'Connell K, Rothman N, Waidyanatha S, Smith MT, Hayes RB, Li G, et al Protein adducts of 1,4-benzoquinone and benzene oxide among smokers and AC C 180 EP 179 D M AN U SC RI PT 163 Winter et al ACCEPTED MANUSCRIPT nonsmokers exposed to benzene in China Cancer Epidemiol Biomarkers Prev 2001; 10:831-8 Ashley CW, Baecher-Allan C Cutting Edge: Responder T cells regulate human DR+ effector regulatory T cell activity via granzyme B J Immunol 2009; 183:4843-7 Hanifin JM, Rajka G Diagnostic features of atopic eczema Acta Derm Venereol 1980; 92:44-7 Hinz D, Bauer M, Roder S, Olek S, Huehn J, Sack U, et al Cord blood Tregs with stable FOXP3 expression are influenced by prenatal environment and associated with atopic dermatitis at the age of one year Allergy 2012; 67:380-9 RI PT 198 199 200 201 202 203 204 205 206 207 208 209 Key words: atopic dermatitis, benzene, Treg, LINA study, IL-10, Granzyme B 211 Acknowledgements 212 We cordially thank the participants of the LINA study and Beate Fink, Anne Hain, 213 Susanne Arnold and Melanie Nowak for their excellent technical assistance and 214 fieldwork 215 Statement of contribution 216 Each named author has substantially contributed to this letter Marcus Winter 217 performed all in vitro assays including Treg expansion and exposure, FACS assays, and 218 qPCR analysis Irina Lehmann, Gunda Herberth, Stefan Röder and Michael Borte were 219 involved in the development of the LINA study design and the field work Gunda 220 Herberth was responsible for cytokine protein analyses in LINA Martin von Bergen was 221 responsible for measurements of indoor benzene concentrations Marcus Winter 222 measured granzyme B in one-year-old child of the LINA cohort Loreen Thürmann 223 performed DNA methylation analysis Zuguang Gu evaluated the publically available 224 RNASeq data Saskia Trump, and Irina Lehmann contributed to the statistical analysis 225 Saskia Trump, Gerrit Schüürmann, Hauke Harms and Roland Eils contributed to the 226 discussion of the data Marcus Winter, Saskia Trump, and Irina Lehmann wrote the 227 manuscript All authors were involved in the revision of the final text AC C EP TE D M AN U SC 210 228 Winter et al ACCEPTED MANUSCRIPT Figure Legend 230 231 232 233 234 235 Figure Effects of 1,4-benzoquinone (1,4-BQ) or benzene exposure (A) CFSE assays reveal a significant impairment in Treg suppressive function by 50 nM 1,4-BQ treatment for 24 h (two-way-ANOVA, p=0.002, n=5) (B) IL-13 and granzyme B concentrations were elevated in the supernatants of these CSFE assays (signed rank sum test, p-value: ** ≤ 0.01, *** ≤ 0.001, n=5) (C) Association of benzene exposure to cytokine secretion and Treg number in LINA AC C EP TE D M AN U SC RI PT 229 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 0.000 OR52E1 polymorphic_pseudogene lincRNA protein_coding antisense lincRNA lincRNA sense_intronic lincRNA lincRNA lincRNA lincRNA lincRNA antisense lincRNA antisense antisense processed_transcript lincRNA lincRNA antisense lincRNA sense_overlapping lincRNA antisense lincRNA lincRNA lincRNA processed_transcript