These studies demonstrate that in ex vivo
human studies, Phe activates the AhR pathway, altering CpG methylation patterns in Treg. Increased CpG methylation in key regulatory regions of the FOXP3
gene destabilizes FOXP3 expression, resulting in the conversion of Treg to the proallergic Th2 Teff phenotype. Our studies indicate that ex vivo
AhR activation via Phe and downstream events resulting in Treg to Th2 conversion are AhR ligand specific. These data demonstrate the important role of Phe in T cell plasticity and pathophysiology of atopic diseases [3
Recently, others have shown that Treg convert to Th2 [47
] in culture; however, in contrast to these previous findings, our data demonstrate that specific environmental insults are needed to induce Treg deviation to Th2 or Th17 phenotypes. In our hands, we did not observe spontaneous conversion of Treg to Th2. The work presented herein is the first to our knowledge to present a detailed mechanism for how Treg conversion to other T cell phenotypes occurs via Phe activation ex vivo
. Our findings provide insight into one mechanistic explanation of PAH-induced modulation of Treg function. We understand that there are several limitations with ex vivo
data, including, but not limited to, the dose of Phe, a type of PAH, and PAH metabolite actually present in vivo,
as well as the involvement of other immune cells and gene loci in the PAH response.
AhR has been shown to modulate Treg differentiation in a ligand specific fashion [5
]. TCDD, an exogenous nondegradable high-affinity AhR ligand, induces functional Treg cells that suppressed experimental autoimmune encephalomyelitis [5
]. In contrast, FICZ, an endogenous rapidly degradable high-affinity AhR ligand, interferes with Treg cell development, boosts Th17 cell differentiation, and increases the severity of disease [5
]. We also tested the TCDD and FICZ ligands and found that both increased human Th17 differentiation ex vivo
without altering FOXP3 methylation (Figures and ). These results further support ligand-specific effects. FICZ has been shown in some cases to promote Th17 differentiation and in some cases not [3
]. These discrepancies between studies may reflect differences in the mouse versus human systems [52
] and/or the involvement of other factors that play a role in skewing Th17/Treg/Th2 development, such as retinoic acid or TGF-β
production by dendritic cells [3
]. Our studies with Phe further support the ligand specific role of AhR in modulating Treg development and its dynamic plasticity while presenting a novel pathway of Treg instability in the human system.
Mice and humans exhibit significant differences in innate and adaptive immunity, including balance of leukocyte subsets, T cell signaling pathway components, and Th1/Th2 differentiation [52
]. This may account for discrepancies in the recent literature debating whether AhR activation leads to Th17, Treg, or other T cell subset differentiation [5
], as well as between our own ex vivo
human assays and in vivo
mouse studies. While our comparison of AhR+/+ to AhR-deficient mice suggests that AhR plays a similar role in regulating DNMT and FOXP3 expression in humans and mice Figures S7(a) and S7(b), differences in mouse versus human ligand binding to AhR continue to be an area of important investigation.
We recognize that while our data point to a possible mechanism involved in Phe-induced Treg conversion to a pro-allergic Teff phenotype (Th2-skewed) in humans, other AhR-ligands could mediate their effects through different mechanisms. A previous study demonstrated that TCDD-induced AhR activation ameliorates inflammation in the mouse colitis model [27
]. Our results indicate that AhR activation leading to Treg to Th2 differentiation may not always be beneficial; during atopic conditions, ligand-specific AhR activation may be detrimental. Duration of AhR activation may also play a role. Persistent ligands that are resistant to degradation, such as TCDD, may induce negative feedback mechanisms that alter the AhR-mediated response [3
]. Our studies provide the basis for further work investigating (i) stability effects of natural and exogenous AhR ligands and (ii) differences in mice that elucidate Phe- and AhR-mediated pathophysiologies.
Although the current study does not address Phe-specific effects and downstream consequences in diseased (atopic) individuals, our previous studies and those from others have reported associations between PAH exposure, increased methylation of certain genes, impaired Treg function, and disease in humans [29
]. The current data strengthen these observations by demonstrating a causative relationship in ex vivo
studies of Treg from healthy individuals and further elucidated the process by identifying genes involved and delineating a mechanistic pathway by which a PAH alters Treg function irreversibly within 3 days ex vivo
A number of studies have shown that deficit in Treg cell function is associated with increasing severity of asthma [29
]. Our findings suggest that this may be due in part to alterations in CCR8 expression, which plays a key role in T cell homing to lung parenchyma and bronchial epithelium and has been implicated as an important factor in allergic inflammation and normal immune homeostasis in asthma [36
]. By decreasing CCR8 expression in Treg, PAHs may limit chemotaxis to lung tissue, where regulatory T cells temper asthmatic and allergic responses. In addition, predominance of Th2-derived IL-4 and IL-13 could induce isotype switching to IgE, which is associated with allergic conditions [59
]. Lack of Treg migration, isotype switching, and Treg to Th2 conversion may all contribute to increases in inflammation and atopic disease.
Our study has important implications for the future of immunomodulatory therapies. Epigenetic changes, particularly aberrant methylation of CpG islands in regulatory sequences of FOXP3, may be prevented or reversed by dynamic DNA remodeling enzymes. Alternatively, pathway targets such as AhR and DNMT may be inhibited by small molecules or other pharmacologic means if specific to Treg cells. Future studies identifying other key players in the conversion of Treg to Teff cells are needed to further understand the mechanism of AhR-ligand-mediated immune changes. Such understanding could lead to improvements in prevention of and treatment for immune-mediated diseases.