bluuebunny/crossref_metadata_2025 huggingface dataset:
Infection Biology of a Novel α-Crystallin of Mycobacterium tuberculosis: Acr2
Authors: Wilkinson Katalin A. , Stewart Graham R. , Newton Sandra M. , Vordermeier H. Martin , Wain John R. , Murphy Helen N. , Horner Katherine , Young Douglas B. , Wilkinson Robert J.
Published: April 2014

Heat shock proteins assist the survival of Mycobacterium tuberculosis (MTB) but also provide a signal to the immune response. The gene most strongly induced by heat shock in MTB is Rv0251c, which encodes Acr2, a novel member of the α-crystallin family of molecular chaperones. The expression of acr2 increased within 1 h after infection of monocytes or macrophages, reaching a peak of 18- to 55-fold by 24 h. Inhibition of superoxide action reduced the intracellular increase in acr2. Despite this contribution to the stress response of MTB, the gene for acr2 appears dispensable; a deletion mutant (Δacr2) was unimpaired in log phase growth and persisted in IFN-γ-activated human macrophages. Acr2 protein was strongly recognized by cattle with early primary Mycobacterium bovis infection and by healthy MTB-sensitized people. Within the latter group, those with recent exposure to infectious tuberculosis had, on average, 2.6 times the frequency of Acr2-specific IFN-γ-secreting T cells than those with more remote exposure (p = 0.009). These data show that, by its up-regulation early after entry to cells, Acr2 gives away the presence of MTB to the immune response. The demonstration that there is infection stage-specific immunity to tuberculosis has implications for vaccine design.
Mastoparan, a G Protein Agonist Peptide, Differentially Modulates TLR4- and TLR2-Mediated Signaling in Human Endothelial Cells and Murine Macrophages
Authors: Lentschat Arnd , Karahashi Hisae , Michelsen Kathrin S. , Thomas Lisa S. , Zhang Wenxuan , Vogel Stefanie N. , Arditi Moshe
Published: April 2014

Previous studies have implicated a role for heterotrimeric G protein-coupled signaling in B cells, monocytes, and macrophages stimulated with LPS and have shown that G proteins coimmunoprecipitate with membrane-bound CD14. In this study, we have extended these observations in human dermal microvessel endothelial cells (HMEC) that lack membrane-bound CD14 and in murine macrophages to define further the role of heterotrimeric G proteins in TLR signaling. Using the wasp venom-derived peptide, mastoparan, to disrupt G protein-coupled signaling, we identified a G protein-dependent signaling pathway in HMEC stimulated with TLR4 agonists that is necessary for the activation of p38 phosphorylation and kinase activity, NF-κB and IL-6 transactivation, and IL-6 secretion. In contrast, HMEC activation by TLR2 agonists, TNF-α, or IL-1β was insensitive to mastoparan. In the murine macrophage cell line, RAW 264.7, and in primary murine macrophages, G protein dysregulation by mastoparan resulted in significant inhibition of LPS-induced signaling leading to both MyD88-dependent and MyD88-independent gene expression, while TLR2-mediated gene expression was not significantly inhibited. In addition to inhibition of TLR4-mediated MAPK phosphorylation in macrophages, mastoparan blunted IL-1R-associated kinase-1 kinase activity induced by LPS, but not by TLR2 agonists, yet failed to affect phosphorylation of Akt by phosphoinositol-3-kinase induced by either TLR2- or TLR4-mediated signaling. These data confirm the importance of heterotrimeric G proteins in TLR4-mediated responses in cells that use either soluble or membrane-associated CD14 and reveal a level of TLR and signaling pathway specificity not previously appreciated.
Structure-Function Relationships among Human Cathelicidin Peptides: Dissociation of Antimicrobial Properties from Host Immunostimulatory Activities
Authors: Braff Marissa H. , Hawkins Mi‘i A. , Nardo Anna Di , Lopez-Garcia Belen , Howell Michael D. , Wong Cathy , Lin Kenneth , Streib Joanne E. , Dorschner Robert , Leung Donald Y. M. , Gallo Richard L.
Published: April 2014

Cathelicidins and other antimicrobial peptides are deployed at epithelial surfaces to defend against infection. These molecules have broad-spectrum killing activity against microbes and can have effects on specific mammalian cell types, potentially stimulating additional immune defense through direct chemotactic activity or induction of cytokine release. In humans, the cathelicidin hCAP18/LL-37 is processed to LL-37 in neutrophils, but on skin it can be further proteolytically processed to shorter forms. The influence of these cathelicidin peptides on keratinocyte function is not known. In the current study, DNA microarray analysis and confirmatory protein analysis showed that LL-37 affects the expression of several chemokines and cytokines by keratinocytes. Analysis of a synthetic peptide library derived from LL-37 showed that antimicrobial activity against bacterial, fungal, and viral skin pathogens resides within specific domains of the parent peptide, but antimicrobial activity does not directly correlate with the ability to stimulate IL-8 production in keratinocytes. IL-8 release was induced by d- and l-amino acid forms of cathelicidin and correlated with membrane permeability, suggesting that highly structure-specific binding to a cell surface receptor is not likely. However, this effect was inhibited by either pertussis toxin or AG1478, an epidermal growth factor receptor tyrosine kinase inhibitor, suggesting that cathelicidin may indirectly stimulate multiple signaling pathways associated with cell surface receptors. Taken together, these observations suggest that proteolytic processing may alter the balance between cathelicidin antimicrobial and host immunostimulatory functions.
Requirement for TLR9 in the Immunomodulatory Activity of Propionibacterium acnes
Authors: Kalis Christoph , Gumenscheimer Marina , Freudenberg Nikolaus , Tchaptchet Sandrine , Fejer György , Heit Antje , Akira Shizuo , Galanos Chris , Freudenberg Marina A.
Published: April 2014

