Invited Perspective: Environmental Chemical-Sensing AHR Remains an Enigmatic Key Player in Toxicology

Abstract

Over the last several years, the aryl hydrocarbon receptor (AHR) has gone from being characterized as not only a receptor that modulates cellular responses to myriad external environmental changes but also as one that regulates intricate aspects of immune homeostasis in response to endogenously produced ligands. Indeed, several endogenous AHR ligands have now been identified, including chemicals derived from tryptophan, phytochemicals, or commensal microbiota. Although AHR might play many roles, no doubt it remains a critical receptor in modulating immune responses within the context of exposure to environmental chemicals. In this issue of Environmental Health Perspectives, Liu et al. provide a detailed characterization of immune effects of fine particulate matter [PM with an aerodynamic diameter of =2:5 lm (PM2:5)] obtained from atmospheric monitoring stations in Taiwan. This PM2:5 contained various polycyclic aromatic hydro-carbons (PAHs), including indeno[1,2,3-cd]pyrene (IP).

Over the last several years, the aryl hydrocarbon receptor (AHR) has gone from being characterized as not only a receptor that modulates cellular responses to myriad external environ-mental changes but also as one that regulates intricate aspects of immune homeostasis in response to endogenously produced ligands. Indeed, several endogenous AHR ligands have now been identified, including chemicals derived from tryptophan, phytochemicals, or commensal microbiota.1,2 Although AHR might play many roles, no doubt it remains a critical receptor in modulating immune responses within the context of exposure to environmental chemicals.

In this issue of Environmental Health Perspectives, Liu et al. provide a detailed characterization of immune effects of fine partic-ulate matter [PM with an aerodynamic diameter of =2:5 lm (PM2:5)] obtained from atmospheric monitoring stations in Taiwan.3 This PM2:5 contained various polycyclic aromatic hydro-carbons (PAHs), including indeno[1,2,3-cd]pyrene (IP).4 They observed that intratracheal administration of PM2:5 or IP alone exa-cerbated pathology and modulated immune responses by using a mouse model for house dust mite (HDM)-mediated asthma.3 Importantly, they used mass cytometry to determine that both PM2:5 and IP exposure increased the percentage and number of TCRb+CD4+CXCR5+Bcl-6+PD-1hi T follicular helper (Tfh) cells in the lung-draining lymph nodes. In addition, the enhancement in Tfh cells was dependent on AHR expression in T cells, at least for PM2:5. Tfh cells are CD4+ T cells that play a critical role in induc-ing isotype-switched antibody responses in germinal centers (GCs).5 Therefore, it was important that they also demonstrated that IP enhanced levels of HDM-specific immunoglobulin E (IgE) and IgG in serum.3 Previously, they had demonstrated that IP-mediated exacerbation in a mouse ovalbumin-induced model of al-lergic lung inflammation was attenuated with the AHR antagonist

CH223191.4

Interestingly, the observation that IP, as an AHR ligand, increased the percentage and number of Tfh cells contradicts another recent paper, in which other AHR ligands suppressed Tfh

cell percentage and number in mice infected with influenza A virus.6 In that paper, the environmental contaminant 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD), the tryptophan derivative 2-(10H-indole-30-carbonyl)-thiazole-4-carboxylic acid methyl ester (ITE), and the endogenous ligand kynurenic acid (KYNA) all suppressed CD4+CD44+CXCR5+PD-1hi Tfh in infected mice.6 Using TCDD, Houser and Lawrence also showed in lung-draining lymph nodes that AHR activation significantly suppressed Tfh cell number as early as day 3 postinfection, that the GC B cell number directly cor-related with Tfh cell number and, further, that AHR activation sup-pressed GC B cells. Consistent with these findings, AHR activation with ligands, including TCDD and ITE, also reduced virus-specific IgG levels in plasma.6

The juxtaposition of the results from these papers displays a common theme in the AHR field: Different AHR ligands pro-duce different, and sometimes opposite, effects in the immune system (Figure 1). The reasons for this remain uncertain, but they could be due to ligand affinity, ligand bioavailability or metabolism, strength of response, or differences in the immune stimulus. Indeed, the two juxtaposed studies used different dis-ease models, with Houser and Lawrence6 using a replicating pathogen (influenza virus), and Liu et al.3 using a representative allergen (HDM).

However, even in the same model system, two different AHR ligands can produce opposite effects. For instance, during influenza A virus infection in which TCDD suppressed Tfh cell

percentage and number, the tryptophan derivative 6-formylindolo (3,2-b)carbazole (FICZ)-which, like TCDD, binds to AHR with high affinity-elevated Tfh cell percentage and number in the lung-draining lymph nodes.7 In addition to this contrasting effect on Tfh cells in influenza virus-infected mice,7 opposite effects of AHR ligands were observed in the experimental autoimmune en-cephalomyelitis model of multiple sclerosis, in which TCDD sup-pressed but FICZ exacerbated disease.8,9 Thus, ligand source might not necessarily be an ideal predictor of whether the out-come will be beneficial or detrimental.

