The aryl hydrocarbon receptor: a rehabilitated target for therapeutic immune modulation.

The AHR has been identified in the earliest multicellular organisms, including nematodes and placozoans 38 , 124 . However, the roles of the AHR in the regulation of inflammation appear to have emerged with the evolution of the first vertebrates 124 . The function of AHR signalling in immune cells has been extensively reviewed elsewhere 125 - 127 so we will provide a brief discussion of these effects, particularly those relevant to targeting AHR therapeutically. DCs are professional antigen-presenting cells, bridging innate and adaptive responses to induce tolerance or immunity 128 . DCs capture endogenous and exogenous antigens, transporting them to secondary lymphoid organs to activate T cells 129 . In this context, AHR regulates DC differentiation and function, impacting T cell activity ( Fig. 2 ). As an example of modulating DC differentiation and activation, AHR inhibition by the synthetic antagonist StemRegenin 1 (SR1) boosts the differentiation of CD34 + haematopoietic progenitor cells into myeloid and plasmacytoid DCs, which exhibit increased interferon-α (IFNα), IL-12 and TNF secretion along with higher expression of co-stimulatory molecules, inducing potent T cell responses 130 . SR1-driven AHR inhibition also stimulates the differentiation of monocytes into monocyte-derived macrophages, whereas AHR activation by FICZ promotes the generation of monocyte-derived DCs via the induction of IRF4 and BLIMP1 (ref. 131 ). Therefore, the AHR stimulates the differentiation of monocytes into both DCs and macrophages. Similarly, AHR activation by TCDD promotes the differentiation of immature DCs into CD11c + MHCII high mature DCs in vitro 132 . The exogenous AHR agonists VAF347, β-naphthoflavone (BNF) and TCDD are also reported to inhibit differentiation of human monocytes into Langerhans DCs 133 . Additionally, oral administration of indoxyl-3-sulfate (I3S) and I3C (Trp-derived metabolites from the diet and microbiota) impaired the differentiation of plasmacytoid DCs that produce type I interferon in mesenteric lymph nodes in response to viral infections 134 . The AHR also impacts antigen presentation in DCs, and hence their ability to activate and polarize T cells. For example, VAF347 inhibits allergic lung inflammation by inducing human tolerogenic DCs that display reduced CD86, HLA-DR and IL-6 expression 135 . Moreover, AHR activation by the agonist 2-(1′ H -indole-3′-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE) in isolated splenic DCs reduced the production of IL-12 for T H 1 cell polarization and IL-6, IL-23 and TGFβ for T H 17 cell polarization 136 . In vivo, ITE administration augmented TGFβ1 and IL-10 production in DCs during EAE, promoting the expansion of Foxp3 + T reg cells and IL-10 + type 1 regulatory T cells (Tr1 cells) to limit central nervous system (CNS) inflammation 136 . Multiple mechanisms contribute to the expansion of Foxp3 + T reg cells and IL-10 + Tr1 cells following AHR activation in DCs. These mechanisms include IL-27 (ref. 137 ) and retinoic acid 136 production, and also upregulation of IDO1 and its paralogue IDO2, which increases Kyn production 138 , 139 and results in AHR activation. AHR signalling in DCs has important consequences for tissue barrier homeostasis. For example, AHR deficiency in DCs impairs Paneth cell function and increases goblet cell differentiation, altering the intestinal epithelial barrier and promoting colitis development 140 . Similarly, environmental chemicals that inhibit AHR worsen inflammation by boosting pro-inflammatory DC and T cell responses 109 . These findings are relevant to multiple mucosal barriers, because mice deprived of dietary AHR ligands also display impaired Langerhans cell migration and exacerbated T cell responses in asthma-like allergic models 141 . Conversely, the adoptive transfer of AHR-induced tolerogenic DCs to a colitis mouse model controls the balance between effector and regulatory T cell responses and ameliorates intestinal inflammation 142 . Although several studies demonstrate an anti-inflammatory role of AHR signalling in DCs, in certain contexts the AHR can participate in proinflammatory responses. AHR stimulation of bone marrow-derived dendritic cells (BMDCs) and splenic DCs with TCDD increased MHC class II and CD86 expression, as well as IL-6 and TNF production in vitro 143 , 144 . In addition, AHR deficiency in Langerhans cells results in the expansion of T H 2 cells and IL-10-producing Tr1 cells upon epicutaneous ovalbumin (OVA) sensitization 145 . Additionally, DC responses can be differentially influenced by various AHR agonists. For example, benzo(a)pyrene — but not FICZ or I3S — downregulates CD83 expression, stabilizing MHC class II expression in the DC membrane. Benzo(a)pyrene-treated DCs exhibit reduced CD80 expression and promote differentiation of Foxp3 + T reg cells 146 . Taken together, these findings suggest that the effects of AHR signalling in DCs involve ligand-specific and context-specific effects. Hence, specific AHR ligands can be designed for the modulation of DC function in vivo, offering therapeutic potential for enhancing immune responses in cancer and infections or reducing reactions in autoimmunity. Designing these ligands requires considering the target, environment and desired outcomes. Understanding AHR signalling in DCs can help identify biomarkers for predicting and monitoring therapy responses. Innate lymphoid cells (ILCs) contribute to immune surveillance and tissue repair, with important functions in autoimmune and infectious diseases 147 . Five ILC subsets have been defined based on the expression of specific transcription factors: T-bet + EOMES − ILCs (ILC1s), GATA3 + ILCs (ILC2s), RORγt + ILCs (ILC3s), T-bet + EOMES + natural killer cells and ID3 + regulatory ILCs 148 . The AHR is crucial for the maintenance and proliferation of CD49a + CD49b − hepatic ILC1s (refs. 149 , 150 ), and human ILC2s express the AHR upon IL-25 and IL-33 stimulation 151 , but the functional role of the AHR in ILC1s and ILC2s remains largely unclear. The AHR enhances ILC3 survival by driving the expression of IL-7R, and the anti-apoptotic proteins Bcl-2 and Ki67, thereby controlling cell proliferation 152 . Moreover, the AHR supports NKp46 + IL-22-producing ILC3s by inducing expression of the Notch transcription factor 153 . Finally, the AHR is critical for the capacity of ILC3s to fight off infections. For example, AHR-driven IL-22-producing ILC3s (refs. 154 , 155 ) and RORγt + ILC3s (ref. 156 ) are essential for clearance of segmented filamentous bacteria and Citrobacter rodentium , respectively. Thus, the AHR plays a critical role in the regulation, maintenance and function of ILCs 157 . T H cells encompass distinct subsets linked to specific functions and molecular phenotypes 158 . As already mentioned, the AHR influences T cell responses indirectly through the modulation of antigen-presenting cells. In addition, AHR signalling within T cells has important effects on their polarization and function ( Fig. 2b ). T H 17 cells, T reg cells and Tr1 cells display the highest AHR expression levels 126 . TGFβ in combination with IL-6 or IL-21 (refs. 159 - 161 ) prompts the differentiation of RORγt-driven T H 17 cells, which are characterized by the production of IL-17A, IL-17F and IL-22 (refs. 25 , 26 , 162 ) ( Fig. 2b ). In addition, IL-23 supports the maturation of T H 17 cells and the development of cellular activities relevant to their pathogenic roles in inflammatory autoimmune disorders 163 ; for example, the suppression of IL-10 production and expression of GM-CSF 164 . Interestingly, IL-6 and IL-21 induce activation of the STAT3 transcription factor, which drives AHR expression in T H 17 cells 87 . Indeed, the AHR cooperates with STAT3 to drive expression of Aiolos, a transcription factor in the Ikaros family, and suppress IL-2 production, thereby alleviating inhibitory effects of IL-2 on the early stages of T H 17 cell differentiation 165 ; the AHR further facilitates T H 17 cell differentiation by inhibiting STAT1 and STAT5 activation 111 , 166 . In addition, the AHR promotes RORγt recruitment to the Il22 promoter, inducing IL-22 production 86 , 167 . Of note, the AHR also cooperates with other transcriptional regulators to drive Il22 expression in CD4 + T H 22 cells, which do not produce IL-17 and IFNγ and play critical roles in mucosal immunity 168 - 171 . Tr1 cells are regulatory cells that do not express the transcription factor Foxp3 but produce high levels of IL-10 (ref. 172 ) ( Fig. 2b ). Tr1 cell differentiation is induced by IL-27 (refs. 161 , 173 , 174 ), which triggers STAT3-driven AHR expression. The AHR cooperates with the transcription factor c-Maf to promote IL-10, IL-21 (ref. 84 ) and CD39 (refs. 87 , 96 ) expression in Tr1 cells; IL-21 stabilizes Tr1 cells in an autocrine manner 175 , whereas IL-10 and CD39 contribute to Tr1 cell immunosuppressive effects. CD39 is an ectonucleotidase that also has an important role in Tr1 cell differentiation 87 . T cell activation triggers the release of ATP to the extracellular medium, and this extracellular ATP (eATP) stabilizes HIF-1α, which promotes AHR degradation by the proteasome 176 . Furthermore, AHR-induced CD39 depletes eATP to facilitate Tr1 cell differentiation, and CD39 also catalyses the degradation of pro-inflammatory eATP into AMP, which is then used by CD73 to