bluuebunny/crossref_metadata_2025 huggingface dataset:
Spi-C Has Opposing Effects to PU.1 on Gene Expression in Progenitor B Cells
Authors: Schweitzer Brock L. , Huang Kelly J. , Kamath Meghana B. , Emelyanov Alexander V. , Birshtein Barbara K. , DeKoter Rodney P.
Published: April 2014

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1−/− fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcγRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcγRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcγRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3′ regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.
Vav1 Controls DAP10-Mediated Natural Cytotoxicity by Regulating Actin and Microtubule Dynamics
Authors: Graham Daniel B. , Cella Marina , Giurisato Emanuele , Fujikawa Keiko , Miletic Ana V. , Kloeppel Tracie , Brim Karry , Takai Toshiyuki , Shaw Andrey S. , Colonna Marco , Swat Wojciech
Published: April 2014

The NK cell-activating receptor NKG2D recognizes several MHC class I-related molecules expressed on virally infected and tumor cells. Human NKG2D transduces activation signals exclusively via an associated DAP10 adaptor containing a YxNM motif, whereas murine NKG2D can signal through either DAP10 or the DAP12 adaptor, which contains an ITAM sequence. DAP10 signaling is thought to be mediated, at least in part, by PI3K and is independent of Syk/Zap-70 kinases; however, the exact mechanism by which DAP10 induces natural cytotoxicity is incompletely understood. Herein, we identify Vav1, a Rho GTPase guanine nucleotide exchange factor, as a critical signaling mediator downstream of DAP10 in NK cells. Specifically, using mice deficient in Vav1 and DAP12, we demonstrate an essential role for Vav1 in DAP10-induced NK cell cytoskeletal polarization involving both actin and microtubule networks, maturation of the cytolytic synapse, and target cell lysis. Mechanistically, we show that Vav1 interacts with DAP10 YxNM motifs through the adaptor protein Grb2 and is required for activation of PI3K-dependent Akt signaling. Based on these findings, we propose a novel model of ITAM-independent signaling by Vav downstream of DAP10 in NK cells.
B7RP-1-ICOS Interactions Are Required for Optimal Infection-Induced Expansion of CD4+ Th1 and Th2 Responses
Authors: Wilson Emma H. , Zaph Colby , Mohrs Markus , Welcher Andy , Siu Jerry , Artis David , Hunter Christopher A.
Published: April 2014

Although initial reports linked the costimulatory molecule ICOS preferentially with the development of Th2 cells, there is evidence that it is not required for protective type 2 immunity to helminths and that it contributes to Th1 and Th2 responses to other parasites. To address the role of ICOS in the development of infection-induced polarized Th cells, ICOS−/− mice were infected with Trichuris muris or Toxoplasma gondii. Wild-type mice challenged with T. muris developed Th2 responses and expelled these helminths by day 18 postinfection, whereas ICOS−/− mice failed to clear worms and produced reduced levels of type 2 cytokines. However, by day 35 postinfection, ICOS−/− mice were able to mount an effective Th2 response and worms were expelled. This delay in protective immunity was associated with a defect in infection-induced increases in the number of activated and proliferating CD4+ T cells. Similarly, following challenge with T. gondii ICOS was required for optimal proliferation by CD4+ T cells. However, the reduced number of activated CD4+ T cells and associated defect in the production of IFN-γ did not result in increased susceptibility to T. gondii, but rather resulted in decreased CNS pathology during the chronic phase of this infection. Taken together, these data are consistent with a model in which ICOS is not involved in dictating polarity of the Th response but rather regulates the expansion of these subsets.
B Cells Expressing a Natural Polyreactive Autoantibody Have a Distinct Phenotype and Are Overrepresented in Immunoglobulin Heavy Chain Transgenic Mice
Authors: Tian Qi , Beardall Michael , Xu Yang , Li Ju , Parker David C. , Casanova Nina , Bakke Anthony C. , Chen Ching
Published: April 2014

Despite stringent regulation of disease-associated autoantibodies, a substantial proportion of circulating Abs in sera of healthy individuals exhibit self-reactivity. These Abs are referred to as naturally occurring or natural autoantibodies (NAAs). To understand the origin and function of NAAs, we have generated a new site-directed transgenic mouse model in which a prerearranged VDJ gene coding for the H chain of a typical polyreactive NAA, ppc1-5, is inserted into the IgH locus. This H chain, when combined with its original L chain, the λ1 L chain, yields a NAA that characteristically binds a variety of self and non-self Ags including ssDNA, actin, ubiquitin, and nitrophenyl phosphocholine. Despite their autoreactivity, B cells expressing ppc1-5H/λ1 NAA are not negatively selected, but rather are overrepresented in the transgenic mice. The shift toward λ1 expression mainly occurs during the transition of immature to mature B cells in the spleen, suggesting a BCR selection process. The ppc1-5H/λ1 B cells exhibit a phenotype that is different from those of the known mature B cell populations, and they are located predominantly in the lymphoid follicles of the spleen and the lymph nodes. These B cells are functionally active, producing high levels of Abs in vivo and responding well to BCR stimulation in vitro. The findings indicate that the ppc1-5/λ1 natural autoantibodies originate from a distinct B cell subset that may be positively selected by virtue of its poly/autoreactivity.
Structure-Activity Profiles of Ab-Derived TNF Fusion Proteins
Authors: Bauer Stefan , Adrian Nicole , Fischer Eliane , Kleber Sascha , Stenner Frank , Wadle Andreas , Fadle Natalie , Zoellner Andy , Bernhardt Rita , Knuth Alexander , Old Lloyd J. , Renner Christoph
Published: April 2014

