bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of Myeloid Cell Function through the CD200 Receptor
Authors: Jenmalm Maria C. , Cherwinski Holly , Bowman Edward P. , Phillips Joseph H. , Sedgwick Jonathon D.
Published: April 2014

Myeloid cells play pivotal roles in chronic inflammatory diseases through their broad proinflammatory, destructive, and remodeling capacities. CD200 is widely expressed on a variety of cell types, while the recently identified CD200R is expressed on myeloid cells and T cells. CD200 deletion in vivo results in myeloid cell dysregulation and enhanced susceptibility to autoimmune inflammation, suggesting that the CD200-CD200R interaction is involved in immune suppression. We demonstrate in this study that CD200R agonists suppress mouse and human myeloid cell function in vitro, and also define a dose relationship between receptor expression and cellular inhibition. IFN-γ- and IL-17-stimulated cytokine secretion from mouse peritoneal macrophages was inhibited by CD200R engagement. Inhibitory effects were not universal, as LPS-stimulated responses were unaffected. Inhibition of U937 cell cytokine production correlated with CD200R expression levels, and inhibition was only observed in low CD200R expressing cells, if the CD200R agonists were further cross-linked. Tetanus toxoid-induced human PBMC IL-5 and IL-13 secretion was inhibited by CD200R agonists. This inhibition was dependent upon cross-linking the CD200R on monocytes, but not on cross-linking the CD200R on CD4+ T cells. In all, we provide direct evidence that the CD200-CD200R interaction controls monocyte/macrophage function in both murine and human systems, further supporting the potential clinical application of CD200R agonists for the treatment of chronic inflammatory diseases.
Induction of Antitumor Immunity by CTL Epitopes Genetically Linked to Membrane-Anchored β2-Microglobulin
Authors: Margalit Alon , Sheikhet Helena M. , Carmi Yaron , Berko Dikla , Tzehoval Esther , Eisenbach Lea , Gross Gideon
Published: April 2014

Level and persistence of antigenic peptides presented by APCs on MHC class I (MHC-I) molecules influence the magnitude and quality of the ensuing CTL response. We recently demonstrated the unique immunological properties conferred on APCs by expressing β2-microglobulin (β2m) as an integral membrane protein. In this study, we explored membrane-anchored β2m as a platform for cancer vaccines using as a model MO5, an OVA-expressing mouse B16 melanoma. We expressed in mouse RMA-S cells two H-2Kb binding peptides from MO5, OVA257–264, and TRP-2181–188, each genetically fused with the N terminus of membranal β2m via a short linker. Specific Ab staining and T cell hybridoma activation confirmed that OVA257–264 was properly situated in the MHC-I binding groove. In vivo, transfectants expressing both peptides elicited stronger CTLs and conferred better protection against MO5 than peptide-saturated RMA-S cells. Cells expressing OVA257–264/β2m were significantly superior to OVA257–264-charged cells in their ability to inhibit the growth of pre-established MO5 tumors. Our results highlight the immunotherapeutic potential of membranal β2m as a universal scaffold for optimizing Ag presentation by MHC-I molecules.
Enhanced Regulatory T Cell Activity Is an Element of the Host Response to Injury
Authors: Choileain Niamh Ni , MacConmara Malcolm , Zang Yan , Murphy Thomas J. , Mannick John A. , Lederer James A.
Published: April 2014

CD4+CD25+ regulatory T cells (Tregs) play a critical role in suppressing the development of autoimmune disease, in controlling potentially harmful inflammatory responses, and in maintaining immune homeostasis. Because severe injury triggers both excessive inflammation and suppressed adaptive immunity, we wished to test whether injury could influence Treg activity. Using a mouse burn injury model, we demonstrate that injury significantly enhances Treg function. This increase in Treg activity is apparent at 7 days after injury and is restricted to lymph node CD4+CD25+ T cells draining the injury site. Moreover, we show that this injury-induced increase in Treg activity is cell-contact dependent and is mediated in part by increased cell surface TGF-β1 expression. To test the in vivo significance of these findings, mice were depleted of CD4+CD25+ T cells before sham or burn injury and then were immunized to follow the development of T cell-dependent Ag-specific immune reactivity. We observed that injured mice, which normally demonstrate suppressed Th1-type immunity, showed normal Th1 responses when depleted of CD4+CD25+ T cells. Taken together, these observations suggest that injury can induce or amplify CD4+CD25+ Treg function and that CD4+CD25+ T cells contribute to the development of postinjury immune suppression.
Virus or TLR Agonists Induce TRAIL-Mediated Cytotoxic Activity of Plasmacytoid Dendritic Cells
Authors: Chaperot Laurence , Blum Ariane , Manches Olivier , Lui Gabrielle , Angel Juliette , Molens Jean-Paul , Plumas Joël
Published: April 2014

Among dendritic cells, plasmacytoid dendritic cells (PDC) represent a functionally distinct lineage. Regarding innate immunity, PDC secrete large amounts of type I IFN upon viral exposure or stimulation by microbial products such as unmethylated CpG-motif containing oligo-DNA due to their selective expression of TLR7 and TLR9. We asked whether they could acquire cytotoxic functions during the early phases of infection or after activation with TLR7 or TLR9 agonists. In the present study, we describe a human PDC cell line called GEN2.2, derived from leukemic PDC, that shares most of the phenotypic and functional features of normal PDC. We show that after contact with the influenza virus, GEN2.2, as well as normal PDC, acquires TRAIL and killer activity against TRAIL-sensitive target cells. Moreover, we show that activation of GEN2.2 cells by CpG-motif containing oligo-DNA or R848 also induces TRAIL and endows them with the ability to kill melanoma cells. Therefore, PDC may represent a major component of innate immunity that could participate to the clearance of infected cells and tumor cells. This phenomenon could be relevant for the efficacy of TLR7 or TLR9 agonists in the therapy of infectious disease and cancer.
Cellular and Molecular Mechanisms of the Selective Regulation of IL-12 Production by 12/15-Lipoxygenase
Authors: Middleton Melissa K. , Rubinstein Tanya , Puré Ellen
Published: April 2014