produce anti-inflammatory adenosine 85 , 87 . Interestingly, the AHR promotes the conversion of T H 17 cells into Tr1 cells 177 , probably as part of a mechanism aimed at limiting immunopathology. AHR activation by TCDD, in the presence of TGFβ, enhances and stabilizes Foxp3 expression in T cells by inducing expression of transcription factors such as SMAD1 and/or Aiolos. Foxp3 and Aiolos inhibit the expression of genes linked to T cell functions, such as IL-2 (ref. 85 ). AHR signalling also impairs STAT1 activation, facilitating polarization of Foxp3 + T reg cells 111 , 136 . AHR expression is linked to Foxp3 + T reg cells that co-express the T cell immunoreceptor with immunoglobulin and ITIM domains (TIGIT), suggesting that it contributes to the regulatory activity of specific Foxp3 + T reg cell subsets 25 . In summary, the AHR acts as a critical regulator of pathogenic and regulatory CD4 + T cell responses. AHR signalling also controls other lymphocyte populations, such as CD8 + T cells. AHR deficiency increased the abundance of short-lived CD8 + effector T cells (CD127 − KLRG1 + ) and central memory cells (CD44 + CD62L + ), while reducing granzyme-B-producing tissue-resident CD8 + CD69 + CD103 + memory T cells 178 . Indeed, AHR signalling supports memory T cells in the skin to help eliminate microbes 179 . On the other hand, the AHR contributes to the exhaustion of tumour-reactive CD8 + T cells in the tumour microenvironment 180 . Similarly, AHR activation by TCDD suppresses CD8 + T cell responses against influenza virus 181 , 182 . Future studies should determine whether specific microenvironments or developmental stages are behind these seemingly opposing results. Finally, the AHR is important for determining the fate of B cells. B cells undergo class-switch recombination, a genetic process enabling alterations in the constant regions of the antibody molecule. These alterations facilitate the production of antibodies across different classes, switching from IgM to IgG, IgA or IgE while maintaining antigen specificity 183 . The AHR negatively regulates class-switch recombination and the differentiation of B cells into plasmablasts and antibody-secreting plasma cells 184 . In addition, the AHR is also essential for the differentiation of IL-10-producing CD19 + CD21 hi CD24 hi regulatory B cells, which limit inflammation in multiple assays 185 . Based on the success of B cell targeting therapies in autoimmune diseases such as MS 118 , the role of the AHR in regulating B cells indicates that it could be targeted for the treatment of T cell-driven autoimmune disorders. However, additional studies are needed to define the effects of AHR modulation on B cell antigen presentation.

As well as the clinical trials with Tapinarof discussed above, 20 other clinical trials have either been completed or are underway to interrogate the clinical effects of AHR modulation, using endogenous and dietary ligands as well as synthetic small molecules ( Table 2 ). Among endogenous ligands, supplementation with l -Trp, an amino acid metabolized into AHR agonists 126 , is being evaluated in individuals with biopsy-confirmed coeliac disease who do not respond to a gluten-free diet ( NCT05576038 ). Another study evaluates the discriminatory potential of endogenous agonists of AHR, such as Kyn, in identifying individuals with hypertension associated with obstructive sleep apnoea ( NCT04646902 ). Inulin is a dietary fibre from plants that induces CYP1A expression by directly interacting with the AHR or enhancing AHR binding to XREs 266 . The impact of inulin on the gut microbiome, gut barrier function, bacterial metabolites and immune cells in individuals with chronic kidney diseases has been investigated ( NCT05071131 ). Both preclinical and early clinical findings support the therapeutic potential of these compounds, but further clinical studies are needed before a definitive conclusion can be drawn about their clinical efficacy. Increased interest in the AHR as a therapeutic target is evidenced by the rise in preclinical drug development programmes targeting the AHR ( Table 3 ). DMVT-506, an AHR agonist with a similar pharmacological profile to tapinarof, is under development by Dermavant Sciences for other inflammatory diseases. In addition to topical formulations, DMVT-506 is being developed as an oral modified-release dosage form for IBD and as a dry powder inhalant for respiratory diseases including asthma and chronic obstructive pulmonary disease. Azora Therapeutics is developing both topical and oral formulations of the natural AHR agonist indirubin for allergic dermatitis 267 and Galileo Biosystems is developing therapeutic AHR modulating agents for the treatment of inflammatory and