The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. ...Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-β-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.
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•Pro-apoptotic Casp8 is essential for lethal LPS shock and E. coli sepsis•Initiation of cytokine production proceeds independent of Casp8 and Casp11•TNF and type 1 IFN drive Casp8 and Casp11 collaboration in target tissues•Combined pro-apoptotic and pro-pyroptotic signaling executes LPS shock
Endotoxic shock requires inflammatory cytokines and cell death; however, initiation and execution signaling pathways remain unresolved. Mandal et al. describe a collaboration between pro-apoptotic caspase-8 and pro-pyroptotic caspase-11, independent of pro-necroptotic RIPK1 kinase activity or RIPK3, to execute TNF- and type I interferon-mediated inflammatory tissue damage underlying endotoxic shock.
Although molecular components that execute noninflammatory apoptotic cell death are well defined, molecular pathways that trigger necrotic cell death remain poorly characterized. Here, we show that ...in response to infection with adenovirus or Listeria monocytogenes, macrophages in vivo undergo rapid proinflammatory necrotic death that is controlled by interferon-regulatory factor 3 (IRF3). The transcriptional activity of IRF3 is, surprisingly, not required for the induction of necrosis, and it proceeds normally in mice deficient in all known regulators of necrotic death or IRF3 activation, including RIPK3, caspases 1, 8, or 11, STING, and IPS1/MAVS. Although L. monocytogenes triggers necrosis to promote the infection, IRF3-dependent necrosis is required for reducing pathogen burden in the models of disseminated infection with adenovirus. Therefore, our studies implicate IRF3 as a principal and nonredundant component of a physiologically regulated necrotic cell-death pathway that operates as an effective innate immune mechanism of host protection against disseminated virus infection.
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•IRF3 is a critical and nonredundant factor that executes necrotic death in vivo•IRF3 executes necrosis independently of its transcription activator function•IRF3-dependent necrotic cell death is distinct from pyroptosis and necroptosis•IRF3-dependent necrosis protects the host from disseminated virus infection
Molecular pathways that execute physiologically regulated necrotic cell death remain poorly characterized. In this study, Shayakhmetov and colleagues implicate transcription factor IRF3 as a critical mediator of necrotic death that is triggered in liver residential macrophages in response to disseminated infections of human adenovirus or Listeria monocytogenes. The transcriptional activity of IRF3 is not required for the execution of macrophage necrosis that operates as an effective innate immune mechanism of host protection against disseminated virus infection.
In contrast to commonly used serotype 5-based adenovirus (Ad) vectors, Ad's containing fibers derived from B-group serotype 35 (Ad5/35) efficiently transduce human DCs ex vivo and appear to target ...antigen-presenting cells after intravenous injection into baboons. Based on this, Ad5/35 vectors could be valuable tools for immunotherapy and vaccination. On the other hand, a number of studies indicate that signaling through the B-group Ad receptor, CD46, can cause tolerance or immunosuppression. Since mice do not express CD46 in a human-like pattern, we studied the in vivo properties of Ad5/35 in transgenic mice that express CD46 in a pattern and at a level similar to those of humans. Hypersensitivity assays and analyses of frequencies of regulatory T cells and T cell responses did not indicate that Ad5/35 injection exerts detrimental effects on the host's immune system. An Ad5/35 vector expressing a model antigen was able to trigger a strong T cell response against the test antigen after intramuscular injection. Overall, compared to Ad5 vectors, Ad5/35 vectors had a better safety profile, reflected by lower serum levels of proinflammatory cytokines.
We have developed a capsid-modified oncolytic adenovirus (Ad) vector (Ad5/35.IR-E1A/TRAIL) that is highly efficient in the elimination of liver metastases derived from various human tumor cell lines ...in mouse models. However, testing for viral efficacy on established tumor cell lines and for safety in mouse models may not be predictive for the performance of these vectors in clinical trials. In contrast to cancer cell lines, the genotype and gene expression profiles of primary short term cultures more closely reflects that of actual tumors. We have therefore tested the oncolytic ability of our vector and a series of control vectors on primary ovarian and cervical cancer cultures. So far we have established two ovarian cancer cultures and three cultures of normal mesothelial cells from ascites. Ad.5/35.IR-E1A/TRAIL caused apoptosis in 100% and ∼80% of ovarian cancer cells in cultures from two different patients, but did not cause cytolysis in mesothelial cells. The efficiency and specificity of oncolysis by Ad5/35.IR-E1a/TRAIL was compared to wild-type Ads. Wild-type Ad35 efficiently caused cytopathic effects in mesothelial cells and ovarian cancer cells (without the characteristic apoptotic features seen in the Ad.5/35.IR-E1A/TRAIL infected cells). Wild-type Ad5 did not efficiently lyse tumor cells, but did cause more CPE in mesothelial cells than wt Ad35, which probably reflects the differential expression of receptors for Ad5 and Ad35. We are in the process of establishing more primary cultures and the results will be reported.For safety studies, baboons were injected through the femoral vein with PBS (mock) or 1 × 10 11 pfu/kg of Ad5/35.IR-E1a/TRAIL (in PBS) and blood samples were monitored for 30 days for biochemical parameters, blood cell counts, vector genome concentrations, and cytokine levels. Upon necropsy, tissue histology, presence of transgene expression, and vector genomes were analyzed in samples of brain, lymph nodes, bone marrow, heart, aorta, digestive tract, liver, pancreas, bladder, testis, muscle, spleen, lung, kidney, peripheral blood cells, and prostate. All laboratory blood test parameters were in normal ranges following injection of the oncolytic vectors. Among the cytokines, IL-6 was elevated at 6 hours after vector infusion, but reached pre-injection levels 24 hours post-infusion. We used real-time PCR for vector genomes to assess the kinetics of Ad clearance from the blood. When we compared the amount of vector DNA associated with blood cells and plasma with the total amount of injected Ad, we found that at 1 min post-infusion, ∼2% of the input Ad dose is associated with blood cells, which could be due to binding to erythrocytes (Ad35 agglutinates monkey erythrocytes). Ad5/35 genomes in plasma and blood cells reached pre-injection levels by 24 hours p.i.. No histopathological changes were found in tissues analyzed 30 days post-infusion. Analysis of vector genomes and transgene expression in tissue samples is ongoing.
Adeno-associated virus (AAV), a defective parvovirus, is considered a promising vector for the delivery of therapeutic genes to cells. Both wild-type and recombinant AAV display a wide tropism and ...integrate into the host genome, in the absence of helper virus, establishing a latent infection. A unique characteristic of wild-type AAV and a potential advantage for use as a delivery system for gene therapy is the site-specific integration of wild-type virus within a small region of chromosome 19, 19q13.3-qter (AAVS1), in up to 85% of cell lines infected with the virus. Although recombinant AAVs, containing only the inverted terminal repeats of wild-type virus, can integrate efficiently into the host genome, specificity for the AAVS1 site appears to be lost. To address this question, the integration characteristics of two recombinant AAVs lacking the rep and cap genes in HeLa cells were examined. Analysis of Southern blots indicated that none of twenty-six cell clones generated after infection with either one of the recombinant AAVs demonstrated integration within the AAVS1 locus on chromo-some 19. Analysis of five of the cell lines by fluorescent chromosome in situ hybridization confirmed the loss of chromosome 19 specificity. Each integration site mapped near a known fragile site and/or location of a proto-oncogene or growth regulatory gene. Retention of site-specific integration of wild-type AAV will require the inclusion of additional AAV-specific sequences within the recombinant vectors.
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