bluuebunny/crossref_metadata_2025 huggingface dataset:
Microglia Initiate Central Nervous System Innate and Adaptive Immune Responses through Multiple TLRs
Authors: Olson Julie K. , Miller Stephen D.
Published: April 2014

Microglia are the resident macrophage-like population in the CNS. Microglia remain quiescent until injury or infection activates the cells to perform effector inflammatory and APC functions. Our previous studies have shown that microglia infected with a neurotropic strain of Theiler’s murine encephalomyelitis virus secreted innate immune cytokines and up-regulated costimulatory molecules and MHC class II, enabling the cells to present viral and myelin Ags to CD4+ T cells. Recently, TLRs have been shown to recognize pathogen-associated molecular patterns and initiate innate immune responses upon interaction with infectious agents. We examined TLR expression on brain microglia and their functional responses upon stimulation with various TLR agonists. We report that mouse microglia express mRNA for all of the recently identified TLRs, TLR1–9, used for recognition of bacterial and viral molecular patterns. Furthermore, stimulation of quiescent microglia with various TLR agonists, including LPS (TLR4), peptidoglycan (TLR2), polyinosinic-polycytidylic acid (TLR3), CpG DNA (TLR9), and infection with viable Theiler’s murine encephalomyelitis virus, activated the cells to up-regulate unique patterns of innate and effector immune cytokines and chemokines at the mRNA and protein levels. In addition, TLR stimulation activated up-regulation of MHC class II and costimulatory molecules, enabling the microglia to efficiently present myelin Ags to CD4+ T cells. Thus, microglia appear to be a unique and important component of both the innate and adaptive immune response, providing the CNS with a means to rapidly and efficiently respond to a wide variety of pathogens.
Analysis of Notch1 Function by In Vitro T Cell Differentiation ofPax5Mutant Lymphoid Progenitors
Authors: Höflinger Sonja , Kesavan Kamala , Fuxa Martin , Hutter Caroline , Heavey Barry , Radtke Freddy , Busslinger Meinrad
Published: April 2014

Signaling through the Notch1 receptor is essential for T cell development in the thymus. Stromal OP9 cells ectopically expressing the Notch ligand Delta-like1 mimic the thymic environment by inducing hemopoietic stem cells to undergo in vitro T cell development. Notch1 is also expressed on Pax5−/− pro-B cells, which are clonable lymphoid progenitors with a latent myeloid potential. In this study, we demonstrate that Pax5−/− progenitors efficiently differentiate in vitro into CD4+CD8+ αβ and γδ T cells upon coculture with OP9-Delta-like1 cells. In vitro T cell development of Pax5−/− progenitors strictly depends on Notch1 function and progresses through normal developmental stages by expressing T cell markers and rearranging TCRβ, γ, and δ loci in the correct temporal sequence. Notch-stimulated Pax5−/− progenitors efficiently down-regulate the expression of B cell-specific genes, consistent with a role of Notch1 in preventing B lymphopoiesis in the thymus. At the same time, Notch signaling rapidly induces cell surface expression of the c-Kit receptor and transcription of the target genes Deltex1 and pre-Tα concomitant with the activation of TCR Vβ germline transcription and the regulatory genes GATA3 and Tcf1. These data suggest that Notch1 acts upstream of GATA3 and Tcf1 in early T cell development and regulates Vβ-DJβ rearrangements by controlling the chromatin accessibility of Vβ genes at the TCRβ locus.
IL-13 Activates a Mechanism of Tissue Fibrosis That Is Completely TGF-β Independent
Authors: Kaviratne Mallika , Hesse Matthias , Leusink Mary , Cheever Allen W. , Davies Stephen J. , McKerrow James H. , Wakefield Lalage M. , Letterio John J. , Wynn Thomas A.
Published: April 2014

Fibrosis is a characteristic feature in the pathogenesis of a wide spectrum of diseases. Recently, it was suggested that IL-13-dependent fibrosis develops through a TGF-β1 and matrix metalloproteinase-9-dependent (MMP-9) mechanism. However, the significance of this pathway in a natural disorder of fibrosis was not investigated. In this study, we examined the role of TGF-β in IL-13-dependent liver fibrosis caused by Schistosoma mansoni infection. Infected IL-13−/− mice showed an almost complete abrogation of fibrosis despite continued and undiminished production of TGF-β1. Although MMP-9 activity was implicated in the IL-13 pathway, MMP-9−/− mice displayed no reduction in fibrosis, even when chronically infected. To directly test the requirement for TGF-β, studies were also performed with neutralizing anti-TGF-β Abs, soluble antagonists (soluble TGF-βR-Fc), and Tg mice (Smad3−/− and TGF-βRII-Fc Tg) that have disruptions in all or part of the TGF-β signaling cascade. In all cases, fibrosis developed normally and with kinetics similar to wild-type mice. Production of IL-13 was also unaffected. Finally, several genes, including interstitial collagens, several MMPs, and tissue inhibitors of metalloprotease-1 were up-regulated in TGF-β1−/− mice by IL-13, demonstrating that IL-13 activates the fibrogenic machinery directly. Together, these studies provide unequivocal evidence of a pathway of fibrogenesis that is IL-13 dependent but TGF-β1 independent, illustrating the importance of targeting IL-13 directly in the treatment of infection-induced fibrosis.
STAT4 Is a Critical Mediator of Early Innate Immune Responses against Pulmonary Klebsiella Infection
Authors: Deng Jane C. , Zeng Xianying , Newstead Michael , Moore Thomas A. , Tsai Wan C. , Thannickal Victor J. , Standiford Theodore J.
Published: April 2014