IL-12 drives type I immune responses and can mediate chronic inflammation that leads to host defense as well as disease. Recently, we discovered a novel role for 12/15-lipoxygenase (12/15-LO) in mediating IL-12p40 expression in atherosclerotic plaque and in isolated macrophages. We now demonstrate that 12/15-LO regulates IL-12 family cytokine production in a cell-type and stimulus-restricted fashion. LPS-stimulated elicited peritoneal macrophages derived from 12/15-LO-deficient (Alox15) mice produced reduced IL-12 and IL-23 levels, but comparable amounts of several other inflammatory mediators tested. Furthermore, LPS stimulation triggered an increase in wild-type macrophage 12/15-LO activity, whereas pharmacological inhibition of 12/15-LO activity suppressed LPS-induced IL-12 production in wild-type macrophages. 12/15-LO-deficient macrophages also produced reduced levels of IL-12 in response to TLR2 stimulation, but not in response to CpG (TLR9) or CD40/CD40L-mediated activation. In contrast to our previous finding of reduced IL-12 production in the setting of atherosclerosis, we found that comparable IL-12 levels were produced in Alox15 and wild-type mice during an acute response to LPS in vivo. This paradox may be explained by normal production of IL-12 by 12/15-LO-deficient neutrophils and dendritic cells, which are major sources of IL-12 during acute inflammation. Finally, we detected selectively decreased association of the transcription factors IFN consensus sequence binding protein and NF-κB with the IL-12p40 promoter in 12/15-LO-deficient macrophages. Taken together, these findings reveal a highly selective pathway to IL-12 production that may prove a useful target in chronic inflammation while sparing the acute response to infection.
PU.1 Regulates Cathepsin S Expression in Professional APCs
Authors: Wang Ying , Baron Rebecca M. , Zhu Guangli , Joo Myungsoo , Christman John W. , Silverman Eric S. , Perrella Mark A. , Riese Richard J. , Cernadas Manuela
Published: April 2014

Cathepsin S (CTSS) is a cysteine protease that is constitutively expressed in APCs and mediates processing of MHC class II-associated invariant chain. CTSS and the Ets family transcription factor PU.1 are highly expressed in cells of both myeloid (macrophages and dendritic cells) and lymphoid (B lymphocytes) lineages. Therefore, we hypothesized that PU.1 participates in the transcriptional regulation of CTSS in these cells. In A549 cells (a human epithelial cell line that does not express either CTSS or PU.1), the expression of PU.1 enhances CTSS promoter activity ∼5- to 10-fold. In RAW cells (a murine macrophage-like cell line that constitutively expresses both CTSS and PU.1), the expression of a dominant-negative PU.1 protein and a short-interfering RNA PU.1 construct attenuates basal CTSS promoter activity, mRNA levels, and protein expression. EMSAs show binding of PU.1 to oligonucleotides derived from the CTSS promoter at two different Ets consensus binding elements. Mutation of these sites decreases the baseline CTSS activity in RAW cells that constitutively express PU.1. Chromatin immunoprecipitation experiments show binding of PU.1 with the CTSS promoter in this same region. Finally, the expression of PU.1, in concert with several members of the IFN regulatory factor family, enhances CTSS promoter activity beyond that achieved by PU.1 alone. These data indicate that PU.1 participates in the regulation of CTSS transcription in APCs. Thus, manipulation of PU.1 expression may directly alter the endosomal proteolytic environment in these cells.
Inflammation Induces Myeloid-Derived Suppressor Cells that Facilitate Tumor Progression
Authors: Bunt Stephanie K. , Sinha Pratima , Clements Virginia K. , Leips Jeff , Ostrand-Rosenberg Suzanne
Published: April 2014

Epidemiological and experimental observations support the hypothesis that chronic inflammation contributes to cancer development and progression; however, the mechanisms underlying the relationship between inflammation and cancer are poorly understood. To study these mechanisms, we have transfected the mouse 4T1 mammary carcinoma with the proinflammatory cytokine IL-1β to produce a chronic inflammatory microenvironment at the tumor site. Mice with 4T1/IL-1β tumors have a decreased survival time and elevated levels of immature splenic Gr1+CD11b+ myeloid-derived cells. These myeloid suppressor cells (MSC) are present in many patients with cancer and inhibit the activation of CD4+ and CD8+ T lymphocytes. 4T1/IL-1β-induced MSC do not express the IL-1R, suggesting that the cytokine does not directly activate MSC. Neither T or B cells nor NKT cells are involved in the IL-1β-induced increase of MSC because RAG2−/− mice and nude mice with 4T1/IL-1β tumors also have elevated MSC levels. MSC levels remain elevated in mice inoculated with 4T1/IL-1β even after the primary tumor is surgically removed, indicating that the IL-1β effect is long lived. Collectively, these findings suggest that inflammation promotes malignancy via proinflammatory cytokines, such as IL-1β, which enhance immune suppression through the induction of MSC, thereby counteracting immune surveillance and allowing the outgrowth and proliferation of malignant cells.
Ly9 (CD229)-Deficient Mice Exhibit T Cell Defects yet Do Not Share Several Phenotypic Characteristics Associated with SLAM- and SAP-Deficient Mice
Authors: Graham Daniel B. , Bell Michael P. , McCausland Megan M. , Huntoon Catherine J. , van Deursen Jan , Faubion William A. , Crotty Shane , McKean David J.
Published: April 2014

