bluuebunny/crossref_metadata_2025 huggingface dataset:
The Oxidation State of Phospholipids Controls the Oxidative Burst in Neutrophil Granulocytes
Authors: Blüml Stephan , Rosc Berit , Lorincz Akos , Seyerl Maria , Kirchberger Stefanie , Oskolkova Olga , Bochkov Valery N. , Majdic Otto , Ligeti Erzsebet , Stöckl Johannes
Published: April 2014

The activation of neutrophil granulocytes has to be carefully controlled to balance desired activity against invading pathogens while avoiding overwhelming activation leading to host tissue damage. We now show that phospholipids are potential key players in this process by either enhancing or dampening the production of reactive oxygen species (ROS) during the oxidative burst. Unoxidized phospholipids induce the production of ROS, and they also work synergistically with FMLP in potentiating the oxidative burst in neutrophil granulocytes. Oxidation of these phospholipids, however, turns them into potent inhibitors of the oxidative burst. OxPls specifically inhibit ROS production by inhibiting the assembly of the phagocyte oxidase complex but do not alter neutrophil viability, nor do they interfere with MAPK activation. Furthermore, up-regulation of the activation marker Mac-1 and phagocytosis of bacteria is not affected. Therefore, phospholipids may act as sensors of oxidative stress in tissues and either positively or negatively regulate neutrophil ROS production according to their oxidation state.
Lung Resident Mesenchymal Stem Cells Isolated from Human Lung Allografts Inhibit T Cell Proliferation via a Soluble Mediator
Authors: Jarvinen Lamis , Badri Linda , Wettlaufer Scott , Ohtsuka Takashi , Standiford Theodore J. , Toews Galen B. , Pinsky David J. , Peters-Golden Marc , Lama Vibha N.
Published: April 2014

Development of allograft rejection continues to be the major determinant of morbidity and mortality postlung transplantation. We have recently demonstrated that a population of donor-derived mesenchymal stem cells is present in human lung allografts and can be isolated and expanded ex vivo. In this study, we investigated the impact of lung resident mesenchymal stem cells (LR-MSCs), derived from allografts of human lung transplant recipients, on T cell activation in vitro. Similar to bone marrow-derived MSCs, LR-MSCs did not express MHC II or the costimulatory molecules CD80 or CD86. In vitro, LR-MSCs profoundly suppressed the proliferative capacity of T cells in response to a mitogenic or an allogeneic stimulus. The immunosuppressive function of LR-MSCs was also noted in the absence of direct cell contact, indicating that LR-MSCs mediated their effect predominantly via a soluble mediator. LR-MSCs isolated from lung transplant recipients demonstrated PGE2 secretion at baseline (385 ± 375 pg/ml), which increased in response to IL-1β (1149 ± 1081 pg/ml). The addition of PG synthesis inhibitors (indomethacin and NS-398) substantially abrogated LR-MSC-mediated immunosuppression, indicating that PGE2 may be one of the major soluble mediators impacting T cell activity. This is the first report to demonstrate that human tissue-derived MSCs isolated from an allogeneic environment have the potential to mediate immunological responses in vitro.
Cutting Edge: IL-1 Controls the IL-23 Response Induced by Gliadin, the Etiologic Agent in Celiac Disease
Authors: Harris Kristina M. , Fasano Alessio , Mann Dean L.
Published: April 2014

IL-23 has been implicated in the pathogenesis of several tissue-specific autoimmune diseases. Currently, celiac disease (CD) is the only autoimmune disease in which both the major genetic (95% HLA-DQ2+) and etiologic factors (dietary glutens) for susceptibility are known. We demonstrate that wheat gliadin induces significantly greater production of IL-23, IL-1β, and TNF-α in PBMC from CD patients compared with HLA-DQ2+ healthy controls, strongly advocating a role for IL-23 in the pathogenesis of CD. Moreover, IL-1β alone triggered IL-23 secretion and the IL-1R antagonist inhibited this response in PBMC and purified monocytes. This sequence of events was replicated by β-glucan, another substance known to induce IL-23 production. Our results suggest that gliadin and β-glucan stimulate IL-23 secretion through induction of the IL-1 signaling pathway and reveal for the first time that the IL-1 system regulates IL-23 production. These findings may provide therapeutic targets for this disease and other inflammatory conditions mediated by IL-23.
Bone Marrow-Derived Dendritic Cells Generated in the Presence of Resolvin E1 Induce Apoptosis of Activated CD4+ T Cells
Authors: Vassiliou Evros K. , Kesler Olga M. , Tadros James H. , Ganea Doina
Published: April 2014

In contrast to the role of dendritic cells (DC) in immunity and tolerance, little is known about their possible role in the resolution of inflammatory processes. In addition to the reduction in the number of infiltrating immune cells, the elimination of effector T cells already present at the inflammatory site represents an essential step toward resolution. Recently, lipid mediators such as the omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid and their metabolites, including resolvin E1 (RvE1), have been shown to accumulate in inflammatory foci during the resolution phase. RvE1 has been reported to reduce immune cell infiltration and proinflammatory cytokine production. In this study we report that DC exposed to RvE1, especially during differentiation, acquire the capacity to induce apoptosis of activated T cells through the induction and activity of indoleamine 2,3-dioxygenase. To our knowledge, this study is the first to report on an omega-3 fatty acid derivative inducing indoleamine 2,3-dioxygenase expression in DC. RvE1-exposed DC maintain an immature chemokine receptor expression pattern even following TLR stimulation, with high CCR5 and no CCR7 expression. This effect implies that DC exposed to RvE1 and pathogens remain at the inflammatory site, instead of migrating to lymph nodes, and induce apoptosis in effector T cells infiltrating the inflammatory site. To our knowledge, the DC described in this study represent a new functional DC subtype, whose essential function resides in the resolution of inflammation.
Early Growth Response Genes Regulate B Cell Development, Proliferation, and Immune Response
Authors: Gururajan Murali , Simmons Alan , Dasu Trivikram , Spear Brett T. , Calulot Christopher , Robertson Darrell A. , Wiest David L. , Monroe John G. , Bondada Subbarao
Published: April 2014

