bluuebunny/crossref_metadata_2025 huggingface dataset:
Differential Calcium Regulation of Proinflammatory Activities in Human Neutrophils Exposed to the Neuropeptide Pituitary Adenylate Cyclase-Activating Protein
Authors: Harfi Issam , Corazza Francis , D’Hondt Stéphanie , Sariban Eric
Published: April 2014

The neuropeptide pituitary adenylate cyclase-activating protein (PACAP) acts via the G protein-coupled receptor vasoactive intestinal peptide/PACAP receptor-1 to induce phospholipase C/calcium and MAPK-dependent proinflammatory activities in human polymorphonuclear neutrophils (PMNs). In this study, we evaluate other mechanisms that regulate PACAP-evoked calcium transients, the nature of the calcium sources, and the role of calcium in proinflammatory activities. Reduction in the activity of PMNs to respond to PACAP was observed after cell exposure to inhibitors of the cAMP/protein kinase A, protein kinase C, and PI3K pathways, to pertussis toxin, genistein, and after chelation of intracellular calcium or after extracellular calcium depletion. Mobilization of intracellular calcium stores was based on the fact that PACAP-associated calcium transient was decreased after exposure to 1) thapsigargin, 2) Xestospongin C, and 3) the protonophore carbonyl cyanide 4-(trifluoromethoxy) phenyl hydrazone; inhibition of calcium increase by calcium channel blockers, by nifedipine and verapamil, indicated that PACAP was also acting on calcium influx. Such mobilization was not dependent on a functional actin cytoskeleton. Homologous desensitization with nanomoles of PACAP concentration and heterologous receptors desensibilization by G protein-coupled receptor agonists were observed. Intracellular calcium depletion modulated PACAP-associated ERK but not p38 phosphorylation; in contrast, extracellular calcium depletion modulated PACAP-associated p38 but not ERK phosphorylation. In PACAP-treated PMNs, reactive oxygen species production and CD11b membrane up-regulation in contrast to lactoferrin release were dependent on both intra- and extracellular calcium, whereas matrix metalloproteinase-9 release was unaffected by extracellular calcium depletion. These data indicate that both extracellular and intracellular calcium play key roles in PACAP proinflammatory activities.
An IL-7 Fusion Protein That Shows Increased Thymopoietic Ability
Authors: Henson Sian M. , Snelgrove Robert , Hussell Tracy , Wells Dominic J. , Aspinall Richard
Published: April 2014

The role of IL-7 during thymopoiesis has led to it being the focus of a number of therapeutic interventions. However, its small size and pleiotropic nature present problems for thymus-directed therapies. We have created a fusion molecule between the extracellular N-terminal domain of CCR9 and IL-7, which has the potential to overcome these difficulties. This novel fusion protein retains the thymopoietic activity of IL-7 and the ligand-binding ability of CCR9. As a thymopoietic agent, compared with IL-7, it shows an enhanced retention in the thymus, increased de novo T cell production, and increased thymic output. Old mice receiving the fusion protein show improved CD8 T cell responses and reduced viral load after infection with influenza virus compared with those receiving IL-7. This chimeric molecule offers a novel therapeutic strategy that may result in the production of an effective immunorestorative agent.
Cutting Edge: Molecular Structure of the IL-1R-Associated Kinase-4 Death Domain and Its Implications for TLR Signaling
Authors: Lasker Michael V. , Gajjar Mark M. , Nair Satish K.
Published: April 2014

IL-1R-associated kinase (IRAK) 4 is an essential component of innate immunity. IRAK-4 deficiency in mice and humans results in severe impairment of IL-1 and TLR signaling. We have solved the crystal structure for the death domain of Mus musculus IRAK-4 to 1.7 Å resolution. This is the first glimpse of the structural details of a mammalian IRAK family member. The crystal structure reveals a six-helical bundle with a prominent loop, which among IRAKs and Pelle, a Drosophila homologue, is unique to IRAK-4. This highly structured loop contained between helices two and three, comprises an 11-aa stretch. Although innate immune domain recognition is thought to be very similar between Drosophila and mammals, this structural component points to a drastic difference. This structure can be used as a framework for future mutation and deletion studies and potential drug design.
Protection against Autoimmunity in Nonlymphopenic Hosts by CD4+CD25+ Regulatory T Cells Is Antigen-Specific and Requires IL-10 and TGF-β
Authors: Huang Xiaopei , Zhu Jiangao , Yang Yiping
Published: April 2014

CD4+CD25+ regulatory T cells (TReg) play a critical role in the control of autoimmunity. However, little is known about how TReg suppress self-reactive T cells in vivo, thus limiting the development of TReg-based therapy for treating autoimmune diseases. This is in large part due to the dependency on a state of lymphopenia to demonstrate TReg-mediated suppression in vivo and the unknown Ag specificity of TReg in most experimental models. Using a nonlymphopenic model of autoimmune pneumonitis and TReg with known Ag specificity, in this study we demonstrated that these TReg can actively suppress activation of self-reactive T cells and protect mice from fatal autoimmune pneumonitis. The protection required TReg with the same Ag specificity as the self-reactive T cells and depended on IL-10 and TGF-β. These results suggest that suppression of autoimmunity by TReg in vivo consists of multiple layers of regulation and advocate for a strategy involving Ag-specific TReg for treating organ-specific autoimmunity, because they do not cause generalized immune suppression.
DX5/CD49b-Positive T Cells Are Not Synonymous with CD1d-Dependent NKT Cells
Authors: Pellicci Daniel G. , Hammond Kirsten J. L. , Coquet Jonathan , Kyparissoudis Konstantinos , Brooks Andrew G. , Kedzierska Katherine , Keating Rachael , Turner Stephen , Berzins Stuart , Smyth Mark J. , Godfrey Dale I.
Published: April 2014

