bluuebunny/crossref_metadata_2025 huggingface dataset:
Recruitment and Activation of Macrophages by Pathogenic CD4 T Cells in Type 1 Diabetes: Evidence for Involvement of CCR8 and CCL1
Authors: Cantor Joseph , Haskins Kathryn
Published: April 2014

Adoptive transfer of diabetogenic CD4 Th1 T cell clones into young NOD or NOD.scid recipients rapidly induces onset of diabetes and also provides a system for analysis of the pancreatic infiltrate. Although many reports have suggested a role for macrophages in the inflammatory response, there has been little direct characterization of macrophage activity in the pancreas. We showed previously that after migration to the pancreas, diabetogenic CD4 T cell clones produce a variety of inflammatory cytokines and chemokines, resulting in the recruitment of macrophages. In this study, we investigated mechanisms by which macrophages are recruited and activated by T cells. Analysis of infiltrating cells after adoptive transfer by the diabetogenic T cell clone BDC-2.5 indicates that large numbers of cells staining for both F4/80 and CD11b are recruited into the pancreas where they are activated to make IL-1β, TNF-α, and NO, and express the chemokine receptors CCR5, CXCR3, and CCR8. Diabetogenic CD4 T cell clones produce several inflammatory chemokines in vitro, but after adoptive transfer we found that the only chemokine that could be detected ex vivo was CCL1. These results provide the first evidence that CCR8/CCL1 interaction may play a role in type 1 diabetes through macrophage recruitment and activation.
Syndecan-4 Mediates the Coinhibitory Function of DC-HIL on T Cell Activation
Authors: Chung Jin-Sung , Dougherty Irene , Cruz Ponciano D. , Ariizumi Kiyoshi
Published: April 2014

Receptor-ligand interactions between APCs and T cells determine whether stimulation of the latter leads to activation or inhibition. Previously, we showed that dendritic cell-associated heparin sulfate proteoglycan-dependent integrin ligand (DC-HIL) on APC can inhibit T cell activation by binding an unknown ligand expressed on activated T cells. Because DC-HIL binds heparin/heparan sulfate and heparin blocks the inhibitory function of DC-HIL, we hypothesized that a heparin/heparan sulfate proteoglycan on activated T cells is the relevant ligand. Screening assays revealed that syndecan-4 (SD-4) is the sole heparan sulfate proteoglycan immunoprecipitated by DC-HIL from extracts of activated T cells and that blocking SD-4 abrogates binding of DC-HIL to activated T cells. Moreover, cell-bound SD-4 ligated by DC-HIL or cross-linked by anti-SD-4 Ab attenuated anti-CD3 responses, whereas knocked-down SD-4 expression led to enhanced T cell response to APC. Blockade of endogenous SD-4 using specific Ab or soluble SD-4 receptor led to augmented T cell reactions to syngeneic and allogeneic stimulation in vitro and exacerbated contact hypersensitivity responses in vivo. We conclude that SD-4 is the T cell ligand through which DC-HIL mediates its negative coregulatory function.
Decay Accelerating Factor Can Control T Cell Differentiation into IFN-γ-Producing Effector Cells via Regulating Local C5a-Induced IL-12 Production
Authors: Lalli Peter N. , Strainic Michael G. , Lin Feng , Medof M. Edward , Heeger Peter S.
Published: April 2014

A newly recognized link between the complement system and adaptive immunity is that decay accelerating factor (DAF), a cell surface C3/C5 convertase regulator, exerts control over T cell responses. Extending these results, we show that cultures of Marilyn TCR-transgenic T cells stimulated with DAF-deficient (Daf1−/−) APCs produce significantly more IL-12, C5a, and IFN-γ compared with cultures containing wild-type APCs. DAF-regulated IL-12 production and subsequent T cell differentiation into IFN-γ-producing effectors was prevented by the deficiency of either C3 or C5a receptor (C5aR) in the APC, demonstrating a link between DAF, local complement activation, IL-12, and T cell-produced IFN-γ. Bone marrow chimera experiments verified that bone marrow cell-expressed C5aR is required for optimal differentiation into IFN-γ-producing effector T cells. Overall, our results indicate that APC-expressed DAF regulates local production/activation of C5a following cognate T cell/APC interactions. Through binding to its receptor on APCs the C5a up-regulates IL-12 production, this in turn, contributes to directing T cell differentiation toward an IFN-γ-producing phenotype. The findings have implications for design of therapies aimed at altering pathologic T cell immunity.
Targeting the Wilms Tumor Antigen 1 by TCR Gene Transfer: TCR Variants Improve Tetramer Binding but Not the Function of Gene Modified Human T Cells
Authors: Thomas Sharyn , Xue Shao-An , Cesco-Gaspere Michela , San José Esther , Hart Daniel P. , Wong Vivian , Debets Reno , Alarcon Balbino , Morris Emma , Stauss Hans J.
Published: April 2014

We have previously described the functional activity of a human TCR specific for an HLA-A2-presented peptide derived from the Wilms tumor Ag 1 (WT1). Recent studies showed that the expression and function of human TCR was improved by the introduction of an additional disulfide bond between the α- and β-chains or by the exchange of the human constant region for murine sequences. In this study, we analyzed the functional activity of WT1-TCR variants expressed in Jurkat cells and in primary T cells. The introduction of cysteine residues or murine constant sequences into the WT1-TCR did not result in a global reduction of mispairing with wild-type TCR chains. Instead, the level of mispairing was affected by the variable region sequences of the wild-type TCR chains. The analysis of freshly transduced peripheral blood T cells showed that the transfer of modified TCR constructs generated a higher frequency of Ag-responsive T cells than the transfer of the wild-type TCR. After several rounds of peptide stimulation this difference was no longer observed, as all transduced T cell populations accumulated ∼90% of Ag-responsive T cells. Although the Ag-responsive T cells expressing the modified TCR bound the HLA-A2/WT1 tetramer more efficiently than T cells expressing the wild-type TCR, this did not improve the avidity of transduced T cells nor did it result in a measurable enhancement in IFN-γ production and cytotoxic activity. This indicated that the enhanced tetramer binding of modified WT1-TCR variants was not associated with improved WT1-specific T cell function.
Cognate Memory CD4+ T Cells Generated with Dendritic Cell Priming Influence the Expansion, Trafficking, and Differentiation of Secondary CD8+ T Cells and Enhance Tumor Control
Authors: Hwang Melissa L. , Lukens John R. , Bullock Timothy N. J.
Published: April 2014

