Mechanisms of cross hyporesponsiveness to toll-like receptor bacterial ligands in intestinal epithelial cells  Jan-Michel Otte, Elke Cario, Daniel K. Podolsky  Gastroenterology  Volume 126, Issue 4, Pages 1054-1070 (April 2004) DOI: 10.1053/j.gastro.2004.01.007

Figure 1 Expression of mRNA encoding TLRs and accessory molecules in primary colonic epithelial and SW480 cells. mRNA expression of TLRs and accessory molecules was analyzed by PCR following reverse transcription of total RNA from freshly isolated primary colonic epithelial cells (A ). Contamination with lymphoid cells was excluded by performing CD45 and CD68 specific PCR. Figure 1B shows results from whole crypts (lane 1), isolated PCR contaminated with a Jurkat 1 cell (lane 2), and isolated IEC free of lymphoid contamination (lanes 3 and 4). Expression of TLRs was also analyzed in cultured cells; shown are results for SW480 cells (C ). Comparable expression patterns were detected in Colo205 cells (not shown). Primary as well as cultured cells express mRNA coding for accessory molecules TIRAP, MD-2, MyD88, and Tollip (D-E; lane 1: SW480 cells; lane 2: Colo205 cells; lane 3: SCPCR cells). Results are of 4 independent experiments. Successful RNA isolation and amplification was analyzed by GAPDH expression. The identity of all fragments was confirmed by sequencing. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 2 Detection of TLR2 and 4 protein on SW480 cells by immunohistochemistry. (A-F) SW480 cells were stained with TLR4 (C and E ) and 2 (D and F ) specific antibodies. Normal serum controls for TLR4 are shown in A and controls for TLR2 in B. Shown are representative results with similar results obtained with Colo205 cells (data not shown). (G-M) Detection of MD-2 on intestinal tissue and isolated and cultured cells by immunohistochemistry. Negative controls included omission of primary antibody (G ) or staining with secondary antibody only (H). MD-2 expression was detected on epithelial cells in tissue sections (I-K ) as well as on the plasma membrane of isolated (L) and cultured (M ) intestinal epithelial cells. (Original magnification (oil): 200× in A-D, 400× in E and F, 100× in G-K, 200× in L and M.)_art> Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 3 TLR ligands induced signaling in IEC. Incubation of SW480 (A ) or Colo205 (B) cells with LPS (2 μg/mL) or LTA (10 μg/mL) induced ERK1/2 phosphorylation as well as IκBα-degradation (C ) in a time-dependent manner; c, unstimulated controls. (D) As assessed with a phospho-specific antibody, stimulation of SW480 cells for 15 minutes with the indicated concentrations of LPS dose dependently induced ERK1/2 phosphorylation. Pretreatment with LPS (2 μg/mL) for 24 hours inhibited LPS induced MAPK phosphorylation. (E ) Following pretreatment for 24 hours with LPS (2 μg/mL), the medium was replaced, and cells were restimulated. After an additional 24 hours, IEC MAPK phosphorylation was again observed. However, no further MAPK activation was detected until the end of the observation period (84 hours) when cells were kept in LPS-enriched medium (F ); c, unstimulated control; +, positive control (2 μg/mL LPS for 30 minutes). Representative blots of n = 5 experiments. Blots were reprobed with anti-total ERK 1/2 MAP kinase to verify equal loading. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 4 TLR ligand induced cross-tolerance. (A ) SW480 Cells remained unstimulated (c), were stimulated for 15 minutes with LPS (2 μg/mL) or LTA (10 μg/mL) for 15 minutes, or, for 24 hours or after 24 hours, cells were restimulated as indicated (LPS [2 μg/mL), LTA (10 μg/mL), TNF-α [10 ng/mL]). Activation of MAPK ERK1/2 was assessed with a phospho-specific antibody. Equal loading was confirmed by reprobing with a total ERK specific antibody. (B and C ) Cells were treated for 24 hours with LPS (2 μg/mL) or LTA (10 μg/mL) (lanes 1 and 2); treated for 30 minutes with LPS, LTA, or whole cell lysate from Salmonella dublin (WCL) o (lanes 3–5); or were pretreated for 24 hours and then subjected to a second stimulus for 30 minutes with LPS or LTA (lanes 6–8). Controls remained unstimulated (c). Cellular extract was prepared, and MAP kinase phosphorylation was examined by Western blot analysis with Ab specific for either the phosphorylated (p) or total