bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipopolysaccharide Initiates a Positive Feedback of Epidermal Growth Factor Receptor Signaling by Prostaglandin E2 in Human Biliary Carcinoma Cells
Authors: Finzi Laetitia , Shao Matt X. G. , Paye François , Housset Chantal , Nadel Jay A.
Published: February 2009

Bacterial products (e.g., LPS) are viewed as critical stimuli in inflammation-associated cancer. Cyclooxygenase 2 (COX-2), a major effector of LPS, and EGFR, are key to carcinogenesis, notably in the hepatobiliary tract. In this study, we tested the hypothesis that LPS can initiate an interaction between the epidermal growth factor receptor (EGFR) and COX-2 pathways. We examined the effect of LPS in biliary carcinoma cells that displayed constitutive COX-2 expression and PGE2 production and in normal human biliary epithelial cells in which COX-2/PGE2 expression was virtually absent. LPS induced early phosphorylation of EGFR and ERK1/2 in both types of cells, which reached maximum levels within 30 min (first phase). However, only the carcinoma cells showed a second significant rise in both EGFR and ERK phosphorylation 6 h after exposure to LPS (second phase). Inhibition of COX-2/PGE2 production prevented the second, but not the first, phase of EGFR and ERK1/2 phosphorylation, implicating COX-2/PGE2 in the second phase of phosphorylation. LPS induced COX-2-derived PGE2 production at 4 h, which was before the rise in the second phosphorylation that occurred at 6 h. Exogenous PGE2 also caused EGFR activation via a signaling pathway involving TACE-dependent TGF-α release. Inhibition of the second phase of EGFR phosphorylation with EGFR or COX-2 inhibitor prevented LPS-induced cell invasion in vitro, demonstrating the biological importance of this COX-2 feedback signaling in cancer cells. We conclude that LPS triggers a positive feedback loop involving COX-2/PGE2 in biliary carcinoma cells and that this second phase of EGFR phosphorylation is implicated in cell invasion by LPS.
Human Basophils Secrete IL-3: Evidence of Autocrine Priming for Phenotypic and Functional Responses in Allergic Disease
Authors: Schroeder John T. , Chichester Kristin L. , Bieneman Anja P.
Published: February 2009

Although IL-3 is commonly recognized for its growth factor-like activity, in vitro studies have long demonstrated a unique capacity for this cytokine to also augment the proinflammatory properties and phenotype of human basophils. In particular, basophils secrete mediators that are hallmarks in allergic disease, including vasoactive amines (e.g., histamine), lipid metabolites (e.g., leukotriene C4), and cytokines (e.g., IL-4/IL-13), which are all markedly enhanced with IL-3 pretreatment. This priming phenomenon is observed in response to both IgE-dependent and IgE-independent stimulation. Additionally, IL-3 directly activates basophils for IL-13 secretion and enhanced CD69 expression, two markers that are elevated in allergic subjects. Lymphocytes are commonly thought to be the source of the IL-3 that primes for these basophil responses. However, we demonstrate herein for the first time that basophils themselves rapidly produce IL-3 (within 4 h) in response to IgE-dependent activation. More importantly, our findings definitively show that basophils rapidly bind and utilize the IL-3 they produce, as evidenced by functional and phenotypic activity that is inhibited in the presence of neutralizing anti-IL-3 receptor (CD123) Abs. We predict that autocrine IL-3 activity resulting from low-level IgE/FcεRI cross-linking by specific allergen represents an important mechanism behind the hyperreactive nature of basophils that has long been observed in allergic disease.
Protein Kinase C-θ Is Required for NK Cell Activation and In Vivo Control of Tumor Progression
Authors: Aguiló Juan I. , Garaude Johan , Pardo Julián , Villalba Martín , Anel Alberto
Published: February 2009

Protein kinase C-θ (PKCθ) was initially isolated as an important PKC isoform expressed in T cells, although its expression is not restricted to these cells. Despite the central function of PKCθ in several immune responses, its role in the antitumor response against MHC class I (MHC-I)-negative cells has not been investigated. This is an important issue because most tumor cells growing in vivo down-regulate MHC-I expression to escape the CTL-mediated response. In the present work, we show that in vivo development of a MHC-I-deficient tumor (RMA-S) is much favored in PKCθ−/− mice compared with wild-type mice. This is associated with a reduced recruitment of NK cells to the site of tumor development and a reduced activation status of recruited NK cells. This correlates with a reduced ex vivo and in vivo cytotoxic potential of NK cells isolated from PKCθ−/− mice treated with polyinosinic:polycytidylic acid. Consistently, polinosinic:cytidilic acid treatment induces PKCθ expression and activation of its enzymatic activity in NK cells in an indirect manner. These observations underline the relevance of PKCθ as a key molecule in NK cell-mediated antitumor immune surveillance.
Recovery from Cyclophosphamide-Induced Lymphopenia Results in Expansion of Immature Dendritic Cells Which Can Mediate Enhanced Prime-Boost Vaccination Antitumor Responses In Vivo When Stimulated with the TLR3 Agonist Poly(I:C)
Authors: Salem Mohamed L. , Díaz-Montero C. Marcela , Al-Khami Amir A. , El-Naggar Sabry A. , Naga Osama , Montero Alberto J. , Khafagy Ahmed , Cole David J.
Published: February 2009