Egr-1 (early growth response gene-1) is an immediate early gene encoding a zinc finger motif-containing transcription factor. Upon cross-linking of BCR, mature B cells undergo proliferation with an increase in Egr-1 message. Immature B lymphoma cells that express Egr-1 message and protein constitutively are growth inhibited when Egr-1 is down-regulated by negative signals from BCR or by antisense oligonucleotides. To test the hypothesis that Egr-1 is important for B cell development, we examined B cells from primary and secondary lymphoid organs in Egr-1−/− mice. Marginal zone B cell development was arrested in these mice, whereas the B cells in all other compartments were increased. To test the hypothesis that Egr-1 function may be partially compensated by other Egr family members, we developed transgenic mice expressing a dominant negative form of Egr-1, which lacks the trans activation domain but retains the DNA-binding domain, in a B cell-specific manner. There was a decrease in B lymphopoiesis in the bone marrow accompanied by a reduction in splenic immature and mature B cells as well as marginal zone B cells in the transgenic mice. Moreover, transgenic mice respond poorly to BCR cross-linking in vitro and T-independent and T-dependent Ags in vivo.
Monocyte Migration and LFA-1-Mediated Attachment to Brain Microvascular Endothelia Is Regulated by SDF-1α through Lyn Kinase
Authors: Malik Mobeen , Chen Ying-Yu , Kienzle Martha F. , Tomkowicz Brian E. , Collman Ronald G. , Ptasznik Andrzej
Published: April 2014

Infiltration of activated monocytes into the brain is a prerequisite for the development of various neurological disorders such as HIV-associated dementia, multiple sclerosis, and other inflammatory processes. In these pathologies, the chemokine SDF-1α (CXCL12) is over-expressed and might attract monocytes into the CNS. We demonstrate here that SDF-1α stimulates migration of monocytes through its receptor, CXCR4, and decreases monocyte adherence to surfaces coated with ICAM-1, a ligand for β2 integrins. SDF-1α also decreases monocyte adherence to brain microvascular endothelial cells (BMVEC) that are activated with TNF-α, IL-1β, or recombinant envelope glycoprotein from HIV-1, which increase BMVEC expression of ICAM-1. The decreased adherence is linked to down-regulation on monocytes of the activation-dependent epitope of the β2 integrin LFA-1 by SDF-1α. Knockdown of Lyn in monocytes using small interfering RNA decreases SDF-1α-mediated migration and prevents the inhibition of monocyte attachment to ICAM-1 and activated BMVEC. Thus, in SDF-1α-stimulated monocytes, Lyn acts as a positive regulator of migration and a negative regulator of adhesion to BMVEC through the LFA-1 integrin. These results provide a novel Lyn-mediated signaling mechanism for the regulation of monocyte movement at the blood-brain barrier.
Type I IFNs Play a Role in Early Resistance, but Subsequent Susceptibility, to the African Trypanosomes
Authors: Lopez Rebecca , Demick Karen P. , Mansfield John M. , Paulnock Donna M.
Published: April 2014

Macrophages express a spectrum of proinflammatory and regulatory mediators during African trypanosomiasis. Microarray analyses revealed similar profiles of induced genes in macrophages stimulated with the trypanosome soluble variant surface glycoprotein in vitro and in macrophages taken from infected mice. Genes associated with the acute phase response and with type I IFN responses were prominent components of the macrophage activation profiles expressed within 72 h in vitro and in vivo. Thus, induction of proinflammatory gene expression is a characteristic of early trypanosome infection that is driven primarily by soluble variant surface glycoprotein exposure, and it may be that IFN-α/β plays a central role in regulation of early resistance to trypanosomes. To test this hypothesis, we assessed parameters of infection in mouse strains with genetic alterations in the IFN-α/β response pathway. We found that Ifnar1−/− mice, which lack the receptor for type I IFNs, exhibited delayed control of parasite burden during the first week of infection and died earlier than did wild-type controls. However, infection of Ubp43−/− mice, which are hyperresponsive to type I IFNs, did not exhibit enhanced resistance to trypanosomes. Instead, these animals also failed to control parasite burden and were more susceptible than wild-type animals. Additionally, the Ubp43−/− mice exhibited a significant defect in IFN-γ production, which is definitively linked to host resistance in trypanosomiasis. These results show that type I IFNs play a role in early control of parasites in infected mice but may contribute to down-regulation of IFN-γ production and subsequent loss of host resistance later in infection.
Cathelicidin Administration Protects Mice from Bacillus anthracis Spore Challenge
Authors: Lisanby Mark W. , Swiecki Melissa K. , Dizon Brian L. P. , Pflughoeft Kathryn J. , Koehler Theresa M. , Kearney John F.
Published: April 2014

