bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of the Chicken C-Type Lectin-Like Receptors B-NK and B-lec Suggests That the NK Complex and the MHC Share a Common Ancestral Region
Authors: Rogers Sally L. , Göbel Thomas W. , Viertlboeck Birgit C. , Milne Sarah , Beck Stephan , Kaufman Jim
Published: April 2014

The sequencing of the chicken MHC led to the identification of two open reading frames, designated B-NK and B-lec, that were predicted to encode C-type lectin domains. C-type lectin domains are not encoded in the MHC of any animal described to date; therefore, this observation was completely unexpected, particularly given that the chicken has a “minimal essential MHC.” In this study, we describe the initial characterization of the B-NK and B-lec genes, and show that they share greatest homology with C-type lectin-like receptors encoded in the human NK complex (NKC), in particular NKR-P1 and lectin-like transcript 1 (LLT1), respectively. In common with NKR-P1 and LLT1, B-NK and B-lec are located next to each other and transcribed in opposite orientation. Like human NKR-P1, B-NK has a functional inhibitory signaling motif in the cytoplasmic tail and is expressed in NK cells. In contrast, B-lec contains an endocytosis motif in the cytoplasmic tail, and like LLT1, is an early activation Ag. Further analysis leads us to propose that there are four subgroups of C-type lectin-like receptors in the NKC, which arose as a result of duplication events. Moreover, this analysis suggests that the NKC may be considered a fifth paralogous region, and therefore shares an ancient common origin with the MHC. This provides evidence that C-type lectin-like receptors were present in the preduplication, primordial MHC region, and suggests that an original function of MHC molecules was for recognition by NK cell receptors encoded nearby.
The Protooncogene c-Maf Is an Essential Transcription Factor for IL-10 Gene Expression in Macrophages
Authors: Cao Shanjin , Liu Jianguo , Song Lihua , Ma Xiaojing
Published: April 2014

IL-10 is an important immunoregulatory factor. However, our understanding of IL-10 gene regulation remains very limited. In this study, following up on our previous novel finding that the protooncogene c-Maf of the basic leucine zipper family of transcription factors is expressed in monocytes and macrophages, we investigate the role of c-Maf in the transcriptional regulation of IL-10 and the underlying molecular mechanism in macrophages. c-Maf-null macrophages exhibit strongly impaired IL-10 protein production and mRNA expression upon LPS stimulation. Ectopic expression of c-Maf stimulates not only exogenously transfected IL-10 promoter-driven luciferase activity in a dose-dependent manner but also enhances endogenous IL-10 gene expression stimulated by LPS. Both in vitro and in vivo experiments identify a c-Maf response element localized to nucleotides −196/−184 relative to the transcription initiation site in the IL-10 promoter. This site represents an atypical 12-O-tetradecanoate-13-acetate-responsive element for musculoaponeurotic fibrosarcoma recognition and functions as an enhancer element in a heterologous and orientation-independent manner. Furthermore, c-Maf is expressed constitutively in resting monocytes/macrophages. IL-4 can up-regulate c-Maf expression, its binding to IL-10 promoter, and dose dependently enhance IL-10 production induced by LPS; moreover, IL-4 failed to enhance LPS-induced IL-10 production in c-Maf-null macrophages. Taken together, these data demonstrate that c-Maf is an indispensable yet constitutive transcription factor for IL-10 gene expression in LPS-activated macrophages, and IL-4 modulates IL-10 production in inflammatory macrophages likely via its ability to induce c-Maf expression. Thus, this study uncovers a novel and important function of c-Maf in macrophages and elucidates its transcriptional mechanism in the regulation of IL-10 gene expression.
Pyocyanin Production byPseudomonas aeruginosaInduces Neutrophil Apoptosis and Impairs Neutrophil-Mediated Host Defenses In Vivo
Authors: Allen Lucy , Dockrell David H. , Pattery Theresa , Lee Daniel G. , Cornelis Pierre , Hellewell Paul G. , Whyte Moira K. B.
Published: April 2014

Clearance of neutrophils from inflamed sites is critical for resolution of inflammation, but pathogen-driven neutrophil apoptosis can impair host defenses. We previously showed that pyocyanin, a phenazine toxic metabolite produced by Pseudomonas aeruginosa, accelerates neutrophil apoptosis in vitro. We compared wild-type and pyocyanin-deficient strains of P. aeruginosa in a murine model of acute pneumonia. Intratracheal instillation of either strain of P. aeruginosa caused a rapid increase in bronchoalveolar lavage neutrophil counts up to 18 h after infection. In wild-type infection, neutrophil numbers then declined steadily, whereas neutrophil numbers increased up to 48 h in mice infected with pyocyanin-deficient P. aeruginosa. In keeping with these differences, pyocyanin production was associated with reduced bacterial clearance from the lungs. Neutrophil apoptosis was increased in mice infected with wild-type compared with the phenazine-deficient strain or two further strains that lack pyocyanin production, but produce other phenazines. Concentrations of potent neutrophil chemokines (MIP-2, KC) and cytokines (IL-6, IL-1β) were significantly lower in wild-type compared with phenazine-deficient strain-infected mice at 18 h. We conclude that pyocyanin production by P. aeruginosa suppresses the acute inflammatory response by pathogen-driven acceleration of neutrophil apoptosis and by reducing local inflammation, and that this is advantageous for bacterial survival.
Febrile-Range Hyperthermia Augments Neutrophil Accumulation and Enhances Lung Injury in Experimental Gram-Negative Bacterial Pneumonia
Authors: Rice Penelope , Martin Erica , He Ju-Ren , Frank Mariah , DeTolla Louis , Hester Lisa , O’Neill Timothy , Manka Cheu , Benjamin Ivor , Nagarsekar Ashish , Singh Ishwar , Hasday Jeffrey D.
Published: April 2014