Recent preclinical studies suggest that vaccination following adoptive transfer of CD8+ T cells into a lymphopenic host can augment the therapeutic antitumor responses of the transferred cells. However, the mechanism by which the lymphopenic microenvironment benefits Ag-specific CD8+ T cell responses remains elusive. We show herein that induction of lymphodepletion by a single 4 mg cyclophosphamide (CTX) treatment induces a marked expansion of immature dendritic cells (DCs) in the peripheral blood on days 8–16 post-CTX (termed restoration phase). In vitro, these DCs were functional, because they showed normal phagocytosis and effective Ag presentation capability upon activation. In vivo, administration of the TLR3 agonist poly(I:C) at the peak of DC expansion (day 12 postlymphopenia) induced inflammatory cytokine production and increases in the number of activated DCs in lymph nodes. Importantly, boosting with gp10025–33 melanoma peptide combined with poly(I:C) 12 days after an initial priming with the same regimen significantly increased the expansion and the antitumor efficacy of adoptively transferred pmel-1 CD8+ T cells. These responses were abrogated after depletion of activated DCs during Ag boosting. In conclusion, our data show that CTX treatment induces, during the restoration phase, expansion of immature DCs, which are functional and can be exploited in vivo to foster more effective antitumor adoptive immunotherapy strategies.
Enhanced In Vivo Growth of Lymphoma Tumors in the Absence of the NK-Activating Receptor NKp46/NCR1
Authors: Halfteck Gili G. , Elboim Moran , Gur Chamutal , Achdout Hagit , Ghadially Hormas , Mandelboim Ofer
Published: February 2009

The in vitro elimination of virus-infected and tumor cells by NK cells is regulated by a balance between signals conveyed via specific inhibitory and activating receptors. Whether NK cells and specifically the NK-activating receptor NKp46 (NCR1 in mice) are directly involved in tumor eradication in vivo is still largely unknown. Since the NKp46/NCR1 tumor ligands have not been identified yet, we use a screening technique to identify functional ligands for NKp46/NCR1 which is based on a cell reporter assay and discover a NCR1 ligand in the PD1.6 lymphoma line. To study whether NKp46/NCR1 is important for the eradication of PD1.6 lymphoma in vivo, we used the Ncr1 knockout Ncr1gfp/gfp mice generated by our group. Strikingly, all Ncr1 knockout mice developed growing PD1.6 tumors, whereas initial tumor growth was observed in the wild-type mice and tumors were completely rejected as time progressed. The growth of other lymphoma cell lines such as B10 and EL4 was equivalent between the Ncr1 knockout and wild-type mice. Finally, we show that PD1.6 lymphoma cells are less killed both in vitro and in vivo in the absence of NKp46/NCR1. Our results therefore reveal a crucial role for NKp46/NCR1 in the in vivo eradication of some lymphoma cells.
Unique Phenotype of Human Tonsillar and In Vitro-Induced FOXP3+CD8+ T Cells
Authors: Siegmund Kerstin , Rückert Beate , Ouaked Nadia , Bürgler Simone , Speiser Andreas , Akdis Cezmi A. , Schmidt-Weber Carsten B.
Published: February 2009

Forkhead box p3 (FOXP3) is known to program the acquisition of suppressive capacities in CD4+ regulatory T cells (Treg), whereas its role in CD8+ T cells is unknown. The current study investigates whether FOXP3 also acts as a Treg master switch in peripheral blood and tonsillar CD8+ T cells. Single-cell analyses reveal the existence of a FOXP3+CD8+ population in human tonsils, whereas FOXP3+CD8+ T cells are rarely detected in peripheral blood. Tonsillar FOXP3+CD8+ T cells exhibit a Treg phenotype with high CTLA-4 and CD45RO and low CD127 and CD69 expression. Interestingly, the tonsillar FOXP3+CD8+ T cells are mostly CD25negative and some cells also express the proinflammatory cytokines TNF-α, IFN-γ, or IL-17A. Particularly, IL-17A-expressing cells are present among FOXP3+CD8+ T cells. Even though FOXP3 expression is at the detection limit in peripheral blood CD8+ T cells ex vivo, it can be induced in vitro in naive CD8+ T cells by polyclonal stimulation. The induced FOXP3+CD8+ T cells are predominantly CD25high and CD28high and similar to tonsillar cells, they produce high levels of TNF-α, IFN-γ, and granzyme B. However, IL-4 expression is mutually exclusive and IL-17A expression is not detectable. These FOXP3+CD8+ T cells suppress the proliferation of CD4+ T cells in cocultures, while showing no direct cytotoxic activity. In conclusion, the current study characterizes FOXP3-expressing CD8+ T cells from human tonsils and shows that in vitro activation leads to FOXP3 expression in CD8+ T cells and gain of suppressive activity.
Mycolactone Inhibits Monocyte Cytokine Production by a Posttranscriptional Mechanism
Authors: Simmonds Rachel E. , Lali Ferdinand V. , Smallie Tim , Small Pamela L. C. , Foxwell Brian M.
Published: February 2009

The virulence and immunosuppressive activity of Mycobacterium ulcerans is attributed to mycolactone, a macrolide toxin synthesized by the bacteria. We have explored the consequence and mechanism of mycolactone pretreatment of primary human monocytes activated by a wide range of TLR ligands. The production of cytokines (TNF, IL-1β, IL-6, IL-10, and IFN-γ-inducible protein-10), chemokines (IL-8), and intracellular effector molecules (exemplified by cyclooxygenase-2) was found to be powerfully and dose dependently inhibited by mycolactone, irrespective of the stimulating ligand. However, mycolactone had no effect on the activation of signaling pathways that are known to be important in inducing these genes, including the MAPK and NF-κB pathways. Unexpectedly, LPS-dependent transcription of TNF, IL-6, and cyclooxygenase-2 mRNA was found not to be inhibited, implying that mycolactone has a novel mechanism of action and must function posttranscriptionally. We propose that mycolactone mediates its effects by inhibiting the translation of a specific subset of proteins in primary human monocytes. This mechanism is distinct from rapamycin, another naturally occurring immunosuppressive lactone. The current findings also suggest that monocyte-derived cytokine transcript and protein levels may not correlate in Buruli ulcer lesions, and urge caution in the interpretation of RT-PCR data obtained from patient biopsy samples.
Hyporesponsiveness of Intestinal Dendritic Cells to TLR Stimulation Is Limited to TLR4
Authors: Cerovic Vuk , Jenkins Christopher D. , Barnes Andrew G. C. , Milling Simon W. F. , MacPherson G. Gordon , Klavinskis Linda S.
Published: February 2009

