bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of Signaling Pathways in Macrophage Exposed to Porphyromonas gingivalis or to Its Purified Cell Wall Components
Authors: Zhou Qingde , Amar Salomon
Published: April 2014

Porphyromonas gingivalis (P. gingivalis) can trigger an inflammatory condition leading to the destruction of periodontal tissues. However P. gingivalis LPS and its fimbriae (FimA) play different roles compared with the live bacteria in the context of intracellular molecule induction and cytokine secretion. To elucidate whether this difference results from different signaling pathways in host immune response to P. gingivalis, its LPS, or its FimA, we examined gene expression profile of human macrophages exposed to P. gingivalis, its LPS, or its FimA. A comparison of gene expression resulted in the identification of three distinct groups of expressed genes. Furthermore, computer-assisted promoter analysis of a subset of each group of differentially regulated genes revealed four putative transcriptional regulation models that associate with transcription factors NFκB, IRF7, and KLF4. Using gene knockout mice and siRNA to silence mouse genes, we showed that both TLR2 and TLR7 are essential for the induction of NFκB-containing genes and NFκB-IFN-sensitive response element (ISRE) cocontaining genes by either P. gingivalis or its purified components. The gene induction via either TLR2 or TLR7 is dependent on both MyD88 and p38 MAPK. However, the unique induction of IFN-β by P. gingivalis LPS requires TLR7 and IFNαβR cosignaling, and the induction of ISRE-containing gene is dependent on the activation of IFN-β autocrine loop. Taken together, these data demonstrate that P. gingivalis and its components induce NFκB-containing genes through either TLR2- or TLR7-MyD88-p38 MAPK pathway, while P. gingivalis LPS uniquely induces ISRE-containing genes, which requires IFNαβR signaling involving IRF7, KLF4, and pY701 STAT1.
Activation of p38 MAPK by Reactive Oxygen Species Is Essential in a Rat Model of Stress-Induced Gastric Mucosal Injury
Authors: Jia Yi-Tao , Wei Wei , Ma Bing , Xu Yu , Liu Wen-Jun , Wang Yu , Lv Kai-Yang , Tang Hong-Tai , Wei Duo , Xia Zhao-Fan
Published: April 2014

Stress ulceration is a common complication in critically ill patients and can result in significant upper gastrointestinal bleeding associated with a high morbidity and mortality. At present, little is known of the molecular mechanisms underlying the incidence of this type of gastric damage. In the present study, we investigated the temporal activation of the redox-sensitive p38 signaling transduction cascade and its roles in a well-defined experimental model of cold immobilization stress-induced gastric ulceration. Exposure of Sprague-Dawley rats to 6 h of cold immobilization stress led to a rapid activation of p38 in the gastric mucosa at as early as 15 min after stress, and this activation was maximal after 1.5 h of stress and still persisted until the end of stress. Selectively blocking p38 by pretreatment with SB 239063, a potent and selective p38 inhibitor, suppressed the stress-promoted TNF-α, IL-1β, and CINC-1 production and then prevented the subsequent neutrophil infiltration, gastric mucosal epithelial necrosis and apoptosis, and the ulcerative lesions formation. Prior administration of the free radical scavengers, tempol and N-acetyl-l-cysteine, abolished the stress induction of p38 activation and the resulting mucosal inflammation and gastric injury. These results demonstrate that reactive oxygen species-mediated p38 activation plays an essential role in the pathogenesis of stress-induced gastric inflammatory damage in the rat model of cold immobilization stress. Our findings suggested that inhibition of p38 activation might be a potential strategy for the prophylaxis and treatment of stress ulceration.
Inhibition of CINC-1 Decreases Right Ventricular Damage Caused by Experimental Pulmonary Embolism in Rats
Authors: Zagorski John , Gellar Michael A. , Obraztsova Maria , Kline Jeffrey A. , Watts John A.
Published: April 2014

Right ventricular (RV) dysfunction is a strong risk factor for poor clinical outcome following pulmonary embolism (PE), the third most prevalent cardiovascular disease. Previous studies in our laboratory demonstrated that RV failure during PE is mediated, in part, by neutrophil-dependant cardiac inflammation. In this study we use DNA microarray analysis of gene expression to demonstrate that PE results in increased expression of the CXC chemokines CINC-1 and CINC-2 between 6 and 18 h after the start of PE in a rat model of PE. Neutrophils accumulate in RV tissue by 18 h, and this inflammation is associated with decreased right heart function. Treatment of rats with Abs to CINC-1 significantly suppressed neutrophil accumulation in RVs during PE (52% reduction in tissue myeloperoxidase) and ameliorated RV failure. In addition, plasma concentration of cardiac troponin I, an established diagnostic marker for cardiac damage, was reduced by 90%. These results suggest that selective anti-inflammatory therapies targeted at neutrophil chemoattractants will reduce cardiac inflammation and reduce RV damage in the setting of PE.
Agrin Signalling Contributes to Cell Activation and Is Overexpressed in T Lymphocytes from Lupus Patients
Authors: Jury Elizabeth C. , Eldridge Jillian , Isenberg David A. , Kabouridis Panagiotis S.
Published: April 2014