NKT cells are typically defined as CD1d-dependent T cells that carry an invariant TCR α-chain and produce high levels of cytokines. Traditionally, these cells were defined as NK1.1+ T cells, although only a few mouse strains express the NK1.1 molecule. A popular alternative marker for NKT cells has been DX5, an Ab that detects the CD49b integrin, expressed by most NK cells and a subset of T cells that resemble NKT cells. Interpretation of studies using DX5 as an NKT cell marker depends on how well DX5 defines NKT cells. Using a range of DX5 and other anti-CD49b Abs, we reveal major differences in reactivity depending on which Ab and which fluorochrome are used. The brightest, PE-conjugated reagents revealed that while most CD1d-dependent NKT cells expressed CD49b, they represented only a minority of CD49b+ T cells. Furthermore, CD49b+ T cell numbers were near normal in CD1d−/− mice that are completely deficient for NKT cells. CD1d tetramer− CD49b+ T cells differ from NKT cells by their activation and memory marker expression, tissue distribution, and CD4/CD8 coreceptor profile. Interestingly, both NKT cells and CD1d tetramer− CD49b+ T cells produce cytokines, but the latter are clearly biased toward Th1-type cytokines, in contrast to NKT cells that produce both Th1 and Th2 cytokines. Finally, we demonstrate that expression of CD49b by NKT cells does not dramatically alter with age, contrasting with earlier reports proposing DX5 as a maturation marker for NKT cells. In summary, our data demonstrate that DX5/CD49b is a poor marker for identifying CD1d-dependent NKT cells.
MD-2 Mediates the Ability of Tetra-Acylated and Penta-Acylated Lipopolysaccharides to AntagonizeEscherichia coliLipopolysaccharide at the TLR4 Signaling Complex
Authors: Coats Stephen R. , Pham Thu-Thao T. , Bainbridge Brian W. , Reife Robert A. , Darveau Richard P.
Published: April 2014

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.
Autoantibodies to Fibrillin-1 Activate Normal Human Fibroblasts in Culture through the TGF-β Pathway to Recapitulate the “Scleroderma Phenotype”
Authors: Zhou Xiaodong , Tan Filemon K. , Milewicz Dianna M. , Guo Xinjian , Bona Constantin A. , Arnett Frank C.
Published: April 2014

Fibroblasts from patients with systemic sclerosis (SSc) are activated producing excessive amounts of extracellular matrix (ECM) components. Recently, we identified a new SSc-specific autoantibody against portions of fibrillin-1, a major component of ECM microfibrils and regulator of TGF-β1 signaling. To examine a potential pathogenic role of anti-fibrillin-1 autoantibodies, normal human fibroblasts were treated with affinity-purified autoantibodies isolated from SSc sera and then examined for alterations in gene and protein expression levels using microarrays, quantitative RT-PCR, immunoblots, and immunofluorescence. Compared with fibroblasts cultured in normal medium or in medium containing normal human IgG, anti-fibrillin-1 autoantibody-treated normal dermal fibroblasts showed increased expression of COL and several other ECM components characteristically overexpressed in SSc fibroblasts. This was accompanied by phosphorylation and nuclear translocation of Smad3. Neutralization of TGF-β1 with anti-TGF-β1 Abs significantly diminished the activation of fibroblasts by anti-fibrillin-1 autoantibodies. These data indicate that anti-fibrillin-1 autoantibodies can induce the activation of normal dermal fibroblasts into a profibrotic phenotype resembling that of SSc by potentially causing the release of sequestered TGF-β1 from fibrillin-1-containing microfibrils in the ECM.
Type I Interferons Trigger Systemic, Partial Lymphocyte Activation in Response to Viral Infection
Authors: Alsharifi Mohammed , Lobigs Mario , Regner Matthias , Lee Eva , Koskinen Aulikki , Müllbacher Arno
Published: April 2014

The vast majority of both T and B cells in mice were found to up-regulate cell surface expression of the early activation markers CD69 and CD86, but not CD25, within 24 h of infection with Semliki Forest virus. Kinetics and magnitude of activation marker expression was dependent on live virus, dose, and correlated with strain virulence. Activation marker expression declined to baseline levels over the next 96 h. This very early “activation” of such a high percentage of lymphocytes required the presence of type I IFN receptor genes, was inducible with poly(I:C), and correlated with IFN-I levels in serum. We conclude that virus-induced IFN-I release systemically affects most of the hosts T and B cells by triggering them rapidly and independently of Ag-reactivity into a semiactivated state.
Synergy between Extracellular Group IIA Phospholipase A2 and Phagocyte NADPH Oxidase in Digestion of Phospholipids of Staphylococcus aureus Ingested by Human Neutrophils
Authors: Femling Jon K. , Nauseef William M. , Weiss Jerrold P.
Published: April 2014

