Phthalates are alkyl diesters of phthalic acid and are used as plasticizers in PVC products, as solvents and fixatives in personal care products and as additives in the enteric coating of some drug tablets. Thus, phthalates are present in a wide array of daily-use products, from flooring and automotive parts to medical devices and even children´s toys. Since these compounds are not covalently bound to the products, they easily migrate from them (6), leading to high human exposure.

Phthalate exposure has been estimated by biomonitoring studies and ranges between 1 and 2 ug/kg bw/day of a single compound for adults (7, 8) and up to 4 ug/kg bw/day for children (9). While oral consumption of contaminated food and dermal absorption from personal care products are considered the main exposure routes (10–12), phthalates are ubiquitously found in environmental matrices such as in the atmosphere, soil and water bodies (13). Of particular interest is the presence of phthalates in the atmospheric aerosol, forming part of the organic content of fine particulate matter (PM_2.5). Phthalate concentrations can range from 1 to 100 ng/m³ outdoors (14, 15) and up to 1,000 ng/m³ indoors (16, 17), being Bis-(2-ethylhexyl) phthalate (DEHP) and Di-n-Butyl phthalate (DBP) the most common.

The biological action induced by phthalates differ from the target cell in which they have an effect. The best described mechanisms involve signaling through the hormone receptors: estrogen (ER) and androgen (AR) receptors; and by the peroxisome proliferator-activated receptor gamma (PPAR-gamma), although other non-transcriptional mechanisms have been described, such as the activation of G protein-coupled receptors (Figure 1).

Figure 1

Molecular signaling induced by phthalates. Phthalates and some phthalate metabolites may exert fast non-transcriptional effects by engaging and activating G protein-coupled receptors (GPCR), which activate downstream pathways that lead to intracellular calcium increases and activation of diverse protein kinases. Phthalates and their metabolites readily cross cell membranes, entering the cytoplasm. Once in the cytoplasm, they may bind to estrogen receptors (ER) and induce their dimerization and therefore their activation, leading to transcriptional activity, which may vary depending on the existing coregulators. Phthalates may also bind androgen receptor (AR), triggering transcriptional effects and/or regulating the activation of kinases and transcription factors. Aryl hydrocarbon receptor (AhR) may also bind phthalates, enter the nucleus and regulate transcription of responsive genes. Upon nucleus localization, phthalates may also act as Peroxisome proliferation-activated receptor gamma (PPARγ) ligands, leading to transcriptional activity.

The immune and endocrine systems are part of the neuroimmunoendocrine network, a recently recognized multisystem circuitry that regulates homeostasis. Regarding the immune system, its cellular components not only express endocrine receptors, but synthetize and respond to several hormones and other endocrine ligands. Some molecular pathways typically associated to the endocrine system play a developmental and regulatory role on the immune system, such as the thymus involution through age, the sexual dimorphism in immune response and the tolerogenic character of immune response during pregnancy.

Estrogen receptors are expressed in most immune cell populations, from hematopoietic precursors to mature B and T lymphocytes, NK cells, monocytes and dendritic cells (42). However, expression patterns are different, with some cells expressing preferentially ER-alpha (e.g. CD4⁺ T cells) and other expressing higher levels of ER-beta (e.g. B cells) (43, 44). In homeostatic conditions, ER-alpha acts on myeloid and lymphoid precursors, inducing developmental pathways (42).

The developmental importance of ER-alpha is depicted by the thymic and splenic hypoplasia observed in ER-alpha-deficient mice (45). ER has a profound effect on T lymphocytes which is sensitive to the dose or physiological levels of estradiol, the natural ligand. A good example is the shift from Th1 response towards Th2 and Treg expansion due to high levels of estrogen, as occurs during pregnancy. B cells are deeply modulated by estrogen, both ER-alpha and ER-beta pathways are involved in maturation, function and survival of this population (42), so that females tend to display a greater humoral response than males (46). Differentiation and maturation of dendritic cells, as well as macrophage effector functions are also modulated by estrogen receptor pathways (44).