TNF application in humans is limited by severe side effects, including life-threatening symptoms of shock. Therefore, TNF can be successfully applied as a tumor therapeutic reagent only under conditions that prevent its systemic action. To overcome this limitation, genetic fusion of TNF to tumor-selective Abs is a favored strategy to increase site-specific cytokine targeting. Because wild-type TNF displays its bioactivity as noncovalently linked homotrimer, the challenge is to define structural requirements for a TNF-based immunokine format with optimized structure-activity profile. We compared toxicity and efficacy of a dimerized CH2/CH3 truncated IgG1-TNF fusion protein and a single-chain variable fragment-coupled TNF monomer recognizing fibroblast-activating protein. The former construct preserves its dimeric structure stabilized by the natural disulfide bond IgG1 hinge region, while the latter trimerizes under native conditions. Analysis of complex formation of wild-type TNF and of both fusion proteins with TNFR type 1 (TNF-R1) using surface plasmon resonance correlated well with in vitro and in vivo toxicity data. There is strong evidence that TNF subunits in a trimeric state display similar toxicity profiles despite genetic fusion to single-chain variable fragment domains. However, LD50 of either immunodeficient BALB/c nu/nu or immunocompetent BALB/c mice was significantly decreased following administration of TNF in the formation of IgG1-derived dimeric fusion protein. Reduction of unspecific peripheral complexation of TNF-R1 resulted in higher anticancer potency by immunotargeting of fibroblast-activating protein-expressing xenografts. The broader therapeutic window of the IgG1-derived TNF fusion protein favors the dimeric TNF-immunokine format for systemic TNF-based tumor immunotherapy.
Conserved Functions of Ikaros in Vertebrate Lymphocyte Development: Genetic Evidence for Distinct Larval and Adult Phases of T Cell Development and Two Lineages of B Cells in Zebrafish
Authors: Schorpp Michael , Bialecki Mike , Diekhoff Dagmar , Walderich Brigitte , Odenthal Jörg , Maischein Hans-Martin , Zapata Agustin G. ,   Tübingen 2000 Screen Consortium,   Freiburg Screening Group, Boehm Thomas
Published: April 2014

Zebrafish has been advocated as an alternative animal model to study lymphocyte development, although the similarities in the genetic requirements of lymphopoiesis between fish and mammals have not yet been investigated. In this study, we examine the role of the transcription factor Ikaros in zebrafish lymphopoiesis. In fish larvae homozygous for an ikaros allele predicted to lack the C-terminal zinc fingers, T lymphopoiesis is absent; the presence of VHDμJμ rearrangements in adolescent fish is delayed in mutants. In adolescent mutant fish, T cells expressing tcrb and tcrd and B cells expressing igm are formed with low efficiency and display an oligoclonal Ag receptor repertoire. By contrast, B cells expressing the igz isotype do not develop, providing genetic evidence for two separate B cell lineages in zebrafish. Thus, Ikaros appears to play similar roles in fish and mammalian lymphopoiesis.
Autoantibodies from Synovial Lesions in Chronic, Antibiotic Treatment-Resistant Lyme Arthritis Bind Cytokeratin-10
Authors: Ghosh Srimoyee , Seward Robert , Costello Catherine E. , Stollar B. David , Huber Brigitte T.
Published: April 2014

Although the causative agent of Lyme disease is definitively known to be the tick-borne spirochete, Borrelia burgdorferi, the etiology of chronic joint inflammation that ensues in a subset of patients remains less well understood. Persistence of arthritis after apparent eradication of the spirochete suggests an autoimmune reaction downstream of the original bacterial infection. We have generated recombinant Ab probes from synovial lesions within affected arthritic joints in an attempt to recapitulate disease-relevant Ag-binding specificities at the site of injury. Using this panel of intra-articular probes, as well as Ab fragments derived from patient peripheral blood, we have identified cytokeratin 10, present in synovial microvascular endothelium, as a target ligand and a putative autoantigen in chronic, antibiotic treatment-resistant Lyme arthritis. Furthermore, there is cross-reactivity between cytokeratin 10 and a prominent B. burgdorferi Ag, outer surface protein A. Release of the self protein in the context of inflammation-induced tissue injury and the resulting in situ response to it could set in motion a feed-forward loop, which amplifies the inflammatory process, thereby rendering it chronic and self-perpetuating, even in the absence of the inciting pathogen.
CD46 (Membrane Cofactor Protein) Acts as a Human Epithelial Cell Receptor for Internalization of Opsonized Uropathogenic Escherichia coli
Authors: Li Ke , Feito Maria Jose , Sacks Steven H. , Sheerin Neil S.
Published: April 2014

