bluuebunny/crossref_metadata_2025 huggingface dataset:
Listeria-Infected Myeloid Dendritic Cells Produce IFN-β, Priming T Cell Activation
Authors: Feng Hanping , Zhang Dong , Palliser Deborah , Zhu Pengcheng , Cai Shenghe , Schlesinger Ann , Maliszewski Laura , Lieberman Judy
Published: April 2014

The intracellular bacterium Listeria monocytogenes infects dendritic cells (DC) and other APCs and induces potent cell-mediated protective immunity. However, heat-killed bacteria fail to do so. This study explored whether DC differentially respond to live and killed Listeria and how this affects T cell activation. To control for bacterial number, a replication-deficient strain, Lmdd, defective in d-alanine biosynthesis, was used. We found that DC internalize both live and heat-killed Lmdd and similarly up-regulate the expression of costimulatory molecules, a necessary step for T cell activation. However, only live Lmdd-infected DC stimulate T cells to express the early activation marker CD69 and enhance T cell activation upon TCR engagement. Infection with live, but not heat-killed, Lmdd induces myeloid DC to secrete copious amounts of IFN-β, which requires bacterial cytosolic invasion. Exposure to high concentrations of IFN-β sensitizes naive T cells for Ag-dependent activation.
ERK Activation Following Macrophage FcγR Ligation Leads to Chromatin Modifications at the IL-10 Locus
Authors: Lucas Mark , Zhang Xia , Prasanna Vikram , Mosser David M.
Published: April 2014

We have previously demonstrated that macrophages stimulated in the presence of immune complexes produce high levels of IL-10. We now examine the mechanism of IL-10 superinduction. We report that the enhanced production of IL-10 correlates with a rapid and enhanced activation of two MAPKs, ERK and p38. The inhibition of either ERK or p38 prevented IL-10 induction, indicating that both MAPKs were required for IL-10 synthesis. By chromatin immunoprecipitation assay, we demonstrate that activation of ERK leads to the phosphorylation of serine 10 on histone H3 at the il-10 gene, making the promoter more accessible to transcription factors generated in response to p38 activation. Inhibition of ERK activation prevented histone modifications, and decreased the binding of Sp1 and STAT3 to the IL-10 promoter. We conclude that the activation of ERK following FcγR ligation leads to a remodeling of the chromatin at the il-10 locus, making it more accessible to transcription factors. The rapid and transient regulation of transcription factor accessibility to the IL-10 promoter by MAPK activation represents a novel way that the production of this cytokine is regulated.
β2-Adrenoceptor Agonists, Like Glucocorticoids, Repress Eotaxin Gene Transcription by Selective Inhibition of Histone H4 Acetylation
Authors: Nie Mei , Knox Alan J. , Pang Linhua
Published: April 2014

Eotaxin is a potent eosinophil chemoattractant implicated in various allergic inflammatory conditions including asthma, but relatively little is known about its regulation. Human airway smooth muscle cells are an important source of eotaxin in the airway. We have previously demonstrated that β2-adrenoceptor agonists (β2-agonists) and glucocorticoids additively inhibit eotaxin production in human airway smooth muscle cells, but the underlying mechanisms are unclear. Here, we studied the molecular mechanisms of their actions and interactions on eotaxin gene transcription. TNF-α-induced eotaxin gene transcription was mediated mainly by the transcription factor NF-κB (p65/p50) as analyzed by luciferase reporter gene assay, Western blotting, EMSA, and electrophoretic mobility supershift assay. Chromatin immunoprecipitation assay demonstrated that TNF-α also induced selective histone H4 acetylation on lysines 5 and 12 at the eotaxin promoter site and p65 binding to the eotaxin promoter, resulting in eotaxin gene transcription. The inhibition of eotaxin production by β2-agonists and glucocorticoids was transcriptional and not due to altered NF-κB nuclear translocation or in vitro promoter binding capability, but due to their inhibition of TNF-α-induced histone H4 acetylation and p65 in vivo binding to the promoter. Additive inhibition was achieved when the two groups of drugs were combined. Our findings reveal a novel mechanism by which β2-agonists, like glucocorticoids, regulate NF-κB-mediated inflammatory gene expression through inhibition of histone acetylation. This provides one explanation for the benefits that result when these agents are combined to treat asthma, and may have important implications in a wide range of inflammatory diseases.
Oxidized Phospholipids Negatively Regulate Dendritic Cell Maturation Induced by TLRs and CD40
Authors: Blüml Stefan , Kirchberger Stefanie , Bochkov Valery N. , Krönke Gerhard , Stuhlmeier Karl , Majdic Otto , Zlabinger Gerhard J. , Knapp Walter , Binder Bernd R. , Stöckl Johannes , Leitinger Norbert
Published: April 2014

Maturation of dendritic cells (DCs) induced by pathogen-derived signals via TLRs is a crucial step in the initiation of an adaptive immune response and therefore has to be well controlled. In this study, we demonstrate that oxidized phospholipids (ox-PLs), which are generated during infections, apoptosis, and tissue damage, interfere with DC activation, preventing their maturation. ox-PLs blocked TLR-3- and TLR-4-mediated induction of the costimulatory molecules CD40, CD80, CD83, and CD86, the cytokines IL-12 and TNF, as well as lymphocyte stimulatory capacity. CD40 and TLR-2-mediated cytokine production was also inhibited, whereas up-regulation of costimulatory molecules via these receptors was not affected by ox-PLs. Thus, formation of ox-PLs during the course of an inflammatory response may represent a negative-feedback loop preventing excessive and sustained immune reactions through regulating DC maturation.
Application of Plasmid DNA Encoding IL-18 Diminishes Development of Herpetic Stromal Keratitis by Antiangiogenic Effects
Authors: Kim Bumseok , Lee Sujin , Suvas Susmit , Rouse Barry T.
Published: April 2014