Signaling lymphocyte activation molecule (SLAM) family receptors are critically involved in modulating innate and adaptive immune responses. Several SLAM family receptors have been shown to interact with the adaptor molecule SAP; however, subsequent intracellular signaling is poorly defined. Notably, mutations in SLAM-associated protein (SAP) lead to X-linked lymphoproliferative disease, a rare but fatal immunodeficiency. Although the SLAM family member Ly9 (CD229) is known to interact with SAP, the functions of this receptor have remained elusive. Therefore, we have generated Ly9−/− mice and compared their phenotype with that of SLAM−/− and SAP−/− mice. We report that Ly9−/− T cells exhibit a mild Th2 defect associated with reduced IL-4 production after stimulation with anti-TCR and anti-CD28 in vitro. This defect is similar in magnitude to the previously reported Th2 defect in SLAM−/− mice but is more subtle than that observed in SAP−/− mice. In contrast to SLAM−/− and SAP−/− mice, T cells from Ly9−/− mice proliferate poorly and produce little IL-2 after suboptimal stimulation with anti-CD3 in vitro. We have also found that Ly9−/− macrophages exhibit no defects in cytokine production or bacterial killing as was observed in SLAM−/− macrophages. Additionally, Ly9−/− mice differ from SAP−/− mice in that they foster normal development of NKT cells and mount appropriate T and B cell responses to lymphocytic choriomeningitis virus. We have identified significant phenotypic differences between Ly-9−/− mice as compared with both SLAM−/− and SAP−/− mice. Although Ly9, SLAM, and SAP play a common role in promoting Th2 polarization, Ly-9 is uniquely involved in enhancing T cell activation.
Molecular, Cellular, and Antigen Requirements for Development of Age-Associated T Cell Clonal Expansions In Vivo
Authors: Messaoudi Ilhem , Warner Jessica , Nikolich-Žugich Dragana , Fischer Miranda , Nikolich-Žugich Janko
Published: April 2014

T cell aging manifests itself both at the cellular (cell-autonomous defects in signaling) and at the population (age-related dysregulation of T cell homeostasis) levels. A prominent contributor to the latter is the appearance of T cell clonal expansions (TCE), with a potential to impair immune defense. In this study, we investigated molecular, cellular, and Ag requirements for TCE development. Of the mutant mice tested, old animals lacking MHC class I exhibited 7-fold fewer TCE than controls, with a 7-fold reduction in TCE. By contrast, animals lacking only one of the MHC class I molecules (Kb or Db), or IL-7R, or devoid of T cell renewal via adult thymectomy, all exhibited significant increases in TCE incidence. This increase directly correlated to lymphopenia, increased CD8 T cell turnover and an accumulation of memory-phenotype T cells. These data suggested that homeostatic cell division in the CD8 compartment enhances the formation of TCE. Repeated immunization with peptide/adjuvant did not result in an increase in Ag-specific TCE; however, adjuvant alone increased TCE incidence. In these experiments, therefore, nonspecific and/or homeostatic proliferation was more efficient in generating TCE in mice than repeated Ag-driven stimulation, suggesting that many, if not most, TCE in specific pathogen-free laboratory mice may be Ag-independent.
An Engineered Human IgG1 Antibody with Longer Serum Half-Life
Authors: Hinton Paul R. , Xiong Joanna M. , Johlfs Mary G. , Tang Meina Tao , Keller Stephen , Tsurushita Naoya
Published: April 2014

The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). By binding to FcRn in endosomes, IgG Abs are salvaged from lysosomal degradation and recycled to the circulation. Several studies have demonstrated a correlation between the binding affinity of IgG Abs to FcRn and their serum half-lives in mice, including engineered Ab fragments with longer serum half-lives. Our recent study extended this correlation to human IgG2 Ab variants in primates. In the current study, several human IgG1 mutants with increased binding affinity to human FcRn at pH 6.0 were generated that retained pH-dependent release. A pharmacokinetics study in rhesus monkeys of one of the IgG1 variants indicated that its serum half-life was ∼2.5-fold longer than the wild-type Ab. Ag binding was unaffected by the Fc mutations, while several effector functions appeared to be minimally altered. These properties suggest that engineered Abs with longer serum half-lives may prove to be effective therapeutics in humans.
Uracil DNA Glycosylase Disruption Blocks Ig Gene Conversion and Induces Transition Mutations
Authors: Saribasak Huseyin , Saribasak Nesibe Nur , Ipek Fatih M. , Ellwart Joachim W. , Arakawa Hiroshi , Buerstedde Jean-Marie
Published: April 2014

Ig gene conversion is most likely initiated by activation-induced cytidine deaminase-mediated cytosine deamination. If the resulting uracils need to be further processed by uracil DNA glycosylase (UNG), UNG inactivation should block gene conversion and induce transition mutations. In this study, we report that this is indeed the phenotype in the B cell line DT40. Ig gene conversion is almost completely extinguished in the UNG-deficient mutant and large numbers of transition mutations at C/G bases accumulate within the rearranged Ig L chain gene (IgL). The mutation rate of UNG-deficient cells is about seven times higher than that of pseudo V gene-deleted (ψV−) cells in which mutations arise presumably after uracil excision. In addition, UNG-deficient cells show relatively more mutations upstream and downstream of the VJ segment. This suggests that hypermutating B cells process activation-induced cytidine deaminase-induced uracils with approximately one-seventh of uracils giving rise to mutations depending on their position.
Nitration and Inactivation of IDO by Peroxynitrite
Authors: Fujigaki Hidetsugu , Saito Kuniaki , Lin Felix , Fujigaki Suwako , Takahashi Kanako , Martin Brian M. , Chen Cai Y. , Masuda Junichi , Kowalak Jeffrey , Takikawa Osamu , Seishima Mitsuru , Markey Sanford P.
Published: April 2014

