Extranuclear DNA accumulates in aged cells and contributes to senescence and inflammation.

Systemic inflammation is central to aging-related conditions. However, the intrinsic factors that induce inflammation are not well understood. We previously identified a cell-autonomous pathway through which damaged nuclear DNA is trafficked to the cytosol where it activates innate cytosolic DNA sensors that trigger inflammation. These results led us to hypothesize that DNA released after cumulative damage contributes to persistent inflammation in aging cells through a similar mechanism. Consistent with this notion, we found that older cells harbored higher levels of extranuclear DNA compared to younger cells. Extranuclear DNA was exported by a leptomycin B-sensitive process, degraded through the autophagosome-lysosomal pathway and triggered innate immune responses through the DNA-sensing cGAS-STING pathway. Patient cells from the aging diseases ataxia and progeria also displayed extranuclear DNA accumulation, increased pIRF3 and pTBK1, and STING-dependent p16 expression. Removing extranuclear DNA in old cells using DNASE2A reduced innate immune responses and senescence-associated (SA) ß-gal enzyme activity. Cells and tissues of Dnase2a-/- mice with defective DNA degradation exhibited slower growth, higher activity of ß-gal, or increased expression of HP-1ß and p16 proteins, while Dnase2a-/- ;Sting-/- cells and tissues were rescued from these phenotypes, supporting a role for extranuclear DNA in senescence. We hypothesize a direct role for excess DNA in aging-related inflammation and in replicative senescence, and propose DNA degradation as a therapeutic approach to remove intrinsic DNA and revert inflammation associated with aging.

Sphingosine 1-phosphate receptor agonism impairs skin dendritic cell migration and homing to secondary lymphoid tissue: association with prolonged allograft survival.

The novel immunomodulator FTY720 is a prototypic sphingosine-1-phosphate (S1P) receptor agonist that regulates lymphocyte migration and prolongs allograft survival. Skin dendritic cells (DC) play important roles in cutaneous immunity. We investigated the migration and function of skin DC exposed to FTY720 in vivo, or to its metabolite FTY720 phosphate (P) in vitro. C57BL/10 (H2(b)) recipient (but not donor) FTY720 treatment prolonged median skin C3H (H2(k)) allograft survival significantly, from 12 to 34.5 days. Non-transplanted, FTY720-treated mice revealed a marked increase in skin DC, although total DC in skin-draining lymph nodes (DLN) were unchanged compared with controls. Fewer allogeneic donor DC migrated to DLN of FTY720-treated graft recipients. DC that migrated from the skin of FTY720-treated mice showed reduced MHC class II, CD86 and CCR7 expression, suggesting impaired migratory potential to secondary lymphoid tissue, that correlated with DC retention in skin, and reduced T cell stimulatory activity. Fewer DC migrated from normal skin explants exposed to the FTY720 metabolite, FTY720P than to control medium. DC that did migrate expressed lower levels of MHC class II, CD86 and CCR7, and inferior T cell stimulatory ability. These data demonstrate S1P signaling regulates skin DC trafficking and modulates MHC class II, costimulatory, and homing receptor molecule expression that impairs their ability to elicit allogeneic T cell responses.

Dnase2a deficiency uncovers lysosomal clearance of damaged nuclear DNA via autophagy.

Deficiencies in DNA-degrading nucleases lead to accumulation of self DNA and induction of autoimmunity in mice and in monogenic and polygenic human diseases. However, the sources of DNA and the mechanisms that trigger immunity remain unclear. We analyzed mice deficient for the lysosomal nuclease Dnase2a and observed elevated levels of undegraded DNA in both phagocytic and nonphagocytic cells. In nonphagocytic cells, the excess DNA originated from damaged DNA in the nucleus based on colocalization studies, live-cell imaging, and exacerbation by DNA-damaging agents. Removal of damaged DNA by Dnase2a required nuclear export and autophagy-mediated delivery of the DNA to lysosomes. Finally, DNA was found to accumulate in Dnase2a(-/-) or autophagy-deficient cells and induce inflammation via the Sting cytosolic DNA-sensing pathway. Our results reveal a cell-autonomous process for removal of damaged nuclear DNA with implications for conditions with elevated DNA damage, such as inflammation, cancer, and chemotherapy.

"Alternatively activated" dendritic cells preferentially secrete IL-10, expand Foxp3+CD4+ T cells, and induce long-term organ allograft survival in combination with CTLA4-Ig.

