<b><i>Introduction:</i></b> Toll-like receptor 4 (TLR4) is a highly conserved immunosurveillance protein of innate immunity, displaying well-established roles in homeostasis and intestinal inflammation. Current evidence shows complex relationships between TLR4 activation, maintenance of health, and disease progression; however, it commonly overlooks the importance of site-specific TLR4 expression. This omission has the potential to influence translation of results as previous evidence shows the differing and distinct roles that TLR4 exhibits are dependent on its spatiotemporal expression. <b><i>Methods:</i></b> An intestinal epithelial TLR4 conditional knockout (KO) mouse line (<i>Tlr4</i>^<i>ΔIEC</i>, <i>n</i> = 6–8) was utilized to dissect the contribution of epithelial TLR4 expression to intestinal homeostasis with comparisons to wild-type (WT) (<i>n</i> = 5–7) counterparts. Functions of the intestinal barrier in the ileum and colon were assessed with tissue resistance in Ussing chambers. Molecular and structural comparisons in the ileum and colon were assessed via histological staining, expression of tight junction proteins (occludin and zonular occludin 1 [ZO-1]), and presence of CD11b-positive immune cells. <b><i>Results:</i></b> There was no impact of the intestinal epithelial TLR4 KO, with no differences in (1) tissue resistance–ileum (mean ± standard error of mean [SEM]): WT 22 ± 7.2 versus <i>Tlr4</i>^<i>ΔIEC</i> 20 ± 5.6 (Ω × cm²) <i>p</i> = 0.831, colon WT 30.8 ± 3.6 versus <i>Tlr4</i>^<i>ΔIEC</i> 45.1 ± 9.5 <i>p</i> = 0.191; (2) histological staining (overall tissue structure); and (3) tight junction protein expression (% area stain, mean ± SEM)–ZO-1: ileum–WT 1.49 ± 0.155 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.17 ± 0.07, <i>p</i> = 0.09; colon–WT 1.36 ± 0.26 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.12 ± 0.18 <i>p</i> = 0.47; occludin: ileum–WT 1.07 ± 0.12 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.95 ± 0.13, <i>p</i> = 0.53; colon–WT 1.26 ± 0.26 versus <i>Tlr4</i>^<i>ΔIEC</i> 1.02 ± 0.16 <i>p</i> = 0.45. CD11b-positive immune cells (% area stain, mean ± SEM) in the ileum were mildly decreased in WT mice: WT 0.14 ± 0.02 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.09 ± 0.01 <i>p</i> = 0.04. However, in the colon, there was no difference in CD11b-positive immune cells between strains: WT 0.53 ± 0.08 versus <i>Tlr4</i>^<i>ΔIEC</i> 0.49 ± 0.08 <i>p</i> = 0.73. <b><i>Conclusions:</i></b> These data have 2 important implications. First, these data refute the assumption that epithelial TLR4 exerts physiological control of intestinal physiology and immunity in health. Second, and most importantly, these data support the use of the <i>Tlr4</i>^<i>ΔIEC</i> line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.

Purpose Irinotecan can cause high levels of diarrhea caused by toxic injury to the gastrointestinal microenvironment. Tolllike receptor 4 (TLR4) and the gut microbiome have previously been implicated in gastrointestinal toxicity and diarrhea; however, the link between these two factors has not been definitively determined. We used a tumor-bearing, intestinal epithelial cell (IEC) TLR4 knockout model (Tlr4 ΔIEC ) to assess microbiome changes following irinotecan treatment. We then determined if a fecal microbiota transplant (FMT) between Tlr4 ΔIEC and wild-type (WT) mice altered irinotecan-induced gastrointestinal toxicity. Methods MC-38 colorectal cancer cells were injected into WT and Tlr4 ΔIEC mice. Fecal samples were collected prior to tumor inoculation, prior to irinotecan treatment and at cull. 16S rRNA gene sequencing was used to assess changes in the microbiome. Next, FMT was used to transfer the microbiome phenotype between Tlr4 ΔIEC and WT mice prior to irinotecan treatment. Gastrointestinal toxicity symptoms were assessed. ResultsIn study 1, there were no compositional differences in the microbiome between Tlr4 ΔIEC and WT mice at baseline. However, predicted functional capacity of the microbiome was different between WT and Tlr4 ΔIEC at baseline and postirinotecan. In study 2, Tlr4 ΔIEC mice were protected from grade 3 diarrhea. Additionally, WT mice who did not receive FMT had more colonic damage in the colon compared to controls (P = 0.013). This was not seen in Tlr4 ΔIEC mice or WT mice who received FMT (P > 0.05). Conclusion Tlr4 ΔIEC and WT had no baseline compositional microbiome differences, but functional differences at baseline and following irinotecan. FMT altered some aspects of irinotecan-induced gastrointestinal toxicity.

