C. Culbreath1, S. Tanner1, T. Berryhill1, R. G. Lorenz2, C. A. Martin1 1University Of Alabama,Pediatric Surgery,Birmingham, Alabama, USA 2University Of Alabama,Pathology,Birmingham, Alabama, USA
Introduction: Maintenance of intestinal homeostasis by protection against pathogenic bacteria and dietary antigens is critical for intestinal homeostasis. Immunoglobulins, specifically IgA, play a key role in coating luminal antigens and preventing translocation of harmful bacteria. The aryl hydrocarbon receptor (AhR) is a basic-helix-loop transcription factor, that when stimulated by exogenous pollutants, microbial products, and dietary components, activates factors important for barrier function and intestinal homeostasis. To date, the function of AhR has not been studied in the developing innate immune system. Furthermore it is not clear how the extrauterine environmental influences the establishment of host/microbial mutualism. We hypothesize that AhR signaling is critical for establishment of intestinal homeostasis in neonates.
Methods: Three groups of mice were used. C57BL/6 (B6) AhR+/+ (WT), B6.AhR-/- (KO) , and B6.AhR+/+ raised on an AhR Ligand Free diet (AhR LF). AhR LF is defined as mice whose parents and the subsequent pups were maintained on a nutritionally balanced diet as well as cage bedding that was free of all exogenous AhR ligands. Enzyme-linked immunosorbent assay (ELISA) was used to measure fecal IgA levels in these groups of mice at 2 and 8 weeks of age. To determine the contribution of IgA from breast milk the gastric contents of 2 week old pups were harvested from the 3 groups of mice. Intestinal homeostasis was measured by culturing the mesenteric lymph nodes (MLN) under aerobic conditions to determine the number of bacterial colony forming units (CFU) after 72 hours. Results were analyzed by the Student’s unpaired T-test and expressed as the mean ± standard error of the mean.
Results: Two week old KO mice had significantly less fecal IgA (37 ± 37 µg/ml) compared to WT (2519 ± 807 µg/ml ) and AhR LF (3043 ± 764 µg/ml), p value = 0.0393. The amount of IgA from the gastric contents of 2 week old mice was not significant, WT (40 ± 14 µg/ml), KO (40 ± 18 µg/ml), AhR LF (24 ± 5 µg/ml), p value = 0.322. At 8 weeks of age AhR LF mice (1769 ± 369 µg/ml) had significantly less fecal IgA than WT (17,574 ± 4916 µg/ml) and KO (12,553 ± 2666 µg/ml), p value = 0.0077. At 2 weeks, KO mice had significantly higher levels of bacterial translocation (158 ± 37 CFU) compared to WT (2 ± 1 CFU) and AhR LF (9 ± 5 CFU), p value = 0 .0132. At 8 weeks AhR LF had significantly higher levels of bacterial translocation (119 ± 57 CFU) compared to WT (15 ± 7) and KO (11 ± 10) , p value = 0.019
Conclusion: In neonatal mice, the lack of AhR signaling is associated with loss of intestinal homeostasis, evidenced by decreased levels of IgA and increased bacterial translocation. In adult mice, exogenous AhR ligand and not receptor signaling is necessary for maintenance of intestinal integrity.