autoimmune diseases. Eli Lilly recently had two patents issued for AHR agonists, suggesting active interest in the area of inflammation 268 , 269 . Examining abandoned clinical programmes is important to identify common pitfalls, refine methodologies, improve future research design, optimize resource allocation and ensure ethical considerations and patient safety, ultimately contributing to more successful clinical research outcomes. For instance, laquinimod is a small molecule AHR agonist that has been in development for neuroinflammatory disease. In the EAE mouse model of MS it had beneficial effects on clinical score, inflammation and demyelination that were not observed in AHR knockout mice 270 . Laquinimod progressed to human clinical studies. In a double-blind randomized phase III clinical trial (ALLEGRO) in patients with relapsing–remitting MS, a 0.6 mg oral dose of laquinimod showed modest benefit compared with placebo in reducing the annualized relapse rate, the primary end-point of the study 271 . A subsequent clinical trial (BRAVO) assessed the safety and efficacy of 0.6 mg oral laquinimod compared with the standard of care (IFNβ 1a once-weekly injection) and with placebo in patients with relapsing–remitting MS. Although secondary measures of clinical benefit were observed, the primary end-point of the annualized relapse rate was not significantly different after laquinimod treatment compared with placebo, in contrast to the benefit seen with IFN-β-1a treatment 272 . These results prompted the termination of laquinimod development by Teva Pharmaceuticals. Interestingly, in the BRAVO study there was a baseline imbalance between the active and placebo groups, with worse conditions in the active groups, which might have influenced the drug effect assessment. Therefore, further investigation of laquinimod mechanisms should provide a clearer understanding of its effects on progressive MS. An eye drop formulation of laquinimod is being developed for the treatment of the inflammatory disease non-infectious uveitis. A phase I clinical study ( NCT05187403 ) established that this formulation is safe and well tolerated, and a phase II study in non-infectious uveitis is planned 273 . Although laquinimod did not demonstrate sufficient efficacy in MS clinical trials, its potential for treating other neuroinflammatory conditions remains under investigation. Indeed, its suppressive effects on astrocyte-intrinsic pathogenic responses 274 suggests potential applications for the therapeutic management of neurodegeneration.

We and others have postulated that the AHR functions as a key regulator of homeostasis that participates in a negative feedback loop to limit immunopathology during viral infections 116 , 244 . These regulatory mechanisms are exploited by pathogens and tumours to evade host immunity. Indeed, targeting the AHR offers a promising therapy for infectious diseases and cancer due to its role in immune regulation and evasion. Given the central role of the AHR in immune regulation, it is not surprising that pathogens activate AHR signalling to evade protective immunity. Therefore, pharmacologic modulation of the AHR might help manage infectious pathologies. AHR activation is detected in multiple infection models, including Zika virus (ZIKV), SARS-CoV-2 and influenza. Using AHR-deficient murine embryonic fibroblasts, Yamada et al. reported that AHR signalling limits type I interferon production induced by various viral pathogens including vesicular stomatitis virus, influenza and herpes simplex virus type 1 (ref. 245 ). The AHR agonists Kyn and FICZ suppressed type I interferon production, whereas inhibitors of IDO1 and TDO (enzymes that catalyse the generation of Trp-derived AHR agonists) enhanced the antiviral response. Furthermore, pharmacologic inhibition of the AHR with the antagonist CH-223191 upregulated the antiviral type I interferon response. These observations suggest that AHR signalling is activated by viruses to downregulate the host antiviral type I interferon response. Indeed, the AHR activates TIPARP, which subsequently inhibits the serine/threonine kinase TANK binding kinase 1 (TBK1), likely by ADP-ribosylation of the kinase domain, and suppresses type I interferon production. This negative feedback loop might have been evolutionarily selected to limit immunopathology but is hijacked by viruses to promote viral replication and infectivity. Thus, both the AHR and TIPARP are potential targets for treating viral infections 245 . Similarly, ZIKV has also been shown to limit type I interferon responses via the AHR. Indeed, pharmacologic inhibition of the AHR using HP163 suppressed ZIKV infection and developmental abnormalities, including intrauterine growth