It is shown in this study that the heparan sulfate proteoglycan agrin is overexpressed in T cells isolated from patients with the autoimmune disease systemic lupus erythematosus (SLE). Freshly isolated CD4+ and CD8+ subpopulations both exhibited higher expression over healthy controls, which however, gradually declined when cells were cultured in vitro. Agrin expression was induced following in vitro activation of cells via their Ag receptor, or after treatment with IFN-α, a cytokine shown to be pathogenic in lupus. Furthermore, serum from SLE patients with active disease was able to induce agrin expression when added to T cells from healthy donors, an increase that was partially blocked by neutralizing anti-IFN-α Abs. Cross-linking agrin with mAbs resulted in rapid reorganization of the actin cytoskeleton, activation of the ERK MAPK cascade, and augmentation of anti-CD3-induced proliferation and IL-10 production, indicating that agrin is a functional receptor in T cells. These results demonstrate that agrin expression in human T cells is regulated by cell activation and IFN-α, and may have an important function during cell activation with potential implications for autoimmunity.
A Novel Role of IL-2 in Organ-Specific Autoimmune Inflammation beyond Regulatory T Cell Checkpoint: Both IL-2 Knockout and Fas Mutation Prolong Lifespan of Scurfy Mice but by Different Mechanisms
Authors: Zheng Lingjie , Sharma Rahul , Gaskin Felicia , Fu Shu Man , Ju Shyr-Te
Published: April 2014

Mutation of the Foxp3 transcription factor in Scurfy (Sf) mice results in complete absence of the CD4+Foxp3+ regulatory T cells (Tregs), severe multiorgan autoimmune syndrome, and early death at 4 wk of age. However, Sf mice simultaneously bearing the Il2−/− (Sf.Il2−/−) or Faslpr/lpr gene (Sf.Faslpr/lpr) have extended lifespan despite totally lacking Tregs, indicating a role of IL-2 and CD95 (Fas) signaling pathways in the multiorgan autoimmune syndrome beyond the Treg checkpoint. IL-2 has been implicated in regulating lymphoproliferation and CD178 (FasL) expression. However, Sf.Il2−/− mice have increased lymphoproliferation and FasL expression. Importantly, the pattern of organ-specific autoimmune response of Sf.Il2−/−mice resembled IL-2 knockout mice whereas that of Sf.Faslpr/lpr was similar to Sf mice, indicating that the distinct and weakened autoimmune manifestation in IL-2 knockout mice was not caused by the residual Tregs. Our study demonstrated a novel role of IL-2 in regulating multiorgan autoimmune inflammation beyond the Treg checkpoint and indicated that both Il2−/− and Faslpr/lpr genes prolong the lifespan of Sf mice but by different mechanisms.
Receptor for Advanced Glycation End Products Expression on T Cells Contributes to Antigen-Specific Cellular Expansion In Vivo
Authors: Moser Bernhard , Desai Dharmesh D. , Downie Matthew P. , Chen Yali , Yan Shi Fang , Herold Kevan , Schmidt Ann Marie , Clynes Raphael
Published: April 2014

Receptor for advanced glycation end products (RAGE) is an activation receptor triggered by inflammatory S100/calgranulins and high mobility group box-1 ligands. We have investigated the importance of RAGE on Ag priming of T cells in murine models in vivo. RAGE is inducibly up-regulated during T cell activation. Transfer of RAGE-deficient OT II T cells into OVA-immunized hosts resulted in reduced proliferative responses that were further diminished in RAGE-deficient recipients. Examination of RAGE-deficient dendritic cells did not reveal functional impairment in Ag presentation, maturation, or migratory capacities. However, RAGE-deficient T cells showed markedly impaired proliferative responses in vitro to nominal and alloantigens, in parallel with decreased production of IFN-γ and IL-2. These data indicate that RAGE expressed on T cells is required for efficient priming of T cells and elucidate critical roles for RAGE engagement during cognate dendritic cell-T cell interactions.
Chronic Antigen Stimulation In Vivo Induces a Distinct Population of Antigen-Specific Foxp3−CD25− Regulatory T Cells
Authors: Hansen Wiebke , Westendorf Astrid M. , Reinwald Simone , Bruder Dunja , Deppenmeier Stefanie , Groebe Lothar , Probst-Kepper Michael , Gruber Achim D. , Geffers Robert , Buer Jan
Published: April 2014

The concept of immune regulation/suppression has been well-established and, besides thymus-derived CD4+CD25+ regulatory T (TR) cells, it became clear that a variety of additional peripherally induced TR cells play vital roles in protection from many harmful immune responses including intestinal inflammation. In the present study, we have analyzed in vivo-induced Ag-specific CD4+ TR cells with respect to their molecular and functional phenotype. By comparative genomics we could show that these Ag-specific TR cells induced by chronic Ag stimulation in vivo clearly differ in their genetic program from naturally occurring thymus-derived CD4+CD25+ TR cells. This distinct population of induced TR cells express neither CD25 nor the TR-associated transcription factor Foxp3. Strikingly, CD25 is not even up-regulated upon stimulation. Despite the lack in Foxp3 expression, these in vivo-induced CD25− TR cells are able to interfere with an Ag-specific CD8+ T cell-mediated intestinal inflammation without significant increase in CD25 and Foxp3 expression. Thus, our results demonstrate that in vivo-induced Ag-specific TR cells represent a distinct population of Foxp3−CD25− TR cells with regulatory capacity both in vitro and in vivo.
IL-22 Is Expressed by Th17 Cells in an IL-23-Dependent Fashion, but Not Required for the Development of Autoimmune Encephalomyelitis
Authors: Kreymborg Katharina , Etzensperger Ruth , Dumoutier Laure , Haak Stefan , Rebollo Angelita , Buch Thorsten , Heppner Frank L. , Renauld Jean-Christophe , Becher Burkhard
Published: April 2014