CD4+ T cells are known to provide support for the activation and expansion of primary CD8+ T cells, their subsequent differentiation, and ultimately their survival as memory cells. However, the importance of cognate memory CD4+ T cells in the expansion of memory CD8+ T cells after re-exposure to Ag has been not been examined in detail. Using bone marrow-derived dendritic cells pulsed with cognate or noncognate MHC class I- and class II-restricted peptides, we examined whether the presence of memory CD4+ T cells with the same Ag specificity as memory CD8+ T cells influenced the quantity and quality of the secondary CD8+ T cell response. After recombinant vaccinia virus-mediated challenge, we demonstrate that, although cognate memory CD4+ T cells are not required for activation of secondary CD8+ T cells, their presence enhances the expansion of cognate memory CD8+ T cells. Cognate CD4+ T cell help results in an approximate 2-fold increase in the frequency of secondary CD8+ T cells in secondary lymphoid tissues, and can be accounted for by enhanced proliferation in the secondary CD8+ T cell population. In addition, cognate memory CD4+ T cells further selectively enhance secondary CD8+ T cell infiltration of tumor-associated peripheral tissue, and this is accompanied by increased differentiation into effector phenotype within the secondary CD8+ T cell population. The consequence of these improvements to the magnitude and phenotype of the secondary CD8+ T cell response is substantial increase in control of tumor outgrowth.
Sphingosine-1 Phosphate Signaling Regulates Positioning of Dendritic Cells within the Spleen
Authors: Czeloth Niklas , Schippers Angela , Wagner Norbert , Müller Werner , Küster Birgit , Bernhardt Günter , Förster Reinhold
Published: April 2014

A successful execution and balance of adaptive immune responses requires a controlled positioning and navigation of dendritic cells (DC) into and inside secondary lymphoid organs. Whereas mechanisms were identified governing the migration of DC from peripheral nonlymphoid organs into their draining lymph nodes, little is known about the molecular cues controlling the proper positioning of spleen or lymph node resident DC. In this study, we show that the sphingosine-1 phosphate (S1P) receptor 1 influences the positioning of immature DC inside the murine spleen. Following treatment with FTY720 or SEW2871, drugs known to interfere with S1P1-mediated signaling, the 33D1+ DC subpopulation homogeneously redistributes from the bridging channels to the marginal zone. In contrast, the CD205+ DC subset remains associated with the T cell zone. Upon in vivo LPS treatment, the maturing DC assemble in the T cell zone. The LPS-driven redistribution occurs in the absence of CCR7 and cannot be prevented by FTY720, indicating that guiding mechanisms differ between immature and mature DC. Along with the observed DC subtype-specific S1P receptor expression pattern as well as the profound up-regulation of S1P1 and S1P3 accompanying DC maturation, these results suggest a decisive contribution of S1P signaling to intrasplenic DC motility and migration.
Essential Role of IL-21 in B Cell Activation, Expansion, and Plasma Cell Generation during CD4+ T Cell-B Cell Collaboration
Authors: Kuchen Stefan , Robbins Rachel , Sims Gary P. , Sheng Chen , Phillips Terence M. , Lipsky Peter E. , Ettinger Rachel
Published: April 2014

During T cell-B cell collaboration, plasma cell (PC) differentiation and Ig production are known to require T cell-derived soluble factors. However, the exact nature of the cytokines produced by activated T cells that costimulate PC differentiation is not clear. Previously, we reported that costimulation of purified human B cells with IL-21 and anti-CD40 resulted in efficient PC differentiation. In this study, we addressed whether de novo production of IL-21 was involved in direct T cell-induced B cell activation, proliferation, and PC differentiation. We found that activated human peripheral blood CD4+ T cells expressed mRNA for a number of cytokines, including IL-21, which was confirmed at the protein level. Using a panel of reagents that specifically neutralize cytokine activity, we addressed which cytokines are essential for B cell activation and PC differentiation induced by anti-CD3-activated T cells. Strikingly, neutralization of IL-21 with an IL-21R fusion protein (IL-21R-Fc) significantly inhibited T cell-induced B cell activation, proliferation, PC differentiation, and Ig production. Inhibition of PC differentiation was observed even when the addition of IL-21R-Fc was delayed until after initial B cell activation and expansion had occurred. Importantly, IL-21 was found to be involved in PC differentiation from both naive and memory B cells. Finally, IL-21R-Fc did not inhibit anti-CD3-induced CD4+ T cell activation, but rather directly blocked T cell-induced B cell activation and PC differentiation. These data are the first to document that B cell activation, expansion, and PC differentiation induced by direct interaction of B cells with activated T cells requires IL-21.
Posttranslational Regulation ofI-Edby Affinity for CLIP
Authors: Rinderknecht Cornelia H. , Belmares Michael P. , Catanzarite Tatiana L. W. , Bankovich Alexander J. , Holmes Tyson H. , Garcia K. Christopher , Nanda Navreet K. , Busch Robert , Kovats Susan , Mellins Elizabeth D.
Published: April 2014

