bluuebunny/crossref_metadata_2025 huggingface dataset:
Toll/IL-1 Receptor Domain-Containing Adaptor Inducing IFN-β (TRIF) Associates with TNF Receptor-Associated Factor 6 and TANK-Binding Kinase 1, and Activates Two Distinct Transcription Factors, NF-κB and IFN-Regulatory Factor-3, in the Toll-Like Receptor Signaling
Authors: Sato Shintaro , Sugiyama Masanaka , Yamamoto Masahiro , Watanabe Yasuyuki , Kawai Taro , Takeda Kiyoshi , Akira Shizuo
Published: April 2014

We previously reported a new Toll/IL-1R (TIR)-containing molecule, named TIR domain-containing adaptor inducing IFN-β (TRIF). Although initial study indicated that TRIF possesses the ability to activate not only the NF-κB-dependent but also the IFN-β promoters, the molecular mechanisms of TRIF-induced signaling are poorly understood. In this study, we investigated the signaling cascades through TRIF. TNF receptor-associated factor (TRAF)6 interacted with TRIF through the TRAF domain of TRAF6 and TRAF6-binding motifs found in the N-terminal portion of TRIF. Disruption of TRAF6-binding motifs of TRIF disabled it from associating with TRAF6, and resulted in a reduction in the TRIF-induced activation of the NF-κB-dependent but not IFN-β promoter. TANK-binding kinase (TBK)-1, which was recently reported to be a kinase of IFN regulatory factor-3, which is an essential transcription factor for IFN-β expression, also associated with the N-terminal region of TRIF. Moreover, the association between TRIF and TBK1 appeared to require the kinase activity of TBK1, as well as phosphorylation of TRIF. Because TRAF6 and TBK1 bind close the region of TRIF, it seems that TRAF6 physically prevents the association between TRIF and TBK1. Taken together, these results demonstrate that TRIF associates with TRAF6 and TBK1 independently, and activates two distinct transcription factors, NF-κB and IFN regulatory factor-3, respectively.
Costimulatory Molecule Immune Enhancement in a Plasmid Vaccine Model Is Regulated in Part Through the Ig Constant-Like Domain of CD80/86
Authors: Agadjanyan Michael G. , Chattergoon Michael A. , Holterman Mark J. , Monzavi-Karbassi Behjatolah , Kim J. Joseph , Dentchev Tzvete , Wilson Darren , Ayyavoo Velpandi , Montaner Luis J. , Kieber-Emmons Thomas , Sekaly Rafick-P. , Weiner David B.
Published: April 2014

There is great interest in understanding the role of costimulatory molecules in immune activation. In both the influenza and HIV DNA immunization models, several groups have reported that coimmunization of mice with plasmids encoding immunogen and CD86, but not CD80, effectively boosts Ag-specific T cell activation. This difference in immune priming provided an opportunity to examine the functional importance of different regions of the B.7 molecules in immune activation. To examine this issue, we developed a series of chimeric CD80 and CD86 constructs as well as deletion mutants, and examined their immune activating potential in the DNA vaccine model. We demonstrate that the lack of an Ig constant-like region in the CD80 molecule is critically important to the enhanced immune activation observed. CD80 C-domain deletion mutants induce a highly inflammatory Ag-specific cellular response when administered as part of a plasmid vaccine. The data suggest that the constant-like domains, likely through intermolecular interactions, are critically important for immune regulation during costimulation and that engineered CD80/86 molecules represent more potent costimulatory molecules and may improve vaccine adjuvant efficacy.
Selective Depletion of Nonspecific T Cells During the Early Stage of Immune Responses to Infection
Authors: Jiang Jiu , Lau Lisa L. , Shen Hao
Published: April 2014

Transient T cell depletion occurs before the development of an effective immune response to infection. In this study we show that most T cells, regardless of specificity, are induced to express early activation markers soon after infection with Listeria monocytogenes or lymphocytic choriomeningitis virus. Ag-specific T cells are further activated to display late activation markers and undergo extensive proliferation. As Ag-specific T cells begin to expand, nonspecific T cells are depleted en masse and exhibit no sign of further activation or proliferation before their depletion. This selective depletion of nonspecific T cells is due to in situ death via apoptosis, as visualized by confocal microscopy. Thus, early activation and subsequent depletion of nonspecific T cells are integral parts of the immune response to proinflammatory infections. These results have important implications for our understanding of early events in the development of a robust T cell response.
Regulation of Eotaxin Gene Expression by TNF-α and IL-4 Through mRNA Stabilization: Involvement of the RNA-Binding Protein HuR
Authors: Atasoy Ulus , Curry Stephanie L. , López de Silanes Isabel , Shyu Ann-Bin , Casolaro Vincenzo , Gorospe Myriam , Stellato Cristiana
Published: April 2014

During inflammatory responses, a major posttranscriptional regulation of early response and inflammatory gene expression occurs through modulation of mRNA turnover. We report that two potent inducers of the CC chemokine eotaxin, TNF-α and IL-4, regulate its production in airway epithelial cells by increasing eotaxin mRNA stability. In experiments using the transcriptional inhibitor actinomycin D, eotaxin mRNA half-life was significantly prolonged by cell stimulation with TNF-α or IL-4, with the combination of the two cytokines being the most effective in extending the mRNA half-life. Involvement of the eotaxin 3′ untranslated region in the mRNA-stabilizing effect was tested by transient transfection of a construct expressing a chimeric transcript carrying a serum-inducible β-globin reporter linked to the eotaxin 3′ untranslated region. The half-life of the chimeric mRNA was markedly increased in cells stimulated with TNF-α and IL-4. Evidence that the mRNA-stabilizing protein HuR participated in the cytokine effect was obtained: first, HuR presence in the cytoplasm, believed to be required for HuR-mediated mRNA stabilization, increased in both transformed (BEAS-2B cell line) and primary bronchial epithelial cells following treatment with TNF-α and IL-4. Second, endogenous eotaxin mRNA was found to bind to HuR in vivo, as detected by immunoprecipitation of HuR-containing messenger ribonucleoprotein complexes followed by real-time RT-PCR analysis; such association increased after cell treatment with TNF-α and IL-4. Third, overexpression of HuR in BEAS-2B cells significantly increased the expression of eotaxin mRNA and protein. Our findings implicate mRNA stabilization in the cytokine-mediated increase in eotaxin expression and strongly suggest a role for HuR in this effect.
Mast Cells Exert Effects Outside the Central Nervous System to Influence Experimental Allergic Encephalomyelitis Disease Course
Authors: Tanzola Melinda B. , Robbie-Ryan Michaela , Gutekunst Claire Anne , Brown Melissa A.
Published: April 2014

