Detection of aryl hydrocarbon receptor agonists in human samples

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor with important functions in the immune response and cancer. AHR agonists are provided by the environment, the commensal flora and the metabolism. Considering AHR physiological functions, AHR agonists may have important effects on health and disease. Thus, the quantification of AHR agonists in biological samples is of scientific and clinical relevance. We compared different reporter systems for the detection of AHR agonists in serum samples of Multiple Sclerosis (MS) patients, and assessed the influence of transfection methods and cell lines in a reporter-based in vitro assay. While the use of stable or transient reporters did not influence the measurement of AHR agonistic activity, the species of the cell lines used in these reporter assays had important effects on the reporter readings. These observations suggest that cell-specific factors influence AHR activation and signaling. Thus, based on the reported species selectivity of AHR ligands and the cell species-of-origin effects that we describe in this manuscript, the use of human cell lines is encouraged for the analysis of AHR agonistic activity in human samples. These findings may be relevant for the analysis of AHR agonists in human samples in the context of inflammatory and neoplastic disorders.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor which was initially described as a molecular sensor for environmental toxins 1 . However, it is now clear that AHR is activated not only by environmental toxins, but also by commensal and endogenous agonists which can initiate ligand-, cell-and tissue-specific biologic responses. Indeed, the role of AHR in inflammatory asnd neoplastic diseases has been studied extensively [1][2][3][4][5][6][7][8][9] . AHR plays an important role in the control of T cells, dendritic cells, gut intraepithelial lymphocytes, tumor cells and astrocytes [1][2][3][4][5][6][7][8][9] . For example, we recently reported that AHR activation in astrocytes limits inflammation in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS) 8 . Tryptophan (Trp) metabolites produced by commensal bacteria in cooperation with host enzymes activate AHR in astrocytes to limit CNS inflammation 8 . Interestingly, we detected decreased AHR activation in MS brain lesions as indicated by the expression of the AHR target gene CYP1B1, concomitant with decreased levels of AHR agonists in MS sera detected using a cell-based reporter assay and mass spectrometry 8 . Moreover, AHR agonistic activity in sera from MS patients was correlated both with disease activity and more severe stages of MS 7 . Collectively, these data identify AHR in astrocytes as a negative regulator of CNS inflammation. Interestingly, decreased AHR agonists were also reported in inflammatory bowel disease (IBD) patients 4 , suggesting that deficits in AHR-driven immune regulation may contribute to multiple human disorders.
AHR agonistic activity is detected in clinical and commercial human samples [10][11][12][13][14][15][16][17][18] . Considering the multiple biological roles of AHR, the quantification of agonists in clinical samples may be of interest to identify potential associations of this pathway with human disorders. However, the total AHR agonistic activity as determined with reporter assays reflects the net activity, which integrates multiple variables such as the levels of AHR agonists of diverse origins (environmental, commensal, dietary and metabolic), their uptake, activation and degradation, as well as the balance between AHR agonists, yet to be characterized physiologic AHR antagonists and other molecules that modulate AHR activation indirectly through their effects on other