retardation and microcephaly, in a mouse model of congenital Zika syndrome 116 . Similar antiviral effects were detected following pharmacologic AHR inhibition in dengue virus-infected human cell cultures 116 . More recently, a similar role for the AHR in coronavirus infection was reported. AHR activity is increased in SARS-CoV-2-infected cells, including in lung tissue from patients infected with SARS-CoV-2. The AHR antagonist CH-223191 suppressed the in vitro replication of several coronaviruses, including HCoV-229E and SARS-CoV-2 (ref. 246 ). Collectively, these observations support the potential of AHR inhibition as a therapeutic strategy in viral infections. The AHR is overexpressed and chronically activated in different types of cancer, including glioblastoma 96 , 247 , breast cancer 248 , and oral and gastric cancers 14 . Chronic AHR activation can suppress tumour-specific immunity and trigger stem-like cancer cell formation, driving tumour migration, proliferation and metastasis 249 - 252 . Cancer cells express high levels of IDO1 and TDO2, generating Trp-derived AHR agonists that suppress protective antitumour immune responses 253 - 255 . For example, we showed that glioblastoma cells activate the AHR in tumour-associated macrophages. AHR activation in tumour-associated macrophages promotes KLF4 expression, suppressing NF-κB activation while driving the expression of CD39, which promotes CD8 + T cell dysfunction 96 . In addition, IFNγ induces cancer cell apoptosis via STAT1; however, stem-like cancer cells that express high levels of AHR and IDO1 develop resistance to cell death 256 . Moreover, the AHR–IDO1 axis upregulates exhaustion pathways in tumour-infiltrating T cells, suppressing immunity during oral squamous cell carcinoma 257 . In addition, IL-2 signalling contributes to CD8 + T cell exhaustion in tumour microenvironments via an AHR-dependent mechanism 180 . Finally, a pathway induced by IL-4-induced 1 (IL4I1) generates AHR-activating indole metabolites and kynurenic acid (KA) 258 . Based on these and other findings, preventing AHR activation constitutes a promising approach to revert tumour immunosuppression 259 - 261 . Indeed, the suppression of AHR activity, either pharmacologically or by genetic deletion, reduced tumour growth by increasing IFNγ + CD8 + T cells within tumours 257 . Interestingly, gut commensal Lactobacilli generate Trp-derived AHR agonists. Ampicillin treatment to decrease Lactobacilli , or the removal of dietary Trp, promoted the intratumour accumulation of CD8 + T cells and reduced the tumour size 262 . In complementary studies, probiotics targeting AHR signalling were used to improve the immune response against tumour cells. In particular, indole-3-carboxylic acid (ICA) produced by Lactobacillus gallinarum can reduce T reg cell differentiation and increase the response of CD8 + T cells to PD1 immune checkpoint blockade in colorectal cancer 263 . Mechanistically, ICA inhibits IDO1, decreasing levels of Kyn available for T reg cell differentiation 263 . Thus, synthetic or microbiome-produced small molecules targeting AHR signalling have the potential to be used in cancer immunotherapy approaches. AHR antagonists are being clinically evaluated. BAY2416964 is a small molecule designed to inhibit the AHR, with the goal of boosting the immune response to cancer. A phase I clinical trial is determining the highest tolerable dose of BAY2416964 in patients with advanced cancer ( NCT04069026 ). Additionally, BAY2416964 is being administered in combination with the anti-PD1 immune checkpoint inhibitor pembrolizumab in patients with advanced solid cancers, such as head and neck cancer, lung cancer and bladder cancer ( NCT04999202 ). Similarly, another AHR antagonist named IK-175 (Ikena Oncology) is being evaluated in early-stage trials as a single agent and in combination with nivolumab, a humanized anti-PD1 antibody, for individuals with advanced or metastatic solid tumours, including urothelial carcinoma ( NCT04200963 ). By contrast, an anti-tumorigenic role of the AHR has been described in recent studies. IL-6 and AHR activation in CD8 + T cells drives the differentiation of an IL-22-producing subset of cells, termed Tc22 cells, which exhibit potent cytolytic activity and robust tumour control, and are associated with improved recurrence-free survival in ovarian cancer 264 . In addition, the probiotic Lactobacillus reuteri is reported to migrate to melanoma, where it secretes the AHR agonist indole-3-aldehyde (I3A), enhancing antitumour immunity by stimulating IFNγ-producing CD8 + T cells within the tumour microenvironment 265 . Future research should address these seemingly contradictory roles of the AHR in tumour-specific immunity.

