1. Volume 141, Issue 4, Pages 1498-1508.e5 (October 2011)
Loss of CFTR Affects Biliary Epithelium Innate Immunity and Causes TLR4–NF-κB— Mediated Inflammatory Response in Mice 
Romina Fiorotto, Roberto Scirpo, Michael Trauner, Luca Fabris, Rafaz Hoque, Carlo Spirli, Mario Strazzabosco 
Gastroenterology 
Volume 141, Issue 4, Pages e5 (October 2011)
DOI: /j.gastro
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2. Figure 1
(A) DSS-induced colitis causes biliary damage in Cftr-KO but not WT mice. WT and Cftr-KO mice received DSS by oral administration as described.7 Liver tissue was stained with the cholangiocyte-specific marker K19. Cftr-KO but not WT mice developed biliary damage, characterized by bile duct proliferation and inflammation. Original magnification 10×; inset: 40×. (B) Computer-assisted morphometric analysis of K19-positive areas, expressed as percentage with respect to the whole area analyzed. Ductular reaction was significantly increased in Cftr-KO mice treated with DSS (Cftr-KO + DSS) (P < .01). (C) Effects of norUDCA versus polymyxin B/neomycin treatment on DSS-induced biliary damage. Cftr-KO mice and WT littermates were exposed to DSS in the presence or absence of norUDCA (15 mg/mouse/day), a bile acid able to stimulate biliary secretion in Cftr-KO mice. Ductular reaction in Cftr-KO mice treated with DSS was not decreased by treatment with norUDCA (P = not significant). On the contrary, treatment with polymyxin B (100 mg/kg body wt/day) plus neomycin (220 mg/kg body wt/day) prevented the increase in ductular reaction (P < .01).
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3. Figure 2
Periportal inflammatory infiltrate in DSS-treated Cftr-KO mice. (A) Immunohistochemical analysis of liver specimens from DSS-treated WT and Cftr-KO mice shows a portal inflammatory cell infiltrate (CD45-positive cells) in Cftr-KO mice. The CD45-positive cell infiltrate is prevalently positive for the neutrophil marker NIMP-R14 and for the macrophage marker F4/80. Original magnification 40×. (B) Fluorescence-activated cell sorter analysis of the nonparenchymal CD45+ population isolated from DSS-treated Cftr-KO mice confirmed the presence of macrophages and showed very low percentages of natural killer, T, and B cells.
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4. Figure 3
Cftr-KO cholangiocytes are more responsive to LPS-stimulated cytokine secretion. Biliary cells isolated from WT and Cftr-KO mice were cultured as a polarized monolayer in Transwell inserts with a 0.4-μm pore semipermeable membrane as previously described.15 Once the confluence of the monolayer was established by measuring transepithelial resistance and membrane potential, cells were exposed to LPS (100 ng/mL) for 6, 12, and 24 hours from the (A) apical or (B) basolateral side. At the end of the treatment, apical and basolateral media were collected and analyzed by Luminex and data were normalized for the cell protein content. Kinetics of cytokine secretion by WT (blue lines) and Cftr-KO (red lines) cholangiocytes in response to LPS exposure (0, 6, 12, and 24 hours) are shown. The plots represent the means of 4 experiments for the 6 cytokines showing the most robust differences between WT and Cftr-KO cells (*P < .05, **P < .01 vs WT at the respective time point). Statistical analysis and raw data are also reported in Supplementary Table 2.
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5. Figure 4
Cftr-KO cholangiocytes respond to LPS with increased TLR4-dependent NF-κB activation. Cells were untreated (Ctrl) or exposed to LPS (100 ng/mL) for 6 and 12 hours. At the end of the treatment, nuclear fractions were extracted. (A) NF-κB activity was assessed by Western blot in nuclear fractions using an antibody against NF-κB component p65. Histone-3 was used to normalize for nuclear protein content. (B) Bar graphs represent optical density quantification of p65 in the nucleus normalized for histone-3 (*P < .05 vs WT; ▴P < .05 vs Cftr-KO 6h). (C) WT and Cftr-KO cells were exposed to LPS (100 ng/mL) for 6 hours in the presence/absence of the TLR4 inhibitor TAK-242; NF-κB activity was measured by luciferin/luciferase reporter gene assay. After LPS stimulation, NF-κB activity significantly increased in Cftr-KO as compared with WT cells (*P < .05 vs WT + LPS); NF-κB activity induced by LPS was significantly inhibited by treatment with TAK 242 (#P < .01 vs LPS). Bar graphs represent mean ± SD of the relative luciferase activity normalized to control in n = 3 different experiments repeated in triplicate for each condition.