Results
The species of the cell lines used influence the detection of AHR agonists in reporter assays. A method for the detection of AHR agonists is the use of cell lines transfected (stably or transiently) with a reporter plasmid containing an AHR responsive promoter element (XRE) which is activated upon ligand-induced AHR recruitment and thereafter drives the expression of reporter molecules such as firefly luciferase or green fluorescent protein (GFP). Thus, as an initial approach to compare different reporter systems we first analyzed the impact of the cell line used in AHR reporter assays. To this end, we transfected human HepG2 hepatoma cells and human HEK293 cells transiently with construct encoding a firefly luciferase under the control of four AHR responsive elements (pGud-Luc1.1) 19 . In both cell lines, we detected decreased AHR agonist activity in MS sera when compared to healthy controls (Fig. 1a,b). Please note that patient treatment status did not affect the AHR agonistic activity detected (Supplemental Fig. 1 and Table 1). Thus, the detection of decreased AHR agonistic activity in MS sera does not depend on the use of a specific human cell line.
Next, we analyzed the effect of the species of the cell line used in the assay. Again, we detected decreased AHR agonistic activity in MS sera when we used human HEK293 cells transiently transfected with the pGud-Luc1.1 AHR reporter (Fig. 1c, left). However, we measured increased AHR agonistic activity in MS sera when we used transiently transfected mouse liver hepatoma HEPA1 cells (Fig. 1c, right). Similarly, mouse H1G1.1C3 cells stably transfected with an AHR reporter 20 detected increased AHR agonistic activity in the same set of MS sera (Fig. 1d). Taken together, these data suggest that the species of the cell line used in the reporter assay influences the detection of AHR agonistic activity, and not whether the cells were stably or transiently transfected.

Cell line species, but not AHR species, affects the quantification of AHR agonistic activity.
Structural differences between mouse and human AHR led to the identification of physiological species-specific agonists that activate human but not murine AHR 19,[21][22][23][24] . Since the transient and stable reporter assays used in these studies are based on the activation of the AHR expressed by the transfected cell, we hypothesized that differences in the sensitivity of mouse and human AHR to agonists in human sera may reflect the activation of human or murine AHR in the cells used in the reporter assay. To address this point, we used human Sum149 breast cancer cells (Sum149 AHR del cells), in which endogenous AHR expression was deleted using CRISPR/ Cas9 technology (Fig. 2a). Human Sum149 AHR del cells were co-transfected with the pGud-Luc1.1 AHR reporter plasmid and a construct coding for either mouse or human AHR, and used to analyze AHR agonistic activity. We detected decreased AHR agonistic activity in MS sera analyzed using human Sum149 AHR del cells expressing either murine or human AHR (Fig. 2b). Collectively, these findings suggest that the cell line species, but not the species of AHR itself, affects the quantification of AHR agonist activity in serum.

TNF-α in MS sera does not interfere with the detection of AHR agonistic activity. TNF-α
is reported to inhibit the activation induced by TCDD of an AHR reporter transiently transfected with Lipofectamine through a mechanism mediated by NF-κB 25 . Thus, it is conceivable that TNF-α in clinical samples interferes with the detection of AHR agonist activity. Indeed, TNF-α is reported to be increased in MS sera by some studies 26,27 , but not consistently 28,29 . To determine whether TNF-α suppresses AHR agonistic activity in MS sera, we first quantified TNF-α in healthy control, definite MS and clinically isolated syndrome (CIS) samples using an ELISA system with a lower limit of detection of 3.2 pg/ml. We did not detect a significant increase of TNF-α in the CIS or MS samples we analyzed (Fig. 3a). TNF-α serum levels were also not correlated with AHR agonistic activity detected with cells transiently transfected (Fig. 3b). Furthermore, TNF-α blockade with specific antibodies did not increase the AHR agonistic activity detected in MS sera using HEK293 cells transiently transfected with the AHR responsive reporter construct (Fig. 3c). Conversely, exogenously added TNF-α did not suppress AHR activation when tested on HEK293 cells transiently transfected (Fig. 3d). These findings suggest that the suppression of AHR activation by TNF-α suggested in previous studies does not affect the detection of AHR agonist activity in sera using human HEK293 cells transfected transiently with the AHR-Luciferase construct.