IDO induction can deplete l-tryptophan in target cells, an effect partially responsible for the antimicrobial activities and antiallogeneic T cell responses of IFN-γ in human macrophages, dendritic cells, and bone marrow cells. l-Tryptophan depletion and NO production are both known to have an antimicrobial effect in macrophages, and the interaction of these two mechanisms is unclear. In this study we found that IDO activity was inhibited by the peroxynitrite generator, 3-(4-morpholinyl)sydnonimine, in PMA-differentiated cytokine-induced THP-1 (acute monocytic leukemia) cells and IFN-γ-stimulated PBMCs, whereas IDO protein expression was unaffected compared with that in untreated cells. Nitrotyrosine was detected in immunoprecipitated (IP)-IDO from PMA-differentiated cytokine-induced THP-1 cells treated with 3-(4-morpholinyl)sydnonimine, but not from untreated cells. Treatment of IP-IDO and recombinant IDO (rIDO) with peroxynitrite significantly decreased enzyme activity. Nitrotyrosine was detected in both peroxynitrite-treated IP-IDO and rIDO, but not in either untreated IP-IDO or rIDO. Peptide analysis by liquid chromatography/electrospray ionization and tandem mass spectrometry demonstrated that Tyr15, Tyr345, and Tyr353 in rIDO were nitrated by peroxynitrite. The levels of Tyr nitration and the inhibitory effect of peroxynitrite on IDO activity were significantly reduced in the Tyr15-to-Phe mutant. These results indicate that IDO is nitrated and inactivated by peroxynitrite and that nitration of Tyr15 in IDO protein is the most important factor in the inactivation of IDO.
Modulation of TLR4 Signaling by a Novel Adaptor Protein Signal-Transducing Adaptor Protein-2 in Macrophages
Authors: Sekine Yuichi , Yumioka Taro , Yamamoto Tetsuya , Muromoto Ryuta , Imoto Seiyu , Sugiyma Kenji , Oritani Kenji , Shimoda Kazuya , Minoguchi Mayu , Akira Shizuo , Yoshimura Akihiko , Matsuda Tadashi
Published: April 2014

Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-κB activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IκB kinase (IKK)-αβ, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-αβ. These interactions augmented MyD88- and/or IKK-αβ-dependent signals, leading to enhancement of the NF-κB activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-κB activation instead of the TNFR-associated factor 6-IL-1R-associated kinase 1 pathway.
Antimicrobial Activity of Native and Synthetic Surfactant Protein B Peptides
Authors: Ryan Marnie A. , Akinbi Henry T. , Serrano Alicia G. , Perez-Gil Jesus , Wu Huixing , McCormack Francis X. , Weaver Timothy E.
Published: April 2014

Surfactant protein B (SP-B) is secreted into the airspaces with surfactant phospholipids where it reduces surface tension and prevents alveolar collapse at end expiration. SP-B is a member of the saposin-like family of proteins, several of which have antimicrobial properties. SP-B lyses negatively charged liposomes and was previously reported to inhibit the growth of Escherichia coli in vitro; however, a separate study indicated that elevated levels of SP-B in the airspaces of transgenic mice did not confer resistance to infection. The goal of this study was to assess the antimicrobial properties of native SP-B and synthetic peptides derived from the native peptide. Native SP-B aggregated and killed clinical isolates of Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus, and group B streptococcus by increasing membrane permeability; however, SP-B also lysed RBC, indicating that the membranolytic activity was not selective for bacteria. Both the antimicrobial and hemolytic activities of native SP-B were inhibited by surfactant phospholipids, suggesting that endogenous SP-B may not play a significant role in alveolar host defense. Synthetic peptides derived from native SP-B were effective at killing both Gram-positive and Gram-negative bacteria at low peptide concentrations (0.15–5.0 μM). The SP-B derivatives selectively lysed bacterial membranes and were more resistant to inhibition by phospholipids; furthermore, helix 1 (residues 7–22) retained significant antimicrobial activity in the presence of native surfactant. These results suggest that the role of endogenous SP-B in host defense may be limited; however, synthetic peptides derived from SP-B may be useful in the treatment of bacterial pneumonias.
Internalizing Antibodies to the C-Type Lectins, L-SIGN and DC-SIGN, Inhibit Viral Glycoprotein Binding and Deliver Antigen to Human Dendritic Cells for the Induction of T Cell Responses
Authors: Dakappagari Naveen , Maruyama Toshiaki , Renshaw Mark , Tacken Paul , Figdor Carl , Torensma Ruurd , Wild Martha A. , Wu Dayang , Bowdish Katherine , Kretz-Rommel Anke
Published: April 2014

The C-type lectin L-SIGN is expressed on liver and lymph node endothelial cells, where it serves as a receptor for a variety of carbohydrate ligands, including ICAM-3, Ebola, and HIV. To consider targeting liver/lymph node-specific ICAM-3-grabbing nonintegrin (L-SIGN) for therapeutic purposes in autoimmunity and infectious disease, we isolated and characterized Fabs that bind strongly to L-SIGN, but to a lesser degree or not at all to dendritic cell-specific ICAM-grabbing nonintegrin (DC-SIGN). Six Fabs with distinct relative affinities and epitope specificities were characterized. The Fabs and those selected for conversion to IgG were tested for their ability to block ligand (HIV gp120, Ebola gp, and ICAM-3) binding. Receptor internalization upon Fab binding was evaluated on primary human liver sinusoidal endothelial cells by flow cytometry and confirmed by confocal microscopy. Although all six Fabs internalized, three Fabs that showed the most complete blocking of HIVgp120 and ICAM-3 binding to L-SIGN also internalized most efficiently. Differences among the Fab panel in the ability to efficiently block Ebola gp compared with HIVgp120 suggested distinct binding sites. As a first step to consider the potential of these Abs for Ab-mediated Ag delivery, we evaluated specific peptide delivery to human dendritic cells. A durable human T cell response was induced when a tetanus toxide epitope embedded into a L-SIGN/DC-SIGN-cross-reactive Ab was targeted to dendritic cells. We believe that the isolated Abs may be useful for selective delivery of Ags to DC-SIGN- or L-SIGN-bearing APCs for the modulation of immune responses and for blocking viral infections.
The Membrane-Bound Chemokine CXCL16 Expressed on Follicle-Associated Epithelium and M Cells Mediates Lympho-Epithelial Interaction in GALT
Authors: Hase Koji , Murakami Takaya , Takatsu Hiroyuki , Shimaoka Takeshi , Iimura Mitsutoshi , Hamura Kimiyo , Kawano Kazuya , Ohshima Sayaka , Chihara Risa , Itoh Kikuji , Yonehara Shin , Ohno Hiroshi
Published: April 2014

