bluuebunny/crossref_metadata_2025 huggingface dataset:
Cytokine-Induced Hepatic Apoptosis Is Dependent on FGL2/Fibroleukin: The Role of Sp1/Sp3 and STAT1/PU.1 CompositecisElements
Authors: Liu Mingfeng , Mendicino Michael , Ning Qin , Ghanekar Anand , He Wei , McGilvray Ian , Shalev Itay , Pivato David , Clark David A. , Phillips M. James , Levy Gary A.
Published: April 2014

Previous studies from our laboratory have shown that fulminant hepatitis caused by the mouse hepatitis virus, MHV-3, is dependent on production of the novel immune coagulant fgl2/fibroleukin. In this study, we investigate the role of IFN-γ and TNF-α in the induction of fgl2 expression and fgl2-dependent hepatic apoptosis. Infusion of IFN-γ in combination with TNF-α through the portal vein of fgl2+/+ mice led to widespread hepatic apoptosis and fibrin deposition. Livers from fgl2−/− mice were normal, although strong expression of the fgl2 knockout reporter gene Lac Z was seen in both resident hepatic macrophages and endothelial cells. In vitro, IFN-γ and TNF-α induced fgl2 expression in a macrophage and endothelial cell-specific manner. In macrophages (peritoneal and RAW 264.7 cells), IFN-γ, but not IFN-α, LPS, TNF-α, or IL-1 induced fgl2 mRNA transcription and protein expression, while in endothelial cells TNF-α, but not IFN-γ, induced fgl2 transcription. In addition, while TNF-α enhanced IFN-γ-induced macrophage fgl2 transcription, IFN-γ also enhanced TNF-α-induced endothelial cell fgl2 transcription. The induction of fgl2 by IFN-γ in macrophages involved a STAT1-dependent pathway, involving the composite cis elements Sp1/Sp3 and GAS/PU.1. The latter interacted with IFN-γ-dependent Sp1/Sp3, STAT1, and the ETS family of transcription factors member PU.1. The interaction of PU.1 with the IFN-γ-activated sequence/ETS family of transcription factors site determined the macrophage-specific induction of fgl2 by IFN-γ. Overall, this study demonstrates that IFN-γ and TNF-α induce hepatocyte apoptosis in vivo, which is dependent on induction of fgl2, and defines the molecular basis of transcription of fgl2 in vitro.
Platelet-Activating Factor-Induced Clathrin-Mediated Endocytosis Requires β-Arrestin-1 Recruitment and Activation of the p38 MAPK Signalosome at the Plasma Membrane for Actin Bundle Formation
Authors: McLaughlin Nathan J. D. , Banerjee Anirban , Kelher Marguerite R. , Gamboni-Robertson Fabia , Hamiel Christine , Sheppard Forest R. , Moore Ernest E. , Silliman Christopher C.
Published: April 2014

Clathrin-mediated endocytosis (CME) is a common pathway used by G protein-linked receptors to transduce extracellular signals. We hypothesize that platelet-activating factor (PAF) receptor (PAFR) ligation requires CME and causes engagement of β-arrestin-1 and recruitment of a p38 MAPK signalosome that elicits distinct actin rearrangement at the receptor before endosomal scission. Polymorphonuclear neutrophils were stimulated with buffer or 2 μM PAF (1 min), and whole cell lysates or subcellular fractions were immunoprecipitated or slides prepared for colocalization and fluorescent resonance energy transfer analysis. In select experiments, β-arrestin-1 or dynamin-2 were neutralized by intracellular introduction of specific Abs. PAFR ligation caused 1) coprecipitation of the PAFR and clathrin with β-arrestin-1, 2) fluorescent resonance energy transfer-positive interactions among the PAFR, β-arrestin-1, and clathrin, 3) recruitment and activation of the apoptosis signal-regulating kinase-1/MAPK kinase-3/p38 MAPK (ASK1/MKK3/p38 MAPK) signalosome, 4) cell polarization, and 5) distinct actin bundle formation at the PAFR. Neutralization of β-arrestin-1 inhibited all of these cellular events, including PAFR internalization; conversely, dynamin-2 inhibition only affected receptor internalization. Selective p38 MAPK inhibition globally abrogated actin rearrangement; however, inhibition of MAPK-activated protein kinase-2 and its downstream kinase leukocyte-specific protein-1 inhibited only actin bundle formation and PAFR internalization. In addition, ASK1/MKK3/p38 MAPK signalosome assembly appears to occur in a novel manner such that the ASK1/p38 MAPK heterodimer is recruited to a β-arrestin-1 bound MKK3. In polymorphonuclear neutrophils, leukocyte-specific protein-1 may play a role similar to fascin for actin bundle formation. We conclude that PAF signaling requires CME, β-arrestin-1 recruitment of a p38 MAPK signalosome, and specific actin bundle formation at the PAFR for transduction before endosomal scission.
Leukotriene A4 Hydrolase Expression in PEL Cells Is Regulated at the Transcriptional Level and Leads to Increased Leukotriene B4 Production
Authors: Arguello Meztli , Paz Suzanne , Hernandez Eduardo , Corriveau-Bourque Catherine , Fawaz Lama M. , Hiscott John , Lin Rongtuan
Published: April 2014