Dendritic cells (DCs) are crucial to intestinal immune regulation because of their roles in inducing protective immunity against pathogens while maintaining tolerance to commensal bacteria. Nonetheless, relatively little is known about intestinal DC responsiveness to innate immune stimuli via TLRs. We have previously shown that DCs migrating from the rat intestine in lymph (iLDCs) are hyporesponsive to LPS stimulation, thus possibly preventing harmful immune responses being induced to commensal flora. In this study, to understand how iLDC function is regulated by innate immune stimuli, we have characterized the expression and function of TLRs in iLDCs isolated from the thoracic duct lymph of mesenteric lymphadenectomized rats and compared these with DCs grown from bone marrow in the presence of Flt3 ligand. We show that iLDCs express mRNAs for all TLRs, but express significantly less TLR4 mRNA than bone marrow-derived DCs. Functionally, iLDCs could be activated by TLR agonists representing intestinal pathogen-associated molecular patterns, with the important exception of the TLR4 agonist LPS. Furthermore, we show that DCs in the intestinal wall interact directly with noninvasive bacteria (Bacillus subtilis spores), leading to an increase in the output of activated iLDCs into lymph, and that DCs containing spores are activated selectively. These data highlight a functional difference between TLR4 and other TLRs. As iLDCs can respond to TLR stimulation in vitro, there must be other mechanisms that prevent their activation by commensal bacteria under steady-state conditions.
Immunosuppressive Myeloid-Derived Suppressor Cells Can Be Converted into Immunogenic APCs with the Help of Activated NKT Cells: An Alternative Cell-Based Antitumor Vaccine
Authors: Ko Hyun-Jeong , Lee Jung-Mi , Kim Yeon-Jeong , Kim Yun-Sun , Lee Kyoo-A , Kang Chang-Yuil
Published: February 2009

Myeloid-derived suppressor cells (MDSCs), which are known to be accumulated in the blood, spleen, and bone marrow of tumor-bearing mice and cancer patients, were tested as APCs for a cellular vaccine because they have phenotypical similarity with inflammatory monocytes and may be differentiated from the same precursors as monocytes. Although MDSCs have immunosuppressive properties, in vivo transferred MDSCs, which present tumor Ag and NKT cell ligand (α-galactosylceramide), significantly prolonged survival time in metastatic tumor-bearing mice in a CD8+ cell-, NK cell-, and NKT cell-dependent manner vs a CD4+ T cell- and host dendritic cell-independent manner. Major concerns about using MDSCs as APCs in a vaccine are their suppression of CTLs and their induction of Foxp3+ regulatory T cells. However, α-galactosylceramide-loaded MDSCs did not suppress CD4+ and CD8+ T cells and allowed for the generation of Ag-specific CTL immunity without increasing the generation of regulatory T cells. Furthermore, stimulation with activated NKT cells induced changes on MDSCs in phenotypical or maturation markers, including CD11b, CD11c, and CD86. Taken together, these findings suggest that NKT cells facilitate the conversion of immunosuppressive MDSCs into immunogenic APCs, eliciting successful antitumor immunity and providing the basis for alternative cell-based vaccines.
Carbon Monoxide Inhibits TLR-Induced Dendritic Cell Immunogenicity
Authors: Rémy Séverine , Blancou Philippe , Tesson Laurent , Tardif Virginie , Brion Régis , Royer Pierre Joseph , Motterlini Roberto , Foresti Roberta , Painchaut Marion , Pogu Sylvie , Gregoire Marc , Bach Jean Marie , Anegon Ignacio , Chauveau Christine
Published: February 2009

Heme oxygenase-1 (HO-1) exerts its functions via the catabolism of heme into carbon monoxide (CO), Fe2+, and biliverdin, as well as by depletion of free heme. We have recently described that overexpression of HO-1 is associated with the tolerogenic capacity to dendritic cells (DCs) stimulated by LPS. In this study, we demonstrate that treatment of human monocyte-derived DCs with CO blocks TLR3 and 4-induced phenotypic maturation, secretion of proinflammatory cytokines, and alloreactive T cell proliferation, while preserving IL-10 production. Treatment of DCs with biliverdin, bilirubin, and deferoxamine or replenishing intracellular heme stores had no effect on DC maturation. HO-1 and CO inhibited LPS-induced activation of the IFN regulatory factor 3 pathway and their effects were independent of p38, ERK, and JNK MAPK. HO-1 and CO treatment also inhibited mouse DC maturation in vitro and mouse DC immunogenic properties in vivo, as shown by adoptive cell transfer in a transgenic model of induced diabetes. Thus, for the first time, our data show that CO treatment inhibits DC immunogenicity induced by TLR ligands and that blockade of IFN regulatory factor 3 is associated with this effect.
TANK-Binding Kinase-1 Plays an Important Role during In Vitro and In Vivo Type I IFN Responses to DNA Virus Infections
Authors: Miyahira Andrea K. , Shahangian Arash , Hwang Seungmin , Sun Ren , Cheng Genhong
Published: February 2009