antisense antisense antisense antisense lincRNA antisense lincRNA lincRNA lincRNA lincRNA antisense antisense lincRNA protein_coding protein_coding lincRNA lincRNA lincRNA sense_intronic lincRNA protein_coding protein_coding protein_coding lincRNA protein_coding antisense lincRNA antisense lincRNA antisense lincRNA lincRNA antisense processed_transcript antisense sense_intronic antisense processed_transcript lincRNA lincRNA lincRNA antisense lincRNA antisense lincRNA antisense lincRNA RI PT RP11-566J3.4 RP11-201K10.3 RP11-78I14.1 AP000255.6 RP11-532L16.3 RP3-508I15.20 RP11-219C20.3 RP11-488L1.1 RP11-302L19.3 AP000569.9 CMP21-97G8.1 RP11-559M23.1 RP11-165A20.3 RP11-416N2.4 RP11-350G8.9 RP11-25K21.6 RP11-367H1.1 KB-1466C5.1 LLfos-48D6.1 AC144652.1 AC079610.1 RP11-128P17.4 RP11-9N20.3 RP11-236B18.5 RP11-115H18.1 RP5-973M2.2 RP6-42F4.1 RP11-350J20.12 RP11-799M12.2 RP3-402G11.28 GS1-293C5.1 XXbac-B444P24.14 RP11-820I16.4 RP11-458F8.4 RP11-525A16.4 CITF22-92A6.1 RP5-1068E13.7 RP11-290D2.6 RP11-449P15.2 RP11-406H21.2 RP4-583P15.15 MRPL30 RP11-127B20.2 RP11-104L21.3 RP11-108L7.15 DGCR10 RP11-1057B6.1 RP11-307N16.6 ANKRD18A CTB-96E2.2 AC108004.2 SNURF RP11-434H6.6 RP11-11N7.4 RP3-510H16.3 RP11-368I23.4 RP11-20I20.4 RP11-335L23.4 RP11-778D9.13 RP11-5C23.2 RP11-212P7.3 RP11-98C1.1 RP11-339B21.10 RP3-402G11.25 CTD-3148I10.15 RP11-255C15.4 CITF22-1A6.3 RP11-523G9.3 RP11-717A5.2 GS1-304P7.3 AP000692.10 AC006946.16 RP4-549L20.3 RP11-107N15.1 SC 0.000 0.000 0.000 0.000 0.000 4.514 2.081 0.000 0.000 0.000 0.000 0.000 0.000 0.288 0.000 0.000 0.000 0.000 0.000 1.780 0.000 0.000 0.000 0.000 0.000 0.000 1.727 0.000 1.486 0.000 0.000 0.000 0.000 0.720 0.000 0.000 1.610 0.000 0.000 0.000 0.265 0.000 2.484 0.000 0.000 0.000 0.922 0.000 0.000 0.000 0.000 2.934 0.000 0.000 0.000 0.000 0.000 0.000 1.332 0.000 0.000 0.000 0.000 0.000 0.000 2.682 0.176 0.000 0.000 0.000 0.000 0.000 0.000 0.000 M AN U D TE EP 0.000 0.000 0.000 0.000 0.000 0.000 10.302 0.000 0.000 0.000 0.797 0.000 0.000 0.000 0.329 0.000 0.000 0.293 0.000 0.376 1.451 0.000 0.000 0.000 0.000 0.000 0.000 2.155 0.000 0.000 1.473 0.000 0.000 0.000 0.890 0.000 0.000 0.919 0.000 0.070 0.943 0.151 0.000 1.418 0.778 0.482 0.000 0.526 0.000 0.092 0.000 0.000 2.511 2.085 0.440 0.612 0.000 0.000 0.000 0.380 1.388 0.000 0.000 0.000 1.041 0.498 2.550 0.201 0.000 0.000 0.000 0.000 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an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 0.000 AP001437.1 antisense lincRNA antisense processed_transcript lincRNA sense_intronic protein_coding lincRNA antisense antisense antisense pseudogene lincRNA sense_intronic lincRNA pseudogene antisense lincRNA antisense lincRNA lincRNA lincRNA antisense lincRNA antisense pseudogene antisense antisense pseudogene lincRNA lincRNA protein_coding antisense antisense lincRNA lincRNA protein_coding antisense protein_coding lincRNA antisense lincRNA protein_coding antisense lincRNA lincRNA sense_intronic lincRNA miRNA antisense lincRNA protein_coding lincRNA protein_coding lincRNA lincRNA processed_transcript