The recently identified CXCL16 has dual functions as a transmembrane adhesion molecule and a soluble chemokine. In this study we found that CXCL16 mRNA and protein were expressed constitutively on the follicle-associated epithelium covering Peyer’s patches (PPs), isolated lymphoid follicles, and cecal patches, but minimally on the villous epithelium in the murine gastrointestinal tract. The CXCL16 receptor CXCR6/Bonzo was constitutively expressed on subpopulations of CD4+ and CD8+ T cells isolated from PPs. The expression of CXCR6/Bonzo on the PP T cells was up-regulated after stimulation with anti-CD3 and anti-CD28 mAbs. The activated PP T cells showed chemotactic migration in response to the soluble N-terminal chemokine domain of CXCL16. Furthermore, the activated PP T cells selectively adhered to cells expressing murine CXCL16. To determine the physiological role of CXCL16 in GALT, we first carefully analyzed T cell distribution in PPs. T cells localized not only in the interfollicular region but also at a lesser frequency in the subepithelial dome (SED) and in the germinal center of lymphoid follicles. Consistently, the majority of the adoptive transferred activated T cells migrated into the SED and the interfollicular region. However, the neutralization of CXCL16 specifically reduced the migration of the adoptive, transferred, activated T cells into the SED of PPs. These data suggest that CXCL16 expressed on the follicle-associated epithelium plays an important role in the recruitment and retention of activated T cells in the SED and should, at least partially, be responsible for lymphocyte compartmentalization in GALT.
Interaction of Mouse Dendritic Cells and Malaria-Infected Erythrocytes: Uptake, Maturation, and Antigen Presentation
Authors: Ing Rebecca , Segura Mariela , Thawani Neeta , Tam Mifong , Stevenson Mary M.
Published: April 2014

Consistent with their seminal role in detecting infection, both mouse bone marrow-derived and splenic CD11c+ dendritic cells (DCs) exhibited higher levels of uptake of Plasmodium chabaudi-parasitized RBCs (pRBCs) than of noninfected RBCs (nRBCs) as determined by our newly developed flow cytometric technique using the dye CFSE to label RBCs before coculture with DCs. To confirm that expression of CFSE by CD11c+ cells following coculture with CFSE-labeled pRBCs represents internalization of pRBC by DCs, we showed colocalization of CFSE-labeled pRBCs and PE-labeled CD11c+ DCs by confocal fluorescence microscopy. Treatment of DCs with cytochalasin D significantly inhibited the uptake of pRBCs, demonstrating that uptake is an actin-dependent phagocytic process. The uptake of pRBCs by splenic CD11c+ DCs was significantly enhanced after infection in vivo and was associated with the induction of DC maturation, IL-12 production, and stimulation of CD4+ T cell proliferation and IFN-γ production. These results suggest that DCs selectively phagocytose pRBCs and present pRBC-derived Ags to CD4+ T cells, thereby promoting development of protective Th1-dependent immune responses to blood-stage malaria infection.
Effects of Immunization with CCR5-Based Cycloimmunogen on Simian/HIVSF162P3 Challenge
Authors: Misumi Shogo , Nakayama Daisuke , Kusaba Masashi , Iiboshi Takaaki , Mukai Ryouzaburo , Tachibana Kuniomi , Nakasone Tadashi , Umeda Mamoru , Shibata Hideaki , Endo Masafumi , Takamune Nobutoki , Shoji Shozo
Published: April 2014

A synthetic cycloimmunogen targeting the HIV-1 coreceptor CCR5 was evaluated for its capacity to induce CCR5-specific Abs with anti-HIV-1 activity in cynomolgus macaques. The cyclic closed-chain dodecapeptide (cDDR5) mimicking the conformation-specific domain of human CCR5 was chemically prepared, in which the Gly-Glu dipeptide links the amino and carboxy termini of the decapeptidyl linear chain (Arg168 to Thr177) derived from the undecapeptidyl arch (Arg168 to Cys178) of extracellular loop-2 in CCR5. The immunization of cynomolgus macaques with the cDDR5-conjugated multiple-Ag peptide (cDDR5-MAP) induced anti-cDDR5 serum production for ∼15 wk after the third immunization. The antisera raised against cDDR5-MAP reacted with both human and macaque CCR5s, and potently suppressed infection by the R5 HIV-1 laboratory isolate (HIVJRFL), R5 HIV-1 primary isolates (clade A:HIV93RW004 and clade C:HIVMJ4), and a pathogenic simian/HIV (SHIVSF162P3) bulk isolate in vitro. To examine the prophylactic efficacy of anti-CCR5 serum Ab for acute HIV-1 infection, cynomolgus macaques were challenged with SHIVSF162P3. The cDDR5-MAP immunization attenuated the acute phase of SHIVSF162P3 replication. The geometric mean plasma viral load in the vaccinated macaques was 217.10 times lower than that of the control macaques at 1 wk postchallenge. Taken together, these results suggest that cDDR5-MAP immunization is an effective prophylactic vaccine strategy that suppresses and delays viral propagation during the initial HIV-1 transmission for the containment of HIV-1 replication subsequent to infection.
CD154 Is Essential for Protective Immunity in Experimental Salmonella Infection: Evidence for a Dual Role in Innate and Adaptive Immune Responses
Authors: al-Ramadi Basel K. , Fernandez-Cabezudo Maria J. , Ullah Azim , El-Hasasna Hussain , Flavell Richard A.
Published: April 2014

