immunotoxicity of organophosphate flame retardants tphp and tdcipp on murine dendritic cells in vitro

Accepted Manuscript Immunotoxicity of organophosphate flame retardants TPHP and TDCIPP on murine dendritic cells in vitro Derya Canbaz, Adrian Logiantara, Ronald van Ree, Leonie S van Rijt PII: S0045-6535(17)30338-7 DOI: 10.1016/j.chemosphere.2017.02.149 Reference: CHEM 18912 To appear in: Chemosphere Received Date: 14 November 2016 Revised Date: 01 February 2017 Accepted Date: 27 February 2017 Please cite this article as: Derya Canbaz, Adrian Logiantara, Ronald van Ree, Leonie S van Rijt, Immunotoxicity of organophosphate flame retardants TPHP and TDCIPP on murine dendritic cells in vitro, Chemosphere (2017), doi: 10.1016/j.chemosphere.2017.02.149 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 ACCEPTED MANUSCRIPT Highlights: -TPHP and TDCIPP can be immunotoxic for dendritic cells -TPHP has an immunomodulatory effect dependent on the maturation status of the dendritic cell -TDCIPP did not have an effect on “steady-state” dendritic cells but was immunosuppressive for house dust mite allergen pulsed dendritic cells -TPHP and TDCIPP induced oxidative stress in dendritic cells ACCEPTED MANUSCRIPT Immunotoxicity of organophosphate flame retardants TPHP and TDCIPP on murine dendritic cells in vitro Derya Canbaza, Adrian Logiantaraa, Ronald van Reea,b, Leonie S van Rijta aDept Center, University of Amsterdam, Meibergdreef 9, 1105AZ, The Netherlands of Experimental Immunology and bDept of Otorhinolaryngology, Academic Medical *To Immunology, Room KO-104, Academic Medical Center, Meibergdreef 9, 1105 AZ whom correspondence should be addressed at Department of Experimental 10 Amsterdam, The Netherlands Phone: 11 l.s.vanrijt@amc.uva.nl 12 Running title: Immuno-modulating effects of PFRs 13 Keywords:, dendritic cells, allergy, house dust mite, phosphorus flame retardants 14 15 16 17 18 +31-205668044, Fax: N/A, E-mail: ACCEPTED MANUSCRIPT 20 Abstract (244) 21 Organophosphate flame retardants (PFRs) are commonly used as alternatives for the 22 banned polybrominated diphenyl ethers (PBDEs) and are ubiquitously detected in indoor 23 dust PFRs can be potentially hazardous to respiratory health via the inhalation of house 24 dust Dendritic cells (DCs) are crucial in the immunological defense against pathogens in 25 the airways In respiratory allergy however, an aberrant immune response is induced 26 against innocuous proteins, like house dust mite allergens In this study, we examined 27 whether exposure to PFRs Triphenylphosphate (TPHP) and Tris(1,3-dichloroisopropyl) 28 phosphate (TDCIPP) affected activation/maturation of DCs at steady-state and during 29 exposure to house dust mite allergens (HDM) Bone marrow-derived dendritic cells 30 (BMDCs) were exposed to a concentration range of each PFR (0.1-100 µM) with or 31 without HDM in vitro to analyze the effect on the expression of major histocompatibility 32 complex class II (MHCII), co-stimulatory molecules and cytokine production 33 Concentrations of TPHP and TDCIPP of ≥50 µM were cytotoxic to BMDCs At these 34 cytotoxic concentrations, TPHP exposure induced an activated phenotype in steady- 35 state DCs, while HDM exposed DCs acquired a tolerogenic phenotype In contrast, 36 TDCIPP exposure had no effect at steady state DCs but suppressed the expression of 37 MHCII, costimulatory molecules, and the IL-6 production in HDM exposed DCs The 38 cytotoxic concentrations induced the anti-oxidant enzyme hemeoxigenase-1, which is a 39 marker for oxidative stress These results demonstrate that PFRs can be immunotoxic 40 for DCs and suggest the necessity to evaluate the effects on the immune system on a 41 cellular level during the risk assessment of these alternative flame retardants ACCEPTED MANUSCRIPT 42 Introduction 43 Flame retardants have become a major indoor pollutant during the last decades Until 44 2004, polybrominated diphenyl ethers (PBDEs) were the primary flame retardants, but 45 they have been phased out in Europe and USA because of their properties of 46 persistence and bio-accumulation as well as their potential to cause adverse health 47 effects The ban of PBDEs has resulted in an increase in the global use of 48 organophosphate flame retardants (PFRs) which are often proposed as alternatives for 49 the banned PBDEs (Van den Eede et al., 2011; van der Veen and de Boer, 2012) 50 Environmental levels of PFRs have been well investigated and concentrations of PFRs 51 such as tris(1,3-dichloroisopropyl)-phosphate (TDCIPP) and triphenylphosphate (TPHP) 52 were found to exceed that of PBDEs (van der