Previous studies using mast cell-deficient mice (W/Wv) revealed that mast cells influence disease onset and severity of experimental allergic/autoimmune encephalomyelitis (EAE), the murine model for multiple sclerosis. The mast cell populations of these mice can be restored by transferring bone marrow-derived mast cells (BMMCs). Studies using the W/Wv reconstitution model have lead to major advances in our understanding of mast cell roles in vivo. However, despite its common use, details regarding the sites and kinetics of mast cell repopulation have remained largely uncharacterized. In this study, we examined the kinetics and tissue distribution of green fluorescent protein+ BMMCs in reconstituted W/Wv mice to identify sites of mast cell influence in EAE. Reconstitution of naive animals with BMMCs does not restore mast cell populations to all organs, notably the brain, spinal cord, lymph nodes, and heart. Despite the absence of mast cells in the CNS, reconstituted mice exhibit an EAE disease course equivalent to that induced in wild-type mice. Mast cells are found adjacent to T cell-rich areas of the spleen and can migrate to the draining lymph node after disease induction. These data reveal that mast cells can act outside the CNS to influence EAE, perhaps by affecting the function of autoreactive lymphocytes.
TNF-α Induces Phosphorylation of p47phox in Human Neutrophils: Partial Phosphorylation of p47phox Is a Common Event of Priming of Human Neutrophils by TNF-α and Granulocyte-Macrophage Colony-Stimulating Factor
Authors: Dewas Cédric , Dang Pham My-Chan , Gougerot-Pocidalo Marie-Anne , El-Benna Jamel
Published: April 2014

Phosphorylation of p47phox is a key event in NADPH oxidase activation. We examined the ability of proinflammatory cytokines such as TNFα, IL-1, and G-CSF to induce this process compared with GM-CSF. Only TNF-α and GM-CSF induced a clear p47phox phosphorylation. This phosphorylation was time dependent and reached its maximum at 20 min. Two-dimensional phosphopeptide mapping of p47phox phosphorylated in neutrophils primed with TNF-α revealed partial phosphorylation of p47phox on the same peptide as for GM-CSF. Neutrophil incubation with TNF-α and subsequent addition of the chemotactic peptide fMLP resulted in more intense phosphorylation of p47phox sites than with each reagent alone. A neutralizing Ab against the p55 TNF receptor, contrary to a neutralizing Ab against the p75 TNF receptor, inhibited TNF-α-induced p47phox phosphorylation. Neutrophil treatment with both TNF-α and GM-CSF resulted in more intense phosphorylation of the same p47phox peptide observed with each cytokine alone, suggesting that they engaged pathways converging on common serines. This additive effect was also obtained on the priming of NADPH oxidase activity. The use of protein kinase inhibitors pointed to the involvement of a protein tyrosine kinase, but not protein kinase C. These findings show that TNF-α, via its p55 receptor, induces a protein tyrosine kinase-dependent selective phosphorylation of p47phox on specific serines. The ability of TNF-α and GM-CSF, two different cytokines with two different receptors to induce this specific p47phox phosphorylation, suggests that this event could be a common element of the priming of neutrophils by TNF-α and GM-CSF.
Activation of Group IV Cytosolic Phospholipase A2 in Human Eosinophils by Phosphoinositide 3-Kinase Through a Mitogen-Activated Protein Kinase-Independent Pathway
Authors: Myou Shigeharu , Leff Alan R. , Myo Saori , Boetticher Evan , Meliton Angelo Y. , Lambertino Anissa T. , Liu Jie , Xu Chang , Munoz Nilda M. , Zhu Xiangdong
Published: April 2014

Activation of group IV cytosolic phospholipase A2 (gIV-PLA2) is the essential first step in the synthesis of inflammatory eicosanoids and in integrin-mediated adhesion of leukocytes. Prior investigations have demonstrated that phosphorylation of gIV-PLA2 results from activation of at least two isoforms of mitogen-activated protein kinase (MAPK). We investigated the potential role of phosphoinositide 3-kinase (PI3K) in the activation of gIV-PLA2 and the hydrolysis of membrane phosphatidylcholine in fMLP-stimulated human blood eosinophils. Transduction into eosinophils of Δp85, a dominant negative form of class IA PI3K adaptor subunit, fused to an HIV-TAT protein transduction domain (TAT-Δp85) concentration dependently inhibited fMLP-stimulated phosphorylation of protein kinase B, a downstream target of PI3K. FMLP caused increased arachidonic acid (AA) release and secretion of leukotriene C4 (LTC4). TAT-Δp85 and LY294002, a PI3K inhibitor, blocked the phosphorylation of gIV-PLA2 at Ser505 caused by fMLP, thus inhibiting gIV-PLA2 hydrolysis and production of AA and LTC4 in eosinophils. FMLP also caused extracellular signal-related kinases 1 and 2 and p38 MAPK phosphorylation in eosinophils; however, neither phosphorylation of extracellular signal-related kinases 1 and 2 nor p38 was inhibited by TAT-Δp85 or LY294002. Inhibition of 1) p70 S6 kinase by rapamycin, 2) protein kinase B by Akt inhibitor, or 3) protein kinase C by Ro-31-8220, the potential downstream targets of PI3K for activation of gIV-PLA2, had no effect on AA release or LTC4 secretion caused by fMLP. We find that PI3K is required for gIV-PLA2 activation and hydrolytic production of AA in activated eosinophils. Our data suggest that this essential PI3K independently activates gIV-PLA2 through a pathway that does not involve MAPK.
IL-7 Stimulates T Cell Renewal Without Increasing Viral Replication in Simian Immunodeficiency Virus-Infected Macaques
Authors: Nugeyre Marie-Thérèse , Monceaux Valérie , Beq Stéphanie , Cumont Marie-Christine , Fang Raphaël Ho Tsong , Chêne Laurent , Morre Michel , Barré-Sinoussi Françoise , Hurtrel Bruno , Israël Nicole
Published: April 2014