Discussion
In this study, we analyzed cell-line based reporter assays for the detection of AHR ligands in human serum. Specifically, we analyzed the effect of the transfection method, the cell line species and TNF-α interference in these reporter systems. While the use of stable versus transient transfection did not affect the quantification of AHR agonistic activity in MS sera, the species of the cell line used had significant effects on the determination of AHR agonistic activity. This result was independent of the species of AHR itself. Finally, TNF-α did not impair the measurement of AHR ligands in transiently transfected human HEK293 cells.
It has recently been reported that AHR agonistic activity varies during the course of inflammatory diseases such as MS and inflammatory bowel disease (IBD), suggesting that changes in circulating AHR agonists reflect alterations in the commensal flora, uptake, degradation or metabolism of physiological AHR agonists 4,7,8 . Thus, the quantification of AHR agonists is of clinical interest. Multiple in vivo and in vitro bioassays exist to quantify AHR agonistic activity in biological samples 30 . Denison and colleagues developed reporter systems based on constructs in which the expression of the firefly luciferase gene is driven by XREs present in the promoter of Cyp1a1, a metabolic gene under control of AHR 31 . Upon binding of ligands to AHR, the AHR-ligand complex translocates to the nucleus and binds to the XRE elements, inducing firefly luciferase expression, the levels of which correlate with the net AHR agonistic activity in the sample. Our findings suggest that the species of the cell lines used for these reporter assays may affect the AHR agonistic activity detected in clinical samples. Moreover, these observations suggest that cell-specific factors influence AHR activation and signaling. This is important to consider when using reporter assays to quantify net AHR agonistic activity, which is influenced not only by the production, uptake and metabolism of AHR agonists, but also by AHR antagonists and other modulators of AHR signaling.
Several factors may contribute to the effects of the species of the cell lines used in reporter assays. First, it is theoretically possible that yet uncharacterized molecules in MS sera activate AHR inhibitory pathways in human but not in murine cells. If present, those inhibitors will decrease net AHR agonistic activity in MS sera, limiting AHR-dependent anti-inflammatory mechanisms. Metabolomic approaches 8 could determine the contribution of specific agonists to the reduced AHR agonistic activity in MS sera. Second, our findings using a human cell line deficient in endogenous AHR reconstituted with mouse or human AHR suggest that species specific mechanisms   affect the sensing of AHR agonists irrespective of the origin of AHR itself. These mechanisms may involve factors controlling AHR interactions in the cytoplasm with the 90-kDa heat shock protein (HSP90), the AHR-interacting protein and the cochaperone p23, which have to release AHR upon ligand binding to allow its translocation to the nucleus 1 . Third, interactions of AHR with additional transcription factors such as nuclear factor kappa B (NF-κB) could be cell-line and species specific, influencing AHR binding to XRE elements 32 . Indeed, we evaluated the potential interference of TNF-α in the reporter assay because TNF-α is a known NF-κB activator, and NF-κB and AHR signaling cross-talk at multiple levels [33][34][35] . TNF-α did not interfere with the detection of AHR ligands in our system, but additional yet unknown serum factors may influence AHR signaling in a species and cell-line specific manner. Taken together, these studies highlight the complexity of measuring AHR agonistic activity in clinical samples. Based on the reported species selectivity of AHR ligands and the cell species effects that we report in this manuscript, the use of human cell lines is encouraged for the analysis of AHR agonistic activity in human samples. Moreover, collectively, these findings further support the use of targeted methods to quantify specific AHR agonists and/or antagonists to study their role in human disease.

Methods
Cell lines. HEK293 cells were purchased from ATCC. HepG2 human hepatoma cells and HEPA1 mouse hepatoma cells were obtained from the ATCC. H1G1.1C3-GFP cells, the generous gift of Dr. M. Denison, have been described previously 20 . Sum149 AHR del cells were generated as follows: the CRISPR vector lentiCRISPR   Samples. All serum samples were collected and stored at −80°C using a standardized protocol as described [37][38][39][40] . Clinical characteristics of the cohorts are shown in Table 1.

Statistical analysis.
Statistical analyses were performed with Prism software (GraphPad), using the statistical tests indicated in the individual figure legends. No samples were excluded. The investigators were blinded as to sample cohorts when performing AHR ligand level measurement and samples were run in duplicates. P values of <0.05 were considered significant. All error bars represent s.e.m.