The importance of murine phospho-MLKL-S345 in situ detection for necroptosis assessment in vivo.

Necroptosis is a caspase-independent modality of cell death implicated in many inflammatory pathologies. The execution of this pathway requires the formation of a cytosolic platform that comprises RIPK1 and RIPK3 which, in turn, mediates the phosphorylation of the pseudokinase MLKL (S345 in mouse). The activation of this executioner is followed by its oligomerisation and accumulation at the plasma-membrane where it leads to cell death via plasma-membrane destabilisation and consequent permeabilisation. While the biochemical and cellular characterisation of these events have been amply investigated, the study of necroptosis involvement in vivo in animal models is currently limited to the use of Mlkl-/- or Ripk3-/- mice. Yet, even in many of the models in which the involvement of necroptosis in disease aetiology has been genetically demonstrated, the fundamental in vivo characterisation regarding the question as to which tissue(s) and specific cell type(s) therein is/are affected by the pathogenic necroptotic death are missing. Here, we describe and validate an immunohistochemistry and immunofluorescence-based method to reliably detect the phosphorylation of mouse MLKL at serine 345 (pMLKL-S345). We first validate the method using tissues derived from mice in which Caspase-8 (Casp8) or FADD are specifically deleted from keratinocytes, or intestinal epithelial cells, respectively. We next demonstrate the presence of necroptotic activation in the lungs of SARS-CoV-infected mice and in the skin and spleen of mice bearing a Sharpin inactivating mutation. Finally, we exclude necroptosis occurrence in the intestines of mice subjected to TNF-induced septic shock. Importantly, by directly comparing the staining of pMLKL-345 with that of cleaved Caspase-3 staining in some of these models, we identify spatio-temporal and functional differences between necroptosis and apoptosis supporting a role of RIPK3 in inflammation independently of MLKL versus the role of RIPK3 in activation of necroptosis.

Identification of FasL as a crucial host factor driving COVID-19 pathology and lethality.

The dysregulated immune response and inflammation resulting in severe COVID-19 are still incompletely understood. Having recently determined that aberrant death-ligand-induced cell death can cause lethal inflammation, we hypothesized that this process might also cause or contribute to inflammatory disease and lung failure following SARS-CoV-2 infection. To test this hypothesis, we developed a novel mouse-adapted SARS-CoV-2 model (MA20) that recapitulates key pathological features of COVID-19. Concomitantly with occurrence of cell death and inflammation, FasL expression was significantly increased on inflammatory monocytic macrophages and NK cells in the lungs of MA20-infected mice. Importantly, therapeutic FasL inhibition markedly increased survival of both, young and old MA20-infected mice coincident with substantially reduced cell death and inflammation in their lungs. Intriguingly, FasL was also increased in the bronchoalveolar lavage fluid of critically-ill COVID-19 patients. Together, these results identify FasL as a crucial host factor driving the immuno-pathology that underlies COVID-19 severity and lethality, and imply that patients with severe COVID-19 may significantly benefit from therapeutic inhibition of FasL.

Zebrafish Xenografts Unveil Sensitivity to Olaparib beyond BRCA Status.

Poly (ADP-ribose) polymerase (PARP) inhibition in BRCA-mutated cells results in an incapacity to repair DNA damage, leading to cell death caused by synthetic lethality. Within the treatment options for advanced triple negative breast cancer, the PARP inhibitor olaparib is only given to patients with BRCA1/2 mutations. However, these patients may show resistance to this drug and BRCA1/2 wild-type tumors can show a striking sensitivity, making BRCA status a poor biomarker for treatment choice. Aiming to investigate if the zebrafish model can discriminate sensitivities to olaparib, we developed zebrafish xenografts with different BRCA status and measured tumor response to treatment, as well as its impact on angiogenesis and metastasis. When challenged with olaparib, xenografts revealed sensitivity phenotypes independent of BRCA. Moreover, its combination with ionizing radiation increased the cytotoxic effects, showing potential as a combinatorial regimen. In conclusion, we show that the zebrafish xenograft model may be used as a sensitivity profiling platform for olaparib in monotherapy or in combinatorial regimens. Hence, this model presents as a promising option for the future establishment of patient-derived xenografts for personalized medicine approaches beyond BRCA status.