bluuebunny/crossref_metadata_2025 huggingface dataset:
Multifunctional, High-Level Cytokine-Producing Th1 Cells in the Lung, but Not Spleen, Correlate with Protection against Mycobacterium tuberculosis Aerosol Challenge in Mice
Authors: Forbes Emily K. , Sander Clare , Ronan Edward O. , McShane Helen , Hill Adrian V. S. , Beverley Peter C. L. , Tchilian Elma Z.
Published: April 2014

Boosting bacillus Calmette-Guérin (BCG)-primed mice with a recombinant adenovirus expressing Mycobacterium tuberculosis Ag 85A by different administration routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to Ag 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast, intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to Ag 85A and an increased response to purified protein derivative. This effect is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensity and integrated median fluorescence intensity for IFN-γ, IL-2, and TNF. In contrast, mice immunized with BCG alone have few Ag-specific cells in the lung and a low proportion of multifunctional cells, although individual cells have high median fluorescence intensity. Successful immunization regimes appear to induce Ag-specific cells with abundant intracellular cytokine staining.
IL-17 Is a Critical Component of Vaccine-Induced Protection against Lung Infection by Lipopolysaccharide-Heterologous Strains of Pseudomonas aeruginosa
Authors: Priebe Gregory P. , Walsh Rebecca L. , Cederroth Terra A. , Kamei Akinobu , Coutinho-Sledge Yamara S. , Goldberg Joanna B. , Pier Gerald B.
Published: April 2014

In a murine model of acute fatal pneumonia, we previously showed that nasal immunization with a live-attenuated aroA deletant of Pseudomonas aeruginosa strain PAO1 elicited LPS serogroup-specific protection, indicating that opsonic Ab to the LPS O Ag was the most important immune effector. Because P. aeruginosa strain PA14 possesses additional virulence factors, we hypothesized that a live-attenuated vaccine based on PA14 might elicit a broader array of immune effectors. Thus, an aroA deletant of PA14, denoted PA14ΔaroA, was constructed. PA14ΔaroA-immunized mice were protected against lethal pneumonia caused not only by the parental strain but also by cytotoxic variants of the O Ag-heterologous P. aeruginosa strains PAO1 and PAO6a,d. Remarkably, serum from PA14ΔaroA-immunized mice had very low levels of opsonic activity against strain PAO1 and could not passively transfer protection, suggesting that an antibody-independent mechanism was needed for the observed cross-serogroup protection. Compared with control mice, PA14ΔaroA-immunized mice had more rapid recruitment of neutrophils to the airways early after challenge. T cells isolated from P. aeruginosa ΔaroA-immunized mice proliferated and produced IL-17 in high quantities after coculture with gentamicin-killed P. aeruginosa. Six hours following challenge, PA14ΔaroA-immunized mice had significantly higher levels of IL-17 in bronchoalveolar lavage fluid compared with unimmunized, Escherichia coli-immunized, or PAO1ΔaroA-immunized mice. Antibody-mediated depletion of IL-17 before challenge or absence of the IL-17 receptor abrogated the PA14ΔaroA vaccine’s protection against lethal pneumonia. These data show that IL-17 plays a critical role in antibody-independent vaccine-induced protection against LPS-heterologous strains of P. aeruginosa in the lung.
Mast Cells Promote Airway Smooth Muscle Cell Differentiation via Autocrine Up-Regulation of TGF-β1
Authors: Woodman Lucy , Siddiqui Salman , Cruse Glenn , Sutcliffe Amanda , Saunders Ruth , Kaur Davinder , Bradding Peter , Brightling Christopher
Published: April 2014

Asthma is a major cause of morbidity and mortality worldwide. It is characterized by airway dysfunction and inflammation. A key determinant of the asthma phenotype is infiltration of airway smooth muscle bundles by activated mast cells. We hypothesized that interactions between these cells promotes airway smooth muscle differentiation into a more contractile phenotype. In vitro coculture of human airway smooth muscle cells with β-tryptase, or mast cells with or without IgE/anti-IgE activation, increased airway smooth muscle-derived TGF-β1 secretion, α-smooth muscle actin expression and agonist-provoked contraction. This promotion to a more contractile phenotype was inhibited by both the serine protease inhibitor leupeptin and TGF-β1 neutralization, suggesting that the observed airway smooth muscle differentiation was driven by the autocrine release of TGF-β1 in response to activation by mast cell β-tryptase. Importantly, in vivo we found that in bronchial mucosal biopsies from asthmatics the intensity of α-smooth muscle actin expression was strongly related to the number of mast cells within or adjacent to an airway smooth muscle bundle. These findings suggest that mast cell localization in the airway smooth muscle bundle promotes airway smooth muscle cell differentiation into a more contractile phenotype, thus contributing to the disordered airway physiology that characterizes asthma.
Evolution of the Inflammatory Response in Vertebrates: Fish TNF-α Is a Powerful Activator of Endothelial Cells but Hardly Activates Phagocytes
Authors: Roca Francisco J. , Mulero Iván , López-Muñoz Azucena , Sepulcre Maria P. , Renshaw Stephen A. , Meseguer José , Mulero Victoriano
Published: April 2014