Lately, IL-17-secreting Th cells have received an overwhelming amount of attention and are now widely held to be the major pathogenic population in autoimmune diseases. In particular, IL-22-secreting Th17 cells were shown to specifically mark the highly pathogenic population of self-reactive T cells in experimental autoimmune encephalomyelitis (EAE). As IL-17A itself was found to only play a minor role during the development of EAE, IL-22 is now postulated to contribute to the pathogenic function of Th17 cells. The goal of this study was to determine the role and function of IL-22 during the development of CNS autoimmunity in vivo. We found that CNS-invading encephalitogenic Th17 cells coexpress IL-22 and that IL-22 is specifically induced by IL-23 in autoimmune-pathogenic CD4+ T cells in a time- and dose-dependent manner. We next generated IL-22−/− mice, which—in contrast to the prediction that expression of inflammatory cytokines by CNS-invading T cells inevitably confers pathogenic function—turned out to be fully susceptible to EAE. Taken together, we show that self-reactive Th cells coexpress IL-17 and IL-22, but that the latter also does not appear to be directly involved in autoimmune pathogenesis of the CNS.
Anti-CD28 Antibodies Modify Regulatory Mechanisms and Reinforce Tolerance in CD40Ig-Treated Heart Allograft Recipients
Authors: Guillonneau Carole , Séveno Céline , Dugast Anne-Sophie , Li Xian-Liang , Renaudin Karine , Haspot Fabienne , Usal Claire , Veziers Joëlle , Anegon Ignacio , Vanhove Bernard
Published: April 2014

Blockade of CD40-CD40 ligand (CD40L) costimulation has been shown to synergize with that of CTLA4/CD28-B7 to promote transplant tolerance. To date, however, CD28-B7 interactions have been prevented using B7-blocking reagents like CTLA4-Ig that inhibit CD28-B7 together with CTLA4-B7 interactions. In this study, we have tested anti-CD28 Abs to prevent selectively CD28-B7 interactions while preserving CTLA4-B7 in addition to CD40-CD40L blockade. In the LEW.1W to LEW.1A rat combination, interfering with CD40-CD40L interactions by CD40Ig administration through gene transfer resulted in indefinite heart allograft survival due to the appearance of clonotypic CD8+CD45RClow regulatory T cells that were capable of transferring the tolerant state to naive animals. However, cardiac transplants in these recipients systematically developed chronic rejection lesions. Whereas anti-CD28 Ab monotherapy only delayed acute rejection and failed to induce tolerance, coadministration of anti-CD28 Abs and CD40Ig resulted in the long-term acceptation of allografts without chronic rejection lesions in 60% of the recipients, reduced the level of intragraft mRNA transcripts for cytokines and immune factors, and fully abrogated alloantibody production. In addition, the nature of regulatory cells was modified: the CD8+CD45RClow clonotypic T cells described in the CD40Ig-treated animals could not be found in cotreated animals, and the other CD8+CD45RClow cells had no regulatory activity and a different cytokine expression profile. Instead, in cotreated recipients we found IDO-dependent non-T cells with regulatory activity in vitro. Thus, the addition of a short-term anti-CD28 treatment with CD40Ig resulted in decreased heart allograft chronic rejection lesions, complete inhibition of Ab production, and modified regulatory mechanisms.
Death Receptor Ligation or Exposure to Perforin Trigger Rapid Egress of the Intracellular Parasite Toxoplasma gondii
Authors: Persson Emma K. , Agnarson Abela Mpobela , Lambert Henrik , Hitziger Niclas , Yagita Hideo , Chambers Benedict J. , Barragan Antonio , Grandien Alf
Published: April 2014

The obligate intracellular parasite Toxoplasma gondii chronically infects up to one-third of the global population, can result in severe disease in immunocompromised individuals, and can be teratogenic. In this study, we demonstrate that death receptor ligation in T. gondii-infected cells leads to rapid egress of infectious parasites and lytic necrosis of the host cell, an active process mediated through the release of intracellular calcium as a consequence of caspase activation early in the apoptotic cascade. Upon acting on infected cells via death receptor- or perforin-dependent pathways, T cells induce rapid egress of infectious parasites able to infect surrounding cells, including the Ag-specific effector cells.
NKT Cells Are Critical to Initiate an Inflammatory Response after Pseudomonas aeruginosa Ocular Infection in Susceptible Mice
Authors: Hazlett Linda D. , Li Qianqian , Liu Jianhua , McClellan Sharon , Du Wenjin , Barrett Ronald P.
Published: April 2014

CD4+ T cells produce IFN-γ contributing to corneal perforation in C57BL/6 (B6) mice after Pseudomonas aeruginosa infection. To determine the role of NK and NKT cells, infected corneas of B6 mice were dual immunolabeled. Initially, more NKT than NK cells were detected, but as disease progressed, NK cells increased, while NKT cells decreased. Therefore, B6 mice were depleted of NK/NKT cells with anti-asialo GM1 or anti-NK1.1 Ab. Either treatment accelerated time to perforation, increased bacterial load and polymorphonuclear neutrophils, but decreased IFN-γ and IL-12p40 mRNA expression vs controls. Next, RAG-1 knockout (−/−; no T/NKT cells), B6.TCR Jα281−/− (NKT cell deficient), α-galactosylceramide (αGalCer) (anergized NKT cells) injected and IL-12p40−/− vs B6 controls were tested. IFN-γ mRNA was undetectable in RAG-1−/−- and αGalCer-treated mice at 5 h and was significantly reduced vs controls at 1 day postinfection. It also was reduced significantly in B6.TCR Jα281−/−, αGalCer-treated, and IL-12p40−/− (activated CD4+ T cells also reduced) vs control mice at 5 days postinfection. In vitro studies tested whether endotoxin (LPS) stimulated Langerhans cells and macrophages (Mφ; from B6 mice) provided signals to activate NKT cells. LPS up-regulated mRNA expression for IL-12p40, costimulatory molecules CD80 and CD86, NF-κB, and CD1d, and addition of rIFN-γ potentiated Mφ CD1d levels. Together, these data suggest that Langerhans cell/Mφ recognition of microbial LPS regulates IL-12p40 (and CD1d) driven IFN-γ production by NKT cells, that IFN-γ is required to optimally activate NK cells to produce IFN-γ, and that depletion of both NKT/NK cells results in earlier corneal perforation.
Expression of CXC Chemokine Ligand 10 from the Mouse Hepatitis Virus Genome Results in Protection from Viral-Induced Neurological and Liver Disease
Authors: Walsh Kevin B. , Edwards Robert A. , Romero Kimberley M. , Kotlajich Matthew V. , Stohlman Stephen A. , Lane Thomas E.
Published: April 2014

