Senataxin suppresses the antiviral transcriptional response and controls viral biogenesis.

The human helicase senataxin (SETX) has been linked to the neurodegenerative diseases amyotrophic lateral sclerosis (ALS4) and ataxia with oculomotor apraxia (AOA2). Here we identified a role for SETX in controlling the antiviral response. Cells that had undergone depletion of SETX and SETX-deficient cells derived from patients with AOA2 had higher expression of antiviral mediators in response to infection than did wild-type cells. Mechanistically, we propose a model whereby SETX attenuates the activity of RNA polymerase II (RNAPII) at genes stimulated after a virus is sensed and thus controls the magnitude of the host response to pathogens and the biogenesis of various RNA viruses (e.g., influenza A virus and West Nile virus). Our data indicate a potentially causal link among inborn errors in SETX, susceptibility to infection and the development of neurologic disorders.

TAOK3, a Regulator of LCK-SHP-1 Crosstalk during TCR Signaling.

Signaling from the T cell receptor for antigen turns on the physiological response of a T cell. The canonical TCR signaling pathway relies on early activation of the Src kinase LCK. This step initiates a cascade of events that lead not only to the phenotypic changes that characterize effector T cells but also to the activation of negative regulatory mechanisms that stop early TCR signaling. These mechanisms ensure qualitative and quantitative fine-tuning of T cell activation. The tyrosine phosphatase SHP-1 is a key player in the downregulation of LCK activation. In this review, we focus on the crosstalk between LCK and SHP-1 and, based on recent data, we introduce the putative kinase TAOK3 as an important regulator of this crosstalk. Given the widespread expression of TAOK3 and SHP-1, we propose that the function of TAOK3 extends beyond T cells and may be fundamental in the regulation of early signaling from receptors that utilize Src kinases.

TAOK3 Regulates Canonical TCR Signaling by Preventing Early SHP-1-Mediated Inactivation of LCK.

Activation of LCK is required for canonical TCR signaling leading to T cell responses. LCK activation also initiates a negative feedback loop mediated by the phosphatase SHP-1 that turns off TCR signaling. In this article, we report that the thousand-and-one amino acid kinase 3 (TAOK3) is a key regulator of this feedback. TAOK3 is a serine/threonine kinase expressed in many different cell types including T cells. TAOK3-deficient human T cells had impaired LCK-dependent TCR signaling resulting in a defect in IL-2 response to canonical TCR signaling but not to bacterial superantigens, which use an LCK-independent pathway. This impairment was associated with enhanced interaction of LCK with SHP-1 after TCR engagement and rapid termination of TCR signals, a defect corrected by TAOK3 reconstitution. Thus, TAOK3 is a positive regulator of TCR signaling by preventing premature SHP-1-mediated inactivation of LCK. This mechanism may also regulate signaling by other Src family kinase-dependent receptors.

Staphylococcal Superantigens Use LAMA2 as a Coreceptor To Activate T Cells.

Canonical Ag-dependent TCR signaling relies on activation of the src-family tyrosine kinase LCK. However, staphylococcal superantigens can trigger TCR signaling by activating an alternative pathway that is independent of LCK and utilizes a Gα11-containing G protein-coupled receptor (GPCR) leading to PLCß activation. The molecules linking the superantigen to GPCR signaling are unknown. Using the ligand-receptor capture technology LRC-TriCEPS, we identified LAMA2, the α2 subunit of the extracellular matrix protein laminin, as the coreceptor for staphylococcal superantigens. Complementary binding assays (ELISA, pull-downs, and surface plasmon resonance) provided direct evidence of the interaction between staphylococcal enterotoxin E and LAMA2. Through its G4 domain, LAMA2 mediated the LCK-independent T cell activation by these toxins. Such a coreceptor role of LAMA2 involved a GPCR of the calcium-sensing receptor type because the selective antagonist NPS 2143 inhibited superantigen-induced T cell activation in vitro and delayed the effects of toxic shock syndrome in vivo. Collectively, our data identify LAMA2 as a target of antagonists of staphylococcal superantigens to treat toxic shock syndrome.

Staphylococcus aureus Downregulates IP-10 Production and Prevents Th1 Cell Recruitment.

