Ing that morphine’s effect on intestinal tightMorphine-induced bacterial translocation is attenuated in TLR2/TLR4 knockout miceTo further determine roles of TLR2 and TLR4 in morphineinduced bacterial translocation, we purchase 370-86-5 implanted C57BL/6 J WT (n = 9), TLR2 knockout (n = 9), TLR4 knockout (n = 9), and TLR2/4double knockout (n = 9) mice with morphine pellets to determine bacterial load in MLN and liver as described previously. Placebo-treated TLR 2, 4 KO mice Dimethylenastron showed very low basal levels of bacterial load in MLN and liver. Morphine-treated WT mice still showed significant bacterial translocation to MLN and liver. In contrast, morphine-treated TLR2, 4 knockout mice showed lower bacterial translocation into MLN and liver than did WT mice (Figure 5) although TLRKO did not show any effects on morphine-induced constipation, suggesting that constipation is not the only dominant factor causing bacterial translocation following morphine treatment and other TLR-dependent mechanisms 25331948 also contribute to the process of TJ disorganization and barrier dysfunction (Figure S3). These findings indicated that both TLR2 and TLR4 are involved in morphine modulation of intestinal barrier function.TLR2/TLR4 knockout protects tight junction organization from morphine-induced disruptionTo further determine the role of TLRs in morphine’s modulation of intestinal tight junction proteins, we isolated the small intestine from WT, TLR2 knockout, TLR4 knockout, and TLR2/4 double 18325633 knockout mice to assess the organization of tight junction proteins, as described previously. In TLR2KO and TLR2/4KO mice, the occludin and ZO-1 staining were continuous and intact following morphine treatment (Figure 6AMorphine Promotes Bacterial TranslocationFigure 4. Morphine treatment upregulates TLR expression in small intestinal epithelial cells. (A) Isolated cells were fixed using eBioscience Fixation and Permeabilization Kit and then incubated with anti-cytokeratin antibody or isotype control. Cytokeratin positive cells were gated in P2 according to isotype control. (B) Real-time PCR analysis of mRNA levels of TLR2 and TLR4 in epithelial cells of small intestine after 24 hour morphine treatment. (C) and (E) Representative expression of TLR2 and TLR4 in epithelial cells of small intestine after 24 hour morphine treatment from 3-time experiments. (D) and (F) Frequencies of TLR2 and TLR4 positive cells within cytokeratin positive cells. * P,0.05 by Student’s t-test. doi:10.1371/journal.pone.0054040.gMorphine Promotes Bacterial TranslocationFigure 5. Morphine-induced bacterial translocation is attenuated in TLR2/TLR4 knockout mice. WT, TLR2 knockout, TLR4 knockout, and TLR2/4 double knockout mice were implanted with 75 mg morphine pellet for 24 hours; MLN(A), liver (B) were cultured on blood agar plates overnight. Bacterial colonies were quantified and described as CFU. ?Mean of CFU *P,0.05, **P,0.01 by ANOVA one-way analysis, followed by Bonferroni post-test (n = 9). doi:10.1371/journal.pone.0054040.gand 6B). In TLR4KO mice, some degree of tight junction disruption was observed following morphine treatment; however, the disruption was not as dramatic as that observed with morphine treatment in WT mice, suggesting a dominant role of TLR2 in morphine modulation of intestinal tight junction organization, which was consistent with our in vitro study: small intestinal cell IEC-6 and colonic epithelial cell CMT-93 were stained for tight junction proteins ZO-1(Figure S4). LPS and LTA but not morphi.Ing that morphine’s effect on intestinal tightMorphine-induced bacterial translocation is attenuated in TLR2/TLR4 knockout miceTo further determine roles of TLR2 and TLR4 in morphineinduced bacterial translocation, we implanted C57BL/6 J WT (n = 9), TLR2 knockout (n = 9), TLR4 knockout (n = 9), and TLR2/4double knockout (n = 9) mice with morphine pellets to determine bacterial load in MLN and liver as described previously. Placebo-treated TLR 2, 4 KO mice showed very low basal levels of bacterial load in MLN and liver. Morphine-treated WT mice still showed significant bacterial translocation to MLN and liver. In contrast, morphine-treated TLR2, 4 knockout mice showed lower bacterial translocation into MLN and liver than did WT mice (Figure 5) although TLRKO did not show any effects on morphine-induced constipation, suggesting that constipation is not the only dominant factor causing bacterial translocation following morphine treatment and other TLR-dependent mechanisms 25331948 also contribute to the process of TJ disorganization and barrier dysfunction (Figure S3). These findings indicated that both TLR2 and TLR4 are involved in morphine modulation of intestinal barrier function.TLR2/TLR4 knockout protects tight junction organization from morphine-induced disruptionTo further determine the role of TLRs in morphine’s modulation of intestinal tight junction proteins, we isolated the small intestine from WT, TLR2 knockout, TLR4 knockout, and TLR2/4 double 18325633 knockout mice to assess the organization of tight junction proteins, as described previously. In TLR2KO and TLR2/4KO mice, the occludin and ZO-1 staining were continuous and intact following morphine treatment (Figure 6AMorphine Promotes Bacterial TranslocationFigure 4. Morphine treatment upregulates TLR expression in small intestinal epithelial cells. (A) Isolated cells were fixed using eBioscience Fixation and Permeabilization Kit and then incubated with anti-cytokeratin antibody or isotype control. Cytokeratin positive cells were gated in P2 according to isotype control. (B) Real-time PCR analysis of mRNA levels of TLR2 and TLR4 in epithelial cells of small intestine after 24 hour morphine treatment. (C) and (E) Representative expression of TLR2 and TLR4 in epithelial cells of small intestine after 24 hour morphine treatment from 3-time experiments. (D) and (F) Frequencies of TLR2 and TLR4 positive cells within cytokeratin positive cells. * P,0.05 by Student’s t-test. doi:10.1371/journal.pone.0054040.gMorphine Promotes Bacterial TranslocationFigure 5. Morphine-induced bacterial translocation is attenuated in TLR2/TLR4 knockout mice. WT, TLR2 knockout, TLR4 knockout, and TLR2/4 double knockout mice were implanted with 75 mg morphine pellet for 24 hours; MLN(A), liver (B) were cultured on blood agar plates overnight. Bacterial colonies were quantified and described as CFU. ?Mean of CFU *P,0.05, **P,0.01 by ANOVA one-way analysis, followed by Bonferroni post-test (n = 9). doi:10.1371/journal.pone.0054040.gand 6B). In TLR4KO mice, some degree of tight junction disruption was observed following morphine treatment; however, the disruption was not as dramatic as that observed with morphine treatment in WT mice, suggesting a dominant role of TLR2 in morphine modulation of intestinal tight junction organization, which was consistent with our in vitro study: small intestinal cell IEC-6 and colonic epithelial cell CMT-93 were stained for tight junction proteins ZO-1(Figure S4). LPS and LTA but not morphi.
