RIPK1 regulates RIPK3-MLKL-driven systemic inflammation and emergency hematopoiesis.

Upon ligand binding, RIPK1 is recruited to tumor necrosis factor receptor superfamily (TNFRSF) and Toll-like receptor (TLR) complexes promoting prosurvival and inflammatory signaling. RIPK1 also directly regulates caspase-8-mediated apoptosis or, if caspase-8 activity is blocked, RIPK3-MLKL-dependent necroptosis. We show that C57BL/6 Ripk1(-/-) mice die at birth of systemic inflammation that was not transferable by the hematopoietic compartment. However, Ripk1(-/-) progenitors failed to engraft lethally irradiated hosts properly. Blocking TNF reversed this defect in emergency hematopoiesis but, surprisingly, Tnfr1 deficiency did not prevent inflammation in Ripk1(-/-) neonates. Deletion of Ripk3 or Mlkl, but not Casp8, prevented extracellular release of the necroptotic DAMP, IL-33, and reduced Myd88-dependent inflammation. Reduced inflammation in the Ripk1(-/-)Ripk3(-/-), Ripk1(-/-)Mlkl(-/-), and Ripk1(-/-)Myd88(-/-) mice prevented neonatal lethality, but only Ripk1(-/-)Ripk3(-/-)Casp8(-/-) mice survived past weaning. These results reveal a key function for RIPK1 in inhibiting necroptosis and, thereby, a role in limiting, not only promoting, inflammation.

The diverse role of RIP kinases in necroptosis and inflammation.

Inflammation is a healthy response to infection or danger and should be rapid, specific and terminated once the threat has passed. Inflammatory diseases, where this regulation fails, cause considerable human suffering. Treatments have successfully targeted pro-inflammatory cytokines, such as tumor-necrosis factor (TNF), that directly induce genes encoding inflammatory products. Inflammatory signals, including TNF, may also directly induce caspase-independent cell death (necroptosis), which can also elicit inflammation. Necroptosis was originally defined as being dependent on the kinase RIPK1 but is now known to be dependent on RIPK3 and the pseudo-kinase MLKL. Therefore, RIPK1, RIPK3 and MLKL are potential therapeutic targets. RIPK1 and RIPK3 also directly regulate inflammatory signaling, which complicates interpretation of their function but might alter their therapeutic utility. This Review examines the role of cell death, particularly necroptosis, in inflammation, in the context of recent insights into the roles of the key necroptosis effector molecules RIPK1, RIPK3 and MLKL.

LUBAC is essential for embryogenesis by preventing cell death and enabling haematopoiesis.

The linear ubiquitin chain assembly complex (LUBAC) is required for optimal gene activation and prevention of cell death upon activation of immune receptors, including TNFR1 1 . Deficiency in the LUBAC components SHARPIN or HOIP in mice results in severe inflammation in adulthood or embryonic lethality, respectively, owing to deregulation of TNFR1-mediated cell death2-8. In humans, deficiency in the third LUBAC component HOIL-1 causes autoimmunity and inflammatory disease, similar to HOIP deficiency, whereas HOIL-1 deficiency in mice was reported to cause no overt phenotype9-11. Here we show, by creating HOIL-1-deficient mice, that HOIL-1 is as essential for LUBAC function as HOIP, albeit for different reasons: whereas HOIP is the catalytically active component of LUBAC, HOIL-1 is required for LUBAC assembly, stability and optimal retention in the TNFR1 signalling complex, thereby preventing aberrant cell death. Both HOIL-1 and HOIP prevent embryonic lethality at mid-gestation by interfering with aberrant TNFR1-mediated endothelial cell death, which only partially depends on RIPK1 kinase activity. Co-deletion of caspase-8 with RIPK3 or MLKL prevents cell death in Hoil-1-/- (also known as Rbck1-/-) embryos, yet only the combined loss of caspase-8 with MLKL results in viable HOIL-1-deficient mice. Notably, triple-knockout Ripk3-/-Casp8-/-Hoil-1-/- embryos die at late gestation owing to haematopoietic defects that are rescued by co-deletion of RIPK1 but not MLKL. Collectively, these results demonstrate that both HOIP and HOIL-1 are essential LUBAC components and are required for embryogenesis by preventing aberrant cell death. Furthermore, they reveal that when LUBAC and caspase-8 are absent, RIPK3 prevents RIPK1 from inducing embryonic lethality by causing defects in fetal haematopoiesis.

The ubiquitin ligase XIAP recruits LUBAC for NOD2 signaling in inflammation and innate immunity.