antisense lincRNA sense_intronic protein_coding lincRNA antisense lincRNA lincRNA sense_intronic sense_intronic antisense lincRNA antisense pseudogene lincRNA antisense lincRNA lincRNA RI PT RP13-131K19.7 AC000068.10 RP5-998N21.10 RP5-1039K5.18 AC002059.10 CTC-432M15.3 LLNLR-246C6.1 RP11-644N4.1 RP5-1180E21.5 RP11-513M16.8 OR5BQ1P RP11-1246C19.1 RP11-370A5.2 RP11-713D19.1 OR2A13P CTB-119C2.1 RP11-1263C18.1 RP11-455J20.3 CTA-217C2.2 KB-7G2.8 RP11-434P11.2 RP11-83A24.2 RP11-399K21.13 LL09NC01-251B2.3 OR7E39P RP3-402G11.26 RP1-100J12.1 OR5BP1P RP11-177J6.1 RP11-394I13.3 RP11-986E7.7 RP11-531F16.4 RP11-18I14.11 RP11-131L23.2 RP11-353K11.1 HOXC4 RP11-338K13.1 RP11-761B3.1 RP11-212P7.2 AP000254.8 CTA-384D8.34 ZBTB8B RP11-474G23.2 RP11-339B21.14 RP5-1013A22.5 RP11-888D10.4 CTA-268H5.14 AC108004.5 RP6-109B7.5 CTC-297N7.8 KRBOX1 RP11-445N20.3 RP11-1084J3.4 AP000350.5 RP11-38M8.1 CTB-13L3.1 AC000068.9 RP11-314B1.2 RP11-493E12.2 RP11-944L7.5 RP11-440D17.4 RP11-527J8.1 RP11-58E21.7 RP11-496H1.2 AC004410.3 DGCR11 RP11-425A6.5 RBAKDN RP5-1136G13.2 AC018755.2 RP11-138A9.2 RP11-22N19.2 RP11-621L6.3 RP1-90G24.11 SC 0.000 0.000 0.000 0.000 0.866 0.000 0.000 0.000 0.000 5.295 0.000 0.000 1.550 0.000 0.000 1.461 0.000 0.000 1.746 0.000 0.778 0.935 0.000 0.000 0.000 0.000 0.649 0.000 1.279 0.000 0.000 1.773 0.000 0.000 2.986 0.000 0.000 0.000 0.644 8.737 0.000 0.032 0.000 3.432 0.000 1.235 0.000 0.000 0.000 1.754 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.522 0.000 0.824 1.320 0.000 0.000 0.000 1.119 0.000 0.000 0.752 0.000 0.517 0.000 0.000 0.000 M AN U D TE EP 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 8.201 0.000 0.078 0.000 0.000 0.000 2.502 0.000 1.576 6.975 0.000 0.000 0.859 0.000 0.000 0.000 0.663 0.000 0.000 0.000 0.000 0.000 3.034 0.000 0.000 4.543 0.000 0.000 0.000 0.468 13.596 0.000 0.000 0.000 1.958 0.000 2.819 0.000 0.000 0.000 3.135 0.000 0.000 0.000 0.000 0.196 0.000 0.000 0.000 0.596 0.000 0.000 3.765 0.000 0.000 0.000 0.532 0.000 0.000 0.429 0.000 1.253 0.000 0.000 0.000 AC C ENSG00000273210.1 ENSG00000273211.1 ENSG00000273212.1 ENSG00000273213.1 ENSG00000273214.1 ENSG00000273216.1 ENSG00000273217.1 ENSG00000273218.1 ENSG00000273219.1 ENSG00000273221.1 ENSG00000273226.1 ENSG00000273228.1 ENSG00000273230.1 ENSG00000273232.1 ENSG00000273233.1 ENSG00000273234.1 ENSG00000273237.1 ENSG00000273238.1 ENSG00000273240.1 ENSG00000273243.1 ENSG00000273244.1 ENSG00000273245.1 ENSG00000273247.1 ENSG00000273248.1 ENSG00000273249.1 ENSG00000273252.1 ENSG00000273253.1 ENSG00000273254.1 ENSG00000273255.1 ENSG00000273257.1 ENSG00000273258.1 ENSG00000273259.1 ENSG00000273261.1 ENSG00000273262.1 ENSG00000273264.1 ENSG00000273265.1 ENSG00000273266.1 ENSG00000273267.1 ENSG00000273269.1 ENSG00000273270.1 ENSG00000273271.1 ENSG00000273272.1 ENSG00000273274.1 ENSG00000273275.1 ENSG00000273281.1 ENSG00000273283.1 ENSG00000273284.1 ENSG00000273287.1 ENSG00000273288.1 ENSG00000273289.1 ENSG00000273290.1 ENSG00000273291.1 ENSG00000273293.1 ENSG00000273294.1 ENSG00000273295.1 ENSG00000273297.1 ENSG00000273299.1 ENSG00000273300.1 