Veen and de Boer, 2012) It has been 53 postulated that this was representing the increased usage However, besides the use of 54 TDCIPP and TPHP as additive flame retardants in polymers, resins, latexes and foams, 55 they are also used in plasticizers, lacquer, paint, lubricant and TPHP in hydraulic fluids 56 (van der Veen and de Boer, 2012) We have recently reported that in mattress dust 57 collected more than two decades ago, PFRs exceeded the levels of PBDEs around sixty 58 fold TDCIPP and TPHP were present in 100% and 91% of the analyzed dust samples, 59 respectively (Canbaz et al., 2016b) Thus, even before the ban, these “alternative” flame 60 retardants were present in greater concentrations than the brominated flame retardants 61 The concentration of TDCIPP in house dust has been correlated with concentrations of 62 its metabolite in human urine (Meeker et al., 2013) ACCEPTED MANUSCRIPT 63 Inhalation of house dust is one of the human exposure routes besides dermal contact 64 and unintended ingestion of dust (Abdallah et al., 2008) Recently, total intake of 65 chlorinated PFRs such as TDCIPP via inhalation has been estimated to exceed intake 66 via dust ingestion (Schreder et al., 2016) The respiratory mucosal immune system is 67 exposed continuously to inhaled proteins which has to result in the appropriate immune 68 response, being either tolerogenic or immunogenic The airways are covered with a 69 network of dendritic cells (DCs) that monitor inhaled air continuously for microorganisms 70 and other substances DCs are able to recognize pathogens via different pattern 71 recognition receptors They process a large variety of antigens for presentation to naïve 72 T cells In this process, they display co-stimulatory molecules and secrete pro- 73 inflammatory cytokines to interact and differentiate naïve T cells into a specific subset of 74 effector T cells (von Garnier and Nicod, 2009; Willart and Hammad, 2010) In house dust 75 mite allergy, a T helper (Th2) type cell response is induced against innocuous proteins 76 derived from the house dust mite feces (Lambrecht and Hammad, 2003, 2009) 77 Environmental co-exposures have been implicated to play an important role in skewing 78 the immune system towards (or away from) an inflammatory immune response to 79 harmless allergens Efforts are being made to elucidate how these co-factors contribute 80 to Th2-skewing (Maes et al., 2010) Respiratory allergic disorders have become very 81 common diseases in Western countries with an increasing prevalence since the 1970s 82 (Eder et al., 2006) Although house dust mite allergy has been associated with the 83 development of respiratory allergic diseases, the underlying mechanism for these 84 immune system disorders are complex and the risk factors are multiple (Eder et al., 85 2006; Beasley et al., 2015) Ubiquitous detection of diverse chemical pollutants in indoor ACCEPTED MANUSCRIPT 86 dust and increasing exposure levels have resulted in many studies aiming to elucidate 87 their possible health effects Indoor air pollutants have been recognized as risk factors 88 for allergic airway diseases such as asthma (Nielsen et al., 2007; Hulin et al., 2012; 89 Patelarou et al., 2015; Jiang et al., 2016) 90 The phosphate backbone is an important characteristic of PFRs, which they share with 91 organophosphate-based pesticides that have been associated with exacerbations in 92 allergic asthma (Proskocil et al., 2008; Hoppin et al., 2009; Hernandez et al., 2011) In 93 concordance, a recent epidemiological study in Japan has revealed an association 94 between increased concentrations of PFRs in house dust with allergic disorders such as 95 allergic asthma, allergic rhinitis and atopic dermatitis (Araki et al., 2014) Although 96 toxicological studies have revealed health threats of PFRs such as neurotoxic effects of 97 TPHP, adverse effects of TPHP and TDCIPP on reproduction, and carcinogenicity of 98 TDCIPP (van der Veen and de Boer, 2012; Bruchajzer et al., 2015), no study has 99 investigated their potential immunomodulating effects 100 Here we investigated whether exposure of DCs to PFRs affects the expression of 101 MHCII, costimulatory molecules and the production of cytokines, at steady-state and 102 during exposure to house dust mite (HDM) allergens 103 104 105 106 ACCEPTED MANUSCRIPT 107 Materials and Methods 108 Mice 6-8 week old female Balb/c mice (Harlan, Horst, The Netherlands) were housed 109 under specific pathogen-free conditions at the animal care facility of the AMC All 110 experiments were approved by the Academic Medical Center Animal Ethics Committee, 