The demonstration that two environmentally relevant ligands exhibit opposite effects on the Tfh cell population (i.e., IP enhanced and TCDD suppressed)3,6 could also be explained by differences in AHR affinity, ligand metabolism, or bioavailability. IP was deter-mined to have a half maximal effective concentration (EC50) for induction of ethoxyresorufin-O-deethylase, an enzymatic measure of AHR-responsive cytochrome P4501A1,of 5:2 × 10-1 lM, which is 10,000 times less effective than TCDD.10 Unlike TCDD, which is relatively resistant to metabolism,11-13 there were several hydroxy-lated metabolites of IP identified in vitro using rat liver enzyme ho-mogenates.14 Further support that metabolism of AHR ligands can influence immune effects, such as CD4+CD44hiCXCR5+PD-1+ Tfh populations in influenza virus infection was shown by compar-ing TCDD and FICZ in mice.7 Specifically, oral TCDD (adminis-tered once) suppressed the percentage and numberof Tfh cells inthe lung-draining lymph nodes, whereas oral FICZ (administered once per day for 8 d) had no effect. However, in cytochrome P 450, family 1, subfamily A, polypeptide 1 (Cyp1a1-=- ) mice infected with influenza virus, both TCDD and FICZ suppressed the number of Tfh cells.7 This indicates that attenuating the clearance of FICZ in vivo produced an effect that was not observed in the presence of normal metabolism. Thus, the metabolism of ligands, regardless of whether the ligands are endogenous or exogenous, likely influen-ces their effects in immune cells.

Another issue that cannot be ignored, regardless of the source of AHR ligand being studied, is the fact that exposure to a single chemical is unlikely. In their paper, Liu et al. acknowledged this critical fact by showing that exposure to IP, both on its own and in a PM2:5 mixture, exacerbated the HDM-induced Tfh populations.3 In addition to drilling down from PM2:5 to PAH to a specific moi-ety, the leveraging of in vitro results strengthened the in vivo find-ings. Specifically, Liu et al. differentiated Tfh cells in vitro and connected altered Tfh cells to changes in gene expression.3 In par-ticular, the associated increase in Il4 and Il21 gene expression was found to be due, in part, to AHR binding to xenobiotic response elements in the promoters of both Il4 and Il21. Importantly, Tfh cells producing interleukin-4 (IL-4) is necessary for IgE class switch15-17 and, as noted, IP increased HDM-mediated IgE.3

Overall, the work by Liu et al.3 advances the field of immunotoxi-cology through the use of mass cytometry to identify a specific immune cell population that was sensitive to regulation by an envi-ronmental contaminant via AHR. Further, although this is likely not the sole consequence of AHR activation by IP, or by PAHs in general, the authors identified a specific role that AHR played in regulating Il4 and Il21 gene expression.3 This work provides a compelling and complete story about how the environmentally relevant AHR ligand IP-induced respiratory disease in vivo identified that exacerbated disease directly correlated with an increased Tfh cell population and IgE production and then showed that AHR was involved in IP-induced Il4 production in vitro, something that is necessary for IgE production.

It will be exciting to see where future work leads, including fur-ther investigation into the mechanisms by which IP alone, and in combination with other contaminants, alters immune responses. For instance, it was recently suggested by using in silico modeling that IP would bind to toll-like receptor 4 with high affinity.18 This is an intriguing finding, considering that Liu et al.3 found that IP exacer-bated airway disease. Hopefully, future research will also identify the mechanistic explanation as to why different AHR ligands sometimes cause similar effects but at other times can cause different, and even completely divergent, consequences. Understanding how differences in AHR affinity, ligand metabolism, and other characteristics influ-ence immune function will be critical to parsing and predicting the immunological consequences of exposure to the myriad ligands of this fascinating environment-sensing transcription factor.

References

References

1. Lin L, Dai Y, Xia Y. 2022. An overview of aryl hydrocarbon receptor ligands in the last two decades (2002-2022): a medicinal chemistry perspective. Eur J Med Chem 244:114845, PMID: 36274276, https://doi.org/10.1016/j.ejmech.2022.114845.

2. Rannug A. 2022. 6-Formylindolo[3,2-b]carbazole, a potent ligand for the aryl hydrocarbon receptor produced both endogenously and by microorganisms, can either promote or restrain inflammatory responses. Front Toxicol 4:775010, PMID: 35295226, https://doi.org/10.3389/ftox.2022.775010.

3. Liu KY, Gao Y, Xiao W, Fu J, Huang S, Han X, et al. 2023. Multidimensional analysis of lung lymph nodes in a mouse model of allergic lung inflammation following PM2.5 and indeno[1,2,3-cd]pyrene exposure. Env Health Perspect 131(3):037014, https://doi.org/10.1289/EHP11580.

