Differential effects of diesel exhaust particles on T cell differentiation and autoimmune disease

Abstract

Background: Exposure to particulate matter (PM) has been associated with increased incidence and severity of autoimmune disease. Diesel PM is primarily composed of an elemental carbon core and adsorbed organic compounds such as polycyclic aromatic hydrocarbons (PAHs) and contributes up to 40% of atmospheric PM. The organic fraction (OF) of PM excludes all metals and inorganics and retains most organic compounds, such as PAHs. Both PM and OF increase inflammation in vitro and aggravate autoimmune disease in humans. PAHs are known aryl hydrocarbon receptor (AHR) ligands. The AHR modulates T cell differentiation and effector function in vitro and in experimental autoimmune encephalomyelitis (EAE), a murine model of autoimmune disease. This study aims to identify whether the total mass or active components of PM are responsible for activating pathways associated with exposure to PM and autoimmune disease. This study tests the hypothesis that active components present in diesel PM and their OF enhance effector T cell differentiation and aggravate autoimmune disease.

Results: Two different diesel samples, each characterized for their components, were tested for their effects on autoimmunity. Both diesel PM enhanced effector T cell differentiation in an AHR-dose-dependent manner and suppressed regulatory T cell differentiation in vitro. Both diesel PM aggravated EAE in vivo. Fractionated diesel OFs exhibited the same effects as PM in vitro, but unlike PM, only one diesel OF aggravated EAE. Additionally, both synthetic PAH mixtures that represent specific PAHs found in the two diesel PM samples enhanced Th17 differentiation, however one lost this effect after metabolism and only one required the AHR.

Conclusions: These findings suggest that active components of PM and not total mass are driving T cell responses in vitro, but in vivo the PM matrix and complex mixtures adsorbed to the particles, not just the OF, are contributing to the observed EAE effects. This implies that examining OF alone may not be sufficient in vivo. These data further suggest that bioavailability and metabolism of organics, especially PAHs, may have an important role in vivo.

Keywords: Aryl hydrocarbon receptor; Autoimmune disease; Cytochrome P450; Diesel exhaust particles; Metabolism; Particulate matter; T cell differentiation.

Fig. 1

EAE Methods. Starting on day − 12 mice every 3 days until day 9, age-matched female mice receive intranasal treatment of SRM1650b PM or OF, SRM2975 PM or OF, and corresponding solvent or PBS control. The mice received 12.5 mg/mL PM or 250 μg PM in 20uL for each dose. On day 0, prior to injections, mice were weighed. A subcutaneous injection of 100 μL MOG_35–55 emulsion was given between the shoulder blades of each mouse and an intraperitoneal injection of 200 ng/mouse pertussis toxin in 500 μL was also given. The emulsion contained 50 μg/mouse MOG_35–55 in CFA and 200 μg/mouse heat-killed M. tuberculosis. Additionally, on day 2 mice received 200 ng/mouse pertussis toxin in 500 μL intraperitoneally. Mice were scored for clinical symptoms and weighed daily starting on day 7 until day 28 when the mice were sacrificed. Abbreviations: EAE, experimental autoimmune encephalomyelitis; SRM, standard reference materials; PM, particulate matter; OF, organic fraction; PBS, phosphate buffered saline; MOG, myelin oligodendrocyte glycoprotein

Fig. 2

Diesel exhaust particles worsen severity of EAE in vivo. Age matched female WT (C57BL/6 J) were exposed intranasal to 12.5 mg/mL PM of SRM1650b PM, SRM2975 PM, or solvent control all diluted in PBS. The mice were dosed intranasal 8 times starting at day − 12 every 3 days until day 9 after induction. Mice were scored daily for severity of disease. a, b SRM1650b and SRM2975 PM worsen severity of EAE. c, d SRM1650b and SRM2975 PM significantly increased peak score. e, f Both DEPs significantly increased cumulative clinical score over the 28-day period. g, h Neither DEP influenced day of disease onset which represents the first day of a clinical score greater than 0. Results are mean ± SEM of (PBS n = 12) (SRM1650b PM n = 13) and (SRM2975 PM n = 13). Significant differences among groups (p < 0.05) are indicated by an asterisk. Abbreviations: SRM, standard reference materials; PM, particulate matter; PBS, phosphate buffered saline; EAE, experimental autoimmune encephalomyelitis; DEP, diesel exhaust particles

