The importance of murine phospho-MLKL-S345 in situ detection for necroptosis assessment in vivo
Abstract
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.
Read more at Cell Death Differ, 2024 May, 21