Probiotics are live microorganisms, primarily bacteria and yeast, found in fermented foods and dietary supplements. They are thought to promote digestive health and a balanced microbiota when consumed in adequate quantities 275 . Additionally, probiotics can be engineered to exert modulatory effects on the immune system 276 , 277 . Indeed, synthetic probiotics have been recently developed as immunotherapeutic approaches for cancer 278 and autoimmunity 279 , 280 . The commensal flora is a physiologic source of AHR agonists, hence probiotic-driven approaches for the modulation of AHR signalling constitute exciting new avenues for immunomodulation ( Fig. 3 ). In 2011, Lactobacillus bulgaricus OLL1181 was shown to induce AHR activation, increasing CYP1A1 and prostaglandin E 2 (PGE 2 ) levels and ameliorating DSS-induced colitis in mice 281 . Since then, several probiotics have been shown to promote intestinal barrier repair and suppress intestinal inflammation by modulating Trp metabolism and AHR activation, including Propionibacterium freudenreichii (strain ET-3) 282 , Lactobacillus acidophilus 283 , Akkermansia muciniphila 284 , Bifidobacterium bifidum 285 , 286 and Ligilactobacillus salivarius (Li01) 287 . The diet and the commensal flora participate in complex interactions that contribute to the regulation of inflammation via the AHR and other mechanisms 288 , 289 . For example, L. reuteri uses Trp as a source of energy. Interestingly, dietary Trp promotes the expansion of L. reuteri , with the subsequent production of Trp metabolites that activate AHR-driven IL-22 production and STAT3 phosphorylation 290 . By maintaining and expanding gut T reg cells 291 , these Trp metabolites promote intestinal epithelial cell proliferation and mucosa homeostasis 292 . In addition, AHR activation by Trp metabolites downregulates the ThPOK transcription factor in intra-epithelial CD4 + T lymphocytes, generating CD4 + CD8αα + cells that exhibit tolerogenic functions in the small intestine 293 ( Fig. 3 ). The gut–brain axis provides an avenue for the modulation of AHR signalling by the commensal flora. Indeed, some metabolites derived from dietary Trp cross the blood–brain barrier and act directly on CNS-resident microglia 216 and astrocytes 215 to activate AHR signalling, limiting the intrinsic pro-inflammatory activities of these cells as well as microglia–astrocyte communication ( Fig. 3 ). A deeper understanding of the specific mechanisms involved in the modulation of AHR signalling by the commensal flora will pave the way for novel strategies in personalized medicine and immune-related disorders.

Historically considered a liability target due to its initial identification as a receptor for man-made environmental toxins, the AHR has now emerged as a viable therapeutic target for inflammatory, infectious, cardiovascular and neoplastic diseases. The development, approval and commercialization of tapinarof has validated the AHR as a drug target, and additional AHR-targeting medicines are expected to be developed in the near future. However, several challenges exist for the development of AHR-targeting drugs. A central goal is to design compounds that maximize the therapeutic benefits of AHR signalling while minimizing potential detrimental effects. Avoiding toxicities linked to chlorinated ligands such as TCDD is critical. The terms SAHRMs and ‘rapidly metabolized AHR ligands’ (RMAHRLs) have been used as definitions to provide insights into the structure and physical chemical properties of AHR ligands, their differential binding properties and their biological effects 303 . SAHRMs preferentially regulate some, but not all, AHR target genes, whereas RMAHRLs are rapidly metabolized, and thus avoid toxicities linked to metabolically persistent agonists such as TCDD. This classification scheme facilitates grouping and mapping the chemical space of AHR ligands to identify useful structural and physical chemical properties for the design of new AHR-targeting molecules that have improved benefit to risk profiles. The dissociation of the therapeutic and toxic effects of AHR signalling is also important to identify compounds that minimize potential drug–drug interactions. One approach is local delivery of a compound, which minimizes systemic exposure and effects on hepatic drug metabolism. Tapinarof and DMVT-506 demonstrate pharmacokinetic profiles that facilitate local delivery to epithelial tissues including the skin (Tapinarof and DMVT-506), gastrointestinal tract (DMVT-506) and lungs (DMVT-506). Nanoparticle-based approaches can also be used to target AHR modulators to specific tissues and/or cell types 302 . Alternatively, the use of probiotics engineered to produce rapidly metabolized AHR agonists can maximize mucosal delivery while limiting systemic exposure 304 . Regarding the potential use of soluble AHR inhibitors as anticancer drugs, several results suggest that additional strategies are required to improve efficacy. Deletion of the AHR in malignant murine oral squamous carcinoma cells blocks the ability of these cells to form tumours in vivo 257 by enhancing tumour-specific immunity. In