Escherichia coli is a common urinary pathogen whose uptake into epithelial cells is mediated by attachment through type 1 fimbriae. In this study, we show by using using human urinary tract epithelial cells that maximal internalization of E. coli is achieved only when bacteria are opsonized with complement. The concentrations of complement proteins in the urine rise sufficiently during infection to allow bacterial opsonization. The complement regulatory protein, CD46 (membrane cofactor protein), acts in cohort with fimbrial adhesion to promote the uptake of pathogenic E. coli. This uptake is inhibited by RNA interference to lower the expression of CD46 and by soluble CD46 that will competitively inhibit opsonized bacteria binding to cell surface CD46. We propose that efficient internalization of uropathogenic E. coli by the human urinary tract depends on cooperation between fimbrial-mediated adhesion and C3 receptor (CD46)–ligand interaction. Complement receptor–ligand interaction could pose a new target for interrupting the cycle of reinfection due to intracellular bacteria.
Heme Oxygenase-1 Mediates the Anti-Inflammatory Effects of Acute Alcohol on IL-10 Induction Involving p38 MAPK Activation in Monocytes
Authors: Drechsler Yvonne , Dolganiuc Angela , Norkina Oxana , Romics Laszlo , Li Weibo , Kodys Karen , Bach Fritz H. , Mandrekar Pranoti , Szabo Gyongyi
Published: April 2014

Inflammation and immunoregulatory cytokines play a central role in alcohol-induced liver damage. We previously reported that acute alcohol treatment augments IL-10 and inhibits TNF-α production in monocytes. Heme oxygenase-1 (HO-1), a stress-inducible protein, also regulates IL-10 and TNF-α production. Here, we report that augmentation of LPS-induced IL-10 production by alcohol was prevented by inhibition of HO-1 activity. Acute ethanol increased LPS-induced enzyme activity and RNA levels of HO-1, and DNA binding of AP-1, a transcription factor essential in HO-1 regulation. LPS-induced phospho-p38 MAPK levels were augmented by ethanol treatment and the p38 inhibitor, SB203580, prevented both the ethanol-induced increase in IL-10 production and the inhibitory effect of ethanol on TNF-α production. Ethanol-induced down-regulation of TNF-α production was abrogated by inhibition of HO-1. We found that LPS-induced activation of NF-κB, a regulator of TNF-α, was inhibited by both ethanol treatment and HO-1 activation, but the ethanol-induced inhibition of NF-κB was HO-1 independent. In LPS-challenged mice in vivo, both acute alcohol administration and HO-1 activation augmented IL-10 and inhibited TNF-α serum levels. These results show that 1) acute alcohol augments HO-1 activation in monocytes, 2) HO-1 activation plays a role in alcohol-induced augmentation of IL-10 production likely via increased p38 MAPK activation, and 3) HO-1 activation is involved in attenuation of TNF-α production by alcohol independent of inhibition of NF-κB activation by alcohol. Thus, HO-1 activation is a key mediator of the anti-inflammatory effects of acute alcohol on monocytes.
Combinatorial SNARE Complexes Modulate the Secretion of Cytoplasmic Granules in Human Neutrophils
Authors: Mollinedo Faustino , Calafat Jero , Janssen Hans , Martín-Martín Belén , Canchado Javier , Nabokina Svetlana M. , Gajate Consuelo
Published: April 2014

Mobilization of human neutrophil granules is critical for the innate immune response against infection and for the outburst of inflammation. Human neutrophil-specific and tertiary granules are readily exocytosed upon cell activation, whereas azurophilic granules are mainly mobilized to the phagosome. These cytoplasmic granules appear to be under differential secretory control. In this study, we show that combinatorial soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes with vesicle-associated membrane proteins (VAMPs), 23-kDa synaptosome-associated protein (SNAP-23), and syntaxin 4 underlie the differential mobilization of granules in human neutrophils. Specific and tertiary granules contained VAMP-1, VAMP-2, and SNAP-23, whereas the azurophilic granule membranes were enriched in VAMP-1 and VAMP-7. Ultrastructural, coimmunoprecipitation, and functional assays showed that SNARE complexes containing VAMP-1, VAMP-2, and SNAP-23 mediated the rapid exocytosis of specific/tertiary granules, whereas VAMP-1 and VAMP-7 mainly regulated the secretion of azurophilic granules. Plasma membrane syntaxin 4 acted as a general target SNARE for the secretion of the distinct granule populations. These data indicate that at least two SNARE complexes, made up of syntaxin 4/SNAP-23/VAMP-1 and syntaxin 4/SNAP-23/VAMP-2, are involved in the exocytosis of specific and tertiary granules, whereas interactions between syntaxin 4 and VAMP-1/VAMP-7 are involved in the exocytosis of azurophilic granules. Our data indicate that quantitative and qualitative differences in SNARE complex formation lead to the differential mobilization of the distinct cytoplasmic granules in human neutrophils, and a higher capability to form diverse SNARE complexes renders specific/tertiary granules prone to exocytosis.
High-Affinity Ligand Probes of CD22 Overcome the Threshold Set by cis Ligands to Allow for Binding, Endocytosis, and Killing of B Cells
Authors: Collins Brian E. , Blixt Ola , Han Shoufa , Duong Bao , Li Hongyi , Nathan Jay K. , Bovin Nicolai , Paulson James C.
Published: April 2014

CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Siaα2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear “masked” from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated “cargo” to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.
Simvastatin Treatment Ameliorates Autoimmune Disease Associated with Accelerated Atherosclerosis in a Murine Lupus Model
Authors: Aprahamian Tamar , Bonegio Ramon , Rizzo Jennifer , Perlman Harris , Lefer David J. , Rifkin Ian R. , Walsh Kenneth
Published: April 2014

Patients with systemic lupus erythematosus develop accelerated atherosclerosis independent of traditional risk factors. The 3-hydroxy-3-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitors are widely prescribed for hyperlipidemia, but they also exhibit anti-inflammatory actions that appear to be independent of their suppressive actions on plasma cholesterol levels. In this study, we analyzed the effect of the HMG-CoA reductase inhibitor simvastatin on disease manifestations in gld.apoE−/− mice that lack functional Fas ligand and apolipoprotein E and exhibit accelerated atherosclerosis and aggravated lupus-like features. Wild-type, gld, apoE−/−, and gld.apoE−/− mice were maintained on a high cholesterol Western diet and received daily simvastatin (0.125 mg/kg) or saline for 12 wk. Serum cholesterol levels were unaffected by simvastatin treatment, but atherosclerotic lesion area was reduced in both apoE−/− and gld.apoE−/− mice treated with simvastatin. Simvastatin also reduced the lymphadenopathy, renal disease, and proinflammatory cytokine production seen in gld.apoE−/−, but not gld, mice. The immunomodulatory effects in gld.apoE−/− mice were associated with enhanced STAT6 and decreased STAT4 induction in submandibular lymph node cells. Along with reductions in serum TNF-α and IFN-γ levels, there was also an increase in IL-4 and IL-10 transcript levels in lymph nodes. These data indicate that HMG-CoA reductase inhibitors ameliorate atherosclerosis and lupus-like autoimmunity independent of their cholesterol-lowering effects via a shift from a Th1 to a Th2 phenotype in the gld.apoE−/− model. Thus, the anti-inflammatory activities of statins may have utility for the treatment of both autoimmunity and atherosclerosis in patients with systemic lupus erythematosus.
Membrane-Associated TGF-β1 Inhibits Human Memory T Cell Signaling in Malignant and Nonmalignant Inflammatory Microenvironments
Authors: Broderick Lori , Bankert Richard B.
Published: April 2014

TGF-β1 is present on cells derived from the microenvironment of human lung tumors and nonmalignant inflammatory tissues. We establish that this cell-associated cytokine mediates hyporesponsiveness of the memory T cells in these microenvironments in situ by blocking TCR signaling. T cells derived from these tissues failed to translocate NF-κB to the nucleus in response to CD3 + CD28 cross-linking. This nonresponsiveness was reversed by an anti-TGF-β1-neutralizing Ab. Refractoriness of the memory T cells to TCR activation was also reversed by the removal of TGF-β1 by briefly pulsing the cells in a low pH buffer. Addition of exogenous TGF-β1 to eluted T cells re-established their nonresponsive state. Neither TGF-β1, anti-TGF-β1 Ab, nor low pH affected TCR signaling potential of peripheral blood T cells. We conclude that TGF-β1 mediates a physiologically relevant regulatory mechanism, selective for memory T cells present in the tumor microenvironment and nonmalignant chronic inflammatory tissues.
Structural Basis for the Interaction of CCR5 with a Small Molecule, Functionally Selective CCR5 Agonist
Authors: Saita Yuji , Kodama Eiichi , Orita Masaya , Kondo Mitsuhiro , Miyazaki Takahiro , Sudo Kenji , Kajiwara Keiko , Matsuoka Masao , Shimizu Yasuaki
Published: April 2014

The chemokine receptor CCR5 is an attractive target for HIV-1 drug development, as individuals whose cells lack surface CCR5 expression are highly resistant to HIV-1 infection. CCR5 ligands, such as CCL5/RANTES, effectively inhibit HIV-1 infection by competing for binding opportunities to the CCR5 and inducing its internalization. However, the inherent proinflammatory activity of the chemotactic response of CCR5 ligands has limited their clinical use. In this study, we found that a novel small molecule, functionally selective CCR5 agonist, 2,2-dichloro-1-(triphenylphosphonio)vinyl formamide perchlorate (YM-370749), down-modulates CCR5 from the cell surface without inducing a chemotactic response and inhibits HIV-1 replication. In molecular docking studies of YM-370749 and a three-dimensional model of CCR5 based on the rhodopsin crystal structure as well as binding and functional studies using various CCR5 mutants, the amino acid residues necessary for interaction with YM-370749 were marked. These results provide a structural basis for understanding the activation mechanism of CCR5 and for designing functionally selective agonists as a novel class of anti-HIV-1 agents.
Human Cytomegalovirus-Encoded UL16 Discriminates MIC Molecules by Their α2 Domains
Authors: Spreu Jessica , Stehle Thilo , Steinle Alexander
Published: April 2014