TNF-α is conserved in all vertebrate classes and has been identified in all taxonomic groups of teleost fish. However, its biological activities and its role in infection are largely unknown. Using two complementary fish models, gilthead seabream and zebrafish, we report here that the main proinflammatory effects of fish TNF-α are mediated through the activation of endothelial cells. Thus, TNF-α promotes the expression of E-selectin and different CC and CXC chemokines in endothelial cells, thus explaining the recruitment and activation of phagocytes observed in vivo in both species. We also found that TLR ligands, and to some extent TNF-α, were able to increase the expression of MHC class II and CD83 in endothelial cells, which might suggest a role for fish endothelial cells and TNF-α in Ag presentation. Lastly, we found that TNF-α increases the susceptibility of the zebrafish to viral (spring viremia of carp virus) and bacterial (Streptococcus iniae) infections. Although the powerful actions of fish TNF-α on endothelial cells suggest that it might facilitate pathogen dissemination, it was found that TNF-α increased antiviral genes and, more importantly, had little effect on the viral load in early infection. In addition, the stimulation of ZF4 cells with TNF-α resulted in increased viral replication. Together, these results indicate that fish TNF-α displays different sorts of bioactivity to their mammalian counterparts and point to the complexity of the evolution that has taken place in the regulation of innate immunity by cytokines.
Prostaglandin E2 Exerts Catabolic Effects in Osteoarthritis Cartilage: Evidence for Signaling via the EP4 Receptor
Authors: Attur Mukundan , Al-Mussawir Hayf E. , Patel Jyoti , Kitay Alison , Dave Mandar , Palmer Glyn , Pillinger Michael H. , Abramson Steven B.
Published: April 2014

Elevated levels of PGE2 have been reported in synovial fluid and cartilage from patients with osteoarthritis (OA). However, the functions of PGE2 in cartilage metabolism have not previously been studied in detail. To do so, we cultured cartilage explants, obtained from patients undergoing knee replacement surgery for advanced OA, with PGE2 (0.1–10 μM). PGE2 inhibited proteoglycan synthesis in a dose-dependent manner (maximum 25% inhibition (p < 0.01)). PGE2 also induced collagen degradation, in a manner inhibitable by the matrix metalloproteinase (MMP) inhibitor ilomastat. PGE2 inhibited spontaneous MMP-1, but augmented MMP-13 secretion by OA cartilage explant cultures. PCR analysis of OA chondrocytes treated with PGE2 with or without IL-1 revealed that IL-1-induced MMP-13 expression was augmented by PGE2 and significantly inhibited by the cycolooygenase 2 selective inhibitor celecoxib. Conversely, MMP-1 expression was inhibited by PGE2, while celecoxib enhanced both spontaneous and IL-1-induced expression. IL-1 induction of aggrecanase 5 (ADAMTS-5), but not ADAMTS-4, was also enhanced by PGE2 (10 μM) and reversed by celecoxib (2 μM). Quantitative PCR screening of nondiseased and end-stage human knee OA articular cartilage specimens revealed that the PGE2 receptor EP4 was up-regulated in OA cartilage. Moreover, blocking the EP4 receptor (EP4 antagonist, AH23848) mimicked celecoxib by inhibiting MMP-13, ADAMST-5 expression, and proteoglycan degradation. These results suggest that PGE2 inhibits proteoglycan synthesis and stimulates matrix degradation in OA chondrocytes via the EP4 receptor. Targeting EP4, rather than cyclooxygenase 2, could represent a future strategy for OA disease modification.
YKL-40 Is Elevated in Patients with Chronic Obstructive Pulmonary Disease and Activates Alveolar Macrophages
Authors: Létuvé Séverine , Kozhich Alexander , Arouche Nassim , Grandsaigne Martine , Reed Jennifer , Dombret Marie-Christine , Kiener Peter A. , Aubier Michel , Coyle Anthony J. , Pretolani Marina
Published: April 2014

YKL-40 is a chitin-binding protein that is elevated in patients with various inflammatory conditions associated with ongoing remodeling. We investigated whether the levels of YKL-40 were up-regulated in the circulation and the airways of patients with chronic obstructive pulmonary disease (COPD), and whether it promoted the production of inflammatory mediators from macrophages. Serum, bronchoalveolar lavage (BAL), bronchial biopsies, lung tissue specimens, and alveolar macrophages from never-smokers (n = 15), smokers without COPD (n = 20), and smokers with COPD (n = 30) were assessed for YKL-40 levels and immunolocalization. In addition, YKL-40-induced mediator release from alveolar macrophages was examined. We found that smokers with COPD had elevated levels of YKL-40 in serum (p ≤ 0.027) and BAL (p ≤ 0.007), more YKL-40-positive cells in bronchial biopsies (p ≤ 0.03), and a greater proportion of alveolar macrophages expressing YKL-40 than smokers without COPD or never-smokers. YKL-40 levels in serum and BAL were associated with airflow obstruction (pre-β2 agonist forced expiratory volume in 1 s, rs = −0.3892, p = 0.0072 and rs = −0.5491, p < 0.0001, respectively) and impaired diffusion lung capacity (transfer factor of the lung for carbon monoxide, rs = −0.4667, p = 0.002 and rs = −0.3854, p = 0.0045, respectively). TNF-α stimulated YKL-40 synthesis in alveolar macrophages from smokers with COPD, and exposure of these cells to YKL-40 promoted the release of IL-8, MCP-1, MIP-1α, and metalloproteinase-9. We conclude that YKL-40 is up-regulated in COPD, in which it may contribute to tissue inflammation and remodeling by sustaining the synthesis of proinflammatory and fibrogenic chemokines and of metalloproteinases by alveolar macrophages.
Thymus Exosomes-Like Particles Induce Regulatory T Cells
Authors: Wang Gui-Jun , Liu Yuelong , Qin Aijian , Shah Spandan V. , Deng Zhong-bin , Xiang Xiaoyu , Cheng Ziqiang , Liu Cunren , Wang Jianhua , Zhang Liming , Grizzle William E. , Zhang Huang-Ge
Published: April 2014