ENSG00000273301.1 ENSG00000273302.1 ENSG00000273303.1 ENSG00000273305.1 ENSG00000273306.1 ENSG00000273307.1 ENSG00000273308.1 ENSG00000273309.1 ENSG00000273311.1 ENSG00000273312.1 ENSG00000273313.1 ENSG00000273314.1 ENSG00000273318.1 ENSG00000273319.1 ENSG00000273320.1 ENSG00000273321.1 ENSG00000273325.1 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 0.000 OR6L2P pseudogene processed_transcript lincRNA pseudogene protein_coding 3prime_overlapping_ncrna lincRNA pseudogene antisense pseudogene lincRNA lincRNA sense_intronic lincRNA processed_transcript lincRNA sense_intronic sense_intronic sense_intronic lincRNA lincRNA antisense antisense lincRNA sense_intronic lincRNA lincRNA antisense sense_intronic antisense lincRNA lincRNA antisense lincRNA lincRNA pseudogene lincRNA antisense lincRNA antisense antisense antisense antisense antisense protein_coding lincRNA protein_coding lincRNA antisense lincRNA lincRNA antisense pseudogene lincRNA antisense lincRNA lincRNA antisense 3prime_overlapping_ncrna pseudogene lincRNA lincRNA processed_transcript processed_transcript pseudogene lincRNA protein_coding lincRNA lincRNA pseudogene antisense antisense lincRNA lincRNA antisense RI PT RP11-141M3.5 RP11-448A19.1 CYP4F36P TM4SF19 XXbac-BPG300A18.13 RP11-61L19.2 OR7M1P RP11-386I14.4 MICE RP5-899E9.1 KB-1440D3.14 XXbac-B444P24.13 PAXIP1-AS1 CTD-2410N18.4 RP11-535A5.1 RP4-539M6.20 RP11-61L19.3 CTA-268H5.12 RP11-672L10.6 RP11-804H8.6 RP11-210G22.1 RP11-378A13.2 KB-67B5.17 RP11-285G1.14 RP11-466F5.10 CTA-989H11.1 RP5-827C21.6 RP11-376P6.3 RP11-700J17.1 RP11-268E23.2 RP11-479O17.10 RP5-1074L1.4 RP11-383I23.2 RP11-803P9.1 OR2Q1P RP11-305L7.7 RP5-1065J22.8 RP5-1098D14.1 RP3-412A9.16 RP11-401O9.4 RP13-514E23.2 RP11-634H22.1 RP11-255E6.6 FLJ00388 RP11-326I19.3 RP11-474G23.1 RP11-408A13.3 RP5-855D21.2 RP11-329B9.3 RP11-114O18.1 RP11-757A13.1 OR5B15P RP11-480C22.1 RP11-96C23.15 RP11-702B10.2 RP4-597N16.4 RP4-751H13.7 CTD-2540B15.13 OR13I1P CTA-223H9.9 RP4-539M6.22 RP5-1165K10.2 RP1-170O19.22 OR8S21P RP11-434H6.7 ZNF8 AC006946.17 RP11-54O7.18 RP5-1147A1.2 RP11-1399P15.1 AC004067.5 RP11-166O4.6 RP11-218F10.3 RP11-76P2.4 SC 0.000 0.000 0.000 0.000 0.000 1.410 0.775 3.952 0.000 0.000 0.000 0.655 0.732 0.000 0.000 0.000 3.850 0.000 0.000 0.401 0.000 0.000 0.000 0.000 0.567 0.000 0.660 0.000 1.873 0.000 0.000 0.725 0.000 1.233 0.000 0.100 0.286 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 4.932 0.000 0.546 0.000 0.000 4.589 2.360 0.000 0.000 0.000 M AN U D TE EP 0.000 0.000 0.233 0.000 0.000 0.000 0.000 1.769 0.000 0.204 0.000 0.534 1.120 2.611 0.000 0.000 0.000 1.255 0.734 0.488 1.945 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.459 2.138 0.000 0.000 0.910 0.000 1.055 0.000 0.171 0.245 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 3.016 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.115 0.000 0.000 0.000 0.000 0.000 0.000 0.000 2.814 0.000 0.000 0.000 1.411 6.110 2.357 0.705 0.254 0.560 AC C ENSG00000273327.1 ENSG00000273328.1 ENSG00000273329.1 ENSG00000273330.1 ENSG00000273331.1 ENSG00000273333.1 ENSG00000273335.1 