bluuebunny/crossref_metadata_2025 huggingface dataset:
Allergy Vaccine Engineering: Epitope Modulation of Recombinant Bet v 1 Reduces IgE Binding but Retains Protein Folding Pattern for Induction of Protective Blocking-Antibody Responses
Authors: Holm Jens , Gajhede Michael , Ferreras Mercedes , Henriksen Anette , Ipsen Henrik , Larsen Jørgen N. , Lund Lise , Jacobi Henrik , Millner Anders , Würtzen Peter A. , Spangfort Michael D.
Published: April 2014

Human type 1 immediate allergic response symptoms are caused by mediator release from basophils and mast cells. This event is triggered by allergens aggregating preformed IgE Abs bound to the high-affinity receptor (FcεRI) on these cells. Thus, the allergen/IgE interaction is crucial for the cascade leading to the allergic and anaphylactic response. Two genetically engineered forms of the white birch pollen major allergen Bet v 1 with point mutations directed at molecular surfaces have been characterized. Four and nine point mutations led to a significant reduction of the binding to human serum IgE, suggesting a mutation-induced distortion of IgE-binding B cell epitopes. In addition, the mutated allergens showed a decrease in anaphylactic potential, because histamine release from human basophils was significantly reduced. Retained α-carbon backbone folding pattern of the mutated allergens was indicated by x-ray diffraction analysis and circular dichroism spectroscopy. The rBet v 1 mutants were able to induce proliferation of T cell lines derived from birch pollen allergic patients. The stimulation indices were similar to the indices of nonmutated rBet v 1 and natural Bet v 1 purified from birch pollen. The ability of anti-rBet v 1 mutant specific mouse IgG serum to block binding of human serum IgE to rBet v 1 demonstrates that the engineered rBet v 1 mutants are able to induce Abs reactive with nonmodified Bet v 1. rBet v 1 mutants may constitute vaccine candidates with improved efficacy/safety profiles for safer allergy vaccination.
IgE-Mediated Mast Cell Activation Induces Langerhans Cell Migration In Vivo
Authors: Jawdat Dunia M. , Albert Eric J. , Rowden Geoffrey , Haidl Ian D. , Marshall Jean S.
Published: April 2014

Langerhans cells and mast cells are both resident in large numbers in the skin and act as sentinel cells in host defense. The ability of mast cells to induce Langerhans cell migration from the skin to the draining lymph node in vivo was examined. Genetically mast cell-deficient (W/Wv) mice and control mice were sensitized with IgE Ab in the ear pinna. Seven to 14 days later, mice were challenged with Ag i.v. After a further 18–24 h, epidermal sheets and draining auricular lymph nodes were examined using Langerin/CD207 immunostaining. In mast cell-containing mice, a significant decrease in the number of Langerhans cells was observed at epidermal sites of mast cell activation. A significant increase in total cellularity and accumulation of Langerin-positive dendritic cells was observed in the auricular lymph nodes, draining the sites of IgE-mediated mast cell activation. These changes were not observed in W/Wv mice, but were restored by local mast cell reconstitution. Treatment of mast cell-containing mice with the H2 receptor antagonist cimetidine significantly inhibited the observed IgE/Ag-induced changes in Langerhans cell location. In contrast, Langerhans cell migration in response to LPS challenge was not mast cell dependent. These data directly demonstrate the ability of mast cells to induce dendritic cell migration to lymph nodes following IgE-mediated activation in vivo by a histamine-dependent mechanism.
Increased Natural Killer Cell Activity in Viremic HIV-1 Infection
Authors: Alter Galit , Malenfant Jessica M. , Delabre Rosemary M. , Burgett Nicole C. , Yu Xu G. , Lichterfeld Mathias , Zaunders John , Altfeld Marcus
Published: April 2014

NK cells are a subset of granular lymphocytes that are critical in the innate immune response to infection. These cells are capable of killing infected cells and secreting integral cytokines and chemokines. The role that this subset of cytolytic cells plays in HIV infection is not well understood. In this study, we dissected the function of NK cells in viremic and aviremic HIV-1-infected subjects, as well as HIV-1-negative control individuals. Despite reduced NK cell numbers in subjects with ongoing viral replication, these cells were significantly more active in secreting both IFN-γ and TNF-α than NK cells from aviremic subjects or HIV-1-negative controls. In addition, NK cells in subjects with detectable viral loads expressed significantly higher levels of CD107a, a marker of lysosomal granule exocytosis. The expression of CD107a correlated with NK cell-mediated cytokine secretion and cytolytic activity as well as with the level of viral replication, suggesting that CD107a represents a good marker for the functional activity of NK cells. Finally, killer Ig-related receptor+ NK cells were stable or elevated in viremic subjects, while the numbers of CD3−/CD56+/CD94+ and CD3−/CD56+/CD161+ NK cells were reduced. Taken together, these data demonstrate that viremic HIV-1 infection is associated with a reduction in NK cell numbers and a perturbation of NK cell subsets, but increased overall NK cell activity.
Blockade of IL-18 Receptor Signaling Delays the Onset of Autoimmune Disease in MRL-Fas
lpr Mice
Authors: Kinoshita Koji , Yamagata Toshiaki , Nozaki Yuji , Sugiyama Masafumi , Ikoma Shinya , Funauchi Masanori , Kanamaru Akihisa
Published: April 2014