Using the recombinant murine coronavirus mouse hepatitis virus (MHV) expressing the T cell-chemoattractant CXCL10 (MHV-CXCL10), we demonstrate a potent antiviral role for CXCL10 in host defense. Instillation of MHV-CXCL10 into the CNS of CXCL10-deficient (CXCL10−/−) mice resulted in viral infection and replication in both brain and liver. Expression of virally encoded CXCL10 within the brain protected mice from death and correlated with increased infiltration of T lymphocytes, enhanced IFN-γ secretion, and accelerated viral clearance when compared with mice infected with an isogenic control virus, MHV. Similarly, viral clearance from the livers of MHV-CXCL10-infected mice was accelerated in comparison to MHV-infected mice, yet was independent of enhanced infiltration of T lymphocytes and NK cells. Moreover, CXCL10−/− mice infected with MHV-CXCL10 were protected from severe hepatitis as evidenced by reduced pathology and serum alanine aminotransferase levels compared with MHV-infected mice. CXCL10-mediated protection within the liver was not dependent on CXC-chemokine receptor 2 (CXCR2) signaling as anti-CXCR2 treatment of MHV-CXCL10-infected mice did not modulate viral clearance or liver pathology. In contrast, treatment of MHV-CXCL10-infected CXCL10−/− mice with anti-CXCL10 Ab resulted in increased clinical disease correlating with enhanced viral recovery from the brain and liver as well as increased serum alanine aminotransferase levels. These studies highlight that CXCL10 expression promotes protection from coronavirus-induced neurological and liver disease.
Type I IFN Induction in Response to Listeria monocytogenes in Human Macrophages: Evidence for a Differential Activation of IFN Regulatory Factor 3 (IRF3)
Authors: Reimer Thornik , Schweizer Matthias , Jungi Thomas W.
Published: April 2014

Listeria monocytogenes is a prototypic bacterium for studying innate and adaptive cellular immunity as well as host defense. Using human monocyte-derived macrophages, we report that an infection with a wild-type strain, but not a listeriolysin O-deficient strain, of the Gram-positive bacterium L. monocytogenes induces expression of IFN-β and a bioactive type I IFN response. Investigating the activation of signaling pathways in human macrophages after infection revealed that a wild-type strain and a hemolysin-deficient strain of L. monocytogenes activated the NF-κB pathway and induced a comparable TNF response. p38 MAPK and activating transcription factor 2 were phosphorylated following infection with either strain, and IFN-β gene expression induced by wild-type L. monocytogenes was reduced when p38 was inhibited. However, neither IFN regulatory factor (IRF) 3 translocation to the nucleus nor posttranslational modifications and dimerizations were observed after L. monocytogenes infection. In contrast, vesicular stomatitis virus and LPS triggered IRF3 activation and signaling. When IRF3 was knocked down using small interfering RNA, a L. monocytogenes-induced IFN-β response remained unaffected whereas a vesicular stomatitis virus-triggered response was reduced. Evidence against the possibility that IRF7 acts in place of IRF3 is provided. Thus, we show that wild-type L. monocytogenes induced an IFN-β response in human macrophages and propose that this response involves p38 MAPK and activating transcription factor 2. Using various stimuli, we show that IRF3 is differentially activated during type I IFN responses in human macrophages.
Direct Interferon-γ Signaling Dramatically Enhances CD4+ and CD8+ T Cell Memory
Authors: Whitmire Jason K. , Eam Boreth , Benning Nicola , Whitton J. Lindsay
Published: April 2014

Studies in IFN-γ-deficient mice suggest that the delivery of IFN-γ to CD8+ T cells early in virus infection programs their eventual contraction, thereby reducing the abundance of CD8+ memory T cells. In this study, we show that such mice fail to completely eliminate virus infection and that, when evaluated without the confounding factor of persisting Ag, both CD4+ and CD8+ T cells undergo profound contraction when they are unable to receive IFN-γ signals. Furthermore, the abundance of CD4+ and CD8+ memory cells that express the IFN-γ receptor is ∼100-fold higher than cells lacking this molecule. Thus, direct IFN-γ signaling is not required for T cell contraction during virus infection, and it enhances, rather than suppresses, the development of virus-specific CD4+ and CD8+ T cell memory.
Human Cytotoxic CD4+ T Cells Recognize HLA-DR1-Restricted Epitopes on Vaccinia Virus Proteins A24R and D1R Conserved among Poxviruses
Authors: Mitra-Kaushik Shibani , Cruz John , Stern Lawrence J. , Ennis Francis A. , Terajima Masanori
Published: April 2014

