Toll-like receptor-4 mediates intestinal barrier breakdown after thermal injury.
OBJECTIVE
Toll-like receptor 4 (TLR-4) activation after sterile injury leads to organ dysfunction at distant sites. We have shown previously that intestinal barrier breakdown and alteration of tight junction proteins follows thermal injury; however, the role of TLR-4 in this process remains unclear. We hypothesized that increased intestinal permeability and barrier breakdown after burns is a TLR-4 dependent process; hence, knocking down the TLR-4 gene would have a protective effect on burn-induced intestinal dysfunction.


METHODS
Male C57BL/6J (TLR-4 wild type [WT]) and C57BL/10ScN (TLR-4 knockout [KO]) mice were assigned randomly to either 30% total body surface area steam burn or sham injury. At 4 h, permeability to intraluminally administered fluorescein isothiocyanate (FITC)-dextran was assessed by measuring the fluorescence of the serum. Intestinal samples were analyzed for the presence of the tight junction protein occludin by immunoblotting and immunohistochemistry. Tumor necrosis factor (TNF)-alpha concentrations in the serum and intestines were measured by enzyme-linked immunosorbent assay at 2 h post-burn.


RESULTS
Serum concentrations of FITC-dextran were decreased in TLR-4 KO mice compared with TLR-4 WT mice after burn injury (92.0 micrograms/mL and 264.5 micrograms/mL, respectively; p < 0.05). After injury, no difference in intestinal permeability was observed between the TLR-4 KO mice and the TLR-4 WT sham-treated mice. The TLR-4 KO mice had preservation of occludin concentrations after thermal injury in both immunoblot and immunohistochemistry assays, but concentrations were decreased in TLR-4 WT animals. The serum concentrations of TNF-alpha serum were higher in TLR-4 WT burned animals than in the sham-treated mice. The TLR-4 KO animals had unmeasurable concentrations of TNF-alpha. No differences in TNF-alpha were observed in the intestinal tissue at 2 h.


CONCLUSIONS
Mice with TLR-4 KO have less intestinal permeability to FITC-dextran than do TLR-4 WT mice after burn injury as a result of alterations in the tight junction protein occludin. These findings suggest that the greater intestinal permeability and barrier breakdown after burn injury is a TLR-4-dependent process. Toll-like receptor 4 may provide a useful target for the prevention and treatment of systemic inflammatory response syndrome and multisystem organ failure after injury.
Peterson
Carrie Y
CY
Division of Trauma, Critical Care and Burns, Department of Surgery, University of California, San Diego, San Diego, California 92103-8896, USA.
Costantini
Todd W
TW
Loomis
William H
WH
Putnam
James G
JG
Wolf
Paul
P
Bansal
Vishal
V
Eliceiri
Brian P
BP
Baird
Andrew
A
Coimbra
Raul
R
D000818:Animals; D002056:Burns / Q000150:complications* / Q000276:immunology*; D003911:Dextrans / Q000493:pharmacokinetics; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives / Q000493:pharmacokinetics; D041981:Gastrointestinal Tract / Q000737:chemistry / Q000276:immunology* / Q000503:physiopathology*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D008565:Membrane Proteins / Q000032:analysis; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D062793:Occludin; D010539:Permeability; D051197:Toll-Like Receptor 4 / Q000172:deficiency / Q000276:immunology*; D014409:Tumor Necrosis Factor-alpha / Q000032:analysis / Q000097:blood
D003911:Dextrans; D008565:Membrane Proteins; C568985:OCLN protein, human; D062793:Occludin; C568986:Ocln protein, mouse; C493487:Tlr4 protein, mouse; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; C015219:fluorescein isothiocyanate dextran; D016650:Fluorescein-5-isothiocyanate