The Effect And Mechanism Of Di-(2-ethylhexyl) Phthalate Exposure On Weanling Mice Humoral Immune Response

Di-(2-ethylhexyl)phthalate(DEHP)is a most abundant and widely-used phthalate plasticizer.DEHP has been considered as a widespread environmental persistent organic pollutant and its potential concern on human health is highlighted on a global scale.Though human exposure routes to DEHP are multiple,including diet,inhalation,skin and medical contact,ingestion of contaminated foods,water and other materials is thought to be a major route.In particular,children are more likely to be exposed to DEHP through gastrointestinal route and consequently are more susceptible to DEHP hazards.Some reports have uncovered a positive association between DEHP exposure and an increased prevalence of anaphylactic diseases including allergic rhinitis,bronchial asthma and atopic dermatitis among genetic stable crowd especially among infants and juveniles in same area.Allergy is a hypersensitive reaction rooted in imbalanced humoral immunity.T follicular helper cell(Tfh),an important CD4~+Th cell subset,until recently has been identified as a key player that potently assists B cells to modulate antibody-mediated humoral immune response in germinal center.Abnormal differentiation and function of Tfh cells cause internal environment immune disorder.Tfh cells may response to stimulation through immunomodulatory receptors.Peroxisome proliferator-actived receptor-γ(PPAR-γ)in nucleus surface and cytoplasm can selectively bind to corresponding ligands.PPAR-γis able to regulate the transcription and expression of target gene after nuclear translocation.Some researches have shown that DEHP is a nuclear receptor regulatory factor.DEHP metabolites may recognize and bind to PPAR-γ,mediating systemic or local toxicity.In the present study,focusing on the newly confirmed Tfh cells,we examined the effects of envirinmental pollutant DEHP on weanling mice humoral immunity and investigated the underlying mechanisms.The experiment methods were designed as follows:(1)Weanling BALB/c mice were gavaged with gradient concentration DEHP and were sensitized with OVA by subcutaneous injection into foot pad.The amount of OVA-specific IgE and IgG1 in the serum was measured by ELISA.The germinal center formation in lymphoid nodule was assessed by immunofluorescence histochemistry.The differentiation of Tfh cells and plasma cells in peripheral lymphoid organ was analyzed by flow cytometry.(2)Th cells including Tfh cells or non-Tfh cells from either DEHP-exposed or normal mice were mixed with B cells from either DEHP-exposed or normal mice,then adoptive transfer into SCID mice via tail vein injection.The production of OVA-specific IgE and IgG1 in the serum was quantified by ELISA after OVA sensitization.Using the above murine model under the condition of gastrointestinal exposure to DEHP,the expression of Tfh cell nuclear receptor PPAR-γand transcription factors Bcl-6 and c-Maf was detected by qRT-PCR and Western Blot.Meanwhile,the expression of Tfh cell transcription factors and synthesis of intracellular cytokines IL-21 and IL-4 was evaluated by flow cytometry.(3)Tfh cells were exposed to DEHP in vitro,adding PPAR-γagonist and antagonist into the cell culture system.The expression of Tfh cell transcription factors Bcl-6 and c-Maf was detected by qRT-PCR and Western Blot.The expression of Tfh cell surface markers and nuclear transcription factors was evaluated by flow cytometry.The secretion of cytokines IL-21 and IL-4 in Tfh cell culture supernatants was measured by ELISA.The experiment results showed as follows:(1)DEHP gastrointestinal exposure acted adjuvant effects to augment OVA-specific IgE and IgG1 production in the serum,amplified germinal center formation in lymphoid nodule,as well as stimulated the expansionofCD4~+CXCR5~+ICOS~+/CD4~+CXCR5~+PD-1~+Tfhcellsand CD19~+CD138~+GL-7~+plasma cells in peripheral lymphoid organ.(2)Serum OVA-specific Ig E and IgG1 in the recipient mice were induced rarely under all combinations of non-Tfh cells and B cells transfusion,while co-transfer of Tfh cells and B cells from normal mice restored basic IgE and IgG1 antibody production.Tfh cells from DEHP-exposed mice co-transferred with B cells from either DEHP-exposed or normal mice boosted OVA-specific IgE and IgG1 antibody production in the recipients,however B cells from DEHP-exposed mice co-transferred with Tfh cells from normal mice did not.DEHP gavage together with OVA sensitization adjuvantly promoted the expression of nuclear receptor PPAR-γand transcription factors(Bcl-6 and c-Maf)and the synthesis of cytokines(IL-21 and IL-4)in Tfh cells.(3)In vitro exposure to DEHP upregulated the expression of Tfh cell transcription factors and surface markers and the synthesis of cytokines.PPAR-γantagonist reduced the expression of Tfh cell transcription factors(Bcl-6 and c-Maf)and surface molecules(CXCR5,ICOS and PD-1),also decreased the secretion of cytokines(IL-21 and IL-4)from Tfh cells.PPAR-γagonist further enhanced the expression of Bcl-6 and c-Maf and the secretion of IL-4.In conclusion,our study demonstrates that:(1)DEHP displays adjuvant toxic effects on inducing the imbalanced humoral immune response in OVA-sensitized weanling mice through gastrointestinal ingestion,promoting the germinal center formation,plasma cells differentiation,and anaphylaxis-related antibodies production.(2)The improper enhancement of humoral immune response in DEHP gastrointestinal exposure model results from Tfh cells intrinsic dysfunction.DEHP has adjuvant toxic effects on Tfh cells by synthesizing an excess of cytokines via over-expression of transcription factors,leading to an increasing secretion of allergy-related immunoglobulins.(3)PPAR-γinside Tfh cells is the target receptor molecule of DEHP metabolites,and PPAR-γparticipates in regulating the differentiation and function of Tfh cells.DEHP metabolites selectively bind to nuclear receptor PPAR-γin Tfh cells,which influences PPAR-γtranscription regulation on downstream genes including transcription factors,surface molecules and cytokines.