Lipopolysaccharide regulation of intestinal tight junction permeability is mediated by TLR-4 signal transduction pathway activation of FAK and MyD88 methods
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Assessment of protein expression by western blot analysis
Protein expression from Caco-2 cells and mouse tissue was assessed by western blot as previously described. Cells and mouse tissue were lysed with lysis buffer (50 mM Tris • HCl (pH 7.5), 150 mM NaCl, 500 µM NaF, 2 mM EDTA, 100 µM vanadate, 100 µM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 40 mM paranitrophenyl phosphate, 1 µg/ml aprotinin, and 1% Triton X-100) on ice for 30 min. The lysates were centrifuged at 10,000 g for 10 min in an Eppendorf Centrifuge (5417R, Hauppauge, NY) to obtain a clear lysate. The supernatant was collected and protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Laemmli gel loading buffer (Bio-Rad Laboratories) was added to the lysate containing 10 - 20 µg of protein and boiled at 100°C for 7 min, after which proteins were separated on an SDS-PAGE...
Cell culture
Caco-2 cells (passage 20) were purchased from the American Type Culture Collection (Manassas, VA) and maintained at 37°C in a culture medium composed of DMEM with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml streptomycin, 4 mM glutamine, 25 mM HEPES, and 10% FBS as previously described. Caco-2 cells were used between passages 22 and 28 in this study. The cells were kept at 37°C in a 5% CO 2 environment. For growth on filters, high-density cells (1 × 10 5 cells) were plated on Transwell filters with 0.4 µm pore (Corning Incorporated, Corning, NY) and monitored regularly by visualization with an inverted microscope (Eclipse TS100/100-F, Nikon, Melville, NY) and by epithelial resistance measurements.
Co-immunoprecipitation analysis
Co-immunoprecipitation analysis was performed using Dynabeads Protein G (Life Technologies AS, Norway). After LPS treatment, Caco-2 cells were lysed in immunoprecipitation buffer (20 mM Tris • HCl (pH 7.5), 0.5 M NaCl, 1% Triton X-100, 50 mM NaF, 2 mM EDTA, 1 mM Na 3 VO 4, 0.1% sodium dodecyl sulfate, 0.5% Nonidet P-40 and 10% glycerol (vol/vol)), containing protease inhibitor cocktail (Roche, Mannheim, Germany). After 4 cycles of freeze-thaw and sonication, lysates were centrifuged at 12,000 g for 10 min. 1.5 mg Dynabeads were incubated with 10 µg TLR-4 antibody for 10 min at room temperature. After washing with washing buffer (PBS pH7.4 with 0.02% Tween-20), 200 µl sample lysates were added into the beads-Ab complex, and incubated with rotation overnight at 4°C. After washing the Dynabeads-Ab-Ag complex 3 times with 200 µl washing buffer, 20 µl elution...
siRNA transfection
Caco-2 monolayers were transiently transfected with siRNA using DharmaFect transfection reagent (Lafayette, Co) as previously described ( ). Briefly, cells (1 × 10 5 /filter) were seeded into a 12-well transwell plate and grown to confluency. Caco-2 monolayers were then washed with PBS twice and 0.5 ml Accell medium (Thermo Scientific, Lafayette, CO) was added to the apical compartment of each filter and 1.5 ml was added to the basolateral compartment of each filter. siRNA (5 ng) of interest and DharmaFect reagent (2 µl) were preincubated in Accell medium. After 5 min of incubation, the two solutions were mixed, and the mixture was added to the apical compartment of each filter. For LPS experiments, Caco-2 cells were transfected with siRNA for 24 h prior to the LPS treatment. The Caco-2 TJ barrier assessments were carried out at the end of day 5 of LPS treatment.
IRAK4 activity measurement
IRAK4 activity was measured using IRAK4 Kinase Enzyme System and ADP-Glo Kinase Assay (Promega, Madison, WI) with modifications. IRAK4 kinase was immunoprecipitated using Dynabeads Protein G as described above. After immunoprecipitation, IRAK4 kinase was eluted with 20 µl Elution buffer (50 mM Glycine pH2.8). For the functional assay, the pH of the eluate was adjusted by adding 20 µl of 1 M Tris (pH7.5). The final pH of the eluate was 7.5. In 96-well plate, the 25 µl total reaction volume included 5 µl 5X kinase buffer, 10 µl enzyme, and 10 µl substrate/ATP mix (2.5 µg MBP protein, 50 µM ATP, and 50 µM DTT). The kinase reaction was incubated at room temperature for 60 min. Then 25 µl ADP-Glo Reagent was added. Followed by incubation at room temperature for 40 min, 50 µl Kinase Detection Reagent was added. After incubation at room...