Cathelicidins are a family of cationic peptides expressed in mammals that possess numerous bactericidal and immunomodulatory properties. In vitro analyses showed that human, mouse, and pig cathelicidins inhibited Bacillus anthracis bacterial growth at micromolar concentrations in the presence or absence of capsule. Combined in vitro analyses of the effects of each peptide on spore germination and vegetative outgrowth by time lapse phase contrast microscopy, transmission electron microscopy, and flow cytometric analysis showed that only the pig cathelicidin was capable of directly arresting vegetative outgrowth and killing the developing bacilli within the confines of the exosporium. C57BL/6 mice were protected from spore-induced death by each cathelicidin in a time- and dose-dependent manner. Protection afforded by the porcine cathelicidin was due to its bactericidal effects, whereas the human and mouse cathelicidins appeared to mediate protection through increased recruitment of neutrophils to the site of infection. These findings suggest that cathelicidins might be utilized to augment the initial innate immune response to B. anthracis spore exposure and prevent the development of anthrax.
Annexin A1 Regulates Intestinal Mucosal Injury, Inflammation, and Repair
Authors: Babbin Brian A. , Laukoetter Mike G. , Nava Porfirio , Koch Stefan , Lee Winston Y. , Capaldo Christopher T. , Peatman Eric , Severson Eric A. , Flower Roderick J. , Perretti Mauro , Parkos Charles A. , Nusrat Asma
Published: April 2014

During mucosal inflammation, a complex array of proinflammatory and protective mechanisms regulates inflammation and severity of injury. Secretion of anti-inflammatory mediators is a mechanism that is critical in controlling inflammatory responses and promoting epithelial restitution and barrier recovery. AnxA1 is a potent anti-inflammatory protein that has been implicated to play a critical immune regulatory role in models of inflammation. Although AnxA1 has been shown to be secreted in intestinal mucosal tissues during inflammation, its potential role in modulating the injury/inflammatory response is not understood. In this study, we demonstrate that AnxA1-deficient animals exhibit increased susceptibility to dextran sulfate sodium (DSS)-induced colitis with greater clinical morbidity and histopathologic mucosal injury. Furthermore, impaired recovery following withdrawal of DSS administration was observed in AnxA1 (−/−) animals compared with wild-type (WT) control mice that was independent of inflammatory cell infiltration. Since AnxA1 exerts its anti-inflammatory properties through stimulation of ALX/FPRL-1, we explored the role of this receptor-ligand interaction in regulating DSS-induced colitis. Interestingly, treatment with an ALX/FPRL-1 agonist, 15-epi-lipoxin A4 reversed the enhanced sensitivity of AnxA1 (−/−) mice to DSS colitis. In contrast, 15-epi-lipoxin A4 did not significantly improve the severity of disease in WT animals. Additionally, differential expression of ALX/FPLR-1 in control and DSS-treated WT and AnxA1-deficient animals suggested a potential role for AnxA1 in regulating ALX/FPRL-1 expression under pathophysiological conditions. Together, these results support a role of endogenous AnxA1 in the protective and reparative properties of the intestinal mucosal epithelium.
STAT4 Isoforms Differentially Regulate Th1 Cytokine Production and the Severity of Inflammatory Bowel Disease
Authors: O'Malley John T. , Eri Rajaraman D. , Stritesky Gretta L. , Mathur Anubhav N. , Chang Hua-Chen , HogenEsch Harm , Srinivasan Mythily , Kaplan Mark H.
Published: April 2014

STAT4, a critical regulator of inflammation in vivo, can be expressed as two alternative splice forms, a full-length STAT4α, and a STAT4β isoform lacking a C-terminal transactivation domain. Each isoform is sufficient to program Th1 development through both common and distinct subsets of target genes. However, the ability of these isoforms to mediate inflammation in vivo has not been examined. Using a model of colitis that develops following transfer of CD4+ CD45RBhigh T cells expressing either the STAT4α or STAT4β isoform into SCID mice, we determined that although both isoforms mediate inflammation and weight loss, STAT4β promotes greater colonic inflammation and tissue destruction. This correlates with STAT4 isoform-dependent expression of TNF-α and GM-CSF in vitro and in vivo, but not Th1 expression of IFN-γ or Th17 expression of IL-17, which were similar in STAT4α- and STAT4β-expressing T cells. Thus, higher expression of a subset of inflammatory cytokines from STAT4β-expressing T cells correlates with the ability of STAT4β-expressing T cells to mediate more severe inflammatory disease.
Stimulatory Role of Lysophosphatidic Acid in Cyclooxygenase-2 Induction by Synovial Fluid of Patients with Rheumatoid Arthritis in Fibroblast-Like Synovial Cells
Authors: Nochi Hiromi , Tomura Hideaki , Tobo Masayuki , Tanaka Nobuyuki , Sato Koichi , Shinozaki Tetsuya , Kobayashi Tsutomu , Takagishi Kenji , Ohta Hideo , Okajima Fumikazu , Tamoto Koichi
Published: April 2014

While inflammatory cytokines are well-recognized critical factors for the induction of cyclooxygenase-2 (COX-2) in activated fibroblast-like synovial cells, the roles of biologically active components other than inflammatory cytokines in synovial fluid remain unknown. Herein, we assessed the role of lysophosphatidic acid (LPA), a pleiotropic lipid mediator, in COX-2 induction using synovial fluid of patients with rheumatoid arthritis (RA) in fibroblast-like RA synovial cells. Synovial fluid from RA patients stimulated COX-2 induction, which was associated with prostaglandin E2 production, in RA synovial cells. The synovial fluid-induced actions were inhibited by Gi/o protein inhibitor pertussis toxin and LPA receptor antagonist 3-(4-[4-([1-(2-chlorophenyl)ethoxy]carbonyl amino)-3-methyl-5-isoxazolyl] benzylsulfanyl) propanoic acid (Ki16425). In fact, LPA alone significantly induced COX-2 expression and enhanced IL-1α- or IL-1β-induced enzyme expression in a manner sensitive to pertussis toxin and Ki16425. RA synovial cells abundantly expressed LPA1 receptor compared with other LPA receptor subtypes. Moreover, synovial fluid contains a significant amount of LPA, an LPA-synthesizing enzyme autotaxin, and its substrate lysophosphatidylcholine. In conclusion, LPA existing in synovial fluid plays a critical role in COX-2 induction in collaboration with inflammatory cytokines in RA synovial cells. Ki16425-sensitive LPA receptors may be therapeutic targets for RA.
Requirements for Effective Antitumor Responses of TCR Transduced T Cells
Authors: de Witte Moniek A. , Jorritsma Annelies , Kaiser Andrew , van den Boom Marly D. , Dokter Maarten , Bendle Gavin M. , Haanen John B. A. G. , Schumacher Ton N. M.
Published: April 2014