CD40-CD154 interactions are of central importance in the induction of humoral and cellular immune responses. In the present study, CD154-deficient (CD154−/−) mice were used to assess the role of CD40-CD154 interactions in regulating the immune response to a systemic Salmonella infection. Compared with C57BL/6 (CD154+/+) controls, CD154−/− mice were hypersusceptible to infection by an attenuated strain of Salmonella enterica serovar Typhimurium (S. typhimurium), as evidenced by decreased survival rate and mean time to death, which correlated with increased bacterial burden and persistence in target organs. CD154−/− mice exhibited a defect both in the production of IL-12, IFN-γ, and NO during the acute phase of the disease and in the generation of Salmonella-specific Ab responses and Ig isotype switching. Furthermore, when CD154−/− animals were administered a sublethal dose of attenuated S. typhimurium and subsequently challenged with a virulent homologous strain, all mice succumbed to an overwhelming infection. Similar treatment of CD154+/+ mice consistently resulted in ≥90% protection. The lack of protective immunity in CD154−/− mice correlated with a decreased T cell recall response to Salmonella Ags. Significant protection against virulent challenge was conferred to presensitized CD154−/− mice by transfer of serum or T cells from immunized CD154+/+ mice. For best protection, however, a combination of immune serum and T cells was required. We conclude that intercellular communications via the CD40-CD154 pathway play a critical role in the induction of type 1 cytokine responses, memory T cell generation, Ab formation, and protection against primary as well as secondary Salmonella infections.
IL-15 Regulates CD8+ T Cell Contraction during Primary Infection
Authors: Yajima Toshiki , Yoshihara Kazufumi , Nakazato Kenji , Kumabe Shino , Koyasu Shigeo , Sad Subash , Shen Hao , Kuwano Hiroyuki , Yoshikai Yasunobu
Published: April 2014

During the course of acute infection with an intracellular pathogen, Ag-specific T cells proliferate in the expansion phase, and then most of the T cells die by apoptosis in the following contraction phase, but the few that survive become memory cells and persist for a long period of time. Although IL-15 is known to play an important role in long-term maintenance of memory CD8+ T cells, the potential roles of IL-15 in CD8+ T cell contraction are not known. Using an adoptive transfer system of OT-I cells expressing OVA257–264/Kb-specific TCR into control, IL-15 knockout (KO) and IL-15 transgenic (Tg) mice followed by challenge with recombinant Listeria monocytogenes expressing OVA, we found that the survival of CD44+CD62L−CD127− effector OT-I cells during the contraction phase is critically dependent on IL-15. In correlation with the expression level of Bcl-2 in OT-I cells, the number of OT-I cells was markedly reduced in IL-15 KO mice but remained at a high level in IL-15 Tg mice during the contraction phase, compared with control mice. In vivo administration of rIL-15 during the contraction phase in IL-15 KO mice inhibited the contraction of effector OT-I cells accompanied by up-regulation of Bcl-2 expression. Furthermore, enforced expression of Bcl-2 protected the majority of effector OT-I cells from death in IL-15 KO mice after infection. These results suggest that IL-15 plays a critical role in protecting effector CD8+ T cells from apoptosis during the contraction phase following a microbial infection via inducing antiapoptotic molecules.
Neuronal Nitric Oxide Synthase Is Necessary for Elimination of Giardia lamblia Infections in Mice
Authors: Li Erqiu , Zhou Ping , Singer Steven M.
Published: April 2014

NO produced by inducible NO synthase (NOS2) is important for the control of numerous infections. In vitro, NO inhibits replication and differentiation of the intestinal protozoan parasite Giardia lamblia. However, the role of NO against this parasite has not been tested in vivo. IL-6-deficient mice fail to control Giardia infections, and these mice have reduced levels of NOS2 mRNA in the small intestine after infection compared with wild-type mice. However, NOS2 gene-targeted mice and wild-type mice treated with the NOS2 inhibitor N-iminoethyl-l-lysine eliminated parasites as well as control mice. In contrast, neuronal NOS (NOS1)-deficient mice and wild-type mice treated with the nonspecific NOS inhibitor NG-nitro-l-arginine methyl ester and the NOS1-specific inhibitor 7-nitroindazole all had delayed parasite clearance. Finally, Giardia infection increased gastrointestinal motility in wild-type mice, but not in SCID mice. Furthermore, treatment of wild-type mice with NG-nitro-l-arginine methyl ester or loperamide prevented both the increased motility and the elimination of parasites. Together, these data show that NOS1, but not NOS2, is necessary for clearance of Giardia infection. They also suggest that increased gastrointestinal motility contributes to elimination of the parasite and may also contribute to parasite-induced diarrhea. Importantly, this is the first example of NOS1 being involved in the elimination of an infection.
Identification and Characterization of U83A Viral Chemokine, a Broad and Potent β-Chemokine Agonist for Human CCRs with Unique Selectivity and Inhibition by Spliced Isoform
Authors: Dewin David R. , Catusse Julie , Gompels Ursula A.
Published: April 2014