Propionibacterium acnes (formerly Corynebacterium parvum) is part of the human flora and, as such, is associated with several human pathologies. It possesses strong immunomodulatory activities, which makes this bacterium interesting for prophylactic and therapeutic vaccination. The bacterial component(s) and the host receptor(s) involved in the induction of these activities are poorly understood. We show in this study that TLR9 is crucial in generating the characteristic effects of killed P. acnes priming in the spleen, such as extramedullary hemopoiesis and organ enlargement, and granuloma formation in the liver. Furthermore, the ability to overproduce TNF-α and IFN-γ in response to LPS, lipid A, synthetic lipopeptide Pam3CysK4, or whole killed bacteria was present in P. acnes-primed wild-type, but not TLR9−/−, mice. Finally, P. acnes priming failed to induce enhanced resistance to murine typhoid fever in TLR9−/− mice. Thus, TLR9 plays an essential role in the induction of immunomodulatory effects by P. acnes. Because IFN-γ is a key mediator of these effects, and enhanced IFN-γ mRNA expression was absent in spleen and liver of P. acnes-primed TLR9−/− mice, we conclude that TLR9 is required for the induction of IFN-γ by P. acnes.
Regulation of p38 MAPK by MAPK Kinases 3 and 6 in Fibroblast-Like Synoviocytes
Authors: Inoue Tomoyuki , Hammaker Deepa , Boyle David L. , Firestein Gary S.
Published: April 2014

The p38 MAPK signal transduction pathway is a key regulator of IL-1 and TNF-α production in rheumatoid arthritis. Previous studies demonstrated that upstream MAPK kinases (MKK3 and MKK6) that regulate p38 are activated in rheumatoid arthritis synovium. However, their functional relevance in fibroblast-like synoviocytes (FLS) has not been determined. To investigate the relative contribution of MKK3 and MKK6 to p38 activation, the effect of dominant-negative (DN) MKK3 and MKK6 constructs on cultured FLS was evaluated. Cultured FLS were stimulated with medium or IL-1β, and immunoblotting was performed. In some experiments, cells were lysed and immunoprecipitated with anti-p38 Ab, followed by in vitro kinase assay with [γ-32P]ATP and GST-activating transcription factor-2 as substrate. IL-1β rapidly induced p38 phosphorylation in cells transfected with empty vector (pcDNA3.1), but was inhibited by 25% in cells expressing DN MKK3 or DN MKK6. Cotransfection with both DN plasmids decreased phospho-p38 by almost 75%. In vitro kinase assays on IL-1-stimulated FLS also showed that the combination of DN MKK3 and DN MKK6 markedly decreased kinase activity compared with empty vector or the individual DN plasmids. Furthermore, IL-1β-induced IL-8, IL-6, and matrix metalloproteinase-3 protein production was significantly inhibited in DN MKK3/DN MKK6-transfected cells. The constructs had no effect on the respective mediator mRNA levels. These data demonstrate that MKK3 and MKK6 make individual contributions to p38 activation in FLS after cytokine stimulation, but that both must be blocked for maximum inhibition.
Activation Pathways Implicate Anti-HLA-DP and Anti-LFA-1 Antibodies as Lead Candidates for Intervention in Chronic Berylliosis
Authors: Chou Yuan K. , Edwards David M. , Weinberg Andrew D. , Vandenbark Arthur A. , Kotzin Brian L. , Fontenot Andrew P. , Burrows Gregory G.
Published: April 2014

CD4+ T cells play a key role in granulomatous inflammation in the lung of patients with chronic beryllium disease. The goal of this study was to characterize activation pathways of beryllium-responsive bronchoalveolar lavage (BAL) CD4+ T cells from chronic beryllium disease patients to identify possible therapeutic interventional strategies. Our results demonstrate that in the presence of APCs, beryllium induced strong proliferation responses of BAL CD4+ T cells, production of superoptimal concentrations of secreted proinflammatory cytokines, IFN-γ, TNF-α,and IL-2, and up-regulation of numerous T cell surface markers that would promote T-T Ag presentation. Ab blocking experiments revealed that anti-HLA-DP or anti-LFA-1 Ab strongly reduced proliferation responses and cytokine secretion by BAL CD4+ T cells. In contrast, anti-HLA-DR or anti-OX40 ligand Ab mainly affected beryllium-induced proliferation responses with little impact on cytokines other than IL-2, thus implying that nonproliferating BAL CD4+ T cells may still contribute to inflammation. Blockade with CTLA4-Ig had a minimal effect on proliferation and cytokine responses, confirming that activation was independent of B7/CD28 costimulation. These results indicate a prominent role for HLA-DP and LFA-1 in BAL CD4+ T cell activation and further suggest that specific Abs to these molecules could serve as a possible therapy for chronic beryllium disease.
Induction of Hyper Th1 Cell-Type Immune Responses by Dendritic Cells Lacking the Suppressor of Cytokine Signaling-1 Gene
Authors: Hanada Toshikatsu , Tanaka Kentaro , Matsumura Yumiko , Yamauchi Moriyasu , Nishinakamura Hitomi , Aburatani Hiroyuki , Mashima Ryuichi , Kubo Masato , Kobayashi Takashi , Yoshimura Akihiko
Published: April 2014