Bacterial pneumonia is a leading cause of morbidity and mortality in the U.S. An effective innate immune response is critical for the clearance of bacteria from the lungs. IL-12, a key T1 cytokine in innate immunity, signals through STAT4. Thus, understanding how STAT4 mediates pulmonary immune responses against bacterial pathogens will have important implications for the development of rational immunotherapy targeted at augmenting innate immunity. We intratracheally administered Klebsiella pneumoniae to wild-type BALB/c and STAT4 knockout (STAT4−/−) mice. Compared with wild-type controls, STAT4−/− mice had decreased survival following intratracheal Klebsiella administration, which was associated with a higher lung and blood bacterial burden. STAT4−/− animals also displayed impaired pulmonary IFN-γ production and decreased levels of proinflammatory cytokines, including the ELR− CXC chemokines IFN-γ-inducible protein-10 and monokine induced by IFN-γ. Although total lung leukocyte populations were similar between STAT4−/− and wild-type animals following infection, alveolar macrophages isolated from infected STAT4−/− mice had decreased production of proinflammatory cytokines, including IFN-γ, compared with infected wild-type mice. The intrapulmonary overexpression of IFN-γ concomitant with the systemic administration of IFN-γ partially reversed the immune deficits observed in STAT4−/− mice, resulting in improved bacterial clearance from the blood. Collectively, these studies demonstrate that STAT4 is required for the generation of an effective innate host defense against bacterial pathogens of the lung.
Low-DoseSalmonellaInfection Evades Activation of Flagellin-Specific CD4 T Cells
Authors: Srinivasan Aparna , Foley Joseph , Ravindran Rajesh , McSorley Stephen J.
Published: April 2014

Many pathogens can establish a lethal infection from relatively small inocula, yet the effect of infectious dose upon CD4 T cell activation is not clearly understood. This issue was examined by tracking Salmonella flagellin-specific SM1 T cells in vivo, after i.v. and oral challenge of mice with virulent Salmonella typhimurium. SM1 T cells rapidly expressed activation markers and expanded in response to high-dose infection but remained completely unresponsive in mice challenged with low doses of Salmonella. SM1 T cells, in these mice, remained unresponsive, despite massive bacterial replication in vivo. Naive SM1 T cells in low-dose Salmonella-infected mice were activated rapidly after the injection of flagellin peptide, demonstrating that these T cells were fully capable of responding, ruling out the possibility of a bacterial-induced suppressive environment. The inability of flagellin-specific SM1 T cells to respond to low-dose infection was not due to Ag down-regulation, because flagellin expression was detected using a functional assay. Together, these data suggest that low-dose Salmonella infection can evade flagellin-specific CD4 T cell activation in vivo.
Reduction of Nitric Oxide Synthase 2 Expression by Distamycin A Improves Survival from Endotoxemia
Authors: Baron Rebecca M. , Carvajal Irvith M. , Liu Xiaoli , Okabe Rachel O. , Fredenburgh Laura E. , Macias Alvaro A. , Chen Yen-Hsu , Ejima Kuniaki , Layne Matthew D. , Perrella Mark A.
Published: April 2014

NO synthase 2 (NOS2) plays an important role in endotoxemia through overproduction of NO. Distamycin A (Dist A) belongs to a class of drugs termed minor-groove DNA binders, which can inhibit transcription factor binding to AT-rich regions of DNA. We and others have previously shown that AT-rich regions of DNA surrounding transcription factor binding sites in the NOS2 promoter are critical for NOS2 induction by inflammatory stimuli in vitro. Therefore, we hypothesized that Dist A would attenuate NOS2 up-regulation in vivo during endotoxemia and improve animal survival. C57BL/6 wild-type (WT) mice treated with Dist A and LPS (endotoxin) showed significantly improved survival compared with animals treated with LPS alone. In contrast, LPS-treated C57BL/6 NOS2-deficient (NOS2−/−) mice did not benefit from the protective effect of Dist A on mortality from endotoxemia. Treatment with Dist A resulted in protection from hypotension in LPS-treated WT mice, but not in NOS2−/− mice. Furthermore, LPS-induced NOS2 expression was attenuated in vivo (WT murine tissues) and in vitro (primary peritoneal and RAW 264.7 murine macrophages) with addition of Dist A. Dist A selectively decreased IFN regulatory factor-1 DNA binding in the enhancer region of the NOS2 promoter, and this IFN regulatory factor-1 site is critical for the effect of Dist A in attenuating LPS induction of NOS2. Our data point to a novel approach in modulating NOS2 expression in vivo during endotoxemia and suggest the potential for alternative treatment approaches for critical illness.
Divergent Trophoblast Responses to Bacterial Products Mediated by TLRs
Authors: Abrahams Vikki M. , Bole-Aldo Paulomi , Kim Yeon Mee , Straszewski-Chavez Shawn L. , Chaiworapongsa Tinnakorn , Romero Roberto , Mor Gil
Published: April 2014

Intrauterine infections have been associated with pregnancy complications that are also linked with increased trophoblast apoptosis. TLRs are key components of the innate immune system which recognize conserved sequences on the surface of pathogens and trigger effector cell functions. We hypothesize that intrauterine infections may cause the excessive trophoblast cell apoptosis observed in abnormal pregnancies and that TLR may provide a mechanism of pathogenesis. In this study we describe the expression and function of TLR-2 and TLR-4 in first trimester trophoblast cells. Although ligation of TLR4 induced cytokine production by trophoblast cells, TLR-2 activation induced apoptosis. TLR-2 mediated apoptosis was dependent upon the Fas-associated death domain, the inactivation of the X-linked inhibitor of apoptosis, and the activation of caspases 8, 9, and 3. These results suggest that certain intrauterine infections may directly induce trophoblast cell death through TLR-2. Our findings provide a novel mechanism of pathogenesis for certain pregnancy complications in which there is engagement of the innate immune system.
Human Plasmacytoid Dendritic Cells Activated by CpG Oligodeoxynucleotides Induce the Generation of CD4+CD25+ Regulatory T Cells
Authors: Moseman E. Ashley , Liang Xueqing , Dawson Amanda J. , Panoskaltsis-Mortari Angela , Krieg Arthur M. , Liu Yong-Jun , Blazar Bruce R. , Chen Wei
Published: April 2014