Leukotropic human herpesvirus 6 (HHV-6) establishes a persistent infection associated with inflammatory diseases and encodes chemokines that could chemoattract leukocytes for infection or inflammation. HHV-6 variant A encodes a distant chemokine homolog, U83A, and a polymorphism promoting a secreted form was identified. U83A and three N-terminal modifications were expressed and purified, and activities were compared with a spliced truncated isoform, U83A-Npep. U83A efficiently and potently induced calcium mobilization in cells expressing single human CCR1, CCR4, CCR6, or CCR8, with EC50 values <10 nM. U83A also induced chemotaxis of Th2-like leukemic cells expressing CCR4 and CCR8. High-affinity binding, 0.4 nM, was demonstrated to CCR1 and CCR5 on monocytic/macrophage cells, and pretreatment with U83A or modified forms could block responses for endogenous ligands. U83A-Npep acted only as antagonist, efficiently blocking binding of CCL3 to CCR1 or CCR5 on differentiated monocytic/macrophage leukemic cells. Furthermore, CCL3 induction of calcium signaling via CCR1 and CCL1 induced chemotaxis via CCR8 in primary human leukocytes was inhibited. Thus, this blocking by the early expressed U83A-Npep could mediate immune evasion before finishing the replicative cycle. However, late in infection, when full-length U83A is made, chemoattraction of CCR1-, CCR4-, CCR5-, CCR6-, and CCR8-bearing monocytic/macrophage, dendritic, and T lymphocyte cells can facilitate dissemination via lytic and latent infection of these cells. This has further implications for neuroinflammatory diseases such as multiple sclerosis, where both cells bearing CCR1/CCR5 plus their ligands, as well as HHV-6A, have been linked. Applications also discussed include novel vaccines/immunotherapeutics for cancer and HIV as well as anti-inflammatories.
Chemotactic Responses of IL-4-, IL-10-, and IFN-γ-Producing CD4+ T Cells Depend on Tissue Origin and Microbial Stimulus
Authors: Debes Gudrun F. , Dahl Martin E. , Mahiny Azita J. , Bonhagen Kerstin , Campbell Daniel J. , Siegmund Kerstin , Erb Klaus J. , Lewis David B. , Kamradt Thomas , Hamann Alf
Published: April 2014

Th1- and Th2-polarized immune responses are crucial in the defense against pathogens but can also promote autoimmunity and allergy. The chemokine receptors CXCR3 and CCR4 have been implicated in differential trafficking of IFN-γ- and IL-4-producing T cells, respectively, but also in tissue and inflammation-specific homing independent of cytokine responses. Here, we tested whether CD4+ T cells isolated from murine tissues under homeostatic or inflammatory conditions exhibit restricted patterns of chemotactic responses that correlate with their production of IFN-γ, IL-4, or IL-10. In uninfected mice, IL-4-producing T cells preferentially migrated to the CCR4 ligand, CCL17, whereas IFN-γ-expressing T cells as well as populations of IL-4+ or IL-10+ T cells migrated to the CXCR3 ligand, CXCL9. All cytokine-producing T cell subsets strongly migrated to the CXCR4 ligand, CXCL12. We assessed chemotaxis of T cells isolated from mice infected with influenza A virus or the nematode Nippostrongylus brasiliensis, which induce a strong Th1 or Th2 response in the lung, respectively. Unexpectedly, the chemotactic responses of IL-4+ T cells and T cells expressing the immunosuppressive cytokine IL-10 were influenced not only by the strongly Th1- or Th2-polarized environments but also by their anatomical localization, i.e., lung or spleen. In contrast, IFN-γ+ T cells exhibited robust chemotaxis toward CXCL9 and had the most consistent migration pattern in both infection models. The results support a model in which the trafficking responses of many effector and regulatory T cells are regulated as a function of the infectious and tissue environments.
Lipoteichoic Acid-Induced Nitric Oxide Production Depends on the Activation of Platelet-Activating Factor Receptor and Jak2
Authors: Han Seung Hyun , Kim Je Hak , Seo Ho Seong , Martin Michael H. , Chung Gook-Hyun , Michalek Suzanne M. , Nahm Moon H.
Published: April 2014

NO production by macrophages in response to lipoteichoic acid (LTA) and a synthetic lipopeptide (Pam3CSK4) was investigated. LTA and Pam3CSK4 induced the production of both TNF-α and NO. Inhibitors of platelet-activating factor receptor (PAFR) blocked LTA- or Pam3CSK4-induced production of NO but not TNF-α. Jak2 tyrosine kinase inhibition blocked LTA-induced production of NO but not TNF-α. PAFR inhibition blocked phosphorylation of Jak2 and STAT1, a key factor for expressing inducible NO synthase. In addition, LTA did not induce IFN-β expression, and p38 mitogen-activated protein serine kinase was necessary for LTA-induced NO production but not for TNF-α production. These findings suggest that Gram-positive bacteria induce NO production using a PAFR signaling pathway to activate STAT1 via Jak2. This PAFR/Jak2/STAT1 signaling pathway resembles the IFN-β, type I IFNR/Jak/STAT1 pathway described for LPS. Consequently, Gram-positive and Gram-negative bacteria appear to have different but analogous mechanisms for NO production.
NF-κB and Activator Protein 1 Response Elements and the Role of Histone Modifications in IL-1β-Induced TGF-β1 Gene Transcription
Authors: Lee Kang-Yun , Ito Kazuhiro , Hayashi Ryuji , Jazrawi Elen P. I. , Barnes Peter J. , Adcock Ian M.
Published: April 2014

Abnormal expression of TGF-β1 is believed to play an important role in the pathogenesis of a number of chronic inflammatory and immune lung diseases, including asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. Gene activation in eukaryotes requires coordinated use of specific cell signals, chromatin modifications, and chromatin remodeling. We studied the roles of the ubiquitous inflammatory transcription factors, NF-κB and AP-1, in activation of the TGF-β1 gene and histone acetylation at the TGF-β1 promoter. IL-1β-induced TGF-β1 protein secretion and mRNA expression were prevented by actinomycin D and were attenuated by the inhibitor of κB kinase 2 inhibitor AS602868 and the JNK inhibitor SP600125, suggesting a degree of transcriptional regulation mediated by the NF-κB and AP-1 pathways. We demonstrated that IL-1β activated the p65 subunit of NF-κB and the c-Jun subunit of AP-1. Using chromatin immunoprecipitation assays, we observed a sequential recruitment of p65 and c-Jun, accompanying ordered elevation of the levels of histone H4 and H3 acetylation and recruitment of RNA polymerase II at distinct regions in the native TGF-β1 promoter. The specific NF-κB and AP-1 binding sites in the TGF-β1 promoter were confirmed by an ELISA-based binding assay, and evidence for histone hyperacetylation in TGF-β1 induction was supported by the observation that the histone deacetylase inhibitor trichostatin A enhanced basal and IL-1β-induced TGF-β1 mRNA expression. Our results suggest that IL-1β-stimulated transcription of TGF-β1 is temporally regulated by NF-κB and AP-1 and involves histone hyperacetylation at distinct promoter sites.
Innate Stimuli Accentuate End-Organ Damage by Nephrotoxic Antibodies via Fc Receptor and TLR Stimulation and IL-1/TNF-α Production
Authors: Fu Yuyang , Xie Chun , Chen Jianlin , Zhu Jiankun , Zhou Hui , Thomas James , Zhou Xin J. , Mohan Chandra
Published: April 2014