Autoimmune disease in Fas-deficient MRL-Faslpr mice is dependent on infiltrating autoreactive leukocytes and autoantibodies, and IFN-γ plays an important role in the pathogenesis. As IL-18 is capable of inducing IFN-γ production in T cells, we hypothesized that signaling through IL-18R is involved in the pathogenesis. To investigate the impact of IL-18 in this autoimmune disease, we generated an MRL-Faslpr strain deficient in IL-18Rα. Compared with the wild-type strain, IL-18Rα-deficient MRL-Faslpr mice survived longer and showed a significant reduction in renal pathology, including glomerular IgG deposits, proteinuria, and serum anti-DNA Abs. Intrarenal transcripts encoding IFN-γ, TNF-α, IL-12, and IL-10, which have been linked to nephritis, were all markedly reduced. Skin lesions, lymphadenopathy, and lung pathology characteristic of the MRL-Faslpr mouse disease were diminished in IL-18Rα-deficient MRL-Faslpr mice. Thus, we conclude that IL-18Rα signaling is critical to the pathogenesis of autoimmune disease in MRL-Faslpr mice.
Cutting Edge: Engagement of CD160 by its HLA-C Physiological Ligand Triggers a Unique Cytokine Profile Secretion in the Cytotoxic Peripheral Blood NK Cell Subset
Authors: Barakonyi Aliz , Rabot Magali , Marie-Cardine Anne , Aguerre-Girr Maryse , Polgar Beata , Schiavon Valérie , Bensussan Armand , Le Bouteiller Philippe
Published: April 2014

CD160 is an Ig-like activating NK cell receptor expressed on the majority of circulating NK cells. This population corresponds to the nonproliferating, highly cytolytic, CD56dimCD16+ subset. CD160 engagement by HLA-C molecules mediates cytotoxic function. In this study, we report that upon specific activation by the physiological ligand HLA-C, or Ab cross-linking, CD160+ peripheral blood NK cells produce IFN-γ, TNF-α, and IL-6. This unique CD160-mediated cytokine production differs from the one observed after CD16 engagement whose expression is also restricted to the CD56dim cytotoxic NK cell subset. As already reported for the CD160-mediated cytotoxic effector function, CD160-mediated cytokine production by peripheral blood-NK cells is negatively controlled by the killer Ig-like receptor CD158b. Thus, the CD160 receptor represents a unique triggering surface molecule expressed by cytotoxic NK cells that participates in the inflammatory response and determines the type of subsequent specific immunity.
Regulation of B Cell Differentiation and Plasma Cell Generation by IL-21, a Novel Inducer of Blimp-1 and Bcl-6
Authors: Ozaki Katsutoshi , Spolski Rosanne , Ettinger Rachel , Kim Hyoung-Pyo , Wang Gang , Qi Chen-Feng , Hwu Patrick , Shaffer Daniel J. , Akilesh Shreeram , Roopenian Derry C. , Morse Herbert C. , Lipsky Peter E. , Leonard Warren J.
Published: April 2014

IL-21 is a type I cytokine whose receptor is expressed on T, B, and NK cells. Within the B cell lineage, IL-21 regulates IgG1 production and cooperates with IL-4 for the production of multiple Ab classes in vivo. Using IL-21-transgenic mice and hydrodynamics-based gene delivery of IL-21 plasmid DNA into wild-type mice as well as in vitro studies, we demonstrate that although IL-21 induces death of resting B cells, it promotes differentiation of B cells into postswitch and plasma cells. Thus, IL-21 differentially influences B cell fate depending on the signaling context, explaining how IL-21 can be proapoptotic for B cells in vitro yet critical for Ag-specific Ig production in vivo. Moreover, we demonstrate that IL-21 unexpectedly induces expression of both Blimp-1 and Bcl-6, indicating mechanisms as to how IL-21 can serve as a complex regulator of B cell maturation and terminal differentiation. Finally, BXSB-Yaa mice, which develop a systemic lupus erythematosus-like disease, have greatly elevated IL-21, suggesting a role for IL-21 in the development of autoimmune disease.
CD28 and Lipid Rafts Coordinate Recruitment of Lck to the Immunological Synapse of Human T Lymphocytes
Authors: Tavano Regina , Gri Giorgia , Molon Barbara , Marinari Barbara , Rudd Christopher E. , Tuosto Loretta , Viola Antonella
Published: April 2014

In T lymphocytes, the Src family kinase Lck associates lipid rafts and accumulates at the immunological synapse (IS) during T cell stimulation by APCs. Using CD4- or CD28-deficient murine T cells, it was suggested that recruitment of Lck to the IS depends on CD4, whereas CD28 sustains Lck activation. However, in human resting T cells, CD28 is responsible for promoting recruitment of lipid rafts to the IS by an unknown mechanism. Thus, we performed a series of experiments to determine 1) whether Lck is recruited to the IS through lipid rafts; and 2) whether Lck recruitment to the IS of human resting T cells depends on CD4 or on CD28 engagement. We found that CD28, but not CD4, stimulation induced recruitment of Lck into detergent-resistant domains as well as its accumulation at the IS. We also found that Lck recruitment to the IS depends on the CD28 COOH-terminal PxxPP motif. Thus, the CD28-3A mutant, generated by substituting the prolines in positions 208, 211, and 212 with alanines, failed to induce Lck and lipid raft accumulation at the synapse. These results indicate that CD28 signaling orchestrates both Lck and lipid raft recruitment to the IS to amplify T cell activation.
β-Defensin-2 Expression Is Regulated by TLR Signaling in Intestinal Epithelial Cells
Authors: Vora Puja , Youdim Adrienne , Thomas Lisa S. , Fukata Masayuki , Tesfay Samuel Y. , Lukasek Katie , Michelsen Kathrin S. , Wada Akihiro , Hirayama Toshiya , Arditi Moshe , Abreu Maria T.
Published: April 2014

