Differential regulation of indoleamine 2,3-dioxygenase by TLRs and CLRs in human DCs: the role of aryl-hydrocarbon receptor Fabian Salazar1, Laurence Hall1, Farouk Shakib1 and Amir Ghaemmaghami1* 1 Division

of Immunology, Faculty of Medicine, University of Nottingham, UK. * Corresponding author: [email protected] INTRODUCTION

Dendritic cells (DCs) are specialized antigen presenting cells that have been shown to play a crucial role in the induction and elicitation of Th2-allergic immune responses. They are able to orchestrate adaptive immune responses by promoting or suppressing T-cell responses. One of the mechanisms that account for their induction of immune-suppression is their expression of the enzyme IDO. Indoleamine 2,3-dioxygenase (IDO) catabolizes tryptophan (TRP), which leads to TRP depletion and production of a series of immune-regulatory metabolites, inducing T cell apoptosis or tolerance. In the context of allergic diseases, there are some controversial studies suggesting that IDO might act either as an inducer or a protector of Th2 responses in models of experimental asthma. Besides these data, there is not much information about the molecular mechanisms of IDO regulation in an allergy context. Data published by our group demonstrated that Der p 1 (a major allergen from house dust mite) could, through engaging mannose receptor (MR) on DCs, down-regulates levels of IDO activity. This in turn could bias immune responses toward a Th2 phenotype. Aryl-hydrocarbon receptor (AhR) is a ligand-dependent transcription factor involved in the detection of intracellular or environmental changes. Previously, AhR has been shown to mediate IDO1 and IDO2 activation in response to TCDD, LPS and CpG in mouse DCs. In addition, kynurenine (KYN), one of the main TRP metabolites produced by IDO, can be recognized by AhR and modulates the immunogenicity of mouse DCs. Here we studied the IDO and AhR regulation by different pattern recognition receptors (PRRs) in human monocyte-derived DCs in order to define the role of these molecules in controlling allergen induced Th2 immune responses. RESULTS

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I FBA I LPS I I I I I I TNF-α IL-10 TNF-α IL-12 I IL-8 IL-6 IL-6 IL-1β IL-10 TLR-4 I MR DC-SIGN IL-1β I I ? DC I IRAK-M ? I TRAF2/3 p50 p50 Bcl3 I I canonical non-canonical I RelA p50 RelB p52 I AhR IDO I I I AhR RelB AhR RelB I ? IDO IL-8 I I MBA: Mannose-based agonist I FBA: Fucose-based agonist MBA

Fig.6. Here we have shown that MBA and FBA can modulates IDO and AhR in human DCs and its cytokine profile depending on the timing of stimulation. Co-stimulation with MBA and LPS leads to down-regulation of IDO and subsequent upregulation of AhR and cytokines. Conversely, priming with MBA leads to down-regulation of both IDO and AhR activity along with the cytokine production, however, AhR expression is augmented. Finally, FBA down-regulate IDO activity in human DCs and modify its cytokine profile regardless the timing of stimulation.

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Fig.4. A. AhR expression and activity in human DCs co-stimulated with mannan (M) (10µg/ml) and LPS (0.01µg/ml). B. AhR expression and activity in human DCs stimulated with mannan (M) (10µg/ml) prior LPS (0.01µg/ml) stimulation.

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2. Priming human DCs with mannose or fucose-based agonists renders them irresponsive to endotoxin driven up-regulation of IDO

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Fig.1. A. IDO activity and expression and cytokine profile in human DCs co-stimulated with House Dust Mite (HDM) extract (10µg/ml) (A), mannan (M) (10µg/ml) (B) or Lewis-X (LeX) (50µg/ml) (C) and LPS (0.01µg/ml) for 24h.

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4. AhR activity is differentially regulated by mannose-based agonists in response to TLR4 stimulation in human DCs

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Fig.3. IDO activity and cytokine profile in human MR-deficient DCs (A) or DC-SIGN-deficient DCs (B) stimulated with mannan (M) (10µg/ml) for 24h prior LPS (0.01µg/ml) stimulation for another 24h.

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1. Mannose and fucose-based agonists both down-regulate IDO while inducing different cytokine profiles in response to TLR4 stimulation in human DCs

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Fig.2. IDO activity and expression and cytokine profile in human DCs stimulated with House Dust Mite (HDM) extract (10µg/ml) (A), mannan (M) (10µg/ml) (B) or Lewis-X (LeX) (50µg/ml) (C) for 24h prior LPS (0.01µg/ml) stimulation for another 24h.

C-type lectin receptors (CLRs) are receptors involved in the recognition and uptake of glyco-antigens. which can facilitate access or modulate PRR-induced activation. Accordingly, different CLRs have been linked to toll-like receptors (TLRs) in allergen recognition and the downstream events leading to Th2 cell polarization. In this line, we have previously shown that MR and DC-SIGN are common receptors for several clinically relevant allergens. Mechanistically, it has been shown that Man-LAM, through MR, mediates IRAK-M induction that negatively regulates TLR signalling. Recognition of fucose-based agonists by DC-SIGN favours Th2 responses via activation of atypical NF-κB family member Bcl3. On the other hand, IDO induction has been showed to be dependent on non-canonical NF-κB pathway.

FUTURE DIRECTIONS NF-κB is a pleiotropic transcription factor controlling many physiological functions and is crucial for generating effective immune responses. RelA, a member of the canonical NF-κB pathway, has been shown to regulates AhR and AhR-dependent gene expression induced by LPS in human DCs. Additionally, AhR has been linked with RelB stabilization, a member of the non-canonical NF-κB pathway. It also has been described a physical and functional association between RelB and AhR that mediates the expression of IL-8. Accordingly, future experiments will aim to elucidate the role of NF-κB pathway in MBA/FBA regulation of IDO/AhR induction by LPS. References: Salazar et al. JACI 2013, Pathak et al. JBC 2005, Gringhuis et al. Nat.Commun. 2014, Puccetti et al. Nat.Rev.Immunol. 2007, Vogel et al. JBC 2013; Mol.Endocrinol. 2007.