The Role of the Aryl Hydrocarbon Receptor (AhR) in the Immune Response against Microbial Infections

Aryl hydrocarbon receptor (AhR), an important nuclear receptor, regulates the cellular response to environmental stressors. It is well known for its critical functions in toxicology, but is currently considered an essential regulator of diseases, with specific modulatory effects on immune, antimicrobial and inflammatory responses. The present chapter discusses AhR’s function and mechanism in the immune response against microbial infections.


Mechanism of AhR action
As a highly conserved nuclear receptor [10], AhR can regulate gene expression after binding to a ligand. AhR binds to its co-chaperones and maintains cytoplasmic localization [16,17]. Ligand binding by AhR results in its release by co-chaperones and translocation into the nucleus, where it forms a heterodimer with the aryl hydrocarbon receptor nuclear translocator (ARNT) [18,19]. Via binding to the genomic DNAusually interacting with AhR response elements (AhREs, 5'-GCGTG-3′) [20,21], also referred to as dioxin (DREs) or xenobiotic (XREs) response elements [9, 10],

AhR expression modulation
The interactions of AhR and its ligands, including polycyclic aromatic hydrocarbons (PAHs), can be used as a cytoplasmic signal sensor. The conformation of AhR changes, and it is transferred from the cytoplasm to the nucleus. The high-affinity ligand TCDD can exert toxic effects by binding with and activating AhR [32,33]. Structural analysis of AhR revealed three domains: 1) The amino-terminal DNA binding domain (DBD) comprises the basic helix-loop-helix (bHLH) region and the nuclear localization signal (NLS); 2) The central PAS region encompasses two degenerate repeats; 3) The carboxy-terminal region features the transactivation domain (TAD) [34]. In addition, phylogenetic data showed that AhR constitutes an ancient protein whose functional orthologues are found in reptiles, amphibians, birds and mammals. However, there are many structural differences between human and murine AhR genes. Sequence analysis revealed approximately 85% structural similarity in the amino-terminal sequence, while the C-terminal region shows a low homology. The TAD or N-terminal domain is the least conservative [34]. The C-terminal domain is a highly unstructured sequence containing a transcriptionally active region and contributes to receptor transformation [35,36].
AhR, heat shock protein 90 and X-associated protein 2 form multiple protein complexes in the cytoplasm. In the presence of ligands or agonists, AhR complexes undergo nuclear translocation and form heterodimers with ARNT. With a core sequence of 5′-GCGTG-3′, the AhR/ARNT complex interacts with DREs in the proximal site of promoters of target genes. Both AhR and ARNT recruit additional transcription co-activators for gene regulation, e.g., CYP and AhRR. Once transferred into the nucleus, AhR undergoes proteasome-induced degradation [37]. AhR function is modulated and weakened by AhRR, another member of the PAS family. After AhR activation, the level of AhRR increases rapidly [38]. Meanwhile, AhRR has a transcriptional repressor domain and can dimerize with ARNT even without an agonist, to fulfill its function [39].