Suppressor of cytokine signaling (SOCS1/JAB) has been shown to play an important role in regulating dendritic cell (DC) function and suppressing inflammatory diseases and systemic autoimmunity. However, role of SOCS1 in DCs for the initiation of Th cell response has not been clarified. Here we demonstrate that SOCS1-deficient DCs induce stronger Th1-type responses both in vitro and in vivo. SOCS1-deficient DCs induced higher IFN-γ production from naive T cells than wild-type (WT) DCs in vitro. Lymph node T cells also produced a higher amount of IFN-γ when SOCS1-deficient bone marrow-derived DCs (BMDCs) were transferred in vivo. Moreover, SOCS1−/− BMDCs raised more effective anti-tumor immunity than WT BMDCs. Microarray analysis revealed that IFN-inducible genes were highly expressed in SOCS1-deficient DCs without IFN stimulation, suggesting hyper STAT1 activation in SOCS1−/− DCs. These phenotypes of SOCS1-deficient DCs were similar to those of CD8α+ DCs, and in the WT spleen, SOCS1 is expressed at higher levels in the Th2-inducing CD4+ DC subset, relative to the Th1-inducing CD8α+ DC subset. We propose that reduction of the SOCS1 gene expression in DCs leads to CD8α+ DC-like phenotype which promotes Th1-type hyperresponses.
HIV-1 Transactivator of Transcription Protein Induces Mitochondrial Hyperpolarization and Synaptic Stress Leading to Apoptosis
Authors: Perry Seth W. , Norman John P. , Litzburg Angela , Zhang Dabao , Dewhurst Stephen , Gelbard Harris A.
Published: April 2014

Despite the efficacy of highly active antiretroviral therapy in reducing viral burden, neurologic disease associated with HIV-1 infection of the CNS has not decreased in prevalence. HIV-1 does not induce disease by direct infection of neurons, although extensive data suggest that intra-CNS viral burden correlates with both the severity of virally induced neurologic disease, and with the generation of neurotoxic metabolites. Many of these molecules are capable of inducing neuronal apoptosis in vitro, but neuronal apoptosis in vivo does not correlate with CNS dysfunction, thus prompting us to investigate cellular and synaptic events occurring before cell death that may contribute to HIV-1-associated neurologic disease. We now report that the HIV-1 regulatory protein transactivator of transcription protein (Tat) increased oxidative stress, ATP levels, and mitochondrial membrane potential in primary rodent cortical neurons. Additionally, a proinflammatory cellular metabolite up-regulated by Tat, platelet-activating factor, also induced oxidative stress and mitochondrial hyperpolarization in neurons, suggesting that this type of metabolic dysfunction may occur on a chronic basis during HIV-1 infection of the CNS. Tat-induced mitochondrial hyperpolarization could be blocked with a low dose of the protonophore FCCP, or the mitochondrial KATP channel antagonist, tolbutamide. Importantly, blocking the mitochondrial hyperpolarization attenuated Tat-induced neuronal apoptosis, suggesting that increased mitochondrial membrane potential may be a causal event in precipitating neuronal apoptosis in cell culture. Finally, Tat and platelet-activating factor also increased neuronal vesicular release, which may be related to increased mitochondrial bioenergetics and serve as a biomarker for early damage to neurons.
Protective CD8 T Cell Immunity Triggered by CpG-Protein Conjugates Competes with the Efficacy of Live Vaccines
Authors: Heit Antje , Schmitz Frank , O’Keeffe Meredith , Staib Caroline , Busch Dirk H. , Wagner Hermann , Huster Katharina M.
Published: April 2014

In contrast to infectious (live) vaccines are those based on subunit Ag that are notoriously poor in eliciting protective CD8 T cell responses, presumably because subunit Ags become insufficiently cross-presented by dendritic cells (DCs) and because the latter need to be activated to acquire competence for cross-priming. In this study, we show that CpG-Ag complexes overcome these limitations. OVA covalently linked to CpG-DNA (CpG-OVA complex), once it is efficiently internalized by DCs via DNA receptor-mediated endocytosis, is translocated to lysosomal-associated membrane protein 1 (LAMP-1)-positive endosomal-lysosomal compartments recently shown to display competence for cross-presentation. In parallel, CpG-OVA complex loaded DCs become activated and acquire characteristics of professional APCs. In vivo, a single s.c. dose of CpG-OVA complex (10 μg of protein) induces primary and secondary clonal expansion/contraction of Ag-specific CD8 T cells similar in kinetics to live vaccines; examples including Listeria monocytogenes genetically engineered to produce OVA (LM-OVA) and two viral vector-based OVA vaccines analyzed. Interestingly, CpG-OVA complex induced almost equal percentages of Ag-specific memory CD8 T cells as did infection with LM-OVA. A single dose vaccination with CpG-OVA complex protected mice against lethal doses of LM-OVA. These data underscore that the synergy imparted by CpG-OVA complex-mediated combined triggering of innate and specific immunity might be key to initiate CD8 T cell-based immunoprotection by synthetic vaccines based on subunit Ag.
In Vitro and In Vivo Characterization of a Novel Antibody-Like Single-Chain TCR Human IgG1 Fusion Protein
Authors: Mosquera Luis A. , Card Kimberlyn F. , Price-Schiavi Shari A. , Belmont Heather J. , Liu Bai , Builes Janette , Zhu Xiaoyun , Chavaillaz Pierre-Andre , Lee Hyung-il , Jiao Jin-an , Francis John L. , Amirkhosravi Ali , Wong Richard L. , Wong Hing C.
Published: April 2014

We have constructed a protein composed of a soluble single-chain TCR genetically linked to the constant domain of an IgG1 H chain. The Ag recognition portion of the protein binds to an unmutated peptide derived from human p53 (aa 264–272) presented in the context of HLA-A2.1, whereas the IgG1 H chain provides effector functions. The protein is capable of forming dimers, specifically staining tumor cells and promoting target and effector cell conjugation. The protein also has potent antitumor effects in an in vivo tumor model and can mediate cell killing by Ab-dependent cellular cytotoxicity. Therefore, single-chain TCRs linked to IgG1 H chains behave like Abs but possess the ability to recognize Ags derived from intracellular targets. These fusion proteins represent a novel group of immunotherapeutics that have the potential to expand the range of tumors available for targeted therapies beyond those currently addressed by the conventional Ab-based approach.
G Protein-Coupled Receptor Kinase 2 in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis
Authors: Vroon Anne , Kavelaars Annemieke , Limmroth Volker , Lombardi Maria Stella , Goebel Marion U. , Van Dam Anne-Marie , Caron Marc G. , Schedlowski Manfred , Heijnen Cobi J.
Published: April 2014