Adoptive transfer of TCR gene-modified T cells has been proposed as an attractive approach to target tumors for which it is difficult or impossible to induce strong tumor-specific T cell responses by vaccination. Whereas the feasibility of generating tumor Ag-specific T cells by gene transfer has been demonstrated, the factors that determine the in vivo effectiveness of TCR-modified T cells are largely unknown. We have analyzed the value of a number of clinically feasible strategies to enhance the antitumor potential of TCR modified T cells. These experiments reveal three factors that contribute greatly to the in vivo potency of TCR-modified T cells. First, irradiation-induced host conditioning is superior to vaccine-induced activation of genetically modified T cells. Second, increasing TCR expression through genetic optimization of TCR sequences has a profound effect on in vivo antitumor activity. Third, a high precursor frequency of TCR modified T cells within the graft is essential. Tumors that ultimately progress in animals treated with this optimized regimen for TCR-based adoptive cell transfer invariably display a reduced expression of the target Ag. This suggests TCR gene therapy can achieve a sufficiently strong selective pressure to warrant the simultaneous targeting of multiple Ags. The strategies outlined in this study should be of value to enhance the antitumor activity of TCR-modified T cells in clinical trials.
Gene Expression Profiling in Autoimmune Noninfectious Uveitis Disease
Authors: Li Zhuqing , Liu Baoying , Maminishkis Arvydas , Mahesh Sankaranarayana P. , Yeh Steven , Lew Julie , Lim Wee Kiak , Sen H. Nida , Clarke Grace , Buggage Ronald , Miller Sheldon S. , Nussenblatt Robert B.
Published: April 2014

Noninfectious uveitis is a predominantly T cell-mediated autoimmune, intraocular inflammatory disease. To characterize the gene expression profile from patients with noninfectious uveitis, PBMCs were isolated from 50 patients with clinically characterized noninfectious uveitis syndrome. A pathway-specific cDNA microarray was used for gene expression profiling and real-time PCR array for further confirmation. Sixty-seven inflammation- and autoimmune-associated genes were found differentially expressed in uveitis patients, with 28 of those genes being validated by real-time PCR. Several genes previously unknown for autoimmune uveitis, including IL-22, IL-19, IL-20, and IL-25/IL-17E, were found to be highly expressed among uveitis patients compared with the normal subjects with IL-22 expression highly variable among the patients. Furthermore, we show that IL-22 can affect primary human retinal pigment epithelial cells by decreasing total tissue resistance and inducing apoptosis possibly by decreasing phospho-Bad level. In addition, the microarray data identified a possible uveitis-associated gene expression pattern, showed distinct gene expression profiles in patients during periods of clinical activity and quiescence, and demonstrated similar expression patterns in related patients with similar clinical phenotypes. Our data provide the first evidence that a subset of IL-10 family genes are implicated in noninfectious uveitis and that IL-22 can affect human retinal pigment epithelial cells. The results may facilitate further understanding of the molecular mechanisms of autoimmune uveitis and other autoimmune originated inflammatory diseases.
Cutting Edge: TLR Ligands Increase TCR Triggering by Slowing Peptide-MHC Class I Decay Rates
Authors: Rudd Brian D. , Brien James D. , Davenport Miles P. , Nikolich-Žugich Janko
Published: April 2014

TLR ligands are among the key stimuli driving the optimal dendritic cell (DC) maturation critical for strong and efficacious T cell priming. In this study, we show that part of this effect occurs via increased TCR triggering. Pretreatment of DCs with TLR ligands resulted in the triggering of many more TCRs in responding CD8+ T cells. Importantly, even when DCs expressed the same amount of cognate peptide-MHC (pMHC) molecules, TLR ligand treatment resulted in down-regulation of larger numbers of TCR molecules. This was independent of the up-regulation of costimulatory, adhesion or cytokine molecules or the amount of noncognate pMHCs. Rather, DCs pretreated with TLR ligands exhibited increased stability of cognate pMHCs, enabling extended TCR triggering. These findings are of potential importance to T cell vaccination.
Cutting Edge: CD47 Controls the In Vivo Proliferation and Homeostasis of Peripheral CD4+CD25+Foxp3+ Regulatory T Cells That Express CD103
Authors: Van Vu Quang , Darwiche Jinane , Raymond Marianne , Lesage Sylvie , Bouguermouh Salim , Rubio Manuel , Sarfati Marika
Published: April 2014