The intestinal epithelium serves as a barrier to the intestinal flora. In response to pathogens, intestinal epithelial cells (IEC) secrete proinflammatory cytokines. To aid in defense against bacteria, IEC also secrete antimicrobial peptides, termed defensins. The aim of our studies was to understand the role of TLR signaling in regulation of β-defensin expression by IEC. The effect of LPS and peptidoglycan on β-defensin-2 expression was examined in IEC lines constitutively or transgenically expressing TLRs. Regulation of β-defensin-2 was assessed using promoter-reporter constructs of the human β-defensin-2 gene. LPS and peptidoglycan stimulated β-defensin-2 promoter activation in a TLR4- and TLR2-dependent manner, respectively. A mutation in the NF-κB or AP-1 site within the β-defensin-2 promoter abrogated this response. In addition, inhibition of Jun kinase prevents up-regulation of β-defensin-2 protein expression in response to LPS. IEC respond to pathogen-associated molecular patterns with expression of the antimicrobial peptide β-defensin-2. This mechanism may protect the intestinal epithelium from pathogen invasion and from potential invaders among the commensal flora.
Transgenic Expression of CTLA-4 Controls Lymphoproliferation in IL-2-Deficient Mice
Authors: Hwang Kwang Woo , Sweatt William B. , Mashayekhi Mona , Palucki David A. , Sattar Hussain , Chuang Ellen , Alegre Maria-Luisa
Published: April 2014

IL-2-deficient mice develop a lymphoproliferative and autoimmune disease characterized by autoimmune hemolytic anemia (AHA) and inflammatory bowel disease. We have previously reported that IL-2 is necessary for optimal up-regulation of CTLA-4, an inducible negative regulator of T cell activation. In this study, we have tested the hypothesis that reduced expression of CTLA-4 in IL-2-deficient T cells contributes to the pathogenesis of disease in IL-2-deficient mice. Expression of CTLA-4 as a transgene completely prevented lymphoaccumulation and AHA in IL-2-deficient mice. The normalization of T cell numbers was due to inhibition of expansion of conventional CD4+CD25− T cells rather than to rescue of the numbers or function of CD4+CD25+ regulatory T cells, suggesting that CTLA-4 expression on conventional T cells plays a role in maintaining normal T cell homeostasis. In addition, the inhibitory effect of the CTLA-4 transgene on T cell expansion was at least in part independent of CD28 expression. Our results suggest that deficient CTLA-4 expression on conventional T cells contributes to the pathophysiology of the lymphoproliferative disease and AHA in IL-2-deficient mice. Thus, restoring CTLA-4 expression in T cells may be an attractive strategy to control clinical autoimmune diseases in which CTLA-4 expression is reduced.
The Regulated Expression of a Diverse Set of Genes during Thymocyte Positive Selection In Vivo
Authors: Mick Verity E. , Starr Timothy K. , McCaughtry Tom M. , McNeil Lisa K. , Hogquist Kristin A.
Published: April 2014

A signal initiated by the newly formed Ag receptor is integrated with microenvironmental cues during T cell development to ensure positive selection of CD4+CD8+ progenitors into functionally mature CD4+ or CD8+ T lymphocytes. During this transition, a survival program is initiated, TCR gene recombination ceases, cells migrate into a new thymic microenvironment, the responsiveness of the Ag receptor is tuned, and the cells commit to a specific T lineage. To determine potential regulators of these processes, we used mRNA microarray analysis to compare gene expression changes in CD4+CD8+ thymocytes from TCR transgenic mice that have received a TCR selection signal with those that had not received a signal. We found 129 genes with expression that changed significantly during positive selection, the majority of which were not previously appreciated. A large number of these changes were confirmed by real-time PCR or flow cytometry. We have combined our findings with gene changes reported in the literature to provide a comprehensive report of the genes regulated during positive selection, and we attempted to assign these genes to positive selection process categories.
B7-H3 Enhances Tumor Immunity In Vivo by Costimulating Rapid Clonal Expansion of Antigen-Specific CD8+ Cytolytic T Cells
Authors: Luo Liqun , Chapoval Andrei I. , Flies Dallas B. , Zhu Gefeng , Hirano Fumiya , Wang Shengdian , Lau Julie S. , Dong Haidong , Tamada Koji , Flies Andrew S. , Liu Yang , Chen Lieping
Published: April 2014

B7-H3 is a B7 family molecule with T cell costimulatory function in vitro. The in vivo role of B7-H3 in the stimulation of tumor immunity is unclear. We report here that expression of B7-H3 by transfection of the mouse P815 tumor line enhances its immunogenicity, leading to the regression of tumors and amplification of a tumor-specific CD8+ CTL response in syngeneic mice. Tumor cells engineered to express B7-H3 elicit a rapid clonal expansion of P1A tumor Ag-specific CD8+ CTL in lymphoid organs in vivo and acquire the ability to directly stimulate T cell growth, division, and development of cytolytic activity in vitro. Our results thus establish a role for B7-H3 in the costimulation of T cell immune responses in vivo.
Specificity-Based Negative Selection of Autoreactive B Cells during Memory Formation
Authors: Guay Heath M. , Panarey Laura , Reed Amy J. , Caton Andrew J.
Published: April 2014