Primary effusion lymphoma (PEL) is a herpesvirus-8-associated lymphoproliferative disease characterized by migration of tumor cells to serous body cavities. PEL cells originate from postgerminal center B cells and share a remarkable alteration in B cell transcription factor expression and/or activation with classical Hodgkin’s disease cells. Comparative analysis of gene expression by cDNA microarray of BCBL-1 cells (PEL), L-428 (classical Hodgkin’s disease), and BJAB cells revealed a subset of genes that were differentially expressed in BCBL-1 cells. Among these, four genes involved in cell migration and chemotaxis were strongly up-regulated in PEL cells: leukotriene A4 (LTA4) hydrolase (LTA4H), IL-16, thrombospondin-1 (TSP-1), and selectin-P ligand (PSGL-1). Up-regulation of LTA4H was investigated at the transcriptional level. Full-length LTA4H promoter exhibited 50% higher activity in BCBL-1 cells than in BJAB or L-428 cells. Deletion analysis of the LTA4H promoter revealed a positive cis-regulatory element active only in BCBL-1 cells in the promoter proximal region located between −76 and −40 bp. Formation of a specific DNA-protein complex in this region was confirmed by EMSA. Coculture of ionophore-stimulated primary neutrophils with BCBL-1 cells leads to an increased production of LTB4 compared with coculture with BJAB and L-428 cells as measured by enzyme immunoassay, demonstrating the functional significance of LTA4H up-regulation.
The Histamine H4 Receptor Mediates Allergic Airway Inflammation by Regulating the Activation of CD4+ T Cells
Authors: Dunford Paul J. , O’Donnell Niall , Riley Jason P. , Williams Kacy N. , Karlsson Lars , Thurmond Robin L.
Published: April 2014

Histamine is an important inflammatory mediator that is released in airways during an asthmatic response. However, current antihistamine drugs are not effective in controlling the disease. The discovery of the histamine H4 receptor (H4R) prompted us to reinvestigate the role of histamine in pulmonary allergic responses. H4R-deficient mice and mice treated with H4R antagonists exhibited decreased allergic lung inflammation, with decreases in infiltrating lung eosinophils and lymphocytes and decreases in Th2 responses. Ex vivo restimulation of T cells showed decreases in IL-4, IL-5, IL-13, IL-6, and IL-17 levels, suggesting that T cell functions were disrupted. In vitro studies indicated that blockade of the H4R on dendritic cells leads to decreases in cytokine and chemokine production and limits their ability to induce Th2 responses in T cells. This work suggests that the H4R can modulate allergic responses via its influence on T cell activation. The study expands the known influences of histamine on the immune system and highlights the therapeutic potential of H4R antagonists in allergic conditions.
Diagnostic and Therapeutic Potential of Amyloid-Reactive IgG Antibodies Contained in Human Sera
Authors: O’Nuallain Brian , Hrncic Rudi , Wall Jonathan S. , Weiss Deborah T. , Solomon Alan
Published: April 2014

Passive immunotherapy using fibril-reactive mAbs has been shown experimentally to reduce amyloid formation and also accelerate amyloidolysis. We now report that human sera, as well as various sources of pooled human IgG, including pharmacologic formulations of immune globulin i.v. (IGIV), contain Abs that specifically recognize fibrils formed from light chains and other amyloidogenic precursor proteins, including serum amyloid A, transthyretin, islet amyloid polypeptide, and amyloid β 1–40 peptide, but notably, do not react with these molecules in their native nonfibrillar forms. After isolation of the Abs from IGIV via fibril-conjugated affinity column chromatography, the EC50-binding value for light chains and amyloid β 1–40 peptide fibrils was ∼15 nM–a magnitude ∼200 and 70 times less than that of the unbound fraction and unfractionated product, respectively. Comparable reactivity was found in the case of those formed from serum amyloid A, transthyretin, and islet amyloid polypeptide. The purified Abs immunostained human amyloid tissue deposits and could inhibit fibrillogenesis, as shown in fibril formation and extension assays. Most importantly, in vivo reactivity was evidenced in a murine model when the enriched Abs were used to image amyloid, as well as expedite its removal. These promising experimental results suggest that fibril affinity-purified IGIV has potential as a diagnostic and therapeutic agent for patients with amyloid-associated disease.
The Role of Noradrenergic Nerves in the Development of the Lymphoproliferative Disease in Fas-Deficient, lpr/lpr Mice
Authors: del Rey Adriana , Roggero Eduardo , Kabiersch Alexa , Schäfer Martin , Besedovsky Hugo O.
Published: April 2014

Lpr/lpr mice develop a lymphoproliferative, autoimmune, lupus-like disease. These mice lack functional Fas (CD95) expression and are resistant to Fas ligand (CD178)-mediated apoptosis, a critical mechanism for the maintenance of peripheral tolerance. In this study, we show that noradrenaline (NA), the main sympathetic neurotransmitter, can induce apoptosis of lymphoid cells independently of functional Fas. Based on this finding, we used lpr/lpr mice as model to study the role of noradrenergic nerves in the expression of a lymphoproliferative disease. Early in ontogeny, the concentration of NA was significantly increased in the spleen of lpr/lpr mice, compared with normal littermates. However, splenic sympathetic innervation gradually declined as the disease progressed, and IgM blood levels and splenic NA concentration inversely correlated when the disease was overtly manifested. When the loss of noradrenergic fibers that occurred naturally during adult life in lpr/lpr mice was experimentally advanced by neonatal sympathectomy, the concentration of IgM and IgG2a in blood was markedly higher than that of control lpr/lpr mice, and the appearance of lymphadenopathy was accelerated. Furthermore, although neonatal denervation did not affect the life span of normal animals, it shortened significantly the survival time of lpr/lpr mice. These data show that, in addition to defects in the Fas pathway, an altered sympathetic innervation in lpr/lpr mice also contributes to the pathogenesis of the autoimmune disease, and strongly support the hypothesis that the sympathetic nervous system can modulate the expression of lymphoproliferative diseases.
CXCR3 and Its Ligands in a Murine Model of Obliterative Bronchiolitis: Regulation and Function
Authors: Medoff Benjamin D. , Wain John C. , Seung Edward , Jackobek Ryan , Means Terry K. , Ginns Leo C. , Farber Joshua M. , Luster Andrew D.
Published: April 2014

