bluuebunny/crossref_metadata_2025 huggingface dataset:
Mouse TCRαβ+CD8αα Intraepithelial Lymphocytes Express Genes That Down-Regulate Their Antigen Reactivity and Suppress Immune Responses
Authors: Denning Timothy L. , Granger Steve , Mucida Daniel , Graddy Ryan , Leclercq Georges , Zhang Weiguo , Honey Karen , Rasmussen Jeffrey P. , Cheroutre Hilde , Rudensky Alexander Y. , Kronenberg Mitchell
Published: April 2014

Mouse small intestine intraepithelial lymphocytes (IEL) that express αβTCR and CD8αα homodimers are an enigmatic T cell subset, as their specificity and in vivo function remain to be defined. To gain insight into the nature of these cells, we performed global gene expression profiling using microarray analysis combined with real-time quantitative PCR and flow cytometry. Using these methods, TCRαβ+CD8αα IEL were compared with their TCRαβ+CD8β+ and TCRγδ+ counterparts. Interestingly, TCRαβ+CD8αα IEL were found to preferentially express genes that would be expected to down-modulate their reactivity. They have a unique expression pattern of members of the Ly49 family of NK receptors and tend to express inhibitory receptors, along with some activating receptors. The signaling machinery of both TCRαβ+CD8αα and TCRγδ+ IEL is constructed differently than other IEL and peripheral T cells, as evidenced by their low-level expression of the linker for activation of T cells and high expression of the non-T cell activation linker, which suppresses T cell activation. The TCRαβ+CD8αα IEL subset also has increased expression of genes that could be involved in immune regulation, including TGF-β3 and lymphocyte activation gene-3. Collectively, these data underscore the fact that, while TCRαβ+CD8αα IEL resemble TCRγδ+ IEL, they are a unique population of cells with regulated Ag reactivity that could have regulatory function.
Activation of Adenosine 2A Receptors Attenuates Allograft Rejection and Alloantigen Recognition
Authors: Sevigny Charles P. , Li Li , Awad Alaa S. , Huang Liping , McDuffie Marcia , Linden Joel , Lobo Peter I. , Okusa Mark D.
Published: April 2014

The current studies investigated the in vitro and in vivo effect of adenosine 2A receptor (A2AR) agonists to attenuate allogenic immune activation. We performed MLRs with spleen T lymphocytes and APCs isolated from wild-type and A2AR knockout mice of both C57BL/6 and BALB/c background strains. Two-way MLR-stimulated T cell proliferation was reduced by ATL313, a selective A2AR agonist in a dose-responsive manner (∼70%; 10 nM), an effect reversed by the A2AR antagonist ZM241385 (100 nM). By one-way MLRs, we observed that ATL313’s inhibitory effect was due to effects on both T cells and APCs. ATL313 suppressed the activation markers CD25 and CD40L and the release of inflammatory cytokines IFN-γ, RANTES, IL-12P70, and IL-2. ATL313 also increased negative costimulatory molecules programmed death-1 and CTLA-4 expressed on T cells. In lymphocytes activated with anti-CD3e mAb, ATL313 inhibited the phosphorylation of Zap70, an effect that was reversed by the protein kinase A inhibitor H-89. In skin transplants, allograft survival was enhanced with ATL313, an effect blocked by ZM241385. These results indicate that A2AR agonists attenuate allogenic recognition by action on both T lymphocytes and APCs in vitro and delayed acute rejection in vivo. We conclude that A2AR agonists may represent a new class of compounds for induction therapy in organ transplantation.
Suppression of Memory CD8 T Cell Generation and Function by Tryptophan Catabolism
Authors: Liu Zhiwei , Dai Hehua , Wan Ni , Wang Tao , Bertera Suzanne , Trucco Massimo , Dai Zhenhua
Published: April 2014

Dendritic cell-derived indoleamine 2,3-dioxygenase (IDO) suppresses naive T cell proliferation and induces their apoptosis by catalyzing tryptophan, and hence is essential for the maintenance of peripheral tolerance. However, it is not known whether memory T cells are subject to the regulation by IDO-mediated tryptophan catabolism, as memory T cells respond more rapidly and vigorously than their naive counterparts and are resistant to conventional costimulatory blockade. In this study, we present the evidence that memory CD8+ T cells are susceptible to tryptophan catabolism mediated by IDO. We found that overexpression of IDO in vivo attenuated the generation of both central memory CD8+ T cells (TCM) and effector memory CD8+ T cells (TEM) while suppressing IDO activity promoted their generation. Moreover, IDO overexpression suppressed the effector function of TCM cells or TCM cell-mediated allograft rejection as well as their proliferation in vivo. Interestingly, TCM cells were resistant to apoptosis induced by tryptophan catabolism. However, IDO overexpression did not suppress the effector function of TEM cells or TEM cell-mediated allograft rejection, suggesting that TEM cells, unlike TCM cells, do not require tryptophan for their effector function once they are generated. This study provides insight into the mechanisms underlying the differential regulation of memory T cell responsiveness and has clinical implications for vaccination or tolerance induction.
Genetic Deletion of Placenta Growth Factor in Mice Alters Uterine NK Cells
Authors: Tayade Chandrakant , Hilchie David , He Hong , Fang Yuan , Moons Lieve , Carmeliet Peter , Foster Robert A. , Croy B. Anne
Published: April 2014