We previously demonstrated that exposure to febrile-range hyperthermia (FRH) accelerates pathogen clearance and increases survival in murine experimental Klebsiella pneumoniae peritonitis. However, FRH accelerates lethal lung injury in a mouse model of pulmonary oxygen toxicity, suggesting that the lung may be particularly susceptible to injurious effects of FRH. In the present study, we tested the hypothesis that, in contrast with the salutary effect of FRH in Gram-negative peritonitis, FRH would be detrimental in multilobar Gram-negative pneumonia. Using a conscious, temperature-clamped mouse model and intratracheal inoculation with K. pneumoniae Caroli strain, we showed that FRH tended to reduce survival despite reducing the 3 day-postinoculation pulmonary pathogen burden by 400-fold. We showed that antibiotic treatment rescued the euthermic mice, but did not reduce lethality in the FRH mice. Using an intratracheal bacterial endotoxin LPS challenge model, we found that the reduced survival in FRH-treated mice was accompanied by increased pulmonary vascular endothelial injury, enhanced pulmonary accumulation of neutrophils, increased levels of IL-1β, MIP-2/CXCL213, GM-CSF, and KC/CXCL1 in the bronchoalveolar lavage fluid, and bronchiolar epithelial necrosis. These results suggest that FRH enhances innate host defense against infection, in part, by augmenting polymorphonuclear cell delivery to the site of infection. The ultimate effect of FRH is determined by the balance between accelerated pathogen clearance and collateral tissue injury, which is determined, in part, by the site of infection.
The Cytokine IL-1β Activates IFN Response Factor 3 in Human Fetal Astrocytes in Culture
Authors: Rivieccio Mark A. , John Gareth R. , Song Xianyuan , Suh Hyeon-Sook , Zhao Yongmei , Lee Sunhee C. , Brosnan Celia F.
Published: April 2014

The cytokine IL-1β is a major activator of primary human fetal astrocytes in culture, leading to the production of a wide range of cytokines and chemokines important in the host defense against pathogens. IL-1β, like TLR4, signals via the MyD88/IL-1βR-associated kinase-1 pathway linked to activation of NF-κB and AP-1. Recent studies have shown that TLR4 also signals independently of MyD88, resulting in the activation of IFN regulatory factor 3 (IRF3), a transcription factor required for the production of primary antiviral response genes such as IFN-β. Using a functional genomics approach, we observed that IL-1β induced in astrocytes a group of genes considered to be IFN-stimulated genes (ISG), suggesting that IL-1β may also signal via IRF3 in these cells. We now show, using real-time PCR, that in astrocytes IL-1β induces the expression of IFN-β, IRF7, CXCL10/IFN-γ-inducible protein-10, and CCL5/RANTES. Chemokine expression was confirmed by ELISA. We also show that IL-1β induces phosphorylation and nuclear translocation of IRF3 and delayed phosphorylation of STAT1. The dependency of IFN-β, IRF7, and CXCL10/IFN-γ-inducible protein-10 gene expression on IRF3 was confirmed using a dominant negative IRF3-expressing adenovirus. The robust induction by IL-1β of additional ISG noted on the microarrays, such as STAT1, 2′5′-oligoadenylate synthetase 2, and ISG15, also supports an active signaling role for IL-1β via this pathway in human fetal astrocytes. These data are the first to show that IL-1β, in addition to TLRs, can stimulate IRF3, implicating this cytokine as an activator of genes involved in innate antiviral responses in astrocytes.
Modulation of Phagocytosis of Apoptotic Neutrophils by Supernatant from Dexamethasone-Treated Macrophages and Annexin-Derived Peptide Ac2–26
Authors: Maderna Paola , Yona Simon , Perretti Mauro , Godson Catherine
Published: April 2014

Phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation. The glucocorticoid-inducible protein annexin 1 and annexin 1-derived peptides show potent anti-inflammatory responses in acute and chronic inflammation. In this study, we report that the annexin 1-derived peptide (Ac2–26) significantly stimulates nonphlogistic phagocytosis of apoptotic polymorphonuclear leukocytes (PMNs) by human monocyte-derived macrophages (Mφ). Peptide Ac2–26-stimulated phagocytosis is accompanied by rearrangement of the Mφ actin cytoskeleton. To investigate the potential role of endogenous annexin on clearance of apoptotic cells, Mφ were cultured for 5 days in the presence of dexamethasone. Supernatants collected from dexamethasone-treated Mφ significantly enhanced the ability of naive Mφ to engulf apoptotic PMNs. This effect was blocked by an annexin blocking Ab, by immunodepletion of the supernatants, and by the formyl peptide receptor/lipoxin receptor antagonist Boc1. In addition, we show that bone marrow-derived Mφ from annexin 1-null mice present a 40% decreased phagocytosis of apoptotic PMNs compared with cells taken from littermate controls. In conclusion, these results emphasize the pivotal role of annexin 1 as mediator for clearance of apoptotic cells and expand its potential therapeutic role in controlling inflammatory diseases.
Alloreactive T Cell Responses and Acute Rejection of Single Class II MHC-Disparate Heart Allografts Are under Strict Regulation by CD4+CD25+ T Cells
Authors: Schenk Soren , Kish Danielle D. , He Chunshui , El-Sawy Tarek , Chiffoleau Eise , Chen Chuangqui , Wu Zihao , Sandner Sigrid , Gorbachev Anton V. , Fukamachi Kiyotaka , Heeger Peter S. , Sayegh Mohamed H. , Turka Laurence A. , Fairchild Robert L.
Published: April 2014