Human CMV infection results in MHC class I down-regulation and induction of NKG2D ligand expression favoring NK recognition of infected cells. However, human CMV-encoded UL16 counteracts surface expression of several NKG2D ligands by intracellular retention. Interestingly, UL16 interacts with MICB, but not with the closely related MICA, and with UL16-binding proteins (ULBP) ULBP1 and ULBP2, which are only distantly related to MICB, but not with ULPB3 or ULBP4, although all constitute ligands for NKG2D. Here, we dissected the molecular basis of MICA-MICB discrimination by UL16 to elucidate its puzzling binding behavior. We report that the UL16-MICB interaction is independent of glycosylation and demonstrate that selective MICB recognition by UL16 is governed by helical structures of the MICB α2 domain. Transplantation of the MICB α2 domain confers UL16 binding capacity to MICA, and thus, diversification of the MICA α2 domain may have been driven by the selective pressure exerted by UL16.
Dengue Virus Induces Expression of CXC Chemokine Ligand 10/IFN-γ-Inducible Protein 10, Which Competitively Inhibits Viral Binding to Cell Surface Heparan Sulfate
Authors: Chen Jia-Perng , Lu Hsin-Lin , Lai Szu-Liang , Campanella Gabriele S. , Sung Jui-Ming , Lu Mei-Yi , Wu-Hsieh Betty A. , Lin Yi-Ling , Lane Thomas E. , Luster Andrew D. , Liao Fang
Published: April 2014

Dengue virus is an arthropod-borne flavivirus that causes a mild febrile illness, dengue fever, or a potentially fatal syndrome, dengue hemorrhagic fever/dengue shock syndrome. Chemokines primarily orchestrate leukocyte recruitment to the areas of viral infection, which makes them critical mediators of immune and inflammatory responses. In the present study, we investigated the induction and function of chemokines in mice early after infection with dengue virus in vivo. We found that CXCL10/IFN-γ-inducible protein 10 (IP-10) expression was rapidly and transiently induced in liver following infection. The expressed CXCL10/IP-10 likely mediates the recruitment of activated NK cells, given that anti-CXCL10/IP-10-treated mice showed diminished NK cell infiltration and reduced hepatic expression of effector molecules in activated NK cells after dengue virus infection. Of particular interest, we found that CXCL10/IP-10 also was able to inhibit viral binding to target cells in vitro. Further investigation revealed that various CXCL10/IP-10 mutants, in which the residues that mediate the interaction between the chemokine and heparan sulfate were substituted, failed to exert the inhibitory effect on dengue binding, which suggests that CXCL10/IP-10 competes with dengue virus for binding to heparan sulfate on the cell surface. Moreover, subsequent plaque assays showed that this inhibition of dengue binding blocked viral uptake and replication. The inhibitory effect of CXCL10/IP-10 on the binding of dengue virus to cells may represent a novel contribution of this chemokine to the host defense against viral infection.
Processing and Presentation of a Mycobacterial Antigen 85B Epitope by Murine Macrophages Is Dependent on the Phagosomal Acquisition of Vacuolar Proton ATPase and In Situ Activation of Cathepsin D
Authors: Singh Christopher R. , Moulton Rachel A. , Armitige Lisa Y. , Bidani Akhil , Snuggs Mark , Dhandayuthapani Subramanian , Hunter Robert L. , Jagannath Chinnaswamy
Published: April 2014

Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.
Bone Marrow Stromal Cell Antigen 2 Is a Specific Marker of Type I IFN-Producing Cells in the Naive Mouse, but a Promiscuous Cell Surface Antigen following IFN Stimulation
Authors: Blasius Amanda L. , Giurisato Emanuele , Cella Marina , Schreiber Robert D. , Shaw Andrey S. , Colonna Marco
Published: April 2014

Type I IFN-producing cells (IPC) are sentinels of viral infections. Identification and functional characterization of these cells have been difficult because of their small numbers in blood and tissues and their complex cell surface phenotype. To overcome this problem in mice, mAbs recognizing IPC-specific cell surface molecules have been generated. In this study, we report the identification of new Abs specific for mouse IPC, which recognize the bone marrow stromal cell Ag 2 (BST2). Interestingly, previously reported IPC-specific Abs 120G8 and plasmacytoid dendritic cell Ag-1 also recognize BST2. BST2 is predominantly specific for mouse IPC in naive mice, but is up-regulated on most cell types following stimulation with type I IFNs and IFN-γ. The activation-induced promiscuous expression of BST2 described in this study has important implications for the use of anti-BST2 Abs in identification and depletion of IPC. Finally, we show that BST2 resides within an intracellular compartment corresponding to the Golgi apparatus, and may be involved in trafficking secreted cytokines in IPC.
Type 1 IFN Deficiency in the Absence of Normal Splenic Architecture during Lymphocytic Choriomeningitis Virus Infection
Authors: Louten Jennifer , van Rooijen Nico , Biron Christine A.
Published: April 2014