Autoreactive B cells are not completely purged from the primary B cell repertoire, and whether they can be prevented from maturation into memory B cells has been uncertain. We show here that a population of B cells that dominates primary immune responses of BALB/c mice to influenza virus A/PR/8/34 hemagglutinin (HA) are negatively selected in transgenic mice expressing PR8 HA as an abundant membrane-bound Ag (HACII mice). However, a separate population of B cells that contains precursors of memory B cells is activated by PR8 virus immunization and is subsequently negatively selected during the formation of the memory response. Negative selection of PR8 HA-specific B cells altered the specificity of the memory B cell response to a mutant virus containing a single amino acid substitution in a B cell epitope. Strikingly, this skewed reactivity resulted from an increase in the formation of memory B cells directed to non-self-epitopes on the mutant virus, which increased 8-fold in HACII mice relative to nontransgenic mice and precisely compensated for the absence of autoreactive PR8 HA-specific memory B cells. Negative selection of PR8 HA-specific B cells was a dominant process, since B cells from HACII mice could induce negative selection of PR8 HA-specific B cells from BALB/c mice. Lastly, HA-specific memory responses were unaffected by self-tolerance in another lineage of HA-transgenic mice (HA104 mice), indicating that the amount and/or cell type in which self-Ags are expressed can determine their ability to prevent autoreactive memory B cell formation.
Viral Macrophage-Inflammatory Protein-II: A Viral Chemokine That Differentially Affects Adaptive Mucosal Immunity Compared with Its Mammalian Counterparts
Authors: Singh Udai P. , Singh Shailesh , Ravichandran Palaniappan , Taub Dennis D. , Lillard James W.
Published: April 2014

Chemokines play a profound role in leukocyte trafficking and the development of adaptive immune responses. Perhaps due to their importance in host defense, viruses have adopted many of the hallmarks displayed by chemokines. In particular, viral MIP-II (vMIP-II) is a human chemokine homologue that is encoded by human herpes virus 8. vMIP-II is angiogenic, selectively chemotactic for Th2 lymphocytes, and a homologue of human I-309 and mouse TCA-3, which also differentially attracts Th2 cells. To better understand the effect of viral chemokines on mucosal immunity, we compared the affects of vMIP-II, I-309, and TCA-3 on cellular and humoral immune responses after nasal immunization with OVA. These CCR8 ligands significantly enhanced Ag-specific serum and mucosal Abs through increasing Th2 cytokine secretion by CD4+ T cells. These alterations in adaptive humoral and cellular responses were preceded (12 h after immunization) by an increase in CD4+ T and B cells in nasal tracts with decreases of these leukocyte populations in the lung. Interestingly, vMIP-II increased neutrophil infiltration in the lung and Ag-specific IL-10-secreting CD4+ T cells after immunization. Although I-309 increased the number of CD28-, CD40L-, and CD30-positive, Ag-stimulated naive CD4+ T cells, vMIP-II and TCA-3 decreased the number of CD28-, CD40L-, and CD30-positive, resting naive CD4+ T cells. Taken together, these studies suggest that CCR8 ligands direct host Th2 responses, and vMIP-II up-regulates IL-10 responses and limits costimulatory molecule expression to mitigate host immunity.
Germline Transcription and Switch Recombination of a Transgene Containing the Entire H Chain Constant Region Locus: Effect of a Mutation in a STAT6 Binding Site in the γ1 Promoter
Authors: Dunnick Wesley A. , Shi Jian , Graves Kevin A. , Collins John T.
Published: April 2014

The switch (S) in H chain class is preceded by germline transcription and then mediated by a DNA recombination event. One of the impediments toward understanding the mechanism is the lack of a system in which a recombinant DNA molecule undergoes cytokine-regulated class S recombination. To study class S recombination, we used transgenic mice with a 230-kb bacterial artificial chromosome that included a rearranged VDJ gene and the entire murine H chain constant region locus. We found that both germline transcription and S recombination to the transgenic γ1 H chain gene were regulated by IL-4 like that of the endogenous genes. In mice with two or more copies of the H chain locus transgene, both germline transcripts and S recombination took place at levels comparable to those from the endogenous loci. We also prepared a version of the transgene with a 4-bp mutation in a STAT6 binding site in the γ1 promoter region. On the average, this mutation reduced germline transcription by 80%, but did not change the amount of S recombination in vitro. Among both the wild-type and mutant transgenes, we found no significant correlation between the amount of germline transcripts and the amount of S recombination. We infer that the physiologic level of germline transcription of the γ1 gene is in excess over the amount required for efficient S recombination.
Complement Inactivation by Recombinant Human C3 Derivatives
Authors: Kölln Johanna , Spillner Edzard , Andrä Jörg , Klensang Katrin , Bredehorst Reinhard
Published: April 2014

From the implications of the complement system in a large number of diseases, an urgent need for therapeutics effecting reduced complement activity in vivo has emerged. In this study we report the design of a novel class of enzymes of human origin that obliterate functional complement by a noninhibitory, catalytic mechanism. Combining the framework of human C3 and the enzymatic mechanism of cobra venom factor, a nontoxic snake venom protein, we established molecules capable of forming stable C3 convertase complexes. Although the half-life of naturally occurring C3 convertase complexes ranges between 1 and 2 min, these complexes exhibit a half-life of up to several hours. Because the overall identity to human C3 could be extended to >90%, the novel C3 derivatives can be assumed to exhibit low immunogenicity and, therefore, represent promising candidates for therapeutic reduction of complement activity in vivo.
NF-κB Regulates Expression of the MHC Class I-Related Chain A Gene in Activated T Lymphocytes
Authors: Molinero Luciana L. , Fuertes Mercedes B. , Girart María Victoria , Fainboim Leonardo , Rabinovich Gabriel A. , Costas Mónica A. , Zwirner Norberto W.
Published: April 2014

