Chemical Sensing in Development and Function of Intestinal Lymphocytes

Abstract

The immune system of the intestinal tract has the challenging task of recognizing and eliminating intestinal pathogens while maintaining tolerance to dietary and commensal antigens; therefore, it must be able to sense environmental cues within the intestine and mount suitable responses dictated by their pathogenic or nonpathogenic nature. The aryl hydrocarbon receptor (AHR) was originally characterized as a chemical sensor of the environmental pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)(1). More recently, AHR has emerged as a major chemical sensor expressed in many intestinal immune cells that enables them to distinguish nutritional and microbial cues and is, therefore, important for development, maintenance and function of the intestinal immune system. In this review, we will highlight recent advances in our knowledge of the role of AHR signaling in intestinal innate lymphoid cells (ILC), T cells and B cells.

Introduction

The aryl hydrocarbon receptor (AHR) belongs to the family of Per-Arnt-Sim (PAS) transcription factors, which are evolutionarily conserved and participate in the sensing of environmental stimuli. They encompass molecules that are involved in chemical sensing, such as AHR, in the regulation of circadian rhythm, such as BMAL1 and BMAL2, and in the detection of oxygen concentrations, such as HIF-1α, HIF-2α and HIF-3α(2–4). AHR is widely expressed in different tissues(5) and its activity is tightly controlled. It is normally present in the cell cytoplasm in an inactive state bound to proteins, including the chaperone Hsp90(6), AHR interacting protein (AIP)(7, 8) and the cochaperone p23(9), that enhance the ability of AHR to bind ligands and prevent its migration to the nucleus. Upon ligand binding, AHR is released from the chaperones, translocates to the nucleus and binds to the AHR-nuclear translocator (ARNT)(10). This heterodimer binds to dioxin responsive elements (DRE) of various enhancers and promoters to induce transcription of target genes(11). Typically, these include the microsomal cytochrome P450-dependent monooxygenases CYP1A1 and CYP1A2, which participate in the metabolism of AHR ligands(2). The AHR signaling pathway has been recently reviewed in detail(12).

Generation and degradation of intestinal AHR ligands

Although the most extensively studied ligands for AHR are pollutants and xenobiotics, such as benzo(a)pyrene, 3-methylcolantrene and TCDD(12), the broad expression of AHR in intestinal immune cells has suggested the presence of intestinal physiological ligands that activate AHR. Several AHR ligands with diverse origins and affinities have been reported (Fig. 1)(13, 14). Exogenous ligands comprise molecules derived from the diet, particularly cruciferous vegetables like broccoli, cauliflowers or cabbages. These vegetables convert tryptophan into glucobrassicin, which is metabolized into indole-3-carbinol (IC3). IC3 dimerizes in the acidic environment of the stomach, generating diindolylmethane (DIM) and indolylcarbazole (ICZ), which activate AHR(15). Other dietary ligands of AHR are natural flavonoids present in fruits and vegetables, such as galangin, genystein, chrysin, apigenin and quercetin(16).

Figure 1. Impact of AHR activation on different lymphocyte populations.

The effect of diverse AHR ligands on lymphocyte populations are depicted. TCDD= 2,3,7,8-tetrachlorodibenzo-p-dioxin, FICZ= 6-formylindolo(3,2-b)carbazole, IC3= indole-3-carbinol (IC3), ITE= 2-(1’H-indole-3’- carbonyl)-thiazole-4-carboxylic acid methyl ester, ILA= Indole-3-lactic acid, IAId= indole-3-aldehyde.

Members of the intestinal microbiota are also able to catabolize tryptophan into AHR ligands, which prolong the healthspan of the host(17). Species of lactobacilli, including Lactobacillus bulgaricus(18) and Lactobacillus reuteri produce AHR ligands, such as indole-3-aldehyde (IAId) and indole-3-lactic acid (ILA), that modulate responses of intestinal innate and adaptive lymphocytes, and ameliorate inflammation(19–21). Pathogenic bacteria, such as Mycobacterium tuberculosis (Mtb) and Pseudomonas aeruginosa (P. aeruginosa), produce pigmented virulence factors that act as AHR ligands. Phenazines from P. aeruginosa and naphthoquinone phthiocol (Pht) from Mtb bind AHR, eliciting an innate immune defense pathway that contains the infection in hematopoietic and epithelial cells(22). Malassezia furfur, a yeast that can be found in the skin, secretes AHR ligands, such as malassezin and ICZ(23, 24). Thus, because it can sense such a wide range of nutritional and microbial molecules from different origins, AHR plays a prominent role in deciphering environmental cues within the intestine.

