P009 Stimulation of Toll-Like Receptor 7 normalises colonic epithelial barrier function during colitis in mice
J. Estevez*, V. Martinez
Universitat Autònoma de Barcelona, Cell biology, physiology and immunology, Bellaterra - Barcelona, Spain
Intestinal inflammation is associated with dysregulated mucosal immune responses and an altered epithelial barrier function (EBF). Stimulators of innate immunity, including Toll-like receptor 7 (TLR7) agonists, have anti-inflammatory activity, with beneficial effects in IBD. We assessed if TLR7 participates in EBF modulation during states of intestinal inflammation.
Colitis was induced in CD1 male mice (dextran sodium sulfate, DSS; 5 % in water for 5 days). At day 7 (acute colitis), the effects of the acute TLR7 stimulation with Imiquimod (IMQ) on colonic epithelial permeability to macromolecules (4 kDa fluorescein isothiocyanate-dextran, FD4) was assessed in vivo (passage of FD4 administered intracolonicaly to blood and urine) and in vitro (apical-to-basolateral flux FD4 in a Ussing chamber system).
Table 2 Flux of FD4 assessed in vitro (Ussing chamber) (data are mean±sem, n=5–10; *: p<0.05 vs Vehicle-vehicle. #: p<0.05 vs. DSS-vehicle)
|Apical-to-basolateral flux of FD4 (% in 60 min)|
|Basolateral addition of vehicle/IMQ||0.00156 ± 0.0005||0.00093 ± 0.0004||0.00314 ± 0.0008 *||0.00240 ± 0.00087|
|Apical addition of vehicle/IMQ||0.00182 ± 0.0005||0.00102 ± 0.0002||0.00408 ± 0.0010 *||0.00190 ± 0.0005 #|
Table 1 Flux of FD4 assessed in vivo (data are mean±sem, n=4–6; *: P<0.05 vs. vehicle-vehicle)
|FD4 in serum (µg/mL)||1.22 ± 0.24||1.71 ± 0.18||22.02 ± 12.65||8.73 ± 4.16|
|FD4 in urine (µg/mL)||5.96 ± 0.91||12.03 ± 2.78||155.80 ± 60.15 *||85.97 ± 35.46|
During DSS-induced colitis, passage of FD4 to blood and urine in in vivo conditions was increased by 18- and 26-fold, respectively (P<0.05 vs. non-inflamed controls; Table 1). Acute treatment with IMQ (300 µg/mouse, intracolonic, 1 h before testing permeability) attenuated the passage of FD4, indicating a partial restoration of EBF (Table 1). Preliminary observations show similar effects during the repeated treatment with IMQ (3 consecutive days).
In colonic sheets from DSS-treated animals the in vitro (Ussing chamber) flux of FD4 was increased by 2-fold vs. non-inflamed tissues (P<0.05; Table 2). In these conditions, apical and basolateral addition of IMQ (300 µg) restored the epithelial permeability to FD4 to basal levels. In non-inflamed tissues, apical or basolateral addition of IMQ reduced the flux of FD4 by 44% and 41%, respectively (Table 2).
Stimulation of TLR7 leads to an improvement of epithelial barrier function, observed mainly in inflammatory states. TLR7-mediated innate immune responses might regulate EBF with a defensive function, preventing the passage of luminal factors and, therefore, reducing antigens exposure and the development of exacerbated immune responses and intestinal inflammation.