Tam, J.S.Y.Coller, J.K.Prestidge, C.A.Bowen, J.M.2025-07-172025-07-172023Inflammation, 2023; 46(1):103-1140360-39971573-2576https://hdl.handle.net/2440/146052Activation of toll-like receptor 4 (TLR4) has been shown to be a major influence on the inflammatory signalling pathways in intestinal mucositis (IM), as demonstrated by TLR4 knock-out mice. Pharmacological TLR4 inhibition has thus been postulated as a potential new therapeutic approach for the treatment of IM but specific TLR4 inhibitors have yet to be investigated. As such, we aimed to determine whether direct TLR4 antagonism prevents inflammation in pre-clinical experimental models of IM. The noncompetitive and competitive TLR4 inhibitors, TAK-242 (10 μM) and IAXO-102 (10 μM), respectively, or vehicle were added to human T84, HT-29, and U937 cell lines and mouse colonic explants 1 h before the addition of lipopolysaccharide (LPS) (in vitro: 100 μg/ mL; ex vivo: 10 μg/mL), SN-38 (in vitro: 1 μM or 1 nM; ex vivo: 2 μM), and/or tumour necrosis factor-alpha (TNF-α) (5 μg/mL). Supernatant was collected for human IL-8 and mouse IL-6 enzyme-linked immunosorbent assays (ELISAs), as a measure of inflammatory signalling. Cell viability was measured using XTT assays. Explant tissue was used in histopathological and RT-PCR analysis for genes of interest: TLR4, MD2, CD14, MyD88, IL-6, IL-6R, CXCL2, CXCR1, CXCR2. SN-38 increased cytostasis compared to vehicle (P < 0.0001). However, this was not prevented by either antagonist (P > 0.05) in any of the 3 cell lines. Quantitative histological assessment scores showed no differences between vehicle and treatment groups (P > 0.05). There were no differences in in vitro IL-8 (P > 0.05, in all 3 cells lines) and ex vivo IL-6 (P > 0.05) concentrations between vehicle and treatment groups. Transcript expression of all genes was similar across vehicle and treatment groups (P > 0.05). TLR4 antagonism using specific inhibitors TAK-242 and IAXO-102 was not effective at blocking IM in these pre-clinical models of mucositis. This work indicates that specific epithelial inhibition of TLR4 with these compounds is insufficient to manage mucositis-related inflammation. Rather, TLR4 signalling through immune cells may be a more important target to prevent IM.en© The Author(s), 2022. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.toll-like receptor 4 (TLR4); TLR4 antagonist; intestinal inflammation; intestinal mucositis; SN-38U937 CellsAnimalsHumansMiceInflammationLipopolysaccharidesInterleukin-8Interleukin-6Toll-Like Receptor 4MucositisIrinotecanJournal article10.1007/s10753-022-01714-02024-02-18617649Coller, J.K. [0000-0002-8273-5048]Bowen, J.M. [0000-0003-0876-0031]