Innate stimuli are well recognized as adjuvants of the systemic immune response. However, their role in driving end-organ disease is less well understood. Whereas the passive transfer of glomerular-targeting Abs alone elicited minimal renal disease, the concomitant delivery of innate stimuli triggered severe nephritis, characterized by proliferative glomerulonephritis with crescent formation, and tubulointerstitial disease. Specifically, stimulating TLR2, TLR3, TLR4, and TLR5 by using peptidoglycan, poly(I:C), LPS, and flagellin, respectively, all could facilitate anti-glomerular Ab-elicited nephritis. In this model, innate and immune triggers synergistically activated several cytokines and chemokines, including IL-1, IL-6, TNF-α, and MCP-1, some of which were demonstrated to be absolutely essential for the development of renal disease. Genetic studies revealed that, whereas the innate trigger is dependent on TLR/IL-1R-associated kinase-mediated signaling, the immune component was contingent on FcR-mediated signals. Importantly, infiltrating leukocytes as well as intrinsic glomerular cells may both serve to integrate these diverse signals. Extrapolating to spontaneous immune-mediated nephritis, although the adaptive immune system may be important in generating end-organ targeting Abs, the extent of damage inflicted by these Abs may be heavily dependent on cues from the innate immune system.
Azathioprine Suppresses Ezrin-Radixin-Moesin-Dependent T Cell-APC Conjugation through Inhibition of Vav Guanosine Exchange Activity on Rac Proteins
Authors: Poppe Daniela , Tiede Imke , Fritz Gerhard , Becker Christoph , Bartsch Brigitte , Wirtz Stefan , Strand Dennis , Tanaka Shinya , Galle Peter R. , Bustelo Xosé R. , Neurath Markus F.
Published: April 2014

We have shown recently that the azathioprine metabolite 6-Thio-GTP causes immunosuppression by blockade of GTPase activation in T lymphocytes. In the present study, we describe a new molecular mechanism by which 6-Thio-GTP blocks GTPase activation. Although 6-Thio-GTP could bind to various small GTPases, it specifically blocked activation of Rac1 and Rac2 but not of closely related Rho family members such as Cdc42 and RhoA in primary T cells upon stimulation with αCD28 or fibronectin. Binding of 6-Thio-GTP to Rac1 did not suppress Rac effector coupling directly but blocked Vav1 exchange activity upon 6-Thio-GTP hydrolysis, suggesting that 6-Thio-GTP loading leads to accumulation of 6-Thio-GDP-loaded, inactive Rac proteins over time by inhibiting Vav activity. In the absence of apoptosis, blockade of Vav-mediated Rac1 activation led to a blockade of ezrin-radixin-moesin dephosphorylation in primary T cells and suppression of T cell-APC conjugation. Azathioprine-generated 6-Thio-GTP thus prevents the development of an effective immune response via blockade of Vav activity on Rac proteins. These findings provide novel insights into the immunosuppressive effects of azathioprine and suggest that antagonists of the Vav-Rac signaling pathway may be useful for suppression of T cell-dependent pathogenic immune responses.
Targeted Expression of the Human Thyrotropin Receptor A-Subunit to the Mouse Thyroid: Insight into Overcoming the Lack of Response to A-Subunit Adenovirus Immunization
Authors: Pichurin Pavel N. , Chen Chun-Rong , Chazenbalk Gregorio D. , Aliesky Holly , Pham Nancy , Rapoport Basil , McLachlan Sandra M.
Published: April 2014

The thyrotropin receptor (TSHR), the major autoantigen in Graves’ disease, is posttranslationally modified by intramolecular cleavage to form disulfide-linked A- and B-subunits. Because Graves’ hyperthyroidism is preferentially induced in BALB/c mice using adenovirus encoding the free A-subunit rather than full-length human TSHR, the shed A-subunit appears to drive the disease-associated autoimmune response. To further investigate this phenomenon, we generated transgenic mice with the human A-subunit targeted to the thyroid. Founder transgenic mice had normal thyroid function and were backcrossed to BALB/c. The A-subunit mRNA expression was confirmed in thyroid tissue. Unlike wild-type littermates, transgenic mice immunized with low-dose A-subunit adenovirus failed to develop TSHR Abs, hyperthyroidism, or splenocyte responses to TSHR Ag. Conventional immunization with A-subunit protein and adjuvants induced TSHR Abs lacking the characteristics of human autoantibodies. Unresponsiveness was partially overcome using high-dose, full-length human TSHR adenovirus. Although of low titer, these induced Abs recognized the N terminus of the A-subunit, and splenocytes responded to A-subunit peptides. Therefore, “non-self” regions in the B-subunit did not contribute to inducing responses. Indeed, transgenic mice immunized with high-dose A-subunit adenovirus developed TSHR Abs with thyrotropin-binding inhibitory activity, although at lower titers than wild-type littermates, suggesting down-regulation in the transgenic mice. In conclusion, in mice expressing a human A-subunit transgene in the thyroid, non-self human B-subunit epitopes are not necessary to induce responses to the A-subunit. Our findings raise the possibility that autoimmunity to the TSHR in humans may not involve epitopes on a cross-reacting protein, but rather, strong adjuvant signals provided in bystander immune responses.