HSV-1 infection of the eye can cause a blinding immunoinflammatory stromal keratitis (SK) lesion. Using the mouse model, we have demonstrated that angiogenesis is an essential step in lesion pathogenesis because its inhibition results in diminished severity. The molecules involved in causing corneal angiogenesis are multiple and include the vascular endothelial growth factor (VEGF) family of proteins. In this report we show that application of plasmid DNA encoding IL-18 to the cornea of mice before HSV-1 ocular infection resulted in reduced angiogenesis and diminished SK immunoinflammatory lesions. The antiangiogenic effects of IL-18 treatment appeared to be mediated by inhibition of VEGF production in the cornea. We also showed that IL-18 controlled VEGF expression in vitro and also decreased CpG oligodeoxynucleotide induced VEGF-dependent neovascularization. In addition the administration of IL-18-binding protein, an IL-18 antagonist, into the inflammatory eye resulted in elevated angiogenesis and increased VEGF expression. Our results indicate that IL-18 is an important endogenous negative regulator of HSV-induced angiogenesis resulting in reduced SK lesion severity. Our results could mean that IL-18 administration may represent a useful approach to manage unwanted angiogenesis.
5-Aminoimidazole-4-Carboxamide Ribonucleoside: A Novel Immunomodulator with Therapeutic Efficacy in Experimental Autoimmune Encephalomyelitis
Authors: Nath Narender , Giri Shailendra , Prasad Ratna , Salem Mohamad Labib , Singh Avtar K. , Singh Inderjit
Published: April 2014

Experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is a Th1-mediated inflammatory demyelinating disease of the CNS. AMP-activated protein kinase was reported recently to have anti-inflammatory activities by negatively regulating NF-κB signaling. In this study, we investigated the prophylactic and therapeutic efficacy of an AMP-activated protein kinase activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), in active and passive EAE induced by active immunization with PLP139–151 or MOG35–55 and in adoptive transfer of PLP139–151-sensitized T cells, respectively. In vivo treatment with AICAR exerted both prophylactic and therapeutic effects on EAE, attenuating the severity of clinical disease. The anti-inflammatory effects of AICAR were associated with the inhibition of the Ag-specific recall responses and inhibition of the Th1-type cytokines IFN-γ and TNF-α, whereas it induced the production of Th2 cytokines IL-4 and IL-10. Treatment of PLP139–151-specific T cells in vitro with AICAR decreased their expression of T-bet in response to IL-12, a Th1 transcription factor, whereas in response to IL-4, it induced the expression and phosphorylation of Th2 transcription factors GATA3 and STAT6, respectively. Moreover, treatment of APCs in vitro with AICAR inhibited their capability to present the proteolipid protein peptide to PLP139–151-specific T cells. In an irrelevant Th1-mediated, OT-2 TCR transgenic mouse model, AICAR impaired in vivo Ag-specific expansion of CD4+ T cells. Together, these findings show for the first time that AICAR is a novel immunomodulator with promising beneficial effects for the treatment of multiple sclerosis and other Th1-mediated inflammatory diseases.
A His-155 to Tyr Polymorphism Confers Gain-of-Function to the Human P2X7 Receptor of Human Leukemic Lymphocytes
Authors: Cabrini Giulio , Falzoni Simonetta , Forchap Sylvia L. , Pellegatti Patrizia , Balboni Alessandra , Agostini Paola , Cuneo Antonio , Castoldi Gianluigi , Baricordi O. Roberto , Di Virgilio Francesco
Published: April 2014

The P2X7R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X7R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X7R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X7R function was monitored by measuring ATP-induced Ca2+ influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X7 allelic variant. Ca2+ influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca2+ flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X7R confirmed an increased ATP-dependent activation of the P2X7 489T mutant with respect to the wild type receptor, as assessed by both by [Ca2+]i influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X7R.
Cutting Edge: Yin-Yang: Balancing Act of Prostaglandins with Opposing Functions to Regulate Inflammation
Authors: Mandal Asim K. , Zhang Zhongjian , Kim Sung-Jo , Tsai Pei-Chih , Mukherjee Anil B.
Published: April 2014

For many years, cyclooxygenase-2 (COX-2), a critical enzyme for PG production, has been the favorite target for anti-inflammatory drug development. However, recent revelations regarding the adverse effects of selective COX-2 inhibitors have stimulated intense debate. Interestingly, in the early phase of inflammation, COX-2 facilitates inflammatory PG production while in the late phase it has anti-inflammatory effects. Moreover, although some PGs are proinflammatory, others have anti-inflammatory effects. Thus, it is likely that PGs with opposing effects maintain homeostasis, although the molecular mechanism(s) remains unclear. We report here that an inflammatory PG, PGD2, via its receptor, mediates the activation of NF-κB stimulating COX-2 gene expression. Most interestingly, an anti-inflammatory PG (PGA1) suppresses NF-κB activation and inhibits COX-2 gene expression. We propose that while pro- and anti-inflammatory PGs counteract each other to maintain homeostasis, selective COX-2 inhibitors may disrupt this balance, thereby resulting in reported adverse effects.
Reciprocal Immunomodulation in a Schistosome and Hepatotropic Virus Coinfection Model
Authors: Edwards Matthew J. , Buchatska Olena , Ashton Miranda , Montoya Maria , Bickle Quentin D. , Borrow Persephone
Published: April 2014

