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. 2017 Dec;187(12):2698-2710.
doi: 10.1016/j.ajpath.2017.08.005.

Lipopolysaccharide-Induced Increase in Intestinal Epithelial Tight Permeability Is Mediated by Toll-Like Receptor 4/Myeloid Differentiation Primary Response 88 (MyD88) Activation of Myosin Light Chain Kinase Expression

Meghali Nighot  1 , Rana Al-Sadi  2 , Shuhong Guo  2 , Manmeet Rawat  1 , Prashant Nighot  2 , Martin D Watterson  3 , Thomas Y Ma  4
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Lipopolysaccharide-Induced Increase in Intestinal Epithelial Tight Permeability Is Mediated by Toll-Like Receptor 4/Myeloid Differentiation Primary Response 88 (MyD88) Activation of Myosin Light Chain Kinase Expression

Meghali Nighot et al. Am J Pathol. 2017 Dec.

Abstract

Lipopolysaccharides (LPSs) are a major component of the Gram-negative bacterial cell wall and play an important role in mediating intestinal inflammatory responses in inflammatory bowel disease. Although recent studies suggested that physiologically relevant concentrations of LPS (0 to 1 ng/mL) cause an increase in intestinal epithelial tight junction (TJ) permeability, the mechanisms that mediate an LPS-induced increase in intestinal TJ permeability remain unclear. Herein, we show that myosin light chain kinase (MLCK) plays a central role in the LPS-induced increase in TJ permeability VSports手机版. Filter-grown Caco-2 intestinal epithelial monolayers and C57BL/6 mice were used as an in vitro and in vivo intestinal epithelial model system, respectively. LPS caused a dose- and time-dependent increase in MLCK expression and kinase activity in Caco-2 monolayers. The pharmacologic MLCK inhibition and siRNA-induced knock-down of MLCK inhibited the LPS-induced increase in Caco-2 TJ permeability. The LPS increase in TJ permeability was mediated by toll-like receptor 4 (TLR-4)/MyD88 signal-transduction pathway up-regulation of MLCK expression. The LPS-induced increase in mouse intestinal permeability also required an increase in MLCK expression. The LPS-induced increase in intestinal permeability was inhibited in MLCK-/- and TLR-4-/- mice. These data show, for the first time, that the LPS-induced increase in intestinal permeability was mediated by TLR-4/MyD88 signal-transduction pathway up-regulation of MLCK. Therapeutic targeting of these pathways can prevent an LPS-induced increase in intestinal permeability. .