Fig. 3

Organic fraction of diesel exhaust particles have differential effects on severity of EAE in vivo. Age matched female C57BL/6 J were exposed intranasally to 12.5 mg/mL PM of SRM1650b OF, SRM2975 OF, or PBS control. The mice were dosed intranasal 8 times starting at day − 12 and continuing every 3 days until day 9. Mice were scored daily for severity of disease. a SRM1650b OF had no effect on severity of EAE. b SRM2975 OF worsens severity of EAE. c SRM1650b OF does not increase peak clinical score. d SRM2975 OF significantly increased peak clinical score. e SRM1650b OF had no effect on cumulative clinical score. (F) SRM2975 OF significantly increased cumulative clinical score over the 28-day period. g, h Neither DEP OF influenced day of disease onset which represents the first day of a clinical score greater than 0. Results are mean ± SEM of (Solvent n = 10), (SRM1650b OF n = 10), and (SRM2975 OF n = 14). Significant differences among groups (p < 0.05) are indicated by an asterisk. Abbreviations: SRM, standard reference materials; PM, particulate matter; OF, organic fraction; PBS, phosphate buffered saline; EAE, experimental autoimmune encephalomyelitis; DEP, diesel exhaust particles

Fig. 4

Diesel exhaust particles alter T cell differentiation in vitro. Naïve CD4⁺ T cells were isolated from WT (C57BL/6 J) or Ahr^−/− mice. At time 0, cells were exposed to a dose-response (5 doses) of SRM1650b PM, SRM2975 PM, or PBS control and cultured for 3 days. a Representative flow plots of WT (top) and Ahr^−/− (bottom) Th17 differentiation at 12.5 μg/mL PM, measured by percent IL-17 positive cells. b SRM1650b PM enhanced Th17 differentiation at 12.5 μg/mL PM compared to PBS control and SRM2975 PM had no effect on Th17 differentiation (top). In Ahr^−/− cells, SRM1650b PM significantly increased % IL-17 positive cells at the two lowest doses. SRM2975 PM suppressed Th17 differentiation in Ahr^−/− cells at 40 μg/mL PM (bottom). c Representative flow plots of WT Treg differentiation at 20 μg/mL PM (top) and 0.78 μg/mL PM (bottom), measured by percent FOXP3 positive cells. d SRM1650b PM suppresses Treg differentiation at 40, 20, and 12.5 μg/mL PM and enhances Treg differentiation at 0.78 μg/mL PM (top). SRM2975 PM enhances Treg differentiation at 3.125 and 0.78 μg/mL PM (bottom). e Representative flow plots of WT (top) and Ahr^−/− (bottom) Th1 differentiation at 12.5 μg/mL PM, measured by percent IFNγ positive cells. f SRM1650b PM had no effect on Th1 differentiation in WT cells (top). SRM2975 PM enhanced Th1 differentiation at 40, 20, and 12.5 μg/mL PM (top). In Ahr^−/− cells, SRM1650b PM suppressed Th1 differentiation at 40 and 20 μg/mL PM but enhanced Th1 differentiation at 3.125 μg/ml PM (bottom). SRM2975 PM suppresses Th1 differentiation in Ahr^−/− cells at 40, 20, 12.5 μg/mL PM (bottom). Results are mean ± SEM of (WT Th17 n = 5), (Ahr^−/− Th17 n = 2), (WT Treg n = 2), (WT Th1 n = 5), and (Ahr^−/− Th1 n = 2). Significant differences among groups (p < 0.05) are indicated by an asterisk. Abbreviations: AHR, aryl hydrocarbon receptor; SRM, standard reference materials; PM, particulate matter; PBS, phosphate buffered saline