TANK-binding kinase-1 (TBK1) and the inducible IκB kinase (IKK-i) have recently been shown to activate type I IFN responses elicited by intracellular detection of RNA or DNA from infecting viruses. Detection of viral RNA is mediated by retinoic acid inducible gene-I or melanoma differentiation-associated gene-5 pathways in which TBK1 and IKK-i have been demonstrated to play redundant roles in IFN activation. In this study, we have examined whether such redundancy occurs in the type I IFN response to DNA viral challenges by examining induction of IFNs and IFN-mediated signaling and gene programs in TBK1−/− macrophages. In contrast to the normal IFN responses in TBK1−/− macrophages infected with an RNA virus, IFN responses were severely abrogated during DNA virus infections in TBK1−/− macrophages. Because both TBK1 and IKK-i are expressed in macrophages, our studies suggest that TBK1 and IKK-i differ functionally in DNA virus-mediated IFN responses; however, they are redundant in RNA virus-mediated IFN responses. Confirmatively, reconstitution of TBK1−/−IKK-i−/− fibroblasts revealed that TBK1 rescued IFN responses to transfected B-DNA to a much stronger degree than IKK-i. Finally, we demonstrate the requirement for the TBK1-IFN regulatory factor-3 pathway in host defense against a DNA virus infection in vivo.
Conditional Inactivation of TACE by a Sox9 Promoter Leads to Osteoporosis and Increased Granulopoiesis via Dysregulation of IL-17 and G-CSF
Authors: Horiuchi Keisuke , Kimura Tokuhiro , Miyamoto Takeshi , Miyamoto Kana , Akiyama Haruhiko , Takaishi Hironari , Morioka Hideo , Nakamura Takashi , Okada Yasunori , Blobel Carl P. , Toyama Yoshiaki
Published: February 2009

The TNF-α converting enzyme (TACE/ADAM17) is involved in the proteolytic release of the ectodomain of diverse cell surface proteins with critical roles in development, immunity, and hematopoiesis. As the perinatal lethality of TACE-deficient mice has prevented an analysis of the roles of TACE in adult animals, we generated mice in which floxed Tace alleles were deleted by Cre recombinase driven by a Sox9 promoter. These mutant mice survived up to 9–10 mo, but exhibited severe growth retardation as well as skin defects and infertility. The analysis of the skeletal system revealed shorter long bones and prominent bone loss, characterized by an increase in osteoclast and osteoblast activity. In addition, these mice exhibited hypercellularity in the bone marrow and extramedullary hematopoiesis in the spleen and liver. Flow cytometric analysis of the bone marrow cells showed a sharp increase in granulopoiesis and in the population of c-Kit-1+ Sca-1+ lineage− cells, and a decrease in lymphopoiesis. Moreover, we found that serum levels of IL-17 and G-CSF were significantly elevated compared with control littermates. These findings indicate that TACE is associated with a regulation of IL-17 and G-CSF expression in vivo, and that the dysregulation in G-CSF production is causally related to both the osteoporosis-like phenotype and the defects in the hematopoietic system.
Role of Invariant NK T Lymphocytes in Immune Responses to CpG Oligodeoxynucleotides
Authors: Paget Christophe , Bialecki Emilie , Fontaine Josette , Vendeville Catherine , Mallevaey Thierry , Faveeuw Christelle , Trottein François
Published: February 2009

Unmethylated CpG oligodeoxynucleotides (ODNs), by activating cells of the innate immune system, such as dendritic cells and NK cells, are potent adjuvants for type 1 immune responses. In the present study, we aimed to investigate the role of invariant NKT (iNKT) cells, a subset of lipid-reactive innate lymphocytes, in CpG ODN-induced innate and acquired type 1 responses. Our data show that, in response to the CpG ODN type B 1826, splenic and hepatic iNKT cells become activated and produce IFN-γ, but not IL-4, both in vitro and in vivo. This Th1 bias is independent from the Ag-presenting molecule CD1d and strongly requires IL-12, at least in vitro. We also report that iNKT cell activation, in response to CpG ODN type B, results in the transactivation of NK cells. To address the potential role of iNKT cells in type 1 innate immunity induced by CpG ODN, a murine model of malignant melanoma was used. We show that CpG ODN type B protects mice against B16F10-induced lung metastasis in wild-type mice, but in a less efficient manner in iNKT cell-deficient animals. Finally, we report that immunization of wild-type mice with CpG ODN type B plus keyhole limpet hemocyanin biases the immune response toward a Th1 direction, an effect strongly mediated by iNKT cells. We conclude that iNKT cells amplify the innate and acquired response to CpG ODN type B, with potentially important consequences for the regulation of immune responses.
Requirement of B Cells for Generating CD4+ T Cell Memory
Authors: Whitmire Jason K. , Asano Mary S. , Kaech Susan M. , Sarkar Surojit , Hannum Lynn G. , Shlomchik Mark J. , Ahmed Rafi
Published: February 2009