Many modulators of inflammation, including chemokines, neuropeptides, and neurotransmitters signal via G protein-coupled receptors (GPCR). GPCR kinases (GRK) can phosphorylate agonist-activated GPCR thereby promoting receptor desensitization. Here we describe that in leukocytes from patients with active relapsing-remitting multiple sclerosis (MS) or with secondary progressive MS, GRK2 levels are significantly reduced. Unexpectedly, cells from patients during remission express even lower levels of GRK2. The level of GRK2 in leukocytes of patients after stroke, a neurological disorder with paralysis but without an autoimmune component, was similar to GRK2 levels in cells from healthy individuals. In addition, we demonstrate that the course of recombinant myelin oligodendrocyte glycoprotein (1–125)-induced experimental autoimmune encephalomyelitis (EAE), an animal model for MS, is markedly different in GRK2+/− mice that express 50% of the GRK2 protein in comparison with wild-type mice. Onset of EAE was significantly advanced by 5 days in GRK2+/− mice. The earlier onset of EAE was associated with increased early infiltration of the CNS by T cells and macrophages. Although disease scores in the first phase of EAE were similar in both groups, GRK2+/− animals did not develop relapses, whereas wild-type animals did. The absence of relapses in GRK2+/− mice was associated with a marked reduction in inflammatory infiltrates in the CNS. Recombinant myelin oligodendrocyte glycoprotein-induced T cell proliferation and cytokine production were normal in GRK2+/− animals. We conclude that down-regulation of GRK2 expression may have important consequences for the onset and progression of MS.
Depletion of Regulatory T Cells in HIV Infection Is Associated with Immune Activation
Authors: Eggena Mark P. , Barugahare Banson , Jones Norman , Okello Martin , Mutalya Steven , Kityo Cissy , Mugyenyi Peter , Cao Huyen
Published: April 2014

Immune activation during chronic HIV infection is a strong clinical predictor of death and may mediate CD4+ T cell depletion. Regulatory T cells (Tregs) are CD4+CD25brightCD62Lhigh cells that actively down-regulate immune responses. We asked whether loss of Tregs during HIV infection mediates immune activation in a cross-sectional study of 81 HIV-positive Ugandan volunteers. We found that Treg number is strongly correlated with both CD4+ and CD8+ T cell activation. In multivariate modeling, this relationship between Treg depletion and CD4+ T cell activation was stronger than any other clinical factor examined, including viral load and absolute CD4 count. Tregs appear to decline at different rates compared with other CD4+ T cells, resulting in an increased regulator to helper ratio in many patients with advanced disease. We hypothesize that this skewing may contribute to T cell effector dysfunction. Our findings suggest Tregs are a major contributor to the immune activation observed during chronic HIV infection.
Detection of Human P2X7 Nucleotide Receptor Polymorphisms by a Novel Monocyte Pore Assay Predictive of Alterations in Lipopolysaccharide-Induced Cytokine Production
Authors: Denlinger Loren C. , Angelini Giuditta , Schell Kathleen , Green Dawn N. , Guadarrama Arturo G. , Prabhu Usha , Coursin Douglas B. , Bertics Paul J. , Hogan Kirk
Published: April 2014

The nucleotide receptor P2X7 is expressed by most leukocytes and initiates signaling events that amplify numerous LPS responses. We tested the hypothesis that loss-of-function polymorphisms in the human P2X7 gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X7 pore assay in whole blood that magnifies the activity from wild-type alleles and preserves the gene dosage effect for the 1513 C polymorphism (AA, 69 ± 4; AC, 42 ± 4; and CC, 6 ± 1-fold stimulation). Thirty of 200 healthy individuals were identified as having low P2X7 pore activity. Seven low pore subjects were 1513 CC, 3 and 11 participants had the other known variants 946 GA and 1729 TA respectively; the remaining 9 volunteers likely have novel polymorphisms. Because platelets are a large source of extracellular ATP during inflammation, whole blood was treated ex vivo with Salmonella typhimurium LPS in the absence of exogenous nucleotides. LPS-stimulated whole blood from individuals in the low pore activity group generated reduced plasma levels of TNF-α (p = 0.036) and higher amounts of IL-10 (p < 0.001) relative to the high pore controls. This reduction in the TNF-α to IL-10 ratio persisted to at least 24 h and is further decreased by cotreatment with 2-methylthio-ATP. The ability of P2X7 polymorphisms to regulate the LPS-induced TNF-α to IL-10 ratio suggests that 15% of healthy adults may exhibit anti-inflammatory mediator responses during major infectious perturbations of the immune system, which can be predicted by P2X7 pore activity.
Mutational Analysis of the Human 2B4 (CD244)/CD48 Interaction: Lys68 and Glu70 in the V Domain of 2B4 Are Critical for CD48 Binding and Functional Activation of NK Cells
Authors: Mathew Stephen O. , Kumaresan Pappanaicken R. , Lee Jae Kyung , Huynh Van T. , Mathew Porunelloor A.
Published: April 2014