Placenta growth factor (PlGF; formerly PGF), a vascular endothelial growth factor gene family member, is expressed in human implantation sites by maternal uterine NK (uNK) and fetal trophoblast cells. Lower than normal concentrations of blood and urinary PlGF have been associated with impending onset of pre-eclampsia, a hypertensive disease of late human gestation characterized by limited intravascular trophoblast invasion. In pregnant rodents, delivery of the PlGF antagonist sFlt-1 or S-endoglin induces pre-eclampsia-like lesions. Mice genetically deleted in PlGF reproduce, but neither their implantation sites nor their uNK cell development are described. We combined real-time PCR of endometrium from nonpregnant and gestation day (gd)6–18 C57BL6/J (B6) mice with immunohistology to analyze PlGF expression in normal mouse pregnancy. To estimate the significance of uNK cell-derived PlGF, PlGF message was quantified in mesometrial decidua from pregnant alymphoid Rag2 null/common γ chain null mice and in laser capture-microdissected B6 uNK cells. Histopathologic consequences from PlGF deletion were also characterized in the implantation sites from PlGF null mice. In B6, decidual PlGF expression rose between gd8–16. uNK cells were among several types of cells transcribing PlGF in decidualized endometrium. Immature uNK cells, defined by their low numbers of cytoplasmic granules, were the uNK cells displaying the greatest number of transcripts. PlGF deletion promoted the early differentiation high numbers of binucleate uNK cells (gd8) but had no other significant, morphometrically detectable impact on implantation sites. Thus, in mice, PlGF plays an important role in successful uNK cell proliferation and/or differentiation.
Activation of Enteroendocrine Cells via TLRs Induces Hormone, Chemokine, and Defensin Secretion
Authors: Palazzo Marco , Balsari Andrea , Rossini Anna , Selleri Silvia , Calcaterra Claudia , Gariboldi Silvia , Zanobbio Laura , Arnaboldi Francesca , Shirai Yuri F. , Serrao Graziano , Rumio Cristiano
Published: April 2014

Enteroendocrine cells are known primarily for their production of hormones that affect digestion, but they might also be implicated in sensing and neutralizing or expelling pathogens. We evaluate the expression of TLRs and the response to specific agonists in terms of cytokines, defensins, and hormones in enteroendocrine cells. The mouse enteroendocrine cell line STC-1 and C57BL/6 mice are used for in vitro and in vivo studies, respectively. The presence of TLR4, 5, and 9 is investigated by RT-PCR, Western blot, and immunofluorescence analyses. Activation of these receptors is studied evaluating keratinocyte-derived chemokine, defensins, and cholecystokinin production in response to their specific agonists. In this study, we show that the intestinal enteroendocrine cell line STC-1 expresses TLR4, 5, and 9 and releases cholecystokinin upon stimulation with the respective receptor agonists LPS, flagellin, and CpG-containing oligodeoxynucleotides. Release of keratinocyte-derived chemokine and β-defensin 2 was also observed after stimulation of STC-1 cells with the three TLR agonists, but not with fatty acids. Consistent with these in vitro data, mice showed increased serum cholecystokinin levels after oral challenge with LPS, flagellin, or CpG oligodeoxynucleotides. In addition to their response to food stimuli, enteroendocrine cells sense the presence of bacterial Ags through TLRs and are involved in neutralizing intestinal bacteria by releasing chemokines and defensins, and maybe in removing them by releasing hormones such as cholecystokinin, which induces contraction of the muscular tunica, favoring the emptying of the distal small intestine.
Enhanced NFATc1 Nuclear Occupancy Causes T Cell Activation Independent of CD28 Costimulation
Authors: Pan Minggui , Winslow Monte M. , Chen Lei , Kuo Ann , Felsher Dean , Crabtree Gerald R.
Published: April 2014

TCR signals induce the nuclear localization of NFATc proteins, which are removed from the nucleus after rephosphorylation by glycogen synthase kinase 3 and other kinases. Rapid nuclear export might allow continuous monitoring of receptor occupancy, making the transcriptional response proportional to the duration of TCR/CD28 signaling. To investigate this possibility, we analyzed mice in which T cells express a NFATc1 variant (NFATc1nuc) with serine-to-alanine changes at the glycogen synthase kinase 3 phosphorylation sites. NFATc1nuc T cells have constitutively nuclear NFATc1, enhanced T cell activation in vivo, and calcineurin-independent proliferation in vitro. NFATc1nuc T cells are hypersensitive to TCR/CD3 stimulation, resulting in enhanced proliferation and cytokine production that is independent of CD28 costimulation. These results support the notion that CD28 inhibits nuclear export of NFATc transcription factors. In addition, NFATc1nuc destabilizes a positive feedback loop in which NFATc1 activates its own transcription as well as its targets, such as CD40 ligand and Th1/Th2 cytokines.
CCL23 Expression Is Induced by IL-4 in a STAT6-Dependent Fashion
Authors: Novak Hermann , Müller Anke , Harrer Nathalie , Günther Claudia , Carballido Jose M. , Woisetschläger Maximilian
Published: April 2014

The chemokine CCL23 is primarily expressed in cells of the myeloid lineage but little information about its regulation is available. In this study, it is demonstrated that IL-4 and IL-13 induced CCL23 expression in human peripheral blood monocytes. GM-CSF had no effect on its own but synergized with IL-4, but not IL-13. CCL23 promoter reporter gene constructs were sensitive to IL-4 stimulation in the presence of the transcription factor STAT6. A canonical STAT6 binding site in the promoter region of the CCL23 gene was critical for the IL-4-inducible phenotype because reporter plasmids with a defective STAT6 binding site were unable to respond to IL-4 stimulation. In addition, two tandem copies of the STAT6 site conferred cytokine responsiveness to a heterologous minimal promoter. Furthermore, IL-4 inducibility of the CCL23 promoter was dependent on the absence of a negatively acting cis-element downstream of the STAT6 binding site. The negative function of this element was operative also on heterologous IL-4-inducible promoters. CCL23 was also expressed in skin from patients suffering from atopic dermatitis at higher levels than in normal individuals. However, no correlation between CCL23 expression in the serum and IgE levels as a diagnostic marker for atopy was found. Collectively, these data suggest a link between the inducible phenotype of CCL23 expression in monocytes by the prototype Th2 molecule pair IL-4/STAT6 and the increased number of CCL23-expressing cells in skin of atopic dermatitis patients.
The Role of Antibody Polyspecificity and Lipid Reactivity in Binding of Broadly Neutralizing Anti-HIV-1 Envelope Human Monoclonal Antibodies 2F5 and 4E10 to Glycoprotein 41 Membrane Proximal Envelope Epitopes
Authors: Alam S. Munir , McAdams Mildred , Boren David , Rak Michael , Scearce Richard M. , Gao Feng , Camacho Zenaido T. , Gewirth Daniel , Kelsoe Garnett , Chen Pojen , Haynes Barton F.
Published: April 2014

