The Aryl Hydrocarbon Receptor as an Immune-Modulator of Atmospheric Particulate Matter-Mediated Autoimmunity

This review examines the current literature on the effects of atmospheric particulate matter (PM) on autoimmune disease and proposes a new role for the aryl hydrocarbon receptor (AHR) as a modulator of T cells in PM-mediated autoimmune disease. There is a significant body of literature regarding the strong epidemiologic correlations between PM exposures and worsened autoimmune diseases. Genetic predispositions account for 30% of all autoimmune disease leaving environmental factors as major contributors. Increases in incidence and prevalence of autoimmune disease have occurred concurrently with an increase in air pollution. Currently, atmospheric PM is considered to be the greatest environmental health risk worldwide. Atmospheric PM is a complex heterogeneous mixture composed of diverse adsorbed organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and dioxins, among others. Exposure to atmospheric PM has been shown to aggravate several autoimmune diseases. Despite strong correlations between exposure to atmospheric PM and worsened autoimmune disease, the mechanisms underlying aggravated disease are largely unknown. The AHR is a ligand activated transcription factor that responds to endogenous and exogenous ligands including toxicants present in PM, such as PAHs and dioxins. A few studies have investigated the effects of atmospheric PM on AHR activation and immune function and demonstrated that atmospheric PM can activate the AHR, change cytokine expression, and alter T cell differentiation. Several studies have found that the AHR modulates the balance between regulatory and effector T cell functions and drives T cell differentiation in vitro and in vivo using murine models of autoimmune disease. However, there are very few studies on the role of AHR in PM-mediated autoimmune disease. The AHR plays a critical role in the balance of effector and regulatory T cells and in autoimmune disease. With increased incidence and prevalence of autoimmune disease occurring concurrently with increases in air pollution, potential mechanisms that drive inflammatory and exacerbated disease need to be elucidated. This review focuses on the AHR as a potential mechanistic target for modulating T cell responses associated with PM-mediated autoimmune disease providing the most up-to-date literature on the role of AHR in autoreactive T cell function and autoimmune disease

The aryl hydrocarbon receptor meets immunology: friend or foe? A little of both

The aryl hydrocarbon receptor (AHR) has long been studied by toxicologists as a ligand-activated transcription factor that is activated by dioxin and other environmental pollutants such as polycyclic aromatic hydrocarbons. The hallmark of AHR activation is the upregulation of the cytochrome P450 enzymes that metabolize many of these toxic compounds. However, recent findings demonstrate that both exogenous and endogenous AHR ligands can alter innate and adaptive immune responses including effects on T-cell differentiation. Kynurenine, a tryptophan breakdown product, is one such endogenous ligand of the AHR. Expression of indoleamine 2,3-dioxygenase by dendritic cells causes accumulation of kynurenine and results in subsequent tolerogenic effects including increased regulatory T cell activity. At the same time, polycyclic aromatic hydrocarbons found in pollution enhance Th17 differentiation in the lungs of exposed mice via the AHR. In this perspective, we will discuss the importance of the AHR in the immune system and the role this might play in normal physiology and response to disease

Exposure to Atmospheric Particulate Matter Enhances Th17 Polarization through the Aryl Hydrocarbon Receptor

<div><p>Lung diseases, including asthma, COPD, and other autoimmune lung pathologies are aggravated by exposure to particulate matter (PM) found in air pollution. IL-17 has been shown to exacerbate airway disease in animal models. As PM is known to contain aryl hydrocarbon receptor (AHR) ligands and the AHR has recently been shown to play a role in differentiation of Th17 T cells, the aim of this study was to determine whether exposure to PM could impact Th17 polarization in an AHR-dependent manner. This study used both cell culture techniques and <i>in vivo</i> exposure in mice to examine the response of T cells to PM. Initially experiments were conducted with urban dust particles from a standard reference material, and ultimately repeated with freshly collected samples of diesel exhaust and cigarette smoke. The readout for the assays was increased T cell differentiation as indicated by increased generation of IL-17A in culture, and increased populations of IL-17 producing cells by intracellular flow cytometry. The data illustrate that Th17 polarization was significantly enhanced by addition of urban dust in a dose dependent fashion in cultures of wild-type but not AHR^-/- mice. The data further suggest that polycyclic aromatic hydrocarbons played a primary role in this enhancement. There was both an increase of Th17 cell differentiation, and also an increase in the amount of IL-17 secreted by the cells. In summary, this paper identifies a novel mechanism whereby PM can directly act on the AHR in T cells, leading to enhanced Th17 differentiation. Further understanding of the molecular mechanisms responsible for pathologic Th17 differentiation and autoimmunity seen after exposure to pollution will allow direct targeting of proteins involved in AHR activation and function for treatment of PM exposures.</p> </div

Investigation of the specific components of SRM1649b that contain Th17 enhancing activity.