contrast, although AHR inhibitors slow tumour growth, they do not completely prevent tumour formation 186 . This lower effectiveness reflects the fact that AHR inhibitors should have multiple effects in order to be efficacious tools for cancer therapy. In particular, AHR inhibitors should target the AHR in malignant cells, where it controls invasiveness, stemness and immunosuppressive genes 250 , 257 , 305 , 306 , and also target the AHR in immune cells, where it promotes immunosuppressive phenotypes 255 , 307 , 308 . Anecdotally, increasing the dose of an AHR inhibitor does not improve cancer outcomes in preclinical models (reviewed elsewhere 187 , 309 ), probably reflecting that a balance between beneficial and detrimental AHR signalling is reached at a certain dose. AHR inhibitors would be expected to limit the production of IDO1-driven immunosuppressive AHR agonists, and decrease levels of suppressive Foxp3 + T reg /Tr1 cells 84 , 85 and CD39 + macrophages 96 . However, in certain contexts AHR inhibitors might also reduce the expression of CD86 (refs. 132 , 143 , 144 ) and MHC molecules 310 , the maturation of antigen-presenting cells 132 , 311 , 312 and levels of granzyme-B + tissue-resident memory T cells 178 . Therefore, additional strategies appear to be required to enhance beneficial effects on the immune system without the deleterious effects, including select AHR modulators, dose optimization and target delivery systems. The AHR also plays a critical role in autoimmune diseases by modulating immune responses. Its activation can stimulate regulatory and anti-inflammatory pathways, whereas its dysregulation can contribute to immune system imbalance and autoimmunity 313 . Although the role of the AHR has been well characterized in inflammatory conditions such as dermatosis, MS/EAE, SLE and IBD, its involvement in many other inflammatory disorders remains underexplored and warrants further investigation. For example, AHR activation by TCDD increases the number of T reg cells in pancreatic lymph nodes, preventing diabetes in the non-obese diabetes mouse model of T1D (ref. 62 ). Moreover, T1D is associated with higher levels of IL-10, decreased AHR gene expression and increased Tr1 cell frequency 314 , highlighting the potential for AHR-targeted immunomodulatory therapies. In summary, once avoided as a liability target, the AHR has now emerged as a druggable target for therapeutic immunomodulation. The identification of important roles for the AHR in the physiological regulation of the immune response has encouraged and guided the development of AHR-targeting drugs. The commercialization of tapinarof has provided validation to the biopharmaceutical industry to pursue additional drugs focused on modulating AHR biology. An improved understanding of structure–activity relationships should lead to more efficient development of safe and efficacious AHR-targeted immunotherapies.

The aryl hydrocarbon receptor (AHR) is a ligand-dependent transcription factor initially recognized as the mediator of the toxic effects of some environmental pollutants. In 1976, Poland and Glover identified a cytosolic protein that bound specifically to 2,3,7,8-tetrachlorodibenzo- p -dioxin (TCDD), a highly toxic dioxin 1 , 2 . They named this protein the aromatic hydrocarbon (Ah) receptor, later identified as the AHR. Further studies revealed that the AHR is a ligand-dependent transcription factor containing a basic helix–loop–helix (bHLH) and a Per–Arnt–Sim (PAS) domain 3 - 8 . Upon ligand binding, the AHR translocates into the nucleus, where it forms a complex with the AHR nuclear translocator (ARNT; also known as HIF-1β) 9 . This complex then binds to specific DNA sequences known as xenobiotic response elements (XREs) or dioxin response elements (DREs) to regulate the expression of target genes 10 . Historically, AHR agonists were often excluded or discarded during drug development campaigns. This avoidance stemmed from concerns about potential adverse effects and toxicity associated with AHR activation, including developmental abnormalities 2 , 11 , 12 and increased cancer risk 13 - 16 . Activation of the AHR by dioxins is known to be teratogenic, causing developmental abnormalities in fetuses 2 . Exposure to dioxins during critical periods of embryonic development can lead to structural malformations in organs and tissues 12 . Indeed, the AHR contributes to the palate and kidney malformations in mouse embryos that are induced by TCDD 11 . Moreover, prolonged AHR activation by dioxins was also found to promote the development of hepatocellular carcinoma 13 and gastric tumours 14 . In addition, decreased expression of the AHR repressor (AHRR) is detected in human tumours, leading to its proposed role as a tumour suppressor 16 . Conversely, the tumorigenic potential of transformed mammary gland primary fibroblasts is reduced by AHR deficiency 15 . These and other findings raised concerns about the safety of drugs that activate the AHR, particularly in pregnant individuals or those of reproductive age. Thus, drug developers have historically been wary of compounds that could trigger sustained