Peripheral CD103+Foxp3+ regulatory T cells (Tregs) can develop both from conventional naive T cells upon cognate Ag delivery under tolerogenic conditions and from thymic-derived, expanded/differentiated natural Tregs. We here show that CD47 expression, a marker of self on hematopoietic cells, selectively regulated CD103+Foxp3+ Treg homeostasis at the steady state. First, the proportion of effector/memory-like (CD44highCD62Llow) CD103+Foxp3+ Tregs rapidly augmented with age in CD47-deficient mice (CD47−/−) as compared with age-matched control littermates. Yet, the percentage of quiescent (CD44lowCD62Lhigh) CD103−Foxp3+ Tregs remained stable. Second, the increased proliferation rate (BrdU incorporation) observed within the CD47−/−Foxp3+ Treg subpopulation was restricted to those Tregs expressing CD103. Third, CD47−/− Tregs maintained a normal suppressive function in vitro and in vivo and their increased proportion in old mice led to a decline of Ag-specific T cell responses. Thus, sustained CD47 expression throughout life is critical to avoid an excessive expansion of CD103+ Tregs that may overwhelmingly inhibit Ag-specific T cell responses.
Spontaneous Colitis Occurrence in Transgenic Mice with Altered B7-Mediated Costimulation
Authors: Kim Gisen , Turovskaya Olga , Levin Matthew , Byrne Fergus R. , Whoriskey John S. , McCabe James G. , Kronenberg Mitchell
Published: April 2014

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4+ T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3+ regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.
The Exocytosis Regulator Synaptotagmin V Controls Phagocytosis in Macrophages
Authors: Vinet Adrien F. , Fukuda Mitsunori , Descoteaux Albert
Published: April 2014

Synaptotagmins (Syts) play a key role in the regulation of Ca2+-triggered exocytosis and membrane fusion events, two crucial events associated to the phagocytic process. In the present study, we investigated the role of Syt V, a regulator of focal exocytosis, in phagocytosis. In macrophages, Syt V is localized on recycling endosomes and on filopodia-like structures and is recruited to the nascent phagosomes independently of the phagocytic receptor engaged. Silencing of Syt V by RNA interference revealed a role for this protein for phagocytosis, particularly under conditions of high membrane demand. In contrast, silencing of Syt V had no effect on the recruitment of the lysosomal marker LAMP1 to phagosomes, indicating that phagosome maturation is not regulated by Syt V. Collectively, these results illustrate the importance of Syt V in the regulation of an important innate function of macrophages. Furthermore, our results are consistent with the concept that focal exocytosis of endocytic organelles is a key event in phagocytosis and suggest that Syt V regulates this process.
Casitas B-Lineage Lymphoma b Inhibits Antigen Recognition and Slows Cell Cycle Progression at Late Times during CD4+ T Cell Clonal Expansion
Authors: Zhang Ruan , Zhang Na , Mueller Daniel L.
Published: April 2014

Optimal clonal expansion of CD4+ T cells during the primary response to Ag requires prolonged TCR recognition of peptide Ag/MHC complexes. In this study, we investigated the capacity of Casitas B-lineage lymphoma b (Cbl-b) to counter-regulate late TCR signals necessary for continued cell division in vivo. During the first 24 h of a primary response to Ag, Cblb−/− 5C.C7 CD4+ T cells demonstrated no alteration in CD69, CD25, and CD71 up-regulation or cell growth as compared with wild-type cells. Nevertheless, beyond 24 h, both the expression of CD71 and the rate of cell division were increased in the genetic absence of Cbl-b, leading to an augmented clonal expansion. This deregulation of late T cell proliferation in the absence of Cbl-b resulted in part from an inability of Cblb−/− T cells to desensitize Akt, PLCγ-1, and ERK phosphorylation events downstream of the TCR/CD3 complex, in addition to their failure to undergo a growth arrest in the absence of Ag. These observations now suggest a novel role for Cbl-b in triggering the exit from cell cycle at the end of a CD4+ T cell clonal expansion.
Imidazoquinoline Acts as Immune Adjuvant for Functional Alteration of Thymic Stromal Lymphopoietin-Mediated Allergic T Cell Response
Authors: Torii Yoshitaro , Ito Tomoki , Amakawa Ryuichi , Sugimoto Hiroyuki , Amuro Hideki , Tanijiri Tsutomu , Katashiba Yuichi , Ogata Makoto , Yokoi Takashi , Fukuhara Shirou
Published: April 2014

Atopic dermatitis is a major allergic disease that develops through dysregulation of Th2-mediated inflammation. Although dendritic cells (DCs) have been thought to play a critical role in the upstream phase of the allergic cascade, conventional drugs such as steroids and chemical mediator antagonists target the effector cells or factors in allergic inflammation. Recently, it has been demonstrated that interaction between thymic stromal lymphopoietin (TSLP) and human DCs plays an essential role in evoking inflammatory Th2 responses in allergy through OX40 ligand expression on DCs. In this study, we provide evidence that R848, an imidazoquinoline compound, which is a TLR ligand and a strong Th1 response-inducing reagent, is a potent adjuvant for the alteration of the Th2-inducing potency of human DCs activated by TSLP (TSLP-DCs). R848 inhibited the inflammatory Th2-inducing capacity of TSLP-DCs and redirected them to possessing an IL-10 and IFN-γ-producing regulatory Th1-inducing capacity. This functional alteration depended on both repression of OX40 ligand expression and induction of IL-12 production from DCs by the addition of R848. Additionally, R848 had the ability to inhibit the TSLP-mediated expansion and maintenance of the Th2 memory response. These findings suggest that imidazoquinoline may be a useful in the treatment of allergic diseases that are triggered by TSLP.
Astrocytic Regulation of Human Monocytic/Microglial Activation
Authors: Kostianovsky Alex M. , Maier Lisa M. , Anderson Richard C. , Bruce Jeffrey N. , Anderson David E.
Published: April 2014