Two neutralizing human mAbs, 2F5 and 4E10, that react with the HIV-1 envelope gp41 membrane proximal region are also polyspecific autoantibodies that bind to anionic phospholipids. To determine the autoantibody nature of these Abs, we have compared their reactivities with human anti-cardiolipin mAbs derived from a primary antiphospholipid syndrome patient. To define the role of lipid polyreactivity in binding of 2F5 and 4E10 mAbs to HIV-1 envelope membrane proximal epitopes, we determined the kinetics of binding of mAbs 2F5 and 4E10 to their nominal gp41 epitopes vs liposome-gp41 peptide conjugates. Both anti-HIV-1 mAbs 2F5 and 4E10 bound to cardiolipin with Kd values similar to those of autoimmune anti-cardiolipin Abs, IS4 and IS6. Binding kinetics studies revealed that mAb 2F5 and 4E10 binding to their respective gp41 peptide-lipid conjugates could best be defined by a two-step (encounter-docking) conformational change model. In contrast, binding of 2F5 and 4E10 mAbs to linear peptide epitopes followed a simple Langmuir model. A mouse mAb, 13H11, that cross-blocks mAb 2F5 binding to the gp41 epitope did not cross-react with lipids nor did it neutralize HIV-1 viruses. Taken together, these data demonstrate the similarity of 2F5 and 4E10 mAbs to known anti-cardiolipin Abs and support the model that mAb 2F5 and 4E10 binding to HIV-1 involves both viral lipid membrane and gp41 membrane proximal epitopes.
T Cell Recognition Patterns of Immunodominant Cytomegalovirus Antigens in Primary and Persistent Infection
Authors: Khan Naeem , Best Donna , Bruton Rachel , Nayak Laxman , Rickinson Alan B. , Moss Paul A. H.
Published: April 2014

Replication of human cytomegalovirus is controlled by a vigorous CD8 T cell response. The persistent nature of infection is believed to periodically stimulate T cell responses resulting in considerable expansions of virus-specific CD8 T cells over time. In this study, we describe the magnitude and breadth of CD8 T cell responses against the immunodominant viral Ags, IE-1 and pp65, in acute and long-term infection using the IFN-γ ELISPOT assay. Simultaneously, we have identified several novel MHC class I restricted CD8 T cell epitopes. Acute phase responses in immunocompetent donors appear to be extremely focused as early as 1 week post diagnosis with dominant peptide-specific responses observed against both proteins. These dominant responses remain detectable at all later time points over a 4-year follow-up. Interestingly the IE-1 responses show an increase over time whereas the pp65 responses do not, which contrasts with data showing that responses against both Ags are elevated in elderly individuals. We also observe the rapid emergence of an effector memory phenotype for virus-specific CD8 T cells as observed in persistent infection. Over time the revertant CD45RApos effector cell population is also expanded, and this is more evident in the preferentially expanded IE-1 responses. We postulate that periodic low-level virus reactivation after the acute infection phase preferentially stimulates these responses whereas pp65-specific T cell expansions probably occur during the infrequent episodes of lytic viral replication or secondary infection.
Resident Vδ1+ γδ T Cells Control Early Infiltration of Neutrophils after Escherichia coli Infection via IL-17 Production
Authors: Shibata Kensuke , Yamada Hisakata , Hara Hiromitsu , Kishihara Kenji , Yoshikai Yasunobu
Published: April 2014

Neutrophils infiltrate the site of infection and play critical roles in host defense, especially against extracellular bacteria. In the present study, we found a rapid and transient production of IL-17 after i.p. infection with Escherichia coli, preceding the influx of neutrophils. Neutralization of IL-17 resulted in a reduced infiltration of neutrophils and an impaired bacterial clearance. Ex vivo intracellular cytokine flow cytometric analysis revealed that γδ T cell population was the major source of IL-17. Mice depleted of γδ T cells by mAb treatment or mice genetically lacking Vδ1 showed diminished IL-17 production and reduced neutrophil infiltration after E. coli infection, indicating an importance of Vδ1+ γδ T cells as the source of IL-17. It was further revealed that γδ T cells in the peritoneal cavity of naive mice produced IL-17 in response to IL-23, which was induced rapidly after E. coli infection in a TLR4 signaling-dependent manner. Thus, although γδ T cells are generally regarded as a part of early induced immune responses, which bridge innate and adaptive immune responses, our study demonstrated a novel role of γδ T cells as a first line of host defense controlling neutrophil-mediated innate immune responses.
Tyk2 Signaling in Host Environment Plays an Important Role in Contraction of Antigen-Specific CD8+ T Cells following a Microbial Infection
Authors: Li Wei , Yamada Hisakata , Yajima Toshiki , Nakagawa Ryusuke , Shimoda Kazuya , Nakayama Keiichi , Yoshikai Yasunobu
Published: April 2014