Staphylococcal superantigens cause toxic shock syndrome, which is characterized by massive T cell activation and a predominant Th1 profile of cytokine production. However, superantigen-producing Staphylococcus aureus strains are often part of the human nasal microbiome, and this carrier state has often been associated with some type 2 immune responses such as chronic sinusitis with polyps and atopic dermatitis. We have previously reported that the S. aureus cell wall downregulates the human T cell response to superantigens through a TLR2-dependent, IL-10-mediated mechanism. In this study, we show that S. aureus also regulates the profile of superantigen-induced T cell recruitment. The staphylococcal superantigen SEE induced the production of Th1 cell-recruiting chemokines, including IP-10, through an IFN-γ-dependent mechanism. Such an induction was suppressed by the concomitant presence of S. aureus The downregulation of IP-10 by S. aureus was mediated by components of its cell wall, but was not due to peptidoglycan-induced IL-10 production. Instead, S. aureus triggered activation of MAPKs p38 and ERK, as well as inhibition of STAT1 signaling in monocytes, altogether contributing to the downregulation of IP-10 and other Th1 cell-recruiting chemokines (e.g., CXCL9 and CXCL11). These effects translated into inhibition of superantigen-induced Th1 cell recruitment. Taken together, our data may explain why colonization of superantigen-producing S. aureus can induce, under some circumstances, mucosal type 2 immune responses.

Correction to: Determinants of Influenza Mortality Trends: Age-Period-Cohort Analysis of Influenza Mortality in the United States, 1959-2016.

First, we use Lexis surfaces based on Serfling models to highlight influenza mortality patterns as well as to identify lingering effects of early-life exposure to specific influenza virus subtypes (e.g., H1N1, H3N2).

Determinants of Influenza Mortality Trends: Age-Period-Cohort Analysis of Influenza Mortality in the United States, 1959-2016.

This study examines the roles of age, period, and cohort in influenza mortality trends over the years 1959-2016 in the United States. First, we use Lexis surfaces based on Serfling models to highlight influenza mortality patterns as well as to identify lingering effects of early-life exposure to specific influenza virus subtypes (e.g., H1N1, H3N2). Second, we use age-period-cohort (APC) methods to explore APC linear trends and identify changes in the slope of these trends (contrasts). Our analyses reveal a series of breakpoints where the magnitude and direction of birth cohort trends significantly change, mostly corresponding to years in which important antigenic drifts or shifts took place (i.e., 1947, 1957, 1968, and 1978). Whereas child, youth, and adult influenza mortality appear to be influenced by a combination of cohort- and period-specific factors, reflecting the interaction between the antigenic experience of the population and the evolution of the influenza virus itself, mortality patterns of the elderly appear to be molded by broader cohort factors. The latter would reflect the processes of physiological capital improvement in successive birth cohorts through secular changes in early-life conditions. Antigenic imprinting, cohort morbidity phenotype, and other mechanisms that can generate the observed cohort effects, including the baby boom, are discussed.

The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L.

The SPFH (stomatin, prohibitin, flotillin, HflC/K) superfamily is composed of scaffold proteins that form ring-like structures and locally specify the protein-lipid composition in a variety of cellular membranes. Stomatin-like protein 2 (SLP2) is a member of this superfamily that localizes to the mitochondrial inner membrane (IM) where it acts as a membrane organizer. Here, we report that SLP2 anchors a large protease complex composed of the rhomboid protease PARL and the i-AAA protease YME1L, which we term the SPY complex (for SLP2-PARL-YME1L). Association with SLP2 in the SPY complex regulates PARL-mediated processing of PTEN-induced kinase PINK1 and the phosphatase PGAM5 in mitochondria. Moreover, SLP2 inhibits the stress-activated peptidase OMA1, which can bind to SLP2 and cleaves PGAM5 in depolarized mitochondria. SLP2 restricts OMA1-mediated processing of the dynamin-like GTPase OPA1 allowing stress-induced mitochondrial hyperfusion under starvation conditions. Together, our results reveal an important role of SLP2 membrane scaffolds for the spatial organization of IM proteases regulating mitochondrial dynamics, quality control, and cell survival.