Several MHC class II alleles linked with autoimmune diseases form unusually low stability complexes with CLIP, leading us to hypothesize that this is an important feature contributing to autoimmune pathogenesis. To investigate cellular consequences of altering class II/CLIP affinity, we evaluated invariant chain (Ii) mutants with varying CLIP affinity for a mouse class II allele, I-Ed, which has low affinity for wild-type CLIP and is associated with a mouse model of spontaneous, autoimmune joint inflammation. Increasing CLIP affinity for I-Ed resulted in increased cell surface and total cellular abundance and half-life of I-Ed. This reveals a post-endoplasmic reticulum chaperoning capacity of Ii via its CLIP peptides. Quantitative effects on I-Ed were less pronounced in DM-expressing cells, suggesting complementary chaperoning effects mediated by Ii and DM, and implying that the impact of allelic variation in CLIP affinity on immune responses will be highest in cells with limited DM activity. Differences in the ability of cell lines expressing wild-type or high-CLIP-affinity mutant Ii to present Ag to T cells suggest a model in which increased CLIP affinity for class II serves to restrict peptide loading to DM-containing compartments, ensuring proper editing of antigenic peptides.
Cyclosporin A Suspends Transplantation Reactions in the Marine SpongeMicrociona prolifera
Authors: Sabella Clarissa , Faszewski Ellen , Himic Lisa , Colpitts Katherine M. , Kaltenbach Jane , Burger Max M. , Fernàndez-Busquets Xavier
Published: April 2014

Sponges are the simplest extant animals but nevertheless possess self-nonself recognition that rivals the specificity of the vertebrate MHC. We have used dissociated cell assays and grafting techniques to study tissue acceptance and rejection in the marine sponge Microciona prolifera. Our data show that allogeneic, but not isogeneic, cell contacts trigger cell death and an increased expression of cell adhesion and apoptosis markers in cells that accumulate in graft interfaces. Experiments investigating the possible existence of immune memory in sponges indicate that faster second set reactions are nonspecific. Among the different cellular types, gray cells have been proposed to be the sponge immunocytes. Fluorescence confocal microscopy results from intact live grafts show the migration of autofluorescent gray cells toward graft contact zones and the inhibition of gray cell movements in the presence of nontoxic concentrations of cyclosporin A. These results suggest that cell motility is an important factor involved in sponge self/nonself recognition. Communication between gray cells in grafted tissues does not require cell contact and is carried by an extracellular diffusible marker. The finding that a commonly used immunosuppressor in human transplantation such as cyclosporin A blocks tissue rejection in marine sponges indicates that the cellular mechanisms for regulating this process in vertebrates might have appeared at the very start of metazoan evolution.
TGF-β1 Induces Progressive Pleural Scarring and Subpleural Fibrosis
Authors: Decologne Nathalie , Kolb Martin , Margetts Peter J. , Menetrier Franck , Artur Yves , Garrido Carmen , Gauldie Jack , Camus Philippe , Bonniaud Philippe
Published: April 2014

Pleural fibrosis is a misunderstood disorder which can cause severe restrictive lung disease with high morbidity and even mortality. The condition can develop in response to a large variety of diseases and tissue injury, among them infectious disease, asbestos, drugs, and radiation therapy. There is no efficient treatment to reverse established pleural fibrosis. TGF-β1 is suspected, even if not proven, as a key cytokine in this process. In this study, we used adenoviral gene transfer of TGF-β1 to the pleural mesothelium in rats. We show that local and transient TGF-β1 overexpression induces homogenous, prolonged, and progressive pleural fibrosis without pleurodesis, associated with severe impairment of pulmonary function. We further demonstrate that pleural fibrosis can expand into the lung parenchyma from the visceral layer, but not into the muscle from the parietal layer. We provide evidence that matrix accumulation and fibrosis within the parenchyma evolved through a process involving “mesothelial-fibroblastoid transformation” and suggest that the pleural mesothelial cell may be an important player involved in the development of the subpleural distribution pattern known to be a hallmark of pulmonary fibrosis. This new model of pleural fibrosis will allow us to better understand the mechanisms of progressive fibrogenesis, and to explore novel antifibrotic therapies in the pleural cavity.
The Novel Endocytic and Phagocytic C-Type Lectin Receptor DCL-1/CD302 on Macrophages Is Colocalized with F-Actin, Suggesting a Role in Cell Adhesion and Migration
Authors: Kato Masato , Khan Seema , d’Aniello Elisabetta , McDonald Kylie J. , Hart Derek N. J.
Published: April 2014

C-type lectin receptors play important roles in mononuclear phagocytes, which link innate and adaptive immunity. In this study we describe characterization of the novel type I transmembrane C-type lectin DCL-1/CD302 at the molecular and cellular levels. DCL-1 protein was highly conserved among the human, mouse, and rat orthologs. The human DCL-1 (hDCl-1) gene, composed of six exons, was located in a cluster of type I transmembrane C-type lectin genes on chromosomal band 2q24. Multiple tissue expression array, RT-PCR, and FACS analysis using new anti-hDCL-1 mAbs established that DCL-1 expression in leukocytes was restricted to monocytes, macrophages, granulocytes, and dendritic cells, although DCL-1 mRNA was present in many tissues. Stable hDCL-1 Chinese hamster ovary cell transfectants endocytosed FITC-conjugated anti-hDCL-1 mAb rapidly (t1/2 = 20 min) and phagocytosed anti-hDCL-1 mAb-coated microbeads, indicating that DCL-1 may act as an Ag uptake receptor. However, anti-DCL-1 mAb-coated microbead binding and subsequent phagocytic uptake by macrophages was ∼8-fold less efficient than that of anti-macrophage mannose receptor (MMR/CD206) or anti-DEC-205/CD205 mAb-coated microbeads. Confocal studies showed that DCL-1 colocalized with F-actin in filopodia, lamellipodia, and podosomes in macrophages and that this was unaffected by cytochalasin D, whereas the MMR/CD206 and DEC-205/CD205 did not colocalize with F-actin. Furthermore, when transiently expressed in COS-1 cells, DCL-1-EGFP colocalized with F-actin at the cellular cortex and microvilli. These data suggest that hDCL-1 is an unconventional lectin receptor that plays roles not only in endocytosis/phagocytosis but also in cell adhesion and migration and thus may become a target for therapeutic manipulation.
Activation-Induced Cytidine Deaminase-Dependent DNA Breaks in Class Switch Recombination Occur during G1 Phase of the Cell Cycle and Depend upon Mismatch Repair
Authors: Schrader Carol E. , Guikema Jeroen E. J. , Linehan Erin K. , Selsing Erik , Stavnezer Janet
Published: April 2014