AhR response to bacterial pathogens
It is known that AhR has a critical function in controlling responses to a variety of microbial pathogens. For example, it is required to effectively clear the while ahr2 only showed an increase in the intestine, suggesting ahr2 may contribute less to immune reactions compared with ahr1a and ahr1b. In SRW-OG1 infected E. coioides, major genes contributing to bacterial recognition, macrophage inflammatory response and gut immunity were overtly upregulated. However, decreased ROS amounts and the downregulation of other associated genes were equally detected, which indicated that SRW-OG1 could prevent ROS production by macrophages through its virulence mechanism. In addition, repression of AhR with an inhibitor or by gene silencing rescued the increases of IL-1 β and IL-8 associated with SRW-OG1 infection, clearly demonstrating that induction of E. coioides macrophages by IL-1 β and IL-8 is controlled by AhR. Nevertheless, AhR exerted no effects on bactericidal permeability-increasing protein/lipopolysaccharide-binding protein (BPI/LBP), reactive oxygen species (ROS) biosynthesis and associated genes. Compared with wild-type macrophages, survival and immune escape rates after SRW-OG1 infection were significantly increased in ahr1a/ahr1b-knockdown and 3′, 4'-DMF treated macrophages. Taken together, ahr1a and ahr1b are necessary for the immune response to SRW-OG1 [44].
Lipopolysaccharide (LPS) stimulation is often utilized to model Gram-negative bacteria-induced sepsis for assessing AhR's functions in infection resistance and septic shock regulation. AhR and TDO2 are required for survival after the initial exposure to LPS [14,20], while subsequent exposures are dependent on AhR and IDO1/2. LPS up-regulates TDO2 and IDO1/2, the rate-limiting enzymes of TRP transformation into KYN, and further induces AhR, thus downregulating proinflammatory cytokines and regulating long-term systemic inflammation [20]. In addition, compared with AhR wild type mice or immune cells, LPS challenged AhR −/− mice or immune cells produce higher concentrations of pro-inflammatory cytokines, including IL-1 β, IL-6, IL-18, IL-12, TNF-α and IFN-γ, as well as NLR Family Pyrin Domain Containing 3 (NLRP3) that regulates multiple pro-inflammatory cytokines. The AhR agonists 3-methylcholine (3-Mc), 6-Formylindolo[3,2-b] carbazole (FICZ), KYN and TCDD could protect AhR WT mice, but conferred no protection to AhR −/− animals, from extremely high amounts of pro-inflammatory cytokines and septic shock [45]. Thus, the immune response to bacterial pathogens requires AhR, and the underlying mechanisms are vital in identifying novel therapeutic agents to combat bacterial pathogens.

AhR response to viral pathogens
AhR is also associated with response to viral pathogens. For example, herpes simplex virus (HSV)-associated eye infection can lead to chronic immune-inflammatory response, causing blindness. However, in a mouse model, a single dose of TCDD could alleviate herpetic keratitis lesions, reduce viral load and decrease pro-inflammatory cytokine levels. However, similar effects were not obtained with FICZ, thus indicating a difference between both AhR ligands [46]. Therefore, response to viral pathogens requires AhR, and nontoxic AhR agonists could be used in the treatment of HSV-induced eye infections.
In influenza virus infection, activation of AhR doubles the number of neutrophils in the airway and interstitium of the lung, which reduces the survival rate from an otherwise sub-lethal infection [47,48]

AhR response to parasitic pathogens
The immune response to parasites also requires AhR. For example, immune response to Toxoplasma gondii, a pathogenic parasite causing toxoplasmosis, requires increased AhR-dependent production of IL-10. Indeed, AhR −/− mice have reduced response to T. gondii and a less pronounced IL-10 increase [56].
After intraperitoneal infection with T. gondii, the death rate of AhR −/− mice was significantly higher than that of WT mice. Moreover, AhR −/− mice showed greater liver injury, and higher levels of NO, IgE and TNF-α, but lower IL-10 secretion in the serum. Interestingly, fewer cysts were found in the brain. The increased mortality was related to reduced IL-10, 5-LOX and GATA-3 expression levels, but increased IFN-γ expression in the spleen. In addition, AhR −/− mice had increased IL-12 and IFN-γ amounts, but decreased TLR2 levels compared with wild-type mice in peritoneal exudate cells. These findings suggest that AhR is vital for limiting inflammation during toxoplasmosis [57].
Therefore, AhR is necessary for parasitic pathogen response. This provides information on a response pathway and can be used to design new treatments.