Interaction between receptors and ligands plays a critical role in the generation of immune responses. The 2B4 (CD244), a member of the CD2 subset of the Ig superfamily, is the high affinity ligand for CD48. It is expressed on NK cells, T cells, monocytes, and basophils. Recent data indicate that 2B4/CD48 interactions regulate NK and T lymphocyte functions. In human NK cells, 2B4/CD48 interaction induces activation signals, whereas in murine NK cells it sends inhibitory signals. To determine the structural basis for 2B4/CD48 interaction, selected amino acid residues in the V domain of the human 2B4 (h2B4) were mutated to alanine by site-directed mutagenesis. Following transient expression of these mutants in B16F10 melanoma cells, their interaction with soluble CD48-Fc fusion protein was assessed by flow cytometry. We identified amino acid residues in the extracellular domain of h2B4 that are involved in interacting with CD48. Binding of CD48-Fc fusion protein to RNK-16 cells stably transfected with wild-type and a double-mutant Lys68Ala-Glu70Ala h2B4 further demonstrated that Lys68 and Glu70 in the V domain of h2B4 are essential for 2B4/CD48 interaction. Functional analysis indicated that Lys68 and Glu70 in the extracellular domain of h2B4 play a key role in the activation of human NK cells through 2B4/CD48 interaction.
Hepatocytes Express Abundant Surface Class I MHC and Efficiently Use Transporter Associated with Antigen Processing, Tapasin, and Low Molecular Weight Polypeptide Proteasome Subunit Components of Antigen Processing and Presentation Pathway
Authors: Chen Ming , Tabaczewski Piotr , Truscott Steven M. , Van Kaer Luc , Stroynowski Iwona
Published: April 2014

Hepatic expression levels of class I MHC Ags are generally regarded as very low. Because the status of these Ags and their ability to present peptides are important for the understanding of pathogen clearance and tolerogenic properties of the liver, we set out to identify the factors contributing to the reported phenotype. Unexpectedly, we found that the surface densities of Kb and Db on C57BL/6 mouse hepatocytes are nearly as high as on splenocytes, as are the lysate concentrations of mRNA encoding H chain and β2-microglobulin (β2m). In contrast, the components of the peptide-loading pathway are reduced in hepatocytes. Despite the difference in the stoichiometric ratios of H chain/β2m/peptide-loading machineries, both cell types express predominantly thermostable class I and are critically dependent on TAP and tapasin for display of surface Ags. Minor differences in the expression patterns in tapasin−/− background suggest cell specificity in class I assembly. Under immunostimulatory conditions, such as exposure to IFN-γ or Listeria monocytogenes, hepatocytes respond with a vigorous mRNA synthesis of the components of the Ag presentation pathway (up to 10-fold enhancement) but up-regulate H chain and β2m to a lesser degree (<2-fold). This type of response should promote rapid influx of newly generated peptides into the endoplasmic reticulum and preferential presentation of foreign/induced Ag by hepatic class I.
Multiple Germline κ Light Chains Generate Anti-Insulin B Cells in Nonobese Diabetic Mice
Authors: Woodward Emily J. , Thomas James W.
Published: April 2014

The highly selective nature of organ-specific autoimmune disease is consistent with a critical role for adaptive immune responses against specific autoantigens. In type 1 diabetes mellitus, autoantibodies to insulin are important markers of the disease process in humans and nonobese diabetic (NOD) mice; however, the Ag-specific receptors responsible for these autoantibodies are obscured by the polyclonal repertoire. NOD mice that harbor an anti-insulin transgene (Tg) (VH125Tg/NOD) circumvent this problem by generating a tractable population of insulin-binding B cells. The nucleotide structure and genetic origin of the endogenous κ L chain (Vκ or IgL) repertoire that pairs with the VH125Tg were analyzed. In contrast to oligoclonal expansion observed in systemic autoimmune disease models, insulin-binding B cells from VH125Tg/NOD mice use specific Vκ genes that are clonally independent and germline encoded. When compared with homologous IgL genes from nonautoimmune strains, Vκ genes from NOD mice are polymorphic. Analysis of the most frequently expressed Vκ1 and Vκ9 genes indicates these are shared with lupus-prone New Zealand Black/BINJ mice (e.g., Vκ1–110*02 and 9–124) and suggests that NOD mice use the infrequent b halpotype. These findings show that a diverse repertoire of anti-insulin B cells is part of the autoimmune process in NOD mice and structural or regulatory elements within the κ locus may be shared with a systemic autoimmune disease.
Several Genes Contribute to the Production of Autoreactive B and T Cells in the Murine Lupus Susceptibility LocusSle1c
Authors: Chen Yifang , Perry Daniel , Boackle Susan A. , Sobel Eric S. , Molina Hector , Croker Byron P. , Morel Laurence
Published: April 2014

The systemic lupus erythematosus 1 (Sle1) locus mediates the loss of tolerance to nuclear Ags in the NZM2410 mouse model of lupus through intrinsic defects in both B and T cells. Congenic analysis has shown that Sle1 corresponds to at least three genetic loci, Sle1a, Sle1b, and Sle1c. Telomeric Sle1c is associated with abnormal B cell responses to subthreshold stimulation with anti-IgM and C3d and with decreased T-dependent humoral immune responses. We have proposed that these phenotypes resulted from polymorphisms in the C3 complement receptor Cr2 gene. We have also found that Sle1c was associated with the production of histone-specific autoreactive CD4+ T cells, which correlated with higher activation and proliferative responses, and a reduction in the CD4+CD25+CD62L+forkhead/winged helix transcription factor gene (Foxp3+) compartment. In this study we showed, using congenic recombinants, that the decreased humoral immune response and impaired GC formation map to the NZM2410 Cr2 allele. A chronic graft-vs-host disease model also showed that Sle1c produces significantly more autoreactive B cells than B6 controls, and that this phenotype maps to two regions excluding the Cr2 gene. Mixed bone marrow chimera demonstrated that the increased activation, proliferative response, and reduced regulatory T cell compartment were intrinsic to Sle1c-expressing CD4+ T cells. These phenotypes mapped to the same two loci identified with the chronic graft-vs-host disease model, excluding the Cr2 region. Overall, these results show that Sle1c results in the production of autoreactive B and T cells through the expression of three different genes, one of which is consistent with Cr2, based on the phenotypes of the Cr2-deficient mice, and the other two corresponding to as yet unidentified genes.
Long-Term Control ofMycobacterium tuberculosisInfection Is Mediated by Dynamic Immune Responses
Authors: Lazarevic Vanja , Nolt Dawn , Flynn JoAnne L.
Published: April 2014