Nucleotide-binding and oligomerization domain (NOD)-like receptors constitute a first line of defense against invading bacteria. X-linked Inhibitor of Apoptosis (XIAP) is implicated in the control of bacterial infections, and mutations in XIAP are causally linked to immunodeficiency in X-linked lymphoproliferative syndrome type-2 (XLP-2). Here, we demonstrate that the RING domain of XIAP is essential for NOD2 signaling and that XIAP contributes to exacerbation of inflammation-induced hepatitis in experimental mice. We find that XIAP ubiquitylates RIPK2 and recruits the linear ubiquitin chain assembly complex (LUBAC) to NOD2. We further show that LUBAC activity is required for efficient NF-κB activation and secretion of proinflammatory cytokines after NOD2 stimulation. Remarkably, XLP-2-derived XIAP variants have impaired ubiquitin ligase activity, fail to ubiquitylate RIPK2, and cannot facilitate NOD2 signaling. We conclude that XIAP and LUBAC constitute essential ubiquitin ligases in NOD2-mediated inflammatory signaling and propose that deregulation of NOD2 signaling contributes to XLP-2 pathogenesis.

Linear ubiquitination prevents inflammation and regulates immune signalling.

Members of the tumour necrosis factor (TNF) receptor superfamily have important functions in immunity and inflammation. Recently linear ubiquitin chains assembled by a complex containing HOIL-1 and HOIP (also known as RBCK1 and RNF31, respectively) were implicated in TNF signalling, yet their relevance in vivo remained uncertain. Here we identify SHARPIN as a third component of the linear ubiquitin chain assembly complex, recruited to the CD40 and TNF receptor signalling complexes together with its other constituents, HOIL-1 and HOIP. Mass spectrometry of TNF signalling complexes revealed RIP1 (also known as RIPK1) and NEMO (also known as IKKγ or IKBKG) to be linearly ubiquitinated. Mutation of the Sharpin gene (Sharpin(cpdm/cpdm)) causes chronic proliferative dermatitis (cpdm) characterized by inflammatory skin lesions and defective lymphoid organogenesis. Gene induction by TNF, CD40 ligand and interleukin-1ß was attenuated in cpdm-derived cells which were rendered sensitive to TNF-induced death. Importantly, Tnf gene deficiency prevented skin lesions in cpdm mice. We conclude that by enabling linear ubiquitination in the TNF receptor signalling complex, SHARPIN interferes with TNF-induced cell death and, thereby, prevents inflammation. Our results provide evidence for the relevance of linear ubiquitination in vivo in preventing inflammation and regulating immune signalling.

Amorphous Formulation and in Vitro Performance Testing of Instantly Disintegrating Buccal Tablets for the Emergency Delivery of Naloxone.

The aim of this study was to develop a freeze-dried buccal tablet for the rapid delivery of naloxone in opioid overdose. The tablet composition was optimized to produce an amorphous matrix, which was confirmed by the absence of peaks associated with crystallinity observed by differential scanning calorimetry and powder X-ray diffraction. Tablets with high gelatin content lacked adequate porosity. Mannitol was added to the formulation to bridge and intercalate gelatin's tight polymer aggregates, however sodium bicarbonate was also required to prevent crystallization within the tablets. A linear reduction in mannitol's recrystallization enthalpy was observed with increasing sodium bicarbonate concentration (ΔrecryH = -20.3[NaHCO3] + 220.9; r(2) = 0.9, n = 18). The minimum sodium bicarbonate concentration for full inhibition of mannitol crystallization was 10.9% w/w. Freeze-dried tablets with lower amounts of sodium bicarbonate possessed a crystalline fraction that PXRD identified as mannitol hemihydrate from the unique peak at 9.7° 2θ. Mannitol's greater affinity for both ions and residual water rather than its affinity for self-association was the mechanism for the inhibition of crystallization observed here. The optimized tablet (composition mannitol 24% w/w (4.26 mg), gelatin 65% w/w (11.7 mg), sodium bicarbonate 11% w/w (1.98 mg), and naloxone 800 µg) formed predominantly amorphous tablets that disintegrated in less than 10 s. Optimized tablets were chemically and physically stable over 9 months storage at 25 °C. As speed of drug liberation is the critical performance attribute for a solid dosage form designed to deliver drug in an emergency, a novel imaging based in vitro disintegration assay for buccal tablets was developed. The assay was optimized with regard to conditions in the buccal cavity: i.e., temperature 33-37 °C, volume of medium (0.1-0.7 mL), and use of mucin-containing biorelevant medium. The disintegration assay was sensitive to temperature, medium volume, and medium composition; naloxone tablet disintegration was extremely rapid, with full disintegration ranging from 5 to 20 s. In conclusion, rapidly disintegrating tablets have been developed which are suitable for proof-of-concept clinical trial in humans to determine the pharmacokinetics of naloxone delivered via the buccal route.