Introduction Gastrointestinal mucositis (GIM) is a side effect of high-dose irinotecan (CPT-11), causing debilitating symptoms that are often poorly managed. The role of TLR4 in the development of GIM has been clearly demonstrated. We, therefore, aimed to investigate the potential of the TLR4 antagonist, IAXO-102, to attenuate gastrointestinal inflammation as well as supress tumour activity in a colorectal-tumour-bearing mouse model of GIM induced by CPT-11. Methods 24 C57BL/6 mice received a vehicle, daily i.p. IAXO-102 (3 mg/kg), i.p. CPT-11 (270 mg/kg) or a combination of CPT-11 and IAXO-102. GIM was assessed using validated toxicity markers. At 72 h, colon and tumour tissue were collected and examined for histopathological changes and RT-PCR for genes of interest; TLR4, MD-2, CD-14, MyD88, IL-6, IL-6R, CXCL2, CXCR1, and CXCR2. Results IAXO-102 prevented diarrhoea in mice treated with CPT-11. Tumour volume in IAXO-102-treated mice was lower compared to vehicle at 48 h (P < 0.05). There were no differences observed in colon and tumour weights between the treatment groups. Mice who received the combination treatment had improved tissue injury score (P < 0.05) in the colon but did not show any improvements in cell proliferation or apoptotic rate. Expression of all genes was similar across all treatment groups in the tumour (P > 0.05). In the colon, there was a difference in transcript expression in vehicle vs. IAXO-102 (P < 0.05) and CPT-11 vs. combination (P < 0.01) in MD-2 and IL-6R, respectively. Conclusion IAXO-102 was able to attenuate symptomatic parameters of GIM induced by CPT-11 as well as reduce tissue injury in the colon. However, there was no effect on cell proliferation and apoptosis. As such, TLR4 activation plays a partial role in GIM development but further research is required to understand the specific inflammatory signals underpinning tissue-level changes.

Inverse agonists of peroxisome proliferator activated receptor γ (PPARγ) have emerged as safer alternatives to full agonists for their reduced side effects while still maintaining impressive insulin-sensitizing properties. To shed light on their molecular mechanism, we characterized the interaction of the PPARγ ligand binding domain with SR10221. X-ray crystallography revealed a novel binding mode of SR10221 in the presence of a transcriptionally repressing corepressor peptide, resulting in much greater destabilization of the activation helix, H12, than without corepressor peptide. Electron paramagnetic resonance provided in-solution complementary protein dynamic data, which revealed that for SR10221-bound PPARγ, H12 adopts a plethora of conformations in the presence of corepressor peptide. Together, this provides the first direct evidence for corepressor-driven ligand conformation for PPARγ and will allow the development of safer and more effective insulin sensitizers suitable for clinical use.

Purpose Toll-like receptor 4 (TLR4) is increasingly recognized for its ability to govern the etiology and prognostic outcomes of colorectal cancer (CRC) due to its profound immunomodulatory capacity. Despite widespread interest in TLR4 and CRC, no clear analysis of current literature and data exists. Therefore, translational advances have failed to move beyond conceptual ideas and suggestions. Methods We aimed to determine the relationship between TLR4 and CRC through a systematic review and analysis of published literature and datasets. Data were extracted from nine studies that reported survival, CRC staging and tumor progression data in relation to TLR4 expression. Primary and metastatic tumor samples with associated clinical data were identified through the Cancer Genome Atlas (TCGA) database. Results Systematic review identified heterogeneous relationships between TLR4 and CRC traits, with no clear theme evident across studies. A total of 448 datasets were identified through the TCGA database. Analysis of TCGA datasets revealed TLR4 mRNA expression is decreased in advanced CRC stages (P < 0.05 for normal vs Stage II, Stage III and Stage IV). Stage-dependent impact of TLR4 expression on survival outcomes were also found, with high TLR4 expression associated with poorer prognosis (stage I vs III (HR = 4.2, P = 0.008) and stage I vs IV (HR = 11.3, P < 0.001)). Conclusion While TLR4 mRNA expression aligned with CRC staging, it appeared to heterogeneously regulate survival outcomes depending on the stage of disease. This underscores the complex relationship between TLR4 and CRC, with unique impacts dependent on disease stage.