Exosomes released from different types of cells have been proposed to contribute to intercellular communication. We report that thymic exosome-like particles (ELPs) released from cells of the thymus can induce the development of Foxp3+ regulatory T (Treg) cells in the lung and liver. Thymic ELPs also induce the conversion of thymic CD4+CD25− T cells into Tregs. Tregs induced by thymic ELPs suppress the proliferation of CD4+CD25− T cells in vitro and in vivo. We further show that neutralization of TGF-β in ELPs partially reverses thymic ELP-mediated induction of CD4+Foxp3+ T cells in the lung and liver. This study demonstrates that thymic ELPs participate in the induction of Foxp3+ Tregs. Also, TGF-β of thymic ELPs might be required for the generation of Tregs in the peripheral tissues.
Splenic Phagocytes Promote Responses to Nucleosomes in (NZB × NZW) F1 Mice
Authors: Okamoto Akiko , Fujio Keishi , van Rooijen Nico , Tsuno Nelson H. , Takahashi Koki , Tsurui Hiromichi , Hirose Sachiko , Elkon Keith B. , Yamamoto Kazuhiko
Published: April 2014

Autoantigen presentation to T cells is crucial for the development of autoimmune disease. However, the mechanisms of autoantigen presentation are poorly understood. In this study, we show that splenic phagocytes play an important role in autoantigen presentation in murine lupus. Nucleosomes are major autoantigens in systemic lupus erythematosus. We found that nucleosome-specific T cells were stimulated dominantly in the spleen, compared with lymph nodes, lung, and thymus. Among splenic APCs, F4/80+ macrophages and CD11b+CD11c+ dendritic cells were strong stimulators for nucleosome-specific T cells. When splenic phagocytes were depleted in (NZB × NZW) F1 (NZB/W F1) mice, nucleosome presentation in the spleen was dramatically suppressed. Moreover, depletion of splenic phagocytes significantly suppressed anti-nucleosome Ab and anti-dsDNA Ab production. Proteinuria progression was delayed and survival was prolonged in phagocyte-depleted mice. The numbers of autoantibody- secreting cells were decreased in the spleen from phagocyte-depleted mice. Multiple injections of splenic F4/80+ macrophages, not those of splenic CD11c+ dendritic cells, induced autoantibody production and proteinuria progression in NZB/W F1 mice. These results indicate that autoantigen presentation by splenic phagocytes including macrophages significantly contributes to autoantibody production and disease progression in lupus-prone mice.
Functional Consequences of Neuromyelitis Optica-IgG Astrocyte Interactions on Blood-Brain Barrier Permeability and Granulocyte Recruitment
Authors: Vincent Thierry , Saikali Philippe , Cayrol Romain , Roth Alejandro D. , Bar-Or Amit , Prat Alexandre , Antel Jack P.
Published: April 2014

Autoantibody neuromyelitis optica-IgG (NMO-IgG) recognizing aquaporin-4 (AQP4) is implicated as playing a central role in the physiopathology of NMO. The aim of this in vitro-based study was to characterize functional consequences of interaction between NMO-IgG and cells of the neurovascular unit (astrocytes and brain endothelium) that would provide insight into recognized features of NMO, namely altered blood-brain barrier (BBB) permeability and granulocyte recruitment. We used sera from NMO and longitudinally extensive transverse myelitis cases shown to bind in a characteristic perivascular pattern to primate cerebellar slices. Using flow cytometry, we found that sera from NMO-IgG-positive patients reacted with CNS-derived human fetal astrocytes, whereas sera from multiple sclerosis patients did not. We demonstrated that NMO-IgG binding to astrocytes alters aquaporin-4 polarized expression and increases permeability of a human BBB endothelium/astrocyte barrier. We further demonstrated that NMO-IgG binding to human fetal astrocytes can result in NK cell degranulation, astrocyte killing by Ab-dependent cellular cytotoxicity and complement-dependent granulocyte attraction through the BBB model. Our study highlights important functional roles for NMO-IgG that could account for pathological lesions and BBB dysfunction observed in NMO.
Subsets of Myeloid-Derived Suppressor Cells in Tumor-Bearing Mice
Authors: Youn Je-In , Nagaraj Srinivas , Collazo Michelle , Gabrilovich Dmitry I.
Published: April 2014

Myeloid-derived suppressor cells (MDSC) are a heterogeneous group of cells that play a critical role in tumor associated immune suppression. In an attempt to identify a specific subset of MDSC primarily responsible for immunosuppressive features of these cells, 10 different tumor models were investigated. All models showed variable but significant increase in the population of MDSC. Variability of MDSC expansion in vivo matched closely the effect of tumor cell condition medium in vitro. MDSC consists of two major subsets of Ly6G+Ly6Clow granulocytic and Ly6G−Ly6Chigh monocytic cells. Granulocytic MDSC have increased level of reactive oxygen species and undetectable level of NO whereas monocytic MDSC had increased level of NO but undetectable levels of reactive oxygen species. However, their suppressive activity per cell basis was comparable. Almost all tumor models demonstrated a preferential expansion of granulocytic subset of MDSC. We performed a phenotypical and functional analysis of several surface molecules previously suggested to be involved in MDSC-mediated suppression of T cells: CD115, CD124, CD80, PD-L1, and PD-L2. Although substantial proportion of MDSC expressed those molecules no differences in the level of their expression or the proportion, positive cells were found between MDSC and cells from tumor-free mice that lack immune suppressive activity. The level of MDSC-mediated T cell suppression did not depend on the expression of these molecules. These data indicate that suppressive features of MDSC is caused not by expansion of a specific subset but more likely represent a functional state of these cells.
Cutting Edge: IFN-γ Enables APC to Promote Memory Th17 and Abate Th1 Cell Development
Authors: Kryczek Ilona , Wei Shuang , Gong Wenrong , Shu Xiaogong , Szeliga Wojciech , Vatan Linhua , Chen Lieping , Wang Guobin , Zou Weiping
Published: April 2014