Acute inflammatory responses to invading bacteria such as Staphylococcus aureus include mobilization of polymorphonuclear leukocytes (PMN) and extracellular group IIA phospholipase A2 (gIIA-PLA2). Although accumulating coincidentally, the in vitro anti-staphylococcal activities of PMN and gIIA-PLA2 have thus far been studied separately. We now show that degradation of S. aureus phospholipids during and after phagocytosis by human PMN requires the presence of extracellular gIIA-PLA2. The concentration of extracellular gIIA-PLA2 required to produce bacterial digestion was reduced 10-fold by PMN. The effects of added gIIA-PLA2 were greater when present before phagocytosis but even apparent when added after S. aureus were ingested by PMN. Related group V and X PLA2, which are present within PMN granules, do not contribute to bacterial phospholipid degradation during and after phagocytosis even when added at concentrations 30-fold higher than that needed for action of the gIIA-PLA2. The action of added gIIA-PLA2 required catalytically active gIIA-PLA2 and, in PMN, a functional NADPH oxidase but not myeloperoxidase. These findings reveal a novel collaboration between cellular oxygen-dependent and extracellular oxygen-independent host defense systems that may be important in the ultimate resolution of S. aureus infections.
Induction of Keratinocyte Migration via Transactivation of the Epidermal Growth Factor Receptor by the Antimicrobial Peptide LL-37
Authors: Tokumaru Sho , Sayama Koji , Shirakata Yuji , Komatsuzawa Hitoshi , Ouhara Kazuhisa , Hanakawa Yasushi , Yahata Yoko , Dai Xiuju , Tohyama Mikiko , Nagai Hiroshi , Yang Lujun , Higashiyama Shigeki , Yoshimura Akihiko , Sugai Motoyuki , Hashimoto Koji
Published: April 2014

The closure of skin wounds is essential for resistance against microbial pathogens, and keratinocyte migration is an important step in skin wound healing. Cathelicidin hCAP18/LL-37 is an innate antimicrobial peptide that is expressed in the skin and acts to eliminate microbial pathogens. Because hCAP18/LL-37 is up-regulated at skin wound sites, we hypothesized that LL-37 induces keratinocyte migration. In this study, we found that 1 μg/ml LL-37 induced the maximum level of keratinocyte migration in the Boyden chamber assay. In addition, LL-37 phosphorylated the epidermal growth factor receptor (EGFR) after 10 min, which suggests that LL-37-induced keratinocyte migration occurs via EGFR transactivation. To test this assumption, we used inhibitors that block the sequential steps of EGFR transactivation, such as OSU8-1, CRM197, anti-EGFR no. 225 Ab, and AG1478. All of these inhibitors completely blocked LL-37-induced keratinocyte migration, which indicates that migration occurs via HB-EGF-mediated EGFR transactivation. Furthermore, CRM197, anti-EGFR no. 225, and AG1478 blocked the LL-37-induced phosphorylation of STAT3, and transfection with a dominant-negative mutant of STAT3 abolished LL-37-induced keratinocyte migration, indicating the involvement of the STAT3 pathway downstream of EGFR transactivation. Finally, we tested whether the suppressor of cytokine signaling (SOCS)/cytokine-inducible Src homology 2-containing protein (CIS) family of negative regulators of STAT3 regulates LL-37-induced keratinocyte migration. Transfection with SOCS1/Jak2 binding protein or SOCS3/CIS3 almost completely abolished LL-37-induced keratinocyte migration. In conclusion, LL-37 induces keratinocyte migration via heparin-binding-EGF-mediated transactivation of EGFR, and SOCS1/Jak 2 binding and SOCS3/CIS3 negatively regulate this migration. The results of this study suggest that LL-37 closes skin wounds by the induction of keratinocyte migration.
Functional Properties and Lineage Relationship of CD8+ T Cell Subsets Identified by Expression of IL-7 Receptor α and CD62L
Authors: Bachmann Martin F. , Wolint Petra , Schwarz Katrin , Jäger Petra , Oxenius Annette
Published: April 2014

Three major subsets of Ag-experienced CD8+ T cells have been identified according to their expression of CD62L and CD127. These markers are associated with central memory T cells (CD62L+CD127+), effector memory T cells (CD162L−CD127+), and effector T cells (CD62L−CD127−). In this study we characterized the development of these three populations during acute and chronic viral infections and after immunization with virus-like particles and determined their lineage relation and functional and protective properties. We found that the balance between the three subsets was critically regulated by the availability of Ag and time. After initial down-regulation of CD127, the responding CD8+ T cell population down-regulated CD62L and re-expressed CD127. Dependent on Ag availability, the cells then further differentiated into CD62L−CD127− effector cells or, in the absence of Ag, re-expressed CD62L to become central memory T cells. Although all three populations efficiently produced effector cytokines such as IFN-γ, CD62L−CD127− effector cells exhibited the highest ex vivo lytic potential. In contrast, CD62L+CD127+ central memory T cells most efficiently produced IL-2 and proliferated extensively in vitro and in vivo upon antigenic restimulation. Strikingly, only effector and effector memory, but not central memory, T cells were able to protect against peripheral infection with vaccinia virus, whereas central memory T cells were most potent at protecting against systemic infection with lymphocytic choriomeningitis virus, indicating that the antiviral protective capacities of specific CD8+ T cell subsets are closely related to the nature of the challenging pathogen.
Zinc-Mediated Inhibition of Cyclic Nucleotide Phosphodiesterase Activity and Expression Suppresses TNF-α and IL-1β Production in Monocytes by Elevation of Guanosine 3′,5′-Cyclic Monophosphate
Authors: von Bülow Verena , Rink Lothar , Haase Hajo
Published: April 2014

The trace element zinc affects several aspects of immune function, such as the release of proinflammatory cytokines from monocytes. We investigated the role of cyclic nucleotide signaling in zinc inhibition of LPS-induced TNF-α and IL-1β release from primary human monocytes and the monocytic cell line Mono Mac1. Zinc reversibly inhibited enzyme activity of phosphodiesterase-1 (PDE-1), PDE-3, and PDE-4 in cellular lysate. It additionally reduced mRNA expression of PDE-1C, PDE-4A, and PDE-4B in intact cells. Although these PDE can also hydrolyze cAMP, only the cellular level of cGMP was increased after incubation with zinc, whereas cAMP was found to be even slightly reduced due to inhibition of its synthesis. To investigate whether an increase in cGMP alone is sufficient to inhibit cytokine release, the cGMP analogues 8-bromo-cGMP and dibutyryl cGMP as well as the NO donor S-nitrosocysteine were used. All three treatments inhibited TNF-α and IL-1β release after stimulation with LPS. Inhibition of soluble guanylate cyclase-mediated cGMP synthesis with LY83583 reversed the inhibitory effect of zinc on LPS-induced cytokine release. In conclusion, inhibition of PDE by zinc abrogates the LPS-induced release of TNF-α and IL-1β by increasing intracellular cGMP levels.
A Novel Role of Hepatocyte Growth Factor as an Immune Regulator through Suppressing Dendritic Cell Function
Authors: Okunishi Katsuhide , Dohi Makoto , Nakagome Kazuyuki , Tanaka Ryoichi , Mizuno Shinya , Matsumoto Kunio , Miyazaki Jun-ichi , Nakamura Toshikazu , Yamamoto Kazuhiko
Published: April 2014