ENSG00000273336.1 ENSG00000273338.1 ENSG00000273340.1 ENSG00000273341.1 ENSG00000273342.1 ENSG00000273343.1 ENSG00000273344.1 ENSG00000273345.1 ENSG00000273348.1 ENSG00000273350.1 ENSG00000273352.1 ENSG00000273353.1 ENSG00000273355.1 ENSG00000273356.1 ENSG00000273360.1 ENSG00000273361.1 ENSG00000273362.1 ENSG00000273363.1 ENSG00000273365.1 ENSG00000273366.1 ENSG00000273367.1 ENSG00000273368.1 ENSG00000273369.1 ENSG00000273370.1 ENSG00000273372.1 ENSG00000273373.1 ENSG00000273374.1 ENSG00000273375.1 ENSG00000273377.1 ENSG00000273381.1 ENSG00000273382.1 ENSG00000273384.1 ENSG00000273387.1 ENSG00000273388.1 ENSG00000273389.1 ENSG00000273391.1 ENSG00000273394.1 ENSG00000273395.1 ENSG00000273396.1 ENSG00000273398.1 ENSG00000273399.1 ENSG00000273402.1 ENSG00000273403.1 ENSG00000273406.1 ENSG00000273407.1 ENSG00000273408.1 ENSG00000273409.1 ENSG00000273413.1 ENSG00000273415.1 ENSG00000273416.1 ENSG00000273419.1 ENSG00000273420.1 ENSG00000273423.1 ENSG00000273424.1 ENSG00000273428.1 ENSG00000273432.1 ENSG00000273433.1 ENSG00000273434.1 ENSG00000273437.1 ENSG00000273439.1 ENSG00000273442.1 ENSG00000273443.1 ENSG00000273444.1 ENSG00000273445.1 ENSG00000273447.1 ENSG00000273448.1 ENSG00000273449.1 ENSG00000273450.1 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT 0.411 RP4-569M23.4 sense_intronic protein_coding lincRNA antisense lincRNA lincRNA pseudogene antisense antisense processed_transcript lincRNA lincRNA antisense lincRNA lincRNA antisense lincRNA antisense pseudogene antisense antisense lincRNA lincRNA antisense lincRNA lincRNA protein_coding protein_coding protein_coding protein_coding protein_coding protein_coding protein_coding protein_coding protein_coding protein_coding protein_coding pseudogene antisense protein_coding protein_coding protein_coding protein_coding protein_coding protein_coding protein_coding rRNA pseudogene lincRNA sense_intronic pseudogene pseudogene antisense pseudogene pseudogene pseudogene pseudogene lincRNA lincRNA pseudogene antisense lincRNA pseudogene lincRNA pseudogene miRNA miRNA miRNA miRNA misc_RNA miRNA miRNA processed_transcript RI PT AC005358.1 RP11-797D24.3 RP11-305O4.3 RP11-686O6.2 RP11-398A8.4 AL138834.1 RP11-313P22.1 RP11-548H3.1 RP13-1039J1.4 RP11-102N12.3 LL09NC01-139C3.1 RP11-295P9.12 RP11-108L7.14 RP11-196O16.1 RP11-465N4.5 RP11-126K1.9 RP4-671G15.2 OR6R2P RP11-225H22.7 RP11-731C17.2 RP4-621B10.8 RP11-114I8.4 RP11-180C16.1 AP000230.1 RP11-80H18.4 CD99 VAMP7 IL9R PPP2R3B SPRY3 DHRSX ASMTL SLC25A6 GTPBP6 P2RY8 PLCXD1 WASH6P WASIR1 IL3RA SHOX ASMT AKAP17A CSF2RA CRLF2 ZBED1 RNA5SP498 TRPC6P RP13-297E16.4 DHRSX-IT1 CD99P1 RPL14P5 LINC00685 DDX11L16 TCEB1P24 LL0YNC03-29C1.1 KRT18P53 LINC00102 RP13-297E16.5 FABP5P13 ASMTL-AS1 LINC00106 DPH3P2 RP11-309M23.1 AMDP1 BX649553.1 BX649553.2 BX649553.3 BX649553.4 RN7SL355P MIR3690 AL732314.1 AJ271736.10 SC 0.000 0.000 0.941 1.227 0.000 0.000 1.939 1.632 0.000 0.000 0.000 1.197 0.000 0.389 1.257 0.000 0.000 0.000 0.000 0.245 0.000 1.832 0.111 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 M AN U D TE EP 0.234 0.000 0.427 0.000 