Detection of microbial DNA is an evolutionarily conserved mechanism that alerts the host immune system to mount a defense response to microbial infections. However, this detection mechanism also poses a challenge to the host as to how to distinguish foreign DNA from abundant self-DNA. Cyclic guanosine monophosphate (GMP)–adenosine monophosphate (AMP) synthase (cGAS) is a DNA sensor that triggers innate immune responses through production of the second messenger cyclic GMP-AMP (cGAMP), which binds and activates the adaptor protein STING. However, cGAS can be activated by double-stranded DNA irrespective of the sequence, including self-DNA. Although how cGAS is normally kept inactive in cells is still not well understood, recent research has provided strong evidence that genomic DNA damage leads to cGAS activation to stimulate inflammatory responses. This review summarizes recent findings on how genomic instability and DNA damage trigger cGAS activation and how cGAS serves as a link from DNA damage to inflammation, cellular senescence, and cancer.

The spores of pathogenic bacteria are involved in host entry and the initial encounter with the host immune system. How bacterial spores interact with host immunity, however, remains poorly understood. Here, we show that the spores of Bacillus anthracis (BA), the etiologic agent of anthrax, possess an intrinsic ability to induce host immune responses. This immunostimulatory activity is attributable to high amounts of RNA present in the spore surface layer. RNA-sensing TLRs, TLR7, and TLR13 in mice and their human counterparts, are responsible for detecting and triggering the host cell response to BA spores, whereas TLR2 mediates the sensing of vegetative BA. BA spores, but not vegetative BA, induce type I IFN (IFN-I) production. Although TLR signaling in itself affords protection against BA, spore RNA–induced IFN-I signaling is disruptive to BA clearance. Our study suggests a role for bacterial spore–associated RNA in microbial pathogenesis and illustrates a little known aspect of interactions between the host and spore-forming bacteria.

Fluid shear stress promotes the emergence of hematopoietic stem cells (HSCs) in the aorta–gonad–mesonephros (AGM) of the developing mouse embryo. We determined that the AGM is enriched for expression of targets of protein kinase A (PKA)–cAMP response element-binding protein (CREB), a pathway activated by fluid shear stress. By analyzing CREB genomic occupancy from chromatin-immunoprecipitation sequencing (ChIP-seq) data, we identified the bone morphogenetic protein (BMP) pathway as a potential regulator of CREB. By chemical modulation of the PKA–CREB and BMP pathways in isolated AGM VE-cadherin + cells from mid-gestation embryos, we demonstrate that PKA–CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors, and is dependent on secreted BMP ligands through the type I BMP receptor. Finally, we observed blunting of this signaling axis using Ncx1 -null embryos, which lack a heartbeat and intravascular flow. Collectively, we have identified a novel PKA–CREB–BMP signaling pathway downstream of shear stress that regulates HSC emergence in the AGM via the endothelial-to-hematopoietic transition.

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A 2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1 + / cmyb + HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A 2b adenosine receptor disrupted scl + hemogenic vascular endothelium and the subsequent EHT process. A 2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates.

The characterization of human dendritic cell (DC) subsets is essential for the design of new vaccines. We report the first detailed functional analysis of the human CD141 + DC subset. CD141 + DCs are found in human lymph nodes, bone marrow, tonsil, and blood, and the latter proved to be the best source of highly purified cells for functional analysis. They are characterized by high expression of toll-like receptor 3, production of IL-12p70 and IFN-β, and superior capacity to induce T helper 1 cell responses, when compared with the more commonly studied CD1c + DC subset. Polyinosine-polycytidylic acid (poly I:C)–activated CD141 + DCs have a superior capacity to cross-present soluble protein antigen (Ag) to CD8 + cytotoxic T lymphocytes than poly I:C–activated CD1c + DCs. Importantly, CD141 + DCs, but not CD1c + DCs, were endowed with the capacity to cross-present viral Ag after their uptake of necrotic virus-infected cells. These findings establish the CD141 + DC subset as an important functionally distinct human DC subtype with characteristics similar to those of the mouse CD8α + DC subset. The data demonstrate a role for CD141 + DCs in the induction of cytotoxic T lymphocyte responses and suggest that they may be the most relevant targets for vaccination against cancers, viruses, and other pathogens.

