Of innate immunity and has to be regulated tightly in gut

Of innate immunity and has to be regulated tightly in gut epithelium to maintain the balance between normal and overexuberant activation due to the presence of large amount of commensal bacteria in the lumen of the gastrointestinal tract [17]. Among all TLRs in the gut, TLR2 and TLR4 play important roles in physiological and pathological processes, and are both involved in intestinal permeability regulation. TLR2 and TLR4 have been shown to regulate the gate-keeping functions of the intestinal follicle-associated epithelium [18]. Paradoxically, activation of TLR4 by 11967625 LPS increases intestinal monolayer permeability in a myosin light chain kinase (MLCK)-dependent manner [19,20]. Meanwhile, there is evidence showing intracellular cross talk between MOR signaling and TLR signaling in various kinds of cells [3]. For example, morphine significantly inhibits tumor necrosis factor-a (TNF- a), but not interleukin-6 (IL-6) production, in a MOR-independent manner in polyglycan-stimulated peripheral blood mononuclear cells [21]. However, the intracellular mechanism underlying how morphine compromises epithelial barrier function via modulating TLRs is still not defined. In the present study, we hypothesize that morphine disrupts the barrier function of gut epithelium by increasing the sensitivity of gut epithelial cells to TLR activation, resulting in bacterial translocation from the gut lumen. We investigated the effects of morphine on gut barrier function in wild type (WT), TLR2 knockout, TLR4 knockout, and TLR2/4 double knockout mice. The direct effects of morphine on gut epithelial cells were further studied with rodent small intestinal and colonic epithelial cell lines, IEC-6 and CMT93, respectively. Our results from in vivo and in vitro studies indicate that morphine treatment compromises gut barrier function in a TLR-dependent manner.Animal treatmentMice received morphine and pellet implantation method as described [23]. Using this method, plasma levels of morphine are in the 0.6?.0-microg/ml range (range seen in opioid abusers and patients on opioids for moderate to severe pain). Furthermore, this model is commonly used in the study of opiate dependence and addiction [23]. Briefly, placebo or 75 mg morphine pellets (National Institutes of Health [NIH]/National Institute on Drug Abuse [NIDA], Bethesda, MD) were inserted in a small pocket created by a small skin incision on the animal’s dorsal side; incisions were closed using surgical wound clips (Stoelting, 9 mm Stainless Steel, Wooddale, IL). Animals were injected with MLCK inhibitor ML-7 (2 mg/kg) overnight before LPS or Lipoteichoic acid (LTA) treatment. At this dose, ML-7 successfully inhibited activity of myosin light chain kinase and protected the barrier function of endothelial cells in mice [24].Intestinal permeabilityAll animals were gavaged with ampicillin-resistant E. coli (26107 CFU suspended in 400 ml of sterile saline) or FITCdextran (600 mg/kg body weight in 20 mg/ml concentration) utilizing a 4-cm long, curved needle with a plastic ball at the tip. After sacrifice, MLN and liver were collected and cultured on LB plates containing 100 mg/ml of ampicillin to measure bacterial translocation. Whole blood FITC-dextran concentration was determined by fluorometry based on a standard curve.ImmunofluorescenceSections of small intestinal and colonic tissue from all mice sacrificed for tight junction staining were frozen in TFMTM tissue freezing medium (TBS, Durham, NC). At least five s.