Gut microbiota shape 'inflamm-ageing' cytokines and account for age-dependent decline in DNA damage repair.

OBJECTIVE: Failing to properly repair damaged DNA drives the ageing process. Furthermore, age-related inflammation contributes to the manifestation of ageing. Recently, we demonstrated that the efficiency of repair of diethylnitrosamine (DEN)-induced double-strand breaks (DSBs) rapidly declines with age. We therefore hypothesised that with age, the decline in DNA damage repair stems from age-related inflammation. DESIGN: We used DEN-induced DNA damage in mouse livers and compared the efficiency of their resolution in different ages and following various permutations aimed at manipulating the liver age-related inflammation. RESULTS: We found that age-related deregulation of innate immunity was linked to altered gut microbiota. Consequently, antibiotic treatment, MyD88 ablation or germ-free mice had reduced cytokine expression and improved DSBs rejoining in 6-month-old mice. In contrast, feeding young mice with a high-fat diet enhanced inflammation and facilitated the decline in DSBs repair. This latter effect was reversed by antibiotic treatment. Kupffer cell replenishment or their inactivation with gadolinium chloride reduced proinflammatory cytokine expression and reversed the decline in DSBs repair. The addition of proinflammatory cytokines ablated DSBs rejoining mediated by macrophage-derived heparin-binding epidermal growth factor-like growth factor. CONCLUSIONS: Taken together, our results reveal a previously unrecognised link between commensal bacteria-induced inflammation that results in age-dependent decline in DNA damage repair. Importantly, the present study support the notion of a cell non-autonomous mechanism for age-related decline in DNA damage repair that is based on the presence of 'inflamm-ageing' cytokines in the tissue microenvironment, rather than an intrinsic cellular deficiency in the DNA repair machinery.

Early age decline in DNA repair capacity in the liver: in depth profile of differential gene expression.

Aging is associated with progressive decline in cell function and with increased damage to macromolecular components. DNA damage, in the form of double-strand breaks (DSBs), increases with age and in turn, contributes to the aging process and age-related diseases. DNA strand breaks triggers a set of highly orchestrated signaling events known as the DNA damage response (DDR), which coordinates DNA repair. However, whether the accumulation of DNA damage with age is a result of decreased repair capacity, remains to be determined. In our study we showed that with age there is a decline in the resolution of foci containing γH2AX and pKAP-1 in diethylnitrosamine (DEN)-treated mouse livers, already evident at a remarkably early age of 6-months. Considerable age-dependent differences in global gene expression profiles in mice livers after exposure to DEN, further affirmed these age related differences in the response to DNA damage. Functional analysis identified p53 as the most overrepresented pathway that is specifically enhanced and prolonged in 6-month-old mice. Collectively, our results demonstrated an early decline in DNA damage repair that precedes 'old age', suggesting this may be a driving force contributing to the aging process rather than a phenotypic consequence of old age.

Macrophages Regulate the Systemic Response to DNA Damage by a Cell Nonautonomous Mechanism.

The DNA damage response (DDR) is a comprehensive and complex network of phosphorylation-mediated signaling pathways that originates endogenously from the DNA lesion and activates intrinsic DNA repair mechanisms. Here we describe a macrophage-dependent mechanism that regulates the response to DNA damage. We demonstrate that human monocytes, by releasing macrophage-derived HB-EGF, enhance DDR in neighboring cells suffering from DNA damage. Consequently, HB-EGF-treated cells exhibit higher double-strand break (DSB) rejoining and display lower levels of residual DSBs. Diethylnitrosamine (DEN) injection induce DSBs along with elevation in the number of macrophages and HB-EGF expression. Significantly, macrophage depletion or blocking HB-EGF activity results in higher levels of nonrepairable DSBs, suggesting that macrophages play a role in the resolution of DNA damage via HB-EGF. This study establishes that macrophages, acting through the activation of the EGFR cascade, constitute an important cell nonautonomous physiologic component of the DDR and points to a unique role played by immune cells in maintaining genome integrity.

MiR-192 directly binds and regulates Dicer1 expression in neuroblastoma.

Neuroblastoma (NB) arises from the embryonic neural crest and is the most common extracranial solid tumor in children under 5 years of age. Reduced expression of Dicer1 has recently been shown to be in correlation with poor prognosis in NB patients. This study aimed to investigate the mechanisms that could lead to the down-regulation of Dicer1 in neuroblastoma. We used computational prediction to identify potential miRs down-regulating Dicer1 in neuroblastoma. One of the miRs that were predicted to target Dicer1 was miR-192. We measured the levels of miR-192 in 43 primary tumors using real time PCR. Following the silencing of miR-192, the levels of dicer1 cell viability, cell proliferation and migration capability were analyzed. Multivariate analysis identified miR-192 as an independent prognostic marker for relapse in neuroblastoma patients (p=0.04). We were able to show through a dual luciferase assay and side-directed mutational analysis that miR-192 directly binds the 3' UTR of Dicer1 on positions 1232-1238 and 2282-2288. An increase in cell viability, proliferation and migration rates were evident in NB cells transfected with miR-192-mimic. Yet, there was a significant decrease in proliferation when NB cells were transfected with an miR-192-inhibitor We suggest that miR-192 might be a key player in NB by regulating Dicer1 expression.