We previously demonstrated that vaccinia virus (VV)-specific CD4+ cytolytic T cells can persist for >50 years after immunization against smallpox in the absence of re-exposure to VV. Nevertheless, there have been few studies focusing on CD4+ T cell responses to smallpox vaccination. To ensure successful vaccination, a candidate vaccine should contain immunodominant CD4+ T cell epitopes as well as CD8+ T and B cell epitopes. In the present study, we established cytotoxic CD4+ T cell lines from VV-immune donors, which recognize epitopes in VV proteins D1R and A24R in association with HLA-DR1 Ags. Comparisons of sequences between different members of the poxvirus family show that both epitopes are completely conserved among VV, variola viruses, and most mammalian poxviruses, including monkeypox, cowpox, and ectromelia. The CD4+ T cell lines lysed VV-infected, Ag- and peptide-pulsed targets, and the lysis was inhibited by concanamycin A. We also detected these peptide-specific cytolytic and IFN-γ-producing CD4+ T cells in short-term bulk cultures of PBMC from each of the three VV-immune donors tested. These are the first VV-specific CD4+ T cell epitopes identified in humans restricted by one of the most common MHC class II molecules, HLA-DR1, and this information may be useful in analyzing CD4+ T cell responses to pre-existing or new generation VV vaccines against smallpox.
Immunomodulatory Dendritic Cells Inhibit Th1 Responses and Arthritis via Different Mechanisms
Authors: van Duivenvoorde Leonie M. , Han Wanda G. H. , Bakker Aleida M. , Louis-Plence Pascale , Charbonnier Louis-Marie , Apparailly Florence , van der Voort Ellen I. H. , Jorgensen Christian , Huizinga Tom W. J. , Toes René E. M.
Published: April 2014

Dendritic cells (DCs) are professional APCs which have the unique ability to present both foreign and self-Ags to T cells and steer the outcome of immune responses. Because of these characteristics, DCs are attractive vehicles for the delivery of therapeutic vaccines. Fully matured DCs are relatively well-defined and even used in clinical trials in cancer. DCs also have the potential to influence the outcome of autoimmunity by modulating the underlying autoimmune response. To gain a better appreciation of the abilities and mechanisms by which immunomodulatory DCs influence the outcome of T cell responses, we studied several immunomodulatory DCs (TNF-, IL-10-, or dexamethasone-stimulated bone marrow-derived DCs) side by side for their ability to modulate T cell responses and autoimmune diseases. Our data show that these differentially modulated DCs display a different composition of molecules involved in T cell activation. Although, all DC subsets analyzed were able to inhibit the induction of collagen-induced arthritis, the modulation of the underlying immune response was different. Vaccination with TNF- or IL-10-modulated DCs altered the Th1/Th2 balance as evidenced by the induction of IL-5- and IL-10-secreting T cells and the concomitant reduction of the IgG2a-IgG1 ratio against the immunizing Ag. In contrast, DCs modulated with dexamethasone did not affect the ratio of IL-5-producing vs IFN-γ-producing T cells and tended to affect the Ab response in a nonspecific manner. These data indicate that distinct mechanisms can be used by distinct DC subsets to change the outcome of autoimmunity.
Cross-Talk between T Cells and Innate Immune Cells Is Crucial for IFN-γ-Dependent Tumor Rejection
Authors: Li Zhiguang , Pradera Felicia , Kammertoens Thomas , Li Bing , Liu Shubai , Qin Zhihai
Published: April 2014

Though the importance of IFN-γ in tumor immunity has been well-demonstrated, little is known about its source and how it is induced. By using various bone marrow chimeric mice, we show here that IFN-γ essential for tumor immunity is solely produced by hemopoietic cells. Surprisingly, IFN-γ derived from T cells was not necessary for tumor immunity in this model. In the immunized mice, in which only innate immune cells have the IFN-γ-producing potential, tumors were efficiently rejected. The innate immune cells, such as NK1.1+ cells and CD11b+ cells, can provide sufficient amounts of IFN-γ which requires, however, the help of T cells. The close cooperation between T cells and innate immune cells during tumor regression is likely mediated by IL-2. Together, our results clearly illustrate how T cells cooperate with innate immune cells for IFN-γ-mediated tumor rejection and this may have important indications for clinical trials of tumor immunotherapy.
Noninvasive Intravital Imaging of Thymocyte Dynamics in Medaka
Authors: Li Jie , Iwanami Norimasa , Hoa Vu Quynh , Furutani-Seiki Makoto , Takahama Yousuke
Published: April 2014

In vivo imaging of thymocytes has not been accomplished due to their localization deep within opaque body and high susceptibility to surgical stress. To overcome these problems, medaka is useful because of transparency and ex-uterine development. We report the noninvasive detection of thymocytes in transgenic medaka that express fluorescent protein under the control of immature-lymphocyte-specific rag1. We show that lymphoid progenitor cells colonize the thymus primordium in an anterior-to-posterior orientation-specific manner, revealing that extrathymic anterior components guide prevascular thymus colonization. We also show that developing thymocytes acquire “random walk motility” along with the expression of Ag receptors and coreceptors, suggesting that thymocyte walking is initiated at the developmental stage for repertoire selection. Thus, transgenic medaka enables real-time intravital imaging of thymocytes without surgical invasion.
Efficient Cross-Presentation by Heat Shock Protein 90-Peptide Complex-Loaded Dendritic Cells via an Endosomal Pathway
Authors: Kurotaki Takehiro , Tamura Yasuaki , Ueda Gosei , Oura Jun , Kutomi Goro , Hirohashi Yoshihiko , Sahara Hiroeki , Torigoe Toshihiko , Hiratsuka Hiroyoshi , Sunakawa Hajime , Hirata Koichi , Sato Noriyuki
Published: April 2014

It is well-established that heat shock proteins (HSPs)-peptides complexes elicit antitumor responses in prophylactic and therapeutic immunization protocols. HSPs such as gp96 and Hsp70 have been demonstrated to undergo receptor-mediated uptake by APCs with subsequent representation of the HSP-associated peptides to MHC class I molecules on APCs, facilitating efficient cross-presentation. On the contrary, despite its abundant expression among HSPs in the cytosol, the role of Hsp90 for the cross-presentation remains unknown. We show here that exogenous Hsp90-peptide complexes can gain access to the MHC class I presentation pathway and cause cross-presentation by bone marrow-derived dendritic cells. Interestingly, this presentation is TAP independent, and followed chloroquine, leupeptin-sensitive, as well as cathepsin S-dependent endosomal pathways. In addition, we show that Hsp90-chaperoned precursor peptides are processed and transferred onto MHC class I molecules in the endosomal compartment. Furthermore, we demonstrate that immunization with Hsp90-peptide complexes induce Ag-specific CD8+ T cell responses and strong antitumor immunity in vivo. These findings have significant implications for the design of T cell-based cancer immunotherapy.
Intrapulmonary Delivery of Bone Marrow-Derived Mesenchymal Stem Cells Improves Survival and Attenuates Endotoxin-Induced Acute Lung Injury in Mice
Authors: Gupta Naveen , Su Xiao , Popov Boris , Lee Jae Woo , Serikov Vladimir , Matthay Michael A.
Published: April 2014