Human coinfection with the helminth parasite Schistosoma mansoni and hepatitis B and hepatitis C viruses is associated with increased hepatic viral burdens and severe liver pathology. In this study we developed a murine S. mansoni/lymphocytic choriomeningitis virus (LCMV) coinfection model that reproduces the enhanced viral replication and liver pathology observed in human coinfections, and used this model to explore the mechanisms involved. Viral coinfection during the Th2-dominated granulomatous phase of the schistosome infection resulted in induction of a strong LCMV-specific T cell response, with infiltration of high numbers of LCMV-specific IFN-γ-producing CD8+ cells into the liver. This was associated with suppression of production of the Th2 cytokines dominant during S. mansoni infection and a rapid increase in morbidity, linked to hepatotoxicity. Interestingly, the liver of coinfected mice was extremely susceptible to viral replication. This correlated with a reduced intrahepatic type I IFN response following virus infection. Schistosome egg Ags were found to suppress the type I IFN response induced in murine bone marrow-derived dendritic cells by polyinosinic-polycytidylic acid. These results suggest that suppression of the antiviral type I IFN response by schistosome egg Ags in vivo predisposes the liver to enhanced viral replication with ensuing immunopathological consequences, findings that may be paralleled in human schistosome/hepatotropic virus coinfections.
Further Characterization of Reproductive Abnormalities in mCd59b Knockout Mice: A Potential New Function of mCd59 in Male Reproduction
Authors: Qin Xuebin , Dobarro Martin , Bedford Sylvia J. , Ferris Sean , Miranda Patricia V. , Song Wenping , Bronson Roderick T. , Visconti Pablo E. , Halperin Jose A.
Published: April 2014

CD59 is a GPI-linked membrane protein that inhibits formation of the membrane attack complex of complement. We reported recently that mice have two CD59 genes (termed mCd59a and mCd59b), and that the targeted deletion of mCd59b (mCd59b−/−) results in spontaneous hemolytic anemia and progressive loss of male fertility. Further studies of the reproductive abnormalities in mCd59b−/− mice reported in this study revealed the presence of abnormal multinucleated cells and increased apoptotic cells within the walls of the seminiferous tubules, and a decrease in the number, motility, and viability of sperm associated with a significant increase in abnormal sperm morphologies. Both the capacitation-associated tyrosine phosphorylation and the ionophore-induced acrosome reaction as well as luteinizing hormone, follicle-stimulating hormone, and testosterone serum levels were similar in mCd59b−/− and mCd59b+/+. Surprisingly, the functional deficiency of the complement protein C3 did not rescue the abnormal reproductive phenotype of mCd59b−/−, although it was efficient in rescuing their hemolytic anemia. These results indicate that the male reproductive abnormalities in mCd59b−/− are complement-independent, and that mCd59 may have a novel function in spermatogenesis that is most likely unrelated to its function as an inhibitor of membrane attack complex formation.
Immune Complex-Dependent Remodeling of the Airway Vasculature in Response to a Chronic Bacterial Infection
Authors: Aurora Arin B. , Baluk Peter , Zhang DongJi , Sidhu Sukhvinder S. , Dolganov Gregory M. , Basbaum Carol , McDonald Donald M. , Killeen Nigel
Published: April 2014

Chronic inflammation in the airways is associated with dramatic architectural changes in the walls of the airways and in the vasculature they contain. In this study, we show that the adaptive immune system is essential for airway remodeling that occurs in mice that are chronically infected with the respiratory pathogen Mycoplasma pulmonis. Angiogenesis, lymphangiogenesis, and epithelial remodeling were greatly reduced in mice that lacked B cells. Substantiating a role for Ab and airway immune complexes, we found that the transfer of immune serum to B cell-deficient mice could reconstitute pathogen-induced angiogenesis. Inflammatory cells recruited to the infected airways were activated by the humoral response, and this activation correlated with the induction of genes for remodeling factors such as vascular endothelial growth factor-D. The results reveal a novel pathway whereby T cell-dependent humoral immunity to a persistent airway infection can induce inflammation-dependent angiogenesis, lymphangiogenesis, and chronic airway pathology.
Critical Requirement of CD11b (Mac-1) on T Cells and Accessory Cells for Development of Experimental Autoimmune Encephalomyelitis
Authors: Bullard Daniel C. , Hu Xianzhen , Schoeb Trenton R. , Axtell Robert C. , Raman Chander , Barnum Scott R.
Published: April 2014