MHC class I-related chain A gene (MICA) is a stress-regulated, HLA-related molecule which exhibits a restricted pattern of expression. MICA protein is up-regulated on different tumor cells, and is recognized by the lectin-like NKG2D molecule expressed by cytotoxic γδ T lymphocytes, CD8+ αβ T lymphocytes, and NK cells. Although MICA is not expressed on resting lymphocytes, we demonstrated that it is induced on activated T cells. Because NF-κB is actively involved in T cell activation, and is constitutively activated in many tumors, here we investigated whether NF-κB may modulate MICA expression. Treatment with the NF-κB inhibitor sulfasalazine (Sz) resulted in a dose-dependent inhibition of MICA expression in anti-CD3- and anti-CD28/PMA-activated T lymphocytes, as assessed by Western blot and RT-PCR analysis. Moreover, Sz also down-regulated MICA expression on epithelial tumor HeLa cells. MICA expression was accompanied by a Sz-sensitive IκBα degradation. EMSA with nuclear extracts from anti-CD3- and anti-CD28/PMA-stimulated T lymphocytes demonstrated the binding of a potential NF-κB family transcription factor to a MICA gene intron 1-derived oligonucleotide that contains a putative κB binding site. Supershift assays demonstrated the presence of p65(RelA)/p50 heterodimers and p50/p50 homodimers in the NF-κB complexes bound to the κB-MICA oligonucleotide. Transient transfection of HeLa cells with p65(RelA) up-regulated MICA expression, as assessed by Western blot and flow cytometry analysis. Hence, we conclude that NF-κB regulates MICA expression on activated T lymphocytes and HeLa tumor cells, by binding to a specific sequence in the long intron 1 of the MICA gene. This constitutes the first description of a transcription factor that regulates MICA gene expression.
The Actin Cytoskeleton Controls the Efficiency of Killer Ig-Like Receptor Accumulation at Inhibitory NK Cell Immune Synapses
Authors: Standeven Leah J. , Carlin Leo M. , Borszcz Peter , Davis Daniel M. , Burshtyn Deborah N.
Published: April 2014

Killer cell Ig-like receptors (KIRs) are MHC class I-specific receptors expressed in NK and T lymphocytes. KIR antagonism of activation signals occurs at the immune synapse between the effector and target cells. The processes that regulate clustering of KIR are not well defined. We have expressed KIR-GFP receptor chimeras in two human NK-like lines, YTS and NK92. In this study, we show that the frequency of KIR enrichment at the synapse was decreased for a KIR that lacks a portion of the cytoplasmic tail. Strikingly, blocking actin polymerization with a high dose of cytochalasin D also substantially decreased clustering of KIR as well as KIR-induced clustering of HLA-C-GFP in target cells. However, the effect of inhibiting actin polymerization was only clearly evident at the earlier time points after cell mixing, and eventually clustering of KIR and HLA-C occurred independently of actin remodeling. Although treatment with anti-LFA-1 also decreased conjugate formation, the frequency of KIR clustering remained normal within the population of conjugates that did form, suggesting that the effect of cytochalasin D is not solely through LFA-1. Collectively, these data suggest that the actin cytoskeleton and the cytoplasmic tail of KIR regulate the efficiency by which KIR accumulates at inhibitory NK cell synapses.
Dendritic Cell-Specific Intercellular Adhesion Molecule 3-Grabbing Nonintegrin Mediates Binding and Internalization of Aspergillus fumigatus Conidia by Dendritic Cells and Macrophages
Authors: Serrano-Gómez Diego , Domínguez-Soto Angeles , Ancochea Julio , Jimenez-Heffernan José A. , Leal Juan Antonio , Corbí Angel L.
Published: April 2014

Aspergillus fumigatus is responsible for a large percentage of nosocomial opportunistic fungal infections in immunocompromised hosts, especially during cytotoxic chemotherapy and after bone marrow transplantation, and is currently a major direct cause of death in leukemia patients. Dendritic cell-specific ICAM-3-grabbing nonintegrin (DC-SIGN) is a type II C-type lectin that functions as an adhesion receptor and is used by viral and bacterial pathogens to gain access to human DC. We report that DC-SIGN specifically interacts with clinical isolates of A. fumigatus. DC-SIGN-dependent binding of A. fumigatus conidia can be demonstrated with stable transfectants and monocyte-derived DC and is inhibited by anti-DC-SIGN Abs. Binding and internalization of A. fumigatus conidia correlates with DC-SIGN cell surface expression levels and is abolished in the presence of A. funigatus-derived cell wall galactomannans. The clinical relevance of this interaction is emphasized by the presence of DC-SIGN in lung DC and alveolar macrophages, and further illustrated by the DC-SIGN-dependent attachment of A. fumigatus conidia to the cell membrane of IL-4-treated monocyte-derived macrophages. Our results suggest the involvement of DC-SIGN in the initial stages of pulmonary infection as well as in fungal spreading during invasive aspergillosis.
Duration of Infection and Antigen Display Have Minimal Influence on the Kinetics of the CD4+ T Cell Response to Listeria monocytogenes Infection
Authors: Corbin Gail A. , Harty John T.
Published: April 2014

The T cell response to infection consists of clonal expansion of effector cells, followed by contraction to memory levels. It was previously thought that the duration of infection determines the magnitude and kinetics of the T cell response. However, recent analysis revealed that transition between the expansion and contraction phases of the Ag-specific CD8+ T cell response is not affected by experimental manipulation in the duration of infection or Ag display. We studied whether the duration of infection and Ag display influenced the kinetics of the Ag-specific CD4+ T cell response to Listeria monocytogenes (LM) infection. We found that truncating infection and Ag display with antibiotic treatment as early as 24 h postinfection had minimal impact on the expansion or contraction of CD4+ T cells; however, the magnitudes of the Ag-specific CD4+ and CD8+ T cell responses were differentially affected by the timing of antibiotic treatment. Treatment of LM-infected mice with antibiotics at 24 h postinfection did not prevent generation of detectable CD4+ and CD8+ memory T cells at 28 days after infection, vigorous secondary expansion of these memory T cells, or protection against a subsequent LM challenge. These results demonstrate that events within the first few days of infection stimulate CD4+ and CD8+ T cell responses that are capable of carrying out the full program of expansion and contraction to functional memory, independently of prolonged infection or Ag display.
Expression of Functional TLR4 Confers Proinflammatory Responsiveness toTrypanosoma cruziGlycoinositolphospholipids and Higher Resistance to Infection withT. cruzi
Authors: Oliveira Ana-Carolina , Peixoto Jaqueline R. , de Arruda Luciana B. , Campos Marco A. , Gazzinelli Ricardo T. , Golenbock Douglas T. , Akira Shizuo , Previato José O. , Mendonça-Previato Lúcia , Nobrega Alberto , Bellio Maria
Published: April 2014

