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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 non-competitive and competitive TLR4 inhibitors, TAK-242 (10\u00a0\u00b5M) and IAXO-102 (10\u00a0\u00b5M), respectively, or vehicle were added to human T84, HT-29, and U937 cell lines and mouse colonic explants 1\u00a0h before the addition of lipopolysaccharide (LPS) (<jats:italic>in vitro<\/jats:italic>: 100\u00a0\u00b5g\/mL; <jats:italic>ex vivo<\/jats:italic>: 10\u00a0\u00b5g\/mL), SN-38 (<jats:italic>in vitro<\/jats:italic>: 1\u00a0\u00b5M or 1\u00a0nM; <jats:italic>ex vivo<\/jats:italic>: 2\u00a0\u00b5M), and\/or tumour necrosis factor-alpha (TNF-\u03b1) (5\u00a0\u00b5g\/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 (<jats:italic>P<\/jats:italic>\u2009&lt;\u20090.0001). However, this was not prevented by either antagonist (<jats:italic>P<\/jats:italic>\u2009&gt;\u20090.05) in any of the 3 cell lines. Quantitative histological assessment scores showed no differences between vehicle and treatment groups (<jats:italic>P<\/jats:italic>\u2009&gt;\u20090.05). There were no differences in <jats:italic>in vitro<\/jats:italic> IL-8 (<jats:italic>P<\/jats:italic>\u2009&gt;\u20090.05, in all 3 cells lines) and <jats:italic>ex vivo<\/jats:italic> IL-6 (<jats:italic>P<\/jats:italic>\u2009&gt;\u20090.05) concentrations between vehicle and treatment groups. Transcript expression of all genes was similar across vehicle and treatment groups (<jats:italic>P<\/jats:italic>\u2009&gt;\u20090.05). 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.\n<\/jats:p>","DOI":"10.1007\/s10753-022-01714-0","type":"journal-article","created":{"date-parts":[[2022,7,22]],"date-time":"2022-07-22T08:06:02Z","timestamp":1658477162000},"page":"103-114","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":16,"title":["Investigation of TLR4 Antagonists for Prevention of Intestinal Inflammation"],"prefix":"10.1007","volume":"46","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2293-4854","authenticated-orcid":false,"given":"Janine S. 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