Ab class switching occurs by an intrachromosomal recombination and requires generation of double-strand breaks (DSBs) in Ig switch (S) regions. Activation-induced cytidine deaminase (AID) converts cytosines in S regions to uracils, which are excised by uracil DNA glycosylase (UNG). Repair of the resulting abasic sites would yield single-strand breaks (SSBs), but how these SSBs are converted to DSBs is unclear. In mouse splenic B cells, we find that AID-dependent DSBs occur in Sμ mainly in the G1 phase of the cell cycle, indicating they are not created by replication across SSBs. Also, G1 phase cells express AID, UNG, and mismatch repair (MMR) proteins and possess UNG activity. We find fewer S region DSBs in MMR-deficient B cells than in wild-type B cells, and still fewer in MMR-deficient/SμTR−/− B cells, where targets for AID are sparse. These DSBs occur predominantly at AID targets. We also show that nucleotide excision repair does not contribute to class switching. Our data support the hypothesis that MMR is required to convert SSBs into DSBs when SSBs on opposite strands are too distal to form DSBs spontaneously.
Mast Cell α and β Tryptases Changed Rapidly during Primate Speciation and Evolved from γ-Like Transmembrane Peptidases in Ancestral Vertebrates
Authors: Trivedi Neil N. , Tong Qiao , Raman Kavita , Bhagwandin Vikash J. , Caughey George H.
Published: April 2014

Human mast cell tryptases vary strikingly in secretion, catalytic competence, and inheritance. To explore the basis of variation, we compared genes from a range of primates, including humans, great apes (chimpanzee, gorilla, orangutan), Old- and New-World monkeys (macaque and marmoset), and a prosimian (galago), tracking key changes. Our analysis reveals that extant soluble tryptase-like proteins, including α- and β-like tryptases, mastins, and implantation serine proteases, likely evolved from membrane-anchored ancestors because their more deeply rooted relatives (γ tryptases, pancreasins, prostasins) are type I transmembrane peptidases. Function-altering mutations appeared at widely separated times during primate speciation, with tryptases evolving by duplication, gene conversion, and point mutation. The α-tryptase Gly216Asp catalytic domain mutation, which diminishes activity, is present in macaque tryptases, and thus arose before great apes and Old World monkeys shared an ancestor, and before the αβ split. However, the Arg−3Gln processing mutation appeared recently, affecting only human α. By comparison, the transmembrane γ-tryptase gene, which anchors the telomeric end of the multigene tryptase locus, changed little during primate evolution. Related transmembrane peptidase genes were found in reptiles, amphibians, and fish. We identified soluble tryptase-like genes in the full spectrum of mammals, including marsupial (opossum) and monotreme (platypus), but not in nonmammalian vertebrates. Overall, our analysis suggests that soluble tryptases evolved rapidly from membrane-anchored, two-chain peptidases in ancestral vertebrates into soluble, single-chain, self-compartmentalizing, inhibitor-resistant oligomers expressed primarily by mast cells, and that much of present numerical, behavioral, and genetic diversity of α- and β-like tryptases was acquired during primate evolution.
A Monocyte-Specific Peptide from Herpes Simplex Virus Type 2 Glycoprotein G Activates the NADPH-Oxidase but Not Chemotaxis through a G-Protein-Coupled Receptor Distinct from the Members of the Formyl Peptide Receptor Family
Authors: Bellner Lars , Karlsson Jennie , Fu Huamei , Boulay François , Dahlgren Claes , Eriksson Kristina , Karlsson Anna
Published: April 2014

We have recently identified a peptide derived from the secreted portion of the HSV-2 glycoprotein G, gG-2p20, to be proinflammatory. Based on its ability to activate neutrophils and monocytes via the formyl peptide receptor (FPR) to produce reactive oxygen species (ROS) that down-regulate NK cell function, we suggested it to be of importance in HSV-2 pathogenesis. We now describe the effects of an overlapping peptide, gG-2p19, derived from the same HSV-2 protein. Also, this peptide activated the ROS-generating NADPH-oxidase, however, only in monocytes and not in neutrophils. Surprisingly, gG-2p19 did not induce a chemotactic response in the affected monocytes despite using a pertussis toxin-sensitive, supposedly G-protein-coupled receptor. The specificity for monocytes suggested that FPR and its homologue FPR like-1 (FPRL1) did not function as receptors for gG-2p19, and this was also experimentally confirmed. Surprisingly, the monocyte-specific FPR homologue FPRL2 was not involved either, and the responsible receptor thus remains unknown so far. However, the receptor shares some basic signaling properties with FPRL1 in that the gG-2p19-induced response was inhibited by PBP10, a peptide that has earlier been shown to selectively inhibit FPRL1-triggered responses. We conclude that secretion and subsequent degradation of the HSV-2 glycoprotein G can generate several peptides that activate phagocytes through different receptors, and with different cellular specificities, to generate ROS with immunomodulatory properties.
Pregnancy Impairs the Innate Immune Resistance toSalmonella typhimuriumLeading to Rapid Fatal Infection
Authors: Pejcic-Karapetrovic Branka , Gurnani Komal , Russell Marsha S. , Finlay B. Brett , Sad Subash , Krishnan Lakshmi
Published: April 2014