Mac-1 (CD18/CD11b) is a member of the β2-integrin family of adhesion molecules and is implicated in the development of many inflammatory diseases. The role of Mac-1 in the development of CNS demyelinating diseases, including multiple sclerosis, is not understood, and Ab inhibition studies in experimental allergic encephalomyelitis (EAE), the animal model for multiple sclerosis, have produced conflicting findings. To clarify these results and to determine Mac-1-mediated mechanisms in EAE, we performed EAE using Mac-1-deficient mice. Mac-1 homozygous-deficient, but not Mac-1 heterozygous-deficient mice, had significantly delayed onset and attenuated EAE. Leukocyte infiltration was similar in both groups of mice in early disease but significantly reduced in spinal cords of receptor-deficient mice in late disease. Adoptive transfer of Ag-restimulated T cells from wild-type to Mac-1-deficient mice produced significantly attenuated EAE, whereas transfer of Mac-1-deficient Ag-restimulated T cells to control mice failed to induce EAE. T cells from myelin oligodendrocyte glycoprotein (MOG)35–55 peptide-primed Mac-1-deficient mice displayed an altered cytokine phenotype with elevated levels of TGF-β and IL-10, but reduced levels of IL-2, IFN-γ, TNF-α, IL-12, and IL-4 compared with control mice. Mac-1-deficient T cells from primed mice proliferated comparably to that of control T cells on MOG35–55 restimulation in vitro. However, the draining lymph nodes of MAC-1-deficient mice on day 10 after MOG35–55 immunization contained lower frequency of blast T cells than in control mice, suggesting poor priming. Our results indicate that Mac-1 expression is critical on both phagocytic cells and T cells for the development of demyelinating disease.
CD1a-, b-, and c-Restricted TCRs Recognize Both Self and Foreign Antigens
Authors: Vincent Michael S. , Xiong Xiaowei , Grant Ethan P. , Peng Wei , Brenner Michael B.
Published: April 2014

Individual CD1-restricted T cells can recognize either endogenous or foreign lipid Ags, but the extent to which the same CD1-restricted TCR can react to both self and microbial lipids is unknown. In this study, we have identified CD1a-, CD1b-, and CD1c-restricted T cells from normal human donors that induce cytolysis and secrete copious IFN-γ in response to self-CD1 expressed on monocyte-derived dendritic cells. Remarkably, microbial Ags presented by CD1 are even more potent agonists for these same T cells. The αβ T cell receptors from such clones are diverse and confer specificity for both self-CD1 and foreign lipid Ags. The dual reactivity of these CD1-restricted cells suggests that the capacity for rapid responses to inflammatory stimuli without memory coexists with the capacity for strong Ag-specific responses and the generation of memory in vivo.
Morphine Induces CD4+ T Cell IL-4 Expression through an Adenylyl Cyclase Mechanism Independent of the Protein Kinase A Pathway
Authors: Roy Sabita , Wang Jinghua , Charboneau Richard , Loh Horace H. , Barke Roderick A.
Published: April 2014

Impaired host defense mechanisms after major operative procedures and trauma are recognized as important factors in the development of infectious complication. Trauma is associated with impaired cellular immunity and CD4+ T cell Th2 differentiation. We have previously implicated morphine treatment as a possible mechanism for Th2 differentiation after injury. In this investigation we first establish that morphine treatment in vivo results in Th2 differentiation and that this effect is mediated through a naltrexone-sensitive opioid receptor. We investigated the intracellular mechanism by which morphine controls CD4+ T cell differentiation and demonstrate that morphine treatment in vitro 1) increases anti CD3/CD28 Ab-induced CD4+ T cell IL-4 protein synthesis, IL-4 mRNA, and GATA-3 mRNA accumulation through a pertussis toxin-sensitive receptor; 2) results in a dose-dependent increase in anti-CD3/CD28 Ab-induced CD4+ T cell cytoplasmic cAMP concentration; and 3) increases the forskolin-stimulated cytoplasmic cAMP level through a pertussis toxin-sensitive receptor. We also demonstrate that chronic morphine treatment increases anti-CD3/CD28 Ab-induced IL-4 promoter activity and IL-4 immunoprotein expression through a p38 MAPK-dependent, but protein kinase A- and Erk1/Erk2-independent, mechanism.
Evaluation of OX40 Ligand as a Costimulator of Human Antiviral Memory CD8 T Cell Responses: Comparison with B7.1 and 4-1BBL
Authors: Serghides Lena , Bukczynski Jacob , Wen Tao , Wang Chao , Routy Jean-Pierre , Boulassel Mohamed-Rachid , Sekaly Rafick-Pierre , Ostrowski Mario , Bernard Nicole F. , Watts Tania H.
Published: April 2014

CTL are important effectors of antiviral immunity. Designing adjuvants that can induce strong cytotoxic T cell responses in humans would greatly improve the effectiveness of an antiviral vaccination or therapeutic strategy. Recent evidence suggests that, in addition to its well-established role in costimulation of CD4 T cell responses, OX40L (CD134) can directly costimulate mouse CD8 T cells. In this study, we evaluated the role of OX40L in costimulation of human antiviral CD8 T cell responses and compared it with two other important costimulators, B7.1 (CD80) and 4-1BBL (CD137L). Delivery of OX40L to human monocytes using a recombinant replication-defective adenovirus led to greater expansion, up-regulation of perforin, enhanced cytolytic activity, and increased numbers of IFN-γ- and TNF-α-producing antiviral memory CD8 T cells in cultures of total T cells. Synergistic or additive effects were observed when OX40L costimulation was combined with 4-1BBL (CD137L) or B7.1 (CD80) costimulation. In total T cell cultures, at low Ag dose, 4-1BBL provided the most potent costimulus for influenza-specific CD8 T cell expansion, followed by B7.1 (CD80) and then OX40L. For isolated CD8 T cells, 4-1BBL was also the most consistent costimulator, followed by B7.1. In contrast, OX40L showed efficacy in direct activation of memory CD8 T cells in only one of seven donors. Thus, OX40L costimulates human antiviral memory CD8 T cell responses largely through indirect effects and can enhance anti-influenza, anti-EBV, and anti-HIV responses, particularly in combination with 4-1BBL or B7.
Functional Analysis of B and T Lymphocyte Attenuator Engagement on CD4+ and CD8+ T Cells
Authors: Krieg Carsten , Han Peggy , Stone Roslynn , Goularte Olivia D. , Kaye Jonathan
Published: April 2014