Th1-derived IFN-γ targets naive T cells and inhibits Th17 development. However, Th1, Th17, and memory but not naive T cells are colocalized in an inflammatory environment. To demonstrate the kinetic relationship between these T cell subsets, we investigated the role of IFN-γ in regulating the development and balance between Th17 and Th1 in humans. We show that IFN-γ stimulates B7-H1 expression on APC subsets and abates their Th1 polarization capacity in a B7-H1-dependent manner. Interestingly, IFN-γ triggers APCs to produce IL-1 and IL-23 and enables them to induce memory Th17 expansion via IL-1 and IL-23 in a B7-H1-independent manner. We propose a novel dynamic between Th1 and Th17 in the course of inflammation as follows: Th1-mediated inflammation is attenuated by IFN-γ-induced B7-H1 on APCs and is evolved toward Th17-mediated chronic inflammation by IFN-γ-induced, APC-derived IL-1 and IL-23. Our study challenges the dogma that IFN-γ suppresses Th17 and enhances Th1 development.
Heterospecific CD4 Help to Rescue CD8 T Cell Killers
Authors: de Goër de Herve Marie-Ghislaine , Cariou Anne , Simonetta Federico , Taoufik Yassine
Published: April 2014

Help from CD4 T cells may be required for optimal generation and maintenance of memory CD8 T cells and also for optimal Ag reactivation. We examined whether the helper cell and the CD8 killer cell need to have the same Ag specificity for help to be effective during interactions of memory T cells with mature APC. This is important because virus and tumor Ag-specific CD4 T cell responses are selectively impaired in several chronic viral infections and malignancies. We performed studies in vitro and in vivo and found that functional memory CD4 T cells generated from a distinct antigenic source (heterospecific helpers) could provide direct and effective help to memory CD8 T cells. Functional heterospecific memory CD4 T cells could also rescue secondary CD8 T cell responses in an experimental tumor model in which homospecific CD4 help was impaired. This could provide a rationale for immunotherapy strategies designed to bypass impaired homospecific help.
Intraclonal Competition Inhibits the Formation of High-Affinity Antibody-Secreting Cells
Authors: Le Thuc-vy L. , Kim Tea Hyun , Chaplin David D.
Published: April 2014

Protective immunity requires a diverse, polyclonal B cell repertoire. We demonstrate that affinity maturation of the humoral response to a hapten is impaired when preexisting clonally restricted cells recognizing the hapten are dominant in the B cell repertoire. B1-8i+/− mice, which feature a high frequency of B cells with nitrophenyl (NP)-binding specificity, respond to NP-haptenated proteins with the production of NP-specific Abs, but affinity maturation is impaired due to insufficient generation of high-affinity Ab-producing cells. We manipulated the frequency of NP-specific B cells by adoptive transfer of B1-8 B cells into naive, wild-type recipients. Remarkably, when 104 B1-8 B cells were transferred, these cells supported efficient affinity maturation and plasma cell differentiation. In contrast, when 106 B1-8 cells were transferred, affinity maturation did not occur. These data indicate that restricting the frequency of clonally related B cells is required to support affinity maturation.
Soluble Frizzled-Related Protein 1 Is Estrogen Inducible in Bone Marrow Stromal Cells and Suppresses the Earliest Events in Lymphopoiesis
Authors: Yokota Takafumi , Oritani Kenji , Garrett Karla P. , Kouro Taku , Nishida Makoto , Takahashi Isao , Ichii Michiko , Satoh Yusuke , Kincade Paul W. , Kanakura Yuzuru
Published: April 2014

It has long been known that lymphopoiesis is transiently suppressed during pregnancy, which can be experimentally simulated by estrogen treatment. We now confirm with Rag1/GFP reporter mice that early lymphoid progenitors in the lineage marker− c-kithigh ScaI+, hematopoietic stem cell-enriched fraction of bone marrow are particularly depressed in these circumstances. Hematopoietic and environmental cells are both potential hormone targets and, because of this complexity, very little is known regarding mechanisms. We have now identified soluble Frizzled-related protein (sFRP)1 as an estrogen-inducible gene in stromal cells, whose expression corresponded to inability to support lymphopoiesis. Bone-lining stromal cells express sFRP1, and the transcripts were elevated by pregnancy or estrogen injection. Estrogen receptor-α was essential for both lymphoid suppression and induction of the sFRP family. SFRP1 has been mainly described as an antagonist for complex Wnt signals. However, we found that sFRP1, like Wnt3a, stabilized β-catenin and blocked early lymphoid progression. Myeloerythroid progenitors were less affected by sFRP1 in culture, which was similar to estrogen with respect to lineage specificity. Hematopoietic stem cells expressed various Frizzled receptors, which markedly declined as they differentiated to lymphoid lineage. Thus, hormonal control of early lymphopoiesis in adults might partly relate to sFRP1 levels.
Polymorphic Sites Away from the Bw4 Epitope That Affect Interaction of Bw4+ HLA-B with KIR3DL1
Authors: Sanjanwala Bharati , Draghi Monia , Norman Paul J. , Guethlein Lisbeth A. , Parham Peter
Published: April 2014

KIR3DL1 is a polymorphic, inhibitory NK cell receptor specific for the Bw4 epitope carried by subsets of HLA-A and HLA-B allotypes. The Bw4 epitope of HLA-B*5101 and HLA-B*1513 is determined by the NIALR sequence motif at positions 77, 80, 81, 82, and 83 in the α1 helix. Mutation of these positions to the residues present in the alternative and nonfunctional Bw6 motif showed that the functional activity of the Bw4 epitopes of B*5101 and B*1513 is retained after substitution at positions 77, 80, and 81, but lost after substitution of position 83. Mutation of leucine to arginine at position 82 led to loss of function for B*5101 but not for B*1513. Further mutagenesis, in which B*1513 residues were replaced by their B*5101 counterparts, showed that polymorphisms in all three extracellular domains contribute to this functional difference. Prominent were positions 67 in the α1 domain, 116 in the α2 domain, and 194 in the α3 domain. Lesser contributions were made by additional positions in the α2 domain. These positions are not part of the Bw4 epitope and include residues shaping the B and F pockets that determine the sequence and conformation of the peptides bound by HLA class I molecules. This analysis shows how polymorphism at sites throughout the HLA class I molecule can influence the interaction of the Bw4 epitope with KIR3DL1. This influence is likely mediated by changes in the peptides bound, which alter the conformation of the Bw4 epitope.
A Novel Role of CD30L/CD30 Signaling by T-T Cell Interaction in Th1 Response against Mycobacterial Infection
Authors: Tang Ce , Yamada Hisakata , Shibata Kensuke , Muta Hiromi , Wajjwalku Worawidh , Podack Eckhard R. , Yoshikai Yasunobu
Published: April 2014