B cells can influence T cell responses by directly presenting Ag or by secreting Ab that binds to Ag to form immunogenic complexes. Conflicting evidence suggests that persisting Ag-Ab complexes propagate long-term T cell memory; yet, other data indicate that memory cells can survive without specific Ag or MHC. In this study, the roles of B cells and Ag-Ab complexes in T cell responses to lymphocytic choriomeningitis virus (LCMV) infection were investigated using B cell-deficient or B cell-competent mice. Despite normal lymphocyte expansion after acute infection, B cell-deficient mice rapidly lost CD4+ T cell memory, but not CD8+ T cell memory, during the contraction phase. To determine whether Ag-Ab complexes sustain CD4+ T cell memory, T cell responses were followed in B cell-transgenic (mIg-Tg) mice that have B cells but neither LCMV-specific Ab nor LCMV-immune complex deposition. In contrast to B cell-deficient mice, mIg-Tg mice retained functional Th cell memory, indicating that B cells selectively preserve CD4+ T cell memory independently of immune complex formation. An in vivo consequence of losing CD4+ T cell memory was that B cell-deficient mice were unable to resolve chronic virus infection. These data implicate a B cell function other than Ab production that induces long-term protective immunity.
Absence of Mucosal Immunity in the Human Upper Respiratory Tract to the Commensal Bacteria Neisseria lactamica but Not Pathogenic Neisseria meningitidis during the Peak Age of Nasopharyngeal Carriage
Authors: Vaughan Andrew T. , Gorringe Andrew , Davenport Victoria , Williams Neil A. , Heyderman Robert S.
Published: February 2009

The normal flora that colonizes the mucosal epithelia has evolved diverse strategies to evade, modulate, or suppress the immune system and avoid clearance. Neisseria lactamica and Neisseria meningitidis are closely related obligate inhabitants of the human upper respiratory tract. N. lactamica is a commensal but N. meningitidis is an opportunistic pathogen that occasionally causes invasive disease such as meningitis and septicemia. We demonstrate that unlike N. meningitidis, N. lactamica does not prime the development of mucosal T or B cell memory during the peak period of colonization. This cannot be explained by the induction of peripheral tolerance or regulatory CD4+CD25+ T cell activity. Instead, N. lactamica mediates a B cell-dependent mitogenic proliferative response that is absent to N. meningitidis. This mitogenic response is associated with the production of T cell-independent polyclonal IgM that we propose functions by shielding colonizing N. lactamica from the adaptive immune system, maintaining immunological ignorance in the host. We conclude that, in contrast to N. meningitidis, N. lactamica maintains a commensal relationship with the host in the absence of an adaptive immune response. This may prolong the period of susceptibility to colonization by both pathogenic and nonpathogenic Neisseria species.
The Activation of P2X7 Receptor Impairs Lysosomal Functions and Stimulates the Release of Autophagolysosomes in Microglial Cells
Authors: Takenouchi Takato , Nakai Masaaki , Iwamaru Yoshifumi , Sugama Shuei , Tsukimoto Mitsutoshi , Fujita Masayo , Wei Jianshe , Sekigawa Akio , Sato Mitsuru , Kojima Shuji , Kitani Hiroshi , Hashimoto Makoto
Published: February 2009

Recently, autophagy has been associated with the TLR signaling pathway to eliminate intracellular pathogens in the innate immune system. However, it is unknown if other pathways regulate autophagy during the immunologic response. Given the critical role of the purinergic P2X7 receptor (P2X7R) pathway during various immunologic functions (i.e., caspase activation and IL-1β secretion), the principal objective here was to determine whether the P2X7R pathway may regulate autophagy in immune cells. We observed in both MG6 mouse microglial cells and primary microglia that activation of P2X7R by ATP increases the expression of microtubule-associated protein 1 light chain 3 (LC3)-II, the autophagosomal membrane-associated form of LC3, in an extracellular Ca2+-dependent manner. Consistent with this, immunohistochemistry showed extensive formation of LC3-immunopositive dots, and electron microscopy demonstrated accumulation of autophagosomes and autophagolysosomes in ATP-treated cells. Importantly, the up-regulation of LC3-II by P2X7R activation was not affected by autophagy inhibitors, such as 3-methyladenine and PI3K inhibitors. Furthermore, while lysosomal functions were impaired by ATP treatment, autophagolysosomal components were released into the extracellular space. Similarly, a phagocytosis assay using Escherichia coli BioParticles showed that phagosome maturation was impaired in ATP-treated cells and a robust release of LC3-immunopositive phagolysosomes was induced along with a radial extension of microtubule bundles. Taken together, the data suggest a novel mechanism whereby the P2X7R signaling pathway may negatively regulate autophagic flux through the impairment of lysosomal functions, leading to stimulation of a release of autophagolysosomes/phagolysosomes into the extracellular space.
No Significant CTL Cross-Priming by Dendritic Cell-Derived Exosomes during Murine Lymphocytic Choriomeningitis Virus Infection
Authors: Coppieters Ken , Barral Ana María , Juedes Amy , Wolfe Tom , Rodrigo Evelyn , Théry Clotilde , Amigorena Sebastian , von Herrath Matthias G.
Published: February 2009

Exosomes are small membrane vesicles of endocytic origin that are secreted by most cells in culture, but are also present in serum. They contain a wide array of protein ligands on their surface, which has led to the hypothesis that they might mediate intercellular communication. Indeed, data support that exosomes can transfer Ags to dendritic cells (DC), and, interestingly, that these DC can subsequently induce T cell priming or tolerance. We have investigated whether this concept can be expanded to antiviral immunity. We isolated exosomes from supernatant of cultured bone marrow-derived DC (BMDC) that were infected with lymphocytic choriomeningitis virus (LCMV) or loaded with an immunodominant LCMV peptide, and characterized them by flow cytometry upon binding to beads. We then incubated the exosome preparations with BMDC and looked at their potential to activate LCMV gp33-specific naive and memory CD8 T cells. We found that exosomes do not significantly contribute to CD8 T cell cross-priming in vitro. Additionally, exosomes derived from in vitro-infected BMDC did not exhibit significant in vivo priming activity, as evidenced by the lack of protection following exosome vaccination. Thus, DC-derived exosomes do not appear to contribute significantly to CTL priming during acute LCMV infection.
Human CD4+ Memory T Cells Are Preferential Targets for Bystander Activation and Apoptosis
Authors: Bangs Sarah C. , Baban Dilair , Cattan Helen J. , Li Chris Ka-Fi , McMichael Andrew J. , Xu Xiao-Ning
Published: February 2009