T cell activation can be profoundly altered by coinhibitory and costimulatory molecules. B and T lymphocyte attenuator (BTLA) is a recently identified inhibitory Ig superfamily cell surface protein found on lymphocytes and APC. In this study we analyze the effects of an agonistic anti-BTLA mAb, PK18, on TCR-mediated T cell activation. Unlike many other allele-specific anti-BTLA mAb we have generated, PK18 inhibits anti-CD3-mediated CD4+ T cell proliferation. This inhibition is not dependent on regulatory T cells, nor does the Ab induce apoptosis. Inhibition of T cell proliferation correlates with a profound reduction in IL-2 secretion, although this is not the sole cause of the block of cell proliferation. In contrast, PK18 has no effect on induction of the early activation marker CD69. PK18 also significantly inhibits, but does not ablate, IL-2 secretion in the presence of costimulation as well as reduces T cell proliferation under limiting conditions of activation in the presence of costimulation. Similarly, PK18 inhibits Ag-specific T cell responses in culture. Interestingly, PK18 is capable of delivering an inhibitory signal as late as 16 h after the initiation of T cell activation. CD8+ T cells are significantly less sensitive to the inhibitory effects of PK18. Overall, BTLA adds to the growing list of cell surface proteins that are potential targets to down-modulate T cell function.
Pharmacological Inhibition of Endotoxin Responses Is Achieved by Targeting the TLR4 Coreceptor, MD-2
Authors: Visintin Alberto , Halmen Kristen A. , Latz Eicke , Monks Brian G. , Golenbock Douglas T.
Published: April 2014

The detection of Gram-negative LPS depends upon the proper function of the TLR4-MD-2 receptor complex in immune cells. TLR4 is the signal transduction component of the LPS receptor, whereas MD-2 is the endotoxin-binding unit. MD-2 appears to activate TLR4 when bound to TLR4 and ligated by LPS. Only the monomeric form of MD-2 was found to bind LPS and only monomeric MD-2 interacts with TLR4. Monomeric MD-2 binds TLR4 with an apparent Kd of 12 nM; this binding avidity was unaltered in the presence of endotoxin. E5564, an LPS antagonist, appears to inhibit cellular activation by competitively preventing the binding of LPS to MD-2. Depletion of endogenous soluble MD-2 from human serum, with an immobilized TLR4 fusion protein, abrogated TLR4-mediated LPS responses. By determining the concentration of added-back MD-2 that restored normal LPS responsiveness, the concentration of MD-2 was estimated to be ∼50 nM. Similarly, purified TLR4-Fc fusion protein, when added to the supernatants of TLR4-expressing cells in culture, inhibited the interaction of MD-2 with TLR4, thus preventing LPS stimulation. The ability to inhibit the effects of LPS as a result of the binding of TLR4-Fc or E5564 to MD-2 highlights MD-2 as the logical target for drug therapies designed to pharmacologically intervene against endotoxin-induced disease.
CD1d-Independent Developmental Acquisition of Prompt IL-4 Gene Inducibility in Thymus CD161(NK1)−CD44lowCD4+CD8− T Cells Is Associated with Complementarity Determining Region 3-Diverse and Biased Vβ2/Vβ7/Vβ8/Vα3.2 T Cell Receptor Usage
Authors: Chen Yi-Ting , Kung John T.
Published: April 2014

Among Ag-inexperienced naive T cells, the CD1d-restricted NKT cell that uses invariant TCR-α-chain is the most widely studied cell capable of prompt IL-4 inducibility. We show in this study that thymus CD161−CD44lowCD4+CD8− T cells promptly produce IL-4 upon TCR stimulation, a response that displays biased Vβ(2/7/8) and Vα3.2 TCR usage. The association of Vβ family bias and IL-4 inducibility in thymus CD161−CD44lowCD4+CD8− T cells is found for B6, B10, BALB/c, CBA, B10.A(4R), and ICR mouse strains. Despite reduced IL-4 inducibility, there is a similarly biased Vβ(2/7/8) TCR usage by IL-4 inducibility+ spleen CD161−CD44lowCD4+CD8− T cells. Removal of α-galacotosylceramide/CD1d-binding cells from CD161−CD44lowCD4+CD8− thymocytes does not significantly affect their IL-4 inducibility. The development of thymus CD161−CD44lowCD4+CD8− T cells endowed with IL-4 inducibility and their associated use of Vβ(2/7/8) are β2-microglobulin-, CD1d-, and p59fyn-independent. Thymus CD161−CD44lowCD4+CD8− T cells produce low and no IFN-γ inducibility in response to TCR stimulation and to IL-12 + IL-18, respectively, and they express diverse complementarity determining region 3 sequences for both TCR-α- and -β-chains. Taken together, these results demonstrate the existence of a NKT cell distinct, TCR-repertoire diverse naive CD4+ T cell subset capable of prompt IL-4 inducibility. This subset has the potential to participate in immune response to a relatively large number of Ags. The more prevalent nature of this unique T cell subset in the thymus than the periphery implies roles it might play in intrathymic T cell development and may provide a framework upon which mechanisms of developmentally regulated IL-4 gene inducibility can be studied.
A High-Affinity Natural Autoantibody from Human Cord Blood Defines a Physiologically Relevant Epitope on the FcεRIα
Authors: Bobrzynski Tomasz , Fux Michaela , Vogel Monique , Stadler Michael B. , Stadler Beda M. , Miescher Sylvia M.
Published: April 2014

