Elise E Crame1, Joanne M Bowen1, Kate R Secombe1, Janet K Coller2, Maxime François3,4, Wayne Leifert3,4, Hannah R Wardill1,5. 1. Discipline of Physiology, School of Biomedicine, The University of Adelaide, Adelaide, South Australia, Australia. 2. Discipline of Pharmacology, School of Biomedicine, The University of Adelaide, Adelaide, South Australia, Australia. 3. CSIRO Health & Biosecurity, Nutrition and Health Program, Molecular Diagnostic Solutions Group, Adelaide, South Australia, Australia. 4. Department of Molecular and Biomedical Science, School of Biological Science, The University of Adelaide, Adelaide, South Australia, Australia. 5. Department of Paediatric Oncology/Haematology, The University of Groningen, University Medical Centre Groningen, Groningen, The Netherlands.
Abstract
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 (Tlr4ΔIEC, 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 Tlr4ΔIEC 20 ± 5.6 (Ω × cm2) p = 0.831, colon WT 30.8 ± 3.6 versus Tlr4ΔIEC 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 Tlr4ΔIEC 1.17 ± 0.07, p = 0.09; colon-WT 1.36 ± 0.26 versus Tlr4ΔIEC 1.12 ± 0.18 p = 0.47; occludin: ileum-WT 1.07 ± 0.12 versus Tlr4ΔIEC 0.95 ± 0.13, p = 0.53; colon-WT 1.26 ± 0.26 versus Tlr4ΔIEC 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 Tlr4ΔIEC 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 Tlr4ΔIEC 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 Tlr4ΔIEC line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.
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 (Tlr4ΔIEC, 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 Tlr4ΔIEC 20 ± 5.6 (Ω × cm2) p = 0.831, colon WT 30.8 ± 3.6 versus Tlr4ΔIEC 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 Tlr4ΔIEC 1.17 ± 0.07, p = 0.09; colon-WT 1.36 ± 0.26 versus Tlr4ΔIEC 1.12 ± 0.18 p = 0.47; occludin: ileum-WT 1.07 ± 0.12 versus Tlr4ΔIEC 0.95 ± 0.13, p = 0.53; colon-WT 1.26 ± 0.26 versus Tlr4ΔIEC 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 Tlr4ΔIEC 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 Tlr4ΔIEC 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 Tlr4ΔIEC line in future models interrogating health and disease, confirming no confounding effects of genetic manipulation.
Authors: Anne Marit de Groot; Kaushik Thanki; Monique Gangloff; Emily Falkenberg; Xianghui Zeng; Djai C J van Bijnen; Willem van Eden; Henrik Franzyk; Hanne M Nielsen; Femke Broere; Nick J Gay; Camilla Foged; Alice J A M Sijts Journal: Mol Ther Nucleic Acids Date: 2018-02-13 Impact factor: 8.886