1.401 0.000 0.000 0.000 1.552 0.000 0.000 0.000 1.366 0.000 0.333 1.435 0.000 0.000 0.000 0.359 1.120 0.000 0.523 0.189 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 AC C ENSG00000273451.1 ENSG00000273452.1 ENSG00000273454.1 ENSG00000273455.1 ENSG00000273456.1 ENSG00000273461.1 ENSG00000273463.1 ENSG00000273464.1 ENSG00000273466.1 ENSG00000273471.1 ENSG00000273472.1 ENSG00000273473.1 ENSG00000273474.1 ENSG00000273476.1 ENSG00000273477.1 ENSG00000273478.1 ENSG00000273481.1 ENSG00000273483.1 ENSG00000273484.1 ENSG00000273485.1 ENSG00000273486.1 ENSG00000273487.1 ENSG00000273488.1 ENSG00000273489.1 ENSG00000273492.1 ENSG00000273493.1 ENSGR0000002586.13 ENSGR0000124333.10 ENSGR0000124334.12 ENSGR0000167393.12 ENSGR0000168939.6 ENSGR0000169084.8 ENSGR0000169093.10 ENSGR0000169100.8 ENSGR0000178605.8 ENSGR0000182162.5 ENSGR0000182378.8 ENSGR0000182484.10 ENSGR0000185203.7 ENSGR0000185291.6 ENSGR0000185960.8 ENSGR0000196433.7 ENSGR0000197976.6 ENSGR0000198223.10 ENSGR0000205755.6 ENSGR0000214717.5 ENSGR0000223274.1 ENSGR0000223484.2 ENSGR0000223511.1 ENSGR0000223571.1 ENSGR0000223773.2 ENSGR0000225661.2 ENSGR0000226179.1 ENSGR0000227159.3 ENSGR0000228410.1 ENSGR0000228572.2 ENSGR0000229232.1 ENSGR0000230542.1 ENSGR0000234622.1 ENSGR0000234958.1 ENSGR0000236017.3 ENSGR0000236871.2 ENSGR0000237040.1 ENSGR0000237531.1 ENSGR0000237801.1 ENSGR0000263835.1 ENSGR0000263980.1 ENSGR0000264510.1 ENSGR0000264819.1 ENSGR0000265350.1 ENSGR0000265658.1 ENSGR0000266731.1 ENSGR0000270726.1 Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT Table E2 Interleukin concentrations in supernatants of CD3/CD28 & IL-2 stimulated Treg exposed to 50 nM 1,4-BQ for 24h DMSO [pg/ml] median (IQR), n=5 1,4-BQ [pg/ml] median (IQR), n=5 p-value* IL-10 206.2 (25.6-2461.7) 153.9 (21.7-2187.0) 0.005 Granzyme A 0.0 (0-71.7) 0.0 (0-69.9) 1.000 Granzyme B 1456.6 (642.4-7850.9) 1209.7 (639.3-7400.9) 0.063 AC C EP TE D M AN U SC *signed rank sum test RI PT Cytokine Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT Table E3 Interleukin concentrations in supernatants of 1,4-benzoquinone exposed Treg after five days of coculture with CFDA-SE labeled autologous CD4+CD25- Tconv Granzyme A Granzyme B DMSO [pg/ml] median (IQR), n=5 1,4-BQ [pg/ml] median (IQR), n=5 4:1 15.6 (8.6-28.0) 26.9 (13.4-48.4) 8:1 30.7 (18.8-42.7) 43.4 (24.9-106.2) 16:1 40.6 (26.9-52.9) 55.2 (35.4-171.4) 4:1 13.1 (8.6-24.8) 11.1 (7.3-33.7) 8:1 32.9 (28.3-38.6) 30.0 (26.5-41.8) 16:1 19.7 (17.3-27.7) 27.9 (13.6-41.2) 4:1 34.6 (21.2-254.6) 283.5 (50.2-607.4) 8:1 149.2 (57.5-170.4) 292.1 (234.5-386.9) 16:1 191.5 (66.6-265.7) 301.5 (263.5-549.4) AC C EP TE D M AN U *two-way ANOVA p-value* 0.009 RI PT IL-13 Tconv:Treg SC Cytokine Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn - 0.004 C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT Table E4 Granzyme B concentrations in supernatants of CD3/CD28 & IL-2 stimulated and non-exposed Treg or Tconv after 24h Cytokine Tconv [pg/ml] median (IQR) 1456.6 (99.1-9071.9) 37001.0 (11856.8-53762.4) AC C EP TE D M AN U SC RI PT Granzyme B Treg [pg/ml] median (IQR) Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT Table E5 Characteristics of the analysed subcohorts for which Treg information was available for (A) mothers and (B) children compared to the entire LINA cohort (A) 305 (48.5) 314 (49.9) 0.768 332 (52.8) 296 (47.1) 0.876 29 (4.6) 191(30.4) 408 (64.9) 0.985 511 (81.2) 94 (14.9) 0.898 389 (61.8) 215 (34.2) 0.844 166 (26.4) 184 (29.3) 186 (29.6) 93 (14.8) 0.983 534 (84.9) 43 (6.8) (0.6) 47 (7.5) 0.999 AC C EP TE D M AN U Maternal age at birth 30 years 312 (51.6) a Maternal history of atopy negative 322 (53.2) positive 282 (46.6) b Maternal education low 29 (4.8) intermediate 182 (30.1) high 393 (65.0) Pet keeping (cat) no 490 (81.0) yes 92 (15.2) Siblings no 371 (61.3) yes 210 (34.7) Season of birth spring 154 (25.5) summer 177 (29.3) fall 183 (30.2) winter 91 (15.0) Tobacco smoke exposure during pregnancy never 512 (84.6) occasionally 42 (6.9) once per week (0.7) daily 46 (7.6) P value* RI PT Analysed subcohort Entire LINA cohort Mothers n (%), N = 605 n (%), N = 629 SC Parameters * P value from chi squared test for cross relationship, subgroup against total cohort a History of atopy is defined as: occurrence of asthma, atopic dermatitis, food allergies or hay fever b low, yrs or less of schooling “Hauptschulabschluss”; intermediate, 10 yrs of schooling “Mittlere Reife”; high, 12 yrs of schooling or more “(Fach-)hochschulreife” Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT (B) Analysed subcohort Entire LINA cohort Children n (%), N = 500 n (%), N = 629 266 (53.2) 234 (46.8) 330 (52.5) 299 (47.5) 0.806 163 (32.6) 242 (48.4) 94 (18.8) 211 (33.5) 296 (47.1) 121 (19.2) 0.901 18 (2.9) 145 (23.1) 466 (74.1) 0.587 417 (83.4) 82 (16.2) 511 (81.2) 94 (14.9) 0.686 434 (86.8) 30 (6.0) (0.6) 32 (6.4) 534 (84.9) 43 (6.8) (0.6) 47 (7.5) 0.830 471 (94.2) 27 (5.2) 568 (90.3) 37 (5.9) 0.624 302 (60.4) 182 (36.4) 389 (61.8) 215 (34.2) 0.494 131 (26.2) 146 (29.2) 151 (30.2) 72 (14.4) 166 (26.4) 184 (29.3) 186 (29.6) 93 (14.8) 0.995 10 (2.0) 122 (24.4) 367 (73.4) D 0.948 RI PT 25 389 214 TE EP AC C P value* 18 310 171 M AN U Gestational week 33-36 weeks 37-40 weeks 41-43 weeks Gender of the child male female Parental history of atopya negative single positive double positive Parental educationb low intermediate high Pet keeping (cat) no yes Prenatal tobacco smoke exposure never occasionally once per week daily Postnatal tobacco smoke exposure no yes Siblings no yes Season of birth spring summer fall winter SC Parameters * P value from chi squared test for cross relationship, subgroup against total cohort a History of atopy is defined as: occurrence of asthma, atopic dermatitis, food allergies or hay fever b low, yrs or less of schooling “Hauptschulabschluss”; intermediate, 10 yrs of schooling “Mittlere Reife”; high, 12 yrs of schooling or more “(Fach-)hochschulreife” Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT Table E6 Characteristics of the children from the analysed subcohort for which Treg information was available compared to the entire LINA cohort