Joseph D. Hernandez

Differential glycosylation of TH1, TH2 and TH-17 effector cells selectively regulates susceptibility to cell death.

Regulated glycosylation controls T cell processes, including activation, differentiation and homing by creating or masking ligands for endogenous lectins. Here we show that stimuli promoting T helper type 1 (TH1), TH2 or interleukin 17–producing T helper (TH-17) differentiation can differentially regulate the glycosylation pattern of T helper cells and modulate their susceptibility to galectin-1, a glycan-binding protein with anti-inflammatory activity. Although TH1- and TH-17–differentiated cells expressed the repertoire of cell surface glycans critical for galectin-1–induced cell death, TH2 cells were protected from galectin-1 through differential sialylation of cell surface glycoproteins. Consistent with those findings, galectin-1–deficient mice developed greater TH1 and TH-17 responses and enhanced susceptibility to autoimmune neuroinflammation. Our findings identify a molecular link among differential glycosylation of T helper cells, susceptibility to cell death and termination of the inflammatory response.

Galectin-1 induces nuclear translocation of endonuclease G in caspase- and cytochrome c-independent T cell death.

AbstractGalectin-1, a mammalian lectin expressed in many tissues, induces death of diverse cell types, including lymphocytes and tumor cells. The galectin-1 T cell death pathway is novel and distinct from other death pathways, including those initiated by Fas and corticosteroids. We have found that galectin-1 binding to human T cell lines triggered rapid translocation of endonuclease G from mitochondria to nuclei. However, endonuclease G nuclear translocation occurred without cytochrome c release from mitochondria, without nuclear translocation of apoptosis-inducing factor, and prior to loss of mitochondrial membrane potential. Galectin-1 treatment did not result in caspase activation, nor was death blocked by caspase inhibitors. However, galectin-1 cell death was inhibited by intracellular expression of galectin-3, and galectin-3 expression inhibited the eventual loss of mitochondrial membrane potential. Galectin-1-induced cell death proceeds via a caspase-independent pathway that involves a unique pattern of mitochondrial events, and different galectin family members can coordinately regulate susceptibility to cell death.

Galectin-1 Binds Different CD43 Glycoforms to Cluster CD43 and Regulate T Cell Death

Galectin-1 kills immature thymocytes and activated peripheral T cells by binding to glycans on T cell glycoproteins including CD7, CD45, and CD43. Although roles for CD7 and CD45 in regulating galectin-1-induced death have been described, the requirement for CD43 remains unknown. We describe a novel role for CD43 in galectin-1-induced death, and the effects of O-glycan modification on galectin-1 binding to CD43. Loss of CD43 expression reduced galectin-1 death of murine thymocytes and human T lymphoblastoid cells, indicating that CD43 is required for maximal T cell susceptibility to galectin-1. CD43, which is heavily O-glycosylated, contributes a significant fraction of galectin-1 binding sites on T cells, as T cells lacking CD43 bound ∼50% less galectin-1 than T cells expressing CD43. Although core 2 modification of O-glycans on other glycoprotein receptors is critical for galectin-1-induced cross-linking and T cell death, galectin-1 bound to CD43 fusion proteins modified with either unbranched core 1 or branched core 2 O-glycans and expression of core 2 O-glycans did not enhance galectin-1 binding to CD43 on T cells. Moreover, galectin-1 binding clustered CD43 modified with either core 1 or core 2 O-glycans on the T cell surface. Thus, CD43 bearing either core 1 or core 2 O-glycans can positively regulate T cell susceptibility to galectin-1, identifying a novel function for CD43 in controlling cell death. In addition, these studies demonstrate that different T cell glycoproteins on the same cell have distinct requirements for glycan modifications that allow recognition and cross-linking by galectin-1.

Different activation signals induce distinct mast cell degranulation strategies

Mast cells (MCs) influence intercellular communication during inflammation by secreting cytoplasmic granules that contain diverse mediators. Here, we have demonstrated that MCs decode different activation stimuli into spatially and temporally distinct patterns of granule secretion. Certain signals, including substance P, the complement anaphylatoxins C3a and C5a, and endothelin 1, induced human MCs rapidly to secrete small and relatively spherical granule structures, a pattern consistent with the secretion of individual granules. Conversely, activating MCs with anti-IgE increased the time partition between signaling and secretion, which was associated with a period of sustained elevation of intracellular calcium and formation of larger and more heterogeneously shaped granule structures that underwent prolonged exteriorization. Pharmacological inhibition of IKK-β during IgE-dependent stimulation strongly reduced the time partition between signaling and secretion, inhibited SNAP23/STX4 complex formation, and switched the degranulation pattern into one that resembled degranulation induced by substance P. IgE-dependent and substance P-dependent activation in vivo also induced different patterns of mouse MC degranulation that were associated with distinct local and systemic pathophysiological responses. These findings show that cytoplasmic granule secretion from MCs that occurs in response to different activating stimuli can exhibit distinct dynamics and features that are associated with distinct patterns of MC-dependent inflammation.