<p>A: Mouse Hepa1 cells that were transfected with luciferase reporter gene fused to the dioxin-responsive elements (DRE) were seeded at 0.6x10⁶ cells per well. Cells were then exposed to media (Control), media + SRM1649b suspended in PBS (SRM/PBS), media + DMSO (DMSO), media + SRM1649b suspended in DMSO (SRM/DMSO) or the organic fraction of SRM1649b suspended in DMSO (fSRM/DMSO) for 4 hours. Luciferase activity was measured on a luminometer. B: Naïve B6 CD4⁺ T cells in Th17 conditions in the presence of fractioned SRM1649b and controls. After 4 days, total RNA was harvested. Using qRT-PCR, the relative levels of IL-17A and Cyp1a1 mRNA were determined. Data presented are the mean and standard deviation of 2 separate experiments and is relative to cells treated with anti-CD3/CD28 antibody stimulation but without mIL-6 and huTGF-β. C: Naïve B6 CD4⁺ T cells in Th17 conditions in the presence varying concentrations of the PAHs benzo[a]pyrene (BaP), benzo[e]pyrene (BeP) and benzo[k]fluoranthene (BkF). Culture supernatant was harvested and ELISA measured the concentration of IL-17A. The line indicates the level of IL-17A expression in cultures exposed to DMSO vehicle only. D: Naïve B6 or AHR^-/- CD4⁺ T cells in Th17 conditions in the presence of BkF. Cells were harvested and subjected to intracellular cytokine analysis to determine the percent of IL-17A-expressing CD4⁺ T cells present after culture. Data from 3 separate experiments was pooled and statistical comparison was made between the cultures with versus cultures without BkF.</p

AHR is required for upregulation of Th17 cells in response to SRM1649b.

<p>A-B: Naïve B6 CD4⁺ T cells were cultured in Th17 conditions (anti-CD3/CD28 antibody + mIL-6 (20 ng/ml) and varying concentrations of huTGF-β (5 ng/ml) for 4 days in the absence or presence of SRM1649b (40 μg/ml) after which total RNA was harvested. Relative levels of AHR and cyp 1a1 mRNA were determined by qRT-PCR. Data are relative to cells treated with anti-CD3/CD28 antibody stimulation but without mIL-6 and huTGF-β. Fold increase results from cultures with (w/) SRM1649b were compared for significance with cultures without (w/o) SRM1649b using the paired student’s t-test. B: Cells were harvested after 4 days and stained with anti-AHR antibody during intracellular cytokine analysis to determine the percent of AHR-expressing CD4⁺ T cells cultured with (solid line) or without (shaded) huTGF-β. C: Naïve CD4⁺ T cells were isolated from the spleens of male B6 or AHR^-/- mice and cultured in Th17 conditions (anti-CD3/CD28 antibody + mIL-6 (20 ng/ml) +/- huTGF-β (5 ng/ml) for 4 days in the varying concentrations SRM1649b. Supernatants were analyzed by ELISA. D - E: Naïve CD4⁺ T cells were isolated from the spleens of AHR^-/- (D) or DREC^-/- (E) mice and cultured in Th17 conditions (anti-CD3/CD28 antibody + mIL-6 (20 ng/ml) +/- huTGF-β (5 ng/ml) for 4 days +/- SRM1649b. Total RNA was harvested and, the relative levels of IL-17A, and IL-22 mRNA were determined by qRT-PCR. Data is relative to cell treated with anti-CD3/CD28 antibody stimulation but without added mIL-6 and huTGF-β. Fold increase results from cultures with (w/) SRM1649b were compared for significance with cultures without (w/o) SRM1649b using the paired student’s t-test. The number in parenthesis indicates the number of individual assays used for that target gene. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

Th17 polarization enhancing activity is found in other PAH containing environmental samples.

<p>A - B: Naïve CD4⁺ T cells were isolated from the spleens of male B6 and cultured in Th17 conditions for 4 days in the presence of varying concentrations of diesel (A) and cigarette (B) extract. Culture media was harvested and the concentration of IL-17A was measured by ELISA. The percent of live/dead stain low CD4⁺ T cells is indicated below the appropriate extract concentration. C - D: Naïve B6 or AHR^-/- CD4⁺ T cells in Th17 conditions in the absence or presence of 8 μg/ml diesel or cigarette extract. Cells were harvested and subjected to intracellular cytokine analysis to determine the percent of IL-17A-expressing CD4⁺ T cells present after culture. For both ELISA and intracellular IL-17A results, data from 3 separate experiments was pooled and statistical comparison was made between the cultures with versus cultures without extract. *, p < 0.05.</p

Th17 differentiation <i>in</i><i>vitro</i> is enhanced with addition of SRM1649b to culture conditions.

<p>A-B: Naïve CD4⁺ T cells were isolated from the spleens of male C57BL/6 mice (B6) and cultured in Th17 conditions (anti-CD3/CD28 antibody + mIL-6 (20 ng/ml) + huTGF-β (5 ng/ml)) for 4 days in the absence or presence of SRM1649b (10, 20 or 40 μg/ml) or FICZ (200 nM). Triplicate culture supernatant was harvested and ELISA measured the concentration of IL-17A and IL-22 (A). Data presented is representative of 3 experiments. In separate experiments, total RNA was harvested from cells and the relative levels of IL-17A, IL-22 and IL-23R mRNA were determined by qRT-PCR (B). Data is relative to cells treated with anti-CD3/CD28 antibody stimulation but without mIL-6 and huTGF-β. Fold increase results from cultures with (w/) SRM1649b were compared for significance with cultures without (w/o) SRM1649b using the paired student’s t-test. The number in parenthesis indicates the number of individual assays used for that target gene. C-D: Naïve B6 CD4⁺ T cells were cultured in Th17 conditions (anti-CD3/CD28 antibody + mIL-6 (20 ng/ml) and varying concentrations of huTGF-β (0 - 5 ng/ml)) for 4 days in the absence or presence of SRM1649b (40 μg/ml). Cells were harvested from and subjected to intracellular cytokine analysis to determine the percent of IL-17A and IFN-γ expressing CD4⁺ T cells present after culture. Data from 3 separate experiments were pooled and statistical comparison was made between the cultures with versus cultures without SRM1649b at each concentration of huTGF-β. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p