The innate immune system responds to unique molecular signatures that are widely conserved among microbes but that are not normally present in host cells. Compounds that stimulate innate immune pathways may be valuable in the design of novel adjuvants, vaccines, and other immunotherapeutics. The cyclic dinucleotide cyclic-di–guanosine monophosphate (c-di-GMP) is a recently appreciated second messenger that plays critical regulatory roles in many species of bacteria but is not produced by eukaryotic cells. In vivo and in vitro studies have previously suggested that c-di-GMP is a potent immunostimulatory compound recognized by mouse and human cells. We provide evidence that c-di-GMP is sensed in the cytosol of mammalian cells via a novel immunosurveillance pathway. The potency of cytosolic signaling induced by c-di-GMP is comparable to that induced by cytosolic delivery of DNA, and both nucleic acids induce a similar transcriptional profile, including triggering of type I interferons and coregulated genes via induction of TBK1, IRF3, nuclear factor κB, and MAP kinases. However, the cytosolic pathway that senses c-di-GMP appears to be distinct from all known nucleic acid–sensing pathways. Our results suggest a novel mechanism by which host cells can induce an inflammatory response to a widely produced bacterial ligand.

Ku is a complex of two proteins, Ku70 and Ku80, and functions as a heterodimer to bind DNA double-strand breaks (DSB) and activate DNA-dependent protein kinase. The role of the Ku70 subunit in DNA DSB repair, hypersensitivity to ionizing radiation, and V(D)J recombination was examined in mice that lack Ku70 ( Ku70 −/− ). Like Ku80 −/− mice, Ku70 −/− mice showed a profound deficiency in DNA DSB repair and were proportional dwarfs. Surprisingly, in contrast to Ku80 −/− mice in which both T and B lymphocyte development were arrested at an early stage, lack of Ku70 was compatible with T cell receptor gene recombination and the development of mature CD4 + CD8 − and CD4 − CD8 + T cells. Our data shows, for the first time, that Ku70 plays an essential role in DNA DSB repair, but is not required for TCR V(D)J recombination. These results suggest that distinct but overlapping repair pathways may mediate DNA DSB repair and V(D)J recombination.

Interleukin-10 (IL-10) is a recently described pleiotropic cytokine secreted mainly by type 2 helper T cells. Previous studies have shown that IL-10 suppresses cytokine expression by natural killer (NK) and type 1 T cells, thus down-regulating cell-mediated immunity and stimulating humoral responses. We here report that injected IL-10 protein is an efficient inhibitor of tumor metastasis in experimental (B16-F10) and spontaneous (M27 and Lox human melanoma) metastasis models in vivo at doses that do not have toxic effects on normal or cancer cells. Histological characterization after IL-10 treatment confirmed the absence of CD8+ and CD4+ T cells and macrophages at the sites of tumor growth, but abundant NK cells were localized at these sites. This unexpected finding was confirmed by showing that IL-10 inhibits most B16-F10 and Lox metastases in mice deficient in T or B cells (SCID and nu/nu mice), but not in those deficient in NK cells (beige mice or NK cell-depleted mice). However, IL-10 downregulation of pro-inflammatory cytokine production and/or recruitment of additional effector cells may also be involved in the anti-tumor effect at higher local concentrations of IL-10, since transfected B16 tumor cells expressing high amounts of IL-10 were rejected by normal, nu/nu, or SCID mice at the primary tumor stage, and there was still a 33% inhibition of tumor metastasis in beige mice.

We have created embryonic stem (ES) cells and mice lacking the predominant isoform (alpha) of the calcineurin A subunit (CNA alpha) to study the role of this serine/threonine phosphatase in the immune system. T and B cell maturation appeared to be normal in CNA alpha -/- mice. CNA alpha -/- T cells responded normally to mitogenic stimulation (i.e., PMA plus ionomycin, concanavalin A, and anti-CD3 epsilon antibody). However, CNA alpha -/- mice generated defective antigen-specific T cell responses in vivo. Mice produced from CNA alpha -/- ES cells injected into RAG-2-deficient blastocysts had a similar defective T cell response, indicating that CNA alpha is required for T cell function per se, rather than for an activity of other cell types involved in the immune response. CNA alpha -/- T cells remained sensitive to both cyclosporin A and FK506, suggesting that CNA beta or another CNA-like molecule can mediate the action of these immunosuppressive drugs. CNA alpha -/- mice provide an animal model for dissecting the physiologic functions of calcineurin as well as the effects of FK506 and CsA.