Typhoid fever and gastroenteritis caused by Salmonella enterica species are increasing globally. Pregnancy poses a high risk, but it is unclear how maternal immunity to infection is altered. In mice, susceptible strains die of S. enterica serovar typhimurium (ST) infection within 7 days whereas resistant mice (129×1/SvJ) develop a chronic infection. We found that virulent ST infection during pregnancy, in normally resistant 129×1/SvJ mice, evoked ∼100% fetal loss and surprisingly >60% host fatality, with a median survival of 6 days. Splenic bacterial load was 1000-fold higher in pregnant mice. This correlated to a diminished splenic recruitment/expansion of innate immune cells: dendritic cells, neutrophils, and NK cells. In particular, the splenic expansion and activation of NK cells postinfection seen in nonpregnant mice was lacking in pregnancy. Most notably, pregnant-infected mice had decreased production of serum IL-12 and increased IL-6 levels. Moreover, uteroplacental tissue of pregnant-infected mice exhibited an ∼40-fold increase in IL-6 mRNA expression relative to noninfected placenta, whereas IL-12p40 was not increased. In vivo blocking of IL-6 significantly reduced the splenic bacterial burden in pregnant mice yet failed to prevent fetal loss. Fetal demise correlated to the rapidity of infection; by 14 h, ST expanded to >105 in the placenta and had reached the fetus. Therefore, the preferential placental expansion of ST plausibly altered the inflammatory response toward IL-6 and away from IL-12, reducing the recruitment/activation of splenic innate immune cells. Thus, highly virulent pathogens may use placental invasion to alter systemic host resistance to infection.
TLR2 and Caspase-8 Are Essential for Group BStreptococcus-Induced Apoptosis in Microglia
Authors: Lehnardt Seija , Wennekamp Julia , Freyer Dorette , Liedtke Christian , Krueger Christina , Nitsch Robert , Bechmann Ingo , Weber Joerg R. , Henneke Philipp
Published: April 2014

Microglia, the resident innate immune cells of the CNS, detect invading pathogens via various receptors, including the TLR. Microglia are involved in a number of neurodegenerative diseases in which their activation may be detrimental to neurons. It is largely unknown how this potentially deleterious action can be countered on a cellular level. We previously found that the interaction of TLR2 with group B Streptococcus (GBS), the most important pathogen in neonatal bacterial meningitis, activates microglia that in turn generate neurotoxic NO. We report in this study that GBS not only activates microglia, but also induces apoptosis in these cells via TLR2 and the TLR-adaptor molecule MyD88. Soluble toxic mediators, such as NO, are not responsible for this form of cell death. Instead, interaction of GBS with TLR2 results in formation and activation of caspase-8, a process that involves the transcription factor family Ets. Whereas caspase-8 plays an essential role in GBS-induced microglial apoptosis, caspase-3 is dispensable in this context. We suggest that TLR2- and caspase-8-mediated microglial apoptosis constitutes an autoregulatory mechanism that limits GBS-induced overactivation of the innate immune system in the CNS.
Stromal Complement Receptor CD21/35 Facilitates Lymphoid Prion Colonization and Pathogenesis
Authors: Zabel Mark D. , Heikenwalder Mathias , Prinz Marco , Arrighi Isabelle , Schwarz Petra , Kranich Jan , von Teichman Adriana , Haas Karen M. , Zeller Nicolas , Tedder Thomas F. , Weis John H. , Aguzzi Adriano
Published: April 2014

We have studied the role of CD21/35, which bind derivatives of complement factors C3 and C4, in extraneural prion replication and neuroinvasion. Upon administration of small prion inocula, CD21/35−/− mice experienced lower attack rates and delayed disease over both wild-type (WT) mice and mice with combined C3 and C4 deficiencies. Early after inoculation, CD21/35−/− spleens were devoid of infectivity. Reciprocal adoptive bone marrow transfers between WT and CD21/35−/− mice revealed that protection from prion infection resulted from ablation of stromal, but not hemopoietic, CD21/35. Further adoptive transfer experiments between WT mice and mice devoid of both the cellular prion protein PrPC and CD21/35 showed that splenic retention of inoculum depended on stromal CD21/35 expression. Because both PrPC and CD21/35 are highly expressed on follicular dendritic cells, CD21/35 appears to be involved in targeting prions to follicular dendritic cells and expediting neuroinvasion following peripheral exposure to prions.
Rectification of Age-Associated Deficiency in Cytotoxic T Cell Response to Influenza A Virus by Immunization with Immune Complexes
Authors: Zheng Biao , Zhang Yongxin , He Hongxia , Marinova Ekaterina , Switzer Kirsten , Wansley Daniel , Mbawuike Innocent , Han Shuhua
Published: April 2014