Natural Abs represent the indigenous immune repertoire and are thus present at birth and persist throughout life. Previously, human autoantibodies to the α domain of the high-affinity IgE receptor (FcεRIα) have been isolated from Ab libraries derived from normal donors and patients with chronic urticaria. To investigate whether these anti-FcεRIα Abs are present in the germline repertoire, we constructed a phage Fab display library from human cord blood, which represents the naive immune repertoire before exposure to exogenous Ags. All isolated clones specific to the FcεRIα had the same sequence. This single IgM Ab, named CBMα8, was strictly in germline configuration and had high affinity and functional in vitro anaphylactogenic activity. Inhibition experiments indicated an overlapping epitope on the FcεRIα recognized by both CBMα8 and the previously isolated anti-FcεRIα Abs from autoimmune and healthy donors. This common epitope on FcεRIα coincides with the binding site for IgE. Affinity measurements demonstrated the presence of Abs showing CBMα8-like specificity, but with a significantly lower affinity in i.v. Ig, a therapeutic multidonor IgG preparation. We propose a hypothesis of escape mutants, whereby the resulting lower affinity IgG anti-FcεRIα Abs are rendered less likely to compete with IgE for binding to FcεRIα.
Leucine Aminopeptidase Is Not Essential for Trimming Peptides in the Cytosol or Generating Epitopes for MHC Class I Antigen Presentation
Authors: Towne Charles F. , York Ian A. , Neijssen Joost , Karow Margaret L. , Murphy Andrew J. , Valenzuela David M. , Yancopoulos George D. , Neefjes Jacques J. , Rock Kenneth L.
Published: April 2014

To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Leucine aminopeptidase (LAP) is an IFN-inducible cytosolic aminopeptidase that can trim precursor peptides to mature epitopes and has been thought to play an important role in Ag presentation. To examine the role of LAP in generating MHC class I peptides in vivo, we generated LAP-deficient mice and LAP-deficient cell lines. These mutant mice and cells are viable and grow normally. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full-length Ag constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. These data demonstrate that LAP is not an essential enzyme for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases.
Tissue-Resident Memory CD8+ T Cells Can Be Deleted by Soluble, but Not Cross-Presented Antigen
Authors: Wei Cheng-Hong , Trenney Rebecca , Sanchez-Alavez Manuel , Marquardt Kristi , Woodland David L. , Henriksen Steven J. , Sherman Linda A.
Published: April 2014

Under noninflammatory conditions, both naive and central memory CD8 T cells can be eliminated in the periphery with either soluble peptide or cross-presented Ag. Here, we assess the tolerance susceptibility of tissue-resident memory CD8 T cells in mice to these two forms of tolerogen. Soluble peptide specifically eliminated the majority of memory CD8 cells present in both lymphoid and extralymphoid tissues including lung and liver, but was unable to reduce the number present in the CNS. In contrast, systemic cross-presentation of Ag by dendritic cells resulted in successful elimination of memory cells only from the spleen, with no significant reduction in the numbers of tissue-resident memory cells in the lung. The fact that tissue-resident memory cells were unable to access cross-presented Ag suggests that either the memory cells in the lung do not freely circulate out of the tissue, or that they circulate through a region in the spleen devoid of cross-presented Ag. Thus, although tissue-resident memory cells are highly susceptible to tolerance induction, both the form of tolerogen and location of the T cells can determine their accessibility to tolerogen and the degree to which they are successfully deleted from specific tissues.
Dimerization of the Major Birch Pollen Allergen Bet v 1 Is Important for its In Vivo IgE-Cross-Linking Potential in Mice
Authors: Schöll Isabella , Kalkura Narayana , Shedziankova Yuliya , Bergmann Alexander , Verdino Petra , Knittelfelder Regina , Kopp Tamara , Hantusch Brigitte , Betzel Christian , Dierks Karsten , Scheiner Otto , Boltz-Nitulescu George , Keller Walter , Jensen-Jarolim Erika
Published: April 2014

In type I allergy, the cross-linking of membrane IgE on B lymphocytes and of cytophilic IgE on effector cells by their respective allergens are key events. For cross-linking two IgE molecules, allergens need at least two epitopes. On large molecules, these could be different epitopes in a multivalent, or identical epitopes in a symmetrical, fashion. However, the availability of epitopes may be limited on small allergens such as Bet v 1, the major birch pollen allergen. The present work analyzes whether dimerization is required for the cross-linking capacity of this allergen. In immunoblots, murine monoclonal and polyclonal human Bet v 1-specific Abs detected, besides a Bet v 1 monomer of 17 kDa, a dimer of 34 kDa. In dynamic light scattering, Bet v 1 appeared as dimers and even multimers, but a single condition could be defined where it behaved exclusively monomerically. Small-angle x-ray scattering of the monomeric and dimeric samples resulted in diagrams agreeing with the calculated models. Circular dichroism measurements indicated that the structure of Bet v 1 was preserved under monomeric conditions. Skin tests in Bet v 1-allergic mice were positive with Bet v 1 dimer, but remained negative using the monomer. Furthermore, in contrast to dimeric Bet v 1, the monomer was less capable of activating murine memory B cells for IgE production in vivo. Our data indicate that the presentation of two identical epitopes by dimerized allergens is a precondition for cross-linking of IgE on mast cells and B lymphocytes.
The Extent of Histone Acetylation Correlates with the Differential Rearrangement Frequency of Individual VH Genes in Pro-B Cells
Authors: Espinoza Celia R. , Feeney Ann J.
Published: April 2014

