Epithelial-Specific TLR4 Knockout Challenges Current Evidence of TLR4 Homeostatic Control of Gut Permeability

Introduction: 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. Methods: An intestinal epithelial TLR4 conditional knockout (KO) mouse line (Tlr4^ΔIEC, n = 6-8) was utilized to dissect the contribution of epithelial TLR4 expression to intestinal homeostasis with comparisons to wild-type (WT) (n = 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. Results: 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 Tlr4^ΔIEC 20 ± 5.6 (Ω × cm²) p = 0.831, colon WT 30.8 ± 3.6 versus Tlr4^ΔIEC 45.1 ± 9.5 p = 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 Tlr4^ΔIEC 1.17 ± 0.07, p = 0.09; colon-WT 1.36 ± 0.26 versus Tlr4^ΔIEC 1.12 ± 0.18 p = 0.47; occludin: ileum-WT 1.07 ± 0.12 versus Tlr4^ΔIEC 0.95 ± 0.13, p = 0.53; colon-WT 1.26 ± 0.26 versus Tlr4^ΔIEC 1.02 ± 0.16 p = 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 Tlr4^ΔIEC 0.09 ± 0.01 p = 0.04. However, in the colon, there was no difference in CD11b-positive immune cells between strains: WT 0.53 ± 0.08 versus Tlr4^ΔIEC 0.49 ± 0.08 p = 0.73. Conclusions: 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 Tlr4^ΔIEC line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.