111 The Netherlands 112 Chemicals TPHP (CAS no 115-86-6; 99% purity) and TDCIPP (CAS no 13674-87-8; 113 95.7% purity) were purchased from Sigma-Aldrich Chemie GmbH (Schnelldorf, 114 Germany) Stock solutions were prepared in dimethyl sulfoxide (DMSO) 115 Generation of bone marrow-derived DCs Bone marrow-derived DCs (BMDCs) were 116 obtained as described earlier (van Rijt et al., 2002) Briefly, BM cells were isolated from 117 the tibiae and femurs of Balb/c mice After red blood cell lysis, BM cells were cultured for 118 ten days in Tissue Culture Medium (RPMI 1640, 5% FCS, 50 µg/ml gentamicin 119 (Invitrogen), 0.05 mM 2-ME) with 20 ng/ml recombinant mouse granulocyte-macrophage 120 colony stimulating factor (GM-CSF, Thermo Scientific, Rockford, IL) 121 In vitro exposure of BMDCs with PFRs On day 9, BMDCs were exposed for 24 hr to 122 0, 0.1, 1, 10, 50 or 100 µM TPHP or TDCIPP with or without 100 µg HDM/ml(Canbaz et 123 al., 2016a), containing µg/ml of Der p 1, a cysteine protease and one of the major 124 allergens of Dermatophagoides pteronyssinus HDM (XPB82D3A2.5, Greer Laboratories; 125 Lenoir, NC) Control exposures contained 0.1% DMSO to match the final concentration 126 achieved in the culture medium in the experimental exposures ACCEPTED MANUSCRIPT 127 BMDC phenotyping and cytokine production At day 10, BMDCs were harvested, 128 washed in FACS buffer (PBS containing 0.25% BSA, 0.05% sodium azide, 0.5 mM 129 EDTA) and stained with antibodies against MHCII(FITC labeled), CD11c(APC labeled), 130 in combination with PE labeled antibodies against costimulatory molecules (cell surface 131 proteins) CD80, CD86 and CD40, or with isotype controls for rat IgG2a and, armenian 132 hamster IgG (eBioscience Inc, San Diego, CA) To prevent non-specific antibody 133 binding, anti-FcRγII/III antibody (clone: 2.4G2, provided by Louis Boon, Bioceros, 134 Utrecht, The Netherlands) was added to the monoclonal antibody mixture 10 µg/ml 135 propidium iodide (PI, Sigma Aldrich Corp St Louis, MO) was used to distinguish 136 between living (PI-) and dead cells (PI+) Flow cytometry was performed using a FACS 137 Calibur (BD Biosciences, San Jose, CA) In all cases, 50000 events/sample was 138 acquired Expression of costimulatory molecules was analyzed on alive dendritic cells 139 (MHCII+CD11c+PI-) with FlowJo software (Tree Star Inc., Ashland, OR) IL-6 and IL-10 140 production by BMDC was determined in culture supernatant by ELISA according to the 141 manufacturer’s instructions (Ready-SET-Go ELISA kit, eBioscience, San Diego, CA) 142 Real Time PCR on BMDCs 143 To determine oxidative stress induced by PFRs, the level of mRNA coding for heme- 144 oxigenase-1 (HO-1), a protective anti-oxidant enzyme, was used as a marker for 145 oxidative stress (Ryter and Tyrrell, 2000) Cultured BMDCs were exposed hours to 146 HDM/PFR (0.1-100 µM) or HDM/DMSO before BMDCs were macerated in Trizol 147 (Invitrogen Life Technologies, Carlsbad, CA, USA) for total RNA extraction according to 148 the manufacturer’s recommendations After synthesis of the complementary DNA ACCEPTED MANUSCRIPT 149 (cDNA) using First strand cDNA Synthesis Kit (Thermo Fisher Scientific, Vilnius, 150 Lithuania), real-time PCR was performed in a 10 µl reaction volume that included µl of 151 SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) and µM of FW 152 and RV primers (HPRT Fw: GTC CTG TGG CCA TCT GCC TAG TA- Rv: AAG TCT 153 GGG GAC GCA GCA ACT; HO-1 Fw: AGC AGA ACC CAG TCT ATG CCC CA- Rv: 154 TGC CAG TGA GGC CCA TAC CAG AA) All reactions were performed in optical 96- 155 well reaction plates using StepOne Plus RealTime PCR System (Applied Biosystems, 156 Warrington, UK) HO-1 mRNA concentrations from each sample were calculated based 157 on the standard curve method as previously described and were normalized to the 158 concentration 159 phosphoribosyltransferase (HPRT) (Silva et al., 2012) In short: duplicate standard 160 curves for HO-1 were obtained by serial dilution (1:5) from a concentrated pool of 161 samples expected to contain HO-1 All samples were normalized to the concentration of 162 the housekeeping gene (HPRT) and the expression was determined in arbitrary units 163 Statistical analysis Independent in vitro experiments were performed 3-4 times The 164 data was presented as the means ± SEM of 3-4 independent experiments The sample 165 size per experimental condition differed from 2, or wells per experiment All data 166 were analyzed by Mann-Whitney U-test using GraphPad Prism software (GraphPad 167 Software Inc., La Jolla, CA) Differences were considered to be significant at a p-value of 168