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MLKL is the essential effector of necroptosis, a form of programmed lytic cell death. We have isolated a mouse strain with a single missense mutation, MlklD139V, that alters the two-helix ‘brace’ that connects the killer four-helix bundle and regulatory pseudokinase domains. This confers constitutive, RIPK3 independent killing activity to MLKL. Homozygous mutant mice develop lethal postnatal inflammation of the salivary glands and mediastinum. The normal embryonic development of MlklD139V homozygotes until birth, and the absence of any overt phenotype in heterozygotes provides important in vivo precedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common human MLKL polymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, chronic recurrent multifocal osteomyelitis (CRMO)., Necroptosis is a regulated form of inflammatory cell death driven by activated MLKL. Here, the authors identify a mutation in the brace region that confers constitutive activation, leading to lethal inflammation in homozygous mutant mice and providing insight into human mutations in this region.

Background Tranexamic acid (TXA) is an antifibrinolytic agent frequently used in elective surgery to reduce blood loss. We recently found it also acts as a potent immune-modulator in patients undergoing cardiac surgery. Methods Patients undergoing lower limb surgery were enrolled into the “Tranexamic Acid in Lower Limb Arthroplasty” (TALLAS) pilot study. The cellular immune response was characterised longitudinally pre- and post-operatively using full blood examination (FBE) and comprehensive immune cell phenotyping by flowcytometry. Red blood cells and platelets were determined in the FBE and levels of T cell cytokines and the plasmin-antiplasmin complex determined using ELISA. Results TXA administration increased the proportion of circulating CD141+ conventional dendritic cells (cDC) on post-operative day (POD) 3. It also reduced the expression of CD83 and TNFR2 on classical monocytes and levels of circulating IL-10 at the end of surgery (EOS) time point, whilst increasing the expression of CCR4 on natural killer (NK) cells at EOS, and reducing TNFR2 on POD-3 on NK cells. Red blood cells and platelets were decreased to a lower extent at POD-1 in the TXA group, representing reduced blood loss. Conclusion In this investigation we have extended our examination on the immunomodulatory effects of TXA in surgery by also characterising the end of surgery time point and including B cells and neutrophils in our immune analysis, elucidating new immunophenotypic changes in phagocytes as well as NK cells. This study enhances our understanding of TXA-mediated effects on the haemostatic and immune response in surgery, validating changes in important functional immune cell subsets in orthopaedic patients.

Mixed lineage kinase domain‐like (MLKL) is the executioner in the caspase‐independent form of programmed cell death called necroptosis. Receptor‐interacting serine/threonine protein kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor the significance of this event has been demonstrated. Here, we show that necroptosis‐specific multi‐mono‐ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin‐insoluble cell fraction comprising organellar and plasma membranes and protein aggregates. Appearance of this ubiquitylated MLKL form can be reduced by expression of a plasma membrane‐located deubiquitylating enzyme. Oligomerization‐induced MLKL ubiquitylation occurs on at least four separate lysine residues and correlates with its proteasome‐ and lysosome‐dependent turnover. Using a MLKL‐DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto‐activation independent of necroptosis signalling in mouse and human cells. Therefore, in addition to the role of ubiquitylation in the kinetic regulation of MLKL‐induced death following an exogenous necroptotic stimulus, it also contributes to restraining basal levels of activated MLKL to avoid unwanted cell death.

Mixed lineage kinase domain-like (MLKL) is the executioner in the caspase-independent form of programmed cell death called necroptosis. Receptor Interacting serine/threonine Protein Kinase 3 (RIPK3) phosphorylates MLKL, triggering MLKL oligomerization, membrane translocation and membrane disruption. MLKL also undergoes ubiquitylation during necroptosis, yet neither the mechanism nor significance of this event have been demonstrated. Here we show that necroptosis-specific, multi-mono-ubiquitylation of MLKL occurs following its activation and oligomerization. Ubiquitylated MLKL accumulates in a digitonin insoluble cell fraction comprising plasma/organellar membranes and protein aggregates. This ubiquitylated form is diminished by a plasma membrane located deubiquitylating enzyme. MLKL is ubiquitylated on at least 4 separate lysine residues once oligomerized, and this correlates with proteasome- and lysosome-dependent turnover. Using a MLKL-DUB fusion strategy, we show that constitutive removal of ubiquitin from MLKL licences MLKL auto-activity independent of necroptosis signalling in mouse and human cells. Therefore, besides its role in the kinetic regulation of MLKL-induced death following an exogenous necroptotic stimulus, ubiquitylation also contributes to the restraint of basal levels of activated MLKL to avoid errant cell death.