Lung transplantation remains the only effective therapy for patients with end-stage lung disease, but survival is limited by the development of obliterative bronchiolitis (OB). The chemokine receptor CXCR3 and two of its ligands, CXCL9 and CXCL10, have been identified as important mediators of OB. However, the relative contribution of CXCL9 and CXCL10 to the development of OB and the mechanism of regulation of these chemokines has not been well defined. In this study, we demonstrate that CXCL9 and CXCL10 are up-regulated in unique patterns following tracheal transplantation in mice. In these experiments, CXCL9 expression peaked 7 days posttransplant, while CXCL10 expression peaked at 1 day and then again 7 days posttransplant. Expression of CXCL10 was also up-regulated in a novel murine model of lung ischemia, and in bronchoalveolar lavage fluid taken from human lungs 24 h after lung transplantation. In further analysis, we found that 3 h after transplantation CXCL10 is donor tissue derived and not dependent on IFN-γ or STAT1, while 24 h after transplantation CXCL10 is from recipient tissue and regulated by IFN-γ and STAT1. Expression of both CXCL9 and CXCL10 7 days posttransplant is regulated by IFN-γ and STAT1. Finally, we demonstrate that deletion of CXCR3 in recipients reduces airway obliteration. However, deletion of either CXCL9 or CXCL10 did not affect airway obliteration. These data show that in this murine model of obliterative bronchiolitis, these chemokines are differentially regulated following transplantation, and that deletion of either chemokine alone does not affect the development of airway obliteration.
Patients with Chronic Granulomatous Disease Have a Reduced Peripheral Blood Memory B Cell Compartment
Authors: Bleesing Jack J. , Souto-Carneiro Margarida M. , Savage William J. , Brown Margaret R. , Martinez Cynthia , Yavuz Sule , Brenner Sebastian , Siegel Richard M. , Horwitz Mitchell E. , Lipsky Peter E. , Malech Harry L. , Fleisher Thomas A.
Published: April 2014

In this study, we have identified an altered B cell compartment in patients with chronic granulomatous disease (CGD), a disorder of phagocyte function, characterized by pyogenic infections and granuloma formation caused by defects in NADPH activity. This is characterized by an expansion of CD5-expressing B cells, and profound reduction in B cells expressing the memory B cell marker, CD27. Both findings were independent of the age, genotype, and clinical status of the patients, and were not accompanied by altered CD5 and CD27 expression on T cells. Focusing on CD27-positive B cells, considered to be memory cells based on somatically mutated Ig genes, we found that the reduction was not caused by CD27 shedding or abnormal retention of CD27 protein inside the cell. Rather, it was determined that CD27-negative B cells were, appropriately, CD27 mRNA negative, consistent with a naive phenotype, whereas CD27-positive B cells contained abundant CD27 mRNA and displayed somatic mutations, consistent with a memory B cell phenotype. Thus, it appears that CGD is associated with a significant reduction in the peripheral blood memory B cell compartment, but that the basic processes of somatic mutation and expression of CD27 are intact. X-linked carriers of CGD revealed a significant correlation between the percentage of CD27-positive B cells and the percentage of neutrophils with normal NADPH activity, reflective of the degree of X chromosome lyonization. These results suggest a role for NADPH in the process of memory B cell formation, inviting further exploration of secondary Ab responses in CGD patients.
Identical TCR β-Chain Rearrangements in Streptococcal Angina and Skin Lesions of Patients with Psoriasis Vulgaris
Authors: Diluvio Laura , Vollmer Sigrid , Besgen Petra , Ellwart Joachim W. , Chimenti Sergio , Prinz Joerg C.
Published: April 2014

Tonsillar infection with Streptococcus pyogenes may induce several nonsuppurative autoimmune sequelae. The precise pathogenetic mechanisms behind this clinically well-established association are still unresolved. Using TCR analysis, we sought to identify a link between streptococcal tonsillitis and the T cell-mediated autoimmune response in psoriasis. Three patients with streptococcal-induced psoriasis underwent tonsillectomy. Using size spectratyping and sequencing of TCR β-chain variable region gene (TCRBV) rearrangements, we compared the TCR usage of psoriatic skin lesions, blood, tonsils, and tonsillar T cells fractionated according to the expression of the skin addressin “cutaneous lymphocyte-associated Ag” (CLA). TCRBV-size spectratype analysis of the blood lymphocytes, tonsils, and the CLA-negative tonsillar T cells revealed largely unselected T cell populations. Instead, TCRBV gene families of the psoriatic lesions and skin-homing CLA-positive tonsillar T cells displayed highly restricted spectratypes. Sequencing of TCRBV cDNA identified various clonal TCRBV rearrangements within the psoriatic lesions that indicated Ag-driven T cell expansion. Several of these clonotypes were also detected within the tonsils and, in one of the patients, within the small subset of CLA-positive tonsillar T cells, suggesting that T cells from the same T cell clones were simultaneously present within skin and tonsillar tissue. Because after tonsillectomy psoriasis cleared in all three patients our observations indicate that T cells may connect psoriatic inflammation to streptococcal angina. They suggest that the chronic streptococcal immune stimulus within the tonsils could act as a source for pathogenic T cells in poststreptococcal disorders, and they may help to explain why eliminating this source with tonsillectomy may improve streptococcal-induced sequelae.
Carriage of DRB1*13 Is Associated with Increased Posttreatment IgE Levels against Schistosoma mansoni Antigens and Lower Long-Term Reinfection Levels
Authors: Booth Mark , Shaw Marie A. , Carpenter Danielle , Joseph Sarah , Kabatereine Narcis B. , Kariuki Henry C. , Mwatha Joseph K. , Jones Frances M. , Vennervald Brigitte J. , Ouma John H. , Dunne David W.
Published: April 2014