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Figures

Figure 1
Figure 1
Time-course effect of lipopolysaccharide (LPS) on Caco-2 myosin light chain kinase (MLCK) protein expression, kinase activity, and phosphorylated (p)-MLC. A: LPS (0.3 ng/mL) caused a time-dependent increase in MLCK protein expression in Caco-2 monolayers, as determined by Western blot analysis. β-Actin was used as an internal control for protein loading. Relative densitometry analysis for MLCK protein levels. B: Graph plot of inulin flux versus MLCK protein expression in Caco-2 monolayers (correlation coefficient r = 0.91). C: LPS caused a time-dependent increase in MLCK activity, as determined by enzyme-linked immunosorbent assay–based in vitro kinase activity measurement. D: LPS treatment (5-day experimental period) resulted in an increase in p-MLC in Caco-2 monolayers, as assayed by immunostaining and visualized by confocal microscopy (x, 40). Control and LPS- and ML-7/LPS–treated cells were stained for actin (red) and p-MLC (green). Minimal changes in actin staining were seen after LPS treatment in x–y plane images (top panels). LPS treatment increased p-MLC staining, which colocalized with actin (yellow). In the x–z plane (bottom panels), the actin staining in LPS-treated cells was irregular, with aggregations colocalizing with p-MLC (yellow) on the basolateral membrane. Inhibition of MLCK with ML-7 (10 μmol/L) prevented LPS-induced actin rearrangement at the perijunctional sites. Nuclei are stained blue (x, 40). Data are expressed as means ± SEM (A and C). n = 3 (A); n = 4 (C). ∗∗P < 0.01, ∗∗∗P < 0.001 versus control. Scale bar = 5 μm (D).
Figure 2
Figure 2
Effect of myosin light chain kinase (MLCK) inhibition on lipopolysaccharide (LPS)–induced increase in Caco-2 MLCK activity and increase in Caco-2 permeability. A: MLCK inhibitor ML-7 (10 μmol/L) significantly prevented the LPS-induced increase in MLCK activity. ML-7 was added 24 hours before LPS treatment, and both LPS and ML-7 were renewed every 24 hours for the 5-day experimental period. B: ML-7 (10 μmol/L) inhibited the LPS-induced increase in inulin flux across Caco-2 monolayers (5-day experimental period). C: ML-7 inhibited the LPS-induced decrease in Caco-2 transepithelial electrical resistance (TER). There was a 5-day experimental period. D: MLCK siRNA transfection resulted in a near complete depletion in MLCK protein expression, as determined by Western blot analysis. siRNA transfection was performed 24 hours before LPS treatment (5-day experimental period). E: MLCK siRNA transfection caused a significant decrease in MLCK mRNA levels, as determined by real-time PCR. siRNA transfection was performed 24 hours before the start of LPS treatment. F: MLCK siRNA transfection prevented the LPS-induced increase in Caco-2 inulin flux. G: MLCK siRNA transfection prevented the LPS-induced decrease in Caco-2 TER. Data are expressed as means ± SEM (A–C and E–G). n = 4 (C and F); n = 5 (E and G). ∗∗P < 0.01, ∗∗∗P < 0.001 versus control; †††P < 0.001 versus LPS treatment. siMLCK, siRNA MLCK; siNT, siRNA nontarget.
Figure 3
Figure 3
The involvement of the toll-like receptor 4 (TLR-4) signaling pathway in lipopolysaccharide (LPS)–induced activation of myosin light chain kinase (MLCK) in Caco-2 monolayers. A: siRNA-induced knock-down of TLR-4 resulted in a near-complete depletion of TLR-4 expression, as assessed by Western blot analysis. Relative densitometry analysis for TLR-4 protein levels is shown. B: siRNA-induced knock-down of TLR-4 prevented the LPS-induced increase in MLCK protein expression. siRNA transfection was performed 24 hours before LPS treatment (5-day experimental period). C: TLR-4–induced silencing by siRNA prevented the LPS-induced phosphorylation of MLC expression, as assayed by immunostaining and visualized by confocal microscopy (5-day experimental period; x, 40). D: siRNA-induced knock-down of MyD88 resulted in a near-complete depletion of MyD88 expression, as assessed by Western blot analysis. Relative densitometry analysis for MyD88 protein levels is shown. E: siRNA-induced knock-down of MyD88 prevented the LPS-induced increase in MLCK protein expression. siRNA transfection was performed 24 hours before LPS treatment (5-day experimental period). Relative densitometry analysis for MLCK protein levels is shown. F: MyD88-induced silencing by siRNA prevented the LPS-induced phosphorylation of MLC expression, as assayed by immunostaining and visualized by confocal microscopy (x, 40). Data are expressed as means ± SEM (A, B, D, and E). n = 3 (A, B, D, and E). ∗∗P < 0.01 versus control; ∗∗∗P < 0.001; ††P < 0.01 versus LPS treatment. Scale bar = 5 μm (C and F).
Figure 4
Figure 4
Effect of lipopolysaccharide (LPS) on myosin light chain kinase (MLCK) expression in mouse intestinal tissues in vivo. A: LPS administration (0.1 mg/kg, i.p.) caused a time-dependent increase in MLCK protein expression. Relative densitometry analysis was based on the upper band shown for MLCK expression. B: Increase in mRNA levels, as assessed by real-time PCR. C: LPS caused an increase in mouse MLCK mRNA levels in a pure population of intestinal epithelial cells (ileum) captured by laser-capture microdissection. D: LPS treatment (5-day experimental period) resulted in an increase in phosphorylated (p)-MLC in mouse intestinal tissues (ileum), as assayed by immunostaining and visualized by confocal microscopy (x, 40). E and F: Effect of i.p. LPS (0.1 mg/kg body weight) on mouse intestinal tissue tumor necrosis factor (TNF)–α (E) and IL-1β (F) expression over the 5-day experimental period. LPS treatment did not cause any change in the mouse tissue level of TNF-α or IL-1β. Data are expressed as means ± SEM (A–C, E, and F). n = 4 (B); n = 5 (C); n = 3 (D). ∗∗P < 0.01, ∗∗∗P < 0.001 versus control. Scale bar = 5 μm (D).
Figure 5
Figure 5
Effect of lipopolysaccharide (LPS) on mouse intestinal permeability in knockout mouse models. A: LPS treatment (0.1 mg/kg) for a 5-day experimental period did not induce an increase in mouse intestinal permeability in myosin light chain kinase (MLCK) knockout (KO) mice. B: MLCK inhibitor (ML-7; 2 mg/kg) significantly prevented the LPS-induced increase in intestinal permeability, as determined by dextran, 10 kDa, flux. C: LPS treatment did not affect the MLCK expression in toll-like receptor 4 (TLR-4) KO mice compared with wild-type (WT) mice, as determined by Western blot analysis. D: LPS treatment did not affect the increase in mouse intestinal permeability in TLR-4 KO mice compared with WT mice, as determined by dextran, 10 kDa, flux. E: LPS treatment did not affect the MLCK expression in MyD88 KO mice compared with WT mice, as determined by Western blot analysis. F: LPS treatment did not affect the increase in mouse intestinal permeability in MyD88 KO mice compared with WT mice, as determined by dextran, 10 kDa, flux. Data are expressed as means ± SEM (A, B, D, and F). n = 4 (A and B). ∗∗P < 0.01, ∗∗∗P < 0.001 versus control WT; ††P < 0.01, †††P < 0.001 versus LPS treatment WT. PBS, phosphate-buffered saline.
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