Background: Tissue-type plasminogen activator (t-PA) has been the mainstay of therapeutic thrombolysis for patients with acute ischaemic stroke (AIS). However, t-PA can cause devastating intracerebral hemorrhage. t-PA can also influence the CNS in part by modulation of BBB permeability. Complement activation also occurs after AIS and has also been reported to increase BBB permeability. The complement components, C3 and C5, can also be activated by t-PA via plasmin formation and cell intrinsic complement may be involved in this process. Tenecteplase (TNK-tPA) is a t-PA variant with a longer plasma half-life, yet the ability of TNK-tPA to modulate the BBB and complement is less clear.Aim: To evaluate the effect of C5 and C5a-receptor 1 (C5aR1) inhibitors on t-PA- and TNK-tPA-mediated opening of the BBB.Methods: We used an in vitro model of the BBB where human brain endothelial cells and human astrocytes were co-cultured on the opposite sides of a porous membrane assembled in transwell inserts. The luminal (endothelial) compartment was stimulated with t-PA or TNK-tPA together with plasminogen, in the presence of PMX205 (a non-competitive C5aR1 antagonist), Avacopan (a competitive C5aR1 antagonist) or Eculizumab (a humanized monoclonal inhibitor of human C5). BBB permeability was assessed 5 and 24 h later. Immunofluorescence was also used to detect changes in C5 and C5aR1 expression in endothelial cells and astrocytes.Results: PMX205, but not Avacopan or Eculizumab, blocked t-PA-mediated increase in BBB permeability at both the 5 and 24 h time points. PMX205 also blocked TNK-tPA-mediated increase in BBB permeability. Immunofluorescence analysis revealed intracellular staining of C5 in both cell types. C5aR1 expression was also detected on the cell surfaces and also located intracellularly in both cell types.Conclusion: t-PA and TNK-tPA-mediated increase in BBB permeability involves C5aR1 receptor activation from cell-derived C5a. Selective inhibitors of C5aR1 may have therapeutic potential in AIS.

Background Tranexamic acid (TXA) is an anti-fibrinolytic agent used to reduce bleeding in various conditions including traumatic brain injury (TBI). As the fibrinolytic system also influences the central nervous system and the immune response, TXA may also modulate these parameters following TBI. Objectives To determine the effect of TXA on blood-brain barrier (BBB) integrity and changes in immune and motor function in male and female mice subjected to TBI. Methods Wild-type and plasminogen deficient (plg-/-) mice were subjected to TBI then administered either TXA/vehicle. The degree of BBB breakdown, intracerebral hemorrhage (ICH), motor dysfunction, and changes in inflammatory subsets in blood and brain were determined. Results and conclusions Tranexamic acid significantly reduced BBB breakdown, and increased blood neutrophils in male mice 3 hours post-TBI. In contrast, TXA treatment of female mice increased BBB permeability and ICH but had no effect on blood neutrophils at the same time-point. TXA improved motor function in male mice but still increased BBB breakdown in female mice 24 hours post-TBI. Brain urokinase-type plasminogen activator (u-PA) antigen and activity levels were significantly higher in injured females compared to males. Because TXA can promote a pro-fibrinolytic effect via u-PA, these sex differences may be related to brain u-PA levels. TXA also increased monocyte subsets and dendritic cells in the injured brain of wild-type male mice 1 week post-TBI. Plg-/- mice of both sexes had reduced BBB damage and were protected from TBI irrespective of treatment indicating that TXA modulation of the BBB is plasmin-dependent. In conclusion, TXA is protective post-TBI but only in male mice.

T cell mediated hypersensitivity to nickel (Ni2+) is one of the most common causes of allergic contact dermatitis. Ni2+ sensitization may also contribute to the failure of Ni2+ containing joint implants, and revision to non-Ni2+ containing hardware can be costly and debilitating. Previously, we identified Ni2+ mimotope peptides, which are reactive to a CD4+ T cell clone, ANi2.3 (Vα1, Vβ17), isolated from a Ni2+ hypersensitive patient with contact dermatitis. This T cell is restricted to the major histocompatibility complex class II (MHCII) molecule, Human Leukocyte Antigen (HLA)-DR52c (DRA, DRB3*0301). However, it is not known if Ni2+ induced T cell responses in sensitized joint replacement failure patients are similar to subjects with Ni2+ induced contact dermatitis. Here, we generated DR52c/Ni2+ mimotope tetramers, and used them to test if the same Ni2+ T cell activation mechanism could be generalized to Ni2+ sensitized patients with associated joint implant failure. We confirmed the specificity of these tetramers by staining of ANi2.3T cell transfectomas. The DR52c/Ni2+ mimotope tetramer detected Ni2+ reactive CD4+ T cells in the peripheral blood mononuclear cells (PBMC) of patients identified as Ni2+ sensitized by patch testing and a positive Ni2+ LPT. When HLA-typed by a DR52 specific antibody, three out of four patients were DR52 positive. In one patient, Ni2+ stimulation induced the expansion of Vβ17 positive CD4+ T cells from 0.8% to 13.3%. We found that the percentage of DR52 positivity and Vβ17 usage in Ni2+ sensitized joint failure patients are similar to Ni sensitized skin allergy patients. Ni2+ independent mimotope tetramers may be a useful tool to identify the Ni2+ reactive CD4+ T cells.