forms of p38 or SAPK/JNK. The results of a representative experiment are shown (n = 4). Comparable results were detected when Colo205 cells were assayed (D and E ). T-84 Cells remained unstimulated (c), were stimulated with whole cell lysate from S. dublin (WCL) for 15 minutes, LPS (2 μg/mL) or LTA (10 μg/mL) for 15 minutes, or, for 24 hours or after 24 hours, cells were restimulated as indicated (LPS [2 μg/mL], LTA [10 μg/mL], TNF-α [10 ng/mL]). Activation of MAPK ERK1/2 was assessed with a phospho-specific antibody. Equal loading was confirmed by reprobing with a total ERK specific antibody. (F and G) Cells were treated for 24 hours with LPS (2 μg/mL) or LTA (10 μg/mL) (lanes 1 and 2); treated for 30 minutes with LPS, LTA, or whole cell lysate from S. dublin (WCL; lanes 3–5); or were pretreated for 24 hours and then subjected to a second stimulus for 30 minutes with LPS or LTA (lanes 6–8). Controls remained unstimulated (c). Cellular extract was prepared, and MAP kinase phosphorylation was examined by Western blot analysis with Ab specific for either the phosphorylated (p) or total forms of p38 or SAPK/JNK. The results of a representative experiment are shown (n = 4). Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 5 IL-8 secretion in untreated and pretreated SW480 cells. (A ) SW480 cells were treated with LPS (2 μg/mL), LTA (10 μg/mL), IL-1β (10 ng/mL), or TNF-α (10 ng/mL) for 12 hours or pretreated with LPS (2 μg/mL) or LTA (10 μg/mL) for 24 hours and then restimulated as indicated for an additional 12 hours; c, unstimulated controls. (B) SW480 cells remained unstimulated (control 0 hours; control 12 hours) or were incubated with E. coli or S. aureus (100 bacteria/cell) for 12 hours. Following removal of bacteria, cells were reexposed with medium, E. coli (100 bacteria/cell), S. aureus (100 bacteria/cell), LPS (2 μg/mL), or LTA (10 μg/mL) for an additional 12 hours as indicated. IL-8 secretion into culture supernatants was assayed by ELISA. Data represent means ± SD of n = 5 independent experiments; ∗P < 0.05; ∗∗P < 0.01. Comparable results were obtained in assays performed with Colo205 cells (data not shown) or differentiated T84 cells (C and D). Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 6 Expression of TLRs and signaling molecules in tolerant SW480 and Colo205 cells. SW480 or Colo205 cells remained unstimulated (c) or were stimulated with LPS (2 μg/mL) or LTA (10 μg/mL) for the times indicated. mRNA of TLR2 (A ) and TLR4 (B) were analyzed by Northern blotting utilizing specific probes, and (C ) protein expression was quantified by immunoprecipitation and Western blotting. Representative blots from n = 5 Northern and Western blot analyses are shown. Protein expression of signaling intermediates in Colo205 cells stimulated with LPS (2 μg/mL) or LTA (10 μg/mL) for the times indicated was analyzed by Western blotting (D). The results are representative of 3 independent experiments with similar results. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 7 Cell surface expression of TLR2 and TLR4 in unstimulated and tolerant SW480 cells. SW480 cells were stimulated with LPS (2 μg/mL; blue bars) or LTA (10 μg/mL; red bars) for up to 24 hours. After 24 hours, the stimulus was removed, and the cells were incubated in medium for another 24 hours (indicated as 48 h). Controls remained unstimulated. TLR2 (A ) or TLR4 (B) expression was determined by FACS analysis and quantified as percentage of unstimulated controls (C and D). SW480 cells were incubated with live S. aureus (E ) or E. coli (F ), respectively, for the indicated times. After 24 hours, the bacteria were removed and the cells incubated in medium for an additional 24 hours (indicated as 48 h). TLR2 (E ) or TLR4 (F ) expression was determined by FACS analysis and is shown in percentage of unstimulated controls. Controls remained unstimulated. Data represent results from n = 4 independent experiments; ∗P < 0.05; ∗∗P < 0.01. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 8 Expression of Tollip in tolerant IEC. Expression of Tollip was analyzed in primary intestinal epithelial cells isolated from various regions of intestinal mucosa by real-time PCR. Results are shown as expression in relation to levels of the housekeeping gene GAPDH (A ). Expression levels of Tollip were also analyzed by Northern blotting (B) or Western blot analysis (C and D) in SW480 and Colo205 cells stimulated with LPS (2 μg/mL) or LTA (10 μg/mL) for the times indicated. Equal loading was confirmed by probing the blots with a GAPDH-specific probe. Assays were also performed utilizing differentiated T84 cells. Following apical stimulation, indicated levels of Tollip were analyzed by Northern blotting (E ) or Western blot analysis (F ). Shown are representative blots of n = 3 experiments. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 9 Overexpression of Tollip inhibits TLR signaling in IEC. (A and B) Cells were transfected with 1 or 2 μg of the HA-Tollip plasmid and, 24 hours later, stimulated with LPS (2 μg/mL) or LTA (10 μg/mL) for 30 minutes. (A ) ERK1/2 MAPK phosphorylation was determined by Western blot analysis using specific antibodies and equal loading was determined by analysis of total ERK. Transfection efficiency of the HA-tagged plasmid was evaluated by reprobing blots with an HA-specific antibody; vc, vector control. (B) Secretion of IL-8 from cells transfected with an empty vector control (vc) or 2 μg of the HA-Tollip plasmid (Tollip) was determined following stimulation with LPS (2 μg/mL) or LTA (10 μg/mL) for 18 hours as described in the Materials and Methods section. Shown are representative results of n = 4 independent experiments; ∗∗P < 0.01. Comparable regulation was detected in Colo205 cells. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 10 Preserved response to TNF-α in cells overexpressing Tollip. IEC were transfected as indicated for Figure 9. Twenty-four hours later, transfected cells were stimulated with LPS (2 μg/mL), LTA (10 μg/mL), or TNF-α (10 ng/mL) for 20 minutes (A ) or 18 hours (B). (A ) ERK1/2 MAPK phosphorylation was determined by Western blot analysis using specific antibodies and equal loading was determined by analysis of total ERK. Transfection efficiency of the HA-tagged plasmid was evaluated by reprobing blots with an HA-specific antibody; vc, vector control. (B) Secretion of IL-8 from cells transfected with an empty vector control (vc) or 2 μg of the HA-Tollip plasmid (Tollip) was determined as described in the Materials and Methods section. Shown are means ± SD of n = 5 independent experiments; ∗∗P < 0.01. Comparable results were obtained with Colo205 cells. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 11 Overexpression of a deletion mutant of Tollip (M Tollip) reverses the regulatory function of wild-type Tollip. IEC were transfected with an empty vector control (vc), 2 μg of the HA-Tollip plasmid, or 5 μg of the HA-mutant Tollip plasmid (m-Tollip). Twenty-four hours later, transfected cells were stimulated with LPS (2 μg/mL) or LTA (10 μg/mL) for 20 minutes (A ), and ERK1/2 MAPK phosphorylation was determined by Western blot analysis using specific antibodies and equal loading was determined by analysis of total ERK. Transfection efficiency of the HA-tagged plasmids was evaluated by reprobing blots with an HA-specific antibody; vc, vector control. Cells transfected with m-Tollip were stimulated with LPS (2 μg/mL) or LTA (10 μg/mL) for 18 hours or restimulated following 24-hour preincubation (B). Secretion of IL-8 into culture supernatants was determined as described in the Materials and Methods section. Shown are means ± SD of n = 5 independent experiments; ∗∗P < 0.01. Comparable results were obtained with Colo205 cells. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)

Figure 12 IRAK activity is impaired in tolerant and Tollip over expressing IEC. SW480 and Colo205 cells were cultured in 10-cm dishes and transfected with the HA-Tollip plasmid (2 μg). Twenty-four hours later, cells were left untreated or stimulated as indicated for 30 minutes. Cells were lysed and proteins subjected to kinase assays or precipitated as indicated in the Materials and Methods section. Phosphorylated IRAK was visualized by autoradiography. Equal loading was confirmed by probing parts of the sample with an IRAK specific antibody, and transfection efficiency was evaluated by reprobing with an HA-specific antibody. Shown are results from experiments with SW480 cells. Comparable data were generated utilizing Colo205 cells. Gastroenterology 2004 126, 1054-1070DOI: (10.1053/j.gastro.2004.01.007)