Praziquantel treatment for Schistosoma mansoni infection enhances Th2 responsiveness against parasite Ags, but also increases the variance in Ab isotype levels. This effect may arise partly from genetic heterogeneity. In this study, associations between HLA polymorphisms at three loci (HLA-DQB1, HLA-DQA1, and HLA-DRB1) and posttreatment Ig responses to S. mansoni Ags were assessed in 199 individuals aged 7–50 years from Uganda. Blood samples were assayed for IgG1, IgG4, and IgE levels against soluble worm Ag (SWA), soluble egg Ag, tegument Ag, and a recombinant tegumental Ag (rSm 22.6) 7 wk after treatment. Multivariate ANOVA analysis initially revealed associations between carriage of DRB1*13 and increased levels of IgG1, IgG4, and IgE against SWA, tegument Ag, and rSM22.6. Subsequent analysis of covariance, which controlled for correlations between isotype levels and also included pretreatment IL-4, IL-5, and IL-13 responsiveness against SWA as covariates, revealed an independent association only between DRB1*13 and a factor score summarizing IgE levels to worm-derived Ags, which was strongest in adults. A post hoc age- and sex-stratified analysis revealed lower reinfection intensities at 1 year, 22 mo, and 6 years after the first round of treatment among carriers of DRB1*13. These results indicate that genetic background has a prominent influence on the posttreatment Th2 immune response to S. mansoni Ags, as well as a downstream association with long-term reinfection levels.
Therapeutic Induction of Regulatory, Cytotoxic CD8+ T Cells in Multiple Sclerosis
Authors: Tennakoon Deepani K. , Mehta Riyaz S. , Ortega Sterling B. , Bhoj Vijay , Racke Michael K. , Karandikar Nitin J.
Published: April 2014

In the setting of autoimmunity, one of the goals of successful therapeutic immune modulation is the induction of peripheral tolerance, a large part of which is mediated by regulatory/suppressor T cells. In this report, we demonstrate a novel immunomodulatory mechanism by an FDA-approved, exogenous peptide-based therapy that incites an HLA class I-restricted, cytotoxic suppressor CD8+ T cell response. We have shown previously that treatment of multiple sclerosis (MS) with glatiramer acetate (GA; Copaxone) induces differential up-regulation of GA-reactive CD8+ T cell responses. We now show that these GA-induced CD8+ T cells are regulatory/suppressor in nature. Untreated patients show overall deficit in CD8+ T cell-mediated suppression, compared with healthy subjects. GA therapy significantly enhances this suppressive ability, which is mediated by cell contact-dependent mechanisms. CD8+ T cells from GA-treated patients and healthy subjects, but not those from untreated patients with MS, exhibit potent, HLA class I-restricted, GA-specific cytotoxicity. We further show that these GA-induced cytotoxic CD8+ T cells can directly kill CD4+ T cells in a GA-specific manner. Killing is enhanced by preactivation of target CD4+ T cells and may depend on presentation of GA through HLA-E. Thus, we demonstrate that GA therapy induces a suppressor/cytotoxic CD8+ T cell response, which is capable of modulating in vivo immune responses during ongoing therapy. These studies not only explain several prior observations relating to the mechanism of this drug but also provide important insights into the natural immune interplay underlying this human immune-mediated disease.
Effects of Aging on the Common Lymphoid Progenitor to Pro-B Cell Transition
Authors: Min Hyeyoung , Montecino-Rodriguez Encarnacion , Dorshkind Kenneth
Published: April 2014

The number of common lymphoid progenitors (CLP) and their pre-pro-B and pro-B cell progeny is reduced in old mice, but the age-related changes responsible for these declines have not been fully elucidated. The aim of this study was to provide additional insights into the impact of senescence on early B cell development by analyzing the CLP and pro-B cell compartments under steady-state conditions and after cytoablation with 5-fluorouracil. 5-Fluorouracil subjects the hemopoietic system to acute stress and has the advantage of revealing defects in progenitors that may otherwise be subtle. The data demonstrate significant, age-related defects in the proliferative potential of early B cell precursors and suggest that the ability of CLP to differentiate into pre-pro-B cells is also compromised by senescence. These age-related changes in early B lymphopoiesis do not result from a general defect in HSC or the bone marrow microenvironment that impairs development in all hemopoietic lineages. Instead, data demonstrating that myeloid progenitor number and developmental potential do not decline with age indicate that B lymphopoiesis is particularly sensitive to defects that accumulate during senescence.
Involvement of TNF Receptor-Associated Factor 6 in IL-25 Receptor Signaling
Authors: Maezawa Yuko , Nakajima Hiroshi , Suzuki Kotaro , Tamachi Tomohiro , Ikeda Kei , Inoue Jun-ichiro , Saito Yasushi , Iwamoto Itsuo
Published: April 2014

IL-25 (IL-17E) induces IL-4, IL-5, and IL-13 production from an unidentified non-T/non-B cell population and subsequently induces Th2-type immune responses such as IgE production and eosinophilic airway inflammation. IL-25R is a single transmembrane protein with homology to IL-17R, but the IL-25R signaling pathways have not been fully understood. In this study, we investigated the signaling pathway under IL-25R, especially the possible involvement of TNFR-associated factor (TRAF)6 in this pathway. We found that IL-25R cross-linking induced NF-κB activation as well as ERK, JNK, and p38 activation. We also found that IL-25R-mediated NF-κB activation was inhibited by the expression of dominant negative TRAF6 but not of dominant negative TRAF2. Furthermore, IL-25R-mediated NF-κB activation, but not MAPK activation, was diminished in TRAF6-deficient murine embryonic fibroblast. In addition, coimmunoprecipitation assay revealed that TRAF6, but not TRAF2, associated with IL-25R even in the absence of ligand binding. Finally, we found that IL-25R-mediated gene expression of IL-6, TGF-β, G-CSF, and thymus and activation-regulated chemokine was diminished in TRAF6-deficient murine embryonic fibroblast. Taken together, these results indicate that TRAF6 plays a critical role in IL-25R-mediated NF-κB activation and gene expression.
Endothelial Protein C Receptor-Dependent Inhibition of Migration of Human Lymphocytes by Protein C Involves Epidermal Growth Factor Receptor
Authors: Feistritzer Clemens , Mosheimer Birgit A. , Sturn Daniel H. , Riewald Matthias , Patsch Josef R. , Wiedermann Christian J.
Published: April 2014

