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.

Cells exposed to sublethal oxidative stress selectively attract monocytes/macrophages via scavenger receptors and MyD88-mediated signaling.

The innate immune system responds to endogenous molecules released during cellular stress or those that have undergone modifications normally absent in healthy tissue. These structures are detected by pattern-recognition receptors, alerting the immune system to "danger." In this study, we looked for early signals that direct immune cells to cells undergoing stress before irreversible damage takes place. To avoid detecting signals emanating from apoptotic or necrotic cells we exposed fibroblasts to sublethal oxidative stress. Our results indicate that both nonenzymatic chemical reactions and aldehyde dehydrogenase-2-mediated enzymatic activity released signals from fibroblasts that selectively attracted CD14(+) monocytes but not T, NK, and NKT cells or granulocytes. Splenocytes from MyD88(-/-) mice did not migrate, and treatment with an inhibitory peptide that blocks MyD88 dimerization abrogated human monocyte migration. Monocyte migration was accompanied by downmodulation of CD14 expression and by the phosphorylation of IL-1R-associated kinase 1, a well-known MyD88-dependent signaling molecule. The scavenger receptor inhibitors, dextran sulfate and fucoidan, attenuated monocyte migration toward stressed cells and IL-1R-associated kinase 1 phosphorylation. Surprisingly, although monocyte migration was MyD88 dependent, it was not accompanied by inflammatory cytokine secretion. Taken together, these results establish a novel link between scavenger receptors and MyD88 that together function as sensors of oxidation-associated molecular patterns and induce monocyte motility. Furthermore, the data indicate that MyD88 independently regulates monocyte activation and motility.

The role of immune checkpoint receptors in the malignant phenotype of cutaneous T cell lymphoma.

Immune checkpoint receptors (ICR) modulate the immune response and are critical hubs for immunotherapy. However, data on their role in T lymphoid malignancies, such as cutaneous T cell lymphoma (CTCL), is sparse. We aimed to explore the role of ICR in the malignant features of transformed T lymphocytes and evaluate the effect of ICR-targeting monoclonal antibodies, often used as immunotherapy for solid tumors. We used the CTCL cell line HH and the Sézary cell line Hut78 to examine ICR expression and the effects of ICR inhibition on cell viability and proliferation. Despite their shared T cell progeny, the different CTCL cell lines exhibit markedly different ICR expression profiles. Programmed cell death-ligand 1 (PD-L1) was expressed by both cell lines, while programmed death-1 (PD-1) was expressed only by the HH cell line. Common to all malignant T cells was an autonomous hyper-proliferative state that did not require T cell receptor stimulation. A monoclonal antibody blocking PD-1 had a small but statistically significant augmenting effect on T cell proliferation. Of note, when the cells were exposed to ionizing radiation, healthy lymphocytes and those derived from the HH cell line were salvaged by anti-PD-L1. We show a regulatory role of ICR, mainly PD-1 and its ligand PD-L1, on cutaneous T cell malignancy.

Soluble SLAMF6 Receptor Induces Strong CD8+ T-cell Effector Function and Improves Anti-Melanoma Activity In Vivo.

SLAMF6, a member of the SLAM (signaling lymphocyte activation molecules) family, is a homotypic-binding immune receptor expressed on NK, T, and B lymphocytes. Phosphorylation variance between T-cell subclones prompted us to explore its role in anti melanoma immunity. Using a 203-amino acid sequence of the human SLAMF6 (seSLAMF6) ectodomain, we found that seSLAMF6 reduced activation-induced cell death and had an antiapoptotic effect on tumor-infiltrating lymphocytes. CD8+ T cells costimulated with seSLAMF6 secreted more IFNγ and displayed augmented cytolytic activity. The systemic administration of seSLAMF6 to mice sustained adoptively transferred transgenic CD8+ T cells in comparable numbers to high doses of IL2. In a therapeutic model, lymphocytes activated by seSLAMF6 delayed tumor growth, and when further supported in vivo with seSLAMF6, induced complete tumor clearance. The ectodomain expedites the loss of phosphorylation on SLAMF6 that occurs in response to T-cell receptor triggering. Our findings suggest that seSLAMF6 is a costimulator that could be used in melanoma immunotherapy. Cancer Immunol Res; 6(2); 127-38. ©2018 AACR.

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.

Inhibition of effector function but not T cell activation and increase in FoxP3 expression in T cells differentiated in the presence of PP14.

BACKGROUND: T-helper polarization of naïve T cells is determined by a complex mechanism that involves many factors, eventually leading to activation of Th1, Th2, or Th17 responses or alternatively the generation of regulatory T cells. Placental Protein 14 (PP14) is a 28 kDa glycoprotein highly secreted in early pregnancy that is able to desensitize T cell receptor (TCR) signaling and modulate T cell activation. METHODOLOGY/PRINCIPAL FINDINGS: Prolonged antigen-specific stimulation of T cells in the presence of PP14 resulted in an impaired secretion of IFN-γ, IL-5 and IL-17 upon restimulation, although the cells proliferated and expressed activation markers. Furthermore, the generation of regulatory CD4(+)CD25(high)Foxp3(+) T cells was induced in the presence of PP14, in both antigen-specific as well as polyclonal stimulation. In accordance with previous reports, we found that the induction of FoxP3 expression by PP14 is accompanied by down regulation of the PI3K-mTOR signaling pathway. CONCLUSIONS/SIGNIFICANCE: These data suggest that PP14 arrests T cells in a unique activated state that is not accompanied with the acquisition of effector function, together with promoting the generation of regulatory T cells. Taken together, our results may elucidate the role of PP14 in supporting immune tolerance in pregnancy by reducing T cell effector functions along with augmenting Treg differentiation.