Uncoupling of pro- and anti-inflammatory properties of Staphylococcus aureus.

Staphylococcus aureus is a Gram-positive bacterium that is carried by a quarter of the healthy human population and that can cause severe infections. This pathobiosis has been linked to a balance between Toll-like receptor 2 (TLR2)-dependent pro- and anti-inflammatory responses. The relationship between these two types of responses is unknown. Analysis of 16 nasal isolates of S. aureus showed heterogeneity in their capacity to induce pro- and anti-inflammatory responses, suggesting that these two responses are independent of each other. Uncoupling of these responses was corroborated by selective signaling through phosphoinositol 3-kinase (PI3K)-Akt-mTOR and extracellular signal-regulated kinase (ERK) for the anti-inflammatory response and through p38 for the proinflammatory response. Uncoupling was also observed at the level of phagocytosis and phagosomal processing of S. aureus, which were required solely for the proinflammatory response. Importantly, the anti-inflammatory properties of an S. aureus isolate correlated with its ability to modulate T cell immunity. Our results suggest the presence of anti-inflammatory TLR2 ligands in the staphylococcal cell wall, whose identification may provide templates for novel immunomodulatory drugs.

The role of IL-10 in microbiome-associated immune modulation and disease tolerance.

Current research on the microbiome of humans and other species is revealing a fundamental role for the interaction between the microbiota and the immune system in determining the health status of the host. In these studies, the cytokine interleukin-10 (IL-10) is emerging as an important player. We present here an overview of the developments in the field emphasizing how the microbiota composition and its interplay with immune cells affect the health of the host through changes in IL-10 production. In addition, we explore the function that IL-10-producing immune cells may have on the qualitative and quantitative changes in the microbiota and thus influence the balance between microbial commensalism and pathogenicity. In the last section of this review, we present a summary of the strategies that target IL-10 for therapeutic purposes using probiotics, purified proteins or biologicals.

Stomatin-like protein 2 deficiency in T cells is associated with altered mitochondrial respiration and defective CD4+ T cell responses.

Stomatin-like protein 2 (SLP-2) is a mostly mitochondrial protein that regulates mitochondrial biogenesis and function and modulates T cell activation. To determine the mechanism of action of SLP-2, we generated T cell-specific SLP-2-deficient mice. These mice had normal numbers of thymocytes and T cells in the periphery. However, conventional SLP-2-deficient T cells had a posttranscriptional defect in IL-2 production in response to TCR ligation, and this translated into reduced CD4(+) T cell responses. SLP-2 deficiency was associated with impaired cardiolipin compartmentalization in mitochondrial membranes, decreased levels of the NADH dehydrogenase (ubiquinone) iron-sulfur protein 3, NADH dehydrogenase (ubiquinone) 1ß subcomplex subunit 8, and NADH dehydrogenase (ubiquinone) 1α subcomplex subunit 9 of respiratory complex I, and decreased activity of this complex as well as of complex II plus III of the respiratory chain. In addition, SLP-2-deficient T cells showed a significant increase in uncoupled mitochondrial respiration and a greater reliance on glycolysis. Based on these results, we propose that SLP-2 organizes the mitochondrial membrane compartmentalization of cardiolipin, which is required for optimal assembly and function of respiratory chain complexes. This function, in T cells, helps to ensure proper metabolic response during activation.

The induction of SHP-1 degradation by TAOK3 ensures the responsiveness of T cells to TCR stimulation.

Thousand-and-one-amino acid kinase 3 (TAOK3) is a serine and threonine kinase that belongs to the STE-20 family of kinases. Its absence reduces T cell receptor (TCR) signaling and increases the interaction of the tyrosine phosphatase SHP-1, a major negative regulator of proximal TCR signaling, with the kinase LCK, a component of the core TCR signaling complex. Here, we used mouse models and human cell lines to investigate the mechanism by which TAOK3 limits the interaction of SHP-1 with LCK. The loss of TAOK3 decreased the survival of naïve CD4+ T cells by dampening the transmission of tonic and ligand-dependent TCR signaling. In mouse T cells, Taok3 promoted the secretion of interleukin-2 (IL-2) in response to TCR activation in a manner that depended on Taok3 gene dosage and on Taok3 kinase activity. TCR desensitization in Taok3-/- T cells was caused by an increased abundance of Shp-1, and pharmacological inhibition of Shp-1 rescued the activation potential of these T cells. TAOK3 phosphorylated threonine-394 in the phosphatase domain of SHP-1, which promoted its ubiquitylation and proteasomal degradation. The loss of TAOK3 had no effect on the abundance of SHP-2, which lacks a residue corresponding to SHP-1 threonine-394. Modulation of SHP-1 abundance by TAOK3 thus serves as a rheostat for TCR signaling and determines the activation threshold of T lymphocytes.