Plasmacytoid dendritic cells (PDCs) are key effectors in host innate immunity and orchestrate adaptive immune responses. CpG oligodeoxynucleotides (ODN) have potent immunostimulatory effects on PDCs through TLR9 recognition and signaling. Little is known about the effects of CpG ODN on human PDC-mediated T cell priming. Here we show that type B CpG ODN effectively promotes PDCs to prime allogeneic naive CD4+CD25− T cells to differentiate into CD4+CD25+ regulatory T (Treg) cells. The CD4+CD25+ T cells induced by CpG ODN-activated PDCs express forkhead transcription factor 3 and produce IL-10, TGF-β, IFN-γ, and IL-6, but low IL-2 and IL-4. These CD4+CD25+ T cells are hyporesponsive to secondary alloantigen stimulation and strongly inhibit proliferation of autologous or allogeneic naive CD4+ T cells in an Ag-nonspecific manner. CpG ODN-activated PDCs require direct contact with T cells to induce CD4+CD25+ Treg cells. Interestingly, IL-10 and TGF-β were undetectable in the supernatants of CpG ODN-stimulated PDC cultures. Both CpG-A and CpG-C ODN-activated PDCs similarly induced the generation of CD4+CD25+ Treg cells with strong immune suppressive function. This study demonstrates that TLR9 stimulation can promote PDC-mediated generation of CD4+CD25+ Treg cells and suggests PDCs may play an important role in the maintenance of immunological tolerance.
CpG DNA Induces IgG Class Switch DNA Recombination by Activating Human B Cells through an Innate Pathway That Requires TLR9 and Cooperates with IL-10
Authors: He Bing , Qiao Xugang , Cerutti Andrea
Published: April 2014

TLRs are pattern recognition receptors that initiate innate immune responses. TLR9 detects microbial DNA with hypomethylated CpG motifs and in humans is preferentially expressed by IFN-α-producing plasmacytoid dendritic cells and B cells. In addition to favoring IFN-α release, TLR9 signals B cell activation, proliferation, and IgM production. Recent findings suggest that CpG DNA-TLR9 interaction plays a key role in systemic lupus erythematosus and rheumatoid arthritis, two autoimmune disorders characterized by dysregulated production of DNA-reactive IgG. We show that CpG DNA initiates germline Cγ1, Cγ2, and Cγ3 gene transcription by activating B cells through a TLR9-mediated NF-κB-Rel-dependent innate pathway that cooperates with IL-10 through STAT proteins and IFN-responsive factors. This pathway is inhibited by chloroquine, a drug that attenuates the clinical manifestations of IgG-mediated autoimmune disorders. Germline Cγ gene transcription is associated with up-regulation of activation-induced cytidine deaminase, a key element of the B cell class switch-inducing machinery, and is followed by class switch DNA recombination from Cμ to Cγ1, Cγ2, and Cγ3. Subsequent IgG production requires additional signals from BCR and a B cell-activating factor of the TNF family (BAFF), produced by dendritic cells upon exposure to IFN-α. Our findings suggest that CpG DNA-TLR9 interaction may be important to initiate or amplify early T cell-independent IgG responses against pathogens. This implies that CpG DNA released during infections may exacerbate autoimmunity by stimulating autoreactive B cells to switch from an IgM to a more pathogenic IgG isotype.
Critical Role of TNF Receptor Type-2 (p75) as a Costimulator for IL-2 Induction and T Cell Survival: A Functional Link to CD28
Authors: Kim Edward Y. , Teh Hung-Sia
Published: April 2014

CD28 provides important signals that lower the threshold of T cell activation, augment the production of IL-2, and promote T cell survival. The recent identification of a second family of costimulatory molecules within the TNFR family has reshaped the “two-signal” model of T cell activation. In this study the role of p75 as a T cell costimulatory molecule in controlling cell fate during TCR/CD28-mediated stimulation was examined. We found that p75-deficient T cells possess a profound defect in IL-2 production in response to TCR/CD28-mediated stimulation. Examination of key signaling intermediates revealed that TCR proximal events such as global tyrosine phosphorylation and ZAP70 phosphorylation, as well as downstream MAPK cascades are unperturbed in p75-deficient T cells. In contrast, p75 is nonredundantly coupled to sustained AKT activity and NF-κB activation in response to TCR/CD28-mediated stimulation. Moreover, p75-deficient T cells possess a defect in survival during the early phase of T cell activation that is correlated with a striking defect in Bcl-xL expression. These data indicate discrete effects of p75 on the intracellular signaling milieu during T cell activation, and reveal the synergistic requirement of TCR, CD28, and p75 toward optimal IL-2 induction and T cell survival. We propose that p75 acts as one of the earliest of the identified costimulatory members of the TNFR family, and is functionally linked to CD28 for initiating and determining T cell fate during activation.
An Important Role for Polymeric Ig Receptor-Mediated Transport of IgA in Protection against Streptococcus pneumoniae Nasopharyngeal Carriage
Authors: Sun Keer , Johansen Finn-Eirik , Eckmann Lars , Metzger Dennis W.
Published: April 2014