The main failure of antiretroviral therapy is the lack of restoration of HIV-specific CD4+ T cells. IL-7, which has been shown to be a crucial cytokine for thymopoiesis, has been envisaged as an additive therapeutic strategy. However, in vitro studies suggest that IL-7 might sustain HIV replication in thymocytes and T lymphocytes. Therefore, in the present study, we evaluated the effect of IL-7 on both T cell renewal and viral load in SIVmac-infected young macaques in the absence of antiretroviral therapy. This evaluation was conducted during the asymptomatic phase in view of a potential treatment of HIV patients. We show that IL-7 induces both a central renewal and a peripheral expansion of T lymphocytes associated with cell activation. No alarming modulation of the other hemopoietic cells was observed. No increase in the viral load was shown in blood or lymph nodes. These data strengthen the rationale for the use of IL-7 as an efficient immunotherapy in AIDS.
Role of IL-12 Receptor β1 in Regulation of T Cell Response by APC in Experimental Autoimmune Encephalomyelitis
Authors: Zhang Guang-Xian , Yu Shuo , Gran Bruno , Li Jifen , Siglienti Ines , Chen Xiaohan , Calida Divina , Ventura Elvira , Kamoun Malek , Rostami Abdolmohamad
Published: April 2014

IL-12 was thought to be involved in the development of experimental autoimmune encephalomyelitis (EAE), a Th1 cell-mediated autoimmune disorder of the CNS. However, we have recently found that IL-12 responsiveness, via IL-12Rβ2, is not required in the induction of EAE. To determine the role of IL-12Rβ1, a key subunit for the responsiveness to both IL-12 and IL-23, in the development of autoimmune diseases, we studied EAE in mice deficient in this subunit of IL-12R. IL-12Rβ1−/− mice are completely resistant to myelin oligodendrocyte glycoprotein (MOG)-induced EAE, with an autoantigen-specific Th2 response. To study the mechanism underlying this Th2 bias, we cocultured purified CD4+ T cells and APCs of MOG-immunized mice. We demonstrate that IL-12Rβ1−/− APCs drive CD4+ T cells of both wild-type and IL-12Rβ1−/− mice to an Ag-induced Th2 phenotype, whereas wild-type APCs drive these CD4+ T cells toward a Th1 type. IL-12Rβ1−/− CD4+ T cells, in turn, appear to exert an immunoregulatory effect on the capacity of wild-type APCs to produce IFN-γ and TNF-α. Furthermore, decreased levels of IL-12p40, p35, and IL-23p19 mRNA expression were found in IL-12Rβ1−/− APCs, indicating an autocrine pathway of IL-12/IL-23 via IL-12Rβ1. IL-18 production and IL-18Rα expression are also significantly decreased in IL-12Rβ1−/− mice immunized with MOG. We conclude that in the absence of IL-12Rβ1, APCs play a prominent regulatory role in the induction of autoantigen-specific Th2 cells.
Interplay between TCR Affinity and Necessity of Coreceptor Ligation: High-Affinity Peptide-MHC/TCR Interaction Overcomes Lack of CD8 Engagement
Authors: Kerry Samantha E. , Buslepp Jennifer , Cramer Lorraine A. , Maile Robert , Hensley Lucinda L. , Nielsen Alma I. , Kavathas Paula , Vilen Barbara J. , Collins Edward J. , Frelinger Jeffrey A.
Published: April 2014

CD8 engagement is believed to be a critical event in the activation of naive T cells. In this communication, we address the effects of peptide-MHC (pMHC)/TCR affinity on the necessity of CD8 engagement in T cell activation of primary naive cells. Using two peptides with different measured avidities for the same pMHC-TCR complex, we compared biochemical affinity of pMHC/TCR and the cell surface binding avidity of pMHC/TCR with and without CD8 engagement. We compared early signaling events and later functional activity of naive T cells in the same manner. Although early signaling events are altered, we find that high-affinity pMHC/TCR interactions can overcome the need for CD8 engagement for proliferation and CTL function. An integrated signal over time allows T cell activation with a high-affinity ligand in the absence of CD8 engagement.
Cell Proliferation Arrest within Intrathymic Lymphocyte Progenitor Cells Causes Thymic Atrophy Mediated by the Aryl Hydrocarbon Receptor
Authors: Laiosa Michael D. , Wyman Amber , Murante Francis G. , Fiore Nancy C. , Staples J. Erin , Gasiewicz Thomas A. , Silverstone Allen E.
Published: April 2014