SUMMARYWe have isolated a mouse strain with a single missense mutation in the gene encoding MLKL, the essential effector of necroptotic cell death. The resulting substitution lies within the two-helix ‘brace’ and confers constitutive, RIPK3 independent, killing activity to MLKL. Mice homozygous forMlklD139Vdevelop lethal inflammation within days of birth, implicating the salivary glands and pericardium as hotspots for necroptosis and inflammatory infiltration. The normal development ofMlklD139Vhomozygotes until birth, and the absence of any overt phenotype in heterozygotes provides importantin vivoprecedent for the capacity of cells to clear activated MLKL. These observations offer an important insight into the potential disease-modulating roles of three common humanMLKLpolymorphisms that encode amino acid substitutions within or adjacent to the brace region. Compound heterozygosity of these variants is found at up to 12-fold the expected frequency in patients that suffer from a pediatric autoinflammatory disease, CRMO.

Necroptosis is a caspase-independent form of programmed cell death that is induced by a variety of different signalling cascades-all culminating in the activation of the pseudokinase mixed lineage kinase domain-like (MLKL). TNF-induced necroptosis is the most intensively studied of these pathways. Here we describe reagents and cell-based techniques that can be used to investigate TNF-mediated necroptosis in the lab.

Necroptosis is a caspase-independent form of programmed cell death that is induced by a variety of different signalling cascades-all culminating in the activation of the pseudokinase mixed lineage kinase domain-like (MLKL). TNF-induced necroptosis is the most intensively studied of these pathways. Here we describe reagents and cell-based techniques that can be used to investigate TNF-mediated necroptosis in the lab.

T cell mediated hypersensitivity to nickel (Ni2+) is one of the most common causes of allergic contact dermatitis. Ni2+ sensitization may also contribute to the failure of Ni2+ containing joint implants, and revision to non-Ni2+ containing hardware can be costly and debilitating. Previously, we identified Ni2+ mimotope peptides, which are reactive to a CD4+ T cell clone, ANi2.3 (Vα1, Vβ17), isolated from a Ni2+ hypersensitive patient with contact dermatitis. This T cell is restricted to major histocompatibility complex class II (MHCII) molecule, Human Leukocyte Antigen (HLA)-DR52c (DRA, DRB3*0301). However, it is not known if Ni2+ induced T cell responses in sensitized joint replacement failure patients are similar to subjects with Ni2+ induced contact dermatitis. Here, we generated DR52c/Ni2+ mimotope tetramers, and used them to test if the same Ni2+ T cell activation mechanism could be generalized to Ni2+ sensitized patients with associated joint implant failure. We confirmed the specificity of these tetramers by staining of ANi2.3 T cell transfectomas. The DR52c/Ni2+ mimotope tetramer detected Ni2+ reactive CD4+ T cells in the peripheral blood mononuclear cells (PBMC) of patients identified as Ni2+ sensitized by patch testing and/or a positive Ni2+ LPT. When HLA-typed by a DR52 specific antibody, three out of four patients were DR52 positive. In one patient, Ni2+ stimulation induced the expansion of Vβ17 positive CD4+ T cells from 0.8% to 13.3%. We found that the percentage of DR52 positivity and Vβ17 usage in Ni2+ sensitized joint failure patients are similar to Ni sensitized skin allergy patients. Ni2+ independent mimotope tetramers may be a useful tool to identify the Ni2+ reactive CD4+ T cells.

Necroptosis is a caspase-independent form of regulated cell death that has been implicated in the development of a range of inflammatory, autoimmune and neurodegenerative diseases. The pseudokinase, Mixed Lineage Kinase Domain-Like (MLKL), is the most terminal known obligatory effector in the necroptosis pathway, and is activated following phosphorylation by Receptor Interacting Protein Kinase-3 (RIPK3). Activated MLKL translocates to membranes, leading to membrane destabilisation and subsequent cell death. However, the molecular interactions governing the processes downstream of RIPK3 activation remain poorly defined. Using a phenotypic screen, we identified seven heat-shock protein 90 (HSP90) inhibitors that inhibited necroptosis in both wild-type fibroblasts and fibroblasts expressing an activated mutant of MLKL. We observed a modest reduction in MLKL protein levels in human and murine cells following HSP90 inhibition, which was only apparent after 15 h of treatment. The delayed reduction in MLKL protein abundance was unlikely to completely account for defective necroptosis, and, consistent with this, we also found inhibition of HSP90 blocked membrane translocation of activated MLKL. Together, these findings implicate HSP90 as a modulator of necroptosis at the level of MLKL, a function that complements HSP90’s previously demonstrated modulation of the upstream necroptosis effector kinases, RIPK1 and RIPK3.