Hepatocyte growth factor (HGF) plays an important role in many biological events such as angiogenesis, cell proliferation, anti-fibrosis and antiapoptosis. It is well known that HGF promotes tumor progression and suppresses development of fibrosis after tissue injury. In contrast, its role in immune-mediated disorders has not been fully clarified. In the present study, we examined the role of HGF in Ag-specific immune response using in vitro studies and an experimental model of allergic airway inflammation. We first confirmed that dendritic cells (DCs) expressed the receptor for HGF, c-met, which was not expressed in T cells. Treatment with HGF both in vitro and in vivo potently suppressed DC functions such as Ag-presenting capacity, thus down-regulating Ag-induced Th1- and Th2-type immune responses. Exogenous administration of the HGF expression plasmid into Ag-primed mice markedly suppressed the development of airway eosinophilia and airway hyperresponsiveness, which was induced by Ag inhalation, with suppression of the Ag-presenting capacity of DCs in the lung. HGF exhibited these immunosuppressive effects without up-regulation of IL-10 or TGF-β. We also found that expression of endogenous HGF in the lung significantly increased following Ag sensitization and inhalation challenges. Finally, neutralization of endogenous HGF in vivo significantly increased airway eosinophilia and airway hyperresponsiveness with up-regulation of the Ag-presenting capacity of DCs in the lung. These results demonstrated a novel, significant, and possibly therapeutic role of HGF as a potent regulator in immune-mediated disorders such as asthma.
CD44 and Annexin A2 Mediate the C5a Chemotactic Cofactor Function of the Vitamin D Binding Protein
Authors: McVoy Lauren A. , Kew Richard R.
Published: April 2014

The vitamin D binding protein (DBP) is a plasma protein that significantly enhances the chemotactic activity of C5a and C5adesArg (cochemotactic activity). The objective of this study was to investigate how DBP mediates this process using neutrophils and U937 cells transfected with the C5a receptor (U937-C5aR cells) and comparing chemotaxis to C-activated serum (DBP dependent) vs purified C5a (DBP independent). Binding to the cell surface is essential for this protein to function as a chemotactic cofactor, and DBP binds to a chondroitin sulfate proteoglycan (CSPG) on neutrophil plasma membrane preparations. To determine whether a CSPG also functions to mediate cochemotactic activity, U937-C5aR cells were grown in chlorate to inhibit CSPG sulfation or treated with chondroitinase AC. Either treatment significantly inhibited chemotaxis only to C-activated serum. CD44 is a major cell surface CSPG on leukocytes, and functions to facilitate chemotaxis. Treatment of cells with anti-CD44 blocks chemotaxis of neutrophils and U937-C5aR cells to C-activated serum but not purified C5a. DBP binds to CD44 on the cell surface as evidenced by coimmunoprecipitation, confocal microscopy, and cell binding studies. Annexin A2 associates with CD44 in lipid rafts; therefore, its potential role in mediating cochemotactic activity was investigated. Results demonstrate that anti-A2 inhibits neutrophil and U937-C5aR chemotaxis specifically to C-activated serum, blocks DBP binding to cells, and colocalizes with anti-DBP on the cell surface. These results provide clear evidence that CD44 and annexin A2 mediate the C5a chemotactic cofactor function of DBP.
Anti-IFN-γ Autoantibodies in Disseminated Nontuberculous Mycobacterial Infections
Authors: Patel Smita Y. , Ding Li , Brown Margaret R. , Lantz Larry , Gay Ted , Cohen Stuart , Martyak Lenna A. , Kubak Bernard , Holland Steven M.
Published: April 2014

Although many patients with disseminated nontuberculous mycobacterial disease have molecular defects in the IFN-γ/IL-12 axis, recent case reports have shown autoantibodies against IFN-γ associated with severe nontuberculous mycobacterial infections. To check this finding in an independent population, we screened 35 patients with either disseminated or pulmonary nontuberculous mycobacterial infections for whom no molecular defect was known. We identified high-titer-neutralizing anti-IFN-γ IgG in the plasma of six patients. All six patients were female, parous, of East Asian descent, and had disseminated infection, predominantly with rapidly growing mycobacteria. The anti-IFN-γ IgG had in vitro biological activity on the IFN-γ-dependent phosphorylation of STAT-1 as well as on the IFN-γ-dependent up-regulation of TNF-α and IL-12. In contrast, this anti-IFN-γ Ab had no effect on IFN-α-dependent STAT-1 phosphorylation. These patients confirm a novel syndrome linking autoimmunity and immunodeficiency.
Induction of Apoptosis by the Hydrocarbon Oil Pristane: Implications for Pristane-Induced Lupus
Authors: Calvani Nicola , Caricchio Roberto , Tucci Marco , Sobel Eric S. , Silvestris Franco , Tartaglia Paola , Richards Hanno B.
Published: April 2014