Fibroblast growth factor-23 and calcium phosphate product in young chronic kidney disease patients: a cross-sectional study.

BACKGROUND: Fibroblast growth factor-23 (FGF-23), a novel marker of bone disease in chronic kidney disease (CKD) has been shown to correlate with vascular calcifications. We aimed to describe the effect of the calcium phosphate product (Ca*P) on FGF-23 concentrations in children and young adults without confounding cardiovascular disease. METHODS: Pediatric and young adult patients with CKD stages I-V were recruited in this cross sectional study to measure FGF-23, cystatin C, vitamin D-metabolites and other serum markers of bone metabolism. FGF-23 levels were determined with an enzyme-linked immunosorbent assay. The association between FGF-23 and (Ca*P) was assessed using non-parametric methods. Patients were divided into two age groups, less than 13 years of age and greater than 13 years of age. RESULTS: This cross-sectional study measured serum FGF-23, in 81 patients (42 females, 51.9%) at London Health Sciences Centre, aged 2 to 25 years, with various stages of CKD (Cystatin C estimated glomerular filtration rate, eGFR=10.7-213.0 ml/min). For the whole entire group of patients, FGF-23 levels were found to correlate significantly with age (Spearman r= 0.26, p=0.0198), Cystatin C eGFR (Spearman r=-0.40 p=0.0002), CKD stage (Spearman r=0.457, p<0.0001), PTH (Spearman r=0.330, p=0.0039), ionized calcium (Spearman r=-0.330, p=0.0049), CysC (Spearman r= 0.404, p=0.0002) and 1,25-dihydroxyvitamin D (Spearman r=-0.345, p=0.0034) concentrations. No significant correlation was found between FGF-23 levels and calcium phosphate product (Spearman r= 0.164, p=0.142). Upon classification of patients into two age groups, less than 13 years of age and more than 13 years of age, correlational results differed significantly. FGF-23 correlated with CysC eGFR( Spearman r= -0.633, p<0.0001), CKD stage (Spearman r=0.731, p<0.0001), phosphate (Spearman r= 0.557, p<0.0001), calcium phosphate product (Spearman r=0.534, p<0.0001), 125(OH)2 Vit D (Spearman r=-0.631, p<0.0001), PTH (Spearman r= 0.475, p=0.0017) and ionized calcium (Spearman r= -0.503, p=0.0015) only in the older group. The relationship between FGF-23 and Ca*P for the older group could be expressed by the exponential model FGF-23= 38.15 e0.4625Ca*P. CONCLUSION: Abnormal values of FGF-23 in adolescents and young adults with CKD correlate with Ca* P in the absence of vascular calcifications, and may serve as a biomarker for the risk of cardiovascular calcifications.

NOD2 Agonism Counter-Regulates Human Type 2 T Cell Functions in Peripheral Blood Mononuclear Cell Cultures: Implications for Atopic Dermatitis.

