An immunohistochemical atlas of necroptotic pathway expression

Necroptosis is a lytic form of regulated cell death reported to contribute to inflammatory diseases of the gut, skin and lung, as well as ischemic-reperfusion injuries of the kidney, heart and brain. However, precise identification of the cells and tissues that undergo necroptotic cell death in vivo has proven challenging in the absence of robust protocols for immunohistochemical detection. Here, we provide automated immunohistochemistry protocols to detect core necroptosis regulators – Caspase-8, RIPK1, RIPK3 and MLKL – in formalin-fixed mouse and human tissues. We observed surprising heterogeneity in protein expression within tissues, whereby short-lived immune barrier cells were replete with necroptotic effectors, whereas long-lived cells lacked RIPK3 or MLKL expression. Local changes in the expression of necroptotic effectors occurred in response to insults such as inflammation, dysbiosis or immune challenge, consistent with necroptosis being dysregulated in disease contexts. These methods will facilitate the precise localisation and evaluation of necroptotic signaling in vivo . Highlights ### Competing Interest Statement KMP, SNY, AH, AL, PG, CRH, JD, IPW, JMH, JS, JMM and ALS have contributed to the development of necroptosis pathway inhibitors in collaboration with Anaxis Pharma Pty Ltd. YP is a co-holder of patent for material arising from this study. All other authors have no additional financial interests. * C8 : Caspase-8 C10 : Caspase-10 cl. C3 : cleaved Caspase-3 TNF : Tumour Necrosis Factor RIPK1 : Receptor-interacting serine/threonine-protein kinase-1 pRIPK1 : phosphorylated RIPK1 RIPK3 : Receptor-interacting serine/threonine-protein kinase-3 pRIPK3 : phosphorylated RIPK3 MLKL : Mixed lineage kinase domain-like protein pMLKL : phosphorylated MLKL R1 : Receptor-interacting serine/threonine-protein kinase-1 R3 : Receptor-interacting serine/threonine-protein kinase-1 ML : Mixed lineage kinase domain-like protein Nec1s : Necrostatin1s WEHI : Walter and Eliza Hall Institute of Medical Research WT : wild-type KO : knockout EV : Expanded View IBD : inflammatory bowel disease UC : ulcerative colitis CD : Crohn’s disease SMA : smooth muscle actin Kpf : Kupffer cell endo : endothelial cell p.o. : oral administration i.p. : intraperitoneal administration CV : central vein PV : portal vein BD : bile duct CA : splenic central artery WP : splenic white pulp MZ : splenic marginal zone RP : splenic red pulp CRP : C-reactive protein TS : Tumour Necrosis Factor and Smac mimetic TSI : Tumour Necrosis Factor and Smac mimetic and IDN-6556 Ab : antibody UMAP : Uniform Manifold Approximation and Projection NP-KLH : 4-hydroxy-3-nitrophenylacetyl hapten conjugated to keyhole limpet hemocyanin CDS : coding sequence