The primary goal of this study was to determine how chronic exposure to Ag influences the functionality of Mycobacterium tuberculosis-specific T cell responses. The frequency of IFN-γ-producing effector CD4+ and CD8+ T cells dynamically changed during persistent M. tuberculosis infection. CD8+ T cells used differential effector functions during acute and chronic phases of the immune response, where CD8+ T cells produced negligible amounts of IFN-γ early in infection, but switched to cytokine production during the chronic stage of infection. Using limiting dilution analysis, CD8+ T cells isolated during the initial phase of infection demonstrated lytic potential, but this waned in the chronic stage. The apparent loss of cytotoxic activity was not associated with the lack of perforin. Ag dose could potentially govern the functional program of CD8+ T cells. Collectively, these results depict a host immune response mounted against M. tuberculosis of a significantly more dynamic nature than previously recognized.
Developmentally Regulated Intestinal Expression of IFN-γ and Its Target Genes and the Age-Specific Response to Enteric Salmonella Infection
Authors: Rhee Sue J. , Walker W. Allan , Cherayil Bobby J.
Published: April 2014

Young infants are highly susceptible to systemic dissemination of enteric pathogens such as Salmonella typhimurium when compared with older individuals. The mechanisms underlying this differential susceptibility have not been defined clearly. To better understand this phenomenon, we examined the responses of adult mice and preweaned pups to oral infection by S. typhimurium. We found clear age-specific differences, namely, an attenuated intestinal inflammatory response and a higher systemic bacterial burden in the pups compared with the adults. To elucidate the molecular basis for these differences, we obtained a microarray-based profile of gene expression in the small intestines of uninfected adult and preweaned animals. The results indicated a striking age-dependent increase in the intestinal expression of a number of IFN-γ-regulated genes involved in antimicrobial defense. This finding was confirmed by real-time quantitative PCR, which also demonstrated an age-dependent increase in intestinal expression of IFN-γ. The developmental up-regulation of the IFN-γ-regulated genes was dependent on both IFN-γ and a normal commensal microflora, as indicated by experiments in IFN-γ-knockout mice and germfree mice, respectively. However, the increase in expression of IFN-γ itself was independent of the commensal flora. The functional importance of IFN-γ in the immunological maturation of the intestine was confirmed by the observation that the response of adult IFN-γ-knockout animals to S. typhimurium infection resembled that of the wild-type pups. Our findings thus reveal a novel role for IFN-γ in the developmental regulation of antimicrobial responses in the intestine.
Immunoproteasome-Deficient Mice Mount Largely Normal CD8+ T Cell Responses to Lymphocytic Choriomeningitis Virus Infection and DNA Vaccination
Authors: Nussbaum Alexander K. , Rodriguez-Carreno Maria P. , Benning Nicola , Botten Jason , Whitton J. Lindsay
Published: April 2014

During viral infection, constitutive proteasomes are largely replaced by immunoproteasomes, which display distinct cleavage specificities, resulting in different populations of potential CD8+ T cell epitope peptides. Immunoproteasomes are believed to be important for the generation of many viral CD8+ T cell epitopes and have been implicated in shaping the immunodominance hierarchies of CD8+ T cell responses to influenza virus infection. However, it remains unclear whether these conclusions are generally applicable. In this study we investigated the CD8+ T cell responses to lymphocytic choriomeningitis virus infection and DNA immunization in wild-type mice and in mice lacking the immunoproteasome subunits LMP2 or LMP7. Although the total number of virus-specific cells was lower in LMP2 knockout mice, consistent with their having lower numbers of naive cells before infection, the kinetics of virus clearance were similar in all three mouse strains, and LMP-deficient mice mounted strong primary and secondary lymphocytic choriomeningitis virus-specific CD8+ T cell responses. Furthermore, the immunodominance hierarchy of the four investigated epitopes (nuclear protein 396 (NP396) > gp33 > gp276 > NP205) was well maintained. We observed a slight reduction in the NP205-specific response in LMP2-deficient mice, but this had no demonstrable biological consequence. DNA vaccination of LMP2- and LMP7-deficient mice induced CD8+ T cell responses that were slightly lower than, although not significantly different from, those induced in wild-type mice. Taken together, our results challenge the notion that immunoproteasomes are generally needed for effective antiviral CD8+ T cell responses and for the shaping of immunodominance hierarchies. We conclude that the immunoproteasome may affect T cell responses to only a limited number of viral epitopes, and we propose that its main biological function may lie elsewhere.
18β-Glycyrrhetinic Acid Triggers Curative Th1 Response and Nitric Oxide Up-Regulation in Experimental Visceral Leishmaniasis Associated with the Activation of NF-κB
Authors: Ukil Anindita , Biswas Aruna , Das Tapasi , Das Pijush K.
Published: April 2014