Recent reports have described reduced immunological responsiveness and stimulatory capacity among monocytes/microglia that infiltrate malignant human gliomas. Herein, we demonstrate that culture of ex vivo human monocytes or primary human microglia with tumor cells isolated from glioblastoma multiforme (GBM) specimens renders them tolerogenic, capable of suppressing the function of ex vivo monocytes in the absence of tumor cells or their soluble factors. We demonstrate that the tolerance induced in monocytes/microglia by GBM tumor cells is not associated with interference with the signaling cascade associated with TLR- or CD40-induced monocyte activation. Rather, these tumor cells appear to up-regulate pathways that antagonize positive signaling pathways, including but not limited to STAT3 and STAT5. Finally, we demonstrate that the tolerogenic properties of GBM tumor cells amplify properties inherent to nontransformed astrocytes. Future studies that identify all of the molecular mechanisms by which astrocytes and malignant gliomas suppress monocyte/microglial function will have dual therapeutic benefits: suppressing these pathways may benefit patients with astrocytic tumors, while enhancing them may benefit patients with autoimmune processes within the CNS, such as multiple sclerosis.
Different Development of Myelin Basic Protein Agonist- and Antagonist-Specific Human TCR Transgenic T Cells in the Thymus and Periphery
Authors: Kawamura Kazuyuki , Yao Karen , Shukaliak-Quandt Jacqueline A. , Huh Jaebong , Baig Mirza , Quigley Laura , Ito Naoko , Necker Antje , McFarland Henry F. , Muraro Paolo A. , Martin Roland , Ito Kouichi
Published: April 2014

Myelin basic protein (MBP)-specific T cells are thought to play a role in the development of multiple sclerosis. MBP residues 111–129 compose an immunodominant epitope cluster restricted by HLA-DRB1*0401. The sequence of residues 111–129 of MBP (MBP111–129) differs in humans (MBP122:Arg) and mice (MBP122:Lys) at aa 122. We previously found that ∼50% of human MBP111–129 (MBP122:Arg)-specific T cell clones, including MS2-3C8 can proliferate in response to mouse MBP111–129 (MBP122:Lys). However, the other half of T cell clones, including HD4-1C2, cannot proliferate in response to MBP111–129 (MBP122:Lys). We found that MBP111–129 (MBP122:Lys) is an antagonist for HD4-1C2 TCR, therefore, MS2-3C8 and HD4-1C2 TCRs are agonist- and antagonist-specific TCRs in mice, respectively. Therefore, we examined the development of HD4-1C2 TCR and MS2-3C8 TCR transgenic (Tg) T cells in the thymus and periphery. We found that dual TCR expression exclusively facilitates the development of MBP111–129 TCR Tg T cells in the periphery of HD4-1C2 TCR/HLA-DRB1*0401 Tg mice although it is not required for their development in the thymus. We also found that MS2-3C8 TCR Tg CD8+ T cells develop along with MS2-3C8 TCR Tg CD4+ T cells, and that dual TCR expression was crucial for the development of MS2-3C8 TCR Tg CD4+ and CD8+ T cells in the thymus and periphery, respectively. These results suggest that thymic and peripheral development of MBP-specific T cells are different; however, dual TCR expression can facilitate their development.
Uncoupling the Roles of HLA-DRB1 and HLA-DRB5 Genes in Multiple Sclerosis
Authors: Caillier Stacy J. , Briggs Farren , Cree Bruce A. C. , Baranzini Sergio E. , Fernandez-Viña Marcelo , Ramsay Patricia P. , Khan Omar , Royal Walter , Hauser Stephen L. , Barcellos Lisa F. , Oksenberg Jorge R.
Published: April 2014

Genetic susceptibility to multiple sclerosis (MS) is associated with the MHC located on chromosome 6p21. This signal maps primarily to a 1-Mb region encompassing the HLA class II loci, and it segregates often with the HLA-DQB1*0602, -DQA1*0102, -DRB1*1501, -DRB5*0101 haplotype. However, the identification of the true predisposing gene or genes within the susceptibility haplotype has been handicapped by the strong linkage disequilibrium across the locus. African Americans have greater MHC haplotypic diversity and distinct patterns of linkage disequilibrium, which make this population particularly informative for fine mapping efforts. The purpose of this study was to establish the telomeric boundary of the HLA class II region affecting susceptibility to MS by assessing genetic association with the neighboring HLA-DRB5 gene as well as seven telomeric single nucleotide polymorphisms in a large, well-characterized African American dataset. Rare DRB5*null individuals were previously described in African populations. Although significant associations with both HLA-DRB1 and HLA-DRB5 loci were present, HLA-DRB1*1503 was associated with MS in the absence of HLA-DRB5, providing evidence for HLA-DRB1 as the primary susceptibility gene. Interestingly, the HLA-DRB5*null subjects appear to be at increased risk for developing secondary progressive MS. Thus, HLA-DRB5 attenuates MS severity, a finding consistent with HLA-DRB5’s proposed role as a modifier in experimental autoimmune encephalomyelitis. Additionally, conditional haplotype analysis revealed a susceptibility signal at the class III AGER locus independent of DRB1. The data underscore the power of the African American MS dataset to identify disease genes by association in a region of high linkage disequilibrium.
T Cell Responses to Whole SARS Coronavirus in Humans
Authors: Li Chris Ka-fai , Wu Hao , Yan Huiping , Ma Shiwu , Wang Lili , Zhang Mingxia , Tang Xiaoping , Temperton Nigel J. , Weiss Robin A. , Brenchley Jason M. , Douek Daniel C. , Mongkolsapaya Juthathip , Tran Bac-Hai , Lin Chen-lung Steve , Screaton Gavin R. , Hou Jin-lin , McMichael Andrew J. , Xu Xiao-Ning
Published: April 2014