A CD30 ligand (CD30L, CD153) is a type II membrane-associated glycoprotein belonging to the TNF family. To illustrate the potential role of CD30L in CD4+ Th1 cell responses, we investigated the fate of Ag-specific CD4+ T cells in CD30L-deficient (CD30L−/−) mice after Mycobacterium bovis bacillus Calmette-Guérin (BCG) infection. The number of bacteria was significantly higher in organs of CD30L−/− mice than in wild-type (WT) mice 4 wk postinfection. The numbers of purified protein derivative- or Ag85B-specific-IFN-γ-producing-CD4+ T cells in spleen, lung, or peritoneal exudate cells were significantly fewer in CD30L−/− mice than in WT mice. During the infection, CD30L was expressed mainly by CD44+CD3+CD4+ T cells but not by CD3+CD8+ T cells, B cells, dendritic cells, or macrophages. Costimulation with agonistic anti-CD30 mAb or coculturing with CD30L-transfected P815 cells restored IFN-γ production by CD4+ T cells from BCG-infected CD30L−/− mice. Coculturing with CD30L+/+CD4+ T cells from BCG-infected WT mice also restored the number of IFN-γ+CD30L−/−CD4+ T cells. When transferred into the CD30L+/+ mice, Ag-specific donor CD30L−/− CD4+ T cells capable of producing IFN-γ were restored to the compared level seen in CD30L+/+ CD4+ T cells on day 10 after BCG infection. When naive CD30L+/+ T cells were transferred into CD30L−/− mice, IFN-γ-producing-CD4+ Th1 cells of donor origin were normally generated following BCG infection, and IFN-γ-producing-CD30L−/−CD4+ Th1 cells of host origin were partly restored. These results suggest that CD30L/CD30 signaling executed by CD30+ T-CD30L+ T cell interaction partly play a critical role in augmentation of Th1 response capable of producing IFN-γ against BCG infection.
Tuberculosis Triggers a Tissue-Dependent Program of Differentiation and Acquisition of Effector Functions by Circulating Monocytes
Authors: Sköld Markus , Behar Samuel M.
Published: April 2014

The origin and function of the different myeloid cell subsets that appear in the lung during pulmonary tuberculosis are unknown. Herein we show that adoptively transferred monocytes give rise to many of the macrophage and dendritic cell (DC) subsets that appear following aerosol infection with virulent Mycobacterium tuberculosis. Monocyte differentiation in infected peripheral tissue is surprisingly heterogeneous and results in the formation of five distinct myeloid subsets, including both classically activated macrophages, that produce inducible NO synthase via an IFN-γ-dependent mechanism, and DC. In contrast, monocytes recruited to draining pulmonary lymph nodes are functionally different and acquire a mature DC phenotype. Thus, while monocytes are recruited to the lungs of uninfected mice, their differentiation and acquisition of myeloid effector functions are dramatically altered in the presence of inflammation and bacteria and are dependent on tissue localization. Therefore, our results support a model in which recruited monocytes are well poised to influence multiple aspects of host immunity to infections in the lungs. This report provides the first direct evidence for monocyte differentiation into both the macrophage and DC lineages in vivo following infection with a live human pathogen.
Ly49h-Deficient C57BL/6 Mice: A New Mouse Cytomegalovirus-Susceptible Model Remains Resistant to Unrelated Pathogens Controlled by the NK Gene Complex
Authors: Fodil-Cornu Nassima , Lee Seung-Hwan , Belanger Simon , Makrigiannis Andrew P. , Biron Christine A. , Buller R. Mark , Vidal Silvia M.
Published: April 2014

Cmv1 was the first mouse cytomegalovirus (MCMV) resistance locus identified in C57BL/6 mice. It encodes Ly49H, a NK cell-activating receptor that specifically recognizes the m157 viral protein at the surface of MCMV-infected cells. To dissect the effect of the Ly49h gene in host-pathogen interactions, we generated C57BL/6 mice lacking the Ly49h region. We found that 36 h after MCMV infection, the lack of Ly49h resulted in high viral replication in the spleen and dramatically enhanced proinflammatory cytokine production in the serum and spleen. At later points in time, we observed that MCMV induced a drastic loss in CD8+ T cells in B6.Ly49h−/− mice, probably reflecting severe histological changes in the spleen. Overall, our results indicate that Ly49H+ NK cells contain a systemic production of cytokines that may contribute to the MCMV-induced pathology and play a central role in maintaining normal spleen cell microarchitecture. Finally, we tested the ability of B6.Ly49h−/− mice to control replication of Leishmania major and ectromelia virus. Resistance to these pathogens has been previously mapped within the NK gene complex. We found that the lack of Ly49H+ NK cells is not associated with an altered resistance to L. major. In contrast, absence of Ly49H+ NK cells seems to afford additional protection against ectromelia infection in C57BL/6 mice, suggesting that Ly49H may recognize ectromelia-infected cells with detrimental effects. Taken together, these results confirm the pivotal role of the Ly49H receptor during MCMV infection and open the way for further investigations in host-pathogen interactions.
Signaling through MyD88 Regulates Leukocyte Recruitment after Brain Injury
Authors: Babcock Alicia A. , Toft-Hansen Henrik , Owens Trevor
Published: April 2014