In-vivo determination of mouse intestinal permeability
The Laboratory Animal Care and Use Committee at the University of New Mexico approved all experimental protocols. Male C57BL/6 mice, TLR-4 knock out and MyD88 knockout mice (9-10 wk) were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were kept two per cage in a temperature-controlled room at 25°C with a 12:12 h light-dark cycle. Diet and drinking water were provided ad libitum.
In-vivo determination of mouse intestinal permeability
LPS effect on intestinal permeability in an in-vivo mouse model system was determined using a re-cycling intestinal perfusion method as previously described. Mice were injected with varying concentrations of LPS intraperitoneally (i.p.) every 24 h for up to 5 days of experimental period. A 6 cm segment of mouse small intestine was isolated and cannulated with a small-diameter plastic tube (in an anesthetized mouse maintained in 1% isoflurane in oxygen) and continuously perfused with 5 ml Krebs-phosphate saline buffer for a 2 h perfusion period. An external recirculating pump (Econo Pump, Bio-Rad) was used to recirculate the perfusate at a constant flow rate (0.75 ml/min). The body temperature of mouse was maintained at 37°C with a temperature-controlled warming blanket. The intestinal permeability was assessed by measuring flux rate of paracellular probe, Texas Red-labeled dextra...
In-vivo transfection of FAK siRNA
The effect of FAK siRNA on mouse small intestinal TJ permeability was determined using the perfusion model described previously. In these studies, mice were fasted for 24 h prior to the surgery. With the abdominal cavity open, a 6-cm segment of mouse small intestine was isolated. The transfection solution (0.5 ml), consisting of FAK siRNA (2.5 nmol) or scramble nontarget siRNA and tansfecting agent Lipofectamine (50 µl), was injected through a 33-gauge needle into the lumen of the small intestine, and the small intestine was cannulated for 1 h. The small intestine was then placed back into the abdominal cavity, and the abdominal cavity was closed with sutures. The FAK siRNA transfection was performed at day 0. After 5 days LPS (0.1 mg/kg body weight, i.p.) treatment, the intestinal permeability was measured using a re-cycling intestinal perfusion method described above. The surge...
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Protein expression by western blot
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Protein expression from Caco-2 cells and mouse tissue was assessed by western blot as previously described. Cells and mouse tissue were lysed with lysis buffer (50 mM Tris • HCl (pH 7.5), 150 mM NaCl, 500 µM NaF, 2 mM EDTA, 100 µM vanadate, 100 µM PMSF, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, 40 mM paranitrophenyl phosphate, 1 µg/ml aprotinin, and 1% Triton X-100) on ice for 30 min. The lysates were centrifuged at 10,000 g for 10 min in an Eppendorf Centrifuge (5417R, Hauppauge, NY) to obtain a clear lysate. The supernatant was collected and protein concentration was determined using the Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA). Laemmli gel loading buffer (Bio-Rad Laboratories) was added to the lysate containing 10 - 20 µg of protein and boiled at 100°C for 7 min, after which proteins were separated on an SDS-PAGE gel. Proteins from the gel were transferred to the membrane (Trans-Blot Transfer Medium, Nitrocellulose Membrane; Bio-Rad) overnight. The membrane was incubated for 2 h in blocking solution (5% dry milk in TBS-Tween 20 buffer). The membrane was then incubated with antibody in blocking solution. Aft...
Caco-2 cells (passage 20) were purchased from the American Type Culture Collection (Manassas, VA) and maintained at 37°C in a culture medium composed of DMEM with 4.5 mg/ml glucose, 50 U/ml penicillin, 50 U/ml streptomycin, 4 mM glutamine, 25 mM HEPES, and 10% FBS as previously described. Caco-2 cells were used between passages 22 and 28 in this study. The cells were kept at 37°C in a 5% CO 2 environment. For growth on filters, high-density cells (1 × 10 5 cells) were plated on Transwell filters with 0.4 µm pore (Corning Incorporated, Corning, NY) and monitored regularly by visualization with an inverted microscope (Eclipse TS100/100-F, Nikon, Melville, NY) and by epithelial resistance measurements.
Co-immunoprecipitation analysis was performed using Dynabeads Protein G (Life Technologies AS, Norway). After LPS treatment, Caco-2 cells were lysed in immunoprecipitation buffer (20 mM Tris • HCl (pH 7.5), 0.5 M NaCl, 1% Triton X-100, 50 mM NaF, 2 mM EDTA, 1 mM Na 3 VO 4, 0.1% sodium dodecyl sulfate, 0.5% Nonidet P-40 and 10% glycerol (vol/vol)), containing protease inhibitor cocktail (Roche, Mannheim, Germany). After 4 cycles of freeze-thaw and sonication, lysates were centrifuged at 12,000 g for 10 min. 1.5 mg Dynabeads were incubated with 10 µg TLR-4 antibody for 10 min at room temperature. After washing with washing buffer (PBS pH7.4 with 0.02% Tween-20), 200 µl sample lysates were added into the beads-Ab complex, and incubated with rotation overnight at 4°C. After washing the Dynabeads-Ab-Ag complex 3 times with 200 µl washing buffer, 20 µl elution buffer (50 mM Glycine pH2.8) and 20 µl SDS sample buffer were added, and heated for 10 min at 95°C. Immunoprecipitates were separated by SDS-PAGE and further analyzed by Western blot using anti-FAK and anti-MyD88 rabbit polyclonal Abs (Abcams).