The protein C pathway is an important regulator of the blood coagulation system. Protein C may also play a role in inflammatory and immunomodulatory processes. Whether protein C or activated protein C affects lymphocyte migration and possible mechanisms involved was tested. Lymphocyte migration was studied by micropore filter assays. Lymphocytes that were pretreated with protein C (Ceprotin) or activated protein C (Xigris) significantly reduced their migration toward IL-8, RANTES, MCP-1, and substance P, but not toward sphingosine-1-phosphate. The inhibitory effects of protein C or activated protein C were reversed by Abs against endothelial protein C receptor and epidermal growth factor receptor. Evidence for the synthesis of endothelial protein C receptor by lymphocytes is shown by demonstration of receptor mRNA expression and detection of endothelial protein C receptor immunoreactivity on the cells’ surface. Data suggest that an endothelial protein C receptor is expressed by lymphocytes whose activation with protein C or activated protein C arrests directed migration. Exposure of lymphocytes to protein C or activated protein C stimulates phosphorylation of Tyr845 of epidermal growth factor receptor, which may be relevant for cytoprotective effects of the protein C pathway.
TNF Receptor Type 2 (p75) Functions as a Costimulator for Antigen-Driven T Cell Responses In Vivo
Authors: Kim Edward Y. , Priatel John J. , Teh Soo-Jeet , Teh Hung-Sia
Published: April 2014

Naive T cells require costimulation for robust Ag-driven differentiation and survival. Members of the TNFR family have been shown to provide costimulatory signals conferring survival at distinct phases of the T cell response. In this study, we show that CD4 and CD8 T cells depend on TNFR type 2 (p75) for survival during clonal expansion, allowing larger accumulation of effector cells and conferring protection from apoptosis for a robust memory pool in vivo. We demonstrate using the MHC class I-restricted 2C TCR and MHC class II-restricted AND TCR transgenic systems that TNFR2 regulates the threshold for clonal expansion of CD4 and CD8 T cell subsets in response to cognate Ag. Using a novel recombinant Listeria monocytogenes (rLM) expressing a secreted form of the 2C agonist peptide (SIY) to investigate the role of TNFR2 for T cell immunity in vivo, we found that TNFR2 controls the survival and accumulation of effector cells during the primary response. TNFR2−/− CD8 T cells exhibit loss of protection from apoptosis that is correlated with diminished survivin and Bcl-2 expression. Null mutant mice were more susceptible to rLM-SIY challenge at high doses of primary infection, correlating with impaired LM-specific T cell response in the absence of TNFR2-mediated costimulation. Moreover, the resulting memory pools specific for SIY and listeriolysin O epitopes derived from rLM-SIY were diminished in TNFR2−/− mice. Thus, examination of Ag-driven T cell responses revealed a hitherto unknown costimulatory function for TNFR2 in regulating T cell survival during the differentiation program elicited by intracellular pathogen in vivo.
T-bet Down-Modulation in Tolerized Th1 Effector CD4 Cells Confers a TCR-Distal Signaling Defect That Selectively Impairs IFN-γ Expression
Authors: Long Meixiao , Slaiby Aaron M. , Hagymasi Adam T. , Mihalyo Marianne A. , Lichtler Alexander C. , Reiner Steven L. , Adler Adam J.
Published: April 2014

When Th1 effector CD4 cells encounter tolerizing Ag in vivo, their capacity to express the effector cytokines IFN-γ and TNF-α is lost more rapidly than noneffector functions such as IL-2 production and proliferation. To localize the relevant intracellular signaling defects, cytokine expression was compared following restimulation with Ag vs agents that bypass TCR-proximal signaling. IFN-γ and TNF-α expression were both partially rescued when TCR-proximal signaling was bypassed, indicating that both TCR-proximal and -distal signaling defects impair the expression of these two effector cytokines. In contrast, bypassing TCR-proximal signaling fully rescued IL-2 expression. T-bet, a transcription and chromatin remodeling factor that is required to direct the differentiation of naive CD4 cells into IFN-γ-expressing Th1 effectors, was partially down-modulated in tolerized Th1 effectors. Enforcing T-bet expression during tolerization selectively rescued the ability to express IFN-γ, but not TNF-α. Conversely, expression of a dominant-negative T-bet in Th1 effectors selectively impaired the ability to express IFN-γ, but not TNF-α. Analysis of histone acetylation at the IFN-γ promoter further suggested that down-modulation of T-bet expression during Th1 effector CD4 cell tolerization does not impair IFN-γ expression potential through alterations in chromatin structure.
Microglia Proliferation Is Regulated by Hydrogen Peroxide from NADPH Oxidase
Authors: Mander Palwinder K. , Jekabsone Aiste , Brown Guy C.
Published: April 2014