The importance of IgA for protection at mucosal surfaces remains unclear, and in fact, it has been reported that IgA-deficient mice have fully functional vaccine-induced immunity against several bacterial and viral pathogens. The role of respiratory Ab in preventing colonization by Streptococcus pneumoniae has now been examined using polymeric IgR knockout (pIgR−/−) mice, which lack the ability to actively secrete IgA into the mucosal lumen. Intranasal vaccination with a protein conjugate vaccine elicited serotype-specific anti-capsular polysaccharide Ab locally and systemically, and pIgR−/− mice produced levels of total serum Ab after vaccination that were similar to wild-type mice. However, pIgR−/− mice had ∼5-fold more systemic IgA and 6-fold less nasal IgA Ab than wild-type mice due to defective transport into mucosal tissues. Wild-type, but not pIgR−/− mice were protected against infection with serotype 14 S. pneumoniae, which causes mucosal colonization but does not induce systemic inflammatory responses in mice. The relative importance of secretory IgA in host defense was further shown by the finding that intranasally vaccinated IgA gene-deficient mice were not protected from colonization. Although secretory IgA was found to be important for protection against nasal carriage, it does not appear to have a crucial role in immunity to systemic pneumococcus infection, because both vaccinated wild-type and pIgR−/− mice were fully protected from lethal systemic infection by serotype 3 pneumococci. The results demonstrate the critical role of secretory IgA in protection against pneumococcal nasal colonization and suggest that directed targeting to mucosal tissues will be needed for effective vaccination in humans.
The Antibiotic Polymyxin B Modulates P2X7 Receptor Function
Authors: Ferrari Davide , Pizzirani Cinzia , Adinolfi Elena , Forchap Sylvia , Sitta Barbara , Turchet Laura , Falzoni Simonetta , Minelli Mattia , Baricordi Roberto , Di Virgilio Francesco
Published: April 2014

The natural peptide polymyxin B (PMB) is a well-known and potent antibiotic that binds and neutralizes bacterial endotoxin (LPS), thus preventing its noxious effects among LPS-mediated endotoxin shock in animal models. We have investigated the effect of PMB on responses mediated by the P2X7R in HEK293 and K562 cells transfected with P2X7 cDNA and in mouse and human macrophages. In addition, in view of the potential exploitation of P2X7-directed agonists in antitumor therapy, we also investigated the effect of PMB in B lymphocytes from patients affected by chronic lymphocytic leukemia. PMB, at an optimal concentration dependent on the given cell type, greatly potentiated the effect of nucleotide-mediated P2X7 stimulation. In particular, ATP-mediated Ca2+ influx, plasma membrane permeabilization, and cytotoxicity were enhanced to an extent that, in the presence of PMB, cells were killed by otherwise ineffective nucleotide concentrations. The synergistic effect due to the combined application of ATP and PMB was prevented by incubation with the irreversible P2X blocker oxidized ATP (oATP), but not with the reversible antagonist 1-(N,O-bis(1,5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl)-4-phenilpiperazine (KN-62). Cells lacking P2X7 were fully insensitive to the combined stimulation with PMB and ATP. Furthermore, PMB at the concentrations used had no untoward effects on cell viability. These results point to PMB as a useful tool for the modulation of P2X7R function and suggest that care should be used in the evaluation of ATP-stimulated immune cell responses in the presence of PMB as they may not solely be affected by removal of contaminating LPS.
Human Parvovirus B19 Transgenic Mice Become Susceptible to Polyarthritis
Authors: Takasawa Naruhiko , Munakata Yasuhiko , Ishii Keiko Kumura , Takahashi Yuichi , Takahashi Minako , Fu Yi , Ishii Tomonori , Fujii Hiroshi , Saito Takako , Takano Hiroshi , Noda Tetsuo , Suzuki Misao , Nose Masato , Zolla-Patzner Suzan , Sasaki Takeshi
Published: April 2014

Human parvovirus B19 (B19) often causes acute polyarthritis in adults. In this paper, we analyzed nucleotide sequences of the B19 genome of patients with rheumatoid arthritis (RA), and then introduced the nonstructual protein 1 (NS1) gene of B19 into C57BL/6 mice that had a genetic origin not susceptible to arthritis. The transgenic mice developed no lesions spontaneously, but were susceptible to type II collagen (CII)-induced arthritis. B19 NS1 was expressed in synovial cells on the articular lesions that were histologically characteristic of granulomatous synovitis and pannus formation in cartilage and bone. Serum levels of anti-CII Abs and TNF-α increased in NS1 transgenic mice to the same levels as those of DBA/1 mice, which were susceptible to polyarthritis. Stimulation with CII increased secretion of Th1-type- and Th2-type cytokines in NS1 transgenic mice, indicating that a nonpermissive H-2b haplotype in the wild type of C57BL/6 mice can be made susceptible to polyarthritis through the expression of NS1. This study is the first to show that a viral agent from the joints in humans can cause CII-induced arthritis resembling RA.
A Transient Post-Translationally Modified Form of Cartilage Type II Collagen Is Ignored by Self-Reactive T Cells
Authors: Yamada Hisakata , Dzhambazov Balik , Bockermann Robert , Blom Thomas , Holmdahl Rikard
Published: April 2014