Intraperitoneal injection of the hydrocarbon oil pristane into normal mice leads to a lupus-like autoimmune syndrome. Although advances in defining the roles of cellular and humoral mediators involved in this syndrome have been made, the mechanisms that initiate a break in tolerance leading to autoimmunity remain unknown. We describe in this study that pristane induces apoptosis both in vivo and in vitro. Pristane arrests cell growth and induces cell death by apoptosis via the mitochondrial pathway of caspase activation in a dose-dependent manner. Nuclear autoantigens created by pristane-induced apoptosis of lymphoid cells within the peritoneal cavity in the setting of a profoundly altered cytokine milieu may be the initiating event in the development of autoimmunity in this syndrome. These findings suggest that apoptosis may be a critical initial event in the pathogenesis of pristane-induced lupus and are of potential relevance for human systemic lupus erythematosus.
Cutting Edge: TLR4 Deficiency Confers Susceptibility to Lethal Oxidant Lung Injury
Authors: Zhang Xuchen , Shan Peiying , Qureshi Salman , Homer Robert , Medzhitov Ruslan , Noble Paul W. , Lee Patty J.
Published: April 2014

TLRs have been studied extensively in pathogen-mediated host responses. We use a murine model of lethal oxidant-mediated injury to demonstrate for the first time that mammalian TLR4 is required for survival and lung integrity. Administering high levels of inspired oxygen, or hyperoxia, is commonly used as a life-sustaining measure in critically ill patients. However, prolonged exposures can lead to respiratory failure and death. TLR4-deficient mice exhibited increased mortality and lung injury during hyperoxia. The enhanced susceptibility of TLR4-deficient mice to hyperoxia was associated with an inability to up-regulate Bcl-2 and phospho-Akt. Restoration of Bcl-2 and phospho-Akt levels by the exogenous transfer of the antioxidant gene heme oxygenase-1 markedly attenuated hyperoxia-induced injury, apoptosis, and mortality in TLR4-deficient mice. Taken together, our results suggest a protective role of TLR4 in oxidant-mediated injury, providing novel mechanistic links among innate immunity, oxidant stress, and apoptosis.
Protection from Abortion by Heme Oxygenase-1 Up-Regulation Is Associated with Increased Levels of Bag-1 and Neuropilin-1 at the Fetal-Maternal Interface
Authors: Sollwedel Andre , Bertoja Annarosa Zambon , Zenclussen Maria Laura , Gerlof Katrin , Lisewski Ulrike , Wafula Paul , Sawitzki Birgit , Woiciechowsky Christian , Volk Hans-Dieter , Zenclussen Ana Claudia
Published: April 2014

Tolerance mechanisms allowing pregnancy success resemble those involved in allograft acceptance. Heme oxygenase (HO) is a tissue-protective molecule, which allows graft acceptance and is known to have antiapoptotic effects on several cell types. We previously reported down-regulated levels of HO-1 and HO-2 in placenta from allopregnant mice undergoing abortion. In this study, we analyzed whether the up-regulation of HO-1 by cobalt-protoporphyrin (Co-PP) during implantation window can rescue mice from abortion. Induction of HO-1 by Co-PP treatment prevented fetal rejection, whereas the down-regulation of HOs by zinc-protoporphyrin application boosted abortion. The beneficial effect of HO-1 induction was not related to a local shift to Th2-profile or to a change in the NO system. Interestingly, the expression of the antiapoptotic/cytoprotective molecule Bag-1 as well as the levels of neuropilin-1, a novel marker for T regulatory cells, were up-regulated after Co-PP treatment. Our data strongly support a very important role for HO-1 in fetal allotolerance and suggest that HO-1 might be protective by up-regulating tissue protective molecules, i.e., Bag-1, and by activating T regulatory cells rather than by changing the local cytokine profile.
Receptor Editing Can Lead to Allelic Inclusion and Development of B Cells That Retain Antibodies Reacting with High Avidity Autoantigens
Authors: Liu Sucai , Velez Maria-Gabriela , Humann Jessica , Rowland Sarah , Conrad Frank J. , Halverson Regina , Torres Raul M. , Pelanda Roberta
Published: April 2014

Receptor editing is a major B cell tolerance mechanism that operates by secondary Ig gene rearrangements to change the specificity of autoreactive developing B cells. In the 3-83Igi mouse model, receptor editing operates in every autoreactive anti-H-2Kb B cell, providing a novel receptor without additional cell loss. Despite the efficiency of receptor editing in generating nonautoreactive Ag receptors, we show in this study that this process does not inactivate the autoantibody-encoding gene(s) in every autoreactive B cell. In fact, receptor editing can generate allelically and isotypically included B cells that simultaneously express the original autoreactive and a novel nonautoreactive Ag receptors. Such dual Ab-expressing B cells differentiate into transitional and mature B cells retaining the expression of the autoantibody despite the high avidity interaction between the autoantibody and the self-Ag in this system. Moreover, we find that these high avidity autoreactive B cells retain the autoreactive Ag receptor within the cell as a consequence of autoantigen engagement and through a Src family kinase-dependent process. Finally, anti-H-2Kb IgM autoantibodies are found in the sera of older 3-83Igi mice, indicating that dual Ab-expressing autoreactive B cells are potentially functional and capable of differentiating into IgM autoantibody-secreting plasma cells under certain circumstances. These results demonstrate that autoreactive B cells reacting with ubiquitous membrane bound autoantigens can bypass mechanisms of central tolerance by coexpressing nonautoreactive Abs. These dual Ab-expressing autoreactive B cells conceal their autoantibodies within the cell manifesting a superficially tolerant phenotype that can be partially overcome to secrete IgM autoantibodies.
A CXCR4-Dependent Chemorepellent Signal Contributes to the Emigration of Mature Single-Positive CD4 Cells from the Fetal Thymus
Authors: Vianello Fabrizio , Kraft Paul , Mok Ying Ting , Hart William K. , White Natasha , Poznansky Mark C.
Published: April 2014