Injury to the CNS provokes an innate inflammatory reaction that engages infiltrating leukocytes with the capacity to repair and/or exacerbate tissue damage. The initial cues that orchestrate leukocyte entry remain poorly defined. We have used flow cytometry to investigate whether MyD88, an adaptor protein that transmits signals from TLRs and receptors for IL-1 and IL-18, regulates leukocyte infiltration into the stab-injured entorhinal cortex (EC) and into sites of axonal degeneration in the denervated hippocampus. We have previously established the kinetics of leukocyte entry into the denervated hippocampus. We now show that significant leukocyte entry into the EC occurs within 3–12 h of stab injury. Whereas T cells showed small, gradual increases over 8 days, macrophage infiltration was pronounced and peaked within 12–24 h. MyD88 deficiency significantly reduced macrophage and T cell recruitment to the stab-injured EC and the denervated hippocampus at 5 days post-injury. Whereas macrophage and T cell entry remained impaired into the denervated hippocampus of MyD88-deficient mice at 8 days, leukocyte infiltration into the stab-injured EC was restored to levels observed in wild-type mice. Transcripts for TNF-α, IL-1β, and CCL2, which increased >50-fold after stab injury in C57BL/6 mice at the time of peak expression, were severely reduced in injured MyD88 knockout mice. Leukocyte recruitment and gene expression were unaffected in TLR2-deficient or TLR4 mutant mice. No significant differences in gene expression were observed in mice lacking IL-1R or IL-18R. These data show that MyD88-dependent signaling mediates proinflammatory gene expression and leukocyte recruitment after CNS injury.
12/15-Lipoxygenase Regulates the Inflammatory Response to Bacterial Products In Vivo
Authors: Dioszeghy Vincent , Rosas Marcela , Maskrey Benjamin H. , Colmont Chantal , Topley Nicholas , Chaitidis Pavlos , Kühn Hartmut , Jones Simon A. , Taylor Philip R. , O'Donnell Valerie B.
Published: April 2014

The peritoneal macrophage (Mφ) is the site of greatest 12/15-lipoxygenase (12/15-LOX) expression in the mouse; however, its immunoregulatory role in this tissue has not been explored. Herein, we show that 12/15-LOX is expressed by 95% of resident peritoneal CD11bhigh cells, with the remaining 5% being 12/15-LOX−. 12/15-LOX+ cells are phenotypically defined by high F4/80, SR-A, and Siglec1 expression, and enhanced IL-10 and G-CSF generation. In contrast, 12/15-LOX− cells are a dendritic cell population. Resident peritoneal Mφ numbers were significantly increased in 12/15-LOX−/− mice, suggesting alterations in migratory trafficking or cell differentiation in vivo. In vitro, Mφ from 12/15-LOX−/− mice exhibit multiple abnormalities in the regulation of cytokine/growth factor production both basally and after stimulation with Staphylococcus epidermidis cell-free supernatant. Resident adherent cells from 12/15-LOX−/− mice generate more IL-1, IL-3, GM-CSF, and IL-17, but less CCL5/RANTES than do cells from wild-type mice, while Staphylococcus epidermidis cell-free supernatant-elicited 12/15-LOX−/− adherent cells release less IL-12p40, IL-12p70, and RANTES, but more GM-CSF. This indicates a selective effect of 12/15-LOX on peritoneal cell cytokine production. In acute sterile peritonitis, 12/15-LOX+ cells and LOX products were cleared, then reappeared during the resolution phase. The peritoneal lavage of 12/15-LOX−/− mice showed elevated TGF-β1, along with increased immigration of monocytes/Mφ, but decreases in several cytokines including RANTES/CCL5, MCP-1/CCL2, G-CSF, IL-12-p40, IL-17, and TNF-α. No changes in neutrophil or lymphocyte numbers were seen. In summary, endogenous 12/15-LOX defines the resident MΦ population and regulates both the recruitment of monocytes/Mφ and cytokine response to bacterial products in vivo.
Reversal of Thymic Stromal Lymphopoietin-Induced Airway Inflammation through Inhibition of Th2 Responses
Authors: Zhou Baohua , Headley Mark B. , Aye Theingi , Tocker Joel , Comeau Michael R. , Ziegler Steven F.
Published: April 2014

Lung-specific thymic stromal lymphopoietin (TSLP) expression is sufficient for the development of an asthma-like chronic airway inflammatory disease. However, the nature of the downstream pathways that regulate disease development are not known. In this study, we used IL-4- and Stat6-deficient mice to establish the role of Th2-type responses downstream of TSLP. IL-4 deficiency greatly reduced, but did not eliminate, TSLP-induced airway hyperresponsiveness, airway inflammation, eosinophilia, and goblet cell metaplasia, while Stat6 deficiency eliminated these asthma-like symptoms. We further demonstrate, using the chronic model of TSLP-mediated airway inflammation, that blockade of both IL-4 and IL-13 responses, through administration of an anti-IL-4Rα mAb, reversed asthma-like symptoms, when given to mice with established disease. Collectively these data provide insight into the pathways engaged in TSLP-driven airway inflammation and demonstrate that simultaneous blockade of IL-4 and IL-13 can reverse established airway disease, suggesting that this may be an effective approach for the therapy of Th2-mediated inflammatory respiratory disease.
Analysis of CD97 Expression and Manipulation: Antibody Treatment but Not Gene Targeting Curtails Granulocyte Migration
Authors: Veninga Henrike , Becker Susann , Hoek Robert M. , Wobus Manja , Wandel Elke , van der Kaa Jos , van der Valk Martin , de Vos Alex F. , Haase Hannelore , Owens Bronwyn , van der Poll Tom , van Lier René A. W. , Verbeek J. Sjef , Aust Gabriela , Hamann Jörg
Published: April 2014