Activation of the aryl hydrocarbon receptor (AHR), a basic helix-loop-helix transcription factor, in lymphocytes by the immunosuppressive environmental contaminant 2,3,7,8,-tetrachlorodibenzo-p-dioxin (TCDD) has been shown to cause thymic atrophy in every species studied. We set out to identify the specific hemopoietic cellular populations in which the AHR was activated to lead to thymic atrophy and to determine the effect of AHR activation in those cellular populations. Initially, we examined whether AHR activation in intrathymic dendritic cells could mediate TCDD-induced thymic atrophy. It was found that thymic atrophy occurred only when the AHR could be activated in the thymocytes but not hemopoietic-derived dendritic cells or other APCs. We next analyzed the effect of TCDD on the proliferation of thymocytes in vivo. There was a significant increase in the percentage of thymocytes in the G1 phase of the cell cycle and a significant decrease in the percentage of S plus G2/M thymocytes, especially in the CD4−CD8−CD3− triple-negative intrathymic progenitor cell population 24 h after exposure to 30 μg/kg TCDD. Furthermore, by 12 h after exposure to TCDD, we observed ∼60% reduction of 5-bromo-2′-deoxyuridine incorporation in specific intrathymic progenitor cell populations. This reduction persisted for at least 6 days. These data indicate that intrathymic progenitor cells are direct targets of TCDD in the thymus and suggest that TCDD causes thymic atrophy by reducing entrance into cell cycle in these populations.
A Murine Locus on Chromosome 18 Controls NKT Cell Homeostasis and Th Cell Differentiation
Authors: Zhang Feng , Liang Zhiyan , Matsuki Naoto , Van Kaer Luc , Joyce Sebastian , Wakeland Edward K. , Aune Thomas M.
Published: April 2014

Th cell differentiation is a critical event in the adaptive immune response. C57BL strains develop predominant Th1 responses while BALB/c develops a predominant Th2 response. To identify quantitative trait loci controlling this variation, we performed Th1/Th2 differentiation assays of F1 × BALB/c progeny. A single strong quantitative trait locus was identified on chromosome 18, with weaker effects detectable on chromosomes 5, 12, and 14. By preparing a congenic BALB.B10.D2c18 strain, we were able to demonstrate that this single locus was sufficient to “repolarize” spleen cell cultures. This difference was not due to intrinsic differences in CD4+ T cells. Rather, introgression of the chromosome 18 locus into BALB/c disrupted Va14Ja18 NKT cell homeostasis resulting in the almost complete absence of this T cell subset. Taken together, these data indicate that genes within chromosome 18 control strain-dependent development of Va14Ja18 NKT cells.
Genomic Organization and Expression Analysis of B7-H4, an Immune Inhibitory Molecule of the B7 Family
Authors: Choi In-Hak , Zhu Gefeng , Sica Gabriel L. , Strome Scott E. , Cheville John C. , Lau Julie S. , Zhu Yuwen , Flies Dallas B. , Tamada Koji , Chen Lieping
Published: April 2014

B7-H4 is a recently identified B7 family member that negatively regulates T cell immunity by the inhibition of T cell proliferation, cytokine production, and cell cycle progression. In this study, we report that the genomic DNA of human B7-H4 is mapped on chromosome 1 comprised of six exons and five introns spanning 66 kb, of which exon 6 is used for alternative splicing to generate two different transcripts. Similar B7-H4 structure is also found in mouse genomic DNA in chromosome 3. A human B7-H4 pseudogene is identified in chromosome 20p11.1 with a single exon and two stop codons in the coding region. Immunohistochemistry analysis using B7-H4-specific mAb demonstrates that B7-H4 is not expressed on the majority of normal human tissues. In contrast, up to 85% (22 of 26) of ovarian cancer and 31% (5 of 16) of lung cancer tissues constitutively express B7-H4. Our results indicate a tight regulation of B7-H4 expression in the translational level in normal peripheral tissues and a potential role of B7-H4 in the evasion of tumor immunity.
Infant and Adult Human B Cell Responses to Rotavirus Share Common Immunodominant Variable Gene Repertoires
Authors: Weitkamp Jörn-Hendrik , Kallewaard Nicole , Kusuhara Koichi , Bures Elizabeth , Williams John V. , LaFleur Bonnie , Greenberg Harry B. , Crowe James E.
Published: April 2014

Ab repertoires exhibit marked restrictions during fetal life characterized by biases of variable gene usage and lack of junctional diversity. We tested the hypothesis that Ab repertoire restriction contributes to the observed poor quality of specific Ab responses made by infants to viral infections. We analyzed the molecular determinants of B cell responses in humans to two Ags of rotavirus (RV), a common and clinically important infection of human infants. We sequenced Ab H and L chain V region genes (VH and VL) of clones expanded from single B cells responding to RV virus protein 6 or virus protein 7. We found that adults exhibited a distinct bias in use of gene segments in the VH1 and VH4 families, for example, VH1–46, VH4–31, and VH4–61. This gene segment bias differed markedly from the VH3 dominant bias seen in randomly selected adult B cells. Recombinant Abs incorporating any of those three immunodominant VH segments bound to RV-infected cells and also to purified RV particles. The RV-specific B cell repertoires of infants aged 2–11 mo and those of adults were highly related when compared by VH, D, JH, VL, and JL segment selection, extent of junctional diversity, and mean H chain complementarity determining region 3 length. These data suggest that residual fetal bias of the B cell repertoire is not a limiting determinant of the quality of Ab responses to viruses of infants beyond the neonatal period.
Th1 Cell-Mediated Resistance to Cutaneous Infection with Leishmania major Is Independent of P- and E-Selectins
Authors: Zaph Colby , Scott Phillip
Published: April 2014

Studies in several models of inflammation have underscored the importance of P- and E-selectins in the migration of T cells to inflamed tissues. However, the role of the endothelial selectins in infection-induced cutaneous inflammation and host-protective immunity has not been investigated. In this study, we demonstrate that CD4+ T cells recruited to the cutaneous compartment during infection with Leishmania major express P- and E-selectin ligands. Furthermore, expression of P- and E-selectin ligands correlates with activated Leishmania-specific Th1 cells and is dependent upon IL-12. To investigate the functional role of the endothelial selectins during leishmaniasis, we infected mice either singly or doubly deficient in the expression of P- and E- selectins. Mice lacking both P- and E-selectins developed significantly less inflammation at the site of a primary and secondary infection, and exhibited an impaired delayed-type hypersensitivity response. Surprisingly, the absence of the endothelial selectins had no effect on the control of parasite replication or immunity to reinfection. Thus, these data demonstrate that although the endothelial selectins contribute to the inflammatory response, they are not required for protective immunity to L. major. Moreover, these data suggest that by blocking P- and E-selectins, the immune pathology associated with cutaneous leishmaniasis might be ameliorated without compromising immunity to infection.
Deficient CD4+ T Cell Priming and Regression of CD8+ T Cell Functionality in Virus-Infected Mice Lacking a Normal B Cell Compartment
Authors: Christensen Jan Pravsgaard , Kauffmann Susanne Ørding , Thomsen Allan Randrup
Published: April 2014