Lysine residues in type II collagen (CII) are normally hydroxylated and subsequently glycosylated in the chondrocyte. The immunodominant T cell epitope of CII involves such post-translationally modified lysine at position 264 that has been shown to be critical in the pathogenesis of murine collagen-induced arthritis and also in human rheumatoid arthritis. In this study we identified a line of transgenic mice expressing a TCR specific for hydroxylated rat CII epitope. They were crossed with transgenic mice expressing the rat CII epitope, either specifically in cartilage (MMC mice) or systemically (TSC mice), to analyze T cell tolerance to a post-translationally modified form of self-CII. The mechanism of T cell tolerance to the hydroxylated CII epitope in TSC mice was found to involve intrathymic deletion and induction of peripheral tolerance. In contrast, we did not observe T cell tolerance in the MMC mice. Analysis of CII prepared from rat or human joint cartilage revealed that most of the lysine 264 is glycosylated rather than remaining hydroxylated. Therefore, we conclude that the transient post-translationally modified form of cartilage CII does not induce T cell tolerance. This lack of T cell tolerance could increase the risk of developing autoimmune arthritis.
Regression of Established Liver Tumor Induced by Monoepitopic Peptide-Based Immunotherapy
Authors: Belnoue Elodie , Guettier Catherine , Kayibanda Michèle , Le Rond Solène , Crain-Denoyelle Anne-Marie , Marchiol Carmen , Ziol Marianne , Fradelizi Didier , Rénia Laurent , Viguier Mireille
Published: April 2014

Most types of cancer are difficult to eradicate, and some, like hepatocellular carcinoma, are almost always fatal. Among various interventions to improve the survival of patients with cancer, immunotherapy seems to hold some promises. However, it requires relevant animal models for preclinical development. In this study we report a new and relevant experimental model where liver tumors grow inside a nontumoral parenchyma of adult mice. This model is based on the intrasplenic injection in syngeneic recipient mice of hepatocytes from transgenic mice expressing SV40 large T oncogene specifically in the liver. Using this model where no apparent spontaneous cellular immune response was observed, immunization using a single injection of monoepitopic SV40 T Ag short peptide was sufficient to provoke liver tumor destruction, leading rapidly to complete remission. Tumor regression was associated with the induction of a long-lasting CD8+ T cell response, observed not only in the spleen but also, more importantly, in the tumoral liver. These results show the efficacy of peptide immunotherapy in the treatment of liver cancer.
The Magnitude of Akt/Phosphatidylinositol 3′-Kinase Proliferating Signaling Is Related to CD45 Expression in Human Myeloma Cells
Authors: Descamps Géraldine , Pellat-Deceunynck Catherine , Szpak Yann , Bataille Régis , Robillard Nelly , Amiot Martine
Published: April 2014

In multiple myeloma, the Akt/PI3K pathway is involved in the proliferation of myeloma cells. In the current study, we have investigated the impact of the CD45 phosphatase in the control of Akt/PI3K activation. We show that Akt activation in response to insulin-like growth factor-1 (IGF-1) is highly variable from one human myeloma cell line to another one. Actually, Akt activation is highly related to whether CD45 is expressed or not. Indeed, both the magnitude and the duration of Akt phosphorylation in response to IGF-1 are more important in CD45− than in CD45+ myeloma cell lines. We next demonstrate a physical association between CD45 and IGF-1 receptor (IGF-1R) suggesting that CD45 could be involved in the dephosphorylation of the IGF-1R. Furthermore, the growth of CD45− myeloma cell lines is mainly or even totally controlled by the PI3K pathway whereas that of CD45+ myeloma cell lines is modestly controlled by it. Indeed, wortmannin, a specific PI3K inhibitor, induced a dramatic growth inhibition in the CD45− myeloma cell lines characterized by a G1 growth arrest, whereas it has almost no effect on CD45+ myeloma cell lines. Altogether, these results suggest that CD45 negatively regulates IGF-1-dependent activation of PI3K. Thus, strategies that block IGF-1R signaling and consequently the Akt/PI3K pathway could be a priority in the treatment of patients with multiple myeloma, especially those lacking CD45 expression that have a very poor clinical outcome.
STAT6-Dependent Differentiation and Production of IL-5 and IL-13 in Murine NK2 Cells
Authors: Katsumoto Takuo , Kimura Motoko , Yamashita Masakatsu , Hosokawa Hiroyuki , Hashimoto Kahoko , Hasegawa Akihiro , Omori Miyuki , Miyamoto Takeshi , Taniguchi Masaru , Nakayama Toshinori
Published: April 2014

NK cells differentiate into either NK1 or NK2 cells that produce IFN-γ or IL-5 and IL-13, respectively. Little is known, however, about the molecular mechanisms that control NK1 and NK2 cell differentiation. To address these questions, we established an in vitro mouse NK1/NK2 cell differentiation culture system. For NK1/NK2 cell differentiation, initial stimulation with PMA and ionomycin was required. The in vitro differentiated NK2 cells produced IL-5 and IL-13, but the levels were 20 times lower than those of Th2 or T cytotoxic (Tc)2 cells. No detectable IL-4 was produced. Freshly prepared NK cells express IL-2Rβ, IL-2RγC, and IL-4Rα. After stimulation with PMA and ionomycin, NK cells expressed IL-2Rα. NK1 cells displayed higher cytotoxic activity against Yac-1 target cells. The levels of GATA3 protein in developing NK2 cells were approximately one-sixth of those in Th2 cells. Both NK1 and NK2 cells expressed large amounts of repressor of GATA, the levels of which were equivalent to CD8 Tc1 and Tc2 cells and significantly higher than those in Th2 cells. The levels of histone hyperacetylation of the IL-4 and IL-13 gene loci in NK2 cells were very low and equivalent to those in naive CD4 T cells. The production of IL-5 and IL-13 in NK2 cells was found to be STAT6 dependent. Thus, similar to Th2 cells, NK2 cell development is dependent on STAT6, and the low level expression of GATA3 and the high level expression of repressor of GATA may influence the unique type 2 cytokine production profiles of NK2 cells.
Murine CD4+CD25+ Regulatory T Cells Fail to Undergo Chromatin Remodeling Across the Proximal Promoter Region of the IL-2 Gene
Authors: Su Leon , Creusot Remi J. , Gallo Elena M. , Chan Steven M. , Utz Paul J. , Fathman C. Garrison , Ermann Joerg
Published: April 2014