AHR ligands also include tryptophan derivatives such as kynurenine, which is generated by an endogenous pathway of tryptophan metabolism that utilizes the enzymes indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO)(25). Although kynurenine is a low affinity AHR agonist, two novel condensation products derived from kynurenine, called trace extended aromatic condensation products (TEACOPs), were recently shown to be active at low picomolar levels; this suggests that kynurenine may act as an AHR pro-ligand that requires yet undefined chemical conversions to become an effective AHR agonist(26). Exposure of endogenous or endogenous tryptophan to UV light also generates a potent metabolite that activates AHR, known as 6-formylindolo(3,2-b)carbazole (FICZ)(27). Whether the endogenous and exogenous pathways that degrade dietary tryptophan to generate AHR ligands affect each other and/or AHR activation remains unclear.

The bioavailability of AHR ligands also depends on AHR activation and subsequent transcriptional activation of monooxygenases, such as CYP1A1, because monooxygenases participate in the metabolism of AHR ligands. Accordingly, constitutive expression of Cyp1a1 in mice in which the Cyp1a1 gene is placed under control of the Rosa26 promoter results in much lower concentrations of AHR ligands in the intestine(28). Thus, AHR activation by AHR ligands triggers a negative feed-back loop that limits the availability of AHR ligands and hence regulates AHR activation.

Impact of AHR on innate lymphoid cells

Innate lymphoid Cells (ILCs) are non-T, non-B lymphocytes that are present throughout the body and enriched in frequency at mucosal surfaces. They are activated primarily through cytokine receptors and respond by producing signature cytokines that mirror adaptive T cell responses. Natural killer cells (NK) represent the innate counterpart of cytotoxic T cells, while ILC1s, ILC2s and ILC3s share key features with Th1, Th2 and Th17/Th22 cells respectively(29, 30). ILC3s secrete IL-22 and promote postnatal development of cryptopatches and isolated lymphoid follicles through expression of lymphotoxin(31). AHR is a central node in ILC3 development (Fig. 1): while Runx3 and its downstream target RorγT promote ILC3 development through AHR induction(32), Ikaros inhibits ILC3 development through inhibition of AHR expression(33). AHR acts by inducing expression of Notch(31), and by stabilizing c-Kit expression (34). AHR-deficient mice harbor very few ILC3s and consequently have poorly developed cryptopatches and isolated lymphoid follicles(34) as well as insufficient production of IL-22 in the intestine; these defects result in susceptibility to Citrobacter rodentium (35). Moreover, minimal intestinal IL-22 permits overgrowth of segmented filamentous bacteria (SFB). This dysbiosis causes a compensatory increase in Th17 that induces colitis(36); inadequate intestinal IL-22 also exacerbates weight loss after Toxoplasma gondii infection due to an enhanced T cell response to T. gondii and microbiota antigens(37). AHR ligands that are produced by members of the microbiota under conditions of unrestricted tryptophan supply (like indole-3-aldehyde generated by Lactobacilli) foster IL-22 production by ILC3s and resistance to Candida albicans colonization(19). Conversely, constitutive expression of Cyp1a1, which severely limits the amount of AHR ligands present in the intestine, results in fewer effective ILC3s and consequent susceptibility to enteric infections(28). In humans, IL-22 producing ILC3s differentiate into IFNγ-producing ILCs if AHR signaling is blocked, suggesting that ongoing AHR signaling is required to maintain the prototypic ILC3 phenotype (38). In addition to ILC3s, AHR ligands promote NK cell cytotoxic activity and IFNγ production, which support NK cell-mediated antitumor activity in vivo(39). Moreover, AHR is required for IL-10 production by NK cells after T. gondii infection(40). Thus, AHR signaling impacts several arms and cell types within the intestinal innate lymphoid system.

Requirement of AHR for differentiation of intestinal T cells

The role of AHR in T cells was initially demonstrated by the induction of T cells with regulatory potential in graft-versus-host-disease after administration of TCDD to mice(41). AHR signaling contributes to the differentiation and function of several T cell populations (Fig. 1). AHR is required for IL-22 production by TH17 cells in vivo and in vitro(42–45), particularly in the presence of TGFβ(46). Moreover, distinct AHR ligands - TCDD and FICZ - promote the differentiation of Tregs and Th17 cells respectively(42, 43). The induction of either Tregs or Th17 cells by high affinity ligands depends on the dose and duration of AHR activation(47).