Skin but not vascularized cardiac allografts from B6.H-2bm12 mice are acutely rejected by C57BL/6 recipients in response to the single class II MHC disparity. The underlying mechanisms preventing acute rejection of B6.H-2bm12 heart allografts by C57BL/6 recipients were investigated. B6.H-2bm12 heart allografts induced low levels of alloreactive effector T cell priming in C57BL/6 recipients, and this priming was accompanied by low-level cellular infiltration into the allograft that quickly resolved. Recipients with long-term-surviving heart allografts were unable to reject B6.H-2bm12 skin allografts, suggesting potential down-regulatory mechanisms induced by the cardiac allografts. Depletion of CD25+ cells from C57BL/6 recipients resulted in 15-fold increases in alloreactive T cell priming and in acute rejection of B6.H-2bm12 heart grafts. Similarly, reconstitution of B6.Rag−/− recipients with wild-type C57BL/6 splenocytes resulted in acute rejection of B6.H-2bm12 heart grafts only if CD25+ cells were depleted. These results indicate that acute rejection of single class II MHC-disparate B6.H-2bm12 heart allografts by C57BL/6 recipients is inhibited by the emergence of CD25+ regulatory cells that restrict the clonal expansion of alloreactive T cells.
Immunization with Lentiviral Vector-Transduced Dendritic Cells Induces Strong and Long-Lasting T Cell Responses and Therapeutic Immunity
Authors: He Yukai , Zhang Jiying , Mi Zhibao , Robbins Paul , Falo Louis D.
Published: April 2014

Dendritic cell (DC) therapies are currently being evaluated for the treatment of cancer. The majority of ongoing clinical trials use DCs loaded with defined antigenic peptides or proteins, or tumor-derived products, such as lysates or apoptotic cells, as sources of Ag. Although several theoretical considerations suggest that DCs expressing transgenic protein Ags may be more effective immunogens than protein-loaded cells, methods for efficiently transfecting DCs are only now being developed. In this study we directly compare the immunogenicity of peptide/protein-pulsed DCs with lentiviral vector-transduced DCs, and their comparative efficacy in tumor immunotherapy. Maturing, bone marrow-derived DCs can be efficiently transduced with lentiviral vectors, and transduction does not affect DC maturation, plasticity, or Ag presentation function. Transduced DCs efficiently process and present both MHC class I- and II-restricted epitopes from the expressed transgenic Ag OVA. Compared with peptide- or protein-pulsed DCs, lentiviral vector-transduced DCs elicit stronger and longer-lasting T cell responses in vivo, as measured by both in vivo killing assays and intracellular production of IFN-γ by Ag-specific T cells. In the B16-OVA tumor therapy model, the growth of established tumors was significantly inhibited by a single immunization using lentiviral vector-transduced DCs, resulting in significantly longer survival of immunized animals. These results suggest that compared with Ag-pulsed DCs, vaccination with lentiviral vector-transduced DCs may achieve more potent antitumor immunity. These data support the further development of lentiviral vectors to transduce DCs with genes encoding Ags or immunomodulatory adjuvants to generate and control systemic immune responses.
Reducing the Stimulation of CD8+ T Cells during Infection with Intracellular Bacteria Promotes Differentiation Primarily into a Central (CD62LhighCD44high) Subset
Authors: van Faassen Henk , Saldanha Marsha , Gilbertson Deanna , Dudani Renu , Krishnan Lakshmi , Sad Subash
Published: April 2014

During infection with lymphocytic choriomeningitis virus, CD8+ T cells differentiate rapidly into effectors (CD62LlowCD44high) that differentiate further into the central memory phenotype (CD62LhighCD44high) gradually. To evaluate whether this CD8+ T cell differentiation program operates in all infection models, we evaluated CD8+ T cell differentiation during infection of mice with recombinant intracellular bacteria, Listeria monocytogenes (LM) and Mycobacterium bovis (BCG), expressing OVA. We report that CD8+ T cells primed during infection with the attenuated pathogen BCG-OVA differentiated primarily into the central subset that correlated to reduced attrition of the primed cells subsequently. CD8+ T cells induced by LM-OVA also differentiated into central phenotype cells first, but the cells rapidly converted into effectors in contrast to BCG-OVA. Memory CD8+ T cells induced by both LM-OVA as well as BCG-OVA were functional in that they produced cytokines and proliferated extensively in response to antigenic stimulation after adoptive transfer. During LM-OVA infection, if CD8+ T cells were guided to compete for access to APCs, then they received reduced stimulation that was associated with increased differentiation into the central subset and reduced attrition subsequently. Similar effect was observed when CD8+ T cells encountered APCs selectively during the waning phase of LM-OVA infection. Taken together, our results indicate that the potency of the pathogen can influence the differentiation and fate of CD8+ T cells enormously, and the extent of attrition of primed CD8+ T cells correlates inversely to the early differentiation of CD8+ T cells primarily into the central CD8+ T cell subset.
Priming in the Presence of IL-10 Results in Direct Enhancement of CD8+ T Cell Primary Responses and Inhibition of Secondary Responses
Authors: Kang Silvia S. , Allen Paul M.
Published: April 2014

Although IL-10 acts as an inhibitory cytokine for APC and CD4+ T cell function, its effects on CD8+ T cells are unclear. Additionally, little is known about whether initial priming in the presence of IL-10 can have long-lasting effects and influence subsequent CD8+ T cell responses that occur in the absence of the cytokine. In the present study, we clarified the role of IL-10 during primary responses and examined whether exposure to IL-10 during initial priming of CD8+ T cells impacted secondary responses. To determine the effect of IL-10 on Ag-specific T cell responses, peptide-pulsed IL-10R2−/− splenic dendritic cells were used to prime T cells from OT-I CD8+ TCR transgenic mice. During the primary response, the presence of IL-10 resulted in enhancement of CD8+ T cell numbers without detectable alterations in the kinetics or percentage of cells that underwent proliferation. A modest increase in survival, not attributable to Bcl-2 or Bcl-xL, was also observed with IL-10 treatment. Other parameters of CD8+ T cell function, including IL-2, IFN-γ, TNF-α, and granzyme production, were unaltered. In contrast, initial exposure to IL-10 during the primary response resulted in decreased OT-I expansion during secondary stimulation. This was accompanied by lowered IL-2 levels and reduced percentages of proliferating BrdU+ cells and OT-I cells that were CD25high. IFN-γ, TNF-α, and granzyme production were unaltered. These data suggest that initial exposure of CD8+ T cells to IL-10 may be temporarily stimulatory; however, programming of the cells may be altered, resulting in diminished overall responses.
Saturated and Polyunsaturated Fatty Acids Reciprocally Modulate Dendritic Cell Functions Mediated through TLR4
Authors: Weatherill Amy R. , Lee Joo Y. , Zhao Ling , Lemay Danielle G. , Youn Hyung S. , Hwang Daniel H.
Published: April 2014