Tyrosine kinase 2 (Tyk2), a member of JAK signal transducer family contributes to the signals triggered by IL-12 for IFN-γ production. To elucidate potential roles of Tyk2 in generation and maintenance of Ag-specific CD8+ T cells, we followed the fate of OVA-specific CD8+ T cells in Tyk2-deficient (−/−) mice after infection with recombinant Listeria monocytogenes expressing OVA (rLM-OVA). Results showed that the numbers of OVA257–264/Kb tetramer-positive CD8+ T cells in Tyk2−/− mice were almost the same as those in Tyk2+/+ mice at the expansion phase on day 7 but were significantly larger in Tyk2−/− mice than those in Tyk2+/+ mice at the contraction phase on day 10 and at the memory phase on day 60 after infection. The intracellular expression level of active caspase-3 was significantly decreased in the OVA-specific CD8+ T cells of Tyk2−/− mice on day 7 compared with those of Tyk2+/+ mice. Adaptive transfer experiments revealed that Tyk2 signaling in other factors rather than CD8+ T cells played a regulatory role in CD8+ T cell contraction following infection. Administration of exogenous IFN-γ from day 6 to day 9 restored the CD8+ T cell contraction in Tyk2−/− mice after infection with rLM-OVA. These results suggest that Tyk2 signaling for IFN-γ production in host environment plays an important role in contraction of effector CD8+ T cells following a microbial infection.
Oncostatin M Secreted by Skin Infiltrating T Lymphocytes Is a Potent Keratinocyte Activator Involved in Skin Inflammation
Authors: Boniface Katia , Diveu Caroline , Morel Franck , Pedretti Nathalie , Froger Josy , Ravon Elisa , Garcia Martine , Venereau Emilie , Preisser Laurence , Guignouard Emmanuel , Guillet Gérard , Dagregorio Guy , Pène Jérôme , Moles Jean-Pierre , Yssel Hans , Chevalier Sylvie , Bernard François-Xavier , Gascan Hugues , Lecron Jean-Claude
Published: April 2014

Cutaneous inflammatory diseases such as psoriasis vulgaris and atopic dermatitis are associated with altered keratinocyte function, as well as with a particular cytokine production profile of skin-infiltrating T lymphocytes. In this study we show that normal human epidermal keratinocytes express a functional type II oncostatin-M (OSM) receptor (OSMR) consisting of the gp130 and OSMRβ components, but not the type I OSMR. The type II OSMR is expressed in skin lesions from both psoriatic patients and those with atopic dermatitis. Its ligand, OSM, induces via the recruitment of the STAT3 and MAP kinase pathways a gene expression profile in primary keratinocytes and in a reconstituted epidermis that is characteristic of proinflammatory and innate immune responses. Moreover, OSM is a potent stimulator of keratinocyte migration in vitro and increases the thickness of a reconstituted epidermis. OSM transcripts are enhanced in both psoriatic and atopic dermatitic skin as compared with healthy skin and mirror the enhanced production of OSM by T cells isolated from diseased lesions. Results from a microarray analysis comparing the gene-modulating effects of OSM with those of 33 different cytokines indicate that OSM is a potent keratinocyte activator similar to TNF-α, IL-1, IL-17, and IL-22 and that it acts in synergy with the latter cytokines in the induction of S100A7 and β-defensin 2 expression, characteristic of psoriatic skin. Taken together, these results demonstrate that OSM and its receptor play an important role in cutaneous inflammatory responses in general and that the specific effects of OSM are associated with distinct inflammatory diseases depending on the cytokine environment.
Cutting Edge: B Cell Receptor (BCR) Cross-Talk: The IL-4-Induced Alternate Pathway for BCR Signaling Operates in Parallel with the Classical Pathway, Is Sensitive to Rottlerin, and Depends on Lyn
Authors: Guo Benchang , Blair Derek , Chiles Thomas C. , Lowell Clifford A. , Rothstein Thomas L.
Published: April 2014

B cell exposure to IL-4 alters subsequent BCR signaling such that ERK phosphorylation becomes signalosome-independent; however, the nature of this new, alternate signaling pathway and its relationship to the classical, signalosome-dependent signaling pathway are not known. In this study, we report that the alternate and classical pathways for BCR signaling are differentially affected by rottlerin, and by Go6976 or LY294002, respectively. Furthermore, in B cells lacking protein kinase C (PKC)β, the classical pathway for BCR signaling is blocked, whereas the alternate pathway is little affected. Conversely, in B cells lacking Lyn, the alternate pathway for BCR signaling is blocked, whereas the classical pathway is little affected. The rottlerin-sensitive element is not PKCδ, inasmuch as the alternate pathway is not blocked in PKCδ-deficient B cells. These results indicate that the rottlerin-sensitive, Lyn-dependent alternate pathway, and the classical pathway, for BCR signaling operate in parallel when BCR engagement follows IL-4 exposure.
IL-7 Is Essential for the Development and the Persistence of Chronic Colitis
Authors: Totsuka Teruji , Kanai Takanori , Nemoto Yasuhiro , Makita Shin , Okamoto Ryuichi , Tsuchiya Kiichiro , Watanabe Mamoru
Published: April 2014

