The Aryl hydrocarbon receptor (AhR) is a ligand-‐ac8vated transcrip8on factor that when ac8vated, results in immunomodula8on. Dendri8c cells (DCs) are important immune cells involved in innate and adap8ve immune responses. Currently, it is not fully understood if different AhR ligands differen8ally affect DCs. We hypothesized that DC ac8va8on will be inhibited following AhR ac8va8on in an AhR ligand-‐specific manner. To test this hypothesis, we evaluated the effects of five disparate AhR ligands: TCDD, Benzo(a)pyrene, FICZ, Indole-‐3-‐carbinol and Indirubin on LPS-‐s8mulated DC 2.4 cells. Altera8ons in DC accessory molecules (CD40, CD80, CD86 and MHC class I) and the produc8on of inflammatory mediators (IL-‐6, TNF-‐α and nitric oxide) were assessed. Cell number and viability were assessed following treatment with all five compounds. All AhR ligands decreased the LPS-‐s8mulated produc8on of the pro-‐inflammatory cytokines, IL-‐6 and TNF-‐α, by the cultured DCs while only Indole-‐3-‐carbinol and Indirubin inhibited the genera8on of nitric oxide. As expected, LPS increased the rela8ve expression of MHC class I, CD40, CD80 and CD86 on the DC2.4 cells. Rela8ve to the vehicle-‐treated controls, TCDD increased the expression of CD40 and CD80; Benzo(a)pyrene decreased CD80; FICZ did not change any of the accessory molecules; Indole-‐3-‐carbinol decreased CD80 and CD86; and Indirubin decreased all four surface molecules. Collec8vely, these results suggest that DCs respond differently to these five dis8nct AhR ligands, effects that are ul8mately expected to alter the genera8on of immune responses mediated by DCs.
Abstract
AhR Ligand-‐Specific Effects on Dendri8c Cell Ac8va8on Kris8na Finsaas, Andrea Miller, and David Shepherd
Center for Environmental Health Sciences, Department of Biomedical & Pharmaceu8cal Sciences, University of Montana, Missoula, MT 59812
DC 2.4 cells
Treatment Groups: Uns8mulated & LPS-‐s8mulated Vehicle control (DMSO 0.01%) AhR ligands: TCDD (10 nM) BaP (10 µM) FICZ (10 nM) IC3 (50 µM) INDR (1 µM)
Pro-‐inflammatory mediators IL-‐6, TNF-‐α and nitric oxide (NO)
Accessory molecules CD40, CD80, CD86 and MHC class I
CD86
DC
CD40
CD80
MHC I
TNF-‐α
IL-‐6 DC
Materials and Methods
Figure 3: DC2.4 cells were seeded at 0.5x106 cells/mL/well into 6-‐well plates (n=4). Cells were either treated with Vehicle, TCDD, BaP, FICZ, I3C and INDR, and incubated for 48 hours. Ajer harvest, inflammatory mediator produc8on and cell surface molecule expression were evaluated by ELISA or flow cytometry, respec8vely.
Introduc<on Dendri8c cells are very important cells in the immune system. They are responsible for the uptake and presenta8on of an8gens from the 8ssue periphery to T cells, enabling the genera8on of the adap8ve immune response and the elimina8on of microbial pathogens. But this process can be interrupted by exposure to AhR ligands, changing how the immune system func8ons. Thus, the AhR plays an integral role in how the adap8ve immune system responds to different chemicals in the environment.
The Aryl hydrocarbon receptor (AhR) is a ligand-‐ac8vated transcrip8on factor found in the cytosol. The AhR is a common pathway that mediates the immunotoxicity of many xenobio8cs. However, it is not currently understood how various AhR ligands affect DCs or the immune system.
• 2,3,7,8-‐Tetrachlorodibenzo-‐p-‐dioxin (TCDD) • Halogenated aroma8c hydrocarbon (HAH) • Formed by industrial processes • Prototypical AhR ligand
• Benzo[a]pyrene (BaP) • Polycyclic aroma8c hydrocarbon (PAH) • By-‐product of automobile exhaust and cigarene smoke • Strong AhR ligand
• 6-‐formylindolo[3,2-‐b]carbazole (FICZ) • Natural AhR ligand • Tryptophan metabolite
• Indole-‐3-‐carbinol (IC3) • Natural AhR ligand • Found in cruciferous vegetables
• Indirubin-‐3’-‐oxime (INDR) • Natural AhR ligand • Found in the indigo plant and commonly used in tradi8onal Chinese medicine
This study inves8gated the effects of TCDD, BaP, FICZ, IC3, and INDR on DCs by evalua8ng the changes that occur following DC ac8va8on by LPS, a cons8tuent of gram-‐nega8ve bacterial cell walls. This study primarily serves to further establish the effects of AhR ac8va8on in DCs and model how immune cells respond to environmental toxic pollutants during bacterial infec8on.
Dendri<c Cell Number and Viability
Figure 4: Cell number and viability was calculated for each treatment group ajer a 48hr incuba8on period. Despite changes in cell number, cell viability stays rela8vely constant across all treatment groups.
The project described was supported by Award Number R25ES016247 and R01ES13847 (DMS) from the Na8onal Ins8tute Of Environmental Health Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the Na8onal Ins8tute Of Environmental Health Sciences or the NIH. Thanks to Jenna Benson and Tom Simones for their assistance in data presenta8on.
