Volume 124, Issue 5, Pages 1395-1407 (May 2003) Enteric flora and lymphocyte-derived cytokines determine expression of heat shock proteins in mouse colonic epithelial cells  Keishi Kojima, Mark W Musch, Hongyu Ren, David L Boone, Barbara A Hendrickson, Averil Ma, Eugene B Chang  Gastroenterology  Volume 124, Issue 5, Pages 1395-1407 (May 2003) DOI: 10.1016/S0016-5085(03)00215-4

Figure 1 Immunohistochemical staining of hsp25 and hsp72 in jejunum and colon of normal C57Bl/6 mice. Paraffin sections of formalin-fixed tissue were stained for hsp25 and hsp72 as described in the Materials and Methods section. Images shown are representative of all intestinal segments from 3 separate mice. (Original magnification 200×.) Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)

Figure 2 Hsp expression in small intestine and colon of Rag-1−/− and wild-type mice. (A ) Differential expression of hsps along the longitudinal axis of the intestine in Rag-1−/− and wild-type mice. Hsp expression was determined in mucosal scrapings from 3 different regions of the intestine by Western blotting as described in the Materials and Methods section. (B) Representative Western blots of colonic mucosal samples taken from 5 mice in each group. Densitometric analysis of colonic hsp25 and hsp72. Densitometric average of values of hsp in colonic mucosa of 5 mice in each group. Values are means ± SE. ∗∗P < 0.01 between longitudinal sections of the Rag-1−/− vs. wild-type by paired t test. Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)

Figure 3 Effect of cytokines on YAMC cell hsp expression. YAMC cells were treated with IL-2 (50 ng/mL), IFN-γ (1000 U/mL), TNF-α (50 ng/mL), IL-4 (50 ng/mL), IL-10 (50 ng/mL), and IL-1β (50 ng/mL) for 48 hours and then hsp expression was analyzed by Western blotting as described in the Materials and Methods section. The image shown is representative of those obtained in 3 separate experiments. Densitometric analysis includes results of the 3 separate experiments. HS lane represents protein from heat-shocked cells as a positive control. Values are means ± SE. ∗P < 0.05, ∗∗P < 0.01 compared with medium control by analysis of variance. Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)

Figure 4 Concentration dependence of IL-2 induction of hsp25 and hsp72. YAMC cells were treated with varying concentrations of IL-2 for 48 hours and then hsp25, hsp72, and hsc73 expressions were analyzed by Western blot as described in the Materials and Methods section. The image shown is representative of those obtained in 3 separate experiments. Densitometric analysis includes results of the 3 separate experiments. Values are means ± SE. ∗P < 0.05, ∗∗P < 0.01 compared with medium control by analysis of variance. Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)

Figure 5 Effect of coculture with LPLs on hsp expression in YAMC cells. YAMC cells were cocultured with nonactivated N-LPLs or A-LPLs (2 × 106 cells/mL) by both anti-CD3 mAb and CD28 mAb, or with IL-2 (100 pg/mL) in the absence or presence of anti IL-2 mAb (2 μg/mL). After 48 hours of culture, hsp25, hsp72, and hsc73 expressions in YAMC cells were analyzed by Western blot as described in the Materials and Methods section. Densitometric analysis includes results of the 3 separate experiments. Values are means ± SE. ∗P < 0.05, ∗∗P < 0.01; N.S., nonsignificant difference. The asterisks immediately above individual data columns were compared with medium. Specific comparisons for use of IL-2 neutralizing antibody are designated by brackets. All comparisons were by analysis of variance. Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)

Figure 6 (A ) Effect of antibiotics on colonic hsp expression. Normal mice were treated with either ciprofloxacin (0.2 mg/mL) or metronidazole (0.6 mg/mL) for 5 days by inclusion in their drinking water. Colonic mucosal scrapings were harvested and 10 μg of protein was analyzed for hsp expression by Western blotting as described in the Materials and Methods section. The image shown and densitometric analysis represent results from 5 separate mice. Values are means ± SE. ∗∗P < 0.01 compared with control mice by analysis of variance. (B) IL-2 and TNF-α mRNA expression in colon mucosa of control and metronidazole-treated mice. Mice were treated with metronidazole included in drinking water for 5 days. The ratio was calculated by densitometric analysis from 5 separate mice. Values are means ± SE. Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)

Figure 7 Effect of bacteria exposure on hsp expression in YAMC cells. YAMC cells were incubated with B. fragilis or DH5α for 3 hours, washed with PBS, and incubated for an additional 48 hours. The hsp expression was analyzed by Western blotting as described in the Materials and Methods section. Image shown is representative of those obtained in 3 different experiments. Densitometric analysis includes results of the 3 experiments. Zero and 48 hours indicate incubation time after 3-hour exposure. HS lane represents protein from heat-shocked cells performed at 0 hours and harvested 2 hours later. Values are means ± SE. ∗P < 0.05, ∗∗P < 0.01 compared with medium 48-hour control by analysis of variance. Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)

Figure 8 The active component of B. fragilis-induced hsp expression in YAMC cells. YAMC cells were incubated with B. fragilis, heat-killed B. fragilis, sonicated B. fragilis, supernatant from an overnight culture of B. fragilis, or the last 2 treated with polymyxin B. YAMC cells were treated with the earlier-listed agents for 3 hours, washed with PBS, and incubated for an additional 48 hours in medium with metronidazole (200 μg/mL) and gentamicin (300 μg/mL). Control indicates untreated cells at the end of the experiment. Image shown is representative of those of 3 separate experiments. Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)

Figure 9 (A ) Metronidazole treatment increases colonic mucosal susceptibility to C. difficile toxin A. Mice were untreated or metronidazole was included in drinking water for 5 days. Colonic mucosa was mounted in Ussing chambers and fluxes of the paracellular permeability marker mannitol were measured as described in the Materials and Methods section. Values are means ± SE for 5 mice in each group. ∗P < 0.05, ∗∗P < 0.01 compared with control or metronidazole treated in the absence of C. difficile toxin A by analysis of variance respectively. □, Control; ■, control + toxin A; ○, metro; •, metro + toxin A. (B) Electrical parameters of metronidazole and untreated control mice: effect of toxin A. Values presented are means ± SE for 5 separate mice, 2 tissues were averaged in each mouse and this value was used to compile means. Values shown before toxin A were at 30 minutes, values after toxin A were after 60 minutes (1 μg/mL). ∗P < 0.05, ∗P < 0.01 by paired t test between either untreated or metronidazole-treated mice with and without toxin. For the transepithelial resistance measurements in B, the asterisk indicates significant difference (P < 0.05 by t test) between control and C. difficile-treated tissues from metronidazole-treated mice. Gastroenterology 2003 124, 1395-1407DOI: (10.1016/S0016-5085(03)00215-4)