AR is expressed in several cell populations from both myeloid and lymphoid origin and has been involved in sexual immunological dimorphism and immunosuppression (47). The majority of the studies regard androgens and and androgen receptor as suppressive or regulatory. AR pathways downregulate T cell and B cell proliferation, as well as the inflammatory response (48). Androgens also modulate the functions of dendritic cells, downregulating expression levels of MHC, HLA and costimulatory molecules, which further modulates T cell response (47). One exception to the predominantly suppressive role of androgens is the neutrophil subpopulation, since androgens (via AR) promotes their differentiation and maturation, probably by enhancing Granulocyte colony-stimulating factor (G-CSF) signaling (48). Though histamine release by mast cells appears to be downregulated by androgens, testosterone has been shown to induce IL-33 expression, which drives the generation of innate lymphoid cells and basophils, which in turn produce Th2 cytokines and drive IgE antibody switch (47). A very interesting observation is that IgE is found to be higher in young males over females affected by allergic rhinitis (49).

PPAR-gamma is expressed in lower amounts in some immune cells such as macrophages, eosinophils, dendritic cells, T and B lymphocytes; however, it has a critical role in both differentiation and activation of these cells in diseases that involve chronic inflammation such as atherosclerosis, inflammatory bowel disease and rheumatoid arthritis (50).

Expression of PPAR-gamma is differentially expressed in different subtypes of macrophages. Resting bone-marrow-derived macrophages express low levels of PPAR-gamma mRNA, whereas activated peritoneal macrophages express high levels of PPAR-gamma (51). However, peritoneal macrophages incubated with 15-dPGJ₂ –a PPAR-gamma agonist- showed morphological features typical of resting cells, even in the presence of IFN-gamma. In addition, it also downregulates iNOS expression and inhibit IFN-gamma-dependent nitrite production. This suggests that might inhibit the expression of genes that become upregulated during macrophage differentiation and activation (51). In contrast, other studies report that activation of PPAR-gama primes primary human monocytes into M2 differentiation, but it is not able to modify marker expression of differentiated M1 or M2 macrophages (52).

Eosinophils, an immune cell population related to allergic responses and asthma, express PPAR-alpha, PPAR-gamma and PPAR-delta. In fact, the administration of PPAR-gamma agonists (rosiglitazone or pioglitazone) in a murine model of allergic asthma, induce a reduction in the number and activation state of eosinophils in the airways, determined by the expression of IL-4, IL-5 and ovalbumin-specific IgE, which suggest that PPAR-gamma may be protective factor in the pathogenesis of the asthma (53). This correlate with in vitro experiments in which the presence of high concentrations of PPAR-gamma agonists reduce the eotaxin-dependent eosinophil migration; while low concentrations of this compounds increase migration (54). Interestingly, this effect is mediated by changes in calcium influx, but not by the expression of CCR3 or phosphorylation of p38 or ERK (55).

As mentioned for eosinophils, PPAR-gamma expression in dendritic cells have been related to allergic responses. Particularly, PPAR-gamma deficient CD11b+ dendritic cells fail to migrate to the lung draining lymph nodes and therefore a reduced capacity to polarize naïve CD4⁺ T cells toward a Th2 phenotype (56, 57). Interestingly, Tuna and collaborators mention that the effect of PPAR-gamma activation depend on the tissue microenvironment. While in the lung it may induce a mucosal phenotype in mDCs and that loss of PPAR-gamma promotes an inflammatory phenotype (determined by the ability of BM-DC to polarize CD4 T cells toward iTregs and to induce CCR9 – a mucosal homing receptor- on T cells); deficiency of this receptor showed no change in the frequency or phenotype of mDC in the colon. This suggest that the intestinal microenvironment can maintain the mucosal DC phenotype of via PPAR-gamma-independent mechanisms (58).

