bluuebunny/crossref_metadata_2025 huggingface dataset:
Enhanced Defense against Pneumocystis carinii Mediated by a Novel Dectin-1 Receptor Fc Fusion Protein
Authors: Rapaka Rekha R. , Goetzman Eric S. , Zheng Mingquan , Vockley Jerry , McKinley Laura , Kolls Jay K. , Steele Chad
Published: April 2014

Pneumocystis carinii (PC) pneumonia is a leading opportunistic infection found among HIV-infected individuals worldwide. Although CD4+ T cell deficiency clearly correlates with susceptibility to PC pneumonia, murine models of disease indicate that PC-directed Abs may prevent infection and/or inhibit growth of existing PC within the lungs. Recognition of PC by alveolar macrophages involves the β-glucan receptor Dectin-1 and macrophage effector function against PC is enhanced by Abs derived from PC-vaccinated hosts. We developed a fusion protein consisting of the extracellular domain of Dectin-1 linked to the Fc portion of murine IgG1, which we hypothesized would enhance host recognition and opsonic phagocytosis of PC. The recombinant protein, Dectin-Fc, is dimeric and the Ag recognition site identifies β-1,3 glucan linkages specifically and with high affinity (KD = 2.03 × 10−7 M). Dectin-Fc enhances RAW264.7 macrophage recognition of the β-glucan containing particulate zymosan in an FcγRII- and FcγRIII-dependent manner and preopsonization of PC organisms with Dectin-Fc increased alveolar and peritoneal macrophage-dependent killing of PC. SCID mice treated with a replication incompetent adenoviral vector expressing Dectin-Fc had attenuated growth of PC within the lungs, overall decreased PC lung burden, and diminished correlates of PC-related lung damage relative to SCID mice receiving a control vector. These findings demonstrate that targeting PC β-glucan with Dectin-Fc enhances host recognition and clearance of PC in the absence of B and T cells, and suggest that FcγR-based targeting of PC, via cell wall carbohydrate recognition, may promote resistance against PC pneumonia in the immunodeficient host.
Staphylococcus aureus-Derived Staphopain B, a Potent Cysteine Protease Activator of Plasma Chemerin
Authors: Kulig Paulina , Zabel Brian A. , Dubin Grzegorz , Allen Samantha J. , Ohyama Takao , Potempa Jan , Handel Tracy M. , Butcher Eugene C. , Cichy Joanna
Published: April 2014

Chemerin is an attractant for cells that express the serpentine receptor CMKLR1, which include immature plasmacytoid dendritic cells (pDC) and macrophages. Chemerin circulates in the blood where it exhibits low biological activity, but upon proteolytic cleavage of its C terminus, it is converted to a potent chemoattractant. Enzymes that contribute to this conversion include host serine proteases of the coagulation, fibrinolytic, and inflammatory cascades, and it has been postulated that recruitment of pDC and macrophages by chemerin may serve to balance local tissue immune and inflammatory responses. In this work, we describe a potent, pathogen-derived proteolytic activity capable of chemerin activation. This activity is mediated by staphopain B (SspB), a cysteine protease secreted by Staphylococcus aureus. Chemerin activation is triggered by growth medium of clinical isolates of SspB-positive S. aureus, but not by that of a SspBnull mutant. C-terminal processing by SspB generates a chemerin isoform identical with the active endogenous attractant isolated from human ascites fluid. Interestingly, SspB is a potent trigger of chemerin even in the presence of plasma inhibitors. SspB may help direct the recruitment of specialized host cells, including immunoregulatory pDC and/or macrophages, contributing to the ability of S. aureus to elicit and maintain a chronic inflammatory state.
TLR4-Mediated Survival of Macrophages Is MyD88 Dependent and Requires TNF-α Autocrine Signalling
Authors: Lombardo Eleuterio , Alvarez-Barrientos Alberto , Maroto Beatriz , Boscá Lisardo , Knaus Ulla G.
Published: April 2014

Modulation of macrophage survival is a critical factor in the resolution of inflammatory responses. Exposure to LPS protects innate immune cells against apoptosis, although the precise pathways responsible for prolongation of macrophage survival remain to be fully established. The goal of this study was to characterize the mechanism of TLR4-mediated survival of murine bone marrow-derived macrophages upon M-CSF withdrawal in more detail. Using a combination of knockout mice and pharmacological inhibitors allowed us to show that TLR4 and TLR2 stimulation promotes long-term survival of macrophages in a MyD88-, PI3K-, ERK-, and NF-κB-dependent manner. LPS-induced long-term, but not short-term, survival requires autocrine signaling via TNF-α and is facilitated by a general cytoprotective program, similar to that mediated by M-CSF. TLR4-mediated macrophage survival is accompanied by a remarkable up-regulation of specific cell surface markers, suggesting that LPS stimulation leads to the differentiation of macrophages toward a mixed macrophage/dendritic cell-like phenotype.
MyD88- and Bruton’s Tyrosine Kinase-Mediated Signals Are Essential for T Cell-Independent Pathogen-Specific IgM Responses
Authors: Alugupalli Kishore R. , Akira Shizuo , Lien Egil , Leong John M.
Published: April 2014

