title: LPS-binding protein enables intestinal epithelial restitution despite LPS exposure.
abstract: OBJECTIVE
  Intestinal epithelial restitution is the first part in the process of mucosal repair after injury in the intestine. Integrity of the intestinal mucosal barrier is important as a first line of defense against bacteria and endotoxin. Necrotizing enterocolitis (NEC) is a major cause of morbidity and mortality in extremely-low-birth-weight infants, but its mechanisms are not well defined. Abnormal bacterial colonization, immature barrier function, innate immunity activation, and inflammation likely play a role. Lipopolysaccharide (LPS)-binding protein (LBP) is secreted by enterocytes in response to inflammatory stimuli and has concentration-dependent effects. At basal concentrations, LBP stimulates the inflammatory response by presenting LPS to its receptor; however, at high concentrations, LBP is able to neutralize LPS and prevent an exaggerated inflammatory response. We sought to determine how LBP would affect wound healing in an in vitro model of intestinal cell restitution and protect against intestinal injury in a rodent model of NEC.
  
  
  METHODS
  Immature intestinal epithelial cells (IEC-6) were seeded in poly-L-lysine-coated 8-chamber slides and grown to confluence. A 500-μm wound was created using a cell scraper mounted on the microscope to achieve uniform wounding. Media was replaced with media containing LPS ± LBP. Slide wells were imaged after 0, 8, and 24 hours and then fixed. Cellular restitution was evaluated via digital images captured on an inverted microscope and wound closure was determined by automated analysis. Toll-like receptor 4 (TLR4) was determined by reverse transcriptase-polymerase chain reaction after RNA isolation from wounded cells 24 hours after treatment.
  
  
  RESULTS
  LPS alone attenuated wound healing in immature intestinal epithelium. This attenuation is reversed by 24 hours with increasing concentrations of LBP so that wound healing is equivalent to control (P < 0.001). TLR4 was increased with LPS alone but levels returned to that of control after addition of LBP in the higher concentrations. LBP had no effect on the development of intestinal injury when given during our rodent model of NEC. Abnormal bacterial colonization and activation of innate immunity by LPS are likely involved in the pathogenesis of NEC.The attenuation of wound healing was reversed when LBP was added to LPS but only in the higher concentrations. At these same concentrations of LBP, TLR4 was decreased to that of control.
  
  
  CONCLUSIONS
  These results indicate that LBP may be a novel therapeutic strategy to facilitate wound healing after the acute phase of NEC and other forms of intestinal injury.
authors: Center for Perinatal Research, The Research Institute at Nationwide Children's Hospital, Nationwide Children's Hospital, Columbus, OH 43205, USA.
authors: Juli M
authors: JM
authors: Richter
authors: Brandon L
authors: BL
authors: Schanbacher
authors: Hong
authors: H
authors: Huang
authors: Jianjing
authors: J
authors: Xue
authors: John A
authors: JA
authors: Bauer
authors: Peter J
authors: PJ
authors: Giannone
mesh_terms: D000208:Acute Disease; D000209:Acute-Phase Proteins / Q000008:administration & dosage* / Q000378:metabolism; D000284:Administration, Oral; D000818:Animals; D002352:Carrier Proteins / Q000008:administration & dosage* / Q000378:metabolism; D002460:Cell Line; D004195:Disease Models, Animal; D020345:Enterocolitis, Necrotizing / Q000188:drug therapy; D020895:Enterocytes / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D007113:Immunity, Innate; D007249:Inflammation / Q000503:physiopathology; D007413:Intestinal Mucosa / Q000378:metabolism*; D007422:Intestines / Q000166:cytology; D008070:Lipopolysaccharides / Q000009:adverse effects* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000008:administration & dosage* / Q000378:metabolism; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism; D014945:Wound Healing / Q000187:drug effects*; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D051197:Toll-Like Receptor 4; D000099231:Lipopolysaccharide-Binding Protein; C495347:Tlr4 protein, rat