Developing thymocytes undergo maturation while migrating through the thymus and ultimately emigrate from the organ to populate peripheral lymphoid tissues. The process of thymic emigration is controlled in part via receptor-ligand interactions between the chemokine stromal-derived factor (SDF)-1, and its cognate receptor CXCR4, and sphingosine 1-phosphate (S1P) and its receptor S1PR. The precise mechanism by which S1P/S1PR and CXCR4/SDF-1 contribute to thymic emigration remains unclear. We proposed that S1P-dependent and -independent mechanisms might coexist and involve both S1P-induced chemoattraction and SDF-1-mediated chemorepulsion or fugetaxis of mature thymocytes. We examined thymocyte emigration in thymi from CXCR4-deficient C57BL/6 embryos in a modified assay, which allows the collection of CD62Lhigh and CD69low recent thymic emigrants. We demonstrated that single-positive (SP) CD4 thymocytes, with the characteristics of recent thymic emigrants, failed to move away from CXCR4-deficient fetal thymus in vitro. We found that the defect in SP CD4 cell emigration that occurred in the absence of CXCR4 signaling was only partially overcome by the addition of the extrathymic chemoattractant S1P and was not associated with abnormalities in thymocyte maturation and proliferative capacity or integrin expression. Blockade of the CXCR4 receptor in normal thymocytes by AMD3100 led to the retention of mature T cells in the thymus in vitro and in vivo. The addition of extrathymic SDF-1 inhibited emigration of wild-type SP cells out of the thymus by nullifying the chemokine gradient. SDF-1 was also shown to elicit a CXCR4-dependent chemorepellent response from fetal SP thymocytes. These novel findings support the thesis that the CXCR4-mediated chemorepellent activity of intrathymic SDF-1 contributes to SP thymocyte egress from the fetal thymus.
Protein Kinase C-θ Is an Early Survival Factor Required for Differentiation of Effector CD8+ T Cells
Authors: Barouch-Bentov Rina , Lemmens Edward E. , Hu Junru , Janssen Edith M. , Droin Nathalie M. , Song Jianxun , Schoenberger Stephen P. , Altman Amnon
Published: April 2014

CD8+ T cells are crucial for host defense against invading pathogens and malignancies. However, relatively little is known about intracellular signaling events that control the genetic program of their activation and differentiation. Using CD8+ T cells from TCR-transgenic mice crossed to protein kinase C-θ (PKCθ)-deficient mice, we report that PKCθ is not required for Ag-induced CD8+ T cell proliferation, but is important for T cell survival and differentiation into functional, cytokine-producing CTLs. Ag-stimulated PKCθ−/− T cells underwent accelerated apoptosis associated with deregulated expression of Bcl-2 family proteins and displayed reduced activation of ERKs and JNKs. Some defects in the function of PKCθ−/− T cells (poor survival and reduced Bcl-2 and Bcl-xL expression, CTL activity, and IFN-γ expression) were partially or fully restored by coculture with wild-type T cells or by addition of exogenous IL-2, whereas others (increased BimEL expression and TNF-α production) were not. These findings indicate that PKCθ, although not essential for initial Ag-induced proliferation, nevertheless plays an important role in promoting and extending T cell survival, thereby enabling the complete genetic program of effector CD8+ differentiation. The requirement for PKCθ in different types of T cell-dependent responses may, therefore, depend on the overall strength of signaling by the TCR and costimulatory receptors and may reflect, in addition to its previously established role in activation, an important, hitherto unappreciated, role in T cell survival.
Diabetes Resistance/Susceptibility in T Cells of Nonobese Diabetic Mice Conferred by MHC and MHC-Linked Genes
Authors: Ueno Aito , Cho Suzanne , Cheng Lu , Wang Zhongying , Wang Bo , Yang Yang
Published: April 2014

Polymorphism of MHC and MHC-linked genes is tightly associated with susceptibility to type 1 diabetes (T1D) in human and animal models. Despite the extensive studies, however, the role of MHC and MHC-linked genes expressed by T cells on T1D susceptibility remains unclear. Because T cells develop from TCR− thymic precursor (pre-T) cells that undergo MHC restriction mediated by thymic stroma cells, we reconstituted the T cell compartment of NOD.scid-RIP-B7.1 mice using pre-T cells isolated from NOD, NOR, AKR, and C57BL/6 (B6) mice. T1D developed rapidly in the mice reconstituted with pre-T cells derived from NOD or NOR donors. In contrast, most of the NOD.scid-RIP-B7.1 mice reconstituted with pre-T cells from AKR or B6 donors were free of T1D. Further analysis revealed that genes within MHC locus of AKR or B6 origin reduced incidence of T1D in the reconstituted NOD.scid-RIP-B7.1 mice. The expression of MHC class I genes of k, but not b haplotype, in T cells conferred T1D resistance. Replacement of an interval near the distal end of the D region in T cells of B6 origin with an identical allele of 129.S6 origin resulted in T1D development in the reconstituted mice. These results provide evidence that the expression of MHC class I and MHC-linked genes in T cells of NOD mice indeed contributes to T1D susceptibility, while expression of specific resistance alleles of MHC or MHC-linked genes in T cells alone would effectively reduce or even prevent T1D.
Increased Expression of TLR3 in Lymph Nodes during Simian Immunodeficiency Virus Infection: Implications for Inflammation and Immunodeficiency
Authors: Sanghavi Sonali K. , Reinhart Todd A.
Published: April 2014