TLRs provide critical signals to induce innate immune responses in APCs such as dendritic cells (DCs) that in turn link to adaptive immune responses. Results from our previous studies demonstrated that saturated fatty acids activate TLRs, whereas n-3 polyunsaturated fatty acids inhibit agonist-induced TLR activation. These results raise a significant question as to whether fatty acids differentially modulate immune responses mediated through TLR activation. The results presented in this study demonstrate that the saturated fatty acid, lauric acid, up-regulates the expression of costimulatory molecules (CD40, CD80, and CD86), MHC class II, and cytokines (IL-12p70 and IL-6) in bone marrow-derived DCs. The dominant negative mutant of TLR4 or its downstream signaling components inhibits lauric acid-induced expression of a CD86 promoter-reporter gene. In contrast, an n-3 polyunsaturated fatty acid, docosahexaenoic acid, inhibits TLR4 agonist (LPS)-induced up-regulation of the costimulatory molecules, MHC class II, and cytokine production. Similarly, DCs treated with lauric acid show increased T cell activation capacity, whereas docosahexaenoic acid inhibits T cell activation induced by LPS-treated DCs. Together, our results demonstrate that the reciprocal modulation of both innate and adaptive immune responses by saturated fatty acid and n-3 polyunsaturated fatty acid is mediated at least in part through TLRs. These results imply that TLRs are involved in sterile inflammation and immune responses induced by nonmicrobial endogenous molecules. These results shed new light in understanding how types of dietary fatty acids differentially modulate immune responses that could alter the risk of many chronic diseases.
A Large Number of T Lymphocytes Recognize Moloney-Murine Leukemia Virus-Induced Antigens, but a Few Mediate Long-Lasting Tumor Immunosurveillance
Authors: Facchinetti Antonella , Santa Silvia Dalla , Mezzalira Silvio , Rosato Antonio , Biasi Giovanni
Published: April 2014

The CD8+ T cell response to Moloney-murine leukemia virus (M-MuLV)-induced Ags is almost entirely dominated by the exclusive expansion of lymphocytes that use preferential TCRVβ chain rearrangements. In mice lacking T cells expressing these TCRVβ, we demonstrate that alternative TCRVβ can substitute for the lack of the dominant TCRVβ in the H-2-restricted M-MuLV Ag recognition. We show that, at least for the H-2b-restricted response, the shift of TCR usage is not related to a variation of the immunodominant M-MuLV epitope recognition. After virus immunization, all the potentially M-MuLV-reactive lymphocytes are primed, but only the deletion of dominant Vβ rescues the alternative Vβ response. The mechanism of clonal T cell “immunodomination” that guides the preferential Vβ expansion is likely the result of a proliferative advantage of T cells expressing dominant Vβ, due to differences in TCR affinity and/or cosignal requirements. In this regard, a CD8 involvement is strictly required for the virus-specific cytotoxic activity of CTL expressing alternative, but not dominant, Vβ gene rearrangements. The ability of T cells expressing alternative TCRVβ rearrangements to mediate tumor protection was evaluated by a challenge with M-MuLV tumor cells. Although T cells expressing alternative Vβ chains were activated and expanded, they were not able to control tumor growth in a long-lasting manner due to their incapacity of conversion and accumulation in the T central memory pool.
Membrane IgE Binds and Activates FcεRI in an Antigen-Independent Manner
Authors: Vangelista Luca , Soprana Elisa , Cesco-Gaspere Michela , Mandiola Paola , Di Lullo Giulia , Fucci Rita N. , Codazzi Franca , Palini Alessio , Paganelli Giovanni , Burrone Oscar R. , Siccardi Antonio G.
Published: April 2014

Interaction of secretory IgE with FcεRI is the prerequisite for allergen-driven cellular responses, fundamental events in immediate and chronic allergic manifestations. Previous studies reported the binding of soluble FcεRIα to membrane IgE exposed on B cells. In this study, the functional interaction between human membrane IgE and human FcεRI is presented. Four different IgE versions were expressed in mouse B cell lines, namely: a truncation at the Cε2-Cε3 junction of membrane IgE isoform long, membrane IgE isoform long (without Igα/Igβ BCR accessory proteins), and both εBCRs (containing membrane IgE isoforms short and long). All membrane IgE versions activated a rat basophilic leukemia cell line transfected with human FcεRI, as detected by measuring the release of both preformed and newly synthesized mediators. The interaction led also to Ca2+ responses in the basophil cell line, while membrane IgE-FcεRI complexes were detected by immunoprecipitation. FcεRI activation by membrane IgE occurs in an Ag-independent manner. Noteworthily, human peripheral blood basophils and monocytes also were activated upon contact with cells bearing membrane IgE. In humans, the presence of FcεRI in several cellular entities suggests a possible membrane IgE-FcεRI-driven cell-cell dialogue, with likely implications for IgE homeostasis in physiology and pathology.
TLR2 Signaling Is Critical for Mycoplasma pneumoniae-Induced Airway Mucin Expression
Authors: Chu Hong Wei , Jeyaseelan Samithamby , Rino John G. , Voelker Dennis R. , Wexler Rachel B. , Campbell Krista , Harbeck Ronald J. , Martin Richard J.
Published: April 2014