Atopic dermatitis (AD) is known as a skin disease; however, T cell immunopathology found in blood is associated with its severity. Skin Staphylococcus aureus (S. aureus) and associated host-pathogen dynamics are important to chronic T helper 2 (Th2)-dominated inflammation in AD, yet they remain poorly understood. This study sought to investigate the effects of S. aureus-derived molecules and skin alarmins on human peripheral blood mononuclear cells, specifically testing Th2-type cells, cytokines, and chemokines known to be associated with AD. We first show that six significantly elevated Th2-related chemokine biomarkers distinguish blood from adult AD patients compared to healthy controls ex vivo; in addition, TARC/CCL17, LDH, and PDGF-AA/AB correlated significantly with disease severity. We then demonstrate that these robust AD-associated biomarkers, as well as associated type 2 T cell functions, are readily reproduced from healthy blood mononuclear cells exposed to the alarmin TSLP and the S. aureus superantigen SEB in a human in vitro model, including IL-13, IL-5, and TARC secretion as well as OX-40-expressing activated memory T cells. We further show that the agonism of nucleotide-binding oligomerization domain-containing protein (NOD)2 inhibits this IL-13 secretion and memory Th2 and Tc2 cell functional activation while inducing significantly increased pSTAT3 and IL-6, both critical for Th17 cell responses. These findings identify NOD2 as a potential regulator of type 2 immune responses in humans and highlight its role as an endogenous inhibitor of pathogenic IL-13 that may open avenues for its therapeutic targeting in AD.

The T cell receptor beta-chain second complementarity determining region loop (CDR2beta governs T cell activation and Vbeta specificity by bacterial superantigens.

Superantigens (SAgs) are microbial toxins defined by their ability to activate T lymphocytes in a T cell receptor (TCR) ß-chain variable domain (Vß)-specific manner. Although existing structural information indicates that diverse bacterial SAgs all uniformly engage the Vß second complementarity determining region (CDR2ß) loop, the molecular rules that dictate SAg-mediated T cell activation and Vß specificity are not fully understood. Herein we report the crystal structure of human Vß2.1 (hVß2.1) in complex with the toxic shock syndrome toxin-1 (TSST-1) SAg, and mutagenesis of hVß2.1 indicates that the non-canonical length of CDR2ß is a critical determinant for recognition by TSST-1 as well as the distantly related SAg streptococcal pyrogenic exotoxin C. Frame work (FR) region 3 is uniquely critical for TSST-1 function explaining the fine Vß-specificity exhibited by this SAg. Furthermore, domain swapping experiments with SAgs, which use distinct domains to engage both CDR2ß and FR3/4ß revealed that the CDR2ß contacts dictate T lymphocyte Vß-specificity. These findings demonstrate that the TCR CDR2ß loop is the critical determinant for functional recognition and Vß-specificity by diverse bacterial SAgs.

A modulatory interleukin-10 response to staphylococcal peptidoglycan prevents Th1/Th17 adaptive immunity to Staphylococcus aureus.

Toll-like receptor (TLR) 2 on antigen-presenting cells (APCs) enables these cells to recognize peptidoglycan-embedded lipopeptides and glycopolymers in the Staphylococcus aureus cell wall and mount an inflammatory response to this microbe. TLR2 signalling can also modulate immunity to S. aureus by inducing an interleukin (IL)-10 response in APCs. What determines the balance between proinflammatory and modulatory responses to S. aureus is unknown. We show that the modulatory IL-10 response preferentially occurs upon CD14- and CD36-independent TLR2 signaling, triggering PI3K activation, and is restricted to monocytes and monocyte-derived macrophages (MΦs). In contrast, monocyte-derived dendritic cells (DCs) produce mostly IL-12 and IL-23. The differential APC polarization induced by staphylococcal peptidoglycan translates into differential T helper responses: MΦs primarily trigger IL-10 and weak IL-17 responses, whereas DCs trigger a robust Th1/Th17 response. Exploitation of TLR2 signalling plasticity by S. aureus may explain the wide range of outcomes of human encounters with this microbe.

CTLA-4Ig blocks the development and progression of citrullinated fibrinogen-induced arthritis in DR4-transgenic mice.

