Updates on the immunopathology and genomics of severe cutaneous adverse drug reactions.

Severe cutaneous adverse reactions (SCARs) such as Stevens-Johnson syndrome, toxic epidermal necrolysis (SJS/TEN), and drug reaction with eosinophilia and systemic symptoms (DRESS)/drug-induced hypersensitivity syndrome (DIHS) cause significant morbidity and mortality and impede new drug development. HLA class I associations with SJS/TEN and drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome have aided preventive efforts and provided insights into immunopathogenesis. In SJS/TEN, HLA class I-restricted oligoclonal CD8+ T-cell responses occur at the tissue level. However, specific HLA risk allele(s) and antigens driving this response have not been identified for most drugs. HLA risk alleles also have incomplete positive and negative predictive values, making truly comprehensive screening currently challenging. Although, there have been key paradigm shifts in knowledge regarding drug hypersensitivity, there are still many open and unanswered questions about SCAR immunopathogenesis, as well as genetic and environmental risk. In addition to understanding the cellular and molecular basis of SCAR at the single-cell level, identification of the MHC-restricted drug-reactive self- or viral peptides driving the hypersensitivity reaction will also be critical to advancing premarketing strategies to predict risk at an individual and drug level. This will also enable identification of biologic markers for earlier diagnosis and accurate prognosis, as well as drug causality and targeted therapeutics.

Neuronal Preconditioning Requires the Mitophagic Activity of C-terminus of HSC70-Interacting Protein.

The C terminus of HSC70-interacting protein (CHIP, STUB1) is a ubiquitously expressed cytosolic E3-ubiquitin ligase. CHIP-deficient mice exhibit cardiovascular stress and motor dysfunction before premature death. This phenotype is more consistent with animal models in which master regulators of autophagy are affected rather than with the mild phenotype of classic E3-ubiquitin ligase mutants. The cellular and biochemical events that contribute to neurodegeneration and premature aging in CHIP KO models remain poorly understood. Electron and fluorescent microscopy demonstrates that CHIP deficiency is associated with greater numbers of mitochondria, but these organelles are swollen and misshapen. Acute bioenergetic stress triggers CHIP induction and relocalization to mitochondria, where it plays a role in the removal of damaged organelles. This mitochondrial clearance is required for protection following low-level bioenergetic stress in neurons. CHIP expression overlaps with stabilization of the redox stress sensor PTEN-inducible kinase 1 (PINK1) and is associated with increased LC3-mediated mitophagy. Introducing human promoter-driven vectors with mutations in either the E3 ligase or tetracopeptide repeat domains of CHIP in primary neurons derived from CHIP-null animals enhances CHIP accumulation at mitochondria. Exposure to autophagy inhibitors suggests that the increase in mitochondrial CHIP is likely due to diminished clearance of these CHIP-tagged organelles. Proteomic analysis of WT and CHIP KO mouse brains (four male, four female per genotype) reveals proteins essential for maintaining energetic, redox, and mitochondrial homeostasis undergo significant genotype-dependent expression changes. Together, these data support the use of CHIP-deficient animals as a predictive model of age-related degeneration with selective neuronal proteotoxicity and mitochondrial failure.SIGNIFICANCE STATEMENT Mitochondria are recognized as central determinants of neuronal function and survival. We demonstrate that C terminus of HSC70-Interacting Protein (CHIP) is critical for neuronal responses to stress. CHIP upregulation and localization to mitochondria is required for mitochondrial autophagy (mitophagy). Unlike other disease-associated E3 ligases such as Parkin and Mahogunin, CHIP controls homeostatic and stress-induced removal of mitochondria. Although CHIP deletion results in greater numbers of mitochondria, these organelles have distorted inner membranes without clear cristae. Neuronal cultures derived from animals lacking CHIP are more vulnerable to acute injuries and transient loss of CHIP renders neurons incapable of mounting a protective response after low-level stress. Together, these data suggest that CHIP is an essential regulator of mitochondrial number, cell signaling, and survival.

