The benzene metabolite 1,4-benzoquinone reduces regulatory T-cell function: A potential mechanism for tobacco smoke–associated atopic dermatitis
Authors: Marcus Winter, Loreen Thürmann, Zuguang Gu, Gerrit Schüürmann, Gunda Herberth, Denise Hinz, Martin von Bergen, Hauke Harms, Sven Olek, Stefan Röder, Michael Borte, Roland Eils, Irina Lehmann, Saskia Trump
Journal: Journal of Allergy and Clinical Immunology (2017)

Exposure to environmental chemicals can contribute to the development or exacerbation of atopic dermatitis (AD). Benzene is one of the chemicals previously been implicated in the etiology of AD.1Herberth 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: 543-550Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar Albeit a ubiquitous environmental chemical, nonoccupational exposure to benzene mainly results from active or passive tobacco smoke exposure in homes or public places. Exposure to benzene has been shown to exert diverse effects on blood cells, in particular on lymphocytes. Although chronic exposure to high levels of benzene may cause leukemia and probably other lymphohematopoietic cancers, already low levels of benzene have been described to decrease the number of peripheral lymphocytes2Casale 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: 392-398Crossref PubMed Scopus (30) Google Scholar in humans including circulating CD4+ T cells, CD4+/CD8+ ratios, B cells, neutrophils, and platelets.3Lan Q. Zhang L. Li G. Vermeulen R. Weinberg R.S. Dosemeci M. et al.Hematotoxicity in workers exposed to low levels of benzene.Science. 2004; 306: 1774-1776Crossref PubMed Scopus (507) Google Scholar Although benzene itself is not regarded as toxic, its metabolization products are believed to be the main source of benzene toxicity. Benzene degradation in humans preferentially occurs via the formation of phenol from the initial oxidation product benzene oxide.4Snyder R. Hedli C.C. An overview of benzene metabolism.Environ Health Perspect. 1996; 104: 1165-1171Crossref PubMed Google Scholar Phenol is then further degraded to the highly reactive electrophile 1,4-benzoquinone (1,4-BQ; see Fig E1 in this article's Online Repository at www.jacionline.org), which can readily react with cellular nucleophiles including proteins or DNA. There is a nonlinear relationship between benzene exposure and metabolite formation, causing a favored production of reactive quinones in case of low-dose benzene exposure (<1 ppm).5Kirkeleit J. Riise T. Gjertsen B.T. Moen B.E. Bratveit M. Bruserud O. Effects of benzene on human hematopoiesis.Open Hematol J. 2008; 2: 87-102Crossref Google Scholar We have previously reported that prenatal benzene exposure is associated with decreased cord blood regulatory T (Treg) cell numbers and a subsequent increased risk for the development of AD in early childhood.1Herberth 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: 543-550Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar This reduction in immunocompetent cells is likely a contributing factor to the increased risk of AD described in relation to benzene/environmental tobacco smoke exposure. To further elucidate the processes underlying the benzene-associated Treg-cell deficiency, we used an in vitro model based on expanded human Treg cells (CD4+CD25hiCD45RA+; for details see Fig E2 in this article's Online Repository at www.jacionline.org) derived from healthy blood donors. As publicly available RNASeq data revealed that neither resting nor activated Treg cells express the necessary enzymes for the degradation of benzene via phenol (see Table E1 in this article's Online Repository at www.jacionline.org), we directly exposed in vitro expanded Treg cells to the benzene metabolite 1,4-BQ. Although Treg-cell exposure (independent donors, n = 5) to 50 nM 1,4-BQ for 24 hours, corresponding to concentrations previously observed in smokers,6Yeowell-O'Connell K. Rothman N. Waidyanatha S. Smith M.T. Hayes R.B. Li G. et al.Protein adducts of 1,4-benzoquinone and benzene oxide among smokers and nonsmokers exposed to benzene in China.Cancer Epidemiol Biomarkers Prev. 2001; 10: 831-838PubMed Google Scholar had no effect on FOXP3 expression as monitored by fluorescence-activated cell sorting (see Fig E4 in this article's Online Repository at www.jacionline.org), it led to a significant decrease in IL-10 production (see Table E2 in this article's Online Repository at www.jacionline.org; signed rank sum test, P = .005) determined by cytometric bead array assays in the cell culture supernatants (cytometric bead array assays performed at least in triplicates; see this article's Online Repository at www.jacionline.org). We hypothesize that this decrease in IL-10 production, a key anti-inflammatory cytokine critical for efficient Treg-cell function, contributed to the impairment in Treg-cell function subsequently observed in in vitro suppression assays (Fig 1, A). Exposed Treg cells were cocultured at different ratios with 5(6)-carboxyfluorescein N-succinimidyl ester (CSFE)–labeled responder T cells (CD4+CD25− as Tconv) of the same donor for 5 days (for details, see this article's Online Repository at www.jacionline.org). 