4. Wong TH, Lee CL, Su HH, Lee CL, Wu CC, Wang CC, et al. 2018. A prominent air pollutant, indeno[1,2,3-cd]pyrene, enhances allergic lung inflammation via aryl hydrocarbon receptor. Sci Rep 8(1):5198, PMID: 29581487, https://doi.org/10. 1038/s41598-018-23542-9.

5. Ribeiro F, Perucha E, Graca L. 2022. T follicular cells: the regulators of germinal center homeostasis. Immunol Lett 244:1-11, PMID: 35227735, https://doi.org/10. 1016/j.imlet.2022.02.008.

6. Houser CL, Lawrence BP. 2022. The aryl hydrocarbon receptor modulates T fol-licular helper cell responses to influenza virus infection in mice. J Immunol 208(10):2319-2330, PMID: 35444027, https://doi.org/10.4049/jimmunol.2100936.

7. Boule LA, Burke CG, Jin GB, Lawrence BP. 2018. Aryl hydrocarbon receptor signaling modulates antiviral immune responses: ligand metabolism rather than chemical source is the stronger predictor of outcome. Sci Rep 8(1):1826, PMID: 29379138, https://doi.org/10.1038/s41598-018-20197-4.

8. Quintana FJ, Basso AS, Iglesias AH, Korn T, Farez MF, Bettelli E, et al. 2008. Control of Treg and TH17 cell differentiation by the aryl hydrocarbon receptor. Nature 453(7191):65-71, PMID: 18362915, https://doi.org/10.1038/nature06880.

9. Veldhoen M, Hirota K, Westendorf AM, Buer J, Dumoutier L, Renauld JC, et al. 2008. The aryl hydrocarbon receptor links TH17-cell-mediated autoimmunity to environmental toxins. Nature 453(7191):106-109, PMID: 18362914, https://doi.org/ 10.1038/nature06881.

10. Bosveld ATC, de Bie PA, van den Brink NW, Jongepier H, Klomp AV. 2002. In vitro EROD induction equivalency factors for the 10 PAHs generally monitored in risk assessment studies in the Netherlands. Chemosphere 49(1):75-83, PMID: 12243332, https://doi.org/10.1016/S0045-6535(02)00161-3.

11. Gasiewicz TA, Geiger LE, Rucci G, Neal RA. 1983. Distribution, excretion, and metabolism of 2,3,7,8-tetrachlorodibenzo-p-dioxin in C57BL/6J, DBA/2J, and B6D2F1/J mice. Drug Metab Dispos 11(5):397-403, PMID: 6138222.

12. Olson JR. 1986. Metabolism and disposition of 2,3,7,8-tetrachlorodibenzo-p-dioxin in guinea pigs. Toxicol Appl Pharmacol 85(2):263-273, PMID: 3764914, https://doi.org/10.1016/0041-008x(86)90121-3.

13. Sorg O, Zennegg M, Schmid P, Fedosyuk R, Valikhnovskyi R, Gaide O, et al. 2009. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) poisoning in Victor Yushchenko: identification and measurement of TCDD metabolites. Lancet 374(9696):1179-1185, PMID: 19660807, https://doi.org/10.1016/S0140-6736(09)60912-0.

14. Rice JE, Coleman DT, Hosted TJ Jr, LaVoie EJ, McCaustland DJ, Wiley JC Jr. 1985. Identification of mutagenic metabolites of indeno[1,2,3-cd]pyrene formed in vitro with rat liver enzymes. Cancer Res 45(11 pt 1):5421-5425, PMID: 4053016.

15. Liang HE, Reinhardt RL, Bando JK, Sullivan BM, Ho IC, Locksley RM. 2012. Divergent expression patterns of IL-4 and IL-13 define unique functions in allergic immunity. Nat Immunol 13(1):58-66, PMID: 22138715, https://doi.org/10.1038/ni.2182.

16. Meli AP, Fontés G, Leung Soo C, King IL. 2017. T follicular helper cell-derived IL-4 is required for IgE production during intestinal helminth infection. J Immunol 199(1):244-252, PMID: 28533444, https://doi.org/10.4049/jimmunol.1700141.

17. Noble A, Zhao J. 2016. Follicular helper T cells are responsible for IgE responses to Der p 1 following house dust mite sensitization in mice. Clin Exp Allergy 46(8):1075-1082, PMID: 27138589, https://doi.org/10.1111/cea.12750.

18. Cabral MB, Dela Cruz CJ, Sato Y, Oyong G, Rempillo O, Galvez MC, et al. 2022. In silico approach in the evaluation of pro-inflammatory potential of polycyclic aromatic hydrocarbons and volatile organic compounds through binding affin-ity to the human toll-like receptor 4. Int J Environ Res Public Health 19(14):8360, PMID: 35886213, https://doi.org/10.3390/ijerph19148360.