As pattern recognition receptors, TLRs signal and induce expression of multiple host defense genes including proinflammatory cytokines and chemokines. To investigate the mechanisms of up-regulation of proinflammatory cytokines and chemokines during SIV infection in rhesus macaques, we measured the relative levels of expression of TLRs 1–10 in lymphoid tissues during different stages of SIV infection. By real-time RT-PCR, TLR3 was determined to be up-regulated in macaque lymph nodes (LN) throughout the course of infection, whereas TLR9 was down-regulated during early stages of infection. CXCL9/Mig, CXCL10/IP-10, IFN-γ, and IFN-α mRNAs were also increased during acute SIV infection and AIDS. Treatment of macaque spleen and LN cells with TLR3 and TLR9 ligands led to the induction of these same genes. TLR3 stimulation had disparate effects on viral transcription and viral replication, because poly(I:C), a model TLR3 ligand, stimulated the viral promoter but potently inhibited SIV replication in primary cultures of macaque spleen and LN cells. These findings identify roles for TLR3 inflammation in lymphoid tissues and in the immunopathogenesis of HIV-1/SIV, and suggest that TLR3 ligands could potentially be used to flush out latently infected cells that persist during antiretroviral therapies.
A Critical Role for Complement C3d and the B Cell Coreceptor (CD19/CD21) Complex in the Initiation of Inflammatory Arthritis
Authors: Del Nagro Christopher J. , Kolla Ravi V. , Rickert Robert C.
Published: April 2014

Complement C3 cleavage products mediate the recognition and clearance of toxic or infectious agents. In addition, binding of the C3d fragment to Ag promotes B lymphocyte activation through coengagment of the BCR and complement receptor 2 (CD21). Signal augmentation is thought to be achieved through enhanced recruitment and activation of CD21-associated CD19. In this study we show, using the DBA/1 collagen-induced arthritis (CIA) model, that conjugation of C3d to heterologous type II collagen is sufficient to cause disease in the absence of the mycobacterial components of CFA. Transient depletion of C3 during the inductive phase of CIA delays and lessens the severity of disease, and DBA/1 mice deficient for coreceptor components CD19 or CD21 are not susceptible to CIA. Adoptive transfer experiments revealed that CD21 expression on either B cells or follicular dendritic cells is sufficient to acquire disease susceptibility. Although CD19−/− and CD21−/− mice produce primary Ab responses to heterologous and autologous type II collagen, they are impaired in the ability to activate T cells, form germinal centers, and produce secondary autoantibody responses. These findings indicate that binding of C3d to self-Ags can promote autoimmunity through enhanced Ag retention and presentation by follicular dendritic cells and B cells, respectively.
T Cell Mimicry and Epitope Specificity of Cross-Reactive T Cell Clones from Rheumatic Heart Disease
Authors: Ellis Nadia M. J. , Li Ya , Hildebrand William , Fischetti Vincent A. , Cunningham Madeleine W.
Published: April 2014

Mimicry between streptococcal M protein and cardiac myosin is important in the pathogenesis of rheumatic heart disease. M protein-specific human T cell clones derived from rheumatic carditis were cross-reactive with human cardiac myosin, and laminin, a valve protein. Among the 11 CD4+ and CD8+ cross-reactive T cell clones, at least 6 different reactivity patterns were distinguished, suggesting different degrees of cross-reactivity and a very diverse T cell repertoire. The latter was confirmed by a heterogeneous Vβ gene and CDR3 usage. HLA restriction and Th1 cytokine production in response to rM6 protein were preserved when the T cell clones were stimulated by human cardiac myosin or other α-helical proteins, such as tropomyosin and laminin. The cross-reactive human T cell clones proliferated to B2 and B3A, dominant peptide epitopes in the B repeat region of streptococcal M protein. In human cardiac myosin, epitopes were demonstrated in the S2 and light meromyosin regions. In our study, T cell mimicry was defined as recognition of structurally related Ags involved in disease and recognized by the same T cell. Mimicry in our study was related to α-helical coiled coil proteins which have a repetitive seven-aa residue periodicity that maintains α-helical structure and thus creates a high number of degenerate possibilities for recognition by T cells. The study of human T cell clones from rheumatic heart disease revealed potential sites of T cell mimicry between streptococcal M protein and human cardiac myosin and represents some of the most well-defined T cell mimicry in human autoimmune disease.
In Vivo Immunomanipulation of Vγ9Vδ2 T Cells with a Synthetic Phosphoantigen in a Preclinical Nonhuman Primate Model
Authors: Sicard Hélène , Ingoure Sophie , Luciani Béatrice , Serraz Claire , Fournié Jean-Jacques , Bonneville Marc , Tiollier Jérôme , Romagné François
Published: April 2014

Vγ9Vδ2+ cells represent the major population of γδ T cells in primate blood and react in an MHC-unrestricted fashion to a set of low m.w. nonpeptide phosphoantigens. Two types of structurally related agonists have been discovered so far: the natural phosphoantigens (hydroxydimethyl allyl-pyrophosphate or isopentenyl-pyrophosphate (IPP)) acting directly on Vγ9Vδ2+ TCR and aminobisphosphonates, which block the mevalonate pathway in target cells, leading to accumulation of natural phosphoantigens that in turn activate Vγ9Vδ2+ cells. We demonstrate in the cynomolgus monkey that Vγ9Vδ2 can be manipulated in vivo with bromohydrin pyrophosphate (BrHPP)/Phosphostim, a potent synthetic agonist for which the mechanism of action is similar to natural phosphoantigens. Although of very short half-life, injection of BrHPP leads to strong activation of Vγ9Vδ2, inducing production of a high level of Th1 cytokines. Combination of BrHPP with low-dose rhIL-2 induces specific amplification of effector-memory peripheral Vγ9Vδ2 in blood in a dose-dependant manner. This transient response returns to baseline within 10–15 days. Successive infusions of BrHPP and rhIL-2 induce less vigorous expansions, suggesting a progressive exhaustion of the response. As no toxicity is detected with or without IL-2, this scheme represents a promising immunotherapeutic strategy for induction of systemic Th1 cytokines and massive expansion of γδ T cell subset with antitumor and anti-infectious properties.
HLA-A*0201, HLA-A*1101, and HLA-B*0702 Transgenic Mice Recognize Numerous Poxvirus Determinants from a Wide Variety of Viral Gene Products
Authors: Pasquetto Valerie , Bui Huynh-Hoa , Giannino Rielle , Mirza Fareed , Sidney John , Oseroff Carla , Tscharke David C. , Irvine Kari , Bennink Jack R. , Peters Bjoern , Southwood Scott , Cerundolo Vincenzo , Grey Howard , Yewdell Jonathan W. , Sette Alessandro
Published: April 2014