Decline in cellular immunity in aging compromises protection against infectious diseases and leads to the increased susceptibility of the elderly to infection. In particular, Ag-specific cytotoxic T lymphocyte (CTL) response against virus is markedly reduced in an aged immune system. It is of great importance to explore novel strategy in eliciting effective antiviral CTL activity in the elderly. In this study, the efficacy and mechanisms of immunization with immune complexes in overcoming age-associated deficiency in cellular immunity were investigated. In this study, we show that the severely depressed CTL response to influenza A in aged mice can be significantly restored by immunization with immune complexes consisting of influenza A virus and mAb to influenza A nucleoprotein. The main mechanisms underlying this recovery of CTL response induced by immune complex immunization in aged mice are enhanced dendritic cell function and elevated production of IFN-γ in both CD4+ Th1 and CD8+ CTLs. Thus, these results demonstrate that immune complex immunization may represent a novel strategy to elicit effective virus-specific cytotoxic response in an aged immune system, and possibly, to overcome age-related immune deficiency in general.
IFN-αβ-Mediated Inflammatory Responses and Antiviral Defense in Liver Is TLR9-Independent but MyD88-Dependent during Murine Cytomegalovirus Infection
Authors: Hokeness-Antonelli Kirsten L. , Crane Meredith J. , Dragoi Ana M. , Chu Wen-Ming , Salazar-Mather Thais P.
Published: April 2014

Chemokine responses critical for inflammation and promotion of effective innate control of murine CMV (MCMV) in liver have been shown to be dependent on immunoregulatory functions elicited by IFN-αβ. However, it remains to be determined whether upstream factors that promote viral sensing resulting in the rapid secretion of IFN-αβ in liver differ from those described in other tissues. Because plasmacytoid dendritic cells (pDCs) are known producers of high levels of systemic IFN-α in response to MCMV, this study examines the in vivo contribution of pDCs to IFN-α production in the liver, and whether production of the cytokine and ensuing inflammatory events are dependent on TLR9, MyD88, or both. We demonstrate that whereas MyD88 deficiency markedly impaired secretion of IFN-α, production of the cytokine was largely independent of TLR9 signaling, in the liver. MyD88 and TLR9 were needed for IFN-α production in the spleen. Moreover, hepatic but not splenic pDCs produced significant amounts of intracellular IFN-α in the absence of TLR9 function during infection. Furthermore, production of CCL2, CCL3, and IFN-γ, as well as the accumulation of macrophages and NK cells, was not affected in the absence of functional TLR9 in the liver. In contrast, these responses were dramatically reduced in MyD88−/− mice. Additionally, MyD88−/− but not TLR9−/− mice exhibited increased sensitivity to virus infection in liver. Collectively, our results define contrasting compartmental functions for TLR9 and MyD88, and suggest that the infected tissue site uniquely contributes to the process of virus sensing and regulation of localized antiviral responses.
Elastin Receptor (Spliced Galactosidase) Occupancy by Elastin Peptides Counteracts Proinflammatory Cytokine Expression in Lipopolysaccharide-Stimulated Human Monocytes through NF-κB Down-Regulation
Authors: Baranek Thomas , Debret Romain , Antonicelli Frank , Lamkhioued Bouchaib , Belaaouaj Azzaq , Hornebeck William , Bernard Philippe , Guenounou Moncef , Le Naour Richard
Published: April 2014

In inflammatory diseases, strong release of elastinolytic proteases results in elastin fiber degradation generating elastin peptides (EPs). Chemotactic activity for inflammatory cells was, among wide range of properties, the former identified biological activity exerted by EPs. Recently, we demonstrated the ability of EPs to favor a Th1 cytokine (IL-2, IFN-γ) cell response in lymphocytes and to regulate IL-1β expression in melanoma cells. We hypothesized that EPs might also influence inflammatory cell properties by regulating cytokine expression by these cells. Therefore, we investigated the influence of EPs on inflammatory cytokine synthesis by human monocytes. We evidenced that EPs down-regulated both at the mRNA and protein levels the proinflammatory TNF-α, IL-1β, and IL-6 expression in LPS-activated monocytes. Such negative feedback loop could be accounted solely for EP-mediated effects on proinflammatory cytokine production because EPs did not affect anti-inflammatory IL-10 or TGF-β secretion by LPS-activated monocytes. Furthermore, we demonstrated that EP effect on proinflammatory cytokine expression by LPS-stimulated monocytes could not be due either to a decrease of LPS receptor expression or to an alteration of LPS binding to its receptor. The inhibitory effects of EPs on cytokine expression were found to be mediated by receptor (spliced galactosidase) occupancy, as being suppressed by lactose, and to be associated with the decrease of NF-κB-DNA complex formation. As a whole, these results demonstrated that EP/spliced galactosidase interaction on human monocytes down-regulated NF-κB-dependent proinflammatory cytokine expression and pointed out the critical role of EPs in the regulation of inflammatory response.
Lipopolysaccharide, IFN-γ, and IFN-β Induce Expression of the Thiol-Sensitive ART2.1 Ecto-ADP-Ribosyltransferase in Murine Macrophages
Authors: Hong Shiyuan , Brass Anette , Seman Michel , Haag Friedrich , Koch-Nolte Friedrich , Dubyak George R.
Published: April 2014

Nicotinamide adenosine dinucleotide (NAD) can act as a modulator of multiple immune and inflammatory responses when released into extracellular compartments. These actions of extracellular NAD are largely mediated by a family of mammalian ecto-ADP-ribosyltransferases (ARTs) that covalently modify target extracellular or cell surface proteins by transferring ADP-ribose to arginine or cysteine residues. In this study, we report that bone marrow-derived macrophages (BMDM) from BALB/c mice lack constitutive expression of any of the six murine ecto-ART subtypes, but selectively up-regulate ART2.1 in response to multiple proinflammatory mediators including agonists for TLR and type I and type II IFN. Stimulation of BMDM with LPS, IFN-β, or IFN-γ induced high expression of ART2.1, but not ART2.2, as a GPI-anchored cell surface ectoenzyme. ART2.1 expression in response to LPS was potentiated by inhibition of ERK1/2 signaling, but inhibited by blockade of the NF-κB, PI3K, and JAK-STAT pathways or the presence of neutralizing anti-IFN-β. The catalytic function of the induced cell surface ART2.1 was strictly dependent on the presence of extracellular thiol-reducing cofactors, suggesting that in vivo activity of ART2.1-expressing macrophages may be potentiated in hypoxic or ischemic compartments. Consistent with the mutated art2a gene in C57BL/6 mice, LPS- or IFN-stimulated BMDM from this strain lacked expression of cell surface ART2 activity in the presence or absence of extracellular thiol reductants. Collectively, these studies identify ART2.1 as a new candidate for linking autocrine/paracrine activation of inflammatory macrophages to the release of NAD, a critical intracellular metabolite.
Exacerbation of Antigen-Induced Arthritis in IFN-γ-Deficient Mice As a Result of Unrestricted IL-17 Response
Authors: Irmler Ingo M. , Gajda Mieczyslaw , Bräuer Rolf
Published: April 2014