CD45 Signals outside of Lipid Rafts to Promote ERK Activation, Synaptic Raft Clustering, and IL-2 Production

CD45 is dynamically repositioned within lipid rafts and the immune synapse during T cell activation, although the molecular consequences of CD45 repositioning remain unclear. In this study we examine the role of CD45 membrane compartmentalization in regulating murine T cell activation. We find that raft-localized CD45 antagonizes IL-2 production by opposing processive TCR signals, whereas raft-excluded CD45 promotes ERK-dependent polarized synaptic lipid raft clustering and IL-2 production. We propose that these dual CD45 activities ensure that only robust TCR signals proceed, whereas signals meeting threshold requirements are potentiated. Our findings highlight membrane compartmentalization as a key regulator of CD45 function and elucidate a novel signal transduction pathway by which raft-excluded CD45 positively regulates T cell activation.

Single-cell systems-level analysis of human Toll-like receptor activation defines a chemokine signature in patients with systemic lupus erythematosus

BACKGROUND Activation of Toll-like receptors (TLRs) induces inflammatory responses involved in immunity to pathogens and autoimmune pathogenesis, such as in patients with systemic lupus erythematosus (SLE). Although TLRs are differentially expressed across the immune system, a comprehensive analysis of how multiple immune cell subsets respond in a system-wide manner has not been described. OBJECTIVE We sought to characterize TLR activation across multiple immune cell subsets and subjects, with the goal of establishing a reference framework against which to compare pathologic processes. METHODS Peripheral whole-blood samples were stimulated with TLR ligands and analyzed by means of mass cytometry simultaneously for surface marker expression, activation states of intracellular signaling proteins, and cytokine production. We developed a novel data visualization tool to provide an integrated view of TLR signaling networks with single-cell resolution. We studied 17 healthy volunteer donors and 8 patients with newly diagnosed and untreated SLE. RESULTS Our data revealed the diversity of TLR-induced responses within cell types, with TLR ligand specificity. Subsets of natural killer cells and T cells selectively induced nuclear factor κ light chain enhancer of activated B cells in response to TLR2 ligands. CD14(hi) monocytes exhibited the most polyfunctional cytokine expression patterns, with more than 80 distinct cytokine combinations. Monocytic TLR-induced cytokine patterns were shared among a group of healthy donors, with minimal intraindividual and interindividual variability. Furthermore, autoimmune disease altered baseline cytokine production; newly diagnosed untreated SLE patients shared a distinct monocytic chemokine signature, despite clinical heterogeneity. CONCLUSION Mass cytometry defined a systems-level reference framework for human TLR activation, which can be applied to study perturbations in patients with inflammatory diseases, such as SLE.

A TNFRSF14-FcɛRI-mast cell pathway contributes to development of multiple features of asthma pathology in mice.

Asthma has multiple features, including airway hyperreactivity, inflammation and remodelling. The TNF superfamily member TNFSF14 (LIGHT), via interactions with the receptor TNFRSF14 (HVEM), can support TH2 cell generation and longevity and promote airway remodelling in mouse models of asthma, but the mechanisms by which TNFSF14 functions in this setting are incompletely understood. Here we find that mouse and human mast cells (MCs) express TNFRSF14 and that TNFSF14:TNFRSF14 interactions can enhance IgE-mediated MC signalling and mediator production. In mouse models of asthma, TNFRSF14 blockade with a neutralizing antibody administered after antigen sensitization, or genetic deletion of Tnfrsf14, diminishes plasma levels of antigen-specific IgG1 and IgE antibodies, airway hyperreactivity, airway inflammation and airway remodelling. Finally, by analysing two types of genetically MC-deficient mice after engrafting MCs that either do or do not express TNFRSF14, we show that TNFRSF14 expression on MCs significantly contributes to the development of multiple features of asthma pathology.