AhR and the intestinal microbiota
AhR is found at high levels in the epithelial barrier [58], and the intestinal barrier of AhR −/− mice is inadequate, suggesting AhR might be important in maintaining or generating a healthy intestinal barrier [19]. In addition, low levels of AhR and AhR's target genes are found in sterile mice [9], and AhR is needed for maintaining the ROR γt+ innate lymphoblastoid cell (ILC) balance in the intestine [18]. In addition, the TRP metabolizing indole biosynthesized by select bacterial components of the intestinal microbiota is an AhR ligand [59, 60]. Diet without indole or antibiotic treatment can lead to the differentiation of mononuclear phagocytes, dependent on AhR, into dendritic cells (DCs) [48], which are more susceptible to gut pathogens in mice [17]. Overall, the above findings suggest AhR might be important in host gut-microbiota interactions.
AhR also plays a role in the reciprocal relationship among intestinal bacteria, bacterial metabolites and the intestinal immune system. AhR-deficient RORγt + ILCs (the main producers of gut IL-22) with lower IL-22 amounts make mice easily die upon Citrobacter rodentium infection. It was pointed out that treatment with FICZ markedly enhances RORγt + ILC accumulation in AhR +/− and AhR +/+ mice, but not in AhR −/− animals [61]. Lactobacillus species (nonpathogenic intestinal bacteria) are capable of producing AhR ligands, including indole-3-aldehydes, from tryptophan in the gut, thus enhancing the production of AhR dependent IL-22 [62]. Indole-3aldehydes induces AhR-associated transcription, but exclusively at elevated concentrations, indicating its low affinity. However, indole-3-acetaldehyde (a product of indole-3-aldehydes) produces the high-affinity ligand FICZ [63], which may be related to the effect reported by Zelante et al. IL-22 affects epithelial cells and causes them to produce antimicrobial peptides, such as type III Reg (regenerating gene product) gamma (RegIIIg), and to stimulate tissue regeneration. Meanwhile, symbiotic bacteria may outperform bacterial pathogens and inhibit Candida albicans colonization [51]. Similar to keratinocyte and skin immune cell levels, AhR amounts are high in IECs and intestinal immune system cells [64].
In AhR-null mice, the number of intraepithelial lymphocytes (IELs) in the small intestine is significantly reduced [6, 64,65], which is related to lower IL-22 amounts, and therefore to downregulated ileal antimicrobial peptides, including RegIIIb and RegIIIg. The microbial loads of the small and large intestines are also elevated. Loss of IELs is cell-intrinsic since AhR-deficient bone marrow cells do not reconstruct the gut in Rag −/− mice [51]. Over time after birth, intestinal Group 3 Innate Lymphoid Cells (ILC3s) [66], ILC22 and CD32NKp46 + lymphoid tissue inducer cells are lost in AhR-deficient mice. Similarly, ILC3's inability to multiply in AhR-deficient mice constitutes an intrinsic function since AhR is required for the transcription of the cell-specific proliferator c-kit [67,68]. As a result, secondary lymphoid structures, including cryptopatches and innate lymphoid follicles, are absent from the gut of AhR-deficient mice, which show susceptibility to C. rodentium. ILC3s feature the secretion of IL-17 and IL-22 [69]. AhR-deficient mice have elevated susceptibility to infection by C. rodentium, as well as dextran sulfate sodium (DSS)-associated colitis. DSS can damage the intestinal epithelium and induce inflammatory reactions and microbial dissemination. AhR-deficient mice containing wild-type IELs are resistant to DSS colitis, indicating IEL role in injury reduction.
AhR-deficient mice have lower amounts of skin and intestinal IELs and intestinal ILCs, thereby increasing susceptibility to C. rodentium infection. These cell types, and the generation of normal gut lymphoid follicles, are regulated by AhR ligands in the diet. In addition, activation of AhR by microbial products equally regulates the production of DP IELs, which constitute another critical group that controls intestinal immunity [70]. It may also be due to the lack of IL-22 that affects the commensal flora [71]. In fact, ID2, a transcription factor, regulates the expression of IL-22 in ILCs via AhR-and IL-23-dependent mechanisms, thereby modulating the intestinal colonization of C. rodentium [72]. In addition, AhR also controls the production of IL-22 by Th22 cells, which protect against intestinal pathogens [73,74]. All these data suggest AhR has a critical function in controlling the interaction at environmental interfaces with microorganisms by regulating IL-22 and other cellular factors. Interestingly, cyp1a1 overexpression leads to the exhaustion of physiological AhR ligands and also increases susceptibility to intestinal bacterial infections [75], highlighting that AhR ligand availability and metabolism are important in controlling AhR-dependent immune effects.