Proinflammatory Th1 responses are believed to be involved in the induction and perpetuation of rheumatoid arthritis. However, the role of IFN-γ, the major cytokine produced by Th1 cells, is still incompletely defined. In the present study, we investigated the effects of IFN-γ deficiency (IFN-γ−/−) on the course of experimental murine Ag-induced arthritis (AIA). In the acute stage of disease, IFN-γ−/− AIA mice showed significantly increased inflammatory responses compared with wild-type C57BL/6 AIA mice, i.e., exacerbated joint swelling, increased delayed-type hypersensitivity reaction, and increased histopathological scores of arthritis. Intraarticular administration of exogenous IFN-γ at induction of AIA significantly suppressed these acute aggravation effects. Stimulated cells isolated from lymph nodes and spleen of IFN-γ−/− AIA mice showed increased production of IL-2, IL-4, IL-5, IL-6, but most prominently of IL-17. These elevations were paralleled by decreased humoral immune responses, with low serum levels of total and Ag-specific IgG (IgG1, IgG2ab, IgG2b, IgG3). At immunohistology, the knee joints of IFN-γ−/− AIA mice showed massive neutrophil granulocyte infiltration. Treatment with mAbs neutralizing IL-17 diminished the acute inflammation. In vitro, Th cell expansion and production of IL-17 upon restimulation were effectively and dose dependently inhibited by IFN-γ. These results clearly demonstrate that IFN-γ has anti-inflammatory properties during the initial phase of AIA, and indicate that IFN-γ deficiency exerts disease-promoting effects, preferentially via IL-17-modulated pathways.
The Role of IκB Kinase 2, but Not Activation of NF-κB, in the Release of CXCR3 Ligands from IFN-γ-Stimulated Human Bronchial Epithelial Cells
Authors: Tudhope Susan J. , Catley Matthew C. , Fenwick Peter S. , Russell Richard E. K. , Rumsey William L. , Newton Robert , Barnes Peter J. , Donnelly Louise E.
Published: April 2014

The severity of chronic obstructive pulmonary disease correlates with increased numbers of cytotoxic CD8+ T lymphocytes in the lung parenchyma. CD8+ T lymphocytes release IFN-γ which stimulates airway epithelial cells to produce CXCR3 chemokines leading to further recruitment of CD8+ T lymphocytes. To evaluate the signaling pathways involved in regulation of CXCR3 ligands, the human bronchial epithelial cell line BEAS-2B was stimulated with IFN-γ and the release of the CXCR3 ligands was measured by ELISA. The release of CXCL9, CXCL10, and CXCL11 was inhibited by an IκB kinase 2 (IKK2) selective inhibitor 2-[(Aminocarbonyl)amino]-5-[4-fluorophenyl]-3-thiophenecarboxamide (TPCA-1) (EC50 values were 0.50 ± 0.03, 0.17 ± 0.06, and 0.45 ± 0.10 μM, respectively (n = 6)) and an IKK1/2 selective inhibitor 2-amino-6-(2′cyclopropylemethoxy-6′-hydroxy-phenyl)-4-piperidin-3-yl-pyridine-3-carbonitrile (EC50 values 0.74 ± 0.40, 0.27 ± 0.06, and 0.88 ± 0.29 μM, respectively (n = 6)). The glucocorticosteroid dexamethasone had no effect on CXCR3 ligand release. The release of CXCL10 was most sensitive to inhibition by IKK2 and a role for IKK2 in CXCL10 release was confirmed by overexpression of dominant-negative adenoviral constructs to IKK2 (68.2 ± 8.3% n = 5), but not of IKK1. Neither phosphorylation of IκBα, translocation of p65 to the nucleus, or activation of a NF-κB-dependent reporter (Ad-NF-κB-luc) were detected following stimulation of BEAS-2B cells with IFN-γ. These data suggest that IKK2 is also involved in the IFN-γ-stimulated release of the CXCR3 ligands through a novel mechanism that is independent NF-κB.
Biomechanical Signals Suppress TAK1 Activation to Inhibit NF-κB Transcriptional Activation in Fibrochondrocytes
Authors: Madhavan Shashi , Anghelina Mirela , Sjostrom Danen , Dossumbekova Anar , Guttridge Denis C. , Agarwal Sudha
Published: April 2014