In this study, we investigate the state of T cell-mediated immunity in B cell-deficient (B−/−) mice infected with two strains of lymphocytic choriomeningitis virus known to differ markedly in their capacity to persist. In B−/− C57BL mice infected with the more persisting virus, virus-specific CD8+ T cells are initially generated that are qualitatively similar to those in wild-type mice. However, although cell numbers are well sustained over time, the capacity to produce cytokines is rapidly impaired. In similarly infected B−/− BALB/c mice, virus-specific CD8+ T cells are completely deleted, indicating that host genotype influences the severity of the T cell defect. In B−/− C57BL mice infected with the less persisting virus, CD8+ T cell dysfunction was not as pronounced, although it was clearly present. Most importantly, the appearance of dysfunctional CD8+ T cells clearly precedes recrudescence of detectable virus, indicating that the T cell defect is not simply a secondary event due to virus buildup resulting from the failure of B−/− mice to produce neutralizing Abs. In contrast with CD8+ T cells, which initially respond almost as in wild-type mice, the priming of virus-specific CD4+ T cells was markedly impaired in B−/− mice infected with either virus strain. Thus, our results indicate that B cells play an important role in antiviral immunity not only as Ab producers, but also in promoting an optimal and sustained T cell response. The T cell defects are likely to contribute to the chronic course of viral infection in B−/− mice.
An Inclusion Membrane Protein from Chlamydia trachomatis Enters the MHC Class I Pathway and Stimulates a CD8+ T Cell Response
Authors: Starnbach Michael N. , Loomis Wendy P. , Ovendale Pam , Regan David , Hess Bruce , Alderson Mark R. , Fling Steven P.
Published: April 2014

During its developmental cycle, the intracellular bacterial pathogen Chlamydia trachomatis remains confined within a protective vacuole known as an inclusion. Nevertheless, CD8+ T cells that recognize Chlamydia Ags in the context of MHC class I molecules are primed during infection. MHC class I-restricted presentation of these Ags suggests that these proteins or domains from them have access to the host cell cytoplasm. Chlamydia products with access to the host cell cytoplasm define a subset of molecules uniquely positioned to interface with the intracellular environment during the pathogen’s developmental cycle. In addition to their use as candidate Ags for stimulating CD8+ T cells, these proteins represent novel candidates for therapeutic intervention of infection. In this study, we use C. trachomatis-specific murine T cells and an expression-cloning strategy to show that CT442 from Chlamydia is targeted by CD8+ T cells. CT442, also known as CrpA, is a 15-kDa protein of undefined function that has previously been shown to be associated with the Chlamydia inclusion membrane. We show that: 1) CD8+ T cells specific for an H-2Db-restricted epitope from CrpA are elicited at a significant level (∼4% of splenic CD8+ T cells) in mice in response to infection; 2) the response to this epitope correlates with clearance of the organism from infected mice; and 3) immunization with recombinant vaccinia virus expressing CrpA elicits partial protective immunity to subsequent i.v. challenge with C. trachomatis.
Inhibition of Neutrophil Leukotriene B4 Production by a Novel Synthetic N-3 Polyunsaturated Fatty Acid Analogue, β-Oxa 21:3n-3
Authors: Robinson Brenton S. , Rathjen Deborah A. , Trout Neil A. , Easton Christopher J. , Ferrante Antonio
Published: April 2014

We recently reported the synthesis and anti-inflammatory properties of a novel long chain polyunsaturated fatty acid (PUFA) with an oxygen atom in the β-position, β-oxa-21:3 n-3 (Z,Z,Z)-(octadeca-9,12,15-trienyloxy) acetic acid). Our data, from studies aimed at elucidating the mechanism of its action, show that pretreatment of human neutrophils with the β-oxa-PUFA substantially depresses the production of leukotriene B4 (LTB4) in response to calcium ionophore, A23187, comparable to standard leukotriene inhibitors such as zileuton and nordihydroguaiaretic acid. Interestingly, the n-6 equivalent, β-oxa 21:3 n-6, is also a strong inhibitor of LTB4 production. In contrast, naturally occurring PUFA only slightly reduce, for eicosapentaenoic (20:5n-3) and docosahexaenoic (22:6n-3) acids, or increase, for arachidonic acid (20:4n-6), the formation of LTB4. The parent β-oxa-21:3n-3 molecule, rather than its derivatives (methyl ester, saturated, monohydroperoxy, or monohydroxy forms), is exclusively responsible for attenuation of LTB4 formation. β-Oxa-21:3n-3 inhibits the conversion of [3H]20:4n-6 to [3H]5-hydroxyeicosatetraenoic acid and [3H]LTB4 by neutrophils in the presence of calcium ionophore and also suppresses the activity of purified 5-lipoxygenase, but not cyclooxygenase 1 and 2. β-Oxa-21:3n-3 is taken up by neutrophils and incorporated into phospholipids and neutral lipids. In the presence of calcium ionophore, the leukocytes convert a marginal amount of β-oxa-21:3n-3 to a 16-monohydroxy-β-oxa-21:3n-3 derivative. After administration to rodents by gavage or i.p. injection, β-oxa-21:3n-3 is found to be incorporated into the lipids of various tissues. Thus, β-oxa-21:3n-3 has the potential to be used in the treatment of inflammatory diseases, which are mediated by products of the lipoxygenase pathway.
CpG-Containing Oligodeoxynucleotide 1826 Converts the Weak Uveitogenic Rat Interphotoreceptor Retinoid-Binding Protein Peptide 1181–1191 into a Strong Uveitogen
Authors: Shao Hui , Lei Song , Sun Sheher L. , Xiang Jim , Kaplan Henry J. , Sun Deming
Published: April 2014