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.

Identification errors in medical research: Privacy at all costs?

In laboratory medicine, a misidentified patient sample can lead to an incorrect tissue diagnosis, a potentially fatal blood transfusion error or other serious adverse events. Although well characterised in routine patient care, the overall impacts of misidentification errors in the clinical research setting are less conspicuous but potentially greater, with downstream effects that may extend beyond care at an individual level. When data discrepancies or queries arise in clinical trial data then a data clarification form (DCF) is issued to the researcher by the overseeing trial coordinator or sponsor. Higher rates of DCF's are sometimes used as a crude surrogate marker of poorer trial quality. However, data is scarce on misidentification rates in clinical trials. In five clinical trials involving 822 histology or blood specimens analysed by our pathology department, DCF's were issued for 21% (174) of specimens. Amongst these 67% (117 / 174) were related to sample identification. Although these errors were recognised before data was compromised or an adverse event occurred, they highlight an alarming lack of stringency of use of patient identifiers in the research setting. We therefore propose the use of an appropriate number of de-identified data points and a formalised specimen accession process as employed in routine care to mitigate misidentification errors and their impact in clinical research. Increased recognition in the research community of the likely effect of truncating or reducing the number of patient identifiers is needed to minimise misidentification errors in the research setting.

In TNF-stimulated cells, RIPK1 promotes cell survival by stabilizing TRAF2 and cIAP1, which limits induction of non-canonical NF-kappaB and activation of caspase-8.

RIPK1 is involved in signaling from TNF and TLR family receptors. After receptor ligation, RIPK1 not only modulates activation of both canonical and NIK-dependent NF-κB, but also regulates caspase-8 activation and cell death. Although overexpression of RIPK1 can cause caspase-8-dependent cell death, when RIPK1(-/-) cells are exposed to TNF and low doses of cycloheximide, they die more readily than wild-type cells, indicating RIPK1 has pro-survival as well as pro-apoptotic activities. To determine how RIPK1 promotes cell survival, we compared wild-type and RIPK1(-/-) cells treated with TNF. Although TRAF2 levels remained constant in TNF-treated wild-type cells, TNF stimulation of RIPK1(-/-) cells caused TRAF2 and cIAP1 to be rapidly degraded by the proteasome, which led to an increase in NIK levels. This resulted in processing of p100 NF-κB2 to p52, a decrease in levels of cFLIP(L), and activation of caspase-8, culminating in cell death. Therefore, the pro-survival effect of RIPK1 is mediated by stabilization of TRAF2 and cIAP1.

Development of a Simple, Serum Biomarker-based Model Predictive of the Need for Early Biologic Therapy in Crohn's Disease.

BACKGROUND: Early or first-line treatment with biologics, as opposed to conventional immunomodulators, is not always necessary to achieve remission in Crohn's disease [CD] and may not be cost-effective. This study aimed to develop a simple model to predict the need for early biologic therapy, in order to risk-stratify CD patients and guide initial treatment selection. METHODS: A model-building study using supervised statistical learning methods was conducted using a retrospective cohort across two tertiary centres. All biologic-naïve CD patients who commenced an immunomodulator between January 1, 2004 and December 31, 2016, were included. A predictive score was derived using Cox regression modelling of immunomodulator failure, and was internally validated using bootstrap resampling. RESULTS: Of 410 patients [median age 37 years, 47% male, median disease duration 4.7 years], 229 [56%] experienced immunomodulator failure [39 required surgery, 24 experienced a new stricture, 44 experienced a new fistula/abscess, 122 required biologic escalation] with a median time to failure of 16 months. Independent predictors of treatment failure included raised C-reactive protein [CRP], low albumin, complex disease behaviour, younger age, and baseline steroids. Highest CRP and lowest albumin measured within the 3 months preceding immunomodulator initiation outperformed baseline measurements. After model selection, only highest CRP and lowest albumin remained and the resultant Crohn's Immunomodulator CRP-Albumin [CICA] index demonstrated robust optimism-corrected discriminative performance at 12, 24, and 36 months (area under the curve [AUC] 0.84, 0.83, 0.81, respectively). CONCLUSIONS: The derived CICA index based on simple, widely available markers is feasible, internally valid, and has a high utility in predicting immunomodulator failure. This requires external, prospective validation.

Dual roles for LUBAC signaling in thymic epithelial cell development and survival.