TLRs function as pattern recognition receptors in mammals and play an essential role in the recognition of microbial components. We found that the injection of glycoinositolphospholipids (GIPLs) from Trypanosoma cruzi into the peritoneal cavity of mice induced neutrophil recruitment in a TLR4-dependent manner: the injection of GIPL in the TLR4-deficient strain of mice (C57BL/10ScCr) caused no inflammatory response. In contrast, in TLR2 knockout mice, neutrophil chemoattraction did not differ significantly from that seen in wild-type controls. GIPL-induced neutrophil attraction and MIP-2 production were also severely affected in TLR4-mutant C3H/HeJ mice. The role of TLR4 was confirmed in vitro by testing genetically engineered mutants derived from TLR2-deficient Chinese hamster ovary (CHO)-K1 fibroblasts that were transfected with CD14 (CHO/CD14). Wild-type CHO/CD14 cells express the hamster TLR4 molecule and the mutant line, in addition, expresses a nonfunctional form of MD-2. In comparison to wild-type cells, mutant CHO/CD14 cells failed to respond to GIPLs, indicating a necessity for a functional TLR4/MD-2 complex in GIPL-induced NF-κB activation. Finally, we found that TLR4-mutant mice were hypersusceptible to T. cruzi infection, as evidenced by a higher parasitemia and earlier mortality. These results demonstrate that natural resistance to T. cruzi is TLR4 dependent, most likely due to TLR4 recognition of their GIPLs.
Rac1 Contributes to Maximal Activation of STAT1 and STAT3 in IFN-γ-Stimulated Rat Astrocytes
Authors: Park Eun Jung , Ji Kyung-Ae , Jeon Sae-Bom , Choi Woo-Hyuck , Han Inn-oc , You Hye-Jin , Kim Jae-Hong , Jou Ilo , Joe Eun-Hye
Published: April 2014

Rac1 GTPase is implicated as a signaling mediator in various cellular events. In this study, we show that Rac1 contributes to IFN-γ-induced inflammatory responses in rat astrocytes. We revealed that IFN-γ rapidly stimulated activation of Rac1 in C6 astroglioma cells by investigating GST-PAK-PBD-binding ability. We also found that Rac1 deficiency led to attenuation of IFN-γ-responsive transcriptional responses. Compared with levels in control cells, IFN-γ-induced IFN-γ-activated sequence promoter activity was markedly reduced in both C6 astroglioma cells and primary astrocytes expressing RacN17, a well-characterized Rac1-negative mutant. The expression of several IFN-γ-responsive genes, such as MCP-1 and ICAM-1, was also reduced in cells expressing RacN17. Consistent with these observations, IFN-γ-induced phosphorylation of STAT1 and STAT3 was lower in C6 cells expressing RacN17 (referred to as C6-RacN17) than in control cells. However, there was no difference in expression level of IFN-γRα subunit and IFN-γ-induced phosphorylation of JAK1 between C6 control and C6-RacN17 cells. Interestingly, Rac1 appeared to associate with IFN-γRα and augment the interaction of IFN-γR with either STAT1 or STAT3 in response to IFN-γ. Taken together, we suggest that Rac1 may serve as an auxiliary mediator of IFN-γ-signaling, at least at the level of STAT activation, thus contributing to maximal activation of IFN-γ-responsive inflammatory signaling in rat astrocytes.
A New Pharmacological Effect of Salicylates: Inhibition of NFAT-Dependent Transcription
Authors: Aceves Mónica , Dueñas Ana , Gómez Cristina , San Vicente Edurne , Crespo Mariano Sánchez , García-Rodríguez Carmen
Published: April 2014

The anti-inflammatory effects of salicylates, originally attributed to inhibition of cyclooxygenase activity, are currently known to involve additional mechanisms. In this study we investigated the possible modulation by salicylates of NFAT-mediated transcription in lymphocytic and monocytic cell lines. RNase protection assays showed that 2-acetoxy-4-trifluoromethylbenzoic acid (triflusal) inhibited, in a dose-dependent manner, mRNA expression of several cytokine genes, most of which are NFAT-regulated and cyclosporin A (CsA)-sensitive. In Jurkat cells, the expression of IL-3, GM-CSF, TNF-α, TGF-β1, IL-2, lymphotactin, MIP-1α, and MIP-1β was inhibited to different extents. In THP-1 cells, inhibition of the expression of M-CSF, G-CSF, stem cell factor, IFN-γ, TNF-α, TGF-β1, lymphotoxin-β1, MIP-1α, MIP-1β, and IL-8 was observed. Sodium salicylate and aspirin only showed significant effects at 5 mM. The transcriptional activity of two genes that contain NFAT sites, a GM-CSF full promoter and a T cell-specific enhancer from the IL-3 locus, was also inhibited by salicylates. Transactivation experiments performed with several NFAT-dependent and AP-1-dependent reporter genes showed that triflusal strongly inhibited NFAT-dependent transcription at concentrations as low as 0.25 mM. Sodium salicylate and aspirin were less potent. The triflusal inhibitory effect was reversible and synergized with suboptimal doses of CsA. Experiments to address the mechanism of action of salicylates in the NFAT activation cascade disclosed a mechanism different from that of CsA, because salicylates inhibited DNA-binding and NFAT-mediated transactivation without affecting phosphorylation or subcellular localization of NFAT. In summary, these data describe a new pharmacological effect of salicylates as inhibitors of NFAT-dependent transcription.
Toll Receptor-Mediated Regulation of NADPH Oxidase in Human Dendritic Cells
Authors: Vulcano Marisa , Dusi Stefano , Lissandrini Daniele , Badolato Raffaele , Mazzi Paola , Riboldi Elena , Borroni Elena , Calleri Angelica , Donini Marta , Plebani Alessandro , Notarangelo Luigi , Musso Tiziana , Sozzani Silvano
Published: April 2014