Aberrant activation of autoreactive T cells is one of the major causes of autoimmune disease. Autoantigens are sequestered and in many cases weak immunogens. For example, in experimental autoimmune uveitis, immunization of naive rats with autologous interphotoreceptor retinoid-binding protein (IRBP) fails to induce intraocular inflammation or a strong T cell response, whereas bovine IRBP is a strong inducer of experimental autoimmune uveitis. Such observations challenge the view that the autoantigen alone is responsible for the development of autoimmunity. Here, we demonstrate that autologous rat IRBP is converted to a strong immunogen in the presence of a small dose of CpG-containing oligodeoxynucleotides. Our results indicate that specific CpG-containing oligodeoxynucleotides may play an important role in the activation and expansion of autoreactive T cells in vivo, leading to autoimmune disease.
In Vivo Impairment of Neutrophil Recruitment during Lentivirus Infection
Authors: Kubes Paul , Heit Bryan , van Marle Guido , Johnston James B. , Knight Derrice , Khan Adil , Power Christopher
Published: April 2014

Evidence indicates that the lentivirus, HIV, infection affects neutrophil response to bacteria and bacterial products in vitro. We used a novel model of rapid onset immunosuppression following infection with a similar lentivirus, feline immunodeficiency virus (FIV), in cats to examine neutrophil function within the microvasculature in vivo and to determine the steps that are impaired in the neutrophil recruitment cascade. In uninfected cats and cats infected neonatally with FIV, the mesentery was exteriorized, but remained autoperfused during intravital microscopy for 4 h. When the tissue was superfused with 10 μg/ml of LPS for 4 h, intravital microscopy displayed a profound increase in neutrophil rolling at both 8 and 12 wk of age in uninfected cats. At 12 wk of age, FIV-infected animals showed a profound decrease in the number of rolling neutrophils. In vitro studies revealed that neutrophils from infected and uninfected animals rolled equally well on surrogate selectin substrata. In addition, in vivo neutrophil adhesion and emigration out of the vasculature were severely reduced, and in vitro neutrophil chemotaxis from FIV-infected animals was significantly impaired in response to fMLP or IL-8. However, FIV infection of neutrophils could not be detected. In summary, in vivo lentivirus infection with immunosuppression leads to a severe impairment in neutrophil rolling, adhesion, and emigration in response to bacterial stimulants potentially involving both endothelial and neutrophil dysfunction. These in vivo studies also indicate that neutrophil dysfunction should be taken into account when treating infections and tissue injury.
Short-Time Infusion of Fish Oil-Based Lipid Emulsions, Approved for Parenteral Nutrition, Reduces Monocyte Proinflammatory Cytokine Generation and Adhesive Interaction with Endothelium in Humans
Authors: Mayer Konstantin , Meyer Sandra , Reinholz-Muhly Marion , Maus Ulrich , Merfels Martina , Lohmeyer Jürgen , Grimminger Friedrich , Seeger Werner
Published: April 2014

Potential impact of ω-3 fatty acids, as contained in fish oil, on immunological function has been suggested because observations of reduced inflammatory diseases in Greenland Inuit were published. A fish oil-based lipid emulsion has recently been approved for parenteral nutrition in many countries. We investigated the influence of a short infusion course of fish oil-based (ω-3) vs conventional (ω-6) lipid emulsion on monocyte function. In a randomized design, twelve healthy volunteers received ω-3 or ω-6 lipid infusion for 48 h, with cross-over repetition of the infusion course after 3 mo. Fatty acid profiles, monocyte cytokine release and adhesive monocyte-endothelium interaction were investigated. Resultant ω-6 lipid emulsion increased plasma-free fatty acids including arachidonic acid, whereas the ω-3/ω-6 fatty acid ratio in monocyte membranes remained largely unchanged. It also caused a tendency toward enhanced monocyte proinflammatory cytokine release and adhesive monocyte-endothelium interaction. In contrast, ω-3 lipid emulsion significantly increased the ω-3/ω-6 fatty acid ratio in the plasma-free fatty acid fraction and in monocyte membrane lipid pool, markedly suppressing monocyte generation of TNF-α, IL-1, IL-6, and IL-8 in response to endotoxin. In addition, it also significantly inhibited both monocyte-endothelium adhesion and transendothelial monocyte migration, although monocyte surface expression of relevant adhesive molecules (CD11b, CD18, CD49 days, CCR2) was unchanged. Although isocaloric, ω-3 and ω-6 lipid emulsions exert differential impact on immunological processes in humans. In addition to its nutritional value, fish oil-based ω-3 lipid emulsion significantly suppresses monocyte proinflammatory cytokine generation and features of monocyte recruitment.
Role of CXCL9/CXCR3 Chemokine Biology during Pathogenesis of Acute Lung Allograft Rejection
Authors: Belperio John A. , Keane Michael P. , Burdick Marie D. , Lynch Joseph P. , Zisman David A. , Xue Ying Ying , Li Kewang , Ardehali Abbas , Ross David J. , Strieter Robert M.
Published: April 2014

