bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of TLR Activation and Up-Regulation of IL-1R-Associated Kinase-M Expression by Exogenous Gangliosides
Authors: Shen Weiping , Stone Kelly , Jales Alessandra , Leitenberg David , Ladisch Stephan
Published: April 2014

Gangliosides, sialic acid-containing glycosphingolipids present in the outer leaflet of plasma membranes, are produced at high levels by some tumors, are actively shed into the tumor microenvironment, and can be detected in high concentrations in the serum of cancer patients. These tumor-shed molecules are known to be immunosuppressive, although mechanisms remain to be fully elucidated. In this study, we show that membrane enrichment of human monocytes with purified exogenous gangliosides potently inhibits ligand-induced activation and proinflammatory cytokine production induced by a broad range of TLRs, including TLR2, TLR3, TLR6, and TLR7/8, in addition to a previously identified inhibitory effect on TLR4 and TLR5. Inhibition of TLR activation is reversible, with complete restoration of TLR signaling within 6–24 h of washout of exogenous gangliosides, and is selective for certain gangliosides (GM1, GD1a, and GD1b), whereas others (GM3) are inactive. To characterize the inhibition, we assessed the expression of the TLR signaling pathway inhibitor, IL-1 receptor associated kinase-M (IRAK-M). In response to ganglioside enrichment alone, we observed striking up-regulation of IRAK-M in monocytes, but without concomitant proinflammatory cytokine production. This contrasts with endotoxin tolerance, in which IRAK-M up-regulation follows proinflammatory cytokine expression caused by LPS exposure. We hypothesize that ganglioside treatment induces a state of tolerance to TLR signaling, leading to blunted activation of innate immune responses. In the tumor microenvironment, shed tumor ganglioside enrichment of APC membranes may likewise cause these cells to bypass the normal TLR signaling response and progress directly to the inhibitory state.
Gastrointestinal Dendritic Cells Promote Th2 Skewing via OX40L
Authors: Blázquez Ana Belén , Berin M. Cecilia
Published: April 2014

Mice can be sensitized to food proteins by oral administration with the adjuvant cholera toxin (CT), such that they undergo anaphylaxis when rechallenged with the sensitizing allergen. In contrast, feeding of Ags alone leads to oral tolerance. Our aim was to define the mechanisms by which gastrointestinal dendritic cells (DCs) participate in the deviation of tolerance to allergic sensitization in the gut in response to CT. BALB/c mice were