Discipline of Physiology, School of Biomedicine, University of Adelaide, Adelaide, South Australia, 5005, Australia.
Discipline of Pharmacology, School of Biomedicine, University of Adelaide, Adelaide, South Australia, Australia.
Inflammation. 2023 Feb;46(1):103-114. doi: 10.1007/s10753-022-01714-0. Epub 2022 Jul 22.
Activation 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 non-competitive 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.
Toll-like receptor 4 (TLR4) 的激活已被证明对肠黏膜炎 (IM) 的炎症信号通路有重大影响，TLR4 敲除小鼠就是证明。因此，抑制 TLR4 已被认为是治疗 IM 的一种有潜力的新治疗方法，但特定的 TLR4 抑制剂仍有待研究。有鉴于此，我们旨在确定直接 TLR4 拮抗是否能预防 IM 的临床前实验模型中的炎症。非竞争性和竞争性 TLR4 抑制剂 TAK-242（10µM）和 IAXO-102（10µM）或载体分别在加入脂多糖（LPS）（体外：100µg/mL；离体：10µg/mL）、SN-38（体外：1µM 或 1nM；离体：2µM）和/或肿瘤坏死因子-α（TNF-α）（5µg/mL）之前 1 小时加入人 T84、HT-29 和 U937 细胞系和小鼠结肠外植体中。收集上清液用于人白细胞介素 8 和小鼠白细胞介素 6 酶联免疫吸附测定（ELISA），作为炎症信号的测量。使用 XTT 测定法测量细胞活力。外植体组织用于感兴趣基因的组织病理学和 RT-PCR 分析：TLR4、MD2、CD14、MyD88、IL-6、IL-6R、CXCL2、CXCR1、CXCR2。与载体相比，SN-38 增加了细胞抑制作用（P<0.0001）。然而，在这 3 种细胞系中，任何一种拮抗剂都没有阻止这种情况（P>0.05）。定量组织学评估评分显示，载体组和治疗组之间无差异（P>0.05）。体外白细胞介素 8（P>0.05，在所有 3 种细胞系中）和离体白细胞介素 6（P>0.05）浓度在载体组和治疗组之间无差异。所有基因的转录表达在载体组和治疗组之间相似（P>0.05）。使用特异性抑制剂 TAK-242 和 IAXO-102 阻断 TLR4 在这些粘膜炎临床前模型中并未有效阻止粘膜炎。这项工作表明，用这些化合物特异性抑制上皮细胞 TLR4 不足以控制粘膜炎相关炎症。相反，通过免疫细胞的 TLR4 信号可能是预防 IM 的更重要靶点。