The heptahelical receptor CD97 is a defining member of the EGF-TM7 family of adhesion class receptors. In both humans and mice, CD97 isoforms are expressed with variable numbers of tandemly arranged N-terminal epidermal growth factor-like domains that facilitate interactions with distinct cellular ligands. Results from treatment of mice with mAbs in various disease models have suggested a role for CD97 in leukocyte trafficking. Here, we aimed to thoroughly characterize the expression profile of CD97, and delineate its biological function. To this end, we applied a novel polyclonal Ab, which is the first antiserum suitable for immunohistochemistry, and combined this analysis with the study of Cd97-lacZ knock-in mice. We show that similar to the situation in humans, hematopoietic, epithelial, endothelial, muscle, and fat cells expressed CD97. Despite this broad expression pattern, the Cd97−/− mouse that we created had no overt phenotype, except for a mild granulocytosis. Furthermore, granulocyte accumulation at sites of inflammation was normal in the absence of CD97. Interestingly, application of CD97 mAbs blocked granulocyte trafficking after thioglycollate-induced peritonitis in wild-type but not in knock-out mice. Hence, we conclude that CD97 mAbs actively induce an inhibitory effect that disturbs normal granulocyte trafficking, which is not perturbed by the absence of the molecule.
αβ T Cell Receptor Transfer to γδ T Cells Generates Functional Effector Cells without Mixed TCR Dimers In Vivo
Authors: van der Veken Lars T. , Coccoris Miriam , Swart Erwin , Falkenburg J. H. Frederik , Schumacher Ton N. , Heemskerk Mirjam H. M.
Published: April 2014

The successful application of T cell-based immunotherapeutic applications depends on the availability of large numbers of T cells with the desired Ag specificity and phenotypic characteristics. Engineering of TCR-transferred T lymphocytes is an attractive strategy to obtain sufficient T cells with an Ag specificity of choice. However, the introduction of additional TCR chains into T cells leads to the generation of T cells with unknown specificity, due to the formation of mixed dimers between the endogenous and introduced TCR chains. The formation of such potentially autoaggressive T cells may be prevented by using γδ T cells as recipient cells, but the in vivo activity of such TCR-engineered γδ T cells has not been established. In the present study, we have investigated the in vivo functionality of TCR-transduced γδ T cells, in particular their Ag specific proliferative capacity, Ag specific reactivity, in vivo persistence, and their capacity to mount recall responses. The results demonstrate that αβ TCR engineering of γδ T cells forms a feasible strategy to generate Ag-specific effector T cells that do not express mixed TCR dimers. In view of increasing concerns on the potential autoimmune consequences of mixed TCR dimer formation, the testing of αβ TCR engineered γδ T cells in clinical trials seems warranted.
CCR5 Expression Levels Influence NFAT Translocation, IL-2 Production, and Subsequent Signaling Events during T Lymphocyte Activation
Authors: Camargo Jose F. , Quinones Marlon P. , Mummidi Srinivas , Srinivas Sowmya , Gaitan Alvaro A. , Begum Kazi , Jimenez Fabio , VanCompernolle Scott , Unutmaz Derya , Ahuja Seema S. , Ahuja Sunil K.
Published: April 2014

Ligands of CCR5, the major coreceptor of HIV-1, costimulate T lymphocyte activation. However, the full impact of CCR5 expression on T cell responses remains unknown. Here, we show that compared with CCR5+/+, T cells from CCR5−/− mice secrete lower amounts of IL-2, and a similar phenotype is observed in humans who lack CCR5 expression (CCR5-Δ32/Δ32 homozygotes) as well as after Ab-mediated blockade of CCR5 in human T cells genetically intact for CCR5 expression. Conversely, overexpression of CCR5 in human T cells results in enhanced IL-2 production. CCR5 surface levels correlate positively with IL-2 protein and mRNA abundance, suggesting that CCR5 affects IL-2 gene regulation. Signaling via CCR5 resulted in NFAT transactivation in T cells that was blocked by Abs against CCR5 agonists, suggesting a link between CCR5 and downstream pathways that influence IL-2 expression. Furthermore, murine T cells lacking CCR5 had reduced levels of intranuclear NFAT following activation. Accordingly, CCR5 expression also promoted IL-2-dependent events, including CD25 expression, STAT5 phosphorylation, and T cell proliferation. We therefore suggest that by influencing a NFAT-mediated pathway that regulates IL-2 production and IL-2-dependent events, CCR5 may play a critical role in T cell responses. In accord with our prior inferences from genetic-epidemiologic studies, such CCR5-dependent responses might constitute a viral entry-independent mechanism by which CCR5 may influence HIV-AIDS pathogenesis.
Genomic Instability Resulting from Blm Deficiency Compromises Development, Maintenance, and Function of the B Cell Lineage
Authors: Babbe Holger , McMenamin Jennifer , Hobeika Elias , Wang Jing , Rodig Scott J. , Reth Michael , Leder Philip
Published: April 2014

The RecQ family helicase BLM is critically involved in the maintenance of genomic stability, and BLM mutation causes the heritable disorder Bloom’s syndrome. Affected individuals suffer from a predisposition to a multitude of cancer types and an ill-defined immunodeficiency involving low serum Ab titers. To investigate its role in B cell biology, we inactivated murine Blm specifically in B lymphocytes in vivo. Numbers of developing B lymphoid cells in the bone marrow and mature B cells in the periphery were drastically reduced upon Blm inactivation. Of the major peripheral B cell subsets, B1a cells were most prominently affected. In the sera of Blm-deficient naive mice, concentrations of all Ig isotypes were low, particularly IgG3. Specific IgG Ab responses upon immunization were poor and mutant B cells exhibited a generally reduced Ab class switch capacity in vitro. We did not find evidence for a crucial role of Blm in the mechanism of class switch recombination. However, a modest shift toward microhomology-mediated switch junction formation was observed in Blm-deficient B cells. Finally, a cohort of p53-deficient, conditional Blm knockout mice revealed an increased propensity for B cell lymphoma development. Impaired cell cycle progression and survival as well as high rates of chromosomal structural abnormalities in mutant B cell blasts were identified as the basis for the observed effects. Collectively, our data highlight the importance of BLM-dependent genome surveillance for B cell immunity by ensuring proper development and function of the various B cell subsets while counteracting lymphomagenesis.
Role of TNF-α Produced by Nonantigen-Specific Cells in a Fulminant Hepatitis Mouse Model
Authors: Ito Hiroyasu , Ando Kazuki , Ishikawa Tetsuya , Saito Kuniaki , Takemura Masao , Imawari Michio , Moriwaki Hisataka , Seishima Mitsuru
Published: April 2014