Recent in vivo and in vitro work suggests that mesenchymal stem cells (MSC) have anti-inflammatory properties. In this study, we tested the effect of administering MSC directly into the airspaces of the lung 4 h after the intrapulmonary administration of Escherichia coli endotoxin (5 mg/kg). MSC increased survival compared with PBS-treated control mice at 48 h (80 vs 42%; p < 0.01). There was also a significant decrease in excess lung water, a measure of pulmonary edema (145 ± 50 vs 87 ± 20 μl; p < 0.01), and bronchoalveolar lavage protein, a measure of endothelial and alveolar epithelial permeability (3.1 ± 0.4 vs 2.2 ± 0.8 mg/ml; p < 0.01), in the MSC-treated mice. These protective effects were not replicated by the use of further controls including fibroblasts and apoptotic MSC. The beneficial effect of MSC was independent of the ability of the cells to engraft in the lung and was not related to clearance of the endotoxin by the MSC. MSC administration mediated a down-regulation of proinflammatory responses to endotoxin (reducing TNF-α and MIP-2 in the bronchoalveolar lavage and plasma) while increasing the anti-inflammatory cytokine IL-10. In vitro coculture studies of MSC with alveolar macrophages provided evidence that the anti-inflammatory effect was paracrine and was not cell contact dependent. In conclusion, treatment with intrapulmonary MSC markedly decreases the severity of endotoxin-induced acute lung injury and improves survival in mice.
Exosomes with Immune Modulatory Features Are Present in Human Breast Milk
Authors: Admyre Charlotte , Johansson Sara M. , Qazi Khaleda Rahman , Filén Jan-Jonas , Lahesmaa Riitta , Norman Mikael , Neve Etienne P. A. , Scheynius Annika , Gabrielsson Susanne
Published: April 2014

Breast milk is a complex liquid with immune-competent cells and soluble proteins that provide immunity to the infant and affect the maturation of the infant’s immune system. Exosomes are nanovesicles (30–100 nm) with an endosome-derived limiting membrane secreted by a diverse range of cell types. Because exosomes carry immunorelevant structures, they are suggested to participate in directing the immune response. We hypothesized that human breast milk contain exosomes, which may be important for the development of the infant’s immune system. We isolated vesicles from the human colostrum and mature breast milk by ultracentrifugations and/or immuno-isolation on paramagnetic beads. We found that the vesicles displayed a typical exosome-like size and morphology as analyzed by electron microscopy. Furthermore, they floated at a density between 1.10 and 1.18 g/ml in a sucrose gradient, corresponding to the known density of exosomes. In addition, MHC classes I and II, CD63, CD81, and CD86 were detected on the vesicles by flow cytometry. Western blot and mass spectrometry further confirmed the presence of several exosome-associated molecules. Functional analysis revealed that the vesicle preparation inhibited anti-CD3-induced IL-2 and IFN-γ production from allogeneic and autologous PBMC. In addition, an increased number of Foxp3+CD4+CD25+ T regulatory cells were observed in PBMC incubated with milk vesicle preparations. We conclude that human breast milk contains exosomes with the capacity to influence immune responses.
Identification and Characterization of ErbB-3-Binding Protein-1 as a Target for Immunotherapy
Authors: Santegoets Saskia J. A. M. , Schreurs Marco W. J. , Reurs Anneke W. , Lindenberg Jelle J. , Kueter Esther W. M. , van den Eertwegh Alfons J. M. , Hooijberg Erik , Brandwijk Ricardo J. , Hufton Simon E. , Hoogenboom Hennie R. , Scheper Rik J. , Somers Veerle A. , de Gruijl Tanja D.
Published: April 2014

Based on immune reactivity in response to a whole-cell colon tumor vaccine and using serological identification of Ags by recombinant cDNA expression cloning, we here describe the molecular and functional identification of a novel human tumor Ag. By screening a cDNA expression library derived from the coloncarcinoma cell line HT-29 with pooled colorectal cancer patients’ sera, 26 clones reactive with IgG Abs could be identified. Characterization of these cDNA clones by sequence analysis and alignment, and detailed serological analysis revealed cancer-related immunoreactivity for the ErbB-3-binding protein-1 (Ebp1). Immunohistochemical staining of colorectal tumors and neighboring normal colon tissue indicated the observed cancer-related immunogenicity of Ebp1 to be related to overexpression. Via reverse immunology, five potential HLA-A2-restricted T cell epitopes were identified, of which two (Ebp145–54 and Ebp159–67) bound HLA-A2 with intermediate and high affinity, respectively. Analysis of their immunogenicity in vitro indicated that only the high-affinity Ebp159 epitope gave rise to CD8+ T cells capable of recognizing both exogenously loaded Ebp1 peptide and endogenously expressed Ebp1 on target cells. In addition, in vivo CD8+ T cell responsiveness against the Ebp159 epitope could be detected in two of nine and three of six cancer patients PBMC and tumor draining lymph nodes, respectively, but not in nine of nine healthy donors tested. These data confirm that Ebp1 is an immunogenic protein, capable of eliciting CD8-mediated responses in vivo and in vitro, providing a rationale for further exploration of Ebp1 as a possible target for anticancer immunotherapy.
Cutting Edge: Primary B Lymphocytes Preferentially Expand Allogeneic FoxP3+ CD4 T Cells
Authors: Chen Xinjian , Jensen Peter E.
Published: April 2014