AhR and T cells
AhR plays an important role in controlling adaptive immunity, and regulating T cell differentiation and direct or indirect functions by affecting antigen presenting cells. It was found that TCDD-activated AhR could inhibit the immune response [76], which is subsequently associated with CD4 + T cell induction [77][78][79]. In addition, the role of AhR in Th17 function and T cell-induced IL-22 biosynthesis have also been determined [74,[80][81][82][83].

AhR and regulatory T cells (Tregs)
AhR shows high expression in Th17 cells, undetectable amounts in Th1 and Th2 cells, and low expression in Tregs. Tregs constitute a T cell subgroup, which helps maintain tolerance to autoantigens, preventing autoimmune pathologies. FoxP3 + Tregs [84,85] and IL-10-producing type 1 regulatory T cells (Tr1 cells) [86] are the most typical Treg entities. Foxp3 + Tregs and Tr1 cells are associated with AhR.
In the presence of TGFβ 1, activating AhR with TCDD can also upregulate SMAD1 in human Tregs, resulting in stable expression of FoxP3 [93]. It was shown in mice with AhR-deficient T cells that AhR could also inhibit the activation of STAT1, which in turn inhibits FoxP3 + Treg differentiation [94]. In addition, AhR regulates the epigenetic modifier Aiolos, which downregulates genes associated with T cell's effector function, such as IL-2 [87]. However, the effect of AhR on FoxP3 + Tregs may be affected by the applied experimental model, which may reflect the different effects of tissue-specific action and/or AhR agonist provided by the symbiotic flora [95].
AhR triggered CD39 equally affects Tr1 cell differentiation. After induction, T cells secrete eATP [103], which then interferes with the differentiation of Tr1 cells through hypoxia inducible factor-1α (HIF1-α). HIF1-α binding is superior to the interaction between AhR and ARNT, and promotes the degradation of AhR through the immune proteasome, thus inhibiting the differentiation of AhR dependent Tr1 cells [101]. The expression of CD39 driven by AhR can deplete eATP and promote the differentiation of Tr1 cells. Therefore, AhR regulates central genes in the Tr1 cell transcription program, while limiting the inhibitory effect of eATP-dependent HIF1-α induction on Tr1 cell differentiation. Overall, the above findings confirm AhR as a potential therapeutic target for immunomodulation.

AhR and T helper 17 (Th17) cells
Th17 cells, forming a unique CD4 + T cell subgroup, can biosynthesize Th17 cytokines and play key roles in the pathogenetic mechanisms of multiple inflammatory ailments. Their differentiation is triggered by IL-6 and transforming growth factor-beta (TGF-β). AhR can modulate Th17 cells by binding to the DRE site in the IL-17 promoter. In addition, AhR and STAT3 can synergistically upregulate Aiolos (IKZF3), an Ikaros family member, which can decrease the expression of IL-2, thus increasing Th17 cell amounts [64].
Th17 cells, producing IL-17A and expressing ROR-γt, are involved in immune responses to extracellular bacterial and fungal pathogens, and participate in the pathological mechanisms of multiple autoimmune diseases [104,105]. Their differentiation involves joint effects of TGF-β and IL-6 or IL-21 [106][107][108]. AhR shows high expression in Th17 cells and is activated by FICZ, which can enhance Th17 cell differentiation and promote IL-22 expression. On the contrary, AhR deficiency can cause Th17 cells to produce IL-22, which may reflect AhR's function in promoting RORγt recruitment to the IL-22 promoter.

