Summary Caspase-8 or cFLIP deficiency leads to embryonic lethality in mice due to defects in endothelial tissues. Caspase-8−/−, RIPK3−/−, but not cFLIP−/−, RIPK3−/−, double-knockout animals develop normally, indicating that caspase-8 antagonizes the lethal effects of RIPK3 during development. Here we show that the acute deletion of caspase-8 in the gut of adult mice induces enterocyte death, disruption of tissue homeostasis and inflammation, resulting in sepsis and mortality. Likewise, acute deletion of caspase-8 in a focal region of the skin induces local keratinocyte death, tissue disruption and inflammation. Strikingly, RIPK3 ablation rescues both phenotypes. Acute loss of cFLIP in the skin produces a similar phenotype, which, however, is not rescued by RIPK3 ablation. TNF neutralization protects from either acute loss of caspase-8 or cFLIP. These results demonstrate that caspase-8-mediated suppression of RIPK3-induced death is required not only during development, but also for adult homeostasis. Furthermore, RIPK3-dependent inflammation is dispensable for the skin phenotype.

Influenza transmission efficiency in ferrets is vital for risk-assessment studies. However, the inability to monitor viral infection and transmission dynamics in real time only provides a glimpse into transmissibility. Here we exploit a replication-competent influenza reporter virus to investigate dynamics of infection/transmission in ferrets. Bioluminescent imaging of ferrets infected with A/California/04/2009 H1N1 virus (CA/09) encoding NanoLuc (NLuc) luciferase provides the first real-time snapshot of influenza infection/transmission. Luminescence in the respiratory tract and in less well-characterized extra-pulmonary sites is observed, and imaging identifies infections in animals that would have otherwise been missed by traditional methods. Finally, the reporter virus significantly increases the speed and sensitivity of virological and serological assays. Thus, bioluminescent imaging of influenza infections rapidly determines intra-host dissemination, inter-host transmission and viral load, revealing infection dynamics and pandemic potential of the virus. These results have important implications for antiviral drug susceptibility, vaccine efficacy, transmissibility and pathogenicity studies.

To streamline standard virological assays, we developed bioluminescent replication-competent influenza reporter viruses that mimic their parental counterparts. These reporter viruses provide a rapid and quantitative readout of viral infection and replication. Moreover, they permit real-time in vivo measures of viral load, tissue distribution, and transmission in the same cohort of animals over the entire course of infection-measurements that were not previously possible. Here we provide detailed protocols using bioluminescent reporter viruses for in vivo imaging in mice and ferrets. We also describe cell culture-based techniques using reporter viruses for quantification of viral titers and performing microneutralization assays. The ease, speed, and adaptability of these approaches have the potential to accelerate multiple areas of influenza virus research.

Neuroblastoma is a common pediatric cancer, where preclinical studies have suggested chemotherapy resistance is driven by a mesenchymal-like gene expression program. However, the poor clinical outcomes imply we need a better understanding of the relationship between patient tumors and preclinical models. Here, we generated single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We developed an unsupervised machine learning approach to compare the gene expression programs found in preclinical models to a large cohort of human neuroblastoma tumors. The dominant adrenergic programs were well preserved in preclinical models, but contrary to previous reports do not unambiguously map to an obvious cell of origin. The mesenchymal-like program was less clearly preserved, and primarily restricted to cancer-associated fibroblasts and Schwann-like cellsin vivo. Surprisingly however, we identified a subtle, weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some high-risk tumors. This program appears distinct from mesenchymal cell lines but was maintained in PDX and a similar program could be chemotherapy-induced in our GEMM after only 24 hours, suggesting an uncharacterized therapy-escape mechanism. Collectively, our findings advance the understanding of neuroblastoma heterogeneity and can inform the development of new treatments.