During B lymphocyte development, Ig heavy and L chain genes are assembled by V(D)J recombination. Individual V, D, and J genes rearrange at very different frequencies in vivo, and the natural variation in recombination signal sequence does not account for all of these differences. Because a permissive chromatin structure is necessary for the accessibility of VH genes for VH to DJH recombination, we hypothesized that gene rearrangement frequency might be influenced by the extent of histone modifications. Indeed, we found in freshly isolated pro-B cells from μMT mice a positive correlation between the level of enrichment of VHS107 genes in the acetylated histone fractions as assayed by chromatin immunoprecipitation, and their relative rearrangement frequency in vivo. In the VH7183 family, the very frequently rearranging VH81X gene showed the highest association with acetylated histones, especially in the newborn. Together, our data show that the extent of histone modifications in pro-B cells should be considered as a mechanism by which accessibility and the rearrangement level of individual VH genes is regulated.
Identification of Soluble N-Ethylmaleimide-Sensitive Factor Attachment Protein Receptor Exocytotic Machinery in Human Plasma Cells: SNAP-23 Is Essential for Antibody Secretion
Authors: Reales Elena , Mora-López Francisco , Rivas Verónica , García-Poley Antonio , Brieva José A. , Campos-Caro Antonio
Published: April 2014

Plasma cells (PC) are B-lymphocytes terminally differentiated in a postmitotic state, with the unique purpose of manufacturing and exporting Igs. Despite the importance of this process in the survival of vertebrates, no studies have been made to understand the molecular events that regulate Ig exocytosis by PC. The present study explores the possible presence of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) system in human PC, and examines its functional role in Ig secretion. Syntaxin-2, Syntaxin-3, Syntaxin-4, vesicle-associated membrane protein (VAMP)-2, VAMP-3, and synaptosome-associated protein (SNAP)-23 could be readily detected in normal human PC obtained from intestinal lamina propria and blood, as well as in human PC lines. Because SNAP-23 plays a central role in SNAREs complex formation, it was chosen to examine possible functional implications of the SNARE system in PC Ig secretion. When recombinant SNAP-23 fusion protein was introduced into the cells, a complete abolishment of Ig production was observed in the culture supernatants of PC lines, as well as in those of normal PC. These results provide insights, for the first time, into the molecular machinery of constitutive vesicular trafficking in human PC Ig secretion and present evidence indicating that at least SNAP-23 is essential for Ab production.
The MHC of the Duck (Anas platyrhynchos) Contains Five Differentially Expressed Class I Genes
Authors: Moon Debra A. , Veniamin Simona M. , Parks-Dely Julie A. , Magor Katharine E.
Published: April 2014

MHC class I proteins mediate a variety of functions in antiviral defense. In humans and mice, three MHC class I loci each contribute one or two alleles and each can present a wide variety of peptide Ags. In contrast, many lower vertebrates appear to use a single MHC class I locus. Previously we showed that a single locus was predominantly expressed in the mallard duck (Anas platyrhynchos) and that locus was adjacent to the polymorphic transporter for the Ag-processing (TAP2) gene. Characterization of a genomic clone from the same duck now allows us to compare genes to account for their differential expression. The clone carried five MHC class I genes and the TAP genes in the following gene order: TAP1, TAP2, UAA, UBA, UCA, UDA, and UEA. We designated the predominantly expressed gene UAA. Transcripts corresponding to the UDA locus were expressed at a low level. No transcripts were found for three loci, UBA, UCA, and UEA. UBA had a deletion within the promoter sequences. UCA carried a stop codon in-frame. UEA did not have a polyadenylation signal sequence. All sequences differed primarily in peptide-binding pockets and otherwise had the hallmarks of classical MHC class I alleles. Despite the presence of additional genes in the genome, the duck expresses predominantly one MHC class I gene. The limitation to one expressed MHC class I gene may have functional consequences for the ability of ducks to eliminate viral pathogens, such as influenza.
MyD88-Dependent and -Independent Murine Cytomegalovirus Sensing for IFN-α Release and Initiation of Immune Responses In Vivo
Authors: Delale Thomas , Paquin André , Asselin-Paturel Carine , Dalod Marc , Brizard Géraldine , Bates Elizabeth E. M. , Kastner Philippe , Chan Susan , Akira Shizuo , Vicari Alain , Biron Christine A. , Trinchieri Giorgio , Brière Francine
Published: April 2014

Antiviral immunity requires early and late mechanisms in which IFN-α and IL-12 play major roles. However, the initial events leading to their production remain largely unclear. Given the crucial role of TLR in innate recognition, we investigated their role in antiviral immunity in vivo. Upon murine CMV (MCMV) infection, both MyD88−/− and TLR9−/− mice were more susceptible and presented increased viral loads compared with C57BL/6, TLR2−/−, TLR3−/−, or TLR4−/− mice. However, in terms of resistance to infection, IFN-α production and in many other parameters of early inflammatory responses, the MyD88−/− mice showed a more defective response than TLR9−/− mice. In the absence of the TLR9/MyD88 signaling pathway, cytokine production was dramatically impaired with a complete abolition of bioactive IL-12p70 serum release contrasting with a high flexibility for IFN-α release, which is initially (36 h) plasmacytoid dendritic cell- and MyD88-dependent, and subsequently (44 h) PDC-, MyD88-independent and, most likely, TLR-independent. NK cells from MCMV-infected MyD88−/− and TLR9−/− mice displayed a severely impaired IFN-γ production, yet retained enhanced cytotoxic activity. In addition, dendritic cell activation and critical inflammatory cell trafficking toward the liver were still effective. In the long term, except for isotype switching to MCMV-specific IgG1, the establishment of Ab responses was not significantly altered. Thus, our results demonstrate a critical requirement of TLR9 in the process of MCMV sensing to assure rapid antiviral responses, coordinated with other TLR-dependent and -independent events that are sufficient to establish adaptive immunity.
A Live Diarrheal Vaccine Imprints a Th2 Cell Bias and Acts as an Anti-Inflammatory Vaccine
Authors: Jun SangMu , Gilmore Wendy , Callis Gayle , Rynda Agnieszka , Haddad Asmahan , Pascual David W.
Published: April 2014