OBJECTIVE: To assess the role of T cells in the mouse model of citrullinated human fibrinogen-induced rheumatoid arthritis (RA) using CTLA-4Ig, an agent that blocks T cell costimulation, which is required for T cell activation. METHODS: Humanized HLA-DRß1*0401-transgenic (DR4-Tg) mice were immunized with Cit-human fibrinogen to induce arthritis. Prior to, and at the onset or peak of, arthritis, the DR4-Tg mice were treated with CTLA-4Ig or control human IgG1 or were left untreated. Arthritis development and progression were monitored by measuring ankle swelling with calipers and by assessing histopathologic changes. The immune responses to the citrullinated antigens and the corresponding unmodified antigens, as well as the arthritogenicity of lymphocytes from these mice, were examined. The latter was performed using lymphocyte transfers from CTLA-4Ig-treated or control mice via intraperitoneal injection into naive DR4-Tg mice. Recipient mice also received an intraarticular injection of Cit-human fibrinogen, unmodified human fibrinogen, or vehicle. RESULTS: CTLA-4Ig-treated, but not human IgG1-treated, arthritic mice had significantly reduced ankle swelling and pathologic joint damage. Treatment with CTLA-4Ig, but not human IgG1, suppressed Cit-human fibrinogen-induced T cell activation, including citrulline-specific T cell activation, when given prior to disease onset. Transfer of splenic lymphocytes from untreated or human IgG1-treated arthritic mice caused arthritis in recipients, and this occurred when Cit-human fibrinogen, but not unmodified fibrinogen, was deposited into the joint. Splenocytes from CTLA-4Ig-treated mice were unable to transfer arthritis. CONCLUSION: Activated citrulline-specific T cells play a direct role in the development and progression of arthritis in this model of Cit-human fibrinogen-induced RA.

A novel, ataxic mouse model of ataxia telangiectasia caused by a clinically relevant nonsense mutation.

Ataxia Telangiectasia (A-T) and Ataxia with Ocular Apraxia Type 1 (AOA1) are devastating neurological disorders caused by null mutations in the genome stability genes, A-T mutated (ATM) and Aprataxin (APTX), respectively. Our mechanistic understanding and therapeutic repertoire for treating these disorders are severely lacking, in large part due to the failure of prior animal models with similar null mutations to recapitulate the characteristic loss of motor coordination (i.e., ataxia) and associated cerebellar defects. By increasing genotoxic stress through the insertion of null mutations in both the Atm (nonsense) and Aptx (knockout) genes in the same animal, we have generated a novel mouse model that for the first time develops a progressively severe ataxic phenotype associated with atrophy of the cerebellar molecular layer. We find biophysical properties of cerebellar Purkinje neurons (PNs) are significantly perturbed (e.g., reduced membrane capacitance, lower action potential [AP] thresholds, etc.), while properties of synaptic inputs remain largely unchanged. These perturbations significantly alter PN neural activity, including a progressive reduction in spontaneous AP firing frequency that correlates with both cerebellar atrophy and ataxia over the animal's first year of life. Double mutant mice also exhibit a high predisposition to developing cancer (thymomas) and immune abnormalities (impaired early thymocyte development and T-cell maturation), symptoms characteristic of A-T. Finally, by inserting a clinically relevant nonsense-type null mutation in Atm, we demonstrate that Small Molecule Read-Through (SMRT) compounds can restore ATM production, indicating their potential as a future A-T therapeutic.

Surface cytotoxic T lymphocyte-associated antigen 4 partitions within lipid rafts and relocates to the immunological synapse under conditions of inhibition of T cell activation.

T cell activation through the T cell receptor (TCR) involves partitioning of receptors into discrete membrane compartments known as lipid rafts, and the formation of an immunological synapse (IS) between the T cell and antigen-presenting cell (APC). Compartmentalization of negative regulators of T cell activation such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) is unknown. Recent crystal structures of B7-ligated CTLA-4 suggest that it may form lattices within the IS which could explain the mechanism of action of this molecule. Here, we show that after T cell stimulation, CTLA-4 coclusters with the TCR and the lipid raft ganglioside GM1 within the IS. Using subcellular fractionation, we show that most lipid raft-associated CTLA-4 is on the T cell surface. Such compartmentalization is dependent on the cytoplasmic tail of CTLA-4 and can be forced with a glycosylphosphatidylinositol-anchor in CTLA-4. The level of CTLA-4 within lipid rafts increases under conditions of APC-dependent TCR-CTLA-4 coligation and T cell inactivation. However, raft localization, although necessary for inhibition of T cell activation, is not sufficient for CTLA-4-mediated negative signaling. These data demonstrate that CTLA-4 within lipid rafts migrates to the IS where it can potentially form lattice structures and inhibit T cell activation.