Mechanism Of Adjuvant Effect Induced By Di-(2-Ethylhexyl) Phthalate In Mouse Asthma Model

Di-(2-ethylhexyl) phthalate (DEHP) is phthalate diester. It is the most commonly used plasticizer due to its softening effect in enhancing plastic flexibility and tensile. As it is widely used in the plastic industry, DEHP is easily released into the environment during the production, handling and disposal process whereby it can enter into the body through many routes, in particular through the oral route.DEHP exposure can cause adverse effects which include reproductive toxicity, genetic toxicity and endocrine toxicity. Especially, DEHP is a well-known antiandrogen, so most of the research has been focused on its reproductive toxicity effects.Allergic diseases are dramatically increasing in all over the world in the recent years, and environmental pollutions attract the researchers' more and more attentions. The exposure of environmental adjuvant chemicals may be one possible reason for the rising incidences of allergic diseases. DEHP belongs to the environmental adjuvant pollutants. Results from epidemiology studies indicate that DEHP exposure in living room is linked to the childhood asthma, wheezing and eczema.In vivo and in vitro studies both demonstrate that the DEHP exposure can regulate the function of human immune system, and is related to the allergic diseases. Animal model studies also show that DEHP incombination with allergen exposure can aggravate allergic airway inflammation, which is so-called adjuvant effect, but DEHP cannot act as a direct allergen trigger the allergic inflammation process The existing animal model data only show a possible correlation between DEHP exposure and allergic inflammation; the specific molecular pathways involved remain to be further studied. We hypothesize that DEHP might aggravate the allergic inflammation responses, as a causative cofactor,via the regulation of key molecules involved in allergy.Oxidative stress is one vital pathway in the pathogenesis of asthma, patients with asthma showed increased levels of oxidative stress. Redox imbalance in patients is associated with the initiation of the nuclear factor kappa B (NF-?B) signal transduction pathway. Activated NF-?B is translocated from the cytoplasm to the nucleus, whereby it combines to the NF-?B promoter. This is followed by elevated expression of many cytokines, include: thymic stromal lymphopoietin (TSLP), interleukin-4(IL-4), IL-1?, tumor necrosis factor a (TNF-a) resulting in the formation of allergic inflammation.TSLP is an important cytokine in Th2 type immune response, it triggers human allergic diseases by inducing the dendritic cells (DCs) to produce specific Th2 cytokines and TNF-a. Furthermore,previous researches using mouse model demonstrate that DEHP co-exposure with allergens augments the oxidative stress levels and upregulation the expression of TSLP in target organs. Consequently, we speculate that oxidative stress,NF-?B and TSLP may be the effective targets to inhibit adjuvant effect of DEHP.The present study aimed to explore the molecular mechanisms of DEHP-induced adjuvant effect in mouse asthma model. Basing on the conclusion that DEHP displays adjuvant effects. In this mouse asthma model, OVA was chosen as the allergen to trigger asthma-like symptoms in BALB/c mice.Meanwhile, DEHP exposure was co-combined with or without OVA sensitization. To further clarify whether TSLP,NF-?B and oxidative stress were the possible pathways for the DEHP-adjuvant effects,TSLP monoclonal antibody, pyrrolidine dithiocarbamate (PDTC) as the specific antagonist of NF-?B and melatonin (MT) as the antioxidant were used by intraperitoneal injection at 60 min before every OVA sensitization.Data showed that DEHP (10 mg/kg/day, 60 days) exposure alone could not significantly induce the asthmatic phenotype. Allergen, OVA, successfully triggered the mouse allergic asthma syndromes:typical airway remodeling, increased OVA specific IgE and increased Th2 cytokines levels. Compared with the OVA group, OVA-combined with DEHP exposure (DEHP+OVA) dramatically upregulated T-IgE, OVA-sIgE and OVA-sIgGl levels (p<0.01). The number of EOS (p<0.01), the contents of Th2 cytokines in BALF (p<0.05), the levels of ECP in lung tissue, the degree of AHR, mucus hypersecretion and collagen fibrosis (p<0.05) were also elevated in this group. When OVA sensitization was combined with DEHP exposure, the asthma-like symptoms are more deteriorative.Also, DEHP co-exposure caused increased contents of TSLP and NF-?B in lung tissue (p<0.05), as well as more activated NF-?B in the nucleus (p<0.01), compared with the OVA group.BALB/c mice showed amazing recovery after the antagonist treatments. Mice in the anti-TSLP group, PDTC group and MT group showed reduced allergic airway inflammation markers: lower levels of IgE in serum (p<0.01), fewer EOS in BALF (p<0.05 or p<0.01), decreased levels of Th2 cytokines in BALF (p<0.05 or p<0.01), attenuated mucus secretion and airway collagen fibrosis in lung tissues. Treatment with TSLP monoclonal antibody, PDTC and MT all effectively alleviated airway inflammation which was worsened by DEHP co-exposure.In summary, we provide strong data to support the notion that DEHP acts as an adjuvant to aggravate airway inflammation responses caused by OVA allergen, we show that the blocking of oxidative stress responses, activation of NF-?B and TSLP effectively prevent the DEHP-induced immunoadjuvant effect.