There is much evidence that T cells may be activated via mechanisms that act independently of direct TCR ligation. Despite this, the question of whether such forms of bystander T cell activation occur during immune responses is hotly debated. To address some outstanding questions, we set up an in vitro system within which to analyze bystander T cell activation in human T cells, in the absence of the possibility for TCR cross-reactivity. In addition, we have investigated the genetic, phenotypic, and functional characteristics of bystander-activated T cells. In this study, we show that bystander T cell activation is, indeed, observed during a specific immune response, and that it occurs preferentially among CD4+ memory T cells. Furthermore, bystander-activated T cells display a distinct gene expression profile. The mechanism for bystander T cell activation involves soluble factors, and the outcome is an elevated level of apoptosis. This may provide an explanation for the attrition of T cell memory pools of heterologous specificity during immune responses to pathogens such as viruses.
Intact Bacteria Inhibit the Induction of Humoral Immune Responses to Bacterial-Derived and Heterologous Soluble T Cell-Dependent Antigens
Authors: Chattopadhyay Gouri , Chen Quanyi , Colino Jesus , Lees Andrew , Snapper Clifford M.
Published: February 2009

During infections with extracellular bacteria, such as Streptococcus pneumoniae (Pn), the immune system likely encounters bacterial components in soluble form, as well as those associated with the intact bacterium. The potential cross-regulatory effects on humoral immunity in response to these two forms of Ag are unknown. We thus investigated the immunologic consequences of coimmunization with intact Pn and soluble conjugates of Pn-derived proteins and polysaccharides (PS) as a model. Coimmunization of mice with Pn and conjugate resulted in marked inhibition of conjugate-induced PS-specific memory, as well as primary and memory anti-protein Ig responses. Inhibition occurred with unencapsulated Pn, encapsulated Pn expressing different capsular types of PS than that present in the conjugate, and with conjugate containing protein not expressed by Pn, but not with 1-μm latex beads in adjuvant. Inhibition was long-lasting and occurred only during the early phase of the immune response, but it was not associated with tolerance. Pn inhibited the trafficking of conjugate from the splenic marginal zone to the B cell follicle and T cell area, strongly suggesting a potential mechanism for inhibition. These data suggest that during infection, bacterial-associated Ags are the preferential immunogen for antibacterial Ig responses.
IL-10-Dependent S100A8 Gene Induction in Monocytes/Macrophages by Double-Stranded RNA
Authors: Endoh Yasumi , Chung Yuen Ming , Clark Ian A. , Geczy Carolyn L. , Hsu Kenneth
Published: February 2009

The S100 calcium-binding proteins S100A8 and S100A9 are elevated systemically in patients with viral infections. The S100A8-S100A9 complex facilitated viral replication in human CD4+ T lymphocytes latently infected with HIV-1- and S100A8-induced HIV-1 transcriptional activity. Mechanisms inducing the S100 genes and the potential source of these proteins following viral activation are unknown. In this study, we show that S100A8 was induced in murine macrophages, and S100A8 and S100A9 in human monocytes and macrophages, by polyinosinic:polycytidylic acid, a dsRNA mimetic. Induction was at the transcriptional level and was IL-10 dependent. Similar to LPS-induced S100A8, induction by dsRNA was dependent on p38 and ERK MAPK. Protein kinase R (PKR) mediates antiviral defense and participates in MyD88-dependent/independent signaling triggered by TLR4 or TLR3. Like IL-10, S100 induction by polyinosinic:polycytidylic acid and by LPS was inhibited by the specific PKR inhibitor 2-aminopurine, indicating a novel IL-10, PKR-dependent pathway. Other mediators such as IFN-β, which synergized with dsRNA, may also be involved. C/EBPβ bound the defined promoter region in response to dsRNA. S100A8 was expressed in lungs of mice infected with influenza virus and was maximal at day 8 with strong immunoreactivity in epithelial cells lining the airways and in mononuclear cells and declined early in the recovery phase, implying down-regulation by mediator(s) up-regulated during resolution of the infection. IL-10 is implicated in viral persistence. Since S100A8/S100A9 levels are likely to be maintained in conditions where IL-10 is raised, these proteins may contribute to viral persistence in patients infected by some RNA viruses.
Down-Regulation of MHC Class II Expression through Inhibition of CIITA Transcription by Lytic Transactivator Zta during Epstein-Barr Virus Reactivation
Authors: Li Dan , Qian Lu , Chen Changguo , Shi Ming , Yu Ming , Hu Meiru , Song Lun , Shen Beifen , Guo Ning
Published: February 2009