AHR activation, as they might pose long-term health risks for patients. It was later found that the AHR can also be activated by agonists generated through multiple metabolic pathways and by external sources such as the diet or commensal microorganisms 17 - 22 ( Table 1 ). The metabolism of tryptophan (Trp) encompasses several biochemical pathways, resulting in the production of multiple AHR agonists. For example, the kynurenine (Kyn) pathway is a major source of AHR agonists, converting Trp into Kyn and other AHR agonists. The conversion of Trp into Kyn involves the enzymes indoleamine 2,3-dioxygenase (IDO1) and tryptophan 2,3-dioxygenase (TDO). However, Trp can also be converted into serotonin and related metabolites bearing AHR agonist activity independently of IDO1 and TDO. Moreover, the microbial metabolism of dietary Trp also produces several AHR agonists, such as tryptamine (TA) and indole-3-acetic acid (IAA). AHR activation results in the induction of specific transcriptional, epigenetic and metabolic responses with important downstream biologic effects 23 , 24 . In 2008, it was reported that activation of the AHR by TCDD resulted in the expansion of the functional regulatory T cell (T reg cell) compartment, whereas AHR activation by the Trp-derived agonist 6-formylindolo[3,2-b]carbazole (FICZ) enhanced T helper 17 cell (T H 17 cell)-driven pathogenic responses in the murine experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis (MS) 25 , 26 . These findings brought attention to the role of the AHR in immune regulation. Most importantly, these studies have paved the way for the development of AHR agonists as potential therapeutic agents for the management of several diseases. For example, 3,5-dihydroxy-4-isopropylstilbene (tapinarof; Dermavant Sciences) was approved for the topical treatment of adult plaque psoriasis and is the first and only therapeutic agent specifically targeting they AHR so far approved by the US Food and Drug Administration (FDA) 27 . This Review describes the mechanisms of AHR-regulated transcription and signal transduction pathways and the capacity of the AHR for modulation of immune responses. We then discuss AHR-targeting strategies for the treatment of inflammation, cancer and infectious diseases, including describing ongoing and historical clinical programmes.

Nanoparticles, typically ranging from 1 to 100 nm, offer unique opportunities for the therapeutic modulation of cells of interest in vivo. Nanoparticles can be prepared from multiple natural sources or synthesized from various materials, including metals and polymers, which endows them with specific functions that enable a myriad of medical applications 294 , 295 . Nanoparticles are being tested in preclinical studies on immunomodulation of the AHR pathway ( Fig. 4a ). For example, in mice, oral administration of nanoparticles loaded with the AHR agonist IAA was shown to normalize gut motility and serotonin secretion and to promote intestinal barrier integrity 296 . Moreover, oral administration of exosome-like nanoparticles derived from the plant Portulaca oleracea promoted the expansion of L. reuteri , increased the indole levels and induced AHR activation in conventional CD4 + T cells, resulting in decreased pro-inflammatory cytokines and the amelioration of DSS-induced intestinal inflammation 297 ( Fig. 4b ). The use of nanoparticles to co-deliver AHR modulators with antigens of interest has emerged as a promising approach to regulate pathogenic immune responses in an antigen-specific manner. For example, the AHR agonist ITE induces a tolerogenic phenotype in DCs and promotes the expansion of T reg cells, ameliorating EAE 136 . Nanoparticles have therefore been developed to co-administer ITE with myelin or β-cell antigens to treat animal models of MS and type 1 diabetes (T1D), respectively. In both models, the administration of these nanoparticles induced tolerogenic DCs in vivo, and promoted the expansion of Foxp3 + T reg cells 298 - 300 . Given that AHR hyperactivation is a mechanism of immune evasion by viruses and tumours, nanoparticles loaded with the AHR inhibitor CH-223191 were found to decrease viral replication and ameliorate congenital Zika syndrome in ZIKV-infected pregnant SJL mice 116 . Similarly, nanoparticles loaded with 6′-bromoindirubin-3′-acetoxime, a synthetic compound that inhibits glycogen synthase kinase 3 (GSK3), reduced IDO1 expression and limited Kyn-driven AHR activation in tumour cells, augmenting the T cell-mediated killing of glioblastoma cells in vitro. In vivo, these nanoparticles improved survival in a preclinical model of glioblastoma 301 . For the successful implementation of nanoparticle-based AHR-targeting approaches, multiple questions need to be addressed, including the optimal nanoparticle design, as well as safety and biocompatibility issues. Designing optimal nanoparticles is challenging due to complex interacting factors including their stability, targeting specificity and variability in patients. However, the convergence of nanoparticle technology and AHR biology offers an innovative tool for antigen-specific immunomodulation 302 .