CD4+CD25+ regulatory T cells (Treg) acquire unique immunosuppressive properties while maintaining an anergy phenotype when activated in vitro under conditions that induce IL-2 production and proliferation in conventional CD4+ T cells. We investigated the mechanism underlying one component of this naturally anergic phenotype, the inability of the Treg cells to produce IL-2 following activation. Analysis of freshly isolated murine CD4+CD25+ Treg and conventional CD4+CD25− T cells following PMA/ionomycin stimulation demonstrated no differences in inducible AP-1 formation, an important transcriptional complex in regulating IL-2 gene expression. Although p38 MAPK and ERK1/2 protein kinases were phosphorylated with similar kinetics, we observed diminished activation of JNK in the CD4+CD25+ Treg cells. However, lentiviral-mediated reconstitution of the JNK pathway using a constitutively active construct did not overcome the block in IL-2 synthesis. Using a PCR-based chromatin accessibility assay we found that the minimal IL-2 promoter region of CD4+CD25+ Treg cells, unlike conventional CD4 T cells, did not undergo chromatin remodeling following stimulation, suggesting that the inability of CD4+CD25+ Treg cells to secrete IL-2 following activation is controlled at the chromatin level.
The Role of CTLs in Persistent Viral Infection: Cytolytic Gene Expression in CD8+ Lymphocytes Distinguishes between Individuals with a High or Low Proviral Load of Human T Cell Lymphotropic Virus Type 1
Authors: Vine Alison M. , Heaps Adrian G. , Kaftantzi Lambrini , Mosley Angelina , Asquith Becca , Witkover Aviva , Thompson Gillian , Saito Mineki , Goon Peter K. C. , Carr Laura , Martinez-Murillo Francisco , Taylor Graham P. , Bangham Charles R. M.
Published: April 2014

The proviral load in human T cell lymphotropic virus type 1 (HTLV-1) infection is typically constant in each infected host, but varies by >1000-fold between hosts and is strongly correlated with the risk of HTLV-1-associated inflammatory disease. However, the factors that determine an individual’s HTLV-1 proviral load remain uncertain. Experimental evidence from studies of host genetics, viral genetics, and lymphocyte function and theoretical considerations suggest that a major determinant of the equilibrium proviral load is the CD8+ T cell response to HTLV-1. In this study, we tested the hypothesis that the gene expression profile in circulating CD8+ and CD4+ lymphocytes distinguishes between individuals with a low proviral load of HTLV-1 and those with a high proviral load. We show that circulating CD8+ lymphocytes from individuals with a low HTLV-1 proviral load overexpressed a core group of nine genes with strong functional coherence: eight of the nine genes encode granzymes or other proteins involved in cell-mediated lysis or Ag recognition. We conclude that successful suppression of the HTLV-1 proviral load is associated with strong cytotoxic CD8+ lymphocyte activity in the peripheral blood.
Protease-Activated Receptor Signaling Increases Epithelial Antimicrobial Peptide Expression
Authors: Chung Whasun O. , Hansen Stephen R. , Rao Divya , Dale Beverly A.
Published: April 2014

Epithelial tissues provide both a physical barrier and an antimicrobial barrier. Antimicrobial peptides of the human β-defensin (hBD) family are part of the innate immune responses that play a role in mucosal defense. hBDs are made in epithelia including oral epithelium where the bacterial load is particularly great. hBD-2 and hBD-3 are up-regulated in response to bacterial stimuli. Previous studies show that hBD-2 expression in human gingival epithelial cells (GEC) is stimulated by both nonpathogenic and pathogenic bacteria, including Porphyromonas gingivalis, a Gram-negative pathogen associated with periodontitis. Present evidence suggests that hBD-2 expression in GEC uses several signaling pathways, including an NF-κB-mediated pathway but without apparent LPS-TLR4 signaling. Protease-activated receptors (PAR) are G-protein-coupled receptors that mediate cellular responses to extracellular proteinases. P. gingivalis secretes multiple proteases that contribute to its virulence mechanisms. To determine whether PAR signaling is used in hBD-2 induction, GEC were stimulated with wild-type P. gingivalis or mutants lacking one or more proteases. hBD-2 mRNA expression was reduced in GEC stimulated with single protease mutants (11–67% compared with wild type), strongly reduced in double mutants (0.1–16%), and restored to wild-type levels (93%) in mutant with restored protease activity. Stimulation by wild type was partially blocked by inhibitors of phospholipase C, a main signaling pathway for PARs. Expression of hBD-3 was unaffected. Peptide agonist of PAR-2, but not PAR-1 activator, also induced hBD-2 in GEC. Thus, P. gingivalis proteases are directly involved in regulation of hBD-2 in cultured GEC, and this induction partially uses the PAR-2 receptor and signaling pathway.
Impaired Immunity to Intestinal Bacterial Infection in Stromelysin-1 (Matrix Metalloproteinase-3)-Deficient Mice
Authors: Li Chris K. F. , Pender Sylvia L. F. , Pickard Karen M. , Chance Victoria , Holloway Judith A. , Huett Alan , Gonçalves Nathalie S. , Mudgett John S. , Dougan Gordon , Frankel Gad , MacDonald Thomas T.
Published: April 2014

Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease. Matrix metalloproteinases (MMPs) have been shown to mediate matrix remodeling and cell migration during tissue injury and repair in the intestine. We have previously shown enhanced pathology in infected TNFRp55−/−, IL-12p40−/−, and IFN-γ−/− mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues. We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3−/− mice. In MMP3−/− mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice. Colonic tissues from MMP3−/− mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12. However, MMP3−/− mice showed delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal lamina propria. CSFE-labeled mesenteric lymph node CD4+ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon of MMP3−/− mice than into those of WT mice. These studies show that mucosal remodeling can occur in the absence of MMP3, but that MMP3 plays a role in the migration of CD4+ T lymphocytes to the intestinal mucosa.
Key Metalloproteinases Are Expressed by Specific Cell Types in Experimental Autoimmune Encephalomyelitis
Authors: Toft-Hansen Henrik , Nuttall Robert K. , Edwards Dylan R. , Owens Trevor
Published: April 2014

Metalloproteinases (MPs) include matrix metalloproteinases (MMPs) and metalloproteinase-disintegrins (ADAMs). Their physiological inhibitors are tissue inhibitor of metalloproteinases (TIMPs). MPs are thought to be mediators of cellular infiltration in the pathogenesis of multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE). We used real-time RT-PCR to profile the expression of all 22 known mouse MMPs, seven ADAMs, and all four known TIMPs in spinal cord from SJL/J mice and mice with adoptively transferred myelin basic protein (MBP)-specific EAE. A significant and >3-fold alteration in expression was observed for MMP-8, MMP-10, MMP-12, ADAM-12, and TIMP-1, which were up-regulated, and for MMP-15, which was down-regulated. Expression levels correlated with disease course, with all but ADAM-12 returning toward control levels in remission. To examine potential cellular sources of these strongly affected proteins in the inflamed CNS, we isolated macrophages, granulocytes, microglia, and T cells by cell sorting from the CNS of mice with EAE and analyzed their expression by real-time RT-PCR. This identified macrophages as a major source of MMP-12 and TIMP-1. Granulocytes were a major source of MMP-8. ADAM-12 was expressed primarily by T cells. Cellular localization of MMP-10, TIMP-1, and ADAM-12 in perivascular infiltrates was confirmed by immunostaining or in situ hybridization. Microglia from control mice expressed strong signal for MMP-15. Strikingly, the expression of MMP-15 by microglia was significantly down-regulated in EAE, which was confirmed by immunostaining. Our study identifies the cellular sources of key MPs in CNS inflammation.
Differential Regulation of Th1/Th2 Cytokine Responses by Placental Protein 14
Authors: Mishan-Eisenberg Galit , Borovsky Zipora , Weber Matthew C. , Gazit Roi , Tykocinski Mark L. , Rachmilewitz Jacob
Published: April 2014

The potency of TCR signaling during primary CD4+ T cell activation influences initial cytokine expression patterns and subsequent polarization toward either Th1 or Th2 subsets. In this study, we demonstrate that the T cell inhibitor placental protein 14 (PP14; glycodelin) preferentially inhibits Th1 cytokine responses and chemokine expression when present during ex vivo priming of CD4+ T cells. PP14 synergizes with exogenously added IL-4 in skewing T cell responses. Significantly, PP14 impairs the down-regulation of GATA-3 transcriptional regulator expression that normally accompanies T cell activation, which is a prerequisite for Th1 development. Taken together, these data document for the first time the ability of PP14 to skew Th responses.
Roles of TNF Receptor-Associated Factor 3 in Signaling to B Lymphocytes by Carboxyl-Terminal Activating Regions 1 and 2 of the EBV-Encoded Oncoprotein Latent Membrane Protein 1
Authors: Xie Ping , Bishop Gail A.
Published: April 2014

TNFR-associated factor (TRAF)3, an adaptor protein that binds the cytoplasmic domains of both CD40 and the EBV-encoded oncoprotein latent membrane protein (LMP)1, is required for positive signaling by LMP1 but not CD40 in B lymphocytes. The present study further investigated how TRAF3 participates in LMP1 signaling. We found that TRAF3 mediates signaling both through direct interactions with the C-terminal activating region (CTAR)1 of LMP1 and through indirect interactions with the CTAR2 region of LMP1 in mouse B cells. Notably, our results demonstrated that the CTAR2 region appears to inhibit the recruitment of TRAF1 and TRAF2 to membrane rafts by the CTAR1 region. Additionally, the absence of TRAF2 in B cells resulted in only a modest reduction in CTAR1-mediated signals and no detectable effect on CTAR2-mediated signals. CTAR1 and CTAR2 cooperated to achieve the robust signaling activity of LMP1 when recruited to the same membrane microdomains in B cells. Interestingly, TRAF3 deficiency completely abrogated the cooperation between CTAR1 and CTAR2, supporting the hypothesis that TRAF3 participates in the physical interaction between CTAR1 and CTAR2 of LMP1. Together, our findings highlight the central importance of TRAF3 in LMP1-mediated signaling, which is critical for EBV persistent infection and EBV-associated pathogenesis.
The B Lymphocyte Adaptor Molecule of 32 Kilodaltons (Bam32) Regulates B Cell Antigen Receptor Internalization
Authors: Niiro Hiroaki , Allam Atef , Stoddart Angela , Brodsky Frances M. , Marshall Aaron J. , Clark Edward A.
Published: April 2014