Despite the unequivocal role of B lymphocytes as effecter cells in humoral immunity, studies have reported that B cells are tolerogenic. The impact of B cell-mediated tolerance and its underlying mechanisms are incompletely understood. Using primary B cells as APCs and allogeneic CD4 T cells as responder cells in mixed leukocyte reactions, we find that B cells preferentially expand FoxP3+ over FoxP3− CD4 T cells in the absence of exogenous cytokines. The preferential expansion of Foxp3+ T cells is further enhanced by a partial blockade of class II MHC-TCR interaction but diminished by stimulatory anti-CD28 Ab or at high B to T cell ratios. Gamma irradiation of B cells selectively abrogates their ability to expand isolated CD25+ but not CD25− CD4 T cells; exogenous IL-2 supplement can partially restore this function. B cell-expanded CD25+ T cells express high levels of FoxP3 and are highly inhibitory in an Ag-specific manner.
Altered B:9–23 Insulin, When Administered Intranasally with Cholera Toxin Adjuvant, Suppresses the Expression of Insulin Autoantibodies and Prevents Diabetes
Authors: Kobayashi Masakazu , Abiru Norio , Arakawa Takeshi , Fukushima Keiko , Zhou Hongbo , Kawasaki Eiji , Yamasaki Hironori , Liu Edwin , Miao Dongmei , Wong F. Susan , Eisenbarth George S. , Eguchi Katsumi
Published: April 2014

Insulin peptide B:9–23 is a major autoantigen in type 1 diabetes that contains two distinct CD4 epitopes (B:9–16 and B:13–23). One of the two epitopes, B:13–23, overlaps with a CTL epitope (B:15–23). In this study, we report that the elimination of the CTL epitope from the B:9–23 peptide by amino acid substitution (with alanine) at positions B:16 and 19 (A16,19 altered peptide ligand) or truncation of the C-terminal amino acids from the peptide (B:9–21), neither of which stimulated the proliferation of insulin B:15–23 reactive CD8 T cells, provided significant intranasally induced suppression of diabetes when coadministered with a potent mucosal adjuvant cholera toxin (CT). Intranasal treatment with A16,19 resulted in the elimination of spontaneous insulin autoantibodies, significant inhibition of insulitis and remission from hyperglycemia, and prevented the progression to diabetes. Intranasal administration of native B:9–23/CT or B:11–23/CT resulted in a significant enhancement of insulin autoantibody expression and severity of insulitis and failed to prevent diabetes. Our present study indicates that elimination of the CTL epitope from the B:9–23 peptide was critically important for mucosally induced diabetes prevention. The A16,19 altered peptide ligand, but not other native insulin peptides, suppresses insulin autoantibodies associated with protection from and remission of diabetes.
Multiple Constraints at the Level of TCRα Rearrangement Impact Vα14i NKT Cell Development
Authors: Hager Elizabeth , Hawwari Abbas , Matsuda Jennifer L. , Krangel Michael S. , Gapin Laurent
Published: April 2014

CD1d-restricted NKT cells that express an invariant Vα14 TCR represent a subset of T cells implicated in the regulation of several immune responses, including autoimmunity, infectious disease, and cancer. Proper rearrangement of Vα14 with the Jα18 gene segment in immature thymocytes is a prerequisite to the production of a TCR that can be subsequently positively selected by CD1d/self-ligand complexes in the thymus and gives rise to the NKT cell population. We show here that Vα14 to Jα rearrangements are temporally regulated during ontogeny providing a molecular explanation to their late appearance in the thymus. Using mice deficient for the transcription factor RORγ and the germline promoters T early-α and Jα49, we show that developmental constraints on both Vα and Jα usage impact NKT cell development. Finally, we demonstrate that rearrangements using Vα14 and Jα18 occur normally in the absence of FynT, arguing that the effect of FynT on NKT cell development occurs subsequent to α-chain rearrangement. Altogether, this study provides evidence that there is no directed rearrangement of Vα14 to Jα18 segments and supports the instructive selection model for NKT cell selection.
A Single Early Activation of Invariant NK T Cells Confers Long-Term Protection against Collagen-Induced Arthritis in a Ligand-Specific Manner
Authors: Coppieters Ken , Van Beneden Katrien , Jacques Peggy , Dewint Pieter , Vervloet Ann , Vander Cruyssen Bert , Van Calenbergh Serge , Chen Guangwu , Franck Richard W. , Verbruggen Gust , Deforce Dieter , Matthys Patrick , Tsuji Moriya , Rottiers Pieter , Elewaut Dirk
Published: April 2014