In this study, we propagated myeloid dendritic cells (DC) from BALB/c (H2(d)) mouse bone marrow progenitors in IL-10 and TGF-beta, then stimulated the cells with LPS. These "alternatively activated" (AA) DC expressed lower TLR4 transcripts than LPS-stimulated control DC and were resistant to maturation. They expressed comparatively low levels of surface MHC class II, CD40, CD80, CD86, and programmed death-ligand 2 (B7-DC; CD273), whereas programmed death-ligand 1 (B7-H1; CD274) and inducible costimulatory ligand expression were unaffected. AADC secreted much higher levels of IL-10, but lower levels of IL-12p70 compared with activated control DC. Their poor allogeneic (C57BL/10; B10) T cell stimulatory activity and ability to induce alloantigen-specific, hyporesponsive T cell proliferation was not associated with enhanced T cell apoptosis. Increased IL-10 production was induced in the alloreactive T cell population, wherein CD4+Foxp3+ cells were expanded. The AADC-expanded allogeneic CD4+CD25+ T cells showed enhanced suppressive activity for T cell proliferative responses compared with freshly isolated T regulatory cells. In vivo migration of AADC to secondary lymphoid tissue was not impaired. A single infusion of BALB/c AADC to quiescent B10 recipients induced alloantigen-specific hyporesponsive T cell proliferation and prolonged subsequent heart graft survival. This effect was potentiated markedly by CTLA4-Ig, administered 1 day after the AADC. Transfer of CD4+ T cells from recipients of long-surviving grafts (>100 days) that were infiltrated with CD4+Foxp3+ cells, prolonged the survival of donor-strain hearts in naive recipients. These data enhance insight into the regulatory properties of AADC and demonstrate their therapeutic potential in vascularized organ transplantation.

The sphingosine-1-phosphate receptor agonist FTY720 modulates dendritic cell trafficking in vivo.

The pro-drug FTY720 is undergoing phase III clinical trials for prevention of allograft rejection. After phosphorylation, FTY720 targets the G protein-coupled-sphingosine-1-phosphate receptor 1 (S1PR1) on lymphocytes, thereby inhibiting their egress from lymphoid organs and their recirculation to inflammatory sites. Potential effects on dendritic cell (DC) trafficking have not been evaluated. Here, we demonstrate the expression of all five S1PR subtypes (S1PR1-5) by murine DCs. Administration of FTY720 to C57BL/10 mice markedly reduced circulating T and B lymphocytes within 24 h, but not blood-borne DCs, which were enhanced significantly for up to 96 h, while DCs in lymph nodes and spleen were reduced. Numbers of adoptively transferred, fluorochrome-labeled syngeneic or allogeneic DCs in blood were increased significantly in FTY720-treated animals, while donor-derived DCs and allostimulatory activity for host naïve T cells within the spleen were reduced. Administration of the selective S1PR1 agonist SEW2871 significantly enhanced circulating DC numbers. Flow analysis revealed that CD11b, CD31/PECAM-1, CD54/ICAM-1 and CCR7 expression on blood-borne DCs was downregulated following FTY720 administration. Transendothelial migration of FTY720-P-treated immature DCs to the CCR7 ligand CCL19 was reduced. These novel data suggest that modulation of DC trafficking by FTY720 may contribute to its immunosuppressive effects.

CCR and CC chemokine expression in relation to Flt3 ligand-induced renal dendritic cell mobilization.

BACKGROUND: We investigated the expression and function of CC chemokine receptors (CCR) on highly-purified kidney and blood dendritic cells isolated from mice in which dendritic cells were mobilized with fms-like tyrosine 3 kinase ligand (Flt3L). METHODS: CCR and CC chemokine expression were determined by RNase protection assay or flow cytometry, and dendritic cell migratory responses assayed using Transwell chambers. Chemokine production in renal tissue was detected by immunofluorescence staining. Trafficking of fluorochrome-labeled dendritic cells was monitored in vivo. RESULTS: Freshly-isolated renal dendritic cells expressed mRNA for CCR1, 2, 5, and 7 and CCR1 and 5 protein. They did not migrate to inducible chemokines--CCL3 [macrophage inflammatory protein (MIP)-1alpha], CCL5 [regulated upon activation, normal T cell expressed and secreted (RANTES)], or CCL20 (MIP-3alpha). Following lipopolysaccharide (LPS) stimulation, the dendritic cells down-regulated CCR1, 2, and 5 expression, up-regulated or sustained signals for CCR7, and migrated to the constitutively expressed ligands CCL19 (MIP-3beta) and CCL21 (secondary lymphoid tissue chemokine). Normal kidneys expressed weak message for CCL2, 3, and 4, with stronger signals for CCL5 and 19. Intrarenal CCL5 production was enhanced by Flt3L administration, in association with marked increases in interstitial CD45+ mononuclear cells. Mobilized blood dendritic cells migrated to CCR2 and CCR5 ligands and trafficked to renal intertubular sites following adoptive (intravenous) transfer. Their migration to the CCR5 ligand MIP-1beta (CCL4) and homing to kidneys of Flt3L-treated recipients were inhibited by CCR5 antagonism. CONCLUSION: These data implicate specific CCR and their ligands in regulation of the dendritic cell constituency of the kidney. CCR5 antagonism inhibits their directed migration and intrarenal accumulation.