Effective vaccines should confer long-term protection against future outbreaks of severe acute respiratory syndrome (SARS) caused by a novel zoonotic coronavirus (SARS-CoV) with unknown animal reservoirs. We conducted a cohort study examining multiple parameters of immune responses to SARS-CoV infection, aiming to identify the immune correlates of protection. We used a matrix of overlapping peptides spanning whole SARS-CoV proteome to determine T cell responses from 128 SARS convalescent samples by ex vivo IFN-γ ELISPOT assays. Approximately 50% of convalescent SARS patients were positive for T cell responses, and 90% possessed strongly neutralizing Abs. Fifty-five novel T cell epitopes were identified, with spike protein dominating total T cell responses. CD8+ T cell responses were more frequent and of a greater magnitude than CD4+ T cell responses (p < 0.001). Polychromatic cytometry analysis indicated that the virus-specific T cells from the severe group tended to be a central memory phenotype (CD27+/CD45RO+) with a significantly higher frequency of polyfunctional CD4+ T cells producing IFN-γ, TNF-α, and IL-2, and CD8+ T cells producing IFN-γ, TNF-α, and CD107a (degranulation), as compared with the mild-moderate group. Strong T cell responses correlated significantly (p < 0.05) with higher neutralizing Ab. The serum cytokine profile during acute infection indicated a significant elevation of innate immune responses. Increased Th2 cytokines were observed in patients with fatal infection. Our study provides a roadmap for the immunogenicity of SARS-CoV and types of immune responses that may be responsible for the virus clearance, and should serve as a benchmark for SARS-CoV vaccine design and evaluation.
Rap1 Activation Is Required for Fcγ Receptor-Dependent Phagocytosis
Authors: Chung Jooho , Serezani Carlos H. , Huang Steven K. , Stern Joel N. H. , Keskin Derin B. , Jagirdar Rajesh , Brock Thomas G. , Aronoff David M. , Peters-Golden Marc
Published: April 2014

Phagocytosis of IgG-opsonized microbes via the Fcγ receptor (FcγR) requires the precise coordination of a number of signaling molecules, including the low-molecular mass GTPases. Little is known about the Ras-family GTPase Rap1 in this process. We therefore investigated its importance in mediating FcγR-dependent phagocytosis in NR8383 rat alveolar macrophages. Pulldown of active Rap1 and fluorescence microscopic analysis of GFP-RalGDS (Ral guanine dissociation stimulator)-transfected macrophages revealed that Rap1 is indeed activated by FcγR crosslinking. Inhibition of Rap1 activity, both by Rap1GAP (GTPase-activating protein) expression and liposome-delivered blocking Ab, severely impaired the ability of cells to ingest IgG-opsonized targets. FcγR-induced Rap1 activation was found to be independent of both cAMP and Ca2+, suggesting a role for the second messenger-independent guanosine exchange factor, C3G. This was supported by the facts that 1) liposome-delivered blocking Ab against C3G inhibited both FcγR-dependent phagocytosis and Rap1 activation, and 2) both active Rap1GTP and C3G were found to translocate to the phagosome. Taken together, our data demonstrate a novel role for Rap1 and its exchange factor C3G in mediating FcγR-dependent phagocytosis.
The Clathrin-Mediated Endocytic Pathway Participates in dsRNA-Induced IFN-β Production
Authors: Itoh Kiyoharu , Watanabe Ayako , Funami Kenji , Seya Tsukasa , Matsumoto Misako
Published: April 2014

TLR3 and cytoplasmic RIG-I-like receptor (RLR) recognize virus-derived dsRNA and induce type I IFN production in a distinct manner. Human TLR3 localizes to the endosomal compartments in myeloid dendritic cells (mDCs), while it localizes to both the cell surface and interior in fibroblasts and epithelial cells. TLR3 signaling arises in the intracellular compartment in both cell types and requires endosomal maturation. The mechanisms by which extracellular dsRNA is delivered to the TLR3-containing organelle remain largely unknown. Among various synthetic dsRNAs, poly(I:C) is preferentially internalized and activates TLR3 in mDCs. In vitro transcribed dsRNAs hardly induce IFN-β production in mDCs. In this study, we demonstrate that the clathrin-dependent endocytic pathway mediates cell entry of poly(I:C) to induce IFN-β gene transcription. Furthermore, poly(I:C)-induced IFN-β production is inhibited by pretreatment of cells with B- and C-type oligodeoxynucleotides (ODNs) but not with TLR7/8 ligands. The binding and internalization of B-type ODNs by mDCs was reduced in the presence of poly(I:C), suggesting that poly(I:C) shares the uptake receptor with B- and C-type ODNs. Hence, foreign dsRNA is recognized by differently categorized receptors, cytoplasmic RIG-I-like receptor, membrane-bound TLR3 and cell-surface RNA capture. The endocytic pathway is critical for dsRNA-induced TLR3-mediated cell activation.
Inhibition of IFN-γ-Induced STAT1 Tyrosine Phosphorylation by Human CMV Is Mediated by SHP2
Authors: Baron Michel , Davignon Jean-Luc
Published: April 2014