Excessive airway mucin production contributes to airway obstruction in lung diseases such as asthma and chronic obstructive pulmonary disease. Respiratory infections, such as atypical bacterium Mycoplasma pneumoniae (Mp), have been proposed to worsen asthma and chronic obstructive pulmonary disease in part through increasing mucin. However, the molecular mechanisms involved in infection-induced airway mucin overexpression remain to be determined. TLRs have been recently shown to be a critical component in host innate immune response to infections. TLR2 signaling has been proposed to be involved in inflammatory cell activation by mycoplasma-derived lipoproteins. In this study, we show that TLR2 signaling is critical in Mp-induced airway mucin expression in mice and human lung epithelial cells. Respiratory Mp infection in BALB/c mice activated TLR2 signaling and increased airway mucin. A TLR2-neutralizing Ab significantly reduced mucin expression in Mp-infected BALB/c mice. Furthermore, Mp-induced airway mucin was abolished in TLR2 gene-deficient C57BL/6 mice. Additionally, Mp was shown to increase human lung A549 epithelial cell mucin expression, which was inhibited by the overexpression of a human TLR2 dominant-negative mutant. These results clearly demonstrate that respiratory Mp infection increases airway mucin expression, which is dependent on the activation of TLR2 signaling.
A CD8+ T Cell Heptaepitope Minigene Vaccine Induces Protective Immunity againstChlamydia pneumoniae
Authors: Pinchuk Irina , Starcher Barry C. , Livingston Brian , Tvninnereim Amy , Wu Shiping , Appella Ettore , Sidney John , Sette Alessandro , Wizel Benjamin
Published: April 2014

An intact T cell compartment and IFN-γ signaling are required for protective immunity against Chlamydia. In the mouse model of Chlamydia pneumoniae (Cpn) infection, this immunity is critically dependent on CD8+ T cells. Recently we reported that Cpn-infected mice generate an MHC class I-restricted CD8+ Tc1 response against various Cpn Ags, and that CD8+ CTL to multiple epitopes inhibit Cpn growth in vitro. Here, we engineered a DNA minigene encoding seven H-2b-restricted Cpn CTL epitopes, the universal pan-DR epitope Th epitope, and an endoplasmic reticulum-translocating signal sequence. Immunization of C57BL/6 mice with this construct primed IFN-γ-producing CD8+ CTL against all seven CTL epitopes. CD8+ T cell lines generated to minigene-encoded CTL epitopes secreted IFN-γ and TNF-α and exhibited CTL activity upon recognition of Cpn-infected macrophages. Following intranasal challenge with Cpn, a 3.6 log reduction in mean lung bacterial numbers compared with control animals was obtained. Using a 20-fold increase in the Cpn challenging dose, minigene-vaccinated mice had a 60-fold reduction in lung bacterial loads, compared with controls. Immunization and challenge studies with β2-microglobulin−/− mice indicated that the reduction of lung Cpn burdens was mediated by the MHC class I-dependent CD8+ T cells to minigene-included Cpn CTL epitopes, rather than by pan-DR epitope-specific CD4+ T cells. This constitutes the first demonstration of significant protection achieved by immunization with a CD8+ T cell epitope-based DNA construct in a bacterial system and provides the basis for the optimal design of multicomponent anti-Cpn vaccines for humans.
Endogenous Attenuation of Allergic Lung Inflammation by Syndecan-1
Authors: Xu Jie , Park Pyong Woo , Kheradmand Farrah , Corry David B.
Published: April 2014

The airway plays a vital role in allergic lung diseases by responding to inhaled allergens and initiating allergic inflammation. Various proinflammatory functions of the airway epithelium have been identified, but, equally important, anti-inflammatory mechanisms must also exist. We show in this study that syndecan-1, the major heparan sulfate proteoglycan of epithelial cells, attenuates allergic lung inflammation. Our results show that syndecan-1-null mice instilled with allergens exhibit exaggerated airway hyperresponsiveness, glycoprotein hypersecretion, eosinophilia, and lung IL-4 responses. However, administration of purified syndecan-1 ectodomains, but not ectodomain core proteins devoid of heparan sulfate, significantly inhibits these inflammatory responses. Furthermore, syndecan-1 ectodomains are shed into the airway when wild-type mice are intranasally instilled with several biochemically distinct inducers of allergic lung inflammation. Our results also show that syndecan-1 ectodomains bind to the CC chemokines (CCL7, CCL11, and CCL17) implicated in allergic diseases, inhibit CC chemokine-mediated T cell migration, and suppress allergen-induced accumulation of Th2 cells in the lung through their heparan sulfate chains. Together, these findings uncover an endogenous anti-inflammatory mechanism of the airway epithelium where syndecan-1 ectodomains attenuate allergic lung inflammation via suppression of CC chemokine-mediated Th2 cell recruitment to the lung.
Inhibition of Mammalian Target of Rapamycin Potentiates Thrombin-Induced Intercellular Adhesion Molecule-1 Expression by Accelerating and Stabilizing NF-κB Activation in Endothelial Cells
Authors: Minhajuddin Mohd , Fazal Fabeha , Bijli Kaiser M. , Amin Md. Ruhul , Rahman Arshad
Published: April 2014

We addressed the regulatory function of mammalian target of rapamycin (mTOR) in the mechanism of thrombin-induced ICAM-1 gene expression in endothelial cells. Pretreatment of HUVECs with rapamycin, an inhibitor of mTOR, augmented thrombin-induced ICAM-1 expression. Inhibition of mTOR by this approach promoted whereas over-expression of mTOR inhibited thrombin-induced transcriptional activity of NF-κB, an essential regulator of ICAM-1 transcription. Analysis of the NF-κB signaling pathway revealed that inhibition of mTOR potentiated IκB kinase activation resulting in a rapid and persistent phosphorylation of IκBα on Ser32 and Ser36, a requirement for IκBα degradation. Consistent with these data, we observed a more efficient and stable nuclear localization of RelA/p65 and, subsequently, the DNA binding activity of NF-κB by thrombin following mTOR inhibition. These data define a novel role of mTOR in down-regulating thrombin-induced ICAM-1 expression in endothelial cells by controlling a delayed and transient activation of NF-κB.
Space, Selection, and Surveillance: Setting Boundaries with BLyS
Authors: Miller Juli P. , Stadanlick Jason E. , Cancro Michael P.
Published: April 2014