Caco-2 monolayers were transiently transfected with siRNA using DharmaFect transfection reagent (Lafayette, Co) as previously described ( ). Briefly, cells (1 × 10 5 /filter) were seeded into a 12-well transwell plate and grown to confluency. Caco-2 monolayers were then washed with PBS twice and 0.5 ml Accell medium (Thermo Scientific, Lafayette, CO) was added to the apical compartment of each filter and 1.5 ml was added to the basolateral compartment of each filter. siRNA (5 ng) of interest and DharmaFect reagent (2 µl) were preincubated in Accell medium. After 5 min of incubation, the two solutions were mixed, and the mixture was added to the apical compartment of each filter. For LPS experiments, Caco-2 cells were transfected with siRNA for 24 h prior to the LPS treatment. The Caco-2 TJ barrier assessments were carried out at the end of day 5 of LPS treatment.
IRAK4 activity was measured using IRAK4 Kinase Enzyme System and ADP-Glo Kinase Assay (Promega, Madison, WI) with modifications. IRAK4 kinase was immunoprecipitated using Dynabeads Protein G as described above. After immunoprecipitation, IRAK4 kinase was eluted with 20 µl Elution buffer (50 mM Glycine pH2.8). For the functional assay, the pH of the eluate was adjusted by adding 20 µl of 1 M Tris (pH7.5). The final pH of the eluate was 7.5. In 96-well plate, the 25 µl total reaction volume included 5 µl 5X kinase buffer, 10 µl enzyme, and 10 µl substrate/ATP mix (2.5 µg MBP protein, 50 µM ATP, and 50 µM DTT). The kinase reaction was incubated at room temperature for 60 min. Then 25 µl ADP-Glo Reagent was added. Followed by incubation at room temperature for 40 min, 50 µl Kinase Detection Reagent was added. After incubation at room temperature for 30 min, the luminescence was recorded with 0.5 s Integration time using PerkinElmer Multilabel Counter.
The Laboratory Animal Care and Use Committee at the University of New Mexico approved all experimental protocols. Male C57BL/6 mice, TLR-4 knock out and MyD88 knockout mice (9-10 wk) were purchased from Jackson Laboratories (Bar Harbor, ME). The mice were kept two per cage in a temperature-controlled room at 25°C with a 12:12 h light-dark cycle. Diet and drinking water were provided ad libitum.
LPS effect on intestinal permeability in an in-vivo mouse model system was determined using a re-cycling intestinal perfusion method as previously described. Mice were injected with varying concentrations of LPS intraperitoneally (i.p.) every 24 h for up to 5 days of experimental period. A 6 cm segment of mouse small intestine was isolated and cannulated with a small-diameter plastic tube (in an anesthetized mouse maintained in 1% isoflurane in oxygen) and continuously perfused with 5 ml Krebs-phosphate saline buffer for a 2 h perfusion period. An external recirculating pump (Econo Pump, Bio-Rad) was used to recirculate the perfusate at a constant flow rate (0.75 ml/min). The body temperature of mouse was maintained at 37°C with a temperature-controlled warming blanket. The intestinal permeability was assessed by measuring flux rate of paracellular probe, Texas Red-labeled dextran (MW = 10,000 g/mol). Water absorption was determined by using a non-absorbable marker sodium ferrocyanide or by measuring the difference between initial and final volume of the perfusate.
The effect of FAK siRNA on mouse small intestinal TJ permeability was determined using the perfusion model described previously. In these studies, mice were fasted for 24 h prior to the surgery. With the abdominal cavity open, a 6-cm segment of mouse small intestine was isolated. The transfection solution (0.5 ml), consisting of FAK siRNA (2.5 nmol) or scramble nontarget siRNA and tansfecting agent Lipofectamine (50 µl), was injected through a 33-gauge needle into the lumen of the small intestine, and the small intestine was cannulated for 1 h. The small intestine was then placed back into the abdominal cavity, and the abdominal cavity was closed with sutures. The FAK siRNA transfection was performed at day 0. After 5 days LPS (0.1 mg/kg body weight, i.p.) treatment, the intestinal permeability was measured using a re-cycling intestinal perfusion method described above. The surgery had no effect on the food intake and the body weight of the animals during the experimental period.
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