The presentation of peptides to T cells by MHC class II molecules is of critical importance in specific recognition to a pathogen by the immune system. The level of MHC class II directly influences T lymphocyte activation. The aim of this study was to identify the possible mechanisms of the down-regulation of MHC class II expression by Zta during EBV lytic cycle. The data in the present study demonstrated that ectopic expression of Zta can strongly inhibit the constitutive expression of MHC class II and CIITA in Raji cells. The negative effect of Zta on the CIITA promoter activity was also observed. Scrutiny of the DNA sequence of CIITA promoter III revealed the presence of two Zta-response element (ZRE) motifs that have complete homology to ZREs in the DR and left-hand side duplicated sequence promoters of EBV. By chromatin immunoprecipitation assays, the binding of Zta to the ZRE221 in the CIITA promoter was verified. Site-directed mutagenesis of three conserved nucleotides of the ZRE221 substantially disrupted Zta-mediated inhibition of the CIITA promoter activity. Oligonucleotide pull-down assay showed that mutation of the ZRE221 dramatically abolished Zta binding. Analysis of the Zta mutant lacking DNA binding domain revealed that the DNA-binding activity of Zta is required for the trans repression of CIITA. The expression of HLA-DRα and CIITA was restored by Zta gene silencing. The data indicate that Zta may act as an inhibitor of the MHC class II pathway, suppressing CIITA transcription and thus interfering with the expression of MHC class II molecules.
Single Nucleotide Changes in the Human Iγ1 and Ιγ4 Promoters Underlie Different Transcriptional Responses to CD40
Authors: Sinquett Frank L. , Dryer Rebecca L. , Marcelli Valentina , Batheja Ameesha , Covey Lori R.
Published: February 2009

Analysis of subclass-specific germline transcription in activated peripheral B cells revealed a highly biased expression pattern of the four Iγ transcripts to signals through CD40 and IL-4. This difference was most pronounced when comparing the profile of Iγ1 and Iγ4 transcripts and was not expected given the very high degree of sequence conservation between promoters. In this report, the influence of sequence differences on the regulation of the Iγ1 and Iγ4 promoters has been investigated given the highly muted transcriptional activity of the Iγ4 promoter. Two regions were analyzed where single nucleotide differences corresponded to major changes in transcriptional activity. These regions were the previously defined CD40 response region containing three putative NF-κB-binding sites and the downstream 36-bp region containing CREB/activating transcription factor and κB6 sites. Mutation of a single nucleotide at position 6 within the Iγ4 κB6 site increased promoter activity to ∼50% of the activity of the Iγ1 promoter. Furthermore, elevated promoter strength corresponded with increased binding of p50, p65, c-Rel, RelB, and p300 proteins to a level comparable with that of Iγ1. Minor nucleotide changes to both the Iγ4 CD40 response region and the 36-bp element resulted in a response undistinguishable from an Iγ1 response, suggesting cooperation between the two regulatory regions for optimal transcriptional activity. Collectively, these mutational analyses suggest that minor sequence differences contribute to the composition and affinity of transcriptional protein complexes regulating subclass-specific germline transcription, which in part impacts the overall level of class switch recombination to targeted CH regions.
The Expression of Heparin-Binding Epidermal Growth Factor-Like Growth Factor by Regulatory Macrophages
Authors: Edwards Justin P. , Zhang Xia , Mosser David M.
Published: February 2009

We previously described a population of regulatory macrophages that produced high levels of IL-10 and low levels of IL-12/23. We now describe and characterize the expression of heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) by these macrophages. HB-EGF has previously been associated with a number of physiological and pathological conditions, including tumor growth and angiogenesis. The induction of HB-EGF in regulatory macrophages is due to new transcription and not to increased mRNA stability. The transcription factor Sp1 is a major factor in HB-EGF production, and knockdown of Sp1 substantially diminishes HB-EGF production. Sp1 was recruited to three sites within the first 2 kb of the HB-EGF promoter following stimulation, and the site located at −83/−54 was required for HB-EGF promoter activity. These regions of the promoter become more accessible to endonuclease activity following macrophage activation, and this accessibility was contingent on activation of the MAPK, ERK. We show that several experimental manipulations that give rise to regulatory macrophages also result in HB-EGF production. These observations indicate that in addition to the secretion of the anti-inflammatory cytokine IL-10, another novel characteristic of regulatory macrophages is the production of angiogenic HB-EGF.
The Sequestosome 1/p62 Attenuates Cytokine Gene Expression in Activated Macrophages by Inhibiting IFN Regulatory Factor 8 and TNF Receptor-Associated Factor 6/NF-κB Activity
Authors: Kim Ji Young , Ozato Keiko
Published: February 2009

Sequestosome 1/p62 (p62) is a scaffold/adaptor protein with multiple functions implicated for neuronal and bone diseases. It carries a ubiquitin binding domain through which it mediates proteasome-dependent proteolysis. In addition, p62 is reported to regulate NF-κB activity in some cells. To date, however, the role of p62 in innate immunity has not been fully elucidated. In this study, we report that IFN-γ plus TLR signaling stimulates late expression of p62 in murine macrophages. Overexpression of p62 inhibited expression of multiple cytokines, IL-12p40, TNF-α, IL-1β, IL-6, and IFN-β, whereas p62 underexpression by small hairpin RNA markedly elevated their expression, indicating that p62 is a broad negative regulator of cytokine expression in stimulated macrophages. We show that p62 interacts with IFN regulatory factor 8 and Ro52, the transcription factor and ubiquitin E3 ligase that are important for IL-12p40 expression. This interaction, detectable at a late stage in stimulated macrophages, led to increased polyubiquitination and destabilization of IFN regulatory factor 8. We also show that upon macrophage stimulation, p62 binds to TNFR-associated factor 6, another E3 ligase important for NF-κB activation, but later this interaction was replaced by the recruitment of the deubiquitinating enzyme, cylindromatosis, an inhibitor of NF-κB activity. Recruitment of cylindromatosis coincided with reduced TNFR-associated factor 6 autoubiquitination and lower NF-κB activation. Our results indicate that p62 orchestrates orderly regulation of ubiquitin modification processes in macrophages to ensure attenuation of cytokine transcription postactivation. Together, p62 may provide a mechanism by which to control excessive inflammatory responses after macrophage activation.
Intestinal Tolerance Is Converted to Autoimmune Enteritis upon PD-1 Ligand Blockade
Authors: Reynoso Erika D. , Elpek Kutlu G. , Francisco Loise , Bronson Roderick , Bellemare-Pelletier Angélique , Sharpe Arlene H. , Freeman Gordon J. , Turley Shannon J.
Published: February 2009