An experimental vaccine for enterotoxigenic Escherichia coli (ETEC) composed of a live, attenuated Salmonella vector-expressing enterotoxigenic E. coli fimbriae, colonization factor Ag I (CFA/I), stimulated a biphasic Th cell response when given orally and suppressed the normally produced proinflammatory response. Such suppression was also evident upon the Salmonella-CFA/I infection of macrophages resulting in diminished TNF-α, IL-1, and IL-6 production and suggesting that the CFA/I fimbrial expression by Salmonella may protect against a proinflammatory disease. To test this hypothesis, SJL/J mice were vaccinated with Salmonella-CFA/I construct 1 or 4 wk before induction of experimental autoimmune encephalomyelitis using an encephalitogenic proteolipid protein peptide, PLP139–151. Mice receiving Salmonella-CFA/I vaccine recovered completely from mild acute clinical disease and showed only mild inflammatory infiltrates in the spinal cord white and gray matter. This protective effect was accompanied by a loss of encephalitogenic IFN-γ-secreting Th cells and was replaced with an increase in IL-4, IL-10, and IL-13 secretion. Collectively, these data suggested that Salmonella-CFA/I is an anti-inflammatory vaccine that down-regulates proinflammatory cells and confers protection against a proinflammatory disease, experimental autoimmune encephalomyelitis, via immune deviation.
Combined Effects of ATP on the Therapeutic Efficacy of Antimicrobial Drug Regimens against Mycobacterium avium Complex Infection in Mice and Roles of Cytosolic Phospholipase A2-Dependent Mechanisms in the ATP-Mediated Potentiation of Antimycobacterial Host Resistance
Authors: Tomioka Haruaki , Sano Chiaki , Sato Katsumasa , Ogasawara Keiko , Akaki Tatsuya , Sano Keisuke , Cai Shan Shan , Shimizu Toshiaki
Published: April 2014

ATP, which serves as a mediator of intramacrophage signaling pathways through purinoceptors, is known to potentiate macrophage antimycobacterial activity. In this study we examined the effects of ATP in potentiating host resistance to Mycobacterium avium complex (MAC) infection in mice undergoing treatment with a drug regimen using clarithromycin and rifamycin and obtained the following findings. First, the administration of ATP in combination with the clarithromycin and rifamycin regimen accelerated bacterial elimination in MAC-infected mice without causing changes in the histopathological features or the mRNA expression of pro- or anti-inflammatory cytokines from those in the mice not given ATP. Second, ATP potentiated the anti-MAC bactericidal activity of macrophages cultivated in the presence of clarithromycin and rifamycin. This effect of ATP was closely related to intracellular Ca2+ mobilization and was specifically blocked by a cytosolic phospholipase A2 (cPLA2) inhibitor, arachidonyl trifluoromethylketone. Third, intramacrophage translocation of membranous arachidonic acid molecules to MAC-containing phagosomes was also specifically blocked by arachidonyl trifluoromethylketone. In the confocal microscopic observation of MAC-infected macrophages, ATP enhanced the intracellular translocation of cPLA2 into MAC-containing phagosomes. These findings suggest that ATP increases the host anti-MAC resistance by potentiating the antimycobacterial activity of host macrophages and that the cPLA2-dependent generation of arachidonic acid from the phagosomal membrane is essential for such a phenomenon.
In Vivo Protective Role of Human Group IIA Phospholipase A2 against Experimental Anthrax
Authors: Piris-Gimenez Alejandro , Paya Miguel , Lambeau Gérard , Chignard Michel , Mock Michèle , Touqui Lhousseine , Goossens Pierre L.
Published: April 2014

Anthrax is an acute disease caused by Bacillus anthracis. Some animal species are relatively resistant to anthrax infection. This trait has been correlated to the extent of the local inflammatory reaction, suggesting innate immunity to be the first line of defense against B. anthracis infection in nonimmunized hosts. Group IIA secreted phospholipase A2 (sPLA2-IIA) is produced in particular by macrophages and possesses potent antibacterial activity especially against Gram-positive bacteria. We have previously shown in vitro that sPLA2-IIA kills both germinated B. anthracis spores and encapsulated bacilli. Here we show that sPLA2-IIA plays in vivo a protective role against experimental anthrax. Transgenic mice expressing human sPLA2-IIA are resistant to B. anthracis infection. In addition, in vivo administration of recombinant human sPLA2-IIA protects mice against B. anthracis infection. The protective effect was observed both with a highly virulent encapsulated nontoxinogenic strain and a wild-type encapsulated toxinogenic strain, showing that toxemia did not hinder the sPLA2-IIA-afforded protection. sPLA2-IIA, a natural component of the immune system, may thus be considered a novel therapeutic agent to be used in adjunct with current therapy for treating anthrax. Its anthracidal activity would be effective even against strains resistant to multiple antibiotics.