Acute allograft rejection is a major complication postlung transplantation and is the main risk factor for the development of bronchiolitis obliterans syndrome. Acute rejection is characterized by intragraft infiltration of activated mononuclear cells. The ELR-negative CXC chemokines CXCL9, CXCL10, and CXCL11) are potent chemoattractants for mononuclear cells and act through their shared receptor, CXCR3. Elevated levels of these chemokines in bronchoalveolar lavage fluid have been associated with human acute lung allograft rejection. This led to the hypothesis that the expression of these chemokines during an allogeneic response promotes the recruitment of mononuclear cells, leading to acute lung allograft rejection. We performed studies in a rat orthotopic lung transplantation model of acute rejection, and demonstrated increased expression of CXCL9 and CXCL10 paralleling the recruitment of mononuclear cells and cells expressing CXCR3 to the allograft. However, CXCL9 levels were 15-fold greater than CXCL10 during maximal rejection. Inhibition of CXCL9 decreased intragraft recruitment of mononuclear cells and cellular expression of CXCR3, resulting in lower acute lung allograft rejection scores. Furthermore, the combination of low dose cyclosporin A with anti-CXCL9 therapy had more profound effects on intragraft leukocyte infiltration and in reducing acute allograft rejection scores. This supports the notion that CXCL9 interaction with cells expressing CXCR3 has an important role in the recruitment of mononuclear cells, a pivotal event in the pathogenesis of acute lung allograft rejection.
Accumulation of Peribronchial Mast Cells in a Mouse Model of Ovalbumin Allergen Induced Chronic Airway Inflammation: Modulation by Immunostimulatory DNA Sequences
Authors: Ikeda Reid K. , Miller Marina , Nayar Jyothi , Walker Linda , Cho Jae Youn , McElwain Kirsti , McElwain Shauna , Raz Eyal , Broide David H.
Published: April 2014

Few peribronchial mast cells are noted either in the lungs of naive mice or in the lungs of OVA-sensitized mice challenged acutely with OVA by inhalation. In this study, we demonstrate that OVA-sensitized mice exposed to repetitive OVA inhalation for 1–6 mo have a significant accumulation of peribronchial mast cells. This accumulation of peribronchial mast cells is associated with increased expression of the Th2 cell-derived mast cell growth factors, including IL-4 and IL-9, but not with the non-Th2 cell-derived mast cell growth factor, stem cell factor. Pretreating mice with immunostimulatory sequences (ISS) of DNA containing a CpG motif significantly inhibited the accumulation of peribronchial mast cells and the expression of IL-4 and IL-9. To determine whether mast cells express Toll-like receptor-9 (TLR-9; the receptor for ISS), TLR-9 expression by mouse bone marrow-derived mast cells (MBMMCs) was assessed by RT-PCR. MBMMCs strongly expressed TLR-9 and bound rhodamine-labeled ISS. However, incubation of MBMMCs with ISS in vitro neither inhibited MBMMC proliferation nor inhibited Ag/IgE-mediated MBMMC degranulation, but they did induce IL-6. Overall these studies demonstrate that mice exposed to repetitive OVA challenge, but not acute OVA challenge, have an accumulation of peribronchial mast cells and express increased levels of mast cell growth factors in the lung. Although mast cells express TLR-9, ISS does not directly inhibit mast cell proliferation in vitro, suggesting that ISS inhibits accumulation of peribronchial mast cells in vivo by indirect mechanism(s), which include inhibiting the lung expression of Th2 cell-derived mast cell growth factors.
CC Chemokine Receptor 2 Expression in Donor Cells Serves an Essential Role in Graft-versus-Host-Disease
Authors: Rao Arun R. , Quinones Marlon P. , Garavito Edgar , Kalkonde Yogeshwar , Jimenez Fabio , Gibbons Caroline , Perez Jennifer , Melby Peter , Kuziel William , Reddick Robert L. , Ahuja Sunil K. , Ahuja Seema S.
Published: April 2014

The complete repertoire of cellular and molecular determinants that influence graft-vs-host disease (GVHD) is not known. Using a well-established murine model of GVHD (B6→bm12 mice), we sought to elucidate the role of the donor non-T cell compartment and molecular determinants therein in the pathogenesis of GVHD. In this model the acute GVHD-inducing effects of purified B6 wild-type (wt) CD4+ T cells was inhibited by wt non-T cells in a dose-dependent manner. Paradoxically, unlike the chronic GVHD phenotype observed in bm12 mice transplanted with B6wt unfractionated splenocytes, bm12 recipients of B6ccr2-null unfractionated splenocytes developed acute GVHD and died of IFN-γ-mediated bone marrow aplasia. This switch from chronic to acute GVHD was associated with increased target organ infiltration of activated CD4+ T cells as well as enhanced expression of Th1/Th2 cytokines, chemokines, and the antiapoptotic factor bfl1. In vitro, ccr2−/− CD4+ T cells in unfractionated splenocytes underwent significantly less activation-induced cell death than B6wt CD4+ T cells, providing another potential mechanistic basis along with enhanced expression of bfl1 for the increased numbers of activated T cells in target organs of B6ccr2−/− splenocyte→bm12 mice. Collectively, these findings have important clinical implications, as they implicate the donor non-T cell compartment as a critical regulator of GVHD and suggest that ccr2 expression in this cellular compartment may be an important molecular determinant of activation-induced cell death and GVHD pathogenesis.
Monoclonal Anti-MAGE-3 CTL Responses in Melanoma Patients Displaying Tumor Regression after Vaccination with a Recombinant Canarypox Virus
Authors: Karanikas Vaios , Lurquin Christophe , Colau Didier , van Baren Nicolas , De Smet Charles , Lethé Bernard , Connerotte Thierry , Corbière Véronique , Demoitié Marie-Ange , Liénard Danielle , Dréno Brigitte , Velu Thierry , Boon Thierry , Coulie Pierre G.
Published: April 2014