with respect to the development of atopic dermatitis Analysed subcohort Entire LINA cohort Children n (%), N = 500 n (%), N = 629 Atopic dermatitis (AD) up to the age of six noa 308 (61.7) 378 (62.4) yesb 191 (38.3) 228 (37.6) thereof … …physician-diagnosed AD up to the age of six no 308 (74.4) 378 (75.4) yes 106 (25.6) 123 (24.6) showing… …symptoms according to Hanifin & Rajka no 308 (63.8) 378(64.4) yes 175 (36.2) 209 (35.6) P value* a 0.873 0.772 0.882 M AN U SC RI PT Parameters children without AD symptoms or a physician diagnosis of AD showing symptoms of AD and/or where diagnosed with AD by a physician AC C EP TE D b children Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an ACCEPTED MANUSCRIPT O O OH benzene RPKM < O CYP2E1 S NH via GST O H3 C HO CYP O O HO O O S-phenylmercapturic acid benzene oxide E,E-muconic acid E,E-muconaldehyde oxepin RPKM = 1,5 nonenzymatic rearrangement RI PT epoxide hydrolase OH OH O OH benzene dihydrodiol phenol RPKM < RPKM < OH HO catechol hydroquinone AC C EP TE D 1,4-benzoquinone M AN U MPO O OH OH NQO1 benzene diol epoxide SC RPKM = 3,4 OH dihydrodiol dehydrogenase CYP2E1 O OH CYP Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn NQO1 MPO O O 1,2-benzoquinone C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an FACS sorting CD4+CD25+CD45RA+ in vitro expansion SC CD25+ enrichment M AN U plate cells/well beads:cell U/ml IL-2 BD FACS Aria SORP - IZKF Leipzig 1st 2nd 96 24 104 5x105 1:1 4:1 300 300 cryopreservation - liquid nitrogen - after weeks expansion & 3d resting (w/o beads) - FoxP3-TSDR methylation - Treg suppressive activity - FoxP3 protein expression TE D - restimulation on d4 & d11 24 well plate 106 cells/well beads:cell = 1:1 300 U/ml IL-2 EP in vitro exposure AC C PBMC isolation RI PT ACCEPTED MANUSCRIPT Treg + - FoxP3-TSDR methylation 50 nM - Treg suppressive activity 24 h - FoxP3 protein expression - cytokine detection (CBA) Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an 92.9 93.5 500 nM 1,4-BQ SC 50 nM 1,4-BQ DMSO 91.4 M AN U Count A RI PT ACCEPTED MANUSCRIPT µM 1,4-BQ 11.1 FVD 450 TE AC C EP 50 nM 1,4-BQ 500 nM 1,4-BQ µM 1,4-BQ vitality [% DMSO of gated lymphocytes] median (IQR) 97.6 (96.2 – 99.0), n = 96.7 (95.7 – 97.7), n = 29.6 (20.8 – 38.3), n = D B Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an DMSO 50 nM 1,4-BQ 97.4 0.58 0.13 1.67 0.06 97.7 CD25 M AN U 0.82 SC A RI PT ACCEPTED MANUSCRIPT 1.67 FoxP3 EP % CD25+FoxP3+ median (IQR) 95.2 (92.3 – 97.5), n = 94.9 (91.8 – 98.0), n = AC C DMSO 50 nM 1,4-BQ TE D B Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn C.33.44.55.54.78.65.5.43.22.2.4 22.Tai lieu Luan 66.55.77.99 van Luan an.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.C.33.44.55.54.78.655.43.22.2.4.55.22 Do an.Tai lieu Luan van Luan an Do an.Tai lieu Luan van Luan an Do an Stt.010.Mssv.BKD002ac.email.ninhd 77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77.77.99.44.45.67.22.55.77.C.37.99.44.45.67.22.55.77t@edu.gmail.com.vn.bkc19134.hmu.edu.vn.Stt.010.Mssv.BKD002ac.email.ninhddtt@edu.gmail.com.vn.bkc19134.hmu.edu.vn