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P047. Differential effects of TLR‑2 heterodimers activation in healthy and inflamed colon in mice


E. Pedrosa1, V. Lorén2, E. Cabré3, C. Sánchez2, P. Romero1, E. Domènech3, J. Manyé2

1Germans Trias i Pujol, Functional Genetics Unit, Badalona, Spain; 2Germans Trias i Pujol, Experimental Digestive Pathology Unit, Badalona, Spain; 3Germans Trias i Pujol Hospital, G‑I & IBD Unit, Badalona, Spain



Background: NOD family receptors play a key role in the maintenance of homeostasis against intestinal microbiota, and their coordinated action is of utmost importance for the host. Although it has been reported that TLR2 stimulation results in a tolerogenic rather than proinflammatory effect if NOD2 is simultaneously stimulated (Watanabe et al. J Clin Invest 2008 & Yang et al. Gastroenterology 2007), little is known about the molecular mechanisms involved in this area of innate mucosal immunity.

Aim: To investigate how the activation of either TLR2/TLR1 or TLR2/TLR6 heterodimers influence NOD2 expression in the colon of healthy and colitic mice.

Methods: Thirty wild-type (WT) and 30 TLR‑2 (−/−) eight-week old female C57BL6j mice were assigned to a DSS-induced or sham colitis. Five days after colitis induction, mice were sacrificed and their colons removed, cleansed, and filled with 1 mL of RPMI 1430 (vehicle) or 1mL of vehicle with 1 µg of either Pam3CSK or lipoteichoic acid (LTA) (ligands of TLR2/TLR1 and TLR2/TLR6, respectively) (n = 5 per group). Colons were sealed and incubated at 37°C in 5%CO2 atmosphere for 3 hours. Then, intraluminal fluid was recovered for cytokine assessment (IL4, IL6, IL8, IL10, IL12p70, IL23, and INFg) by Luminex platform. RNA was obtained from colonic tissue for quantification of TLR1, TLR2, TLR6, NOD2, and 2b-microglobulin, by qPCR. Results of qPCR were normalized to the housekeeping gene, and expressed as folds of change regarding their respective vehicle-treated counterparts. All results refer to WT mice as compared to their respective TLR2 (−/−) experimental group.

Results: The expression of TLR2 was enhanced by both ligands in healthy colons, and decreased in inflamed ones (p < 0.04 in every case). The activation of TLR2/TLR1 heterodimer (by Pam3CSK) enhanced and decreased the expression of NOD2 in healthy and inflamed colons, respectively (p = 0.016 in both cases), while no changes in NOD expression with TLR2/TLR6 activation (by LTA) were observed. LTA incubation resulted in TLR6 enhanced expression, as well as in increased IL‑6, IL‑8, and IL‑10 levels, in inflamed colons.

Conclusions:

  1. Activation of both TLR2 heterodimers induce opposite effects in TLR2 expression in healthy and inflamed colon.
  2. This is associated to parallel effects on NOD2 expression only after TLR2/TLR1 activation.