Interleukin-2 (IL-2)-deficient (IL-2-/-) mice develop anemia and colonic inflammatory bowel disease. To elucidate the mechanism of this disease, we have bred IL-2-/- mice to two strains of immunodeficient mice, RAG-2-deficient (RAG-2-/-, lacking B and T cells) and JH-deficient mice (JH-/-, lacking B cells). IL-2-/-, RAG-2-/- double-mutant mice are disease free, while IL-2-/-, JH-/- double-mutant mice succumb to bowel disease at the same rate as IL-2-/- littermates. IL-2-/-, JH-/- mice do not, however, succumb to anemia. Thus, spontaneous intestinal inflammation in IL-2-/- mice requires mature T cells, not B cells, while anemia is dependent on B cells.

The costimulatory signal provided to T cells through CTLA-4-ligand interactions is required for T cell activation resulting in increased interleukin 2 (IL-2) production in vitro, but its role in the production of IL-4 and other cytokines is unclear and few in vivo studies have been performed to confirm results of in vitro experiments. We have examined the in vivo effects of blocking CTLA-4 ligands on the T helper cell 2 (Th2)-associated mucosal immune response that follows oral infection of mice with the nematode parasite, Heligmosomoides polygyrus. CTLA-4Ig administration inhibited H. polygyrus-induced increases in mesenteric lymph node (MLN) B cell major histocompatibility complex class II expression and size and T cell-derived IL-4 gene expression. In addition, CTLA-4 immunoglobulin (Ig) partially blocked increased IL-3, IL-5, and IL-9 cytokine gene expression in Peyer's patch (PP) and MLN 8 d after primary inoculation of mice with the parasite. Increases in the number of IL-4- but not IL-5-secreting cells were also inhibited by CTLA-4Ig. H. polygyrus-induced elevations in serum IgE levels but not blood eosinophils, were markedly inhibited by CTLA-4Ig. These results suggest that stimulation of CD28 and/or CTLA-4 is required for T cell priming leading to IL-4 cytokine production, B cell activation, and IgE secretion during a Th2-like, mucosal immune response to a nematode parasite.

We have studied contributions to hematopoiesis of genetically distinct stem cell populations in allophenic mice. Chimeras were made by aggregating embryos of inbred strains known to differ with respect to stem cell population kinetics. One partner strain (DBA/2) has previously been shown to normally have a stem cell (CFU-S) population of which 24% are in S-phase of the cell cycle, whereas the homologous population of the other partner strain (C57BL/6) was characterized by having only 2.6% in cycle (7). Contributions of the chimeric stem cell population to mature blood cell pools were studied throughout the life of the mice and intrinsic differences in stem cell function and aging were reflected in dynamic patterns of blood cell composition. The DBA/2 stem cell population was eclipsed by stem cells of the C57BL/6 genotype and, after 1.5-3 yr, the hemato-lymphoid composition of 22 of 27 mice studied for this long had shifted by at least 25 percentage points toward the C57BL/6 genotype. 8 of the 27 had hematolymphoid populations solely of C57BL/6 origin. To test whether or not a population of stem cells with an inherently higher cycling rate (DBA/2) might have a competitive advantage during repopulation, we engrafted allophenic marrow into lethally irradiated (C57BL/6 x DBA/2)F1 recipients. DBA/2 hematopoiesis was predominant early, far outstripping its representation in the marrow graft. Perhaps as a consequence of inherently greater DBA/2 stem cell proliferation, the populations of developmentally more restricted precursor populations (CFU-E, BFU-E, CFU-GM, CFU-GEMM) showed an overwhelming DBA/2 bias in the first 2-3 mo after engraftment. However, as in the allophenic mice themselves during the aging process, the C57BL/6 genotypic representation was ascendant over the subsequent months. The shift toward C57BL/6 genotype was first documented in the marrow and spleen precursor cell populations and was subsequently reflected in the circulating, mature blood cells. Bone marrow-derived stromal cell cultures from engrafted mice were studied and genotypic analyses showed donor representation in stromal cell populations that reflected donor hematopoietic contributions in the same recipient. Results from these studies involving two in vivo settings (allophenic mice and engraftment by allophenic marrow) are consistent with the notion that a cell autonomous difference in stem cell proliferation confers on one population a competitive repopulating advantage, but at the expense of longevity.