In previous studies, the mechanisms of acute liver injury and virus exclusion have been examined using a model wherein HBsAg-specific CTL are injected into HBsAg transgenic (Tg) mice. The importance of the role of TNF-α in virus exclusion was shown, but its role in liver injury was unclear. We crossed the TNF-α knockout mouse and HBsAg-Tg mouse to establish the HBsAg-Tg/TNF-α KO mouse, and examined the influence of TNF-α on liver injury. The severity of liver damage, as determined by serum alanine aminotransferase activity, was ∼100 times greater in HBsAg-Tg/TNF-α+/+ than in HBsAg-Tg/TNF-α−/− mice after i.v. administration of 5 × 106 CTLs. This liver damage reached the peak of its severity within 24–48 h, and was restored 7 days later. Histopathological examination showed hepatocellular necrosis and inflammatory cell infiltrate 24 h after the CTL injection in HBsAg-Tg/TNF-α+/+ mice but not in HBsAg-Tg/TNF-α−/− mice. The liver damage was fatal for all HBsAg-Tg/TNF-α+/+ mice that received 1.5 × 107 CTLs. In contrast, 1.5 × 107 CTLs could not kill the HBsAg-Tg/TNF-α−/− mice. The TNF-α production level was enhanced after the CTL injection in not only intrahepatic macrophages but also other types of mononuclear cells from non-HBsAg-Tg/TNF-α+/+ mice. An adoptive transfer examination revealed that severe liver damage occurred in HBsAg-Tg/TNF-α−/− mice that had received mononuclear cells from TNF-α+/+ mice. In conclusion, the present study provides evidence that TNF-α produced by intrahepatic non-Ag-specific inflammatory cells is critical in the development of lethal necroinflammatory liver disease.
Two Structurally Different Rituximab-Specific CD20 Mimotope Peptides Reveal That Rituximab Recognizes Two Different CD20-Associated Epitopes
Authors: Perosa Federico , Favoino Elvira , Vicenti Chiara , Guarnera Andrea , Racanelli Vito , De Pinto Vito , Dammacco Franco
Published: April 2014

Peptide mimotopes of the CD20 epitope recognized by rituximab are useful tools for studying this therapeutic mAb’s functional properties. We previously identified two structurally different peptides that are both effective mimotopes: a 7-mer cyclic peptide (Rp15-C) bearing the antigenic motif a/sNPS that matches 170ANPS173 of the extracellular loop of CD20, and a 12-mer linear peptide (Rp5-L) containing the antigenic motif WPxWLE lacking sequence homology to CD20. In this study, we investigated whether the different structures of Rp15-C and Rp5-L reflect the mimicry of the same or different CD20 epitopes recognized by rituximab. Using immunochemical methods, we found that, like Rp15-C, Rp5-L mimics the raft-associated form of CD20 (by inhibiting rituximab binding to CD20 in vitro). Rp5-L and Rp15-C elicit, in immunized mice, anti-CD20 Abs that stain CD20+ cells with a punctate pattern similar to that of rituximab. However, only anti-Rp5-L Abs recognize denatured CD20. When phage-display peptide libraries were panned with anti-Rp5-L, phage clones were enriched that expressed the consensus qWPxwL, similar to the antigenic motif WPxWLE, but not matching a/sNPS. Finally, WPxWLE and ANPS share some, but not all, contact sites within the rituximab Ag-combining site, indicating that WPxWLE is not an exact replica of Rp15-C (or CD20) ANPS. Altogether, these results indicate that the two structurally different peptides are also conformationally different, and suggest that rituximab recognizes two different CD20-associated epitopes.
Programmed Necrotic Cell Death Induced by Complement Involves a Bid-Dependent Pathway
Authors: Ziporen Lea , Donin Natalie , Shmushkovich Taisia , Gross Atan , Fishelson Zvi
Published: April 2014

The membrane attack complex (MAC) of the complement system induces a necrotic-type cell death. Earlier findings suggested that Bcl-2 protects cells from MAC-induced necrosis. Here we examined the involvement of Bid, a proapoptotic protein, in MAC-induced cytotoxicity. Bid knockout (Bid−/−) mouse embryonic fibroblasts (MEF) and primary fibroblasts were damaged by complement but to a significantly lower extent than wild-type (WT) fibroblasts. Bid silencing with small interfering RNA duplexes led to elevated resistance of mouse fibroblasts, human K562, and Jurkat cells to lysis by complement. Bid−/− MEF were also resistant to toxic doses of streptolysin O, melittin, and A23187. Analysis of complement protein deposition on fibroblasts demonstrated that less complement C3 and C9 bound to Bid−/− than to WT cells, even though expression of the membrane complement inhibitors Crry and CD59 was relatively reduced on Bid−/− cells. Bid was rapidly cleaved in WT MEF subjected to lytic doses of MAC. Pretreatment of the cells with the pan-caspase inhibitor z-Val-Ala-Asp(OMe)-fluoromethylketone reduced Bid cleavage and cell lysis. These results indicate that complement MAC activates two cell death pathways, one involving caspases and Bid and one that is Bid-independent.