Age-associated epigenetic modifications in human DNA increase its immunogenicity

Abstract

Chronic inflammation, increased reactivity to self-antigens and incidences of cancer are hallmarks of aging. However, the underlying mechanisms are not well understood. Age-associated alterations in the DNA either due to oxidative damage, defects in DNA repair or epigenetic modifications such as methylation that lead to mutations and changes in the expression of genes are thought to be partially responsible. Here we report that epigenetic modifications in aged DNA also increase its immunogenicity rendering it more reactive to innate immune system cells such as the dendritic cells. We observed increased upregulation of costimulatory molecules as well as enhanced secretion of IFN-alpha from dendritic cells in response to DNA from aged donors as compared to DNA from young donors when it was delivered intracellularly via Lipofectamine. Investigations into the mechanisms revealed that DNA from aged subjects is not degraded, neither is it more damaged compared to DNA from young subjects. However, there is significantly decreased global level of methylation suggesting that age-associated hypomethylation of the DNA may be the cause of its increased immunogenicity. Increased immunogenicity of self DNA may thus be another mechanism that may contribute to the increase in age-associated chronic inflammation, autoimmunity and cancer.

Figure 1.. DNA from aged subjects is more immunogenic than DNA from young subjects.

(A) DCs were activated with aged and young DNA complexed with lipofectamine. The expression of costimulatory molecules CD80 and CD86 and the maturation molecule (CD83) in the unactivated and activated DCs was measured by flow cytometry. Figure is representative of ten such experiments using fifteen separate aged and young DNA. (B) Bar graph represents the mean fluorescence intensity of CD80, CD86 and CD83 of the same. (C) Supernatants collected from DCs activated with aged and young DNA were assayed for IFN-α using specific ELISA. Bar diagrams depict the concentration of IFN-α secreted by the DCs. Figure is mean + S.E. of thirty separate aged and young DNA tested.

Figure 2.. DNA from aged subjects is demethylated compared to DNA from young subjects.

(A) FlashGel showing the molecular weight of Aged and young DNA. Figure is representative of eight such experiments. (B) Bar diagram depicting the damage in DNA from aged and young subjects as determined by ELISA that measures the number of abasic sites per 10⁵ base pairs. Figure is mean + S.E. of twenty five separate aged and young DNA tested. (C) Bar diagram depicts the percent of global methylation in aged and young DNA as measured by ELISA. The methylated fraction of DNA is recognized by 5-methylcytosine antibody and quantified through an ELISA-like reaction. Figure is mean + S.E. of twenty four separate aged and young DNA tested.

Figure 3.. Immunogenicity of mammalian DNA correlates inversely with DNA methylation.

(A) DNA was methylated using a methyl transferase enzyme and percent global methylation was measured by ELISA. Bar diagram shows the percent of global methylation at 0h, 4h and 16h after the reaction. Figure is mean + S.E. of five separate DNA tested. (B) The immunogenicity of the methylated DNA was determined by measuring the IFN-α secretion by DCs. Bar diagram shows the level of IFN-α secreted by DCs in response to the DNA. Figure is mean + S.E. of five separate DNA tested. (C) PBMCs were treated with hydrogen peroxide to induce DNA damage. Damaged DNA was extracted and the extent of damage was determined by ELISA. Bar diagram shows the level of DNA damage before and after treatment. Figure is mean + S.E. of five separate DNA tested (D) The immunogenicity of the H₂O₂ damaged DNA was determined by measuring the IFN-α secretion by DCs. Bar diagram shows the level of IFN-α secreted by DCs in response to the DNA. Figure is mean + S.E. of five separate DNA tested.