T cells express PPAR-gamma 1, but not PPAR-gamma 2 and it is upregulated on activated T cells. Interestingly, its activation through agonists (such as 15-deoxy-Δ12, 14-prostaglandin J2 or troglitazone) inhibits PMA- mediated proliferation and induce a decrement in cell viability (59). This effect was also described in human and B cell lines. Incubation of B lineage cells with PPAR-gamma against but not with PPAR-alpha agonists, induce apoptosis as demonstrated by the increase of AnnexinV staining and the evaluation of DNA fragmentation in TUNEL assays (60).

Because of its function as an environmental sensor, AhR is widely expressed in the body and the immune system. However, it is highly expressed at the barrier sites such as the skin, lung and gut. In the immune system, the expression of AhR has been described in dendritic cells, B cells and some subpopulations of T cells such as Th17, and in lesser amount, in regulatory T cells (61).

In the T cell subpopulations, AhR is highly expressed in Th17 and in a lesser amount in regulatory T cells (Tregs), but it has not been found in naïve, Th1 or Th2 cells (41). The role of this receptor in Th17 cells remains mostly elusive, but some studies suggest that AhR interacts with NF-κB and STAT1, inhibiting those proinflammatory pathways (62) and promoting the Th17 phenotype by enabling cells to produce high levels of IL-17 and IL-22 (63). Regarding B cells, it has been reported that AhR is expressed in several subpopulations, although at different levels (41). AhR role in B cell function is still not well understood, but an in vitro study reported B cell maturation impairment and antibody production suppression when LPS stimulation occurred in presence of a potent AhR ligand (64). AhR is also expressed by antigen presenting cells, such as dendritic cells, whose differentiation and maturation can be affected by AhR activation. Furthermore, activation of AhR decreases the expression of class II MHC, as well as costimulatory molecules, promoting regulatory T cells differentiation (65).

As summarized in Table 1, most studies about the impact of phthalates in the immune system are developed in murine macrophages either cell lines or in primary cultures of peritoneal exudates. In these cells, it was found that some phthalates such as DEHP, DEP and MEHP promoted a pro-inflammatory state characterized by an increase in the expression and secretion of IL-1, IL-6, TNF-alpha, and the chemokine CXCL1 and ROS (66). Other studies found that the secretion of IL-6, IL-10 and the chemokine CXCL8 was enhanced, while TNF-alpha was impaired by DEP and DnBP presence during stimulation (67). They are also capable of increase the phagocytic index of rabbit lung macrophages, which is accompanied by an increment in the release of lysosomal hydrolases (68). However, Shertzer found that even when the phagocytic index after the infection with S. aureous was increased, there was a reduction in the rate of pathogen destruction; which may impact as a deficient response in subsequent exposures (69). On the contrary, other phthalates such as the DBP, BzBP, DEP and DPrP inhibit the production of IL-1beta, IL-6 and IFN-beta (70, 71), apparently by the inactivation of the IFN-beta promoter (70).

In T cells, phthalates seem to promote the polarization to the Th2 phenotype. Hansen et al. showed that 100 uM of DEP and DnBP were capable to suppress the secretion of IL-2 and IL-4, TNF-alpha and IFN-gamma, with no impact in cell viability. It was found that DEHP stimulation might also increase the expression of TCR and CD3 molecules and antigen-induced proliferation (72). In total splenocyte cultures, DEHP significantly increased IL-4 and IFN-gamma concentrations, while MEHP increased IL-6 in supernatants. Interestingly, the effect of DEHP was mediated by the activation of the Ca²⁺/CaN/NF-AT signaling pathway, which was not observed with MEHP. These data suggest that both phthalates may induce the same outcome but by altering different cellular mechanisms (73). DEHP also induces B cell proliferation; activate Ca²⁺ flux, membrane PKC translocation, ERK1/2 phosphorylation and NF-kappa B activation after 2 hrs (74).

In murine thymocytes, DEHP also increases B cell proliferation as demonstrated by Yamashita et al. (75). In contrast, MEHP was found to suppress B cell ((3)H) thymidine incorporation in both bone marrow-derived B cells and the B cell line BU-11 at doses of 25 or 100 uM. This was explained by an increase in B-cell apoptosis at high doses and an apparent arrest without death at low doses (76).