In virus models explored in detail in mice, CTL typically focus on a few immunodominant determinants. In this study we use a multipronged approach to understand the diversity of CTL responses to vaccinia virus, a prototypic poxvirus with a genome ∼20-fold larger than that of the model RNA viruses typically studied in mice. Based on predictive computational algorithms for peptide binding to HLA supertypes, we synthesized a panel of 2889 peptides to begin to create an immunomic map of human CTL responses to poxviruses. Using this panel in conjunction with CTLs from vaccinia virus-infected HLA transgenic mice, we identified 14 HLA-A*0201-, 4 HLA-A*1101-, and 3 HLA-B*0702-restricted CD8+ T cell determinants distributed over 20 distinct proteins. These peptides were capable of binding one or multiple A2, A3, and B7 supertype molecules with affinities typical of viral determinants. Surprisingly, many of the viral proteins recognized are predicted to be late gene products, in addition to the early intermediate gene products expected. Nearly all of the determinants identified have identical counterparts encoded by modified vaccinia virus Ankara as well as variola virus, the agent of smallpox. These findings have implications for the design of new smallpox vaccines and the understanding of immune responses to large DNA viruses in general.
IL-13 Acutely Augments HIV-Specific and Recall Responses from HIV-1-Infected Subjects In Vitro by Modulating Monocytes
Authors: Papasavvas Emmanouil , Sun Junwei , Luo Qi , Moore Elizabeth C. , Thiel Brian , MacGregor Rob Roy , Minty Adrian , Mounzer Karam , Kostman Jay R. , Montaner Luis J.
Published: April 2014

We show in this study that acute exposure of PBMCs derived from HIV-infected subjects to IL-13 results in increased recall T cell lymphoproliferative responses against HIV-1 p24 (n = 30, p < 0.0001) and other recall Ags (influenza, n = 43, p < 0.0001; purified protein derivative tuberculin, n = 6, p = 0.0299). This effect is due to a mechanism that acutely targets APC function in the adherent monocyte subset, as shown by the expansion of CD4+ T cell responses following coculture of IL-13-treated enriched CD14+ monocytes with donor-matched enriched CD4+ T cells and Ag. Exposure to IL-13 over 18–72 h resulted in a significant enhancement of monocyte endocytosis (n = 11, p = 0.0005), CD86 expression (n = 12, p = 0.001), and a significant decrease in spontaneous apoptosis (n = 8, p = 0.008). Moreover, IL-13 exposure induced a significant decrease of significantly elevated constitutive levels of PBMC-secreted TNF-α (n = 14, p < 0.001) and IL-10 (n = 29, p < 0.001) within 18 h of exposure ex vivo, also reflected by decreased gene expression in the adherent cell population. Our data show that IL-13 is able to acutely enhance the function of the CD14+ cell subset toward supporting Ag-specific cell-mediated responses in chronic HIV-1 infection.
The Devil in the Details: The Emerging Role of Anticitrulline Autoimmunity in Rheumatoid Arthritis
Authors: van Gaalen Floris , Ioan-Facsinay Andreea , Huizinga Tom W. J. , Toes René E. M.
Published: April 2014

Rheumatoid arthritis is a chronic inflammatory autoimmune disease of unknown cause. The immune response against citrullinated Ags has recently become the prime suspect for disease pathogenesis. Immunity against citrullinated Ags is thought to play a pivotal role in the disease for several reasons: 1) citrullinated Ags are expressed in the target organ, the inflamed joint; 2) anti-citrullinated protein Abs are present before the disease becomes manifest; and 3) these Abs are highly specific for rheumatoid arthritis. In this review, data from clinical, genetic, biochemical, and animal studies is combined to create a profile of this remarkable autoantibody response. Moreover, a model is proposed of how the anti-citrullinated proteins response is generated and how it could eventually lead to chronic inflammation.
Cutting Edge: Early IFN-γ Signaling Directly Enhances Primary Antiviral CD4+ T Cell Responses
Authors: Whitmire Jason K. , Benning Nicola , Whitton J. Lindsay
Published: April 2014

IFN-γ drives CD4+ T cell differentiation toward the Th1 phenotype (Th1) and suppresses Th2 development. Current evidence indicates that IFN-γ inhibits T cell proliferation and decreases T cell survival. In contrast to the above, we show here that antiviral CD4+ T cell generation after infection is reduced in the absence of IFN-γ signals. The deficient expansion of cells was not due to perturbations in T cell sensitivity to peptide or to altered migratory patterns through nonlymphoid tissues. Instead, IFN-γ enhanced early antiviral CD4 responses largely through direct signals into these cells. Our data challenge prevailing dogma and have implications for how the sizes of the CD8+ and CD4+ T cell responses are established.