ALBERT

Beschreibung: Introduction The regulation of neutrophil lifespan is critical for a circumscribed immune response. Neutrophils are sensitive to Fas/CD95 death receptor signaling in vitro but it is unknown if Fas regulates neutrophil lifespan in vivo. We hypothesized that FasL-expressing CD8+ T cells, which kill antigen-stimulated T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection, can also induce neutrophil death in tissues during infection. Infection of Fas- and FasL-deficient mice with the enteropathogenic-like mouse pathogen Citrobacter rodentium (C.rodentium) is associated with neutrophil infiltration and severe diarrhoea. We hypothesized that a deficiency of Fas in neutrophils prolongs neutrophil lifespan and contributes to the accumulation of neutrophils in C.rodentium-infected mice. Methods Fas signaling can drive IL-1β and IL-18 production and thereby affect neutrophil accumulation in tissues, so mixed bone marrow chimeras were generated to compare the accumulation of WT and Fas-deficient neutrophils in vivo. For mixed bone-marrow chimeras, irradiated Ly5.1 mice were reconstituted with 2.5 x 106 bone marrow cells from ubiquitin-GFP or Ly5.1/5.2 mice combined with LysM-Cre Fasfl/fl at a 1:1 ratio. For infection studies, mice were infected with LCMV docile or C.rodentium. For in vitro assays, neutrophils were primed for 1 h with GM-CSF prior to treatment with Toll-like receptor (TLR) ligands or IL-18 and FcFasL. Neutrophil viability was measured by live cell imaging and automated image analysis. Results Using LysM-Cre Fasfl/fl mice, which lack Fas expression in macrophages and neutrophils, we show that Fas regulates neutrophil lifespan in the colon during C.rodentium infection, and during LCMV infection in the lung, peripheral blood and spleen. To examine if pathogen-derived molecules can modulate Fas signaling in neutrophils, we primed neutrophils with TLR ligands, which ablated Caspase-8 processing and Fas signaling. Treatment of neutrophils with the TLR ligands Pam3CSK4, Pam2CSK4 or LPS or the MyD88-dependent cytokine IL-18 strongly protected neutrophils against Fas-induced death. Conclusion These data provide the first in vivo genetic evidence that neutrophil lifespan is controlled by death receptor signaling and provides a mechanism to account for neutrophil resistance to Fas stimulation during infection. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 1213 The inflammasome is an innate immune complex that recognizes a wide range of pathogen, cellular stress and damage signals. It triggers the Caspase-1-dependent production of inflammatory cytokines including IL-1β and IL-18. Sensors for the inflammasome include the nucleotide-binding oligomerisation domain, leucine-rich repeat (NLR) proteins. The best-characterised of these is NLRP3, which mediates antibacterial, viral, fungal and parasitic immune responses, and is mutated in a spectrum of autoinflammatory diseases. Activation of mammalian NLRs can also result in a Caspase-1-dependent form of cell death termed pyroptosis, the importance of which in disease states remains unclear. For example, it is known that NLRP3 activating mutations in humans can be effectively treated by neutralising IL-1β, suggesting that cell death induced by NLRP3 activation does not play a significant role in pathology. Similarly, the NLRP1b inflammasome is activated by anthrax lethal toxin to cause macrophage pyroptosis, but this does not play a role in anthrax sensitivity in vivo. Here we define a role for NLRP1 in autoinflammatory disease and the pyroptotic death of hematopoietic progenitor cells. We began by generating two mouse models, one carrying a point mutation in NLRP1a that results in constitutive activation of the protein, and another harbouring a deletion of the entire NLRP1 locus. Animals homozygous for the NRLP1a activating mutation developed a multi-organ neutrophilic inflammatory disease characterised by meningitis, hepatitis, pneumonitis, pancreatitis, pulmonary peri-arteritis, myocarditis and inflammatory bowel disease. Mean survival was approximately 3 months of age. Genetic crosses established that this inflammatory disease was driven by Caspase-1 and IL-1β, but was independent of ASC and Caspase-11, and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, constitutively active NLRP1a triggered the Caspase-1-dependent death of hematopoietic progenitor cells resulting in leukopenia at steady state. To evaluate the effect of activated NLRP1a during hematopoietic stress, IL-1 receptor-deficient mice homozygous for the NRLP1a activating mutation were challenged with 5-fluorouracil. Strikingly, these animals succumbed shortly after the nadir of leukopenia at 12 days post-injection. They exhibited hypoplastic bone marrow, lymphopenia, monocytopenia and a deficit of reticulocytes consistent with a functional deficiency in hematopoietic progenitor cells. Conversely, in mice lacking NLRP1, we observed improved recovery of the hematopoietic compartment following hemoablative chemotherapy, with increases in the numbers of platelets, lymphocytes and monocytes relative to littermate controls. Severe infections are commonly associated with a range of cytopenias including anemia, lymphopenia, neutropenia and thrombocytopenia, however, the etiological triggers for these conditions have not been elucidated. We therefore infected IL-1 receptor-deficient mice homozygous for the NRLP1a activating mutation with lymphocytic choriomeningitis virus (LCMV). Relative to controls, more severe cytopenia and bone marrow hypoplasia was observed. In contrast, NLRP1-deficient animals showed enhanced recovery from LCMV. Our results suggest that activation of the NLRP1 inflammasome in hematopoietic progenitors may contribute to cytopenias induced by hematopoietic stresses such as chemotherapy or infection. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 389 The survival of megakaryocytes and platelets is regulated by the intrinsic apoptosis pathway. Both cell types express Bak and Bax, the essential mediators of intrinsic apoptosis, which must be kept in check for cellular viability to be maintained. In platelets, Bak and Bax are restrained by the pro-survival protein Bcl-xL. Mutations that reduce the pro-survival activity of Bcl-xL cause dose-dependent cell-intrinsic reductions in circulating platelet life span in mice. Accordingly, pharmacological blockade of Bcl-xL with the BH3 mimetic drugs ABT-737 or ABT-263 (Navitoclax) triggers platelet death and thrombocytopenia in mice, dogs and humans. In mice, loss of Bak and Bax almost doubles platelet life span, and renders platelets refractory to the effects of ABT-737. In megakaryocytes, we and others have recently demonstrated that Bcl-xLand its pro-survival relative Mcl-1 are essential for restraint of the intrinsic apoptosis pathway. Their loss triggers Bak/Bax-mediated death. Conversely, ablation of Bak and Bax can protect megakaryocytes from acute apoptotic insults, such as treatment with carboplatin. Combined with the fact that platelet production is normal in the absence of Bak and Bax, these studies have brought into question the long-standing theory that megakaryocytes deliberately undergo apoptosis in order to shed platelets. However, whilst it is clear that the intrinsic apoptosis pathway is not required for thrombopoiesis, the role of the extrinsic pathway—the other major route to apoptotic cell death—has not been established. In the current study we examined the functionality of, and physiological requirement for, the extrinsic apoptosis pathway in megakaryocytes and platelets. The extrinsic pathway is triggered when members of the tumor necrosis factor (TNF) superfamily such as Fas ligand (FasL) bind to cell surface death receptors (e.g. Fas). This induces receptor multimerization, recruitment of death domain adaptor proteins (e.g. FADD) and subsequent activation of Caspase-8, which is the essential mediator of extrinsic pathway-mediated cell death. We found that both megakaryocytes and platelets express critical components of the pathway, including FADD, Caspase-8 and Bid. Megakaryocytes, but not platelets, also expressed the death receptor Fas. Mature fetal liver-derived megakaryocytes treated with soluble FasL exhibited activation of Caspase-8 and the effector Caspases-3/7. This was accompanied by mitochondrial damage and a failure of pro-platelet formation. To establish the requirement for the extrinsic pathway in megakaryocyte development and platelet production, we conditionally deleted Caspase-8 from the megakaryocyte lineage. Platelet counts and platelet life span in Casp8Pf4Δ/Pf4Δ mice were indistinguishable from those of wild-type littermates. Megakaryocyte numbers, morphology, ploidy and in vitro pro-platelet formation capacity were also normal. Caspase-8-deficient megakaryocytes were resistant to FasL treatment. Casp8Pf4Δ/Pf4Δ animals responded to experimentally-induced thrombocytopenia in a manner similar to wild-type. Collectively, these data indicate that the extrinsic apoptosis pathway is dispensable for the generation and survival of platelets. To examine any potential redundancy between the extrinsic and intrinsic apoptosis pathways, we generated Bak−/−BaxPf4Δ/Pf4ΔCasp8Pf4Δ/Pf4Δ triple knockout mice. The ability of these animals to produce platelets, both at steady state and under conditions of thrombopoietic stress, was unperturbed. Megakaryocyte numbers and morphology were normal. Thus, platelet shedding by megakaryocytes does not require the intrinsic or extrinsic apoptosis pathways. Together with recent work demonstrating that the apoptotic initiator caspase, Caspase-9, is also dispensable for platelet production, we conclude that platelet biogenesis is not an apoptotic process. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Type I interferon (IFN-I) strongly inhibits viral replication and is a crucial factor in controlling virus infections and diseases. Cellular activation through pattern recognition receptors induces interferon production in a wide variety of hematopoietic and nonhematopoietic cell types, including dendritic cells, fibroblasts, hepatocytes, and cells of neuronal origin. The relative contribution of hematopoietic and nonhematopoietic cells to the overall interferon response is an important issue which has not been fully addressed. Using irf7−/− and wild-type bone marrow chimeras we analyzed the contribution of IFN-I from bone marrow–derived sources in the control of viral infections and immunopathology in mice. We found that during systemic cytopathic virus infection, hematopoietic cells were essential for production of IFN-I, inhibition of viral spread to peripheral organs, and limiting cell damage. In a model of autoimmune diabetes induced by noncytopathic virus infection, hematopoietic cell–derived IFN-I was essential for CD8+ T cell–dependent cytotoxicity in pancreatic β-islet cells and induction of diabetes. These data suggest that during systemic viral infection primarily hematopoietic cell–derived IFN-I controls viral replication and viral-induced disease.

Beschreibung: The role of caspase-8 and its adaptor Fas-associated death domain (FADD) in lymphocyte apoptosis is well defined, but their functions in other hemopoietic lineages are not clear. We were unable to generate transgenic mice expressing dominant inhibitors of FADD or caspase-8 in hemopoietic cells, possibly because their expression may have precluded production of vital hemopoietic cells. When using a retroviral gene delivery system, fetal liver stem cells expressing a dominant-negative mutant of FADD (FADD-DN) were unable to generate myeloid or lymphoid cells upon transplantation into lethally irradiated mice. However, fetal liver stem cells expressing very low levels of the caspase-8 inhibitor cytokine response modifier A (CrmA) could reconstitute the hemopoietic system. This level of CrmA expression provided some protection against Fas ligand (FasL)–induced apoptosis and promoted accumulation of myeloid cells in the bone marrow, but it did not inhibit mitogen-induced proliferation of B or T lymphocytes. Using an in vitro colony formation assay, we found that fetal liver stem cells expressing FADD-DN, CrmA, or a dominant-negative mutant of caspase-8 could not proliferate in response to cytokine stimulation. These data demonstrate that the enzymatic activity of caspase-8 and its adaptor FADD are required for cytokine-induced proliferation of hemopoietic progenitor cells.