Interestingly, these alterations are also seen in other organisms such as the rainbow trout, in which, in vitro DEHP exposition of B cells inhibited in a dose-dependent way the proliferation of these cells. Also, authors found that there were changes in the differentiation that generates deficient plasmablast expansion and a reduced number of IgM-secreting plasma cells (77).

Regarding to other immune cell types, there is also evidence in which the effect of some phthalates modifies the function of bone marrow-derived dendritic cells (BM-DC) and basophils. In DC’s, the presence of 10 uM of DEHP during the differentiation of BM-derived macrophages increased the expression of maturation markers such as MHC-II, CD80/86, CD11c and DEC205. It also increased their capacity to induce secretion of IFN-gamma, IL-4 and IL-10 by T cells. This effect was also observed when DC’s were differentiated in normal conditions and DEHP was added during the activation. Interestingly, at 100 uM of DEHP during activation, the percentage of maturation/activations markers were reduced and their antigen presenting activity is not affected as observed by the reduced proliferation of co-cultured T cells. Authors suggest that the enhancement on these markers and function might contribute to the aggravating effect of DEHP on allergic disorders, which also may be useful for the treatment in this kind of pathologies (72).

Finally, since air pollutants have been related to airway diseases, the study of immune cell populations related to allergies has gained relevance. The RBL-2H3 cells, a basophil-derived cell line, which has been described also as a mast cell model, was exposed to different phthalates -DPB, DiPB and DEHP- at concentrations of 50-500 uM. Authors described that in the absence of antigen stimulation, none of those compounds induce β-hexosaminidase release. However, when cells were pre-sensitized and then stimulated with an antigen, all of the phthalates increased the degranulation of these cells (78). Another report found an interest correlation with the number of carbon atoms present in the phthalates and the histamine-release of human basophils. In this work, PMBC’s were incubated in the presence different phthalates and the stimulated with any of the following stimuli: anti-IgE, the bacterial derived peptide fMLP, a calcium Ionophore or an allergen (cat hair extract). As described before, there was no effect on the histamine release in the absence of activation. Interestingly, only the phthalates and monophthalates with an 8-carbon atoms alkyl chain length were the stronger histamine-release potentiators, when there were no differences for the 4-, 9- or 10-carbon chain (79).

Some studies have also assessed the effect of phthalates on human cell lines and primary cultures (Table 2). As in the former studies, heterogeneity in exposure times and concentrations is observed, but some general features can be distinguished. It is interesting to note that, in general terms, a more reactive or proinflammatory response is observed in human cells in vitro exposed to different phthalates, regardless of the heterogeneity of models and exposure schemes.

Studies with THP, a macrophage cell line seem to concur in that phthalates and their metabolites increase the cytokine and chemokine production (79, 91, 92). Tetz and cols. Also observed a proinflammatory effect of phthalate exposure, with activation of COX pathway and increased prostaglandin production (93). An interesting study by Teixeira noted that the effect of phthalate exposure showed differential features, depending on the macrophage phenotype, either M1 or M2 (94).

One of the first studies that documented the effect of phthalates on immune cells was derived from the concern of donated blood being storage in plastic bags; this study found decreased chemotaxis and bactericidal capacity of neutrophils (95). However, more recent studies using human granulocytes have found rather an increase in chemotaxis, IL-8 (96, 97), and promotion of inflammatory response (88).

Mast cells are of particular interest, given their role in allergic disease. Lee and cols. Reported an increase in IFN-gamma and IL-4 production upon DEHP exposure, which is linked to a more reactive phenotype. However, more studies regarding mast cells differentiation, migration and function are desirable.

Given the complex multidirectional regulation that occurs between immune and endocrine system, it is clear that, although they offer valuable information, in vitro studies don´t fully represent what occurs in a whole organism, and it is why in vivo studies are of such importance. Data from the full literature review is detailed in Table 3.