Assembly Dynamics and Stoichiometry of the Apoptosis Signal-regulating Kinase (ASK) Signalosome in Response to Electrophile Stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a key sensor kinase in the mitogen-activated protein kinase pathway that transduces cellular responses to oxidants and electrophiles. ASK1 is regulated by a large, dynamic multiprotein signalosome complex, potentially including over 90 reported ASK1-interacting proteins. We employed both shotgun and targeted mass spectrometry assays to catalogue the ASK1 protein-protein interactions in HEK-293 cells treated with the prototypical lipid electrophile 4-hydroxy-2-nonenal (HNE). Using both epitope-tagged overexpression and endogenous expression cell systems, we verified most of the previously reported ASK1 protein-protein interactions and identified 14 proteins that exhibited dynamic shifts in association with ASK1 in response to HNE stress. We used precise stable isotope dilution assays to quantify protein stoichiometry in the ASK signalosome complex and identified ASK2 at a 1:1 stoichiometric ratio with ASK1 and 14-3-3 proteins (YWHAQ, YWHAB, YWHAH, and YWHAE) collectively at a 0.5:1 ratio with ASK1 as the main components. Several other proteins, including ASK3, PARK7, PRDX1, and USP9X were detected with stoichiometries of 0.1:1 or less. These data support an ASK signalosome comprising a multimeric core complex of ASK1, ASK2, and 14-3-3 proteins, which dynamically engages other binding partners needed to mediate diverse stress-response signaling events. This study further demonstrates the value of combining global and targeted MS approaches to interrogate multiprotein complex composition and dynamics.

Dynamic Phosphorylation of Apoptosis Signal Regulating Kinase 1 (ASK1) in Response to Oxidative and Electrophilic Stress.

Apoptosis signal-regulating kinase 1 (ASK1) is a critical cellular stress sensor that senses diverse reactive chemotypes and integrates these chemical signals into a single biological pathway response. It is unknown whether ASK1 senses all stressors in the same way or if unique stress-specific mechanisms detect distinct chemotypes. In order to answer this question, we treated ASK1-expressing cells with two distinct stress activators, H2O2 and 4-hydroxy-2-nonenal (HNE), and monitored the phosphorylation state of ASK1. Phosphorylation is an important regulator for the activity of ASK1, and we hypothesized that these two chemically distinct molecules may produce differences in the phosphorylation state of ASK1. Shotgun mass spectrometry and manual validation identified 12 distinct ASK1 phosphosites. Targeted parallel reaction monitoring assays were used to track the phosphorylation dynamics of each confirmed site in response to treatment. Eleven phosphosites exhibited dynamic response to one or both treatments. Six of these sites were identified in both H2O2- and HNE-treated cells, and four of these exhibited a consistent response between the two molecules. The results confirm that different chemotypes produce distinct phosphorylation patterns in concert with activation of a common MAPK pathway.

Multiomic single-cell sequencing defines tissue-specific responses in Stevens-Johnson Syndrome and Toxic epidermal necrolysis.

Stevens-Johnson syndrome and toxic epidermal necrolysis (SJS/TEN) is a rare but life-threatening cutaneous drug reaction mediated by human leukocyte antigen (HLA) class I-restricted CD8+ T-cells. To obtain an unbiased assessment of SJS/TEN cellular immunopathogenesis, we performed single-cell (sc) transcriptome, surface proteome, and TCR sequencing on unaffected skin, affected skin, and blister fluid from 17 SJS/TEN patients. From 119,784 total cells, we identified 16 scRNA-defined subsets, confirmed by subset-defining surface protein expression. Keratinocytes upregulated HLA and IFN-response genes in the affected skin. Cytotoxic CD8+ T-cell subpopulations of expanded and unexpanded TCRαß clonotypes were shared in affected skin and blister fluid but absent or unexpanded in SJS/TEN unaffected skin. SJS/TEN blister fluid is a rich reservoir of oligoclonal CD8+ T-cells with an effector phenotype driving SJS/TEN pathogenesis. This multiomic database will act as the basis to define antigen-reactivity, HLA restriction, and signatures of drug-antigen-reactive T-cell clonotypes at a tissue level.

The role of central nervous system development in late-onset neurodegenerative disorders.