The innate immune system uses different mechanisms to respond to infectious pathogens. Experiments evaluating the requirements for a type 1 IFN (IFN-αβ) response to lymphocytic choriomeningitis virus (LCMV) resulted in the surprising discovery that mice deficient in B and T cell development, i.e., RAG-deficient and SCID, had profoundly reduced levels of IFN-αβ in serum and spleen, despite high viral replication. In addition to lacking an adaptive immune system, these strains exhibit aberrant splenic architecture, and the defect in type 1 IFN production was also observed in mice lacking normal splenic marginal zone (MZ) organization due to genetic deficiencies in B cell development or in cytokine functions required for development of the MZ, i.e., μMT, lymphotoxin-α, and TNFR1. Interestingly, the IFN-αβ reduction was not observed after murine CMV infection. Depletion of phagocytic cells from normally developed spleens by treatment with clodronate-containing liposomes demonstrated that these populations were required for the type 1 IFN response to LCMV, but not to murine CMV, and for control of viral replication. Complete repopulation of the MZ was necessary to restore normal IFN-αβ production. In contrast, control of LCMV replication correlated with the return of CD11c+ cells. Taken together, these results demonstrate the complexity and sophistication of the splenic MZ in sensing and responding to particular pathogens and reveal the importance of organ architecture in the production of type 1 IFN.
Synchrony of High Endothelial Venules and Lymphatic Vessels Revealed by Immunization
Authors: Liao Shan , Ruddle Nancy H.
Published: April 2014

The mature phenotype of peripheral lymph node (LN) high endothelial venules (HEVs), defined as MAdCAM-1lowPNAdhighLTβRhigh HEC-6SThigh, is dependent on signaling through the lymphotoxin-β receptor (LTβR). Plasticity of PLN HEVs during immunization with oxazolone was apparent as a reversion to an immature phenotype (MAdCAM-1highPNAdlowLTβRlow HEC-6STlow) followed by recovery to the mature phenotype. The recovery was dependent on B cells and was inhibited by LTβR-Ig treatment. Concurrent with HEV reversion, at day 4 following oxazolone or OVA immunization, reduced accumulation of Evans blue dye and newly activated DCs in the draining LNs revealed a temporary afferent lymphatic vessel (LV) functional insufficiency. T cell priming to a second Ag was temporarily inhibited. At day 7, lymphangiogenesis peaked in both the skin and draining LN, and afferent LV function was restored at the same time as HEV phenotype recovery. This process was delayed in the absence of B cells. LV and HEV both express the LTβR. During lymphangiogenesis in the draining LN, HEV, and LV were directly apposed; some vessels appeared to express both PNAd and LYVE-1. Pretreatment with LTβR-Ig drastically reduced the number of PNAd+LYVE-1+ vessels, suggesting a reduction in LV and HEV cross-talk. The concordance in time and function and the close physical contact between LVs and HEVs in the remodeling process after immunization indicate that the two vascular systems are in synchrony and engage in cross-talk through B cells and LTβR.
Leukotriene B4 Receptors BLT1 and BLT2: Expression and Function in Human and Murine Mast Cells
Authors: Lundeen Katherine A. , Sun Binggang , Karlsson Lars , Fourie Anne M.
Published: April 2014

Leukotriene B4 (LTB4) is a potent activator and chemoattractant for leukocytes and is implicated in several inflammatory diseases. The actions of LTB4 are mediated by two cell surface receptors, BLT1, which is predominantly expressed in peripheral blood leukocytes, and BLT2, which is expressed more ubiquitously. Recently, BLT1 expression and LTB4-dependent chemotaxis have been reported in immature mast cells (MCs). We now show the first evidence for BLT2 mRNA expression, in addition to BLT1, in murine bone marrow-derived MCs (mBMMCs) and in a human MC line (HMC-1). Protein expression of BLT1 was confirmed by mAb staining in HMC-1 cells and shown to be predominantly intracellular. Both HMC-1 cells and mBMMCs migrated to LTB4 in a dose-dependent manner in chemotaxis assays. Migration to LTB4 could be inhibited by either a BLT1- or BLT2-selective antagonist. Significant dose-dependent migration of mBMMCs also was observed to 12-(S)-hydroxyeicosotetraenoic acid, a BLT2-selective agonist, demonstrating functional BLT2 activity in these cells. Stimulation of mBMMCs with LTB4 induced transient, dose-dependent, ERK phosphorylation and changes in Akt phosphorylation. Dose-dependent ERK phosphorylation also was observed in response to 12-(S)-hydroxyeicosotetraenoic acid, indicating signaling downstream of BLT2. Pretreatment of mBMMCs with stem cell factor significantly down-regulated expression of BLT1 and BLT2 mRNA and inhibited their migration to LTB4. This study demonstrates expression of functional LTB4 receptors, both BLT1 and BLT2, in murine and human MCs and a regulatory role for stem cell factor in their expression. These receptors may mediate recruitment and accumulation of MCs in response to LTB4 production in areas of inflammation.
Cooperative Induction of a Tolerogenic Dendritic Cell Phenotype by Cytokines Secreted by Pancreatic Carcinoma Cells
Authors: Bellone Graziella , Carbone Anna , Smirne Carlo , Scirelli Tiziana , Buffolino Alessandra , Novarino Anna , Stacchini Alessandra , Bertetto Oscar , Palestro Giorgio , Sorio Claudio , Scarpa Aldo , Emanuelli Giorgio , Rodeck Ulrich
Published: April 2014

