biomolecules Review The Role of the Key Effector of Necroptotic Cell Death, MLKL, in Mouse Models of Disease Emma C. Tovey Crutchfield 1,2,3 , Sarah E. Garnish 2,3 and Joanne M. Hildebrand 2,3,* 1 Department of Medical Education, University of Melbourne, Parkville, VIC 3052, Australia; [email protected] 2 The Walter and Eliza Hall Institute of Medical Research, Parkville, VIC 3052, Australia; [email protected] 3 Department of Medical Biology, University of Melbourne, Parkville, VIC 3052, Australia * Correspondence: [email protected] Abstract: Necroptosis is an inflammatory form of lytic programmed cell death that is thought to have evolved to defend against pathogens. Genetic deletion of the terminal effector protein—MLKL— shows no overt phenotype in the C57BL/6 mouse strain under conventional laboratory housing conditions. Small molecules that inhibit necroptosis by targeting the kinase activity of RIPK1, one of the main upstream conduits to MLKL activation, have shown promise in several murine models of non-infectious disease and in phase II human clinical trials. This has triggered in excess of one billion dollars (USD) in investment into the emerging class of necroptosis blocking drugs, and the potential utility of targeting the terminal effector is being closely scrutinised. Here we review murine models of disease, both genetic deletion and mutation, that investigate the role of MLKL. We summarize a series of examples from several broad disease categories including ischemia reperfusion injury, Citation: Tovey Crutchfield, E.C.; sterile inflammation, pathogen infection and hematological stress. Elucidating MLKL’s contribution Garnish, S.E.; Hildebrand, J.M. The to mouse models of disease is an important first step to identify human indications that stand to Role of the Key Effector of Necroptotic Cell Death, MLKL, in benefit most from MLKL-targeted drug therapies. Mouse Models of Disease. Biomolecules 2021, 11, 803. https:// Keywords: necroptosis; MLKL; programmed cell death doi.org/10.3390/biom11060803 Academic Editors: Ivan Poon, Georgia Atkin-Smith, Rochelle Tixeira 1. Introduction and Amy A. Baxter Necroptosis and Disease Mixed Lineage Kinase Domain-Like (MLKL) was shown to be the essential effector of a Received: 1 May 2021 pro-inflammatory, lytic form of programmed cell death called necroptosis in 2012 [1,2]. Like Accepted: 24 May 2021 other forms of lytic cell death (e.g., pyroptosis), necroptosis is characterised by the release Published: 28 May 2021 of Damage-Associated Molecular Patterns (DAMPs) and IL-33, IL-1α and IL-1β production, as recently reviewed by [3]. Unlike apoptosis, necroptosis and the canonical signalling Publisher’s Note: MDPI stays neutral pathway RIPK1-RIPK3-MLKL are not essential to the development and homeostasis of with regard to jurisdictional claims in multicellular organisms [4]. The two most downstream effectors of necroptosis, MLKL and published maps and institutional affil- iations. its obligate activating kinase RIPK3, can be deleted at the genetic level in laboratory mice without any overt developmental consequences in the absence of challenge [5–7]. Thus, it is widely held that MLKL and necroptosis have evolved primarily to defend against pathogenic insult to cells and tissues. This important role of necroptosis in pathogen defence is written in the DNA of many bacteria and viruses alike, which together encode Copyright: © 2021 by the authors. several genes that disarm different facets of the necroptotic signalling pathway [8–10]. Licensee MDPI, Basel, Switzerland. In evolution, genetic deletion of MLKL and/or RIPK3 in the ancestors of modern day This article is an open access article carnivora, metatheria (marsupials) and aves (birds) show that complex vertebrates can distributed under the terms and conditions of the Creative Commons survive without necroptosis when faced with infectious challenges of the ‘real world’ [11]. Attribution (CC BY) license (https:// This adds to the precedent for the well-honed flexibility, redundancy and co-operation creativecommons.org/licenses/by/ of different programmed cell death pathways in the defence against pathogens [12] and 4.0/). offers some biological guide that inhibiting MLKL pharmacologically in humans would not Biomolecules 2021, 11, 803. https://doi.org/10.3390/biom11060803 https://www.mdpi.com/journal/biomolecules Biomolecules 2021, 11, 803 2 of 21 Biomolecules 2021, 11, 803 2 of 22 [12] and offers some biological guide that inhibiting MLKL pharmacologically in humans would not compromise pathogenic defence. Interestingly, recent studies suggest that the suppression of necroptosis may even reduce the resulting inflammatory response that is oftencompromise more dangerous pathogenic than defence. the infection Interestingly, itself [13]. recent Mlkl studies gene suggest knockout that (abbreviated the suppression to bothof necroptosis Mlkl−/− and Mlkl may KO) even mice reduce are distinguishable the resulting inflammatory from wild-type response mice in that numerous is often mod- more −/− elsdangerous of disease, than a broad the infection sampling itself of which [13]. Mlkl are genepresented knockout (Table (abbreviated 1) and illustrated to both (FigureMlkl 1)and here.Mlkl KO) mice are distinguishable from wild-type mice in numerous models of disease, a broadThe therapeutic sampling of potential which are for presented necroptosis-targ (Table1)eted and drugs illustrated lies largely (Figure in1 non-infectious) here. indications.The therapeutic The first human potential clinical for necroptosis-targeted trials of RIPK1-targetted drugs small lies largely molecule in non-infectious compounds wereindications. conducted The in first cohorts human of rheumatoid clinical trials arthri of RIPK1-targettedtis, ulcerative colitis small and molecule psoriasis compounds patients [14].were While conducted these showed in cohorts some of promise rheumatoid in phase arthritis, II, these ulcerative were returned colitis and to the psoriasis research pa- tients [14]. While these showed some promise in phase II, these were returned to the phase in late 2019 by their licensee GlaxoSmithKline [15]. At the time of writing, there are research phase in late 2019 by their licensee GlaxoSmithKline [15]. At the time of writing, only three active clinical trials in progress: a phase I trial assessing the safety of a new there are only three active clinical trials in progress: a phase I trial assessing the safety RIPK1 inhibitor (GFH312), a phase II study utilising RIPK1-binding compound, of a new RIPK1 inhibitor (GFH312), a phase II study utilising RIPK1-binding compound, SAR443122, in cutaneous lupus erythematosus patients and a phase I/II study utilising SAR443122, in cutaneous lupus erythematosus patients and a phase I/II study utilising RIPK1 inhibitor GSK2982772 in psoriasis [16]. There are currently no ‘first in human’ trials RIPK1 inhibitor GSK2982772 in psoriasis [16]. There are currently no ‘first in human’ trials of RIPK3- or MLKL-binding compounds listed on http://clinicaltrials.gov (accessed on 28 of RIPK3- or MLKL-binding compounds listed on http://clinicaltrials.gov (accessed on May 21). 28 May 2021). Neurological Sciatic nerve injury Focal cortical ischemia Autoimmune encephalomyelitis CPZ-demyelination Salivary glands Parkinson’s disease Focal inflammation1 Hematological Musculoskeletal Casp8-/- & Fadd-/- Acute muscle injury lymphadenopathy & autoimmunity 1 MYC-KO Arthritis (A20 ) Lymphoproliferative disease1 Prolonged bleeding time VTE Hematopoietic defects 1 Cardiac remodelling, Hepatic IAV convalescence IRI Respiratory Asymptomatic S.pneumoniae S.marcescens1 IAV, lethal dose1 Renal Secondary S.pneumoniae to IAV Cholesterol crystal embolism IRI1 AKI (sodium oxalate) AKI (cisplatin driven) 1 ANCA vasculitis Gastrointestinal L.monocytogenes 1 Acute colitis (DSS) 1 S.enterica Metabolic Colitis, ileitis Diet-induced obesity 1 (FaddIEC-KO, Casp8IEC-KO, Tsc1IEC-KO, cFLIPs)1 Atherosclerosis Pancreatitis (cerulein) 1 NAFLD (high fat) NAFLD (FFC diet)1 Systemic inflammation Casp8D387A/D387A S.aureus sepsis1 TNFα, high dose1 Skin SHARPIN deficiency Subcutaneous S.aureus Lethal neonatal Inflammation (Ripk1EKO)1 inflammatory syndrome (MlklD139V)1 Necroptosis protects against disease. Necroptosis contributes to disease. FigureFigure 1. 1. TheThe role role of of necroptosis necroptosis in in infective infective and and non-in non-infectivefective challenges challenges spanning spanning a awide wide array array of of 1 physiologicalphysiological systems. systems. Littermate1 Littermate controls controls utilised. utilised. Biomolecules 2021, 11, 803 3 of 22 Table 1. Understanding the role of necroptosis in the aetiology of disease using Mlkl−/− and mutant mice. −/− −/− Challenge Method Outcome in Mlkl (Mutant or Knock-Down) Mouse Relative to Mlkl Mouse and Wild-Type Control Reference Wild-Type Control Details Ischemia and reperfusion injury (IRI) −/− Reduced TUNEL positive hepatocytes and serum ALT levels in Mlkl −/− Hepatic IRI 45 min ischemia in chow mice, irrespective of diet. Hepatic neutrophil infiltration and TNF, IL-1, Sex-matched Mlkl (CRISPR-Cas9) and [17] or Western diet fed mice. Wt mice utilised. MIP-2, and IL-6 mRNA reduced post I/R in Mlkl−/− mice. −/− Renal Cholesterol crystal Mlkl mice protected from infarction (measured by infarct size, kidney infarction, no −/− [18] embolism. injury and neutrophil infiltration). No difference in the extent of acute Mlkl [5] and Wt mice utilised. reperfusion kidney injury (measured by eGFR) and kidney