The glycosphingolipid α-galactosylceramide (α-GalCer) has been shown to be a potent activator of invariant NKT (iNKT) cells, rapidly inducing large amounts of both Th1 and Th2 cytokines upon injection in mice. The C-glycoside analog of α-GalCer (α-C-GalCer), by contrast, results in an enhanced Th1-type response upon activation of iNKT cells. We administered a single dose of these Ags to DBA/1 mice during the early induction phase of collagen-induced arthritis and demonstrated therapeutic efficacy of α-GalCer when administered early rather than late during the disease. Surprisingly, the Th1-polarizing analog α-C-GalCer also conferred protection. Furthermore, a biphasic role of IFN-γ in the effect of iNKT cell stimulation was observed. Whereas in vivo neutralization of IFN-γ release induced by either α-GalCer or α-C-GalCer early during the course of disease resulted in partial improvement of clinical arthritis symptoms, blockade of IFN-γ release later on resulted in a more rapid onset of arthritis. Although no phenotypic changes in conventional T cells, macrophages, or APCs could be detected, important functional differences in T cell cytokine production in serum were observed upon polyclonal T cell activation, 2 wk after onset of arthritis. Whereas α-GalCer-treated mice produced significantly higher amounts of IL-10 upon systemic anti-CD3 stimulation compared with PBS controls, T cells from α-C-GalCer-treated mice, by contrast, produced substantially lower levels of cytokines, suggesting the involvement of different protective mechanisms. In conclusion, these findings suggest long-term, ligand-specific, time-dependent, and partially IFN-γ-dependent immunomodulatory effects of iNKT cells in collagen-induced arthritis.
Development of Sjögren’s Syndrome in Nonobese Diabetic-Derived Autoimmune-Prone C57BL/6.NOD-Aec1Aec2 Mice Is Dependent on Complement Component-3
Authors: Nguyen Cuong Q. , Kim Hyuna , Cornelius Janet G. , Peck Ammon B.
Published: April 2014

The role of complement in the etiology of Sjögren’s syndrome (SjS), a human autoimmune disease manifested primarily by salivary and lacrimal gland dysfunction resulting in dry mouth/dry eye syndrome, remains ill-defined. In the present study, we examined the role of complement component-3 (C3) using a newly constructed C3-gene knockout mouse, C57BL/6.NOD-Aec1Aec2.C3−/−. Inactivation of C3 in the parental C57BL/6.NOD-Aec1Aec2 strain, a model of primary SjS, resulted in a diminished or total absence of both preclinical and clinical manifestations during development and onset of disease, including reduced acinar cell apoptosis, reduced levels of caspase-3, lack of leukocyte infiltration of submandibular glands, reduced synthesis of disease-associated autoantibodies, maintenance of normal glandular architecture, and retention of normal saliva secretion. In addition, C57BL/6-NOD.Aec1Aec2.C3−/− mice did not exhibit increased numbers of marginal zone B cells, a feature of SjS-prone C57BL/6-NOD.Aec1Aec2 mice. Interestingly, C57BL/6-NOD.Aec1Aec2.C3−/− mice retained some early pathological manifestations, including activation of serine kinases with proteolytic activity for parotid secretory protein. This improvement in the clinical manifestations of SjS-like disease in C57BL/6.NOD-Aec1Aec2.C3−/− mice, apparently a direct consequence of C3 deficiency, supports a much more important role for complement in the adaptive autoimmune response than previously recognized, possibly implicating an essential role for innate immunity.
Fimbrial Proteins of Porphyromonas gingivalis Mediate In Vivo Virulence and Exploit TLR2 and Complement Receptor 3 to Persist in Macrophages
Authors: Wang Min , Shakhatreh Muhamad-Ali K. , James Deanna , Liang Shuang , Nishiyama So-ichiro , Yoshimura Fuminobu , Demuth Donald R. , Hajishengallis George
Published: April 2014

Porphyromonas gingivalis is an oral/systemic pathogen implicated in chronic conditions, although the mechanism(s) whereby it resists immune defenses and persists in the host is poorly understood. The virulence of this pathogen partially depends upon expression of fimbriae comprising polymerized fimbrillin (FimA) associated with quantitatively minor proteins (FimCDE). In this study, we show that isogenic mutants lacking FimCDE are dramatically less persistent and virulent in a mouse periodontitis model and express shorter fimbriae than the wild type. Strikingly, native fimbriae allowed P. gingivalis to exploit the TLR2/complement receptor 3 pathway for intracellular entry, inhibition of IL-12p70, and persistence in macrophages. This virulence mechanism also required FimCDE; indeed, mutant strains exhibited significantly reduced ability to inhibit IL-12p70, invade, and persist intracellularly, attributable to failure to interact with complement receptor 3, although not with TLR2. These results highlight a hitherto unknown mechanism of immune evasion by P. gingivalis that is surprisingly dependent upon minor constituents of its fimbriae, and support the concept that pathogens evolved to manipulate innate immunity for promoting adaptive fitness and thus their capacity to cause disease.
Complement Receptor 3 Blockade Promotes IL-12-Mediated Clearance of Porphyromonas gingivalis and Negates Its Virulence In Vivo
Authors: Hajishengallis George , Shakhatreh Muhamad-Ali K. , Wang Min , Liang Shuang
Published: April 2014

The ability of certain pathogens to exploit innate immune function allows them to undermine immune clearance and thereby increase their persistence and capacity to cause disease. Porphyromonas gingivalis is a major pathogen in periodontal disease and is associated with increased risk of systemic conditions. We have previously shown that the fimbriae of P. gingivalis interact with complement receptor 3 (CR3; CD11b/CD18) in monocytes/macrophages, resulting in inhibition of IL-12p70 production in vitro. The in vivo biological implications of this observation were investigated in this study using a CR3 antagonist (XVA143). XVA143 was shown to block CR3 binding of P. gingivalis fimbriae and reverse IL-12p70 inhibition; specifically, CR3 blockade resulted in inhibition of ERK1/2 phosphorylation and up-regulation of IL-12 p35 and p40 mRNA expression. Importantly, mice pretreated with XVA143 elicited higher IL-12p70 and IFN-γ levels in response to P. gingivalis i.p. infection and displayed enhanced pathogen clearance, compared with similarly infected controls. The notion that CR3 is associated with reduced IL-12p70 induction and impaired P. gingivalis clearance was confirmed using i.p. infected wild-type and CR3-deficient mice. Moreover, XVA143 dramatically attenuated the persistence and virulence of P. gingivalis in experimental mouse periodontitis, as evidenced by reduced induction of periodontal bone loss. Therefore, CR3 blockade may represent a promising immunomodulatory approach for controlling human periodontitis and possibly associated systemic diseases.