Bacteremia is one of the leading causes of death by infectious disease. To understand the immune mechanisms required for the rapid control of bacteremia, we studied Borrelia hermsii, a bacterial pathogen that colonizes the blood stream of humans and rodents to an extremely high density. A T cell-independent IgM response is essential and sufficient for controlling B. hermsii bacteremia. Mice deficient in Bruton’s tyrosine kinase (Btk), despite their known defect in BCR signaling, generated B. hermsii-specific IgM and resolved bacteremia, suggesting that an alternative activation or costimulatory pathway remained functional for T cell-independent B cells in Btk−/− mice. B. hermsii contains putative ligands for TLRs, and we found that mice deficient in TLR1, TLR2, or the TLR adaptor MyD88 generated anti-B. hermsii IgM with delayed kinetics and suffered more severe episodes of bacteremia. In striking contrast to the anti-B. hermsii IgM response in mice deficient only in Btk, mice deficient in both Btk and MyD88 were entirely incapable of generating B. hermsii-specific Ab or resolving bacteremia. The response to a T cell-dependent model Ag was unaffected in Btk−/− × MyD88−/− mice. These results suggest that MyD88 specifically promotes T cell-independent BCR signaling and that, in the absence of Btk, this TLR-mediated stimulation is a required component of this signal.
Epitope Cross-Reactivity Frequently Differs between Central and Effector Memory HIV-Specific CD8+ T Cells
Authors: McKinnon Lyle R. , Ball T. Blake , Wachihi Charles , McLaren Paul J. , Waruk Jillian L. M. , Mao Xiaojuan , Ramdahin Sue , Anzala A. Omu , Kamene Jane , Luo Ma , Fowke Keith R. , Plummer Francis A.
Published: April 2014

HIV diversity may limit the breadth of vaccine coverage due to epitope sequence differences between strains. Although amino acid substitutions within CD8+ T cell HIV epitopes can result in complete or partial abrogation of responses, this has primarily been demonstrated in effector CD8+ T cells. In an HIV-infected Kenyan cohort, we demonstrate that the cross-reactivity of HIV epitope variants differs dramatically between overnight IFN-γ and longer-term proliferation assays. For most epitopes, particular variants (not the index peptide) were preferred in proliferation in the absence of corresponding overnight IFN-γ responses and in the absence of the variant in the HIV quasispecies. Most proliferating CD8+ T cells were polyfunctional via cytokine analyses. A trend to positive correlation was observed between proliferation (but not IFN-γ) and CD4 counts. We present findings relevant to the assessment of HIV vaccine candidates and toward a better understanding of how viral diversity is tolerated by central and effector memory CD8+ T cells.
Role of the GALT in Scrapie Agent Neuroinvasion from the Intestine
Authors: Glaysher Bridget R. , Mabbott Neil A.
Published: April 2014

Following oral exposure, some transmissible spongiform encephalopathy (TSE) agents accumulate first upon follicular dendritic cells (FDCs) in the GALT. Studies in mice have shown that this accumulation is obligatory for the efficient delivery of the TSE agent to the brain. However, which GALTs are crucial for disease pathogenesis is uncertain. Mice deficient in specific GALT components were used here to determine their separate involvement in scrapie agent neuroinvasion from the intestine. In the combined absence of the GALTs and FDCs (lymphotoxin (LT)α−/− mice and LTβ−/− mice), scrapie agent transmission was blocked. When FDC maturation was induced in remaining lymphoid tissues, mice that lacked both Peyer’s patches (PPs) and mesenteric lymph nodes (wild-type (WT)→LTα−/− mice) or PPs alone (WT→LTβ−/− mice) remained refractory to disease, demonstrating an important role for the PPs. Although early scrapie agent accumulation also occurs within the mesenteric lymph nodes, their presence in WT→LTβ−/− mice did not restore disease susceptibility. We have also shown that isolated lymphoid follicles (ILFs) are important novel sites of TSE agent accumulation in the intestine. Mice that lacked PPs but contained numerous FDC-containing mature ILFs succumbed to scrapie at similar times to control mice. Because the formation and maturation status of ILFs is inducible and influenced by the gut flora, our data suggest that such factors could dramatically affect susceptibility to orally acquired TSE agents. In conclusion, these data demonstrate that following oral exposure TSE agent accumulation upon FDCs within lymphoid tissue within the intestine itself is critically required for efficient neuroinvasion.
IL-17-Mediated Regulation of Innate and Acquired Immune Response against Pulmonary Mycobacterium bovis Bacille Calmette-Guérin Infection
Authors: Umemura Masayuki , Yahagi Ayano , Hamada Satoru , Begum Mst Dilara , Watanabe Hisami , Kawakami Kazuyoshi , Suda Takashi , Sudo Katsuko , Nakae Susumu , Iwakura Yoichiro , Matsuzaki Goro
Published: April 2014

IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against intracellular bacterial infection remains unclear. In the present study, we demonstrate that IL-17 is an important cytokine not only in the early neutrophil-mediated inflammatory response, but also in T cell-mediated IFN-γ production and granuloma formation in response to pulmonary infection by Mycobacterium bovis bacille Calmette-Guérin (BCG). IL-17 expression in the BCG-infected lung was detected from the first day after infection and the expression depended on IL-23. Our observations indicated that γδ T cells are a primary source of IL-17. Lung-infiltrating T cells of IL-17-deficient mice produced less IFN-γ in comparison to those from wild-type mice 4 wk after BCG infection. Impaired granuloma formation was also observed in the infected lungs of IL-17-deficient mice, which is consistent with the decreased delayed-type hypersensitivity response of the infected mice against mycobacterial Ag. These data suggest that IL-17 is an important cytokine in the induction of optimal Th1 response and protective immunity against mycobacterial infection.
The Granulocyte Receptor Carcinoembryonic Antigen-Related Cell Adhesion Molecule 3 (CEACAM3) Directly Associates with Vav to Promote Phagocytosis of Human Pathogens
Authors: Schmitter Tim , Pils Stefan , Sakk Vadim , Frank Ronald , Fischer Klaus-Dieter , Hauck Christof R.
Published: April 2014