The BLyS family of ligands and receptors governs B cell homeostasis by controlling survival, differentiation, and lifespan. This family consists of multiple receptors and ligands, allowing independent regulation of different B cell subsets by varying the combination and levels of receptors expressed. Multiple downstream signaling pathways are implicated in these activities, reflecting this receptor complexity as well as cross-talk with other B cell signaling systems. BLyS levels are associated with multiple forms of humoral autoimmunity and can modulate tolerogenic elimination at the transitional checkpoint. BLyS responsiveness thus balances peripheral selection against cell numbers, providing an elastic system that varies selective stringency based on homeostatic demands.
Cutting Edge: T Cell Ig Mucin-3 Reduces Inflammatory Heart Disease by Increasing CTLA-4 during Innate Immunity
Authors: Frisancho-Kiss Sylvia , Nyland Jennifer F. , Davis Sarah E. , Barrett Masheka A. , Gatewood Shannon J. L. , Njoku Dolores B. , Cihakova Daniela , Silbergeld Ellen K. , Rose Noel R. , Fairweather DeLisa
Published: April 2014

Autoimmune diseases can be reduced or even prevented if proinflammatory immune responses are appropriately down-regulated. Receptors (such as CTLA-4), cytokines (such as TGF-β), and specialized cells (such as CD4+CD25+ T regulatory cells) work together to keep immune responses in check. T cell Ig mucin (Tim) family proteins are key regulators of inflammation, providing an inhibitory signal that dampens proinflammatory responses and thereby reducing autoimmune and allergic responses. We show in this study that reducing Tim-3 signaling during the innate immune response to viral infection in BALB/c mice reduces CD80 costimulatory molecule expression on mast cells and macrophages and reduces innate CTLA-4 levels in CD4+ T cells, resulting in decreased T regulatory cell populations and increased inflammatory heart disease. These results indicate that regulation of inflammation in the heart begins during innate immunity and that Tim-3 signaling on cells of the innate immune system critically influences regulation of the adaptive immune response.
Translocation of Active Heparanase to Cell Surface Regulates Degradation of Extracellular Matrix Heparan Sulfate upon Transmigration of Mature Monocyte-Derived Dendritic Cells
Authors: Benhamron Sandrine , Nechushtan Hovav , Verbovetski Inna , Krispin Alon , Abboud-Jarrous Ghada , Zcharia Eyal , Edovitsky Evgeny , Nahari Efrat , Peretz Tamar , Vlodavsky Israel , Mevorach Dror
Published: April 2014

After Ag capture and exposure to danger stimuli, maturing dendritic cells (DCs) migrate to regional lymph nodes, where antigenic peptides are presented to T lymphocytes. To migrate from peripheral tissue such as the epidermis to regional lymph nodes, Ag-bearing epidermal Langerhans cells must move through an extracellular matrix (ECM) of various compositions. The nature of their capacity to transmigrate via ECM is not well understood, although MIP-3β and CCR7 play critical roles. We were interested in verifying whether heparanase, a heparan sulfate-degrading endo-β-d-glucuronidase that participates in ECM degradation and remodeling, is expressed and functional in monocyte-derived DCs. Using immunohistochemistry, confocal microscopy, RT-PCR, Western blot analysis, assays for heparanase activity, and Matrigel transmigration, we show that heparanase is expressed in both nuclei and cytoplasm of immature DCs, and that gene expression and synthesis take place mainly in monocytes and early immature DCs. We also found that both nuclear and cytoplasm fractions show heparanase activity, and upon LPS-induced maturation, heparanase translocates to the cell surface and degrades ECM heparan sulfate. Matrigel transmigration assays showed a MIP-3β-comparable role for heparanase. Because heparan sulfate glycosaminoglycans play a key role in the self-assembly, insolubility, and barrier properties of the ECM, the results of this study suggest that heparanase is a key enzyme in DC transmigration through the ECM.
Monocyte/Macrophage Dysfunctions Do Not Impair the Promotion of Myelofibrosis by High Levels of Thrombopoietin
Authors: Wagner-Ballon Orianne , Chagraoui Hédia , Prina Eric , Tulliez Micheline , Milon Geneviève , Raslova Hana , Villeval Jean-Luc , Vainchenker William , Giraudier Stéphane
Published: April 2014

Several lines of evidence indicate that the megakaryocyte/platelet lineage is crucial in myelofibrosis induction. The demonstration that NOD/SCID mice with functionally deficient monocytes do not develop fibrotic changes when exposed to thrombopoietin (TPO) also suggests an important role for monocyte/macrophages. However, in this animal model, the development of myelofibrosis is dependent on the level of TPO. This study was conducted to investigate whether NOD/SCID mice exposed to high TPO levels mediated by a retroviral vector would be refractory to the development of bone marrow fibrosis. We show that TPO and TGF-β1 in plasma from NOD/SCID and SCID mice engrafted with TPO-overexpressing hemopoietic cells reach levels similar to the ones reached in immunocompetent mice, and all animals develop a myeloproliferative disease associated with a dense myelofibrosis at 8 wk posttransplantation. Monocytes in NOD/SCID mice are functionally deficient to secrete cytokines such as IL-1α in response to stimuli, even under TPO expression. Surprisingly, the plasma of these mice displays high levels of IL-α, which was demonstrated to originate from platelets. Together, these data suggest that completely functional monocytes are not required to develop myelofibrosis and that platelets are able, under TPO stimulation, to synthesize inflammatory cytokines, which may be involved in the pathogenesis of myelofibrosis and osteosclerosis.
Glucocorticoid-Induced TNF Receptor Family Related Gene Activation Overcomes Tolerance/Ignorance to Melanoma Differentiation Antigens and Enhances Antitumor Immunity
Authors: Ramirez-Montagut Teresa , Chow Andrew , Hirschhorn-Cymerman Daniel , Terwey Theis H. , Kochman Adam A. , Lu Sydney , Miles Randy C. , Sakaguchi Shimon , Houghton Alan N. , van den Brink Marcel R. M.
Published: April 2014