The B lymphocyte adaptor molecule of 32 kDa (Bam32) is an adaptor that plays an indispensable role in BCR signaling. In this study, we found that upon BCR ligation, Bam32 is recruited to the plasma membrane where it associates with BCR complexes and redistributes and internalizes with BCRs. BCR ligation induced colocalization of Bam32 with lipid rafts, clathrin, and actin filaments. An inhibitor of Src family protein tyrosine kinases (PTKs) blocked both BCR-induced tyrosine phosphorylation of Bam32 and BCR internalization. Moreover, BCR internalization is impaired in Bam32−/− and Lyn−/− cells, and expression of Bam32 with a mutation of its tyrosine phosphorylation site (Y139F) inhibited BCR internalization. These data suggest that Bam32 functions downstream of Src family PTKs to regulate BCR internalization. Bam32 deficiency does not affect tyrosine phosphorylation of clathrin or the association of clathrin with lipid rafts upon BCR cross-linking. However, BCR-induced actin polymerization is impaired in Bam32−/− cells. Collectively, these findings indicate a novel role of Bam32 in connecting Src family PTKs to BCR internalization by an actin-dependent mechanism.
Polymorphonuclear Cell Transmigration Induced by Pseudomonas aeruginosa Requires the Eicosanoid Hepoxilin A3
Authors: Hurley Bryan P. , Siccardi Dario , Mrsny Randall J. , McCormick Beth A.
Published: April 2014

Lung inflammation resulting from bacterial infection of the respiratory mucosal surface in diseases such as cystic fibrosis and pneumonia contributes significantly to the pathology. A major consequence of the inflammatory response is the recruitment and accumulation of polymorphonuclear cells (PMNs) at the infection site. It is currently unclear what bacterial factors trigger this response and exactly how PMNs are directed across the epithelial barrier to the airway lumen. An in vitro model consisting of human PMNs and alveolar epithelial cells (A549) grown on inverted Transwell filters was used to determine whether bacteria are capable of inducing PMN migration across these epithelial barriers. A variety of lung pathogenic bacteria, including Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa are indeed capable of inducing PMN migration across A549 monolayers. This phenomenon is not mediated by LPS, but requires live bacteria infecting the apical surface. Bacterial interaction with the apical surface of A549 monolayers results in activation of epithelial responses, including the phosphorylation of ERK1/2 and secretion of the PMN chemokine IL-8. However, secretion of IL-8 in response to bacterial infection is neither necessary nor sufficient to mediate PMN transepithelial migration. Instead, PMN transepithelial migration is mediated by the eicosanoid hepoxilin A3, which is a PMN chemoattractant secreted by A549 cells in response to bacterial infection in a protein kinase C-dependent manner. These data suggest that bacterial-induced hepoxilin A3 secretion may represent a previously unrecognized inflammatory mechanism occurring within the lung epithelium during bacterial infections.
Protein Kinase Cδ Is Required for p47phox Phosphorylation and Translocation in Activated Human Monocytes
Authors: Bey Erik A. , Xu Bo , Bhattacharjee Ashish , Oldfield Claudine M. , Zhao Xiaoxian , Li Qing , Subbulakshmi Venkita , Feldman Gerald M. , Wientjes Frans B. , Cathcart Martha K.
Published: April 2014

Our laboratory is interested in understanding the regulation of NADPH oxidase activity in human monocyte/macrophages. Protein kinase C (PKC) is reported to be involved in regulating the phosphorylation of NADPH oxidase components in human neutrophils; however, the regulatory roles of specific isoforms of PKC in phosphorylating particular oxidase components have not been determined. In this study calphostin C, an inhibitor for both novel PKC (including PKCδ, -ε, -θ, and -η) and conventional PKC (including PKCα and -β), inhibited both phosphorylation and translocation of p47phox, an essential component of the monocyte NADPH oxidase. In contrast, GF109203X, a selective inhibitor of classical PKC and PKCε, did not affect the phosphorylation or translocation of p47phox, suggesting that PKCδ, -θ, or -η is required. Furthermore, rottlerin (at doses that inhibit PKCδ activity) inhibited the phosphorylation and translocation of p47phox. Rottlerin also inhibited O⨪2 production at similar doses. In addition to pharmacological inhibitors, PKCδ-specific antisense oligodeoxyribonucleotides were used. PKCδ antisense oligodeoxyribonucleotides inhibited the phosphorylation and translocation of p47phox in activated human monocytes. We also show, using the recombinant p47phox-GST fusion protein, that p47phox can serve as a substrate for PKCδ in vitro. Furthermore, lysate-derived PKCδ from activated monocytes phosphorylated p47phox in a rottlerin-sensitive manner. Together, these data suggest that PKCδ plays a pivotal role in stimulating monocyte NADPH oxidase activity through its regulation of the phosphorylation and translocation of p47phox.
The Inhibitory Receptor PIR-B Negatively Regulates Neutrophil and Macrophage Integrin Signaling
Authors: Pereira Shalini , Zhang Hong , Takai Toshiyuki , Lowell Clifford A.
Published: April 2014

The Ig-like receptor family member, PIR-B, has been shown to play an inhibitory role in receptor signaling within B cells, mast cells, and dendritic cells. As it has been implicated in integrin-mediated responses, we investigated the effect of loss of the PIR-B protein on integrin-mediated signaling in primary murine myeloid cells. The pir-b−/− neutrophils displayed enhanced respiratory burst, secondary granule release, and a hyperadhesive phenotype when plated on surfaces coated with either extracellular matrix proteins or cellular adhesion molecules in the presence or absence of the soluble inflammatory agonist TNF-α. The pir-b−/− and wild-type cells responded equivalently when stimulated with TNF-α in suspension, indicating that the hyperresponsive phenotype of the pir-b−/− cells during adhesion was due to enhanced integrin signaling. Both wild-type and pir-b−/− neutrophils expressed similar levels of integrin subunits. Primary bone marrow-derived macrophages from pir-b−/− mice were also hyperadhesive and spread more rapidly than wild-type cells following plating on surfaces that cross-linked cellular β2 integrins. Biochemical analysis of macrophages from pir-b−/− mice revealed enhanced phosphorylation and activation of proteins involved in integrin signaling. These observations point to a nonredundant role for PIR-B in the regulation of leukocyte integrin signaling.