Although IL-7 has recently emerged as a key cytokine involved in controlling the homeostatic turnover and the survival of peripheral resting memory CD4+ T cells, its potential to be sustained pathogenic CD4+ T cells in chronic immune diseases, such as inflammatory bowel diseases, still remains unclear. In this study, we demonstrate that IL-7 is essential for the development and the persistence of chronic colitis induced by adoptive transfer of normal CD4+CD45RBhigh T cells or colitogenic lamina propria (LP) CD4+ memory T cells into immunodeficient IL-7+/+ × RAG-1−/− and IL-7−/− × RAG-1−/− mice. Although IL-7+/+ × RAG-1−/− recipients transferred with CD4+CD45RBhigh splenocytes developed massive inflammation of the large intestinal mucosa concurrent with massive expansion of Th1 cells, IL-7−/− × RAG-1−/− recipients did not. Furthermore, IL-7−/− × RAG-1−/−, but not IL-7+/+ × RAG-1−/−, mice transferred with LP CD4+CD44highCD62L−IL-7Rαhigh effector-memory T cells (TEM) isolated from colitic CD4+CD45RBhigh-transferred mice did not develop colitis. Although rapid proliferation of transferred colitogenic LP CD4+ TEM cells was observed in the in IL-7−/− × RAG-1−/− mice to a similar extent of those in IL-7+/+ × RAG-1−/− mice, Bcl-2 expression was significantly down-modulated in the transferred CD4+ T cells in IL-7−/− × RAG-1−/− mice compared with those in IL-7+/+ × RAG-1−/− mice. Taken together, IL-7 is essential for the development and the persistence of chronic colitis as a critical survival factor for colitogenic CD4+ TEM cells, suggesting that therapeutic approaches targeting IL-7/IL-7R signaling pathway may be feasible in the treatment of inflammatory bowel diseases.
Regulation of Oxidative Stress Responses by Ataxia-Telangiectasia Mutated Is Required for T Cell Proliferation
Authors: Bagley Jessamyn , Singh Gyanesh , Iacomini John
Published: April 2014

Mutations in the gene encoding ataxia-telangiectasia (A-T) mutated (Atm) cause the disease A-T, characterized by immunodeficiency, the molecular basis of which is not known. Following stimulation through the TCR, Atm-deficient T cells and normal T cells in which Atm is inhibited undergo apoptosis rather than proliferation. Apoptosis is prevented by scavenging reactive oxygen species (ROS) during activation. Atm therefore plays a critical role in T cell proliferation by regulating responses to ROS generated following T cell activation. The inability of Atm-deficient T cells to control responses to ROS is therefore the molecular basis of immunodeficiency associated with A-T.
Spontaneous Autoreactive Memory B Cell Formation Driven by a High Frequency of Autoreactive CD4+ T Cells
Authors: Guay Heath M. , Larkin Joseph , Picca Cristina Cozzo , Panarey Laura , Caton Andrew J.
Published: April 2014

Although somatically mutated autoantibodies are characteristic of many autoimmune diseases, the processes that can lead to their development remain poorly understood. We have examined the formation of autoreactive memory B cells in PevHA mice, which express the influenza virus PR8 hemagglutinin (HA) as a transgenic membrane bound neo-self-Ag. Using a virus immunization strategy, we show that PR8 HA-specific memory B cell formation can occur in PevHA mice, even though a major subset of PR8 HA-specific B cells is negatively selected from the primary repertoire. Moreover, PR8 HA-specific memory B cells develop spontaneously in TS1 × PevHA mice, which coexpress a transgenic PR8 HA-specific TCR and contain a high frequency of HA-specific CD4+ T cells. Notably, autoreactive memory B cell formation occurred in TS1 × PevHA mice even though approximately half of the HA-specific CD4+ T cells were CD25+Foxp3+ cells that could significantly attenuate, but did not completely abolish HA-specific autoantibody production in an adoptive transfer setting. The findings provide evidence that a high frequency of autoreactive CD4+ T cells can be sufficient to promote autoreactive memory B cell formation in the absence of signals provided by overt immunization or infection and despite the presence of abundant autoantigen-specific CD4+CD25+Foxp3+ regulatory T cells.
Aggregated Ursolic Acid, a Natural Triterpenoid, Induces IL-1β Release from Murine Peritoneal Macrophages: Role of CD36
Authors: Ikeda Yasutaka , Murakami Akira , Fujimura Yoshinori , Tachibana Hirofumi , Yamada Koji , Masuda Daisaku , Hirano Ken-ichi , Yamashita Shizuya , Ohigashi Hajime
Published: April 2014

IL-1β has been shown to play a pivotal role in the development of inflammatory disorders. We recently found that a natural triterpene, ursolic acid (UA), enhanced MIF release from nonstimulated macrophages. In this study, we examined the effects of UA on the production of several cytokines in resident murine peritoneal macrophages (pMφ). UA increased the protein release of IL-1β, IL-6, and MIF, but not of TNF-α, in dose- and time-dependent manners. This triterpene also strikingly induced the activation of p38 MAPK and ERK1/2 together with that of upstream kinases. The release of UA-induced IL-1β was significantly inhibited by the inhibitors of p38 MAPK, MEK1/2, ATP-binding cassette transporter, and caspase-1. Furthermore, UA induced intracellular ROS generation for IL-1β production, which was suppressed by an antioxidant. Pretreatment with an anti-CD36 Ab significantly suppressed IL-1β release, and surface plasmon resonance assay results showed that UA bound to CD36 on macrophages. In addition, the amount of IL-1β released from UA-treated pMφ of CD36-deficient mice was markedly lower than that from those of wild-type mice. Interestingly, UA was found to aggregate in culture medium, and the aggregates were suggested to be responsible for IL-1β production. In addition, i.p. administration of UA increased the levels of IL-1β secretion and MPO activity in colonic mucosa of ICR mice. Taken together, our results indicate that aggregated UA is recognized, in part, by CD36 on macrophages for generating ROS, thereby activating p38 MAPK, ERK1/2, and caspase-1, as well as releasing IL-1β protein via the ATP-binding cassette transporter.
β1-Adrenergic Receptors on Immune Cells Impair Innate Defenses against Listeria
Authors: Emeny Rebecca T. , Gao Donghong , Lawrence David A.
Published: April 2014