The human brain is dependent upon successfully maintaining ionic, energetic and redox homeostasis within exceptionally narrow margins for proper function. The ability of neurons to adapt to genetic and environmental perturbations and evoke a 'new normal' can be most fully appreciated in the context of neurological disorders in which clinical impairments do not manifest until late in life, although dysfunctional proteins are expressed early in development. We now know that proteins controlling ATP generation, mitochondrial stability, and the redox environment are associated with neurological disorders such as Parkinson's disease and amyotrophic lateral sclerosis. Generally, focus is placed on the role that early or long-term environmental stress has in altering the survival of cells targeted by genetic dysfunctions; however, the central nervous system undergoes several periods of intense stress during normal maturation. One of the most profound periods of stress occurs when 50% of neurons are removed via programmed cell death. Unfortunately, we have virtually no understanding of how these events proceed in individuals who harbor mutations that are lethal later in life. Moreover, there is a profound lack of information on circuit formation, cell fate during development and neurochemical compensation in either humans or the animals used to model neurodegenerative diseases. In this review, we consider the current knowledge of how energetic and oxidative stress signaling differs between neurons in early versus late stages of life, the influence of a new group of proteins that can integrate cell stress signals at the mitochondrial level, and the growing body of evidence that suggests early development should be considered a critical period for the genesis of chronic neurodegenerative diseases.

Practical Implementation of Genetics: New Concepts in Immunogenomics to Predict, Prevent, and Diagnose Drug Hypersensitivity.

Delayed drug hypersensitivities are CD8+ T cell-mediated reactions associated with up to 50% mortality. Human leukocyte antigen (HLA) alleles are known to predispose disease and are specific to drug, reaction, and patient ethnicity. Pretreatment screening is recommended for a handful of the strongest associations to identify and prevent drug use in high-risk patients. However, an incomplete predictive value implicates other HLA-imposed risk factors, and low carriage of many identified HLA-risk alleles combined with the high cost of sequence-based typing has limited economic viability for similar recommendation of screening across drugs and health care systems. For mitigation, an expanding armory of low-cost polymerase chain reaction-based screens is being developed, and HLA-imposed risk factors are being discovered. These include (1) polymorphic variants of metabolic and endoplasmic reticulum aminopeptidase enzymes toward multiallelic screening with increased predictivity; (2) regulation by immune checkpoint inhibitors, enabling detolerized animal models of human disease; and (3) immunodominant T cell receptors (TCR) on clonally expanded CD8+ T cells. For the latter, HLA risk-restricted TCR provides immunogenomic strategies and samples from a single patient to identify novel HLA-risk associations in underserved minority populations, tissue-relevant effector biomarkers toward earlier diagnosis and treatment, and HLA-TCR-presented immunogenic structures to aid future drug development.

Genomic Risk Factors Driving Immune-Mediated Delayed Drug Hypersensitivity Reactions.

Adverse drug reactions (ADRs) remain associated with significant mortality. Delayed hypersensitivity reactions (DHRs) that occur greater than 6 h following drug administration are T-cell mediated with many severe DHRs now associated with human leukocyte antigen (HLA) risk alleles, opening pathways for clinical prediction and prevention. However, incomplete negative predictive value (NPV), low positive predictive value (PPV), and a large number needed to test (NNT) to prevent one case have practically prevented large-scale and cost-effective screening implementation. Additional factors outside of HLA contributing to risk of severe T-cell-mediated DHRs include variation in drug metabolism, T-cell receptor (TCR) specificity, and, most recently, HLA-presented immunopeptidome-processing efficiencies via endoplasmic reticulum aminopeptidase (ERAP). Active research continues toward identification of other highly polymorphic factors likely to impose risk. These include those previously associated with T-cell-mediated HLA-associated infectious or auto-immune disease such as Killer cell immunoglobulin-like receptors (KIR), epistatically linked with HLA class I to regulate NK- and T-cell-mediated cytotoxic degranulation, and co-inhibitory signaling pathways for which therapeutic blockade in cancer immunotherapy is now associated with an increased incidence of DHRs. As such, the field now recognizes that susceptibility is not simply a static product of genetics but that individuals may experience dynamic risk, skewed toward immune activation through therapeutic interventions and epigenetic modifications driven by ecological exposures. This review provides an updated overview of current and proposed genetic factors thought to predispose risk for severe T-cell-mediated DHRs.