Novel tools for primary immunodeficiency diagnosis: making a case for deep profiling

Purpose of reviewThis review gives an overview of the systems-immunology single-cell proteomic and transcriptomic approaches that can be applied to study primary immunodeficiency. It also introduces recent advances in multiparameter tissue imaging, which allows extensive immune phenotyping in disease-affected tissue. Recent findingsMass cytometry is a variation of flow cytometry that uses rare earth metal isotopes instead of fluorophores as tags bound to antibodies, allowing simultaneous measurement of over 40 parameters per single-cell. Mass cytomety enables comprehensive single-cell immunophenotyping and functional assessments, capturing the complexity of the immune system, and the molecularly heterogeneous consequences of primary immunodeficiency defects. Protein epitopes and transcripts can be simultaneously detected allowing immunophenotype and gene expression evaluation in mixed cell populations. Multiplexed epitope imaging has the potential to provide extensive phenotypic characterization at the subcellular level, in the context of 3D tissue microenvironment. SummaryMass cytometry and multiplexed epitope imaging can complement genetic methods in diagnosis and study of the pathogenesis of primary immunodeficiencies. The ability to understand the effect of a specific defect across multiple immune cell types and pathways, and in affected tissues, may provide new insight into tissue-specific disease pathogenesis and evaluate effects of therapeutic interventions.

Allergic Diseases and Immune-Mediated Food Disorders in Pediatric Acute-Onset Neuropsychiatric Syndrome

Background: The prevalence and impact of allergic and immune-mediated food disorders in pediatric acute-onset neuropsychiatric syndrome (PANS) are mostly unknown. Objective: We sought to explore the prevalence of atopic dermatitis (AD), asthma, allergic rhinitis (AR), IgE-mediated food allergies (FAs), and other immune-mediated food disorders requiring food avoidance in patients with PANS. In addition, to further understand the extent of food restriction in this population, we investigated the empiric use of dietary measures to improve PANS symptoms. Methods: Pediatric patients in a PANS Clinic and Research Program were given surveys regarding their caregiver burdens, allergic and food-related medical history, and whether food elimination resulted in perception of improvement of PANS symptoms. A review of health records was conducted to confirm that all responses in the survey were concordant with documentation of each patients medical chart. Results: Sixty-nine (ages 4-20 years) of 80 subjects who fulfilled PANS criteria completed the surveys. Thirteen (18.8%) had AD, 11 (15.9%) asthma, 33 (47.8%) AR, 11 (15.9%) FA, 1 (1.4%) eosinophilic gastrointestinal disorders, 1 (1.4%) food protein-induced enterocolitis syndrome, 3 (4.3%) milk protein-induced proctocolitis syndrome, and 3 (4.3%) celiac disease. Thirty subjects (43.5%) avoided foods due to PANS; elimination of gluten and dairy was most common and was associated with perceived improvement of PANS symptoms (by parents). This perceived improvement was not confirmed with objective data. Conclusions: The prevalence of allergic and immune-mediated food disorders in PANS is similar to the general population as reported in the literature, with the exception of AR that appears to be more prevalent in our PANS cohort. More research will be required to establish whether diet or allergies influence PANS symptoms.

Anaphylaxis to invasive chlorhexidine administration despite tolerance of topical chlorhexidine use

Perioperative anaphylaxis is a potentially life-threatening hypersensitivity reaction (HSR) caused by drugs used during anesthesia or surgery. Early symptoms of anaphylaxis including pruritus and dyspnea are unattainable in anesthetized patients. Diagnosis is thereby delayed until hypotension and inability to ventilate the patient are present, likely contributing to the 3% to 9% mortality rate. The incidence of perioperative anaphylaxis differs regionally, ranging between 1 in 5,000 to 1 in 20,000 anesthetic procedures. Because numerous medications are rapidly administered, a causative agent is identified in less than 60% of cases, with neuromuscular-blocking agents (NMBAs) and antibiotics being the most commonly implicated. Chlorhexidine, a preferred antiseptic in both household and hospital products, is an underrecognized cause of immediate HSRs. Previously used mostly in topical preparations, chlorhexidine is now frequently impregnated in catheters and implantable devices to mitigate infections. The ubiquitous use of chlorhexidine has been implicated in the increasing rates of sensitization and subsequent severe reactions. As of 2015, 68 distinct perioperative anaphylactic reactions to chlorhexidine were reported in the literature and most were preceded by minor HSRs such as rash. In addition, there is limited information on delayed skin prick test (SPT) positivity with chlorhexidine and the dangers of false-negative results with subsequent intradermal test (IDT). We report 2 cases of chlorhexidine-induced perioperative anaphylaxis after invasive exposure, who were completely asymptomatic with topical use and demonstrated delayed SPT positivity 30 minutes after application.