The efficacy of 18β-glycyrrhetinic acid (GRA), a pentacyclic triterpene belonging to the β-amyrin series of plant origin, was evaluated in experimental visceral leishmaniasis. GRA is reported to have antitumor and immunoregulatory activities, which may be attributable in part to the induction of NO. Indeed, an 11-fold increase in NO production was observed with 20 μM GRA in mouse peritoneal macrophages infected with Leishmania donovani promastigotes. In addition to having appreciable inhibitory effects on amastigote multiplication within macrophages (IC50, 4.6 μg/ml), complete elimination of liver and spleen parasite burden was achieved by GRA at a dose of 50 mg/kg/day, given three times, 5 days apart, in a 45-day mouse model of visceral leishmaniasis. GRA treatment resulted in reduced levels of IL-10 and IL-4, but increased levels of IL-12, IFN-γ, TNF-α, and inducible NO synthase, reflecting a switch of CD4+ differentiation from Th2 to Th1. This treatment is likely to activate immunity, thereby imparting resistance to reinfection. GRA induced NF-κB migration into the nucleus of parasite-infected cells and caused a diminishing presence of IκB in the cytoplasm. The lower level of cytoplasmic IκBα in GRA-treated cells resulted from increased phosphorylation of IκBα and higher activity of IκB kinase (IKK). Additional experiments demonstrated that GRA does not directly affect IKK activity. These results suggest that GRA exerts its effects at some level upstream of IKK in the signaling pathway and induces the production of proinflammatory mediators through a mechanism that, at least in part, involves induction of NF-κB activation.
An Intact Phosphocholine Binding Site Is Necessary for Transgenic Rabbit C-Reactive Protein to Protect Mice against Challenge with Platelet-Activating Factor
Authors: Black Steven , Wilson Augusta , Samols David
Published: April 2014

C-reactive protein (CRP), an acute phase protein in humans and rabbits, is part of the innate immune system. The role of CRP in host defense has been thought to be largely due to its ability to bind phosphocholine, activate complement, and interact with IgGRs (FcγRs). We have shown previously that transgenic rabbit CRP (rbCRP) protects mice from lethal challenges with platelet-activating factor (PAF). To investigate the mechanism of this protection, we created additional lines of transgenic mice that express either wild-type rbCRP, a variant of rbCRP with altered complement activation activity (Y175A), or a variant of rbCRP unable to bind phosphocholine (F66Y/E81K). In the current study, these lines were challenged with a single injection of PAF and their survival monitored. Mice expressing wild-type and Y175A rbCRP were protected against challenge by PAF whereas mice expressing F66Y/E81K rbCRP were not. Treatment with cobra venom factor did not affect survival, confirming the results with the Y175A rbCRP variant and indicating that complement activation was not required to mediate protection. Both wild-type rbCRP and Y175A rbCRP were capable of binding PAF in vitro whereas F66Y/E81K rbCRP was not. Although other interpretations are possible, our results suggest that the protective effect of rbCRP against PAF is due to sequestration of PAF.
Macrophages Induce Invasiveness of Epithelial Cancer Cells Via NF-κB and JNK
Authors: Hagemann Thorsten , Wilson Julia , Kulbe Hagen , Li Ningfeng Fiona , Leinster David A. , Charles Kellie , Klemm Florian , Pukrop Tobias , Binder Claudia , Balkwill Frances R.
Published: April 2014

Tumor-associated macrophages may influence tumor progression, angiogenesis and invasion. To investigate mechanisms by which macrophages interact with tumor cells, we developed an in vitro coculture model. Previously we reported that coculture enhanced invasiveness of the tumor cells in a TNF-α- and matrix metalloprotease-dependent manner. In this report, we studied intracellular signaling pathways and induction of inflammatory genes in malignant cells under the influence of macrophage coculture. We report that coculture of macrophages with ovarian or breast cancer cell lines led to TNF-α-dependent activation of JNK and NF-κB pathways in tumor cells, but not in benign immortalized epithelial cells. Tumor cells with increased JNK and NF-κB activity exhibited enhanced invasiveness. Inhibition of the NF-κB pathway by TNF-α neutralizing Abs, an NF-κB inhibitor, RNAi to RelA, or overexpression of IκB inhibited tumor cell invasiveness. Blockade of JNK also significantly reduced invasiveness, but blockade of p38 MAPK or p42 MAPK had no effect. Cocultured tumor cells were screened for the expression of 22 genes associated with inflammation and invasion that also contained an AP-1 and NF-κB binding site. EMMPRIN and MIF were up-regulated in cocultured tumor cells in a JNK- and NF-κB-dependent manner. Knocking down either MIF or EMMPRIN by RNAi in the tumor cells significantly reduced tumor cell invasiveness and matrix metalloprotease activity in the coculture supernatant. We conclude that TNF-α, via NF-κB, and JNK induces MIF and EMMPRIN in macrophage to tumor cell cocultures and this leads to increased invasive capacity of the tumor cells.
Elimination of Senescent Neutrophils by TNF-Related Apoptosis-Inducing Ligand
Authors: Lum Julian J. , Bren Gary , McClure Rebecca , Badley Andrew D.
Published: April 2014

Neutrophils are phagocytic effectors which are produced in the bone marrow and released into the circulation. Thereafter, they are either recruited to sites of inflammation or rapidly become senescent, return to the bone marrow, and undergo apoptosis. Stromal cell-derived factor 1 (SDF-1) coordinates the return of senescent neutrophils to the bone marrow by interacting with CXCR4 that is preferentially expressed on senescent neutrophils. We demonstrate that CXCR4 ligation by SDF-1 or other CXCR4 agonists significantly increases the expression of both TNF-related apoptosis-inducing ligand (TRAIL) and of the death-inducing TRAIL receptors on neutrophils, which confers an acquired sensitivity to TRAIL-mediated death and results in TRAIL-dependent apoptosis. In vivo administration of TRAIL antagonists results in neutrophilic accumulation within the bone marrow and a reduction in neutrophil apoptosis; conversely recombinant TRAIL administration reduced neutrophil number within bone marrow. Thus, SDF-1 ligation of CXCR4 causes the parallel processes of chemotaxis and enhanced TRAIL and TRAIL death receptor expression, resulting in apoptosis of senescent neutrophils upon their return to the bone marrow.
P-Selectin Glycoprotein Ligand 1 Is Not Required for the Development of Experimental Autoimmune Encephalomyelitis in SJL and C57BL/6 Mice
Authors: Engelhardt Britta , Kempe Birgit , Merfeld-Clauss Stephanie , Laschinger Melanie , Furie Bruce , Wild Martin K. , Vestweber Dietmar
Published: April 2014