The human granulocyte-specific receptor carcinoembryonic antigen-related cell adhesion molecule (CEACAM)3 is critically involved in the opsonin-independent recognition of several bacterial pathogens. CEACAM3-mediated phagocytosis depends on the integrity of an ITAM-like sequence within the cytoplasmic domain of CEACAM3 and is characterized by rapid stimulation of the GTPase Rac. By performing a functional screen with CEACAM3-expressing cells, we found that overexpression of a dominant-negative form of the guanine nucleotide exchange factor Vav, but not the dominant-negative versions SWAP70, Dock2, or ELMO1 interfered with CEACAM3-initiated phagocytosis. Moreover, small interfering RNA-mediated silencing of Vav reduced uptake and abrogated the stimulation of Rac in response to bacterial CEACAM3 engagement. In Vav1/Vav2-deficient cells, CEACAM3-mediated internalization was only observed after re-expression of Vav. Vav colocalized with CEACAM3 upon bacterial infection, coimmunoprecipitated in a complex with CEACAM3, and the Vav Src homology 2 domain directly associated with phosphorylated Tyr230 of CEACAM3. In primary human granulocytes, TAT-mediated transduction of dominant-negative Vav, but not SWAP70, severely impaired the uptake of CEACAM3-binding bacteria. These data support the view that, different from canonical ITAM signaling, the CEACAM3 ITAM-like sequence short-wires bacterial recognition and Rac stimulation via a direct association with Vav to promote rapid phagocytosis and elimination of CEACAM-binding human pathogens.
H2-M3-Restricted CD8+ T Cells Induced by Peptide-Pulsed Dendritic Cells Confer Protection against Mycobacterium tuberculosis
Authors: Doi Takehiko , Yamada Hisakata , Yajima Toshiki , Wajjwalku Worawidh , Hara Toshiro , Yoshikai Yasunobu
Published: April 2014

One of the oligopolymorphic MHC class Ib molecules, H2-M3, presents N-formylated peptides derived from bacteria. In this study, we tested the ability of an H2-M3-binding peptide, TB2, to induce protection in C57BL/6 mice against Mycobacterium tuberculosis. Immunization with bone marrow-derived dendritic cell (BMDC) pulsed with TB2 or a MHC class Ia-binding peptide, MPT64190–198 elicited an expansion of Ag-specific CD8+ T cells in the spleen and the lung. The number of TB2-specific CD8+ T cells reached a peak on day 6, contracted with kinetics similar to MPT64190–198-specific CD8+ T cells and was maintained at an appreciable level for at least 60 days. The TB2-specific CD8+ T cells produced less effector cytokines but have stronger cytotoxic activity than MPT64190–198-specific CD8+ T cells. Mice immunized with TB2-pulsed BMDC as well as those with MPT64190–198-pulsed BMDC showed significant protection against an intratracheal challenge with M. tuberculosis H37Rv. However, histopathology of the lung in mice immunized with TB2-pulsed BMDC was different from mice immunized with MPT64190–198-pulsed BMDC. Our results suggest that immunization with BMDC pulsed with MHC class Ib-restricted peptides would be a useful vaccination strategy against M. tuberculosis.
Catalase Overexpression Fails to Attenuate Allergic Airways Disease in the Mouse
Authors: Reynaert Niki L. , Aesif Scott W. , McGovern Toby , Brown Amy , Wouters Emiel F. M. , Irvin Charles G. , Janssen-Heininger Yvonne M. W.
Published: April 2014

Oxidative stress is a hallmark of asthma, and increased levels of oxidants are considered markers of the inflammatory process. Most studies to date addressing the role of oxidants in the etiology of asthma were based on the therapeutic administration of low m.w. antioxidants or antioxidant mimetic compounds. To directly address the function of endogenous hydrogen peroxide in the pathophysiology of allergic airway disease, we comparatively evaluated mice systemically overexpressing catalase, a major antioxidant enzyme that detoxifies hydrogen peroxide, and C57BL/6 strain matched controls in the OVA model of allergic airways disease. Catalase transgenic mice had 8-fold increases in catalase activity in lung tissue, and had lowered DCF oxidation in tracheal epithelial cells, compared with C57BL/6 controls. Despite these differences, both strains showed similar increases in OVA-specific IgE, IgG1, and IgG2a levels, comparable airway and tissue inflammation, and identical increases in procollagen 1 mRNA expression, following sensitization and challenge with OVA. Unexpectedly, mRNA expression of MUC5AC and CLCA3 genes were enhanced in catalase transgenic mice, compared with C57BL/6 mice subjected to Ag. Furthermore, when compared with control mice, catalase overexpression increased airway hyperresponsiveness to methacholine both in naive mice as well as in response to Ag. In contrast to the prevailing notion that hydrogen peroxide is positively associated with the etiology of allergic airways disease, the current findings suggest that endogenous hydrogen peroxide serves a role in suppressing both mucus production and airway hyperresponsiveness.
Y402H Polymorphism of Complement Factor H Affects Binding Affinity to C-Reactive Protein
Authors: Laine Matti , Jarva Hanna , Seitsonen Sanna , Haapasalo Karita , Lehtinen Markus J. , Lindeman Nina , Anderson Don H. , Johnson Patrick T. , Järvelä Irma , Jokiranta T. Sakari , Hageman Gregory S. , Immonen Ilkka , Meri Seppo
Published: April 2014

Complement factor H (FH) is an important regulator of the alternative complement pathway. The Y402H polymorphism within the seventh short consensus repeat of FH was recently shown to be associated with age-related macular degeneration, the most common cause of irreversible blindness in the Western world. We examined the effects of this polymorphism on various FH functions. FH purified from sera of age-related macular degeneration patients homozygous for the FH402H variant showed a significantly reduced binding to C-reactive protein (CRP), an acute phase protein, as compared with FH derived from unaffected controls homozygous for the FH402Y variant. Strongly reduced binding to CRP was also observed with a recombinant fragment of FH (short consensus repeat 5–7) containing the same amino acid change. Because the interaction of CRP and FH promotes complement-mediated clearance of cellular debris in a noninflammatory fashion, we propose that the reduced binding of FH402H to CRP could lead to an impaired targeting of FH to cellular debris and a reduction in debris clearance and enhanced inflammation along the macular retinal pigmented epithelium-choroid interface in individuals with age-related macular degeneration.
A Novel Pathway That Regulates Inflammatory Disease in the Respiratory Tract
Authors: Niu Naiqian , Le Goff Marc K. , Li Fangyong , Rahman Marina , Homer Robert J. , Cohn Lauren
Published: April 2014