In general terms, phthalate exposure leads to higher immunoglobulin levels. In some models, an increase of IgG -mainly IgG, has been observed (106–110), while in sensitized animals is the IgE class which is augmented (111). For one of the most studied phthalates – DEHP, the increase in IgE seems to be mediated by the promotion of differentiation of B cells into plasma cells (112) and by the stimulation of follicular T helper cells (Thf), producing higher IL-21 and IL-4 (106).

In diverse pathologies that are characterized by hypersensitivity, such as asthma, dermatitis and rhinitis, phthalate exposure has been shown to increase Th2 (DBP, DPP, DEHP and DINP) and Th17 cytokines (DBP) (110, 113, 114). In animal asthma models, the most studied phthalates -DEHP and DINP- have been shown to increase pulmonary inflammation, antibody production (specially IgE), and IL-4 production. This Th2 polarization has also been observed in an allergic rhinitis model, where DEHP exposure leads to higher IL-13 levels in the nasal epithelium (114). In cutaneous hypersensitivity models, DBP, DPP and DINP have been shown to promote Th2 response, as denoted by a high IL-4 production (115, 116).

Moreover, phthalate exposure also stimulates the production of chemoattractant molecules, such as TSLP, CCL17 and CCL22 (115, 117, 118), which coincides with another common finding: a higher recruitment of antigen presenting cells like dendritic cells and macrophages. This phenomenon, known as the adjuvant effect, has been observed in skin and other tissues, including adipose tissue, and for various phthalates (119, 120).

One of the most typical target organs regarding phthalate toxicity is the male gonad. The effect of phthalates on this tissue has been mainly attributed to pure endocrine mechanisms, but it seems that immunomodulatory mechanisms may as well play an important role. It has been observed that exposure to some phthalates, fundamentally DEHP, causes an increased infiltration of macrophages and other leukocytes in the testis, together with a higher expression of proinflammatory cytokines, such as IL-1β and IFN-gamma, which leads to germinal cell apoptosis and lower testosterone levels (121–124).

Epidemiological studies, albeit heterogeneous, support the immunomodulatory effects of phthalates, particularly due to prenatal exposure. Data from the full literature review is detailed in Table 4. A 70% higher risk of asthma diagnosis at age 5-11 was observed in children born to women with higher urinary levels of BBzP and DnBP metabolites during their pregnancies (132). Another study, carried out in a Spaniard cohort, also found an association between asthma diagnosis at age 7 and maternal urinary levels of MBzP and the sum of DEHP metabolites (133). In a similar fashion, a study in Taiwan reported an association between high maternal DEHP exposition and asthma incidence, particularly in boys (134). In a French cohort, a study found a correlation between maternal urinary concentrations of DiBP and DiNP metabolites and eczema occurrence; interestingly, this association was stronger for boys (135).

At the molecular level, Ashley-Martin didn’t find an association between maternal MCPP (a nonspecific secondary metabolite) and levels of IgE, nor IL-33 in neonates (136). However, another study where MEP, MEHP, MBP and MBzP were measured, found a positive association between maternal urinary levels of those metabolites and IgE levels of children at age 2 and 5 (137). Interestingly, this association was only significative in boys.

Regarding current exposure in adults, the NHANES 2005-2006 study found an association between urinary levels of high molecular weight phthalate metabolites and allergic sensitization (defined by the response to at least 19 specific IgE antigens) (138). Furthermore, MBzP concentration correlated with the presence of allergic symptoms at the moment of the sampling. However, this same study found that in children there was an inverse relationship between urinary concentrations of phthalate metabolites and the incidence of asthma and hay fever. In another study, in adults allergic to dust mite, inhalation of DEHP-containing dust modified the expression of G-CSF, IL-5 and IL-6 in a differential fashion, depending on the concentration (139). At low levels, those cytokines increased, while at high concentrations both G-CSF and IL-6 decreased. It is interesting to note that, similarly to what is observed with some hormones, the dose-response relationship seems to be non-monotonic.