Thymic epithelial cells (TECs) form a unique microenvironment that orchestrates T cell differentiation and immunological tolerance. Despite the importance of TECs for adaptive immunity, there is an incomplete understanding of the signalling networks that support their differentiation and survival. We report that the linear ubiquitin chain assembly complex (LUBAC) is essential for medullary TEC (mTEC) differentiation, cortical TEC survival and prevention of premature thymic atrophy. TEC-specific loss of LUBAC proteins, HOIL-1 or HOIP, severely impaired expansion of the thymic medulla and AIRE-expressing cells. Furthermore, HOIL-1-deficiency caused early thymic atrophy due to Caspase-8/MLKL-dependent apoptosis/necroptosis of cortical TECs. By contrast, deficiency in the LUBAC component, SHARPIN, caused relatively mild defects only in mTECs. These distinct roles for LUBAC components in TECs correlate with their function in linear ubiquitination, NFκB activation and cell survival. Thus, our findings reveal dual roles for LUBAC signaling in TEC differentiation and survival.

First study of safety and tolerability of 3,4-methylenedioxymethamphetamine-assisted psychotherapy in patients with alcohol use disorder.

BACKGROUND: 3,4-methylenedioxymethamphetamine (MDMA) therapy has qualities that make it potentially well suited for patients with addictions, but this has never been explored in a research study. We present data from the Bristol Imperial MDMA in Alcoholism (BIMA) study. This is the first MDMA addiction study, an open-label safety and tolerability proof-of-concept study investigating the potential role for MDMA therapy in treating patients with alcohol use disorder (AUD). AIMS: This study aimed to assess if MDMA-assisted psychotherapy can be delivered safely and can be tolerated by patients with AUD post detoxification. Outcomes regarding drinking behaviour, quality of life and psychosocial functioning were evaluated. METHODS: Fourteen patients with AUD completed a community alcohol detoxification and received an eight-week course of recovery-based therapy. Participants received two sessions with MDMA (187.5 mg each session). Psychological support was provided before, during and after each session. Safety and tolerability were assessed alongside psychological and physiological outcome measures. Alcohol use behaviour, mental well-being and functioning data were collected for nine months after alcohol detoxification. RESULTS: MDMA treatment was well tolerated by all participants. No unexpected adverse events were observed. Psychosocial functioning improved across the cohort. Regarding alcohol use, at nine months post detox, the average units of alcohol consumption by participants was 18.7 units per week compared to 130.6 units per week before the detox. This compares favourably to a previous observational study (the 'Outcomes' study) by the same team with a similar population of people with AUD. CONCLUSIONS: This study provides preliminary support for the safety and tolerability of a novel intervention for AUD post detox. Further trials to examine better the therapeutic potential of this approach are now indicated.

Thiopurines vs methotrexate: Comparing tolerability and discontinuation rates in the treatment of inflammatory bowel disease.

BACKGROUND: There are safety concerns regarding immunomodulators (thiopurines and methotrexate) for treatment of inflammatory bowel disease (IBD). AIM: To compare the long-term tolerability, and persistence of thiopurine and methotrexate therapy in IBD. METHODS: A retrospective cohort study was performed at two hospitals between 1 January 2004 and 31 December 2016 for patients commenced on thiopurines or methotrexate for IBD. Treatment discontinuation rates, intolerances and disease activity were obtained from medical records. RESULTS: There were 782 patients commenced on immunomodulator therapy; 244 (31%) on methotrexate with folate (67% subcutaneous therapy) and 538 (69%) on thiopurine (73% azathioprine). Median follow-up was 42 vs 47 months (P = 0.09). In patients on thiopurines, median 6-TGN was 298 pmol/8 x 108 RBCs, while the median dose of methotrexate was 25 mg weekly. Methotrexate recipients had a higher rate of prior immunomodulator intolerance, were typically older and had a longer disease duration (54% vs 3%, median 43 vs 36 years, 6 vs 5 years, respectively, each P < 0.05). Overall, 208 (27%) discontinued therapy due to adverse events, (40% on methotrexate vs 19% on thiopurines, P < 0.001), including nausea (18% vs 4%), fatigue (7% vs 2%) and hepatotoxicity (8% vs 2%, each P < 0.001). Hospitalisations from adverse events (0.8% vs 0.9%) and serious infections (9% vs 12%), and deaths (1% vs 0%) were comparable between groups (all P > 0.05). Discontinuation due to adverse events occurred later in patients on methotrexate than on thiopurines (median 7 vs 5 months, P = 0.08). CONCLUSION: Discontinuation of methotrexate occurred at rates twice that of dose-optimised thiopurine therapy.