In animals with acute airway inflammation followed by repeated exposure to inhaled Ag, inflammation wanes over time and thus limits the study of chronic airway inflammatory diseases such as asthma. We developed a model of airway inflammation and inhalational exposure to investigate regulatory pathways in the respiratory tract. We show that Th1- and Th2-induced airway inflammation followed by repeated exposure to inhaled Ag leads to a state of immunosuppression. Challenge of these animals with a marked population of TCR transgenic effector Th1 or Th2 cells results in a striking inhibition of inflammation and effector Th cells. In Th2 models, airway hyperresponsiveness, mucus, and eosinophilia are reduced. The inhibitory effects observed are Ag nonspecific, can be induced in lymphocyte-deficient mice, and are associated with a population of TGF-β1-expressing macrophages. Induction of this pathway may offer potent localized treatment of chronic T cell-mediated respiratory illnesses and provide insights into the development of such diseases.
A Cardiovascular Drug Rescues Mice from Lethal Sepsis by Selectively Attenuating a Late-Acting Proinflammatory Mediator, High Mobility Group Box 1
Authors: Li Wei , Li Jianhua , Ashok Mala , Wu Rongqian , Chen Dazhi , Yang Lihong , Yang Huan , Tracey Kevin J. , Wang Ping , Sama Andrew E. , Wang Haichao
Published: April 2014

The pathogenesis of sepsis is mediated in part by bacterial endotoxin, which stimulates macrophages/monocytes to sequentially release early (e.g., TNF, IL-1, and IFN-γ) and late (e.g., high mobility group box 1 (HMGB1) protein) proinflammatory cytokines. The recent discovery of HMGB1 as a late mediator of lethal sepsis has prompted investigation for development of new experimental therapeutics. We found that many steroidal drugs (such as dexamethasone and cortisone) and nonsteroidal anti-inflammatory drugs (such as aspirin, ibuprofen, and indomethacin) failed to influence endotoxin-induced HMGB1 release even at superpharmacological concentrations (up to 10–25 μM). However, several steroid-like pigments (tanshinone I, tanshinone IIA, and cryptotanshinone) of a popular Chinese herb, Danshen (Salvia miltiorrhiza), dose dependently attenuated endotoxin-induced HMGB1 release in macrophage/monocyte cultures. A water-soluble tanshinone IIA sodium sulfonate derivative (TSNIIA-SS), which has been widely used as a Chinese medicine for patients with cardiovascular disorders, selectively abrogated endotoxin-induced HMGB1 cytoplasmic translocation and release in a glucocorticoid receptor-independent manner. Administration of TSNIIA-SS significantly protected mice against lethal endotoxemia and rescued mice from lethal sepsis even when the first dose was given 24 h after the onset of sepsis. The therapeutic effects were partly attributable to attenuation of systemic accumulation of HMGB1 (but not TNF and NO) and improvement of cardiovascular physiologic parameters (e.g., decrease in total peripheral vascular resistance and increase in cardiac stroke volume) in septic animals. Taken together, these data re-enforce the pathogenic role of HMGB1 in lethal sepsis, and support a therapeutic potential for TSNIIA-SS in the treatment of human sepsis.
VCAM-1 Activation of Endothelial Cell Protein Tyrosine Phosphatase 1B
Authors: Deem Tracy L. , Abdala-Valencia Hiam , Cook-Mills Joan M.
Published: April 2014

Lymphocytes migrate from the blood into tissue by binding to and migrating across endothelial cells. One of the endothelial cell adhesion molecules that mediate lymphocyte binding is VCAM-1. We have reported that binding to VCAM-1 activates endothelial cell NADPH oxidase for the generation of reactive oxygen species (ROS). The ROS oxidize and stimulate an increase in protein kinase C (PKC)α activity. Furthermore, these signals are required for VCAM-1-dependent lymphocyte migration. In this report, we identify a role for protein tyrosine phosphatase 1B (PTP1B) in the VCAM-1 signaling pathway. In primary cultures of endothelial cells and endothelial cell lines, Ab cross-linking of VCAM-1 stimulated an increase in serine phosphorylation of PTP1B, the active form of PTP1B. Ab cross-linking of VCAM-1 also increased activity of PTP1B. This activation of PTP1B was downstream of NADPH oxidase and PKCα in the VCAM-1 signaling pathway as determined with pharmacological inhibitors and antisense approaches. In addition, during VCAM-1 signaling, ROS did not oxidize endothelial cell PTP1B. Instead PTP1B was activated by serine phosphorylation. Importantly, inhibition of PTP1B activity blocked VCAM-1-dependent lymphocyte migration across endothelial cells. In summary, VCAM-1 activates endothelial cell NADPH oxidase to generate ROS, resulting in oxidative activation of PKCα and then serine phosphorylation of PTP1B. This PTP1B activity is necessary for VCAM-1-dependent transendothelial lymphocyte migration. These data show, for the first time, a function for PTP1B in VCAM-1-dependent lymphocyte migration.
The Src Family Kinases Hck and Fgr Regulate Neutrophil Responses to N-Formyl-Methionyl-Leucyl-Phenylalanine
Authors: Fumagalli Laura , Zhang Hong , Baruzzi Anna , Lowell Clifford A. , Berton Giorgio
Published: April 2014