Fig. 5

Organic fraction of diesel exhaust particles impact T cell differentiation in vitro. Naïve CD4⁺ T cells were isolated from WT (C57BL/6 J) or Ahr^−/− mice. At time 0 cells were exposed to a dose-response (8 doses) of SRM1650b OF, SRM2975 OF, or solvent control and cultured for 3 days. a Representative flow plots of WT (left) and Ahr^−/− (right) Th17 differentiation at 5.0 μg/mL OC, measured by percent IL-17 positive cells. b SRM1650b OF enhanced Th17 differentiation at 10 and 5 μg/mL OC compared to solvent control (top left) and SRM2975 OF enhanced Th17 differentiation at 5μg/mL OC (bottom left). In Ahr^−/− cells, SRM1650b OF lost theTh17 differentiation effect (top right) and SRM2975 OF had no effect on Th17 differentiation (bottom right). c Representative flow plots of WT Treg differentiation at 0.00001 μg/mL OC measured by percent FOXP3 positive cells. d SRM1650b OF has no effect on Treg differentiation (top). SRM2975 OF suppresses Treg differentiation at 0.00001 μg/mL OC (bottom). Results are mean ± SEM of (WT Th17 SRM1650b OF n = 5), (Ahr^−/− Th17 SRM1650b OF n = 4), (WT Th17 SRM2975 OF n = 6), (Ahr^−/− Th17 SRM2975 OF n = 3), and (WT Treg SRM1650b OF and SRM2975 OF n = 4), Significant differences among groups (p < 0.05) are indicated by an asterisk. Abbreviations: AHR, aryl hydrocarbon receptor; SRM, standard reference materials; OF, organic fraction; OC, organic carbon

Fig. 6

Diesel exhaust PAH mixtures alter Treg differentiation and have AHR and CYP dependent effects on Th17 differentiation. Naïve CD4⁺ T cells were isolated from WT (C57BL/6 J), Ahr^−/−, or CypTKO mice. At time 0 cells were exposed to a dose-response (8 doses) of SRM1650b PAH mixtures, SRM2975 PAH mixtures, or solvent control and cultured for 3 days. a Representative flow plots of WT Treg differentiation at 0.00001 μg/mL OC, measured by percent FOXP3 positive cells. b SRM2975 PAH mixtures suppressed Treg differentiation at the lowest dose compared to solvent control. c Representative flow plots of WT (left), Ahr^−/− (middle), and CypTKO (right) at 5 μg/ml OC. d SRM1650b PAH mixtures enhanced Th17 differentiation at 40 μg/ml OC (top) and SRM2975 PAH mixtures had no effect (bottom). e In Ahr^−/− cells, SRM1650b (top) and SRM2975 (bottom) PAH mixtures had no effect on percent IL-17 positive cells. f In CypTKO cells, SRM1650b PAH mixtures enhanced Th17 differentiation at 5μg/ml OC (top) and SRM2975 PAH mixtures enhanced Th17 differentiation at the three highest doses (bottom). Results are mean ± SEM of n = 2 (WT Treg), n = 7 (WT Th17), n = 4 (Ahr^−/− Th17), and n = 6 (CypTKO Th17). Significant differences among groups (p < 0.05) are indicated by an asterisk. Abbreviations: AHR, aryl hydrocarbon receptor; CYP, cytochrome P450; TKO, triple knockout; SRM, standard reference materials; PAH, polycyclic aromatic hydrocarbons; OC, organic carbon

Fig. 7

Summary of DEP mediated autoimmune disease. Two DEPs, SRM1650b from a 4-cylinder diesel engine, and SRM2975 from a 2-cylinder diesel engine, were tested for the effects on T cell differentiation and autoimmune disease. SRM1650b enters the T cell, binds AHR, which then translocate to the nucleus and binds DNA, driving transcription of CYP enzymes (top). SRM1650b enhances Th17 differentiation in an AHR-dependent manner and worsens autoimmune disease (top). Based on the in vivo EAE data, SRM1650b requires the particle to aggravate autoimmune disease because of bioavailability of the PAHs and their ability to activate the AHR. Similarly, SRM1975 enters the T cell, binds AHR, moves to the nucleus, binds DNA, and drives transcription of CYP enzymes (bottom). SRM2975 enhances Th1 differentiation in an AHR-dependent manner and worsens autoimmune disease (bottom). Based on the in vivo EAE data demonstrating SRM2975 worsens autoimmune disease in PM and OF forms and the in vitro data showing a role of CYP enzymes in T cell differentiation, metabolism of SRM2975 plays a role in its ability to worsen autoimmune disease in that CYP metabolism of PAHs may lead to more potent intermediate that drives the response in vivo. Additionally, in the presence of PAHs and AHR activation, enhanced effector differentiation by both samples results in increase in Th17 or Th1 cells and a reduction in Treg cells. However, when PAHs are diluted at low doses, enhanced effector differentiation is no longer observed, and the balance is switched to enhanced Treg differentiation. Abbreviations: SRM, standard reference materials; DEPs, diesel exhaust particles; AHR, aryl hydrocarbon receptor; CYP, cytochrome P450; PAH, polycyclic aromatic hydrocarbons