Exercise/joint mobilization is therapeutic for inflammatory joint diseases like rheumatoid and osteoarthritis, but the mechanisms underlying its actions remain poorly understood. We report that biomechanical signals at low/physiological magnitudes are potent inhibitors of inflammation induced by diverse proinflammatory activators like IL-1β, TNF-α, and lipopolysaccharides, in fibrochondrocytes. These signals exert their anti-inflammatory effects by inhibiting phosphorylation of TAK1, a critical point where signals generated by IL-1β, TNF-α, and LPS converge to initiate NF-κB signaling cascade and proinflammatory gene induction. Additionally, biomechanical signals inhibit multiple steps in the IL-1β-induced proinflammatory cascade downstream of IκB kinase activation to regulate IκBα and IκBβ degradation and synthesis, and promote IκBα shuttling to export nuclear NF-κB and terminate its transcriptional activity. The findings demonstrate that biomechanical forces are but another important signal that uses NF-κB pathway to regulate inflammation by switching the molecular activation of discrete molecules involved in proinflammatory gene transcription.
Vasoactive Hormone Adrenomedullin and Its Binding Protein: Anti-Inflammatory Effects by Up-Regulating Peroxisome Proliferator-Activated Receptor-γ
Authors: Miksa Michael , Wu Rongqian , Cui Xiaoxuan , Dong Weifeng , Das Padmalaya , Simms H. Hank , Ravikumar Thanjavur S. , Wang Ping
Published: April 2014

Sepsis is a critical inflammatory condition from which numerous patients die due to multiple organ failure and septic shock. The vasoactive hormone adrenomedullin (AM) and its binding protein (AMBP-1) are beneficial in sepsis by abrogating the progression to irreversible shock and decreasing proinflammatory cytokine release. To investigate the anti-inflammatory mechanism, we studied to determine the effect of the AM/AMBP-1 complex on peroxisome proliferator-activated receptor-γ (PPAR-γ) expression and activation by using RAW264.7 cells and a rat endotoxemia model. LPS treatment significantly decreased PPAR-γ expression in vivo and in vitro and was associated with increased TNF-α production. Treatment with AM/AMBP-1 for 4 h completely restored PPAR-γ levels in both models, resulting in TNF-α suppression. In a knockdown model using small interfering RNA in RAW264.7 macrophages, AM/AMBP-1 failed to suppress TNF-α production in the absence of PPAR-γ. LPS caused the suppression of intracellular cyclic AMP (cAMP), which was prevented by simultaneous AM/AMBP-1 treatment. Although incubation with dibutyryl cAMP significantly decreased LPS-induced ΤΝF-α release, it did not alter PPAR-γ expression. Through inhibition studies using genistein and PD98059 we found that the Pyk-2 tyrosine kinase-ERK1/2 pathway is in part responsible for the AM/AMBP-1-mediated induction of PPAR-γ and the anti-inflammatory effect. We conclude that AM/AMBP-1 is protective in sepsis due to its vasoactive properties and direct anti-inflammatory effects mediated through both the cAMP-dependent pathway and Pyk-2-ERK1/2-dependent induction of PPAR-γ.
Topically Applied 1,25-Dihydroxyvitamin D3 Enhances the Suppressive Activity of CD4+CD25+ Cells in the Draining Lymph Nodes
Authors: Gorman Shelley , Kuritzky L. Alexandra , Judge Melinda A. , Dixon Katie M. , McGlade Jacqueline P. , Mason Rebecca S. , Finlay-Jones John J. , Hart Prue H.
Published: April 2014

The immunomodulatory effects of vitamin D have been described following chronic oral administration to mice or supplementation of cell cultures with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), the active form of vitamin D. In this study, topically applied 1,25(OH)2D3, enhanced the suppressive capacity of CD4+CD25+ cells from the draining lymph nodes. The effects of topical 1,25(OH)2D3 were compared with those of UVB irradiation, which is the environmental factor required for 1,25(OH)2D3 production in skin. CD4+ cells from the skin-draining lymph nodes (SDLN) of either 1,25(OH)2D3-treated or UVB-irradiated mice had reduced capacity to proliferate to Ags presented in vitro, and could suppress Ag-specific immune responses upon adoptive transfer into naive mice. This regulation was lost upon removal of CD4+CD25+ cells. Furthermore, purified CD4+CD25+ cells from the SDLN of 1,25(OH)2D3-treated or UVB-irradiated mice compared with equal numbers of CD4+CD25+ cells from control mice had increased capacity to suppress immune responses in both in vitro and in vivo assay systems. Following the sensitization of recipient mice with OVA, the proportion of CD4+Foxp3+ cells of donor origin significantly increased in recipients of CD4+CD25+ cells from the SDLN of 1,25(OH)2D3-treated mice, indicating that these regulatory T cells can expand in vivo with antigenic stimulation. These studies suggest that 1,25(OH)2D3 may be an important mediator by which UVB-irradiation exerts some of its immunomodulatory effects.
Estradiol Attenuates Lipopolysaccharide-Induced CXC Chemokine Ligand 8 Production by Human Peripheral Blood Monocytes
Authors: Pioli Patricia A. , Jensen Amy L. , Weaver Lehn K. , Amiel Eyal , Shen Zheng , Shen Li , Wira Charles R. , Guyre Paul M.
Published: April 2014

Regulation of the inflammatory response is imperative to the maintenance of immune homeostasis. Activated monocytes elaborate a broad variety of proinflammatory cytokines that mediate inflammation, including CXCL8. Release of this chemokine attracts neutrophils to sites of bacterial invasion and inflammation; however, high levels of CXCL8 may result in excessive neutrophil infiltration and subsequent tissue damage. In this study, we demonstrate that 17β-estradiol (E2) attenuates LPS-induced expression of CXCL8 in human peripheral blood monocytes. Treatment of monocytes with estradiol before administration of LPS reduces CXCL8 message and protein production through an estrogen receptor-dependent mechanism, and luciferase reporter assays demonstrate that this inhibition is mediated transcriptionally. Importantly, the ability of estradiol-pretreated LPS-activated monocytes to mobilize neutrophils is impaired. These results implicate a role for estradiol in the modulation of the immune response, and may lead to an enhanced understanding of gender-based differences in inflammatory control mechanisms.