Glucocorticoid-induced TNF receptor family related protein (GITR) is present on many different cell types. Previous studies have shown that in vivo administration of an anti-GITR agonist mAb (DTA-1) inhibits regulatory T cells (Treg)-dependent suppression and enhances T cell responses. In this study, we show that administration of DTA-1 induces >85% tumor rejection in mice challenged with B16 melanoma. Rejection requires CD4+, CD8+, and NK1.1+ cells and is dependent on IFN-γ and Fas ligand and independent of perforin. Depletion of Treg via anti-CD25 treatment does not induce B16 rejection, whereas 100% of the mice depleted of CD25+ cells and treated with DTA-1 reject tumors, indicating a predominant role of GITR on effector T cell costimulation rather than on Treg modulation. T cells isolated from DTA-1-treated mice challenged with B16 are specific against B16 and several melanoma differentiation Ags. These mice develop memory against B16, and a small proportion of them develop mild hypopigmentation. Consistent with previous studies showing that GITR stimulation increases Treg proliferation in vitro, we found in our model that GITR stimulation expanded the absolute number of FoxP3+ cells in vivo. Thus, we conclude that overall, GITR stimulation overcomes self-tolerance/ignorance and enhances T cell-mediated antitumor activity with minimal autoimmunity.
Suppression of Alveolar Macrophage Apoptosis Prolongs Survival of Rats and Mice withPneumocystisPneumonia
Authors: Lasbury Mark E. , Durant Pamela J. , Ray Chad A. , Tschang Dennis , Schwendener Reto , Lee Chao-Hung
Published: April 2014

The number of alveolar macrophages is decreased in patients or animals with Pneumocystis pneumonia (Pcp). This loss of alveolar macrophages is in part due to apoptosis caused by Pneumocystis infection. The mechanism of apoptosis induction is unknown. Cell-free bronchoalveolar lavage fluids from Pneumocystis-infected rats or mice have the ability to induce apoptosis in normal alveolar macrophages. To characterize the mechanisms by which apoptosis proceeds in alveolar macrophages during Pcp, specific caspase inhibitors are tested for their ability to suppress the apoptosis. In vitro induction of apoptosis can be inhibited by the caspase-9 inhibitor (Z-LEHD-FMK) but not by the inhibitor to caspase-8 or -10. The caspase-9 inhibitor can also inhibit apoptosis of alveolar macrophages in vivo when it is intranasally instilled into dexamethasone-immunosuppressed, Pneumocystis-infected rats or L3T4 cell-depleted, Pneumocystis-infected mice. The number of alveolar macrophages rebounds in caspase-9 inhibitor-treated Pcp animals. Phagocytic activity of alveolar macrophages in treated animals is also recovered, and organism burden in these animals is reduced. Administration of caspase-9 inhibitor also clears the exudate that normally fills the alveoli during Pcp and decreases lung inflammation. Furthermore, caspase-9-treated Pcp animals survive for the entire 70-day period of the study, whereas nontreated Pcp animals die 40–60 days after initiation of infection. Depletion of recovered alveolar macrophages by intranasal administration of clodronate-containing liposomes in caspase-9 inhibitor-treated animals abrogates the effects of the inhibitor. Together, these results indicate that immunomodulation of the host response may be an alternative to current treatments for Pcp.
NK Lytic-Associated Molecule, Involved in NK Cytotoxic Function, Is an E3 Ligase
Authors: Fortier Julie M. , Kornbluth Jacki
Published: April 2014

NK lytic-associated molecule (NKLAM) is a protein involved in the cytolytic function of NK cells and CTLs. It has been localized to the cytolytic granules in NK cells and is up-regulated when cells are exposed to cytokines IL-2 or IFN-β. We report in this study that NKLAM contains a cysteine-rich really interesting new gene (RING) in between RING-RING domain, and that this domain possesses strong homology to the RING domain of the known E3 ubiquitin ligase, Dorfin. To determine whether NKLAM functions as an E3 ligase, we performed coimmunoprecipitation binding assays with ubiquitin conjugates (Ubcs) UbcH7, UbcH8, and UbcH10. We demonstrated that both UbcH7 and UbcH8 bind to full-length NKLAM. We then performed a similar binding assay using endogenous NKLAM and UbcH8 expressed by human NK clone NK3.3 to show that the protein interaction occurs in vivo. Using the yeast two-hybrid system, we identified uridine kinase like-1 (URKL-1) protein as a substrate for NKLAM. We confirmed that NKLAM and URKL-1 interact in mammalian cells by using both immunoprecipitation and confocal microscopy. We demonstrated decreased protein expression and enhanced ubiquitination of URKL-1 in the presence of NKLAM. These data indicate that NKLAM is a RING finger protein that binds Ubcs and has as one of its substrates, URKL-1, thus defining this cytolytic protein as an E3 ubiquitin ligase.
Selective Export of HLA-F by Its Cytoplasmic Tail
Authors: Boyle Louise H. , Gillingham Alison K. , Munro Sean , Trowsdale John
Published: April 2014

