Abstract 3443: Aryl hydrocarbon receptor and its ligands influence the formation of colonic tertiary lymphoid tissues
Authors: Allred Kimberly F. , Garcia-Villatoro Erika L. , DeLuca Jennifer , Tepe Victoria , Bomstein Zachary , Ufondu Arinzechukwu , Safe Stephen H. , Chapkin Robert S. , Jayaraman Arul , Allred Clinton D. 
Published: April 2023

Abstract
Background: In the large intestine, tertiary lymphoid tissues (TLTs) serve as localized centers of adaptive immune responses. These structures often form in response to inflammation and infection during adulthood. However, more recently TLTs have become a focal point of colon caner development and progression as their presence is often associated with positive clinical outcomes. Interestingly, several studies have demonstrated that the aryl hydrocarbon receptor (AhR) influences the development of secondary lymphoid tissues, but minimal studies have focused on its role in TLT formation. The purpose of the presented studies was to test the hypothesis that activation of AhR in intestinal epithelial cells (IECs) induces the formation of TLTs in the colon.
Methods: Wild type (WT) and IEC-specific AhR knockout (CDX2PCreT2 x AhRf/f- iAhRKO) mice were used in several different experimental models. In the first study, azoxymethane (AOM) was used to induce precancerous lesions in the colon and TLT formation was evaluated in the presence and absence of AhR. Then, experiments were conducted to determine the necessity of AhR expression in IECs to influence the formation of TLTs following induction of acute inflammation induced by Dextran sodium sulfate (DSS) exposure. Finally, the ability of known, naturally occurring and microbial-derived AhR ligands (indole-3-aldehyde (I3A), 3,3ʹ-diindolylmethane (DIM), and indole-3-acetic acid (IAA)) were tested for their ability to induce TLT formation in the colon. Intestinal permeability (FITC-Dextran), expression of IEC-associated genes (real-time qPCR), and TLT formation/composition (H&E staining/Immunofluorescence) were evaluated.
Results: Results from the first study demonstrated that IEC-specific AhR KO mice exposed to AOM developed significantly fewer TLTs when compared to WT controls, while expression of Il-22 and other chemokines involved in TLT formation were also significantly downregulated. In the acute inflammation study, sex specific results were found. In females, loss of AhR activity in IECs reduced the formation of TLTs without significant changes in immune cell composition within their TLTs. Conversely, in males, loss of AhR lowered expression of functional-IEC genes (Ocln, Il-22), increased number of TLTs, increased T-cell density, and lower B: T cell ratio. Finally, in the other experiments, I3A induced TLT formation and DIM promoted intestinal barrier integrity through the upregulation of various tight junction genes and genes involved in the signaling associated with TLT formation. Evaluation of IAA which is a microbial-derived, AhR ligand is ongoing.
Conclusion: These data indicate that AhR plays a distinct role in the formation of TLTs in the colon and these effects are likely ligand specific and may have a significant effect on colon tumor formation.
Citation Format: Kimberly F. Allred, Erika L. Garcia-Villatoro, Jennifer DeLuca, Victoria Tepe, Zachary Bomstein, Arinzechukwu Ufondu, Stephen H. Safe, Robert S. Chapkin, Arul Jayaraman, Clinton D. Allred. Aryl hydrocarbon receptor and its ligands influence the formation of colonic tertiary lymphoid tissues [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3443.