Microglia are resident brain macrophages that become activated and proliferate following brain damage or stimulation by immune mediators, such as IL-1β or TNF-α. We investigated the mechanisms by which microglial proliferation is regulated in primary cultures of rat glia. We found that basal proliferation of microglia was stimulated by proinflammatory cytokines IL-1β or TNF-α, and this proliferation was completely inhibited by catalase, implicating hydrogen peroxide as a mediator of proliferation. In addition, inhibitors of NADPH oxidase (diphenylene iodonium or apocynin) also prevented microglia proliferation, suggesting that this may be the source of hydrogen peroxide. IL-1β and TNF-α rapidly stimulated the rate of hydrogen peroxide produced by isolated microglia, and this was inhibited by diphenylene iodonium, implying that the cytokines were acting directly on microglia to stimulate the NADPH oxidase. Low concentrations of PMA or arachidonic acid (known activators of NADPH oxidase) or xanthine/xanthine oxidase or glucose oxidase (generating hydrogen peroxide) also increased microglia proliferation and this was blocked by catalase, showing that NADPH oxidase activation or hydrogen peroxide was sufficient to stimulate microglia proliferation. In contrast to microglia, the proliferation of astrocytes was unaffected by the presence of catalase. In conclusion, these findings indicate that microglial proliferation in response to IL-1β or TNF-α is mediated by hydrogen peroxide from NADPH oxidase.
Destructive Cleavage of Antigenic Peptides Either by the Immunoproteasome or by the Standard Proteasome Results in Differential Antigen Presentation
Authors: Chapiro Jacques , Claverol Stéphane , Piette Fanny , Ma Wenbin , Stroobant Vincent , Guillaume Benoît , Gairin Jean-Edouard , Morel Sandra , Burlet-Schiltz Odile , Monsarrat Bernard , Boon Thierry , Van den Eynde Benoît J.
Published: April 2014

The immunoproteasome (IP) is usually viewed as favoring the production of antigenic peptides presented by MHC class I molecules, mainly because of its higher cleavage activity after hydrophobic residues, referred to as the chymotrypsin-like activity. However, some peptides have been found to be better produced by the standard proteasome. The mechanism of this differential processing has not been described. By studying the processing of three tumor antigenic peptides of clinical interest, we demonstrate that their differential processing mainly results from differences in the efficiency of internal cleavages by the two proteasome types. Peptide gp100209–217 (ITDQVPSFV) and peptide tyrosinase369–377 (YMDGTMSQV) are destroyed by the IP, which cleaves after an internal hydrophobic residue. Conversely, peptide MAGE-C2336–344 (ALKDVEERV) is destroyed by the standard proteasome by internal cleavage after an acidic residue, in line with its higher postacidic activity. These results indicate that the IP may destroy some antigenic peptides due to its higher chymotrypsin-like activity, rather than favor their production. They also suggest that the sets of peptides produced by the two proteasome types differ more than expected. Considering that mature dendritic cells mainly contain IPs, our results have implications for the design of immunotherapy strategies.
I-Ep-Bound Self-Peptides: Identification, Characterization, and Role in Alloreactivity
Authors: Felix Nathan J. , Suri Anish , Walters James J. , Horvath Stephen , Gross Michael L. , Allen Paul M.
Published: April 2014

T cell recognition of peptide/allogeneic MHC complexes is a major cause of transplant rejection. Both the presented self-peptides and the MHC molecules are involved; however, the molecular basis for alloreactivity and the contribution of self-peptides are still poorly defined. The murine 2.102 T cell is specific for hemoglobin(64–76)/I-Ek and is alloreactive to I-Ep. The natural self-peptide/I-Ep complex recognized by 2.102 remains unknown. In this study, we characterized the peptides that are naturally processed and presented by I-Ep and used this information to define the binding motif for the murine I-Ep class II molecule. Interestingly, we found that the P9 anchor residue preferred by I-Ep is quite distinct from the residues preferred by other I-E molecules, although the P1 anchor residue is conserved. A degree of specificity for the alloresponse was shown by the lack of stimulation of 2.102 T cells by 19 different identified self-peptides. The binding motif was used to search the mouse genome for candidate 2.102 reactive allopeptides that contain strong P1 and P9 anchor residues and possess previously identified allowable TCR contact residues. Two potential allopeptides were identified, but only one of these peptides, G protein-coupled receptor 128, was able to stimulate 2.102 T cells. Thus, the G protein-coupled receptor 128 peptide represents a candidate allopeptide that is specifically recognized by 2.102 T cells bound to I-Ep and was identified using bioinformatics. These studies highlight the specific involvement of self-peptides in alloreactivity.
The Ret Finger Protein Inhibits Signaling Mediated by the Noncanonical and Canonical IκB Kinase Family Members
Authors: Zha Jikun , Han Ke-Jun , Xu Liang-Guo , He Wei , Zhou Qianhe , Chen Danying , Zhai Zhonghe , Shu Hong-Bing
Published: April 2014

IFN regulatory factor-3 is a transcription factor that is required for the rapid induction of type I IFNs in the innate antiviral response. Two noncanonical IκB kinase (IKK) family members, IKKε and TRAF family-associated NF-κB activator-binding kinase-1, have been shown to phosphorylate IFN regulatory factor-3 and are critically involved in virus-triggered and TLR3-mediated signaling leading to induction of type I IFNs. In yeast two-hybrid screens for potential IKKε-interacting proteins, we identified Ret finger protein (RFP) as an IKKε-interacting protein. Coimmunoprecipitation experiments indicated that RFP interacted with IKKε and TRAF family-associated NF-κB activator-binding kinase-1 as well as the two canonical IKK family members, IKKβ and IKKα. RFP inhibited activation of the IFN-stimulated response element and/or NF-κB mediated by the IKK family members and triggered by TNF, IL-1, polyinosinic-polycytidylic acid (ligand for TLR3), and viral infection. Moreover, knockdown of RFP expression by RNA interference-enhanced activation of IFN-stimulated response element and/or NF-κB triggered by polyinosinic-polycytidylic acid, TNF, and IL-1. Taken together, our findings suggest that RFP negatively regulates signaling involved in the antiviral response and inflammation by targeting the IKKs.
Identification and Characterization of DC-SCRIPT, a Novel Dendritic Cell-Expressed Member of the Zinc Finger Family of Transcriptional Regulators
Authors: Triantis Vassilis , Trancikova Dagmar Eleveld , Looman Maaike W. G. , Hartgers Franca C. , Janssen Richard A. J. , Adema Gosse J.
Published: April 2014