Ag presentation by dendritic cells (DC) is essential to effective antitumor T cell responses in cancer patients. Depending on their origin, maturation state, and the ambient cytokine milieu, DC can differentiate into distinct subpopulations, which preferentially either induce Th1 cell activation (CD11c+,CD123− myeloid DC (MDC)) or immunosuppressive T cell development (CD11c−,CD123+ plasmacytoid DC (PDC)). The present study was undertaken to characterize the effects of pancreatic carcinoma cell-derived cytokines on immature monocyte-derived DC (iMo-DC) in vitro and in vivo. Medium conditioned by human pancreatic carcinoma cells inhibited iMo-DC proliferation, expression of costimulatory molecules (CD80 and CD40) and of HLA-DR, and functional activity as assessed by MLR and IL-12p70 production. iMo-DC generated from pancreatic carcinoma patients in advanced stages of the disease similarly showed decreased levels of HLA-DR expression and reduced ability to stimulate MLR in response to CD40L and IFN-γ. Moreover, in tumor-patient peripheral blood, the ratio of MDC to PDC cells was lower than in healthy controls due to reduced numbers of MDC CD11c+ cells. Importantly, rather than a single cytokine, a combination of tumor-derived cytokines was responsible for these effects; these were primarily TGF-β, IL-10, and IL-6, but not vascular endothelial growth factor. In summary, we have identified an array of pancreatic carcinoma-derived cytokines that cooperatively affect iMo-DC activation in a manner consistent with ineffective antitumor immune responses.
Nucleotide-Binding Oligomerization Domain-Like Receptors: Intracellular Pattern Recognition Molecules for Pathogen Detection and Host Defense
Authors: Franchi Luigi , McDonald Christine , Kanneganti Thirumala-Devi , Amer Amal , Núñez Gabriel
Published: April 2014

The nucleotide binding oligomerization domain-like receptor (NLR) family of pattern recognition molecules is involved in a diverse array of processes required for host immune responses against invading pathogens. Unlike TLRs that mediate extracellular recognition of microbes, several NLRs sense pathogens in the cytosol and upon activation induce host defense signaling pathways. Although TLRs and NLRs differ in their mode of pathogen recognition and function, they share similar domains for microbial sensing and cooperate to elicit immune responses against the pathogen. Genetic variation in several NLR genes is associated with the development of inflammatory disorders or increased susceptibility to microbial infection. Further understanding of NLRs should provide critical insight into the mechanisms of host defense and the pathogenesis of inflammatory diseases.
Thrombospondin/CD47 Interaction: A Pathway to Generate Regulatory T Cells from Human CD4+CD25− T Cells in Response to Inflammation
Authors: Grimbert Philippe , Bouguermouh Salim , Baba Nobuyasu , Nakajima Toshiharu , Allakhverdi Zoulfia , Braun Deborah , Saito Hirohisa , Rubio Manuel , Delespesse Guy , Sarfati Marika
Published: April 2014

Thymus-derived CD4+ CD25+ T regulatory cells (Tregs) are essential for the maintenance of self-tolerance. What critical factors and conditions are required for the extra-thymic development of Tregs remains an important question. In this study, we show that the anti-inflammatory extracellular matrix protein, thrombospondin-1, promoted the generation of human peripheral regulatory T cells through the ligation of one of its receptor, CD47. CD47 stimulation by mAb or a thrombospondin-1 peptide induced naive or memory CD4+CD25− T cells to become suppressive. The latter expressed increased amounts of CTLA-4, OX40, GITR, and Foxp3 and inhibited autologous Th0, Th1, and Th2 cells. Their regulatory activity was contact dependent, TGF-β independent, and partially circumvented by IL-2. This previously unknown mechanism to induce human peripheral Tregs in response to inflammation may participate to the limitation of collateral damage induced by exacerbated responses to self or foreign Ags and thus be relevant for therapeutic intervention in autoimmune diseases and transplantation.
CD94/NKG2A Inhibits NK Cell Activation by Disrupting the Actin Network at the Immunological Synapse
Authors: Masilamani Madhan , Nguyen Connie , Kabat Juraj , Borrego Francisco , Coligan John E.
Published: April 2014

An adequate immune response is the result of the fine balance between activation and inhibitory signals. The exact means by which inhibitory signals obviate activation signals in immune cells are not totally elucidated. Human CD94/NKG2A is an ITIM-containing inhibitory receptor expressed by NK cells and some CD8+ T cells that recognize HLA-E. We show that the engagement of this receptor prevents NK cell activation by disruption of the actin network and exclusion of lipid rafts at the point of contact with its ligand (inhibitory NK cell immunological synapse, iNKIS). CD94/NKG2A engagement leads to recruitment and activation of src homology 2 domain-bearing tyrosine phosphatase 1. This likely explains the observed dephosphorylation of guanine nucleotide exchange factor and regulator of actin, Vav1, as well as ezrin-radixin-moesin proteins that connect actin filaments to membrane structures. In contrast, NK cell activation by NKG2D induced Vav1 and ezrin-radixin-moesin phosphorylation. Thus, CD94/NKG2A prevents actin-dependent recruitment of raft-associated activation receptors complexes to the activating synapse. This was further substantiated by showing that inhibition of actin polymerization abolished lipid rafts exclusion at the iNKIS, whereas cholesterol depletion had no effect on actin disruption at the iNKIS. These data indicate that the lipid rafts exclusion at the iNKIS is an active process which requires an intact cytoskeleton to maintain lipid rafts outside the inhibitory synapse. The net effect is to maintain an inhibitory state in the proximity of the iNKIS, while allowing the formation of activation synapse at distal points within the same NK cell.
Different Neurotropic Pathogens Elicit Neurotoxic CCR9- or Neurosupportive CXCR3-Expressing Microglia
Authors: Li He , Gang Zhou , Yuling He , Luokun Xie , Jie Xiong , Hao Lei , Li Wei , Chunsong Hu , Junyan Liu , Mingshen Jiang , Youxin Jin , Feili Gong , Boquan Jin , Jinquan Tan
Published: April 2014