MHC class I molecules exit the endoplasmic reticulum (ER) by an unknown mechanism. Although a selective export mechanism has been proposed for the anterograde transport of class I, a motif responsible for export has never been identified. Although classical class I molecules lacking their cytoplasmic tail are expressed on the cell surface, we found that HLA-F was entirely dependent on its cytoplasmic tail for export from the ER. Two known export motifs were recognizable in HLA-F. A C-terminal valine residue functioned in ER export and interacted with coat complex (COP)II, while an RxR motif also played an important role in anterograde transport and bound to 14-3-3 proteins. This divergent trafficking of HLA-F implicates an alternative function for HLA-F, independent of loading with peptides in the ER.
Functional and Molecular Comparison of Anergic and Regulatory T Lymphocytes
Authors: Knoechel Birgit , Lohr Jens , Zhu Shirley , Wong Lisa , Hu Donglei , Ausubel Lara , Abbas Abul K.
Published: April 2014

Tolerance in vivo is maintained by multiple mechanisms that function to prevent autoimmunity. An encounter of CD4+ T cells with a circulating self-Ag leads to partial thymic deletion, the development of CD25+ regulatory T cells (Tregs), and functional anergy in the surviving CD25− population. We have compared anergic and regulatory T cells of the same Ag specificity generated in vivo by the systemic self-Ag. Anergic cells are unresponsive to the self-Ag that induces tolerance, but upon transfer into a new host and immunization, anergic cells can induce a pathologic autoimmune reaction against tissue expressing the same Ag. Tregs, in contrast, are incapable of mediating harmful reactions. To define the basis of this functional difference, we have compared gene expression profiles of anergic and regulatory T cells. These analyses show that Tregs express a distinct molecular signature, but anergic cells largely lack such a profile. Anergic cells express transcripts that are associated with effector differentiation, e.g., the effector cytokines IL-4 and IFN-γ. Anergic cells do not produce these cytokines in response to self-Ag, because the cells exhibit a proximal signaling block in response to TCR engagement. Thus, anergy reflects an aborted activation pathway that can readily be reversed, resulting in pathologic effector cell responses, whereas Treg development follows a distinct developmental pathway that extinguishes effector functions.
Cervical Thymus in the Mouse
Authors: Dooley James , Erickson Matthew , Gillard Geoffrey O. , Farr Andrew G.
Published: April 2014

Although thymic ectopy has long been recognized in humans, the functional activity or potential immunological significance of this thymic tissue is unknown. In this study, we describe murine thymic ectopy, cervical thymic tissue that possesses the same general organization as the thoracic thymus, that is able to support T cell differentiation, and that can export T cells to the periphery. Unexpectedly, the pattern of autoantigen expression by ectopic thymic tissue differs from that of the thoracic thymus, raising the possibility that these two thymic environments may project different versions of self.
Exogenous and Endogenous TLR Ligands Activate Anti-Chromatin and Polyreactive B Cells
Authors: Fields Michele L. , Metzgar Michele H. , Hondowicz Brian D. , Kang Sun-Ah , Alexander Shawn T. , Hazard Kristin D. , Hsu Alice C. , Du Yang-Zhu , Prak Eline Luning , Monestier Marc , Erikson Jan
Published: April 2014

Autoreactive B cells may become activated in a T-independent manner via synergistic engagement of the BCR and TLRs. Using the VH3H9 Ig H chain transgene to track anti-chromatin B cells, we demonstrate that VH3H9/Vλ1 anti-chromatin B cells proliferate in response to stimulatory oligodeoxynucleotides containing CpG motifs, suggesting that these autoreactive B cells are responsive to TLR9 signaling. Strikingly, some VH3H9 B cells, but not the well-characterized VH3H9/Vλ1 B cells, proliferate spontaneously in culture medium. This proliferation is blocked by inhibitory CpG oligodeoxynucleotides, implicating the TLR9 (or possibly TLR7) pathway. Most hybridomas generated from the proliferating cells are polyreactive, and one exhibits binding to nuclear Ags but not to the other Ags tested. Thus, B cells carrying autoreactive and/or polyreactive specificities may be susceptible to T cell-independent activation via dual engagement of the BCR and TLRs.
Conditional Ablation of MHC-II Suggests an Indirect Role for MHC-II in Regulatory CD4 T Cell Maintenance
Authors: Shimoda Michiko , Mmanywa Faith , Joshi Sunil K. , Li Tao , Miyake Katsuya , Pihkala Jeanene , Abbas Jonathan A. , Koni Pandelakis A.
Published: April 2014

Although the importance of MHC class II (MHC-II) in acute homeostatic proliferation of regulatory T (Treg) cells has been established, we considered here the maintenance and state of Treg cells in mice that are almost completely devoid of MHC-II in their periphery but still make their own CD4 T cells and Treg cells. The latter was accomplished by conditional deletion of a loxP-flanked MHC-II β-chain allele using a TIE2Cre transgene, which causes a very high degree of deletion in hemopoietic/endothelial progenitor cells but without deletion among thymic epithelial cells. Such conditional MHC-II-deficient mice possess their own relatively stable levels of CD4+CD25+ cells, with a normal fraction of Foxp3+ Treg cells therein, but at a level ∼2-fold lower than in control mice. Thus, both Foxp3low/− CD4+CD25+ cells, said to be a major source of IL-2, and IL-2-dependent Foxp3+ Treg cells are reduced in number. Furthermore, CD25 expression is marginally reduced among Foxp3+ Treg cells in conditional MHC-II-deficient mice, indicative of a lack of MHC-II-dependent TCR stimulation and/or IL-2 availability, and IL-2 administration in vivo caused greatly increased cell division among adoptively transferred Treg cells. This is not to say that IL-2 can cause Treg cell division in the complete absence of MHC-II as small numbers of MHC-II-bearing cells do remain in conditional MHC-II-deficient mice. Rather, this suggests only that IL-2 was limiting. Thus, our findings lend support to the proposal that Treg cell homeostasis depends on a delicate balance with a population of self-reactive IL-2-producing CD4+CD25+ cells which are themselves at least in part MHC-II-dependent.