A missense mutation in the MLKL brace region promotes lethal neonatal inflammation and hematopoietic dysfunction.

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).

The Asian clam Corbicula fluminea as a biomonitor of trace element contamination: accounting for different sources of variation using an hierarchical linear model.

In the present study, specimens of the invasive clam, Corbicula fluminea, were collected above and below possible sources of potentially toxic trace elements (As, Cd, Cr, Cu, Hg, Pb, and Zn) in the Altamaha River system (Georgia, U.S.A.). Bioaccumulation of these elements was quantified, along with environmental (water and sediment) concentrations. Hierarchical linear models were used to account for variability in tissue concentrations related to environmental (site water chemistry and sediment characteristics) and individual (growth metrics) variables while identifying the strongest relations between these variables and trace element accumulation. The present study found significantly elevated concentrations of Cd, Cu, and Hg downstream of the outfall of kaolin-processing facilities, Zn downstream of a tire cording facility, and Cr downstream of both a nuclear power plant and a paper pulp mill. Models of the present study indicated that variation in trace element accumulation was linked to distance upstream from the estuary, dissolved oxygen, percentage of silt and clay in the sediment, elemental concentrations in sediment, shell length, and bivalve condition index. By explicitly modeling environmental variability, the Hierarchical linear modeling procedure allowed the identification of sites showing increased accumulation of trace elements that may have been caused by human activity. Hierarchical linear modeling is a useful tool for accounting for environmental and individual sources of variation in bioaccumulation studies.

RIPK1 prevents TRADD-driven, but TNFR1 independent, apoptosis during development.

RIPK1 is an essential downstream component of many pattern recognition and death receptors. RIPK1 can promote the activation of caspase-8 induced apoptosis and RIPK3-MLKL-mediated necroptosis, however, during development RIPK1 limits both forms of cell death. Accordingly, Ripk1-/- mice present with systemic cell death and consequent multi-organ inflammation, which is driven through the activation of both FADD-caspase-8 and RIPK3-MLKL signaling pathways causing perinatal lethality. TRADD is a death domain (DD) containing molecule that mediates signaling downstream of TNFR1 and the TLRs. Following the disassembly of the upstream receptor complexes either RIPK1 or TRADD can form a complex with FADD-caspase-8-cFLIP, via DD-DD interactions with FADD, facilitating the activation of caspase-8. We show that genetic deletion of Ripk1 licenses TRADD to complex with FADD-caspase-8 and activates caspase-8 during development. Deletion of Tradd provided no survival advantage to Ripk1-/- animals and yet was sufficient to reduce the systemic cell death and inflammation, rescue the intestinal and thymic histopathologies, reduce cleaved caspases in most tissues and rescue the anemia observed in Ripk1-/- neonates. Furthermore, deletion of Ripk3 is sufficient to rescue the neonatal lethality of Ripk1-/-Tradd-/- animals and delays but does not completely prevent early mortality. Although Ripk3 deletion provides a significant survival advantage, Ripk1-/-Tradd-/-Ripk3-/- animals die between 22 and 49 days, are runty compared to littermate controls and present with splenomegaly. These findings reveal a new mechanism by which RIPK1 limits apoptosis through blocking TRADD recruitment to FADD and preventing aberrant activation of caspase-8.

Screening and Prophylaxis to Prevent Hepatitis B Reactivation: Introduction and Immunology.

Current recommendations concerning hepatitis C virus (HBV) reactivation are limited, with nearly all guidelines focused on its occurrence in patients with hematological malignancies or some solid tumors, who are treated with immunosuppressive therapies. Few of the guidelines address reactivation in patients receiving immunosuppression with organ transplants or treatment with any of the many immunosuppressive agents in use today for the treatment of multiple different diseases, or in patients receiving the direct-acting antivirals used in the treatment of hepatitis C virus (HCV). This article covers the immunology of HBV reactivation, mechanisms of viral clearance, and recommendations for screening and prophylaxis.

Screening and Prophylaxis to Prevent Hepatitis B Reactivation: Transplant Recipients.

Organ transplantation is a lifesaving procedure for many patients. To prevent rejection or graft-versus-host disease, recipients require long-term immunosuppression. In patients who have ever been exposed to hepatitis B, it is possible for reactivation to occur; this includes patients who are anti-hepatitis B core antibody-positive only or both anti-hepatitis B core antibody-positive and hepatitis B surface antibody-positive. The susceptibility to this varies with the nature of the transplant. Hepatitis B can be transmitted from donor to recipient. It is important to assess the hepatitis B status and formulate a strategy to prevent transmission and prevent reactivation.