Activation of NADPH oxidase represents an essential mechanism of defense against pathogens. Dendritic cells (DC) are phagocytic cells specialized in Ag presentation rather than in bacteria killing. Human monocyte-derived DC were found to express the NADPH oxidase components and to release superoxide anions in response to phorbol esters and phagocytic agonists. The NADPH oxidase components p47phox and gp91phox were down-regulated during monocyte differentiation to DC, and maturation of DC with pathogen-derived molecules, known to activate TLRs, increased p47phox and gp91phox expression and enhanced superoxide anions release. Similar results were obtained with plasmacytoid DC following maturation with influenza virus. In contrast, activation of DC by immune stimuli (CD40 ligand) did not regulate NADPH oxidase components or respiratory burst. NADPH oxidase-derived oxygen radicals did not play any role in DC differentiation, maturation, cytokine production, and induction of T cell proliferation, as based on the normal function of DC generated from chronic granulomatous disease patients and the use of an oxygen radical scavenger. However, NADPH oxidase activation was required for DC killing of intracellular Escherichia coli. It is likely that the selective regulation of oxygen radicals production by pathogen-activated DC may function to limit pathogen dissemination during DC trafficking to secondary lymphoid tissues.
Selective Blockade of NF-κB Activity in Airway Immune Cells Inhibits the Effector Phase of Experimental Asthma
Authors: Desmet Christophe , Gosset Philippe , Pajak Bernard , Cataldo Didier , Bentires-Alj Mohamed , Lekeux Pierre , Bureau Fabrice
Published: April 2014

Knockout mice studies have revealed that NF-κB plays a critical role in Th2 cell differentiation and is therefore required for induction of allergic airway inflammation. However, the questions of whether NF-κB also plays a role in the effector phase of airway allergy and whether inhibiting NF-κB could have therapeutic value in the treatment of established asthma remain unanswered. To address these issues, we have assessed in OVA-sensitized wild-type mice the effects of selectively antagonizing NF-κB activity in the lungs during OVA challenge. Intratracheal administration of NF-κB decoy oligodeoxynucleotides to OVA-sensitized mice led to efficient nuclear transfection of airway immune cells, but not constitutive lung cells and draining lymph node cells, associated with abrogation of NF-κB activity in the airways upon OVA provocation. NF-κB inhibition was associated with strong attenuation of allergic lung inflammation, airway hyperresponsiveness, and local production of mucus, IL-5, IL-13, and eotaxin. IL-4 and OVA-specific IgE and IgG1 production was not reduced. This study demonstrates for the first time that activation of NF-κB in local immune cells is critically involved in the effector phase of allergic airway disease and that specific NF-κB inhibition in the lungs has therapeutic potential in the control of pulmonary allergy.
Green Tea Epigallocatechin-3-Gallate Mediates T Cellular NF-κB Inhibition and Exerts Neuroprotection in Autoimmune Encephalomyelitis
Authors: Aktas Orhan , Prozorovski Timour , Smorodchenko Alina , Savaskan Nicolai E. , Lauster Roland , Kloetzel Peter-Michael , Infante-Duarte Carmen , Brocke Stefan , Zipp Frauke
Published: April 2014

Recent studies in multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), point to the fact that even in the early phase of inflammation, neuronal pathology plays a pivotal role in the sustained disability of affected individuals. We show that the major green tea constituent, (−)-epigallocatechin-3-gallate (EGCG), dramatically suppresses EAE induced by proteolipid protein 139–151. EGCG reduced clinical severity when given at initiation or after the onset of EAE by both limiting brain inflammation and reducing neuronal damage. In orally treated mice, we found abrogated proliferation and TNF-α production of encephalitogenic T cells. In human myelin-specific CD4+ T cells, cell cycle arrest was induced, down-regulating the cyclin-dependent kinase 4. Interference with both T cell growth and effector function was mediated by blockade of the catalytic activities of the 20S/26S proteasome complex, resulting in intracellular accumulation of IκB-α and subsequent inhibition of NF-κB activation. Because its structure implicates additional antioxidative properties, EGCG was capable of protecting against neuronal injury in living brain tissue induced by N-methyl-d-aspartate or TRAIL and of directly blocking the formation of neurotoxic reactive oxygen species in neurons. Thus, a natural green tea constituent may open up a new therapeutic avenue for young disabled adults with inflammatory brain disease by combining, on one hand, anti-inflammatory and, on the other hand, neuroprotective capacities.
Modulation of Ca2+ Signaling by Na+/Ca2+ Exchangers in Mast Cells
Authors: Aneiros Eduardo , Philipp Stephan , Lis Annette , Freichel Marc , Cavalié Adolfo
Published: April 2014