Dendritic cells (DC) compose a heterogeneous population of cells that hold a leading role in initiating and directing immune responses. Although their function in recognizing, capturing, and presenting Ags is well defined, the molecular mechanisms that control their differentiation and immune functions are still largely unknown. In this study, we report the isolation and characterization of DC-SCRIPT, a novel protein encoded by an 8-kb mRNA that is preferentially expressed in DC. DC-SCRIPT is expressed in multiple DC subsets in vivo, including myeloid DC, plasmacytoid DC, and Langerhans cells. At the protein level, DC-SCRIPT consists of a proline-rich region, 11 C2H2-type zinc fingers, and an acidic region. Localization studies reveal that DC-SCRIPT resides in the nucleus and that nuclear localization is critically dependent on the zinc fingers. The protein displays no transcriptional activation properties according to assorted transactivation assays, but interacts with the corepressor C-terminal binding protein 1. Taken together, our results show that we have isolated a novel DC marker that could be involved in transcriptional repression. In contrast to other DC molecules, DC-SCRIPT identifies all DC subsets tested to date.
Aspartic Acid Homozygosity at Codon 57 of HLA-DQ β Is Associated with Susceptibility to Pulmonary Tuberculosis in Cambodia
Authors: Delgado Julio C. , Baena Andres , Thim Sok , Goldfeld Anne E.
Published: April 2014

After infection with Mycobacterium tuberculosis, clinical disease usually remains latent, contained by the host immune response. Although polymorphisms of HLA loci have been hypothesized to play a major role in the breakdown of latency, a functional link has not been established. Molecular-based HLA-typing methods were used to test the association of sets of HLA alleles encoding an aspartic acid at codon 57 of the HLA-DQ β-chain (HLA-DQ β57-Asp) with susceptibility to tuberculosis in a cohort of 436 pulmonary tuberculosis patients and 107 healthy controls from Cambodia. HLA class II null cells were transduced with HLA-DQ β57-Asp or HLA-DQ β57-Ala and evaluated for their ability to bind peptides from two immunogenic M. tuberculosis specific proteins, ESAT-6 and CFP-10. In this study, we report a highly significant association between progressive pulmonary tuberculosis and homozygosity for HLA-DQ β57-Asp alleles. The presence of HLA-DQ β57-Asp resulted in a significantly reduced ability to bind a peptide from the central region of the ESAT-6 protein. Furthermore, when this peptide was presented by an HLA-DQ β57-Asp allele, Ag-specific IFN-γ production from CD4+ T cells from tuberculosis patients was significantly less than when this peptide was presented by an HLA-DQ-β allele encoding an alanine at codon 57. Multiple genetic loci and ethnic-specific factors are likely involved in the human immune response to tuberculosis. The data presented here provide a functional explanation for a highly significant association between an HLA polymorphism and tuberculosis in a highly characterized group of patients with susceptibility to progressive tuberculosis infection in Cambodia.
IL-23 Enhances the Inflammatory Cell Response in Cryptococcus neoformans Infection and Induces a Cytokine Pattern Distinct from IL-12
Authors: Kleinschek Melanie A. , Muller Uwe , Brodie Scott J. , Stenzel Werner , Kohler Gabriele , Blumenschein Wendy M. , Straubinger Reinhard K. , McClanahan Terrill , Kastelein Robert A. , Alber Gottfried
Published: April 2014

IL-23, a heterodimeric cytokine composed of the p40 subunit of IL-12 and a novel p19 subunit, has been shown to be a key player in models of autoimmune chronic inflammation. To investigate the role of IL-23 in host resistance during chronic fungal infection, wild-type, IL-12- (IL-12p35−/−), IL-23- (IL-23p19−/−), and IL-12/IL-23- (p40-deficient) deficient mice on a C57BL/6 background were infected with Cryptococcus neoformans. Following infection, p40-deficient mice demonstrated higher mortality than IL-12p35−/− mice. Reconstitution of p40-deficient mice with rIL-23 prolonged their survival to levels similar to IL-12p35−/− mice. IL-23p19−/− mice showed a moderately reduced survival time and delayed fungal clearance in the liver. Although IFN-γ production was similar in wild-type and IL-23p19−/− mice, production of IL-17 was strongly impaired in the latter. IL-23p19−/− mice produced fewer hepatic granulomata relative to organ burden and showed defective recruitment of mononuclear cells to the brain. Moreover, activation of microglia cells and expression of IL-1β, IL-6, and MCP-1 in the brain was impaired. These results show that IL-23 complements the more dominant role of IL-12 in protection against a chronic fungal infection by an enhanced inflammatory cell response and distinct cytokine regulation.
Innate Immune Dysfunction in HIV Infection: Effect of HIV Envelope-NK Cell Interactions
Authors: Kottilil Shyam , Shin Kyungmin , Jackson Julia O. , Reitano Kristin N. , O’Shea Marie Angeline , Yang Jun , Hallahan Claire W. , Lempicki Richard , Arthos James , Fauci Anthony S.
Published: April 2014