Cold restraint (CR) for 1 h elicits a psychological and physiological stress that inhibits host defenses against Listeria monocytogenes (LM). Previous analyses indicated that this inhibition is not due to depletion of B or T cells but is instead dependent on signaling through β-adrenoceptors (βARs). We now show that impaired host resistance by CR cannot be accounted for by a decrease in LM-specific (listeriolysin O91–99 tetramer+) effector CD8+ T cells; this result is consistent with previous observations that CR-induced effects are mainly limited to early anti-LM responses. β2-Adrenoceptor (β2AR)−/− FVB/NJ and wild-type FVB/NJ mice had equivalent anti-LM defenses, whereas β1-adrenoceptor (β1AR)−/− FVB/NJ mice had lower levels of LM even when subjected to CR treatment. Additionally, host-resistance competency of β1AR−/− mice could be transferred to irradiated wild-type mice reconstituted with β1AR−/− bone marrow progenitors and spleen cells, indicating that β1AR signaling on immune cells reduces anti-LM responses. β1AR−/− mice had improved cellular (delayed-type hypersensitivity) responses while β2AR−/− mice had improved humoral responses (IgG1, IgG2, and IgM), a result that further explains the strain differences in LM defenses. CR-induced expression of β1AR and β2AR mRNA was assessed by real-time PCR. CR treatment significantly increased βAR mRNAs in Ficoll-purified and F4/80+-enhanced liver but not splenic homogenates, demonstrating an organ-specific effect of stress that alters host defenses. Finally, CR treatment induced early increases in perforin expression that may enhance immune cell apoptosis and interfere with LM clearance. In conclusion, β1AR signaling has immunomodulatory effects on early cell-mediated immune responses; a lack of β1AR signaling improves antilisterial defenses and cell-mediated immunity, in general.
TLR2-Mediated Survival of Staphylococcus aureus in Macrophages: A Novel Bacterial Strategy against Host Innate Immunity
Authors: Watanabe Ikuko , Ichiki Manami , Shiratsuchi Akiko , Nakanishi Yoshinobu
Published: April 2014

TLR2 plays a role as a pattern-recognition receptor in the innate immune response involving secreted proteins against microbial pathogens. To examine its possible involvement in the cellular response, we determined the levels of the engulfment and subsequent killing of bacteria by macrophages prepared from TLR2-deficient and wild-type mice. The level of the engulfment of Staphylococcus aureus or Escherichia coli was almost the same between TLR2-lacking and wild-type macrophages. However, the colony-forming ability of engulfed S. aureus, but not of E. coli, decreased to a greater extent in TLR2-lacking macrophages than in the wild-type control. The incubation with S. aureus caused activation of JNK in wild-type macrophages but not in TLR2-lacking macrophages, and the pretreatment of wild-type macrophages with a JNK inhibitor increased the rate of killing of engulfed S. aureus, but again not of E. coli. In addition, the number of colonies formed by engulfed S. aureus increased in the JNK-dependent manner when TLR2-lacking macrophages were pretreated with LPS. Furthermore, JNK seemed to inhibit the generation of superoxide, not of NO, in macrophages. These results collectively suggested that the level of superoxide is reduced in macrophages that have engulfed S. aureus through the actions of TLR2-activated JNK, resulting in the prolonged survival of the bacterium in phagosomes. The same regulation did not influence the survival of E. coli, because this bacterium was more resistant to superoxide than S. aureus. We propose a novel bacterial strategy for survival in macrophages involving the hijacking of an innate immune receptor.
Intestinal Lamina Propria Retaining CD4+CD25+ Regulatory T Cells Is A Suppressive Site of Intestinal Inflammation
Authors: Makita Shin , Kanai Takanori , Nemoto Yasuhiro , Totsuka Teruji , Okamoto Ryuichi , Tsuchiya Kiichiro , Yamamoto Masafumi , Kiyono Hiroshi , Watanabe Mamoru
Published: April 2014

It is well known that immune responses in the intestine remain in a state of controlled inflammation, suggesting that not only does active suppression by regulatory T (TREG) cells play an important role in the normal intestinal homeostasis, but also that its dysregulation of immune response leads to the development of inflammatory bowel disease. In this study, we demonstrate that murine CD4+CD25+ T cells residing in the intestinal lamina propria (LP) constitutively express CTLA-4, glucocorticoid-induced TNFR, and Foxp3 and suppress proliferation of responder CD4+ T cells in vitro. Furthermore, cotransfer of intestinal LP CD4+CD25+ T cells prevents the development of chronic colitis induced by adoptive transfer of CD4+CD45RBhigh T cells into SCID mice. When lymphotoxin (LT)α-deficient intercrossed Rag2 double knockout mice (LTα−/− × Rag2−/−), which lack mesenteric lymph nodes and Peyer’s patches, are transferred with CD4+CD45RBhigh T cells, they develop severe wasting disease and chronic colitis despite the delayed kinetics as compared with the control LTα+/+ × Rag2−/− mice transferred with CD4+CD45RBhigh T cells. Of note, when a mixture of splenic CD4+CD25+ TREG cells and CD4+CD45RBhigh T cells are transferred into LTα−/− × Rag2−/− recipients, CD4+CD25+ TREG cells migrate into the colon and prevent the development of colitis in LTα−/− × Rag2−/− recipients as well as in the control LTα+/+ × Rag2−/− recipients. These results suggest that the intestinal LP harboring CD4+CD25+ TREG cells contributes to the intestinal immune suppression.
CD56brightCD16− Killer Ig-Like Receptor− NK Cells Display Longer Telomeres and Acquire Features of CD56dim NK Cells upon Activation
Authors: Romagnani Chiara , Juelke Kerstin , Falco Michela , Morandi Barbara , D’Agostino Antonella , Costa Roberta , Ratto Giovanni , Forte Giuseppe , Carrega Paolo , Lui Gabrielle , Conte Romana , Strowig Till , Moretta Alessandro , Münz Christian , Thiel Andreas , Moretta Lorenzo , Ferlazzo Guido
Published: April 2014