The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck−/−fgr−/− neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck−/−fgr−/− mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-μm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-μm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck−/−fgr−/− neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca2+ concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck−/−fgr−/− neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck−/−fgr−/− neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.
Variable Requirement of Dendritic Cells for Recruitment of NK and T Cells to Different TLR Agonists
Authors: Uchida Takefumi , Scumpia Philip O. , Murasko Donna M. , Seki Shuhji , Woulfe Susan , Clare-Salzler Michael J. , Moldawer Lyle L.
Published: April 2014

TLRs initiate the host immune response to microbial pathogens by activating cells of the innate immune system. Dendritic cells (DCs) can be categorized into two major groups, conventional DCs (including CD8+ and CD8− DCs) and plasmacytoid DCs. In mice, these subsets of DCs express a variety of TLRs, with conventional DCs responding in vitro to predominantly TLR3, TLR4, TLR5, and TLR9 ligands, and plasmacytoid DCs responding mainly to TLR7 and TLR9 ligands. However, the in vivo requirement of DCs to initiate immune responses to specific TLR agonists is not fully known. Using mice depleted of >90% of CD11c+ MHC class II+ DCs, we demonstrate that cellular recruitment, including CD4+ T cell and CX5+DX5+ NK cell recruitment to draining lymph nodes following the footpad administration of TLR4 and TLR5 agonists, is dramatically decreased upon reduction of DC numbers, but type I IFN production can partially substitute for DCs in response to TLR3 and TLR7 agonists. Interestingly, TLR ligands can activate T cells and NK cells in the draining lymph nodes, even with reduced DC numbers. The findings reveal considerable plasticity in the response to TLR agonists, with TLR4 and TLR5 agonists sharing the requirement of DCs for subsequent lymph node recruitment of NK and T cells.
Resolvin E1 Selectively Interacts with Leukotriene B4 Receptor BLT1 and ChemR23 to Regulate Inflammation
Authors: Arita Makoto , Ohira Taisuke , Sun Yee-Ping , Elangovan Siva , Chiang Nan , Serhan Charles N.
Published: April 2014

Resolvin E1 (RvE1) is a potent anti-inflammatory and proresolving mediator derived from omega-3 eicosapentaenoic acid generated during the resolution phase of inflammation. RvE1 possesses a unique structure and counterregulatory actions that stop human polymorphonuclear leukocyte (PMN) transendothelial migration and PMN infiltration in several murine inflammatory models. To examine the mechanism(s) underlying anti-inflammatory actions on PMNs, we prepared [3H]RvE1 and characterized its interactions with human PMN. Results with membrane fractions of human PMN demonstrated specific binding with a Kd of 48.3 nM. [3H]RvE1 specific binding to human PMN was displaced by leukotriene B4 (LTB4) and LTB4 receptor 1 (BLT1) antagonist U-75302, but not by chemerin peptide, a ligand specific for another RvE1 receptor ChemR23. Recombinant human BLT1 gave specific binding with [3H]RvE1 with a Kd of 45 nM. RvE1 selectively inhibited adenylate cyclase with BLT1, but not with BLT2. In human PBMC, RvE1 partially induced calcium mobilization, and blocked subsequent stimulation by LTB4. RvE1 also attenuated LTB4-induced NF-κB activation in BLT1-transfected cells. In vivo anti-inflammatory actions of RvE1 were sharply reduced in BLT1 knockout mice when given at low doses (100 ng i.v.) in peritonitis. In contrast, RvE1 at higher doses (1.0 μg i.v.) significantly reduced PMN infiltration in a BLT1-independent manner. These results indicate that RvE1 binds to BLT1 as a partial agonist, potentially serving as a local damper of BLT1 signals on leukocytes along with other receptors (e.g., ChemR23-mediated counterregulatory actions) to mediate the resolution of inflammation.
Relevance of C1 and C2 Epitopes for Hemopoietic Stem Cell Transplantation: Role for Sequential Acquisition of HLA-C-Specific Inhibitory Killer Ig-Like Receptor
Authors: Fischer Johannes C. , Ottinger Hellmut , Ferencik Stanislav , Sribar Martina , Punzel Michael , Beelen Dietrich W. , Schwan M. Alexander , Grosse-Wilde Hans , Wernet Peter , Uhrberg Markus
Published: April 2014

Killer Ig-like receptors (KIR) and HLA class I ligands were studied in unrelated hemopoietic stem cell transplantation for chronic myeloid leukemia (n = 108). Significantly improved overall survival was observed in patients, which were homozygous for HLA-C-encoded group 1 (C1) ligands compared with those with group 2 (C2) ligands. Favorable outcome in the former patient group was an early effect that was highly significant in patients transplanted with G-CSF-mobilized peripheral blood and patients with advanced disease stages. In contrast, presence of C1 ligands in the donor was associated with significantly reduced patient survival. The differential roles of the two HLA-C ligands are explained in the context of a biased NK cell reconstitution, which is generally dominated by the presence of C1- but absence of C2-specific NK cells. The clinical observations are corroborated by in vitro experiments showing that NK cells derived from hemopoietic progenitor cells generally acquire the C1-specific inhibitory KIR2DL2/3 at earlier time points and with higher frequency than the C2-specific KIR2DL1. These findings define a novel determinant for understanding the role of NK cells in clinical hemopoietic stem cell transplantation.
Fever-Like Hyperthermia Controls T Lymphocyte Persistence by Inducing Degradation of Cellular FLIPshort
Authors: Meinander Annika , Söderström Thomas S. , Kaunisto Aura , Poukkula Minna , Sistonen Lea , Eriksson John E.
Published: April 2014