AHR signaling also promotes the development of Tr1 cells in mice and humans. Specifically, stimulation of T cells with TGFβ and IL-27 induces AHR, which binds to the transcription factor c-Maf. AHR and c-Maf transactivate the IL-10 and IL-21 promoters, resulting in the differentiation of Tr1 cells(48). Moreover, AHR contributes to metabolic programming of Tr1 cells: while HIF1-α promotes the early glycolytic metabolism of Tr1 cells, AHR promotes HIF1-α degradation and takes control of late Tr1 cell glycolysis(49). Finally, AHR enhances Tr1 expression of genes involved in intestinal homing, such as CCR9(50). In the small intestine, AHR signaling is required for development and maintenance of disparate T cell populations located underneath the epithelial cell layer of the small intestine, collectively defined as intraepithelial lymphocytes (IEL) (Fig. 1). TCRγδ IELs (TCR Vγ5 cells) and TCRαβ CD8αα+ IELs require dietary AHR ligands. While these IELs can develop, home to their target organs, and proliferate in the absence of AHR, they require AHR to persist at epithelial sites in both the intestine and skin(51). More recently, AHR signaling was found to be critical for the development of another IEL population located in the small intestine. The differentiation of TCRαβ CD4+CD8αα+ IELs (DP IELs) from TCRαβ CD4+ IELs requires cell-intrinsic AHR activation by indole −3-lactic acid produced by L. reuteri(20) together with TGFβ(52). Collectively, these findings illustrate how AHR signaling bridges environmental cues with intestinal T cell responses.

Impact of AHR on B cells

The suppression of humoral immune responses by TCDD exposure has been known since the 1970s(53). B cells express low levels of AHR, which increase after BCR activation and IL-4 treatment through a STA6-dependent pathway(54, 55). Notably, marginal zone B cells, B1 B cells and plasma cells have higher levels of AHR than do other subsets(56). TCDD binding to AHR induces BACH2 expression, which inhibits PRDM1 expression and B cell differentiation(57). The AHR ligand 2-(1’H-indole-3’- carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), which derives from tryptophan metabolism, suppresses the expression of IgM, IgG1, and IgE in purified B cells activated with anti-CD40 antibody and IL-4(58). Interestingly, in vivo, adoptively transferred AHR-deficient B cells proliferate poorly compared to AHR sufficient cells, due to their inability to express cyclin O(56). Thus, endogenous ligands may have a different impact on B cells than do exogenous ligands like TCDD. In humans, TCDD stimulation of B cells activates SHP-1, augments BCL-6 protein levels and curtails CD80 and CD69 expression, which collectively indicate impaired cellular activation(59). TCDD also enhances transcription of BZLF1, an immediate-early gene of the Epstein-Barr virus (EBV) that mediates the switch from the latent to the lytic form of infection in B cell lines and in a salivary gland epithelial cell line(57). AHR activation in human B cells can also reduce IgM production; a combination of 3 variants (P517S+R554K+V570I) is necessary to impair TCDD-mediated suppression of IgM secretion(60). While the studies outlined above have shown that AHR can temper B cell responses, extensive work still needs to be done to understand how AHR ligands affect humoral responses at mucosal sites.

Concluding Remarks

Research from the past few years has emphasized the crucial role of AHR signaling in several intestinal populations. While we now know that diverse T cell populations, as well as ILC3s rely on AHR ligand binding to develop, produce cytokines and/or remain at mucosal sites, more research is needed to determine how the vast array of AHR ligands have such disparate impacts on immune cell populations. Several factors likely affect the outcome of the interaction of an AHR ligand with the immune system: 1) the origin of the ligand - is it is an endogenous ligand, produced by the microbiota, a pathogen or a product of the diet? 2) the concentration of the AHR ligand as well as its localization in the organism – is it present at similar concentrations throughout the intestine or is produced in particular areas? 3) how susceptible is the AHR ligand to degradation by enzymes like Cyp1a1 - can it be metabolized into compounds with higher or lower affinity?

While the AHR pathway may be an attractive candidate to modify the intestinal immune response in order to control the overt inflammation characteristic of inflammatory bowel disease, more studies are needed to understand the nature of each AHR ligand so that we can identify specific ligands that render the desired effect.

Highlights.

• AHR senses environmental cues that modulate intestinal immune cells.

• AHR signaling affects multiple immune cell types, including innate lymphoid cells, T cells and B cells.

• AHR is activated by a vast array of exogenous and endogenous ligands that engender diverse outcomes depending on the specific ligand and cell type involved.