Human NK cells can be divided into CD56dimCD16+ killer Ig-like receptors (KIR)+/− and CD56brightCD16− KIR− subsets that have been characterized extensively regarding their different functions, phenotype, and tissue localization. Nonetheless, the developmental relationship between these two NK cell subsets remains controversial. We report that, upon cytokine activation, peripheral blood (PB)-CD56bright NK cells mainly gain the signature of CD56dim NK cells. Remarkably, KIR can be induced not only on CD56bright, but also on CD56dim KIR− NK cells, and their expression correlates with lower proliferative response. In addition, we demonstrate for the first time that PB-CD56dim display shorter telomeres than PB- and lymph node (LN)-derived CD56bright NK cells. Along this line, although human NK cells collected from nonreactive LN display almost no KIR and CD16 expression, NK cells derived from highly reactive LN, efferent lymph, and PB express significant amounts of KIR and CD16, implying that CD56bright NK cells could acquire these molecules in the LN during inflammation and then circulate through the efferent lymph into PB as KIR+CD16+ NK cells. Altogether, our results suggest that CD56brightCD16− KIR− and CD56dimCD16+KIR+/− NK cells correspond to sequential steps of differentiation and support the hypothesis that secondary lymphoid organs can be sites of NK cell final maturation and self-tolerance acquisition during immune reaction.
A Unique Thymic Fibroblast Population Revealed by the Monoclonal Antibody MTS-15
Authors: Gray Daniel H. D. , Tull Dedreia , Ueno Tomoo , Seach Natalie , Classon Brendan J. , Chidgey Ann , McConville Malcolm J. , Boyd Richard L.
Published: April 2014

T cell differentiation in the thymus is dependent upon signals from thymic stromal cells. Most studies into the nature of these signals have focused only on the support provided by the thymic epithelium, but there is an emerging view that other stromal cells such as mesenchymal fibroblasts may also be involved. Study of the latter has been hindered by a lack of appropriate markers, particularly those allowing their isolation. In this study, we describe a new surface marker of thymic stroma, MTS-15, and demonstrate its specificity for fibroblasts and a subset of endothelial cells. Coculture experiments showed that the determinant could be transferred between cells. Extensive biochemical analysis demonstrated that the Ag bound by MTS-15 was the glycosphingolipid Forssman determinant, consistent with the distribution observed. Transcriptional analysis of purified MTS-15+ thymic fibroblasts revealed a unique expression profile for a number of chemokines and growth factors important to thymocyte and epithelial cell development. In a model of cyclophosphamide-induced thymic involution and regeneration, fibroblasts were found to expand extensively and express growth factors important to epithelial proliferation and increased T cell production just before thymic regeneration. Overall, this study identifies a useful marker of thymic fibroblasts and highlights this subpopulation as a key player in thymic function by virtue of their support of both thymocytes and epithelial cells.
A Critical Role for p53 in the Control of NF-κB-Dependent Gene Expression in TLR4-Stimulated Dendritic Cells Exposed to Genistein
Authors: Dijsselbloem Nathalie , Goriely Stanislas , Albarani Valentina , Gerlo Sarah , Francoz Sarah , Marine Jean-Christophe , Goldman Michel , Haegeman Guy , Berghe Wim Vanden
Published: April 2014

Considerable research has focused on the anti-inflammatory and antiproliferative activities exhibited by the soy isoflavone genistein. We previously demonstrated that genistein suppresses TNF-α-induced NF-κB-dependent IL-6 gene expression in cancer cells by interfering with the mitogen- and stress-activated protein kinase 1 activation pathway. However, effects of isoflavones on immune cells, such as dendritic cells, remain largely unknown. Here we show that genistein markedly reduces IL-6 cytokine production and transcription in LPS-stimulated human monocyte-derived dendritic cells. More particularly, we observe that genistein inhibits IL-6 gene expression by modulating the transcription factor NF-κB. Examination of NF-κB-related events downstream of TLR4 demonstrates that genistein affects NF-κB subcellular localization and DNA binding, although we observe only a minor inhibitory impact of genistein on the classical LPS-induced signaling steps. Interestingly, we find that genistein significantly increases p53 protein levels. We also show that overexpression of p53 in TLR4/MD2 HEK293T cells blocks LPS-induced NF-κB-dependent gene transcription, indicating the occurrence of functional cross-talk between p53 and NF-κB. Moreover, analysis of IL-6 mRNA levels in bone marrow-derived p53 null vs wild-type dendritic cells confirms a role for p53 in the reduction of NF-κB-dependent gene expression, mediated by genistein.
Genome-Wide Network Analysis Reveals the Global Properties of IFN-β Immediate Transcriptional Effects in Humans
Authors: Fernald Guy Haskin , Knott Simon , Pachner Andrew , Caillier Stacy J. , Narayan Kavitha , Oksenberg Jorge R. , Mousavi Parvin , Baranzini Sergio E.
Published: April 2014

IFN-β effectively controls clinical exacerbations and magnetic resonance imaging activity in most multiple sclerosis patients. However, its mechanism of action has not been yet fully elucidated. In this study we used DNA microarrays to analyze the longitudinal transcriptional profile of blood cells within a week of IFN-β administration. Using differential expression and gene ontology analyses we found evidence of a general decrease in the cellular activity of T lymphocytes resembling the endogenous antiviral response of IFNs. In contrast, most of the differentially expressed genes (DEGs) from untreated individuals were involved in cellular physiological processes. We then used mutual information (MI) to build networks of coregulated genes in both treated and untreated individuals. Interestingly, the connectivity distribution (k) of networks generated with high MI values displayed scale-free properties. Conversely, the observed k for networks generated with suboptimal MI values approximated a Poisson distribution, suggesting that MI captures biologically relevant interactions. Gene networks from individuals treated with IFN-β revealed a tight core of immune- and apoptosis-related genes associated with higher values of MI. In contrast, networks obtained from untreated individuals primarily reflected cellular housekeeping functions. Finally, we trained a neural network to reverse engineer the directionality of the main interactions observed at the biological process level. This is the first study that incorporates network analysis to investigate gene regulation in response to a therapeutic drug in humans. Implications of this method in the creation of personalized models of response to therapy are discussed.
Mannose-Binding Lectin A-Deficient Mice Have Abrogated Antigen-Specific IgM Responses and Increased Susceptibility to a Nematode Infection
Authors: Carter Tim , Sumiya Michiko , Reilly Kerri , Ahmed Rubina , Sobieszczuk Peter , Summerfield John A. , Lawrence Rachel A.
Published: April 2014

