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Cell death coordinates repair programs following pathogen attack and tissue injury. However, aberrant cell death can interfere with such programs and cause organ failure. cFLIP is a crucial regulator of cell death and a substrate of Caspase-8. Yet, the physiological role of cFLIP cleavage by Caspase-8 remains elusive. Here, we discovered an essential role for cFLIP cleavage in restraining cell death in different pathophysiological scenarios. Mice expressing a cleavage-resistant cFLIP mutant,CflipD377A, exhibited increased sensitivity to SARS-CoV-induced lethality, impaired skin wound healing and increased tissue damage caused bySharpindeficiency.In vitro, abrogation of cFLIP cleavage sensitizes cells to TNF-induced necroptosis and apoptosis by favoring complex-II formation. Mechanistically, the cell death-sensitizing effect of the D377A mutation depends on Gln(Q)469. These results reveal a crucial role for cFLIP cleavage in controlling the amplitude of cell death responses occurring upon tissue stress, to ensure the execution of repair programs.

SUMMARYThe cytotoxic proteases Granzymes A (GzmA) and B (GzmB) are important components of the arsenal used by cytotoxic immune cells to kill virally infected/damaged cells. Until now, there has been limited evidence that GzmA/B proteins contribute to combating intracellular bacterial pathogens in mammals. Here, we find that the route of infection of the intracellular bacterial pathogen Salmonella enterica serovar Typhimurium reveals the distinct roles that Granzymes play in defending against bacterial infection. We used Gzma-/-Gzmb-/- mice to discover that Granzymes are required to protect mice against oral infection with Salmonella. However, Granzymes do not play a role in systemic infection. We investigated the tissue-specific expression of Granzymes and determined that intestinal intraepithelial lymphocytes (IEL) are the only cell types that express Granzymes in healthy non-infected mice. In fact, IEL are sufficient to mediate the protective effects of Granzymes against Salmonella infection. Intriguingly, we found that GzmA and GzmB play opposing roles in Salmonella control, with GzmA being protective against infection whilst GzmB promoted infection. Both GzmA and GzmB proteins functioned independently of the pore-forming molecule Perforin, suggesting extracellular action. Our study reveals that IEL-expressed Granzymes play significant and distinct functions in host defense from oral bacterial infection.

BackgroundTumor-associated macrophages (TAMs) may both promote and suppress tumor development. Therefore, molecules that are able to activate and repolarize these cells into a tumoricidal phenotype could be of great interest as a new strategy for cancer immunotherapy. Fungal β-glucans have been suggested as a promising way of activating TAMs, but most of the research has been carried out on particulate β-glucans of large sizes (> 500 kDa), which potentially have different biological properties than smaller, water-soluble molecules with similar structures.MethodsBone marrow-derived mouse macrophages were treated with 6 different purified polysaccharides isolated from the medicinal fungus Inonotus obliquus. Nitric oxide concentration was quantified using the Griess assay and by qPCR of iNOS mRNA. IL-6 and TNF-α concentrations were quantified using Luminex ELISA technology (using human monocyte-derived macrophages and mouse bone-marrow derived macrophages). Growth inhibition of cancer cells was measured using radiolabeled thymidine. Receptor interaction was determined using HEK-Blue™ reporter cell lines and TLR4 KO macrophages.Main findingsThe acidic, water-soluble polysaccharides AcF1, AcF2 and AcF3 induced nitric oxide (NO) production by mouse macrophages when combined with IFN-γ, leading to a strong subsequent tumoricidal activity by the macrophages. Tumoricidal activity of AcF1 and AcF3 was fully retained in TLR4 knockout macrophages, demonstrating that the macrophage activation was not dependent on TLR4. Further, AcF3 induced high levels of the pro-inflammatory cytokines IL-6 and TNF-α in human and mouse macrophages, independent of co-activation with IFN-γ. The polysaccharides were shown to bind TLR2, TLR4 and Dectin-1a to varying degrees, and these receptors were likely to be responsible for the macrophage activation. The acidic polysaccharides AcF1, AcF2 and AcF3 strongly activated TLR2, while AcF3 and AcF1 activated TLR4. The acidic polysaccharides had low affinity to Dectin-1a compared to the polysaccharides IWN, EWN and A1, which suggests that this receptor is not the main receptor for the pro-inflammatory activity observed.ConclusionFor the first time, this study demonstrates that I. obliquus polysaccharides are able to bind multiple pattern recognition receptors to activate macrophages into a pro-inflammatory anti-tumor phenotype. The induction of tumoricidal activity in the macrophages as well as the interaction with TLR2, TLR4 and Dectin-1a suggest that the I. obliquus polysaccharides may have unique ways of interacting with macrophages, which could open up for new treatment options in cancer immunotherapy.