Trp53 deficiency protects against acute intestinal inflammation

Abstract

The p53 protein has not only important tumor suppressor activity but also additional immunological and other functions, whose nature and extent are just beginning to be recognized. In this article, we show that p53 has a novel inflammation-promoting action in the intestinal tract, because loss of p53 or the upstream activating kinase, ATM, protects against acute intestinal inflammation in murine models. Mechanistically, deficiency in p53 leads to increased survival of epithelial cells and lamina propria macrophages, higher IL-6 expression owing to enhanced glucose-dependent NF-κB activation, and increased mucosal STAT3 activation. Blockade or loss of IL-6 signaling reverses the protective effects of p53 deficiency. Conversely, IL-6 treatment protects against acute colitis in a manner dependent on STAT3 signaling and induction of cytoprotective factors in epithelial cells. Together, these results indicate that p53 promotes inflammation in the intestinal tract through suppression of epithelium-protective factors, thus significantly expanding the spectrum of physiological and immunological p53 activities unrelated to cancer formation.

FIGURE 1

Attenuation of acute colitis in the absence of p53. (A) Immunoblots of phospho-p53 (serine 15) and total p53 of whole colon extracts from mice treated with DSS for the indicated times. Whole colon from mice exposed to 4 Gy γ-irradiation and from Trp53^−/− mice were used as controls. A second experiment gave similar results. (B) Expression levels of the indicated p53 target genes, and the p53-independent control, ICAM-1, were assayed by real-time PCR in RNA extracted from the colon of wild-type (Trp53^+/+) and p53-deficient (Trp53^−/−) mice, either treated with DSS (day 7) or left untreated, and are expressed as ratio in DSS-treated over untreated mice, normalized to GAPDH. Data are mean ± SD (n=3; t-test). (C) Trp53^−/− (n=5), Trp53^+/− (n=16), and Trp53^+/+ mice (n=7) were treated with DSS. Body weights are shown as mean ± SD of the percentage of the initial weight. All three groups were significantly different from each other (p<0.01), as calculated by repeated-measures ANOVA for the entire weight curves. (D) Kaplan-Meier survival curves for Trp53^−/− (n=45, dotted line), Trp53^+/− (n=74, dashed line), and Trp53^+/+ mice (n=77, solid line), with p-values calculated by log rank test (*p<0.05). (E) Paraffin sections of the colon from DSS-treated mice (day 7–10) were prepared and stained with hematoxylin and eosin. (F) Histological inflammation scores (range 0–12) and total colon ulcerations were evaluated on the colon sections. Each data point represents one animal, with means shown as horizontal lines (p-values were determined by Wilcoxon rank sum test). (G) MPO levels were assayed in colon lysates on days 0 and 7 of DSS treatment. Data are mean ± SD (n=3/group; p-values were determined by two-way ANOVA and Tukey post-test).

FIGURE 2

Function of ATM in acute colitis. Wild-type (Atm^+/+) and ATM-deficient (Atm^−/−) mice were treated with 3% DSS in the drinking water for 6 days and returned to regular water. (A) Lysates of colon tissue were prepared at the indicated times and examined by immunoblotting for phospho-p53 (serine 15) and total p53. The results are representative of three separate experiments. (B) Body weights were determined daily and are shown as mean ± SD of the percentage of initial weight (n=9–10 mice/group). The curves were significantly different (p<0.05) as determined by repeated-measures ANOVA. (C) Kaplan-Meier survival curves after DSS treatment (n=20 for Atm^+/+ and n=16 for Atm^−/− mice); *p<0.05 by log rank test. (D) Colon sections were prepared after 10 days and stained with hematoxylin and eosin. (E) Histological inflammation scores and total mucosal ulcerations were determined on H&E stained colon sections of DSS-treated mice (day 7–10). Each data point represents one animal, with means shown as horizontal lines (p-values were determined by rank sum test). (F) MPO levels were assayed in colon lysates on days 0 and 10 of DSS treatment. Data are mean ± SD (n=3/group; p-values were calculated by t-test).

FIGURE 3

p53 deficiency attenuates apoptosis in acute colitis. Wild-type (Trp53^+/+) and Trp53^−/− mice were left untreated (day 0) or were treated with 3% DSS for 6 days followed by one day of regular drinking water. (A) Levels of total and cleaved caspase 3 in total colon lysates were determined by immunoblotting. Blots are from one representative out of two independent experiments. (B) Apoptotic cells were identified in paraffin sections of the colon by TUNEL staining. Representative images from one out of three independent experiments are shown. Apoptotic cells were quantitated per high-power field (HPF) with a fluorescence microscope using a 20x lens. Data are mean + SD (n=5; p-value was determined by t-test). (C) Flow cytometric analysis of CD45⁺ F4/80⁺ lamina propria macrophages isolated from the colon and stained with fluorochrome-labeled annexin V and propidium iodide. The percentage of cells in each quadrant is shown in the corners of each dot plot. Data are representative of three independent experiments.

FIGURE 4

Lamina propria cells mediate protection against acute colitis in the absence of p53. Conditional knock-out mice for p53 in the intestinal epithelium (Trp53^IEC-KO, open circles) and controls (Trp53^IEC-WT, closed circles) were generated by crossing floxed p53 mice with villin-Cre transgenic mice. (A) Total p53 expression was examined by immunoblotting in intestinal epithelial cells (IEC) isolated from the colon. (B) Mice were treated with DSS. Body weights were recorded daily and are displayed as mean ± SD of the percentage of the initial weight (n=13 to 15 mice/group; analysis by repeated-measures ANOVA showed no significant difference between the groups). (C) Histological inflammation scores and total mucosal ulcerations were determined on colon sections on day 7. Each data point represents one animal, with means shown as horizontal lines (n= 13 to 15 mice/group); analysis by t-test or rank sum test yield no significant (N.S.) difference. (D) Numbers of apoptotic cells were determined by TUNEL staining and counting per high-power field (HPF) under a fluorescence microscope using a 20x lens. Data are mean + SD (significance evaluated by t-test).