What mechanism that determines microglia accomplishing destructive or constructive role in CNS remains nebulous. We report here that intracranial priming and rechallenging with Toxoplasma gondii in mice elicit neurotoxic CCR9+Irg1+ (immunoresponsive gene 1) microglia, which render resistance to apoptosis and produce a high level of TNF-α; priming and rechallenging with lymphocytic choriomeningitis virus elicit neurosupportive CXCR3+Irg1− microglia, which are sensitive to apoptosis and produce a high level of IL-10 and TGF-β. Administration of CCR9 and/or Irg1 small interfering RNA alters the frequency and functional profiles of neurotoxic CCR9+Irg1+ and neurosupportive CXCR3+Irg1− microglia in vivo. Moreover, by using a series of different neurotropic pathogens, including intracellular parasites, chronic virus, bacteria, toxic substances, and CNS injury to intracranially prime and subsequent rechallenge mice, the bi-directional elicitation of microglia has been confirmed as neurotoxic CCR9+Irg1+ and neurosupportive CXCR3+Irg1− cells in these mouse models. These data suggest that there exist two different types of microglia, providing with a novel insight into microglial involvement in neurodegenerative and neuroinflammatory pathogenesis such as Alzheimer’s disease and AIDS dementia.
Mechanisms Regulating the Proliferative Potential of Human CD8+ T Lymphocytes Overexpressing Telomerase
Authors: Menzel Olivier , Migliaccio Marco , Goldstein Darlene R. , Dahoun Sophie , Delorenzi Mauro , Rufer Nathalie
Published: April 2014

In human somatic cells, including T lymphocytes, telomeres progressively shorten with each cell division, eventually leading to a state of cellular senescence. Ectopic expression of telomerase results in the extension of their replicative life spans without inducing changes associated with transformation. However, it is yet unknown whether somatic cells that overexpress telomerase are physiologically indistinguishable from normal cells. Using CD8+ T lymphocyte clones overexpressing telomerase, we investigated the molecular mechanisms that regulate T cell proliferation. In this study, we show that early passage T cell clones transduced or not with human telomerase reverse transcriptase displayed identical growth rates upon mitogenic stimulation and no marked global changes in gene expression. Surprisingly, reduced proliferative responses were observed in human telomerase reverse transcriptase-transduced cells with extended life spans. These cells, despite maintaining high expression levels of genes involved in the cell cycle progression, also showed increased expression in several genes found in common with normal aging T lymphocytes. Strikingly, late passage T cells overexpressing telomerase accumulated the cyclin-dependent inhibitors p16Ink4a and p21Cip1 that have largely been associated with in vitro growth arrest. We conclude that alternative growth arrest mechanisms such as those mediated by p16Ink4a and p21Cip1 still remained intact and regulated the growth potential of cells independently of their telomere status.
The Major Glycoprotein Allergen from Arachis hypogaea, Ara h 1, Is a Ligand of Dendritic Cell-Specific ICAM-Grabbing Nonintegrin and Acts as a Th2 Adjuvant In Vitro
Authors: Shreffler Wayne G. , Castro Russell R. , Kucuk Z. Yesim , Charlop-Powers Zachary , Grishina Galina , Yoo Steven , Burks A. Wesley , Sampson Hugh A.
Published: April 2014

Nonmammalian glycan structures from helminths act as Th2 adjuvants. Some of these structures are also common on plant glycoproteins. We hypothesized that glycan structures present on peanut glycoallergens act as Th2 adjuvants. Peanut Ag (PNAg), but not deglycosylated PNAg, activated monocyte-derived dendritic cells (MDDCs) as measured by MHC/costimulatory molecule up-regulation, and by their ability to drive T cell proliferation. Furthermore, PNAg-activated MDDCs induced 2- to 3-fold more IL-4- and IL-13-secreting Th2 cells than immature or TNF/IL-1-activated MDDCs when cultured with naive CD4+ T cells. Human MDDCs rapidly internalized Ag in a calcium- and glycan-dependent manner consistent with recognition by C-type lectin. Dendritic cell (DC)-specific ICAM-grabbing nonintegrin (DC-SIGN) (CD209) was shown to recognize PNAg by enhanced uptake in transfected cell lines. To identify the DC-SIGN ligand from unfractionated PNAg, we expressed the extracellular portion of DC-SIGN as an Fc-fusion protein and used it to immunoprecipitate PNAg. A single glycoprotein was pulled down in a calcium-dependent manner, and its identity as Ara h 1 was proven by immunolabeling and mass spectrometry. Purified Ara h 1 was found to be sufficient for the induction of MDDCs that prime Th2-skewed T cell responses. Both PNAg and purified Ara h 1 induced Erk 1/2 phosphorylation of MDDCs, consistent with previous reports on the effect of Th2 adjuvants on DCs.