1,4-BQ-exposed Treg cells showed a significant decrease in suppressive function compared with vehicle control–treated Treg cells of the same donor (Fig 1, A; 2-way ANOVA, P = .002). Evaluation of cytokine secretion during the duration of the suppression assay revealed a significant increase in IL-13 and granzyme B (GrzB; Fig 1, B; see Table E3 in this article's Online Repository at www.jacionline.org). Elevated GrzB concentrations were most likely related to enhanced production by responder T cells (Tconv) because they seem to be the main source of GrzB, secreting about 25-fold higher amounts of GrzB compared with Treg cells (see Table E4 in this article's Online Repository at www.jacionline.org). This increase in GrzB as an important mediator of cell death and Treg-cell activity might point to an enhanced apoptosis rate in Treg cells, which—in addition to their diminished IL-10 secretion—could account for the relative decrease in Treg-cell suppression by 1,4-BQ.7Ashley C.W. Baecher-Allan C. Cutting edge: responder T cells regulate human DR+ effector regulatory T cell activity via granzyme B.J Immunol. 2009; 183: 4843-4847Crossref PubMed Scopus (39) Google Scholar To evaluate whether these in vitro observations can be related to the in vivo situation, we additionally studied the effect of benzene exposure in our prospective mother-child cohort (LINA: Lifestyle and Environmental Factors and Their Influence on Newborns Allergy Risk; Table E5 in this article's Online Repository at www.jacionline.org). As already earlier shown for cord blood Treg cells,1Herberth 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: 543-550Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar we can provide a link between indoor benzene concentrations—a good proxy for active or passive tobacco smoke exposure—and peripheral Treg-cell numbers in mothers and 1-year-old children. Benzene exposure during pregnancy was associated with a decrease in maternal peripheral Treg-cell numbers (Fig 1, C; adjusted mean ratio [MR] = 0.89; 95% CI, 0.81-0.97, n = 561; for confounders, see this article's Online Repository at www.jacionline.org). This inverse relationship between perinatal benzene exposure, considering benzene measurements during pregnancy and 1 year after birth, and Treg-cell numbers was also observed in 1-year-old children (Fig 1, C; MR = 0.94; 95% CI, 0.88-0.99, n = 480; for confounders, see this article's Online Repository). Similar to the increase in IL-13, which we observed in our in vitro coculture assays (see Table E3), a positive association was found between indoor benzene concentrations and PHA-induced IL-13 concentrations in maternal or infantile blood samples, respectively (Fig 1, C; mothers: MR = 1.10, 95% CI, 1.01-1.20, n = 570; children: MR = 1.39, 95% CI, 1.15-1.68, n = 489). In addition, GrzB concentrations measured in PHA-stimulated whole blood samples were positively associated with benzene concentrations (see Fig E5 in this article's Online Repository at www.jacionline.org; adjusted MR = 1.20; 95% CI, 1.00-1.44; n = 264) and negatively associated with the number of Treg cells in these children (see Fig E5; adjusted MR = 0.94; 95% CI, 0.89-0.99; n = 261), further corroborating our in vitro findings. In children, the high concentrations of PHA-stimulated IL-13 were associated with an increased risk for these children to develop symptoms according to Hanifin and Rajka8Hanifin J.M. Rajka G. Diagnostic features of atopic eczema.Acta Derm Venereol. 1980; 92: 44-47Google Scholar and/or physician-diagnosed AD up to the age of 6 years (adjusted odds ratio, 1.31; 95% CI, 1.11-1.55, per ln(1 pg/mL) increase in PHA-IL-13; n = 490; see this article's Online Repository and Table E6 in this article's Online Repository at www.jacionline.org). Our results support the notion that even low levels of the reactive benzene metabolite 1,4-BQ can significantly impair the suppressive function of Treg cells by reducing their potential to secrete the anti-inflammatory cytokine IL-10. This functional impairment most likely reduces Treg-cell suppressive capacity, resulting in higher GrzB production by conventional T cells and a subsequent further loss in Treg-cell numbers presumably due to higher apoptosis rates induced by GrzB. In addition to the earlier reported induction of miR-223 under benzene exposure leading to reduced Treg-cell numbers1Herberth 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: 543-550Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 9Hinz 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: 380-389Crossref PubMed Scopus (157) Google Scholar in an indirect manner, we here demonstrate an additional direct inhibitory effect of the benzene metabolite 1,4-BQ on Treg-cell functional activity, providing further mechanistic insight into how benzene/tobacco smoke exposure can contribute to early childhood AD by reducing Treg-cell activity. We cordially thank the participants of the LINA study and Beate Fink, Anne Hain, Susanne Arnold, and Melanie Nowak for their excellent technical assistance and fieldwork. 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