The B7 family member programmed death-1 ligand (PD-L1) has been shown to play an inhibitory role in the regulation of T cell responses in several organs. However, the role of PD-L1 in regulating tolerance to self-Ags of the small intestine has not been previously addressed. In this study, we investigated the role of PD-L1 in CD8+ T cell tolerance to an intestinal epithelium-specific Ag using the iFABP-tOVA transgenic mouse model, in which OVA is expressed as a self-Ag throughout the small intestine. Using adoptive transfer of naive OVA-specific CD8+ T cells, we show that loss of PD-1:PD-L1 signaling, by either Ab-mediated PD-L1 blockade or transfer of PD-1−/− T cells, leads to considerable expansion of OVA-specific CD8+ T cells and their differentiation into effector cells capable of producing proinflammatory cytokines. A fatal CD8+ T cell-mediated inflammatory response develops rapidly against the small bowel causing destruction of the epithelial barrier, severe blunting of intestinal villi, and recruitment and activation of myeloid cells. This response is highly specific because immune destruction selectively targets the small intestine but not other organs. Collectively, these results indicate that loss of the PD-1:PD-L1 inhibitory pathway breaks CD8+ T cell tolerance to intestinal self-Ag, thus leading to severe enteric autoimmunity.
Regulation of Mature ADAM17 by Redox Agents for L-Selectin Shedding
Authors: Wang Yue , Herrera Amy H. , Li Ying , Belani Kiran K. , Walcheck Bruce
Published: February 2009

L-selectin is constitutively expressed by neutrophils and plays a key role in directing these cells to sites of inflammation. Upon neutrophil activation, L-selectin is rapidly and efficiently down-regulated from the cell surface by ectodomain shedding. We have directly shown that A disintegrin and metalloprotease 17 (ADAM17) is a primary and nonredundant sheddase of L-selection by activated neutrophils in vivo. Following cell activation, intracellular signals lead to the induction of ADAM17’s enzymatic activity; however, the target of this inducer mechanism remains unclear. Our study provides evidence of an activation mechanism that involves the extracellular region of the mature form of cell surface ADAM17 and not its intracellular region. We demonstrate that the catalytic activity of purified ADAM17 lacking a prodomain and its intracellular region is diminished under mild reducing conditions by DTT and enhanced by H2O2 oxidation. Moreover, H2O2 reversed ADAM17 inhibition by DTT. The treatment of neutrophils with H2O2 also induced L-selectin shedding in an ADAM17-dependent manner. These findings suggest that thiol-disulfide conversion occurring in the extracellular region of ADAM17 may be involved in its activation. An analysis of ADAM17 revealed that within its disintegrin/cysteine-rich region are two highly conserved, vicinal cysteine sulfhydryl motifs (cysteine-X-X-cysteine), which are well-characterized targets for thiol-disulfide exchange in various other proteins. Using a cell-based ADAM17 reconstitution assay, we demonstrate that the cysteine-X-X-cysteine motifs are critical for L-selectin cleavage. Taken together, our findings suggest that reduction-oxidation modifications of cysteinyl sulfhydryl groups in mature ADAM17 may serve as a mechanism for regulating the shedding of L-selectin following neutrophil stimulation.
Asthma-Related Environmental Fungus, Alternaria, Activates Dendritic Cells and Produces Potent Th2 Adjuvant Activity
Authors: Kobayashi Takao , Iijima Koji , Radhakrishnan Suresh , Mehta Vinay , Vassallo Robert , Lawrence Christopher B. , Cyong Jong-Chol , Pease Larry R. , Oguchi Katsuji , Kita Hirohito
Published: February 2009

Asthma is thought to result from dysregulated Th2-like airway inflammatory responses to the environment. Although the etiology of asthma is not fully understood in humans, clinical and epidemiological evidence suggest a potential link between exposure to environmental fungi, such as Alternaria, and development and/or exacerbation of asthma. The goal of this project was to investigate the mechanisms of airway Th2 responses by using Alternaria as a clinically relevant model for environmental exposure. Airway exposure of naive animals to an experimental Ag, OVA, or a common allergen, short ragweed pollen, induced no or minimal immune responses to these Ags. In contrast, mice developed strong Th2-like immune responses when they were exposed to these Ags in the presence of Alternaria extract. Extracts of other fungi, such as Aspergillus and Candida, showed similar Th2 adjuvant effects, albeit not as potently. Alternaria stimulated bone marrow-derived dendritic cells (DCs) to express MHC class II and costimulatory molecules, including OX40 ligand, in vitro. Importantly, Alternaria inhibited IL-12 production by activated DCs, and DCs exposed to Alternaria enhanced Th2 polarization of CD4+ T cells. Furthermore, adoptive airway transfer of DCs, which had been pulsed with OVA in the presence of Alternaria, showed that the recipient mice had enhanced IgE Ab production and Th2-like airway responses to OVA. Thus, the asthma-related environmental fungus Alternaria produces potent Th2-like adjuvant effects in the airways. Such immunogenic properties of certain environmental fungi may explain their strong relationships with human asthma and allergic diseases.