We have analyzed the T cell responses of HLA-A1 metastatic melanoma patients with detectable disease, following vaccination with a recombinant ALVAC virus, which bears short MAGE-1 and MAGE-3 sequences coding for antigenic peptides presented by HLA-A1. To evaluate the anti-MAGE CTL responses, we resorted to antigenic stimulation of blood lymphocytes under limiting dilution conditions, followed by tetramer analysis and cloning of the tetramer-positive cells. The clones were tested for their specific lytic ability and their TCR sequences were obtained. Four patients who showed tumor regression were analyzed, and an anti-MAGE-3.A1 CTL response was observed in three of these patients. Postvaccination frequencies of anti-MAGE-3.A1 CTL were 3 × 10−6, 3 × 10−3, and 3 × 10−7 of the blood CD8 T cells, respectively. These three responses were monoclonal. No anti-MAGE-1.A1 CTL response was observed. These results indicate that, like peptide immunization, ALVAC immunization produces monoclonal responses. They also suggest that low-level antivaccine CTL responses can initiate a tumor regression process. Taken together, our analysis of anti-MAGE-3.A1 T cell responses following peptide or ALVAC vaccination shows a degree of correlation between CTL response and tumor regression, but firm conclusions will require larger numbers.
Gene Transfer of a Cell Cycle Modulator Exerts Anti-Inflammatory Effects in the Treatment of Arthritis
Authors: Nonomura Yoshinori , Kohsaka Hitoshi , Nagasaka Kenji , Miyasaka Nobuyuki
Published: April 2014

Forced expression of a cyclin-dependent kinase inhibitor gene, p21Cip1 in the synovial tissues was effective in treating animal models of rheumatoid arthritis. Synovial hyperplasia in the treated joints was suppressed, reflecting the inhibitory effect of p21Cip1 on cell cycle progression. Additionally, lymphocyte infiltration, expression of inflammatory cytokines, and destruction of the bone and cartilage were inhibited. To determine why the cell cycle regulator gene exerted such anti-inflammatory effects, we investigated gene expression by rheumatoid synovial fibroblasts with or without the p21Cip1 gene transferred. We have found that p21Cip1 gene transfer down-regulates expression of various inflammatory mediators and tissue-degrading proteinases that are critically involved in the pathology of rheumatoid arthritis. These molecules included IL-6, -8, type I IL-1R (IL-1R1), monocyte chemoattractant protein-1, macrophage inflammatory protein-3α, cathepsins B and K, and matrix metalloproteinases-1 and -3. Down-regulation of IL-1R1 by p21Cip1 resulted in attenuated responsiveness to IL-1. Inhibition of the inflammatory gene expression by p21Cip1 was seen even when IL-1 is absent. This IL-1R1-independent suppression was accompanied by reduced activity of c-Jun N-terminal kinase, which was associated with p21Cip1, and inactivation of NF-κB and AP-1. These multiple regulatory effects should work in concert with the primary effect of inhibiting cell cycle in ameliorating the arthritis, and suggest a heretofore unexplored relationship between cyclin-dependent kinase inhibitor gene and inflammatory molecules.
Up-Regulation of Hlx in Immature Th Cells Induces IFN-γ Expression
Authors: Zheng Wei-ping , Zhao Qian , Zhao Xinyan , Li Baiyong , Hubank Mike , Schatz David G. , Flavell Richard A.
Published: April 2014

Differentiation of Th cells from naive precursors is a dynamic process that involves multiple transcription factors acting at specific time points to regulate gene expression. In this study we show that the homeobox transcription factor Hlx is up-regulated early in Th1 cell differentiation. Mice constitutively expressing an Hlx transgene driven by a CD4 promoter showed marked reduction in the CD4+CD8+ thymocyte population. The Hlx transgenic mice generated increased numbers of Th1 cells in response to keyhole limpet hemocyanin immunization. After differentiation under Th2-polarizing conditions in vitro, the transgenic CD4 T cells expressed high levels of IFN-γ. Intracellular cytokine staining revealed that in addition to Th2 cells, large numbers of Th0 and Th1 cells were generated from such in vitro differentiated transgenic CD4 T cells. Retrovirally overexpressed Hlx also induced the aberrant expression of IFN-γ in normal CD4 T cells differentiated under Th2-polarizing conditions. This effect was apparent only when Hlx was introduced into the cells by retroviral infection at an early time point that led to the expression of the retrovirally transferred Hlx gene at a time comparable to that of the up-regulation of the endogenous Hlx during Th1 cell differentiation. Later infection with Hlx-expressing retrovirus showed no effect. Thus, the induction of IFN-γ expression by Hlx depends on a permissive epigenetic state of the IFN-γ gene locus and/or the molecular context of the immature Th cells.
NK Cells Regulate CD8+ T Cell Effector Function in Response to an Intracellular Pathogen
Authors: Vankayalapati Ramakrishna , Klucar Peter , Wizel Benjamin , Weis Stephen E. , Samten Buka , Safi Hassan , Shams Homayoun , Barnes Peter F.
Published: April 2014

We studied the role of NK cells in regulating human CD8+ T cell effector function against mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Depletion of NK cells from PBMC of healthy tuberculin reactors reduced the frequency of M. tuberculosis-responsive CD8+IFN-γ+ cells and decreased their capacity to lyse M. tuberculosis-infected monocytes. The frequency of CD8+IFN-γ+cells was restored by soluble factors produced by activated NK cells and was dependent on IFN-γ, IL-15, and IL-18. M. tuberculosis-activated NK cells produced IFN-γ, activated NK cells stimulated infected monocytes to produce IL-15 and IL-18, and production of IL-15 and IL-18 were inhibited by anti-IFN-γ. These findings suggest that NK cells maintain the frequency of M. tuberculosis-responsive CD8+IFN-γ+ T cells by producing IFN-γ, which elicits secretion of IL-15 and IL-18 by monocytes. These monokines in turn favor expansion of Tc1 CD8+ T cells. The capacity of NK cells to prime CD8+ T cells to lyse M. tuberculosis-infected target cells required cell-cell contact between NK cells and infected monocytes and depended on interactions between the CD40 ligand on NK cells and CD40 on infected monocytes. NK cells link the innate and the adaptive immune responses by optimizing the capacity of CD8+ T cells to produce IFN-γ and to lyse infected cells, functions that are critical for protective immunity against M. tuberculosis and other intracellular pathogens.