To investigate the role of mannose-binding lectin-A (MBL-A) in protection against infectious disease, MBL-A−/−-deficient mice were generated. Using a well-characterized mouse model of human filarial nematode infection, nematode survival and protective immune responses were tested in vivo. Blood-borne Brugia malayi microfilariae survived for significantly longer time periods in MBL-A−/− than in wild-type (WT) mice. However, no differences in either splenic cytokine responses or induction of leukocytes in the blood were observed. A profound abrogation of Ag-specific IgM levels was measured in B. malayi-infected MBL-A−/− mice, and some IgG isotypes were higher than those observed in WT animals. To establish whether there was a defect in Ab responses per se in MBL-A−/− mice or the effect was specific to filarial infection, we immunized these mice with OVA or a carbohydrate-free protein. Significantly, Ag-specific IgM responses were defective to both of these Ags, and Ag-specific IgG responses were largely unaffected. Furthermore, in naive mice, total IgM levels did not differ between MBL-A−/− and WT mice. This article describes the first demonstration that MBL-A may function independently of MBL-C and suggests that MBL-A, like other C-type lectins and members of the complement cascade, is intimately involved in the priming of the humoral Ab response.
Impaired Host Defense in Mice Lacking ONZIN
Authors: Ledford Julie G. , Kovarova Martina , Koller Beverly H.
Published: April 2014

ONZIN is a small, cysteine-rich peptide of unique structure that is conserved in all vertebrates examined to date. We show that ONZIN is expressed at high levels in epithelial cells of the intestinal tract, the lung, and in cells of the immune system including macrophages and granulocytes. Because this pattern of expression is suggestive of a role in innate immune function, we have generated mice lacking this protein and examined their ability to respond to challenge with infectious agents. Onzin−/− mice show a heightened innate immune response after induction of acute peritonitis with Klebsiella pneumoniae. This increased response is consistent with an increased bacterial burden in the Onzin−/− mice. Ex vivo studies show that, whereas phagocytosis is not altered in Onzin−/− neutrophils, phagocytes lacking this protein kill bacteria less effectively. This result identifies ONZIN as a novel class of intracellular protein required for optimal function of the neutrophils after uptake of bacteria.
Role of MyD88 in Route-Dependent Susceptibility to Vesicular Stomatitis Virus Infection
Authors: Zhou Shenghua , Kurt-Jones Evelyn A. , Fitzgerald Katherine A. , Wang Jennifer P. , Cerny Anna M. , Chan Melvin , Finberg Robert W.
Published: April 2014

TLRs are important components of the innate immune response. The role of the TLR signaling pathway in host defense against a natural viral infection has been largely unexplored. We found that mice lacking MyD88, an essential adaptor protein in TLR signaling pathway, were extremely sensitive to intranasal infection with vesicular stomatitis virus, and this susceptibility was dose dependent. We demonstrated that this increased susceptibility correlates with the impaired production of IFN-α and defective induction and maintenance of neutralizing Ab. These studies outline the important role of the TLR signaling pathway in nasal mucosae-respiratory tracts-neuroepithelium environment in the protection against microbial pathogen infections. We believe that these results explain how the route of infection, probably by virtue of activating different cell populations, can lead to entirely different outcomes of infection based on the underlying genetics of the host.
Dynamic Relationship between IFN-γ and IL-2 Profile of Mycobacterium tuberculosis-Specific T Cells and Antigen Load
Authors: Millington Kerry A. , Innes John A. , Hackforth Sarah , Hinks Timothy S. C. , Deeks Jonathan J. , Dosanjh Davinder P. S. , Guyot-Revol Valerie , Gunatheesan Rubamalaar , Klenerman Paul , Lalvani Ajit
Published: April 2014

Distinct IFN-γ and IL-2 profiles of Ag-specific CD4+ T cells have recently been associated with different clinical disease states and Ag loads in viral infections. We assessed the kinetics and functional profile of Mycobacterium tuberculosis Ag-specific T cells secreting IFN-γ and IL-2 in 23 patients with untreated active tuberculosis when bacterial and Ag loads are high and after curative treatment, when Ag load is reduced. The frequencies of M. tuberculosis Ag-specific IFN-γ-secreting T cells declined during 28 mo of follow-up with an average percentage decline of 5.8% per year (p = 0.005), while the frequencies of Ag-specific IL-2-secreting T cells increased during treatment (p = 0.02). These contrasting dynamics for the two cytokines led to a progressive convergence of the frequencies of IFN-γ- and IL-2-secreting cells over 28 mo. Simultaneous measurement of IFN-γ and IL-2 secretion at the single-cell level revealed a codominance of IFN-γ-only secreting and IFN-γ/IL-2 dual secreting CD4+ T cells in active disease that shifted to dominance of IFN-γ/IL-2-secreting CD4+ T cells and newly detectable IL-2-only secreting CD4+ T cells during and after treatment. These distinct T cell functional signatures before and after treatment suggest a novel immunological marker of mycobacterial load and clinical status in tuberculosis that now requires validation in larger prospective studies.