Atm-deficient mice exhibit increased sensitivity to dextran sulfate sodium-induced colitis characterized by elevated DNA damage and persistent immune activation

Abstract

The role of ataxia telangiectasia mutated (ATM), a DNA double-strand break recognition and response protein, in inflammation and inflammatory diseases is unclear. We have previously shown that high levels of systemic DNA damage are induced by intestinal inflammation in wild-type mice. To determine the effect of Atm deficiency in inflammation, we induced experimental colitis in Atm(-/-), Atm(+/-), and wild-type mice via dextran sulfate sodium (DSS) administration. Atm(-/-) mice had higher disease activity indices and rates of mortality compared with heterozygous and wild-type mice. Systemic DNA damage and immune response were characterized in peripheral blood throughout and after three cycles of treatment. Atm(-/-) mice showed increased sensitivity to levels of DNA strand breaks in peripheral leukocytes, as well as micronucleus formation in erythroblasts, compared with heterozygous and wild-type mice, especially during remission periods and after the end of treatment. Markers of reactive oxygen and nitrogen species-mediated damage, including 8-oxoguanine and nitrotyrosine, were present both in the distal colon and in peripheral leukocytes, with Atm(-/-) mice manifesting more 8-oxoguanine formation than wild-type mice. Atm(-/-) mice showed greater upregulation of inflammatory cytokines and significantly higher percentages of activated CD69+ and CD44+ T cells in the peripheral blood throughout treatment. ATM, therefore, may be a critical immunoregulatory factor dampening the deleterious effects of chronic DSS-induced inflammation, necessary for systemic genomic stability and homeostasis of the gut epithelial barrier.

Fig. 1

Disease activity indices (DAIs) of Atm^−/−, Atm^+/−, and wildtype mice. Atm^−/− mice exhibit higher DAIs (**: p<0.01) by Student’s unpaired t-test compared to Atm^+/− and wildtype mice. Two Atm^−/− mice died; one at end of cycle 2, and one at end of cycle 3. Non-treated mice of all genotypes had DAIs of 0 throughout the entire study (not shown).

Fig. 2

A. Mean olive tail moments of peripheral leukocytes with and without hOgg1 incubation. A portion of cells were treated with H₂O₂ for 20min as a positive control. Two-way ANOVA with Dunn’s multiple comparison test demonstrate significant (p<0.001) differences between genotypes. B. Percent positive cells for γH2AX in peripheral leukocytes of Atm^−/−, Atm^+/−, and wildtype mice. A portion of cells were treated with H₂O₂ for 20min before staining as a positive control. Two-way ANOVA with Dunn’s multiple comparison test demonstrated significant treatment effects. Genotype differences are shown *: p<0.05, **: p<0.01.

Fig. 3

Micronucleated normochromatic erythrocytes (MN-NCEs) per 1000 NCEs. ANOVA of a linear regression model for all three genotypes and treatment cycle effects were **: p<0.01, *: p<0.05 for Atm^−/− versus Atm^+/− and wildtype mice unless indicated otherwise.

Fig. 4

8-oxoguanine and nitrotyrosine formation in peripheral leukocytes and the distal colon. A.,B. 8-oxoguanine (green) and nitrotyrosine (red) staining in peripheral leukocytes, respectively (x100). C., D. Percent positive cells for 8-oxoguanine and nitrotyrosine, respectively, in peripheral leukocytes of Atm^−/− and wildtype mice. *: p<0.05, **: p<0.01 by Student’s unpaired t-test. E., F. Staining in the distal colon of wildtype mice for 8-oxoguanine and nitrotyrosine, respectively, treated with DSS for 7 days. (x10) G., H. Staining in the distal colon of Atm^−/− mice for 8-oxoguanine and nitrotyrosine, respectively, treated with DSS for 7 days. (x10) I. Quantification of 8-oxoguanine and nitrotyrosine staining in wildtype and Atm^−/− mice expressed in pixel area with brightness value above a set threshold (arbitraty units). **: p<0.01 by Student’s unpaired t-test.

Fig. 5

Cytokine panel in peripheral blood by quantitative real-time PCR. A., B., C. Th1 cytokine panel of TNF-α, MCP-1, and IFN-γ, respectively. D., E., F. IL-12, IL-23, and IL-6, respectively. G.,H.,I. Th2 cytokine panel of TGF-β, IL-10, and IL-4, respectively. Data are mean expression of gene over expression of TBP, the internal control gene. *: p<0.05, **: p<0.01 by two-way ANOVA for genotype comparisons.

Fig. 6

Flow cytometric analysis of peripheral leukocytes. A.,B. Percent gated CD69⁺ T-cells (CD4 or CD8α positive) and CD44⁺ T-cells, respectively, in peripheral blood. 15,000 cells were counted per mouse. *: p<0.05, **: p<0.01 by Student’s unpaired t-test with Welch correction for genotype comparisons. C. Baseline CD4⁺ and CD8α⁺ peripheral blood T-cells of Atm^−/−, Atm^+/−, and wildtype mice. D. Mean fluorescent intensities of CD44⁺ T-cells in Atm^−/−, Atm^+/−, and wildtype mice after cycle 2 and before cycle 3, respectively. Filled line represents isotype control.