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An immunohistochemical atlas of necroptotic pathway expression
Shene Chiou1,2, Aysha H Al-Ani1,2,3, Yi Pan1
1Walter and Eliza Hall Institute of Medical Research, Parkville, Australia.
Abstract:
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.
Insights
New immunohistochemistry protocols enable detection of necroptosis regulators (Caspase-8, RIPK1, RIPK3, MLKL) in tissues. This reveals immune barrier cells express necroptotic effectors, aiding disease research.
Area of Science:
- Cell Biology
- Immunology
- Pathology
Background:
- Necroptosis, a regulated cell death, is implicated in inflammatory and ischemic diseases.
- Identifying necroptotic cells in vivo is challenging due to a lack of reliable detection methods.
Purpose of the Study:
- To develop and validate automated immunohistochemistry protocols for detecting key necroptosis regulators (Caspase-8, RIPK1, RIPK3, MLKL).
- To investigate the in vivo expression patterns of these regulators in various mouse and human tissues.
Main Methods:
- Automated immunohistochemistry protocols were established for formalin-fixed tissues.
- Detection of Caspase-8, RIPK1, RIPK3, and MLKL proteins was performed on mouse and human tissue samples.
Main Results:
- Significant heterogeneity in necroptosis effector protein expression was observed within tissues.
- Short-lived immune barrier cells showed high expression of necroptotic effectors, unlike long-lived cells.
- Expression levels of these proteins changed locally in response to inflammation, dysbiosis, or immune challenges.
Conclusions:
- The developed protocols enable precise localization and evaluation of necroptosis signaling in vivo.
- Necroptosis dysregulation is associated with various disease contexts.
- Immune barrier cells are key sites of necroptotic effector expression.
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