Human CMV (HCMV) is a ubiquitous β-herpesvirus which has developed several mechanisms of escape from the immune system. IFN-γ-induced signaling relies on the integrity of the JAK/STAT pathway which is regulated by phosphorylation steps and leads to nuclear translocation of tyrosine-phosphorylated STAT1 (STAT1-P-Tyr), and its binding to IFN-γ activation site sequences of IFN-γ-inducible promoters. Activation of those promoters leads to the expression of genes involved in the immune response and in the antiviral effects of IFN-γ. Src homology region 2 domain-containing phosphatase 2 (SHP2) is a ubiquitous phosphatase involved in the regulation of IFN-γ-mediated tyrosine phosphorylation. Several mechanisms account for the inhibition IFN-γ signaling pathway by HCMV. In this study, we have identified a new mechanism that involved the inhibition of STAT1 tyrosine phosphorylation within 12–24 h postinfection. This defect was dependent on HCMV transcription. Consequences were impaired nuclear translocation of STAT1-P-Tyr, inhibition of IFN-γ activation site-STAT1 interaction, and inhibition of HLA-DR expression. Expression of indoleamine-2,3-dioxygenase which is involved in the antiviral effects of IFN-γ was also inhibited. Treatment of cells with sodium orthovanadate rescued STAT1 tyrosine phosphorylation, suggesting that a tyrosine phosphatase was involved in this inhibition. Coimmunoprecipitation of STAT1 and SHP2 was induced by HCMV infection, and SHP2 small interfering RNA restored the expression of STAT1-P-Tyr. Our data suggest that SHP2 activation induced by HCMV infection is responsible for the down-regulation of IFN-γ-induced STAT1 tyrosine phosphorylation.
Binding of Complement Inhibitor C4b-Binding Protein Contributes to Serum Resistance of Porphyromonas gingivalis
Authors: Potempa Michal , Potempa Jan , Okroj Marcin , Popadiak Katarzyna , Eick Sigrun , Nguyen Ky-Anh , Riesbeck Kristian , Blom Anna M.
Published: April 2014

The periodontal pathogen Porphyromonas gingivalis is highly resistant to the bactericidal activity of human complement, which is present in the gingival crevicular fluid at 70% of serum concentration. All thirteen clinical and laboratory P. gingivalis strains tested were able to capture the human complement inhibitor C4b-binding protein (C4BP), which may contribute to their serum resistance. Accordingly, in serum deficient of C4BP, it was found that significantly more terminal complement component C9 was deposited on P. gingivalis. Moreover, using purified proteins and various isogenic mutants, we found that the cysteine protease high molecular weight arginine-gingipain A (HRgpA) is a crucial C4BP ligand on the bacterial surface. Binding of C4BP to P. gingivalis appears to be localized to two binding sites: on the complement control protein 1 domain and complement control protein 6 and 7 domains of the α-chains. Furthermore, the bacterial binding of C4BP was found to increase with time of culture and a particularly strong binding was observed for large aggregates of bacteria that formed during culture on solid blood agar medium. Taken together, gingipains appear to be a very significant virulence factor not only destroying complement due to proteolytic degradation as we have shown previously, but was also inhibiting complement activation due to their ability to bind the complement inhibitor C4BP.
Interleukin-10 Promotes Mycobacterium tuberculosis Disease Progression in CBA/J Mice
Authors: Beamer Gillian L. , Flaherty David K. , Assogba Barnabe D. , Stromberg Paul , Gonzalez-Juarrero Mercedes , de Waal Malefyt Rene , Vesosky Bridget , Turner Joanne
Published: April 2014

IL-10 is a potent immunomodulatory cytokine that affects innate and acquired immune responses. The immunological consequences of IL-10 production during pulmonary tuberculosis (TB) are currently unknown, although IL-10 has been implicated in reactivation TB in humans and with TB disease in mice. Using Mycobacterium tuberculosis-susceptible CBA/J mice, we show that blocking the action of IL-10 in vivo during chronic infection stabilized the pulmonary bacterial load and improved survival. Furthermore, this beneficial outcome was highly associated with the recruitment of T cells to the lungs and enhanced T cell IFN-γ production. Our results indicate that IL-10 promotes TB disease progression. These findings have important diagnostic and/or therapeutic implications for the prevention of reactivation TB in humans.
Yersinia pestis Evades TLR4-dependent Induction of IL-12(p40)2 by Dendritic Cells and Subsequent Cell Migration
Authors: Robinson Richard T. , Khader Shabaana A. , Locksley Richard M. , Lien Egil , Smiley Stephen T. , Cooper Andrea M.
Published: April 2014

At the temperature of its flea vector (∼20–30°C), the causative agent of plague, Yersinia pestis, expresses a profile of genes distinct from those expressed in a mammalian host (37°C). When dendritic cells (DC) are exposed to Y. pestis grown at 26°C (Y. pestis-26°), they secrete copious amounts of IL-12p40 homodimer (IL-12(p40)2). In contrast, when DCs are exposed to Y. pestis grown at 37°C (Y. pestis-37°), they transcribe very little IL-12p40, which is secreted as IL-12p40 monomer (IL-12p40). Y. pestis-26° also induces migration of DCs to the homeostatic chemokine CCL19, whereas Y. pestis-37° does not; migratory DCs are positive for IL-12p40 transcription and secrete mostly IL-12(p40)2; DCs lacking IL-12p40 do not migrate. Expression of acyltransferase LpxL from Escherichia coli in Y. pestis-37° results in the production of a hexa-acylated lipid A, also seen in Y. pestis-26°, rather than tetra-acylated lipid A normally seen in Y. pestis-37°. The LpxL-expressing Y. pestis-37° promotes DC IL-12(p40)2 production and induction of DC migration. In addition, absence of TLR4 ablates production of IL-12(p40)2 in DC exposed to Y. pestis-26°. The data demonstrate the molecular pathway by which Y. pestis evades induction of early DC activation as measured by migration and IL-12(p40)2 production.