We have previously described a number of NK cell dysfunctions in HIV-viremic individuals. In the present study, we performed DNA microarray analysis followed by phenotypic and functional characterization in an effort to investigate which HIV envelope glycoproteins (gp120) affect the physiologic functions of NK cells. Upon treatment of NK cells with HIV gp120, DNA microarray analyses indicated up-regulation of several categories of genes that are associated with apoptosis, suppression of both cellular proliferation and survival, as well as down-regulation of genes that play a vital role in cell proliferation, innate immune defense mechanism, and cell survival. Both subtypes of gp120 suppressed NK cell cytotoxicity, proliferation, and the ability to secrete IFN-γ. NK cells exposed to X4-subtype HIV gp120 showed a significant decrease in the levels of CC chemokines, while exposure to R5-subtype HIV gp120 had minimal effect. Extended exposure to HIV gp120 resulted in apoptosis of NK cells, further validating the microarray data. Our data demonstrate that exposure of NK cells to HIV envelope proteins results in profound cellular abnormalities at the level of gene expression as well as generic cell functions. These findings are likely to be a consequence of a direct HIV gp120-mediated effect on NK cells. Identification of specific surface receptors on NK cells that interact with HIV envelope proteins might explain how HIV is capable of circumventing innate immune defense mechanisms and establishing infection in susceptible individuals.
Impairment of Alternative Macrophage Activation Delays Cutaneous Leishmaniasis in Nonhealing BALB/c Mice
Authors: Hölscher Christoph , Arendse Berenice , Schwegmann Anita , Myburgh Elmarie , Brombacher Frank
Published: April 2014

Expressed on various cell types, the IL-4Rα is a component of both receptors for IL-4 and IL-13. Susceptibility of BALB/c mice to Leishmania major is believed to be dependent on the development of IL-4- and IL-13-producing Th2 cells, while IFN-γ secretion by Th1 cells is related to resistance. Despite a sustained development of Th2 cells, IL-4Rα-deficient BALB/c mice are able to control acute cutaneous leishmaniasis, suggesting that IL-4Rα-bearing cells other than Th2 cells contribute to susceptibility. To analyze the contribution of the IL-4Rα on macrophages, recently generated macrophage/neutrophil-specific IL-4Rα-deficient mice on a susceptible BALB/c genetic background were infected with L. major. Strikingly, macrophage/neutrophil-specific IL-4Rα-deficient mice showed a significantly delayed disease progression with normal Th2 and type 2 Ab responses but improved macrophage leishmanicidal effector functions and reduced arginase activity. Together, these results suggest that alternative macrophage activation contributes to susceptibility in cutaneous leishmaniasis.
Modified Pulmonary Surfactant Is a Potent Adjuvant That Stimulates the Mucosal IgA Production in Response to the Influenza Virus Antigen
Authors: Mizuno Dai , Ide-Kurihara Mikiko , Ichinomiya Tomoko , Kubo Itsuka , Kido Hiroshi
Published: April 2014

The intranasal administration of influenza hemagglutinin (HA) vaccine with Surfacten, a modified pulmonary surfactant free of antigenic c-type lectins, as a mucosal adjuvant induced the highest protective mucosal immunity in the airway. The intranasal immunization of mice with HA vaccine (0.2 μg)-Surfacten (0.2 μg) selectively induced the neutralizing anti-HA IgA, but not IgG, and conferred nearly maximal protection in the airway, without inducing a systemic response. In contrast, intranasal inoculation of vaccine with 0.2 μg of the potent mucosal adjuvant cholera toxin B* (CT-B*), prepared by adding 0.2% native CT to the B subunit of CT, induced both anti-HA IgA and IgG in the airway and in the serum. The intranasal administration of HA vaccine alone induced a limited amount of mucosal IgA against influenza virus. Although the s.c. administration of HA vaccine prominently induced serum IgG and IgA, Surfacten and CT-B* did not enhance their induction, and the concentrations of Abs leaking into the airways were insufficient to prevent viral multiplication. The intranasal administration of HA-Surfacten stimulated the expression of MHC class II, CD40, and CD86 molecules in the CD11c-positive cells isolated from the nasal mucosa, but not the expression of cells from the lungs or spleens. Lymphocytes isolated from the airway mucosa after intranasal HA-Surfacten immunization prominently induced TGF-β1 which, compared with inoculation without Surfacten, promoted an Ag-specific mucosal IgA response. Surfacten alone, however, did not induce TGF-β1. Our observations suggest that Surfacten, by mimicking the natural surfactant, is an effective mucosal adjuvant in the process of airway immunization.
Selectin Ligand-Independent Priming and Maintenance of T Cell Immunity during Airborne Tuberculosis
Authors: Schreiber Tanja , Ehlers Stefan , Aly Sahar , Hölscher Alexandra , Hartmann Sven , Lipp Martin , Lowe John B. , Hölscher Christoph
Published: April 2014

Immunity to Mycobacterium tuberculosis infection is critically dependent on the timely priming of T effector lymphocytes and their efficient recruitment to the site of mycobacterial implantation in the lung. E-, P-, and L-selectin counterreceptors control lymphocyte homing to lymph nodes and leukocyte trafficking to peripheral sites of acute inflammation, their adhesive function depending on fucosylation by fucosyltransferases (FucT) IV and VII. To address the relative importance of differentially glycosylated selectin counterreceptors for priming of T cell effector functions in a model of mycobacteria-induced granulomatous pulmonary inflammation, we used aerosol-borne M. tuberculosis to infect FucT-IV−/−, FucT-VII−/−, FucT-IV−/−/FucT-VII−/−, or wild-type control mice. In lymph nodes, infected FucT-IV−/−/FucT-VII−/− and, to a lesser extent, FucT-VII−/− mice had severely reduced numbers of T cells and reduced Ag-specific effector responses. By contrast, recruitment of activated T cells into the lungs was similar in all four groups of mice during infection and expression of T cell, and macrophage effector functions were only delayed in lungs of FucT-IV−/−/FucT-VII−/− mice. Importantly, lungs from all groups expressed CXCL13, CCL21, and CCL19 and displayed organized follicular neolymphoid structures after infection with M. tuberculosis, which suggests that the lung served as a selectin ligand-independent priming site for immune responses to mycobacterial infection. All FucT-deficient strains were fully capable of restricting M. tuberculosis growth in infected organs until at least 150 days postinfection. Our observations indicate that leukocyte recruitment functions dictated by FucT-IV and FucT-VII-dependent selectin ligand activities are not critical for inducing or maintaining T cell effector responses at levels necessary to control pulmonary tuberculosis.