AhR and other T cells
Th22 cells are a CD4 + T cell subpopulation. They produce IL-22 without IL-17's intervention and their differentiation is induced by IL-6, IL-21 or IL-23. AhR controls the production of IL-22 in Th22 cells, and other cellular factors are essential for their mucosal immune functions [73,[109][110][111].
The AhR pathway also significantly affects CD8 + T cells. Activation of AhR by TCDD indirectly inhibits the primary response of CD8 + T cells to influenza virus through the regulatory mechanism of DC function [112]. In addition, CD8 + T cells of mouse models administered the AhR agonist TCDD in the developmental stage show a weak response to influenza virus infection later in life [113]. The above data indicate epigenetic alterations that can lead to prolonged functional defects in CD8 + T cells detectable after viral attack. Compared with other CD8 + T cell subsets, AhR expression is much higher in tissue resident CD8 + memory cells (TRMs). Taken together, these findings indicate that, similar to previously reported CD4 + T cell data, the AhR pathway plays a major role in regulating specific CD8 + T cell subgroups, such as TRMs and DP IELs.
AhR equally regulates γδ T cells, which are tissue resident lymphocytes. It regulates first-line immune response at epithelial sites and controls tissue homeostasis [114]. Despite AhR expression in the totality of γδ T-cell subgroups, AhR-deficiency significantly reduces the amounts of skin intraepithelial lymphocytes, mostly composed of Vγ3 and Vγ5 γδ T cells in the intestine and CD8αα αβ T cells [115]. AhR also regulates IL-22 expression by γδ T cells that produce IL-17 [116,117]. The above data indicate that AhR has a significant effect on T cells residing in tissues, which supports further investigation of AhR's function in non-CD4 + T cells.
In conclusion, AhR controls T cell responses at many levels and regulates transcription factors, enzymes, epigenetic modifiers and effector molecules that modulate T cell stability and metabolism. Lineage-specific responses to AhR induction may lead to ligand-specific effects, which are combined with cytokine-driven activities on the genome, thereby regulating AhR-interacting chaperones and controlling the accessibility of AhR's direct and indirect transcription targets [118].
Comprehensive studies of these interactions should provide insights into the design of immune-modulators against AhR.

AhR and B cells
The B lymphocyte is an important part of humoral immunity, which has high specificity against a variety of pathogens. After stimulation via an antigen receptor, activation of immature B cells leads to clonal expansion, antibody isotype conversion and differentiation into antibody-secreting plasma cells, thus producing strong immune reactions [119]. In the process of infection, mature B cells in the lymph nodes and secondary lymphoid organs undergo somatic hypermutation and produce plasma cells featuring elevated antigen affinity and unique effector function [120].
It seems that all B cells produce AhR, but specific subsets, e.g., marginal B cell and B1 B cell subsets, have higher levels than the others. Li and collaborators demonstrated that AhR contributes to the development of B lymphocytes, based on cord blood CD34 and feeder cells, which promote B cell development. Meanwhile, AhR induction inhibits the formation of early B cells and pro-B cells. AhR controls B cell differentiation by transcriptionally suppressing the early B cell genes EBF1 and PAX5 [121].
AhR, overtly induced after activation of B cells, has a critical function in regulating the fate of activated cells. Vaidyanathan and colleagues revealed AhR suppresses switch-like recombination by changing the amounts of activated cytidine deaminase. These authors showed that AhR suppresses B cell transformation into plasmablasts and plasma cells that secrete antibodies [122]. In addition, Villa et al.
provided evidence of a role for AhR in B cells, revealing that AhR expression is increased after administration of IL-4 as well as B cell receptor engagement. Nevertheless, the proliferation of AhR-deficient B cells is decreased, and cells could not progress to the S-phase. Furthermore, AhR-deficient B cells could not compete with the decreased AhR +/+ B cell capability of reconstructing the empty host, and could not induce antigen-dependent proliferation in mice. Gene expression profile analysis showed that AhR excision downregulates cyclin O, an important gene controlling the cell cycle [123].