In multiple sclerosis and in its animal model experimental autoimmune encephalomyelitis (EAE), inflammatory cells migrate across the endothelial blood-brain barrier and gain access to the CNS. The involvement of P-selectin glycoprotein ligand 1 (PSGL-1) and of its major endothelial ligand P-selectin in this process have been controversial. In this study we demonstrate that although encephalitogenic T cells express functional PSGL-1, which can bind to soluble and immobilize P-selectin if presented in high concentrations, PSGL-1 is not involved T cell interaction with P-selectin expressing brain endothelial cells in vitro. Furthermore, neither anti-PSGL-1 Abs nor the lack of PSGL-1 in PSGL-1-deficient mice inhibits the recruitment of inflammatory cells across the blood-brain barrier or the development of clinical EAE. Taken together, our findings demonstrate that PSGL-1 is not required for the pathogenesis of EAE.
Mucosal B Cell Deficiency in IgA−/− Mice Abrogates the Development of Allergic Lung Inflammation
Authors: Arnaboldi Paul M. , Behr Melissa J. , Metzger Dennis W.
Published: April 2014

We have investigated the consequence of lack of IgA on host immunity using a murine model of allergic lung inflammation. Mice with a targeted disruption of the α-switch region and 5′ H chain gene (IgA−/− mice), which lack total IgA, developed significantly reduced pulmonary inflammation with fewer inflammatory cells in lung tissue and bronchoalveolar lavage fluids, as well as reduced levels of total and IgG1 OVA-specific Abs and decreased IL-4 and IL-5 in bronchoalveolar lavage fluids compared with IgA+/+ controls, following allergen sensitization and challenge. This defect was attributable to fewer B cells in the lungs of IgA−/− mice. Polymeric IgR-deficient (pIgR−/−) mice, which lack the receptor that transports polymeric IgA across the mucosal epithelium where it is cleaved to form secretory IgA, were used to assess the contribution of secretory IgA vs total IgA in the induction of allergic lung inflammation. pIgR−/− and pIgR+/+ mice had comparable levels of inflammation, demonstrating that IgA bound to secretory component is not necessary for the development of allergic lung inflammation, although this does not necessarily rule out a role for transudated IgA in lung secretions because of “mucosal leakiness” in these mice. The results indicate that Ag-specific B cells are required at mucosal surfaces for induction of inflammation and likely function as major APCs in the lung for soluble protein Ags.
Contribution of the Lymphotoxin β Receptor to Liver Regeneration
Authors: Anders Robert A. , Subudhi Sumit K. , Wang Jing , Pfeffer Klaus , Fu Yang-Xin
Published: April 2014

The liver has an enormous capacity to regenerate in response to insults, but the cellular events and molecules involved in liver regeneration are not well defined. In this study, we report that ligands expressed on the surface of lymphocytes have a substantial effect on liver homeostasis. We demonstrate that a T cell-restricted ligand, homologous to lymphotoxin, exhibits inducible expression, competes with herpesvirus glycoprotein D for herpesvirus entry mediator on T cells (LIGHT), signaling through the lymphotoxin receptor (LTβR) expressed on mature hepatocytes induces massive hepatomegaly. Using genetic targeting and a receptor fusion protein, we further show that mice deficient in LTβR signaling have a severe defect in their ability to survive partial hepatectomy with marked liver damage and failure to initiate DNA synthesis after partial hepatectomy. We further show that mice deficient in a LTβR ligand, LTα, also show decreased ability to survive partial hepatectomy with similar levels of liver damage and decreased DNA synthesis. Therefore, our study has revealed an unexpected role of lymphocyte-restricted ligands and defined a new pathway in supporting liver regeneration.
Targeting Positive Regulatory Domain I-Binding Factor 1 and X Box-Binding Protein 1 Transcription Factors by Multiple Myeloma-Reactive CTL
Authors: Lotz Carina , Mutallib Sarah Abdel , Oehlrich Nicole , Liewer Ulrike , Ferreira Edite Antunes , Moos Marion , Hundemer Michael , Schneider Sandra , Strand Susanne , Huber Christoph , Goldschmidt Hartmut , Theobald Matthias
Published: April 2014

Growing evidence indicates that multiple myeloma (MM) and other malignancies are susceptible to CTL-based immune interventions. We studied whether transcription factors inherently involved in the terminal differentiation of mature B lymphocytes into malignant and nonmalignant plasma cells provide MM-associated CTL epitopes. HLA-A*0201 (A2.1) transgenic mice were used to identify A2.1-presented peptide Ag derived from the plasma cell-associated transcriptional regulators, positive regulatory domain I-binding factor 1 (PRDI-BF1) and X box-binding protein 1 (XBP-1). A2.1-restricted CTL specific for PRDI-BF1 and XBP-1 epitopes efficiently killed a variety of MM targets. PRDI-BF1- and XBP-1-reactive CTL were able to recognize primary MM cells from A2.1+ patients. Consistent with the expression pattern of both transcription factors beyond malignant and nonmalignant plasma cells, PRDI-BF1- and XBP-1-specific CTL activity was not entirely limited to MM targets, but was also associated with lysis of certain other malignancies and, in defined instances, with low-to-intermediate level recognition of a few types of normal cells. Our results also indicate that the A2.1-restricted, PRDI-BF1- and XBP-1-specific human CD8+ T cell repertoire is affected by partial self tolerance and may thus require the transfer of high-affinity TCR to break tolerance. We conclude that transcription factors governing terminal cellular differentiation may provide MM- and tumor-associated CTL epitopes.