Mast cells rely on Ca2+ signaling to initiate activation programs leading to release of proinflammatory mediators. The interplay between Ca2+ release from internal stores and Ca2+ entry through store-operated Ca2+ channels has been extensively studied. Using rat basophilic leukemia (RBL) mast cells and murine bone marrow-derived mast cells, we examine the role of Na+/Ca2+ exchangers. Calcium imaging experiments and patch clamp current recordings revealed both K+-independent and K+-dependent components of Na+/Ca2+ exchange. Northern blot analysis indicated the predominant expression of the K+-dependent sodium-calcium exchanger NCKX3. Transcripts of the exchangers NCX3 and NCKX1 were additionally detected in RBL cells with RT-PCR. The Ca2+ clearance via Na+/Ca2+ exchange represented ∼50% of the total clearance when Ca2+ signals reached levels ≥200 nM. Ca2+ signaling and store-operated Ca2+ entry were strongly reduced by inverting the direction of Na+/Ca2+ exchange, indicating that Na+/Ca2+ exchangers normally extrude Ca2+ ions from cytosol and prevent the Ca2+-dependent inactivation of store-operated Ca2+ channels. Working in the Ca2+ efflux mode, Na+/Ca2+ exchangers such as NCKX3 and NCX3 might, therefore, play a role in the Ag-induced mast cell activation by controlling the sustained phase of Ca2+ mobilization.
TLR Activation Synergizes with Kilham Rat Virus Infection to Induce Diabetes in BBDR Rats
Authors: Zipris Danny , Lien Egil , Xie Jenny X. , Greiner Dale L. , Mordes John P. , Rossini Aldo A.
Published: April 2014

Virus infection is hypothesized to be an important environmental “trigger” of type 1 diabetes in humans. We used the BBDR rat model to investigate the relationship between viral infection and autoimmune diabetes. BBDR rats are diabetes-free in viral Ab-free housing, but the disease develops in ∼30% of BBDR rats infected with Kilham rat virus (KRV) through a process that does not involve infection of pancreatic β cells. Pretreatment with polyinosinic-polycytidylic (poly(I:C)), a ligand of TLR3, acts synergistically to induce diabetes in 100% of KRV-infected rats. The mechanisms by which KRV induces diabetes and TLR3 ligation facilitates this process are not clear. In this study, we demonstrate that activation of the innate immune system plays a crucial role in diabetes induction. We report that multiple TLR agonists synergize with KRV infection to induce diabetes in BBDR rats, as do heat-killed Escherichia coli or Staphylococcus aureus (natural TLR agonists). KRV infection increases serum IL-12 p40 in a strain-specific manner, and increases IL-12 p40, IFN-γ-inducible protein-10, and IFN-γ mRNA transcript levels, particularly in the pancreatic lymph nodes of BBDR rats. Infection with vaccinia virus or H-1 parvovirus induced less stimulation of the innate immune system and failed to induce diabetes in BBDR rats. Our results suggest that the degree to which the innate immune system is activated by TLRs is important for expression of virus-induced diabetes in genetically susceptible hosts.
Enhanced Tolerance to Autoimmune Uveitis in CD200-Deficient Mice Correlates with a Pronounced Th2 Switch in Response to Antigen Challenge
Authors: Taylor Neil , McConnachie Karen , Calder Claudia , Dawson Rosemary , Dick Andrew , Sedgwick Jonathon D. , Liversidge Janet
Published: April 2014

A single exposure to inhaled Ag 10 days before immunization leads to long term, Ag-specific tolerance. Respiratory tract myeloid APCs are implicated, but how regulation is invoked, and how tolerance is sustained are unclear. This study examines the in vivo function of the myeloid regulatory molecule CD200 in the process of tolerance induction. Despite earlier onset of experimental autoimmune uveitis in sham-tolerized, CD200-deficient mice, disease incidence and subsequent severity were actually reduced compared with those in wild-type mice. Protection was more effective and long term, lasting at least 28 days. Halting disease progression and tolerance in CD200−/− mice correlated with a marked increase in Th2-associated cytokine production by Ag-challenged splenocytes. Reduced overall disease and enhanced tolerance in the CD200-deficient mice in this model system were unexpected and may be related to altered populations of MHC class IIlow APC in the respiratory tract compared with wild-type mice together with associated activation of STAT6 in draining lymph nodes of tolerized mice. These data indicate that in the absence of default inhibitory CD200 receptor signaling, alternative, powerful regulatory mechanisms are invoked. This may represent either permissive dominant Th2 activation or an altered hierarchy of negative signaling by other myeloid cell-expressed regulatory molecules.
Uncoupling of IL-2 Signaling from Cell Cycle Progression in Naive CD4+ T Cells by Regulatory CD4+CD25+ T Lymphocytes
Authors: Duthoit Christine T. , Mekala Divya J. , Alli Rajshekkhar S. , Geiger Terrence L.
Published: April 2014

Prior reports have shown that CD4+CD25+ regulatory T cells suppress naive T cell responses by inhibiting IL-2 production. In this report, using an Ag-specific TCR transgenic system, we show that naive T cells stimulated with cognate Ag in the presence of preactivated CD4+CD25+ T cells also become refractory to the mitogenic effects of IL-2. T cells stimulated in the presence of regulatory T cells up-regulated high affinity IL-2R, but failed to produce IL-2, express cyclins or c-Myc, or exit G0-G1. Exogenous IL-2 failed to break the mitotic block, demonstrating that the IL-2 production failure was not wholly responsible for the proliferation defect. This IL-2 unresponsiveness did not require the continuous presence of CD4+CD25+ regulatory T cells. The majority of responder T cells reisolated after coculture with regulatory cells failed to proliferate in response to IL-2, but were not anergic and proliferated in response to Ag. The mitotic block was also dissociated from the antiapoptotic effects of IL-2, because IL-2 still promoted the survival of T cells that had been cocultured with CD4+CD25+ T cells. IL-2-induced STAT5 phosphorylation in the cocultured responder cells was intact, implying that the effects of the regulatory cells were downstream of receptor activation. Our results therefore show that T cell activation in the presence of CD4+CD25+ regulatory T cells can induce an alternative stimulation program characterized by up-regulation of high affinity IL-2R, but a failure to produce IL-2, and uncoupling of the mitogenic and antiapoptotic effects of IL-2.