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6. Figure 5
Increased TLR4 phosphorylation is caused by increased Src activity in Cftr-KO cholangiocytes. (A) WT cells were exposed to LPS (100 ng/mL) for 12 hours; Cftr-KO cells were exposed to LPS (100 ng/mL) in the presence or absence of PP2 (10 μmol/L) (Src inhibitor) for 12 hours; untreated cells served as control (Ctrl). TLR4 phosphorylation was determined by Western blot using an antibody against the TLR4 tyrosine phosphorylation site (Y674). (B) Bar graph represents the ratio between p-TLR4 (pY674) and total TLR4. WT cholangiocytes show a significant tyrosine phosphorylation of TLR4 after 12 hours of exposure to LPS, while in CF cholangiocytes the level of TLR4 phosphorylation is significantly higher at basal condition and increased on LPS stimulation. Pretreatment with PP2 (10 μmol/L) completely blunted TLR4 phosphorylation in Cftr-KO cells. (C) Src activity was determined by Western blot using an antibody against the active form of Src (pY418) in cells at basal conditions. (D) Bar graphs represent the optical density ratio of p-Src (pY418) versus total Src. Cftr-KO cells show an increased amount of p-Src (P < .01) as compared with WT cells, suggesting an increase in the activity of the kinase. Data represent mean ± SD of n = 3 different experiments.
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7. Figure 6
CFTR expression in HEK-Blue cells decreases NF-κB activation after LPS stimulation. HEK-Blue cells were transfected with WT-CFTR or with an empty vector plasmid complementary DNA. (A) As shown in the Western blot, on the contrary to cells transfected with WT-CFTR DNA, there is no endogenous expression of CFTR protein in cells transfected with the empty vector. (B) Bar graph represents percent changes in NF-κB activation in response to LPS in HEK-Blue cells transfected with WT-CFTR as compared with the increase in NF-κB activation measured in control cells transfected with empty vector (average ± SD, n = 3 independent experiments, *P < .05).
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8. Supplementary Figure 1
Effect of DSS-induced colitis on colonic histology. Colonic tissue from WT and Cftr-KO mice untreated or treated with DSS were H&E stained. As compared with (A) untreated mice, the degree of colitis induced by DSS treatment was similar between (B and C) WT and (D and E) Cftr-KO mice as determined by the presence of mononuclear cell infiltrates, loss of crypts, and mucosal ulcerations.
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9. Supplementary Figure 2
Effect of DSS on portal blood endotoxin level. Plasma endotoxin was measured in WT and Cftr-KO mice treated with DSS in the presence and absence of antibiotics. Endotoxin level was unchanged in WT versus Cftr-KO mice after DSS treatment but was significantly decreased in both groups in the presence of antibiotics (*P < .05). Bar graph represents averages ± SD of n = 3 mice.
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10. Supplementary Figure 3
Effect of DSS on serum alanine aminotransferase levels. Mean alanine aminotransferase levels (U/L) ± SD are shown for WT and Cftr-KO mice untreated or treated with DSS or DSS plus antibiotics. Treatment with DSS induces a significant increase in alanine aminotransferase levels only in Cftr-KO mice (P < .05) that are back to normal levels after treatment with antibiotics (P < .05).
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11. Supplementary Figure 4
A20 and IRAK-M, negative regulators of TLR4, were not reduced in Cftr-KO cells. Biliary cells were isolated and cultured as described in Figure 4. WT and Cftr-KO were untreated (Ctrl) or exposed to LPS (100 ng/mL) for 6 and 12 hours. To understand if the altered TLR4 response elicited by LPS was a consequence of lack of negative feedback control of the TLR4 signaling pathway, we measured by Western blot the expression of (A) A20 and (B) IRAK-M in total protein lysates from Cftr-KO and WT cholangiocytes. Bar graphs represent optical density quantification of A20 and IRAK-M, normalized for β-actin. No significant reduction of IRAK-M and A20 was found in CF cholangiocytes. On the contrary, both regulators showed a higher expression at basal condition in Cftr-KO cells, consistent with a physiologic regulatory response to the excessive TLR4 activation. Data represent averages of n = 3 experiments. *P < .05 versus WT control, #P < .05 versus WT 6h.
Gastroenterology  , e5DOI: ( /j.gastro )
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