FIGURE 5

Increased production of cytoprotective cytokines in acute colitis in p53-deficient mice. Wild-type (Trp53^+/+, closed bars) and p53-deficient (Trp53^−/−, open bars) mice were treated with DSS or were left untreated. (A) Left: Expression levels were assayed by real-time PCR in whole colon RNA, and are expressed as ratio in DSS-treated (day 7) over untreated mice, normalized to β-actin. Middle and right: Cytokine levels in whole colon lysates were determined by ELISA. All expression data are mean + SD (n=3; p-values relative to p53-proficient controls were determined by t-test). (B) Single cell suspensions of epithelium and lamina propria from the colon of untreated and DSS-treated wild-type mice were FACS-sorted into epithelial cells (Epi), macrophages (Mac), and dendritic cells (DC). Cytokine mRNA levels were analyzed by real-time PCR, and are expressed relative to the levels in cells from untreated mice. Data are mean + SD (n=3). (C) Activation of STAT3 was examined in lysates from isolated colon epithelial cells (IEC) and whole colon by immunoblotting for phospho-STAT3 and, as a control, total STAT3. (D) None marrow-derived macrophages and dendritic cells were stimulated with the indicated agonists for 8 hours. Supernatants were analyzed by ELISA. Data are mean + SD from three mice in one experiment out of five independent experiments (p-values were determined by t-test). (E) Wild-type and p53 deficient bone marrow-derived macrophages were stimulated with LPS in the absence or presence of 2-Deoxy-glucose (2-DG), and mRNA levels were determined by real-time PCR and IL-6 levels in the supernatants were assayed by ELISA. Data are mean + SD (n=6; p-values relative to controls were calculated by t-test). (F) Macrophages were stimulated with LPS for 4 h, with and without 2-DG, and NF-κB/p65 DNA-binding activity was determined by ELISA. Data are mean + SD (n=3; p-values relative to controls and relative to 2-DG treatment were determined by t-test).

FIGURE 6

Hematopoietic cells are responsible for colitis protection due to p53 deficiency. (A) Mice lacking p53 in macrophages and polymorphonuclear neutrophils (Mac/PMN) were generated by crossing floxed Trp53 mice with LysM-Cre mice, and subjected to DSS-induced colitis. Histological inflammation scores were determined after 7 days. Analysis by rank sum test showed no significant (N.S.) difference. (B–D) Bone marrow chimeric mice were generated with wild-type (Trp53^+/+) recipients and bone marrow from wild-type (closed circles/bars) or p53-deficient donors (Trp53^−/−, open circles/bars). After 8 weeks, mice were subjected to DSS treatment. (B) Body weights are shown as mean ± SD of the percentage of the initial weight (n=10 mice/group). The curves were significantly different (p<0.05) as determined by repeated-measures ANOVA. (C) Histological inflammation scores and total ulcerations were determined on colon sections on day 7–10. The horizontal lines represent the means (n=16 to 17 mice/group), significances were calculated by rank sum test (histology scores) or t-test (ulcerations). (D) MPO levels were determined in colon lysates on days 0 and 10 of DSS treatment. Data are mean + SD (n=3, p-value was determined by t-test).

FIGURE 7

Role of IL-6 in mediating protection against acute colitis in the absence of p53. (A,B) Double knock-out mice for p53 and IL-6 (Trp53^−/− x IL-6^−/−), single knock-out mice for p53 (Trp53^−/− x IL-6^+/+), and/or IL-6 (Trp53^+/+− x IL-6^−/−), and wild-type (Trp53^+/+ x IL-6^+/+) were subjected to DSS colitis. Some mice were treated i.p. with 3 µg of a monoclonal rat antibody against IL-6, soluble gp130/Fc fusion protein, or rat IgG, as indicated, every 3 days during DSS treatment for 6 days. As a control, Trp53^+/+ mice were given rat IgG. Histological inflammation scores and total ulceration were determined on colon sections on day 7–10. Each data point represents one animal, with means shown as horizontal lines. P-values were calculated by rank sum test (histology scores) or t-test (ulcerations). (C–E) DSS colitis was induced in wild-type (Trp53^+/+) mice and conditional knock-out mice for STAT3 in the intestinal epithelium (STAT3^IEC-KO) and their controls (STAT3^IEC-WT), with or without parallel injection with recombinant IL-6 (5 µg i.p.) or vehicle every 3 days from days 0–6 (C,D,E) or days 7–13 (E). Body weights (C) are expressed as means ± SD (n=5) of the percentages of the initial weight. The weight curves were significantly different (p<0.01) as determined by repeated-measures ANOVA. (D) Colon sections were prepared on days 7 or 14, stained with hematoxylin/eosin, and (E) analyzed for histological inflammation scores and total ulcerations. Each data point represents one animal, with means shown as horizontal lines. P-values were calculated by rank sum test (histology scores) or t-test (ulcerations); N.S. not significant. (F) Intestinal epithelial cells (IEC) were isolated from the colon of the indicated mice at 12 h after i.p. injection of 5 µg recombinant IL-6 or PBS as a control. Levels of the indicated gene products were examined by immunoblotting. The blots are representative of two separate experiments. Expression levels were determined by real-time PCR in IEC RNA obtained 2 h after IL-6 injection, and are expressed as mean + SD (n=3) of the ratio of mRNA in treated over untreated cells (x Control); p-values were determined by t-test.