Acknowledgements
Conclusions • I3C and INDR inhibit DC prolifera8on. • At the concentra8ons tested, all five AhR ligands are not cytotoxic. • AhR ac8va8on in DCs anenuates the produc8on of IL-‐6 and TNF-‐α. • Accessory molecules were most sensi8ve to INDR>TCDD=I3C>BaP>FICZ. • DCs are sensi8ve to AhR ac8va8on. Pro-‐inflammatory Mediator Produc<on
Figure 5: The effects of a panel of AhR ligands on the produc8on of IL-‐6, TNF-‐α and nitric oxide (NO) by DC2.4 cells. DCs were uns8mulated or s8mulated with LPS for 48 hrs, supernatants collected and analyzed by ELISA (IL-‐6 and TNF-‐α) or the Greiss reac8on (NO).
* significant difference uns8mulated vs. LPS # significant difference treated groups vs. respec8ve control
RESULTS Summary
• I3C and INDR increased DC2.4 cell numbers following LPS ac8va8on. • No effects were observed on DC viability following treatment with all five AhR ligands. • All AhR ligands examined decreased the LPS-‐s8mulated produc8on of the pro-‐inflammatory cytokines, IL-‐6 and TNF-‐α. • I3C and INDR inhibited the produc8on of nitric oxide. • TCDD increased expression of CD40 and CD80. • BaP decreased CD80. • FICZ did not affect any of the accessory molecules tested. • I3C decreased CD80 and CD86. • INDR decreased MHCI, CD40, CD80 and CD86.
Future Direc<ons • Examine bone marrow-‐derived dendri8c cells (BMDCs) to establish if primary DCs are affected similar to DC2.4 cells. • Determine if the AhR ligand-‐specific effects in DCs are AhR-‐dependent by using BMDCs from AhR knockout mice. • Determine if these different ligands mediate their specific effects via the canonical or non-‐canonical AhR signaling pathways. • Assess cell cycle changes in the DCs ajer AhR ac8va8on.
AhR Signaling Pathway
Figure 2: When bound by an AhR ligand, the AhR can be ac8vated by two signaling pathways. In the canonical pathway, AhR dissociates from its chaperone proteins and migrates to the nucleus where it heterodimerizes with the Aryl hydrocarbon receptor nuclear translocator (ARNT) and modulates gene transcrip8on via Dioxin response elements (DREs). In the non-‐canonical pathway, AhR can alter NF-‐κB signaling, ul8mately affec8ng transcrip8on.
transcrip8on mRNA
Hsp90
Hsp90 TCDD TCDD
TCDD
AhR
AhR
ARNT
transla8on
TOXIC RESPONSE Cell membrane
nucleus
NF-‐κB
Role of Dendri<c Cells in the Immune System
Figure 1: Dendri8c cells are integral cellular contributors to innate and adap8ve immune responses.
Experiment 1 Experiment 2
IL-6 Production
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TNF-! Production
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NO- Production
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Cos<mulatory Molecule Expression
US=388.5±42 LPS=508.25±33
US=510.75±22 LPS=1630.25±134
US= 584.75±55 LPS=1047.75±57
US=706.25±36 LPS=740.75±96
US=791±29 LPS=708±31
US=1620±28 LPS=2352±70 #
US=1114±18 LPS=1522±17 #
US=914±18 LPS=3863±84
US=753±71 # LPS=603±10 #
US=1073±58 LPS=2357±78 #
US=1142±80 LPS=1551±60 #
US=1826±45 # LPS=2901±92 #
US=391±20 LPS=542.75±28
US=522.75±19 LPS=1944.5±201 # US=678±27
LPS=624.75±65 # US=624.25±18 LPS=1124.5±60
US=439±92 LPS=464±45
US=516.75±44 LPS=1223.5±221 #
US=8029.25±731 # LPS=8772±1053 #
US=1005.25±415 LPS=835.75±172
US=357.5±28 LPS=575±120
US=460±16 LPS=1564.5±37
US=648±15 LPS=1066.5±22
US=778.75±129 LPS=831±368
* significant difference uns8mulated vs. LPS # significant difference treated groups vs. respec8ve control
Num
ber of events
Rela8ve Expression
Figure 6: The effects of a panel of AhR ligands on the expression of CD40, CD80, CD86 and MHC I by DC 2.4 cells. DCs were treated with LPS and Vehicle, TCDD, BaP, FICZ, I3C, INDR for 48hrs. Cells were harvested, blocked for non-‐specific staining, and labeled with op8mally 8trated monoclonal an8bodies specific for the accessory molecules. DC2.4 cells were then analyzed on a BD Biosciences Aria Flow cytometer and representa8ve histograms generated using FlowJo sojware. Individual histograms are shown with corresponding mean values ± SEM for all treatment groups.
IL-6 Production
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TNF-! Production
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3500 *
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BD
NO- Production
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# • GREEN – AhR dependent increase • RED – AhR dependent decrease
Activation
T cells
Cytokine Produc8on
Cytokine Produc8on
Innate Immunity
Adap8ve Immunity
Pathogen Uptake
Immature Dendri8c Cell
Mature Dendri8c Cell
Adap<ve Immunity
NO