Immunopharmacogenomics: Mechanisms of HLA-Associated Drug Reactions.

The human leukocyte antigen (HLA) system is the most polymorphic in the human genome that has been associated with protection and predisposition to a broad array of infectious, autoimmune, and malignant diseases. More recently over the last two decades, HLA class I alleles have been strongly associated with T-cell-mediated drug hypersensitivity reactions. In the case of abacavir hypersensitivity and HLA-B*57:01, the 100% negative predictive value and low number needed to test to prevent a single case has led to a durable and effective global preprescription screening strategy. However, HLA associations are still undefined for most drugs clinically associated with different delayed drug hypersensitivity phenotypes, and an HLA association relevant to one population is not generalizable across ethnicities. Furthermore, while a specific risk HLA allele is necessary for drug-induced T-cell activation, it is not sufficient. The low and incomplete positive predictive value has hindered efforts at clinical implementation for many drugs but has provided the impetus to understand the mechanisms of HLA class I restricted T-cell-mediated drug hypersensitivity reactions. Current research has focused on defining the contribution of additional elements of the adaptive immune response and other genetic and ecologic risk factors that contribute to drug hypersensitivity risk. In this review we focus on new insights into immunological, pharmacological, and genetic mechanisms underpinning HLA-associated drug reactions and the implications for future translation into clinical care.

Visual Genomics Analysis Studio as a Tool to Analyze Multiomic Data.

Type B adverse drug reactions (ADRs) are iatrogenic immune-mediated syndromes with mechanistic etiologies that remain incompletely understood. Some of the most severe ADRs, including delayed drug hypersensitivity reactions, are T-cell mediated, restricted by specific human leukocyte antigen risk alleles and sometimes by public or oligoclonal T-cell receptors (TCRs), central to the immunopathogenesis of tissue-damaging response. However, the specific cellular signatures of effector, regulatory, and accessory immune populations that mediate disease, define reaction phenotype, and determine severity have not been defined. Recent development of single-cell platforms bringing together advances in genomics and immunology provides the tools to simultaneously examine the full transcriptome, TCRs, and surface protein markers of highly heterogeneous immune cell populations at the site of the pathological response at a single-cell level. However, the requirement for advanced bioinformatics expertise and computational hardware and software has often limited the ability of investigators with the understanding of diseases and biological models to exploit these new approaches. Here we describe the features and use of a state-of-the-art, fully integrated application for analysis and visualization of multiomic single-cell data called Visual Genomics Analysis Studio (VGAS). This unique user-friendly, Windows-based graphical user interface is specifically designed to enable investigators to interrogate their own data. While VGAS also includes tools for sequence alignment and identification of associations with host or organism genetic polymorphisms, in this review we focus on its application for analysis of single-cell TCR-RNA-Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE)-seq, enabling holistic cellular characterization by unbiased transcriptome and select surface proteome. Critically, VGAS does not require user-directed coding or access to high-performance computers, instead incorporating performance-optimized hidden code to provide application-based fast and intuitive tools for data analyses and production of high-resolution publication-ready graphics on standard specification laptops. Specifically, it allows analyses of comprehensive single-cell TCR sequencing (scTCR-seq) data, detailing (i) functional pairings of α-ß heterodimer TCRs, (ii) one-click histograms to display entropy and gene rearrangements, and (iii) Circos and Sankey plots to visualize clonality and dominance. For unbiased single-cell RNA sequencing (scRNA-seq) analyses, users extract cell transcriptome signatures according to global structure via principal component analysis, t-distributed stochastic neighborhood embedding, or uniform manifold approximation and projection plots, with overlay of scTCR-seq enabling identification and selection of the immunodominant TCR-expressing populations. Further integration with similar sequence-based detection of surface protein markers using oligo-labeled antibodies (CITE-seq) provides comparative understanding of surface protein expression, with differential gene or protein analyses visualized using volcano plot or heatmap functions. These data can be compared to reference cell atlases or suitable controls to reveal discrete disease-specific subsets, from epithelial to tissue-resident memory T-cells, and activation status, from senescence through exhaustion, with more finite transcript expression displayed as violin and box plots. Importantly, guided tutorial videos are available, as are regular application updates based on the latest advances in bioinformatics and user feedback.