AhR and dendritic cells (DCs)
DCs are essential in controlling T cell response and regulating immune tolerance [124]. AhR regulates DC differentiation and function, thereby profoundly affecting T cell-dependent immune reactions. AhR also affects antigen presentation by DCs. Bone marrow derived DCs (BMDCs) exposed to TCDD show decreased CD11c amounts, but increased production of MHC-II, CD86, IL6 and TNFα [125]. Similar findings were reported in TCDD treated splenic DCs [126]. However, different results were observed by using the AhR agonist ITE. The expression of MHC-II and co-stimulatory molecules and the production of Th1 and Th17 polarization cytokines in splenic DCs were decreased by ITE stimulation of AhR.
Recent experiments in ovalbumin-induced asthma models provide additional evidence for the physiological regulation of AhR in DCs, with AhR-deficient mice exhibiting enhanced inflammatory reactions, elevated Th2 differentiation and higher DC MHC-II and CD86 amounts [127]. In addition, AhR signaling has been reported to regulate the activity of CD103 + /CD11b + DCs during influenza virus infection, thereby reducing induction in protective CD8 + T cells [128]. Overall, this evidence confirms that AhR is a potential therapeutic target for regulating T cell responses in DC.
Multiple mechanisms are involved in AhR-associated regulation of DC function. AhR upregulates IDO 1 and 2 [129, 130], which catalyze the production of KYN, thus promoting the differentiation of FoxP3 + Tregs [131]. Indeed, AhR-deficient DCs could not induce Treg differentiation and Th17 cell proliferation in culture. It is consistent with the immunosuppressive effect of AhR in DCs. Recently, it was reported that IDO expression is maintained by an autocrine loop involving AhR and KYN in tumor infiltrating tolerogenic DCs [132]. Additionally, AhR induction in DCs induces a retinoic acid-dependent enzymatic mechanism, thus promoting FoxP3 + Treg differentiation and inhibiting effector T cells [133][134][135][136][137].

Conclusions
Studies evaluating AhR's functions in immune cell development, immune response modulation and immune tolerance have aroused great interest. Originally, AhR was considered a protein sensing environmental substances and regulating drug metabolism. Recently, the role of AhR in regulating normal physiological processes has attracted increasing attention. The organism must perceive and mount substantial responses to environmental changes. Indeed, AhR senses biochemical, chemical and physical environments. Combined with a small amount of high-affinity physiological ligands, including FICZ and ICZ, AhR plays a role in cell proliferation, differentiation and function.
Current evidence indicates that AhR has a critical function in host response to bacterial pathogens. It also overtly influences resistance to infections by extracellular and intracellular bacteria. AhR is considered the best resistance factor for LM. It may have a new function in the innate immunity of LM infection, and AhR-deficient mice have increased sensitivity to LM. Activation of AhR can protect mice from the deadly attack of S. pneumoniae, inhibit bacterial growth and fight infection. AhR can also react with viral pathogens and parasitic infections. After infection by viruses and parasites, lack of AhR aggravates the host's inflammatory response. AhR regulates host's immune cells, confirming that AhR is a regulatory molecule with essential functions in the activation and induction of immune cells, e.g., T cells and inflammatory factors. Barrier organs are critical in immunity; specifically, large amounts of ahr are expressed in the intestine, which has a high potential for preventive and treatment interventions. AhR has a critical function in controlling the degree of inflammation in response to symbiotic microbiota and tissue destruction. Progress is being made in determining the molecular mechanisms by which AhR affects different cell types. To understand the complex process of AhR in immunity and antibacterial, to mitigate risks, and to develop novel treatment and prevention tools, more research is needed.