Fever has a major impact on immune responses by modulating survival, proliferation, and endurance of lymphocytes. Lymphocyte persistence in turn is determined by the equilibrium between death and survival-promoting factors that regulate death receptor signaling in these cells. A potential integrator of death receptor signaling is the caspase-8 inhibitor c-FLIP, the expression of which is dynamically regulated, either rapidly induced or down-regulated. In this study, we show in activated primary human T lymphocytes that hyperthermia corresponding to fever triggered down-regulation of both c-FLIP-splicing variants, c-FLIPshort (c-FLIPS) and c-FLIPlong, with consequent sensitization to apoptosis mediated by CD95 (Fas/APO-1). The c-FLIP down-regulation and subsequent sensitization was specific for hyperthermic stress. Additionally, we show that the hyperthermia-mediated down-regulation was due to increased ubiquitination and proteasomal degradation of c-FLIPS, the stability of which we have shown to be regulated by its C-terminal splicing tail. Furthermore, the induced sensitivity to CD95 ligation was independent of heat shock protein 70, as thermotolerant cells, expressing substantially elevated levels of heat shock protein 70, were not rescued from the effect of hyperthermia-mediated c-FLIP down-regulation. Our findings indicate that fever significantly influences the rate of lymphocyte elimination through depletion of c-FLIPS. Such a general regulatory mechanism for lymphocyte removal has broad ramifications for fever-mediated regulation of immune responses.
Disruption of CD36 Impairs Cytokine Response to Plasmodium falciparum Glycosylphosphatidylinositol and Confers Susceptibility to Severe and Fatal Malaria In Vivo
Authors: Patel Samir N. , Lu Ziyue , Ayi Kodjo , Serghides Lena , Gowda D. Channe , Kain Kevin C.
Published: April 2014

CD36 is a scavenger receptor that has been implicated in malaria pathogenesis as well as innate defense against blood-stage infection. Inflammatory responses to Plasmodium falciparum GPI (pfGPI) anchors are believed to play an important role in innate immune response to malaria. We investigated the role of CD36 in pfGPI-induced MAPK activation and proinflammatory cytokine secretion. Furthermore, we explored the role of this receptor in an experimental model of acute malaria in vivo. We demonstrate that ERK1/2, JNK, p38, and c-Jun became phosphorylated in pfGPI-stimulated macrophages. In contrast, pfGPI-induced phosphorylation of JNK, ERK1/2, and c-Jun was reduced in Cd36−/− macrophages and Cd36−/− macrophages secreted significantly less TNF-α in response to pfGPI than their wild-type counterparts. In addition, we demonstrate a role for CD36 in innate immune response to malaria in vivo. Compared with wild-type mice, Cd36−/− mice experienced more severe and fatal malaria when challenged with Plasmodium chabaudi chabaudi AS. Cd36−/− mice displayed a combined defect in cytokine induction and parasite clearance with a dysregulated cytokine response to infection, earlier peak parasitemias, higher parasite densities, and higher mortality rates than wild-type mice. These results provide direct evidence that pfGPI induces TNF-α secretion in a CD36-dependent manner and support a role for CD36 in modulating host cytokine response and innate control of acute blood-stage malaria infection in vivo.
T Cell TRAIL Promotes Murine Lupus by Sustaining Effector CD4 Th Cell Numbers and by Inhibiting CD8 CTL Activity
Authors: Rus Violeta , Nguyen Vinh , Puliaev Roman , Puliaeva Irina , Zernetkina Valentina , Luzina Irina , Papadimitriou John C. , Via Charles S.
Published: April 2014

T cells play an essential role in driving humoral autoimmunity in lupus. Molecules such as TRAIL exhibit strong T cell modulatory effects and are up-regulated in lupus, raising the possibility that they may influence disease severity. To address this possibility, we examined the role of TRAIL expression on pathogenic T cells in an induced model of murine lupus, the parent-into-F1 (P→F1) model of chronic graft-vs-host disease (GVHD), using wild-type or TRAIL-deficient donor T cells. Results were compared with mice undergoing suppressive acute GVHD. Although chronic GVHD mice exhibited less donor T cell TRAIL up-regulation and IFN-α-inducible gene expression than acute GVHD mice, donor CD4+ T cell TRAIL expression in chronic GVHD was essential for sustaining effector CD4+ Th cell numbers, for sustaining help to B cells, and for more severe lupus-like renal disease development. Conversely, TRAIL expression on donor CD8+ T cells had a milder, but significant down-regulatory effect on CTL effector function, affecting the perforin/granzyme pathway and not the Fas ligand pathway. These results indicate that, in this model, T cell-expressed TRAIL exacerbates lupus by the following: 1) positively regulating CD4+ Th cell numbers, thereby sustaining T cell help for B cells, and 2) to a lesser degree by negatively regulating perforin-mediated CD8+ CTL killing that could potentially eliminate activated autoreactive B cells.
Cutting Edge: IL-2 Is Essential for TGF-β-Mediated Induction of Foxp3+ T Regulatory Cells
Authors: Davidson Todd S. , DiPaolo Richard J. , Andersson John , Shevach Ethan M.
Published: April 2014

TGF-β is a pluripotent cytokine that is capable of inducing the expression of Foxp3 in naive T lymphocytes. TGF-β-induced cells are phenotypically similar to thymic-derived regulatory T cells in that they are anergic and suppressive. We have examined the cytokine and costimulatory molecule requirements for TGF-β-mediated induction and maintenance of Foxp3 by CD4+Foxp3− cells. IL-2 plays a non-redundant role in TGF-β-induced Foxp3 expression. Other common γ-chain-utilizing cytokines were unable to induce Foxp3 expression in IL-2-deficient T cells. The role of CD28 in the induction of Foxp3 was solely related to its capacity to enhance the endogenous production of IL-2. Foxp3 expression was stable in vitro and in vivo in the absence of IL-2. As TGF-β-induced T regulatory cells can be easily grown in vitro, they may prove useful for the treatment of autoimmune diseases, for the prevention of graft rejection, and graft versus host disease.
Follicular Lymphoma Intratumoral CD4+CD25+GITR+ Regulatory T Cells Potently Suppress CD3/CD28-Costimulated Autologous and Allogeneic CD8+CD25− and CD4+CD25− T Cells
Authors: Hilchey Shannon P. , De Asit , Rimsza Lisa M. , Bankert Richard B. , Bernstein Steven H.
Published: April 2014

Regulatory T cells (TR) play a critical role in the inhibition of self-reactive immune responses and as such have been implicated in the suppression of tumor-reactive effector T cells. In this study, we demonstrate that follicular lymphoma (FL)-infiltrating CD8+ and CD4+ T cells are hyporesponsive to CD3/CD28 costimulation. We further identify a population of FL-infiltrating CD4+CD25+GITR+ TR that are significantly overrepresented within FL nodes (FLN) compared with that seen in normal (nonmalignant, nonlymphoid hyperplastic) or reactive (nonmalignant, lymphoid hyperplastic) nodes. These TR actively suppress both the proliferation of autologous nodal CD8+CD25− and CD4+CD25− T cells, as well as cytokine production (IFN-γ, TNF-α and IL-2), after CD3/CD28 costimulation. Removal of these cells in vitro by CD25+ magnetic bead depletion restores both the proliferation and cytokine production of the remaining T cells, demonstrating that FLN T cell hyporesponsiveness is reversible. In addition to suppressing autologous nodal T cells, these TR are also capable of suppressing the proliferation of allogeneic CD8+CD25− and CD4+CD25− T cells from normal lymph nodes as well as normal donor PBL, regardless of very robust stimulation of the target cells with plate-bound anti-CD3 and anti-CD28 Abs. The allogeneic suppression is not reciprocal, as equivalent numbers of CD25+FOXP3+ cells derived from either normal lymph nodes or PBL are not capable of suppressing allogeneic CD8+CD25− and CD4+CD25− T cells, suggesting that FLN TR are more suppressive than those derived from nonmalignant sources. Lastly, we demonstrate that inhibition of TGF-β signaling partially restores FLN T cell proliferation suggesting a mechanistic role for TGF-β in FLN TR-mediated suppression.
Functional Plasticity in Memory T Helper Cell Responses
Authors: Krawczyk Connie M. , Shen Hao , Pearce Edward J.
Published: April 2014

Following activation, naive CD4+ Th cells can differentiate to selectively produce either the Th1 lineage-specific cytokine IFN-γ or the Th2 cytokine IL-4 and, in so doing, lose the capacity to produce cytokines of the alternative lineage. Lineage commitment of murine CD4+ T cells has largely been considered to be absolute with little flexibility to produce cytokines of the opposing lineage. In this study, we demonstrate that cells within Th2 memory populations can produce IFN-γ if reactivated in vivo in the context of an innate response that favors Th1 cell development. Likewise, cells within Th1 memory populations produce IL-4 when challenged under conditions that promote Th2 responses. Both effector and unpolarized central memory cells retain the potential to produce cytokines that were not made during the primary response. These findings reveal that both effector and central memory Th1 and Th2 cells possess the capacity to respond to environmental cues to produce pathogen-appropriate cytokines of the opposing lineage.
Prostaglandin E2 Induces the Expression of IL-1α in Colon Cancer Cells
Authors: Shao Jinyi , Sheng Hongmiao
Published: April 2014

PGE2 has been shown to exert pro-oncogenic effects in colorectal neoplasia through producing autocrine or paracrine growth factors. In the present study, we demonstrate that PGE2 induced the expression of IL-1α in colon cancer cells, which plays critical roles in tumor metastasis and neoangiogenesis in a variety of cancers. PGE2 increased the levels of both IL-1α mRNA and protein, suggesting a positive feedback loop between the IL-1 pathway and PGE2 signaling. Mechanistically, PGE2 induced the expression of IL-1α at both transcriptional and posttranscriptional levels. PGE2 stimulated the transcriptional activity of the IL-1α promoter and significantly stabilized IL-1α mRNA. Moreover, we show that IL-1α enhanced colorectal neoplasia, stimulating cell migration and neoangiogenesis. Knockdown of the expression of IL-1α by small-interfering RNA resulted in a reduction of vascular endothelial growth factor secretion in colon cancer cells and an inhibition of tube formation by HUVECs. Thus, our results suggest that PGE2 induces the expression of proinflammatory cytokine IL-1α, which may potentially enhance the proneoplastic actions of the cyclooxygenase-2/PGE2 signaling pathway.
Different Kinetics of Blimp-1 Induction in B Cell Subsets Revealed by Reporter Gene
Authors: Fairfax Kirsten A. , Corcoran Lynn M. , Pridans Clare , Huntington Nicholas D. , Kallies Axel , Nutt Stephen L. , Tarlinton David M.
Published: April 2014

The transcriptional repressor Blimp-1 (B lymphocyte-induced maturation protein 1) has been described as a “master regulator” of B cell differentiation into Ab-secreting cells (ASCs). Although there is mounting evidence for the importance and necessity of Blimp-1 in plasma cell development, there is uncertainty as to the role it plays in B cell differentiation of B cell subsets and the way in which it may interact with other transcription factors such as Pax5 and Bcl6 during ASC differentiation. Using a mouse expressing GFP under the control of the Blimp-1 regulatory elements (Blimp-1GFP/+), we examined the kinetics of Blimp-1 up-regulation in purified B cell subsets following activation. B1 cells showed the most rapid and pronounced up-regulation of Blimp-1 in response to the mitogens tested, followed by marginal zone B cells and then conventional B2 cells. Interestingly, only B1 cells substantially up-regulated Blimp-1 expression in response to CpG. B1 cells secreted negligible Ig upon isolation but were able to up-regulate Blimp-1 and initiate Ig secretion within 28 h of stimulation. Also of interest, B1 cells have a transcriptional factor profile that is intermediate between a naive B cell and an ASC, indicative of the semiactivated state of B1 cells. Transferred naive Blimp-1GFP/+ B1 and B2 cells both gave rise to ASCs in the bone marrow, suggesting no intrinsic barriers to B1 cell entry into the long-lived ASC compartment.
Four Functionally Distinct Populations of Human Effector-Memory CD8+ T Lymphocytes
Authors: Romero Pedro , Zippelius Alfred , Kurth Isabel , Pittet Mikaël J. , Touvrey Cédric , Iancu Emanuela M. , Corthesy Patricia , Devevre Estelle , Speiser Daniel E. , Rufer Nathalie
Published: April 2014

In humans, the pathways of memory and effector T cell differentiation remain poorly defined. We have dissected the functional properties of ex vivo effector-memory (EM) CD45RA−CCR7− T lymphocytes present within the circulating CD8+ T cell pool of healthy individuals. Our studies show that EM T cells are heterogeneous and are subdivided based on differential CD27 and CD28 expression into four subsets. EM1 (CD27+CD28+) and EM4 (CD27−CD28+) T cells express low levels of effector mediators such as granzyme B and perforin and high levels of CD127/IL-7Rα. EM1 cells also have a relatively short replicative history and display strong ex vivo telomerase activity. Therefore, these cells are closely related to central-memory (CD45RA−CCR7+) cells. In contrast, EM2 (CD27+CD28−) and EM3 (CD27−CD28−) cells express mediators characteristic of effector cells, whereby EM3 cells display stronger ex vivo cytolytic activity and have experienced larger numbers of cell divisions, thus resembling differentiated effector (CD45RA+CCR7−) cells. These data indicate that progressive up-regulation of cytolytic activity and stepwise loss of CCR7, CD28, and CD27 both characterize CD8+ T cell differentiation. Finally, memory CD8+ T cells not only include central-memory cells but also EM1 cells, which differ in CCR7 expression and may therefore confer memory functions in lymphoid and peripheral tissues, respectively.
Peroxisome Proliferator-Activated Receptor γ (PPARγ) and Immunoregulation: Enhancement of Regulatory T Cells through PPARγ-Dependent and -Independent Mechanisms
Authors: Wohlfert Elizabeth A. , Nichols Frank C. , Nevius Erin , Clark Robert B.
Published: April 2014

Peroxisome proliferator-activated receptor (PPAR)γ is a nuclear hormone receptor primarily characterized for its effect on insulin metabolism. PPARγ ligands, used to treat human type 2 diabetes, also down-regulate most immune system cells including APCs and pathogenic T cells. These effects putatively underlie the efficacy of PPARγ ligands in treating animal models of autoimmunity, leading to projections of therapeutic potential in human autoimmunity. However, the relationship between PPARγ ligands and CD4+CD25+ regulatory T cells (Tregs) has not been examined. Specifically, no studies have examined the role of Tregs in mediating the in vivo immunoregulatory effects of PPARγ ligands, and there have been no investigations of the use of PPARγ ligands to treat autoimmunity in the absence of Tregs. We now characterize the novel relationship between ciglitazone, a thiazolidinedione class of PPARγ ligand, and both murine natural Tregs (nTregs) and inducible Tregs (iTregs). In vitro, ciglitazone significantly enhances generation of iTregs in a PPARγ-independent manner. Surprisingly, and contrary to the current paradigm, we find that, in a model of graft-vs-host disease, the immunotherapeutic effect of ciglitazone requires the presence of nTregs that express PPARγ. Overall, our results indicate that, unlike its down-regulatory effect on other cells of the immune system, ciglitazone has an enhancing effect on both iTregs and nTregs, and this finding may have important implications for using PPARγ ligands in treating human autoimmune disease.
Minor Antigen H60-Mediated Aplastic Anemia Is Ameliorated by Immunosuppression and the Infusion of Regulatory T Cells
Authors: Chen Jichun , Ellison Felicia M. , Eckhaus Michael A. , Smith Aleah L. , Keyvanfar Keyvan , Calado Rodrigo T. , Young Neal S.
Published: April 2014

Human bone marrow (BM) failure mediated by the immune system can be modeled in mice. In the present study, infusion of lymph node (LN) cells from C57BL/6 mice into C.B10-H2b/LilMcd (C.B10) recipients that are mismatched at multiple minor histocompatibility Ags, including the immunodominant Ag H60, produced fatal aplastic anemia. Declining blood counts correlated with marked expansion and activation of CD8 T cells specific for the immunodominant minor histocompatibility Ag H60. Infusion of LN cells from H60-matched donors did not produce BM failure in C.B10 mice, whereas isolated H60-specific CTL were cytotoxic for normal C.B10 BM cells in vitro. Treatment with the immunosuppressive drug cyclosporine abolished H60-specific T cell expansion and rescued animals from fatal pancytopenia. The development of BM failure was associated with a significant increase in activated CD4+CD25+ T cells that did not express intracellular FoxP3, whereas inclusion of normal CD4+CD25+ regulatory T cells in combination with C57BL/6 LN cells aborted H60-specific T cell expansion and prevented BM destruction. Thus, a single minor histocompatibility Ag H60 mismatch can trigger an immune response leading to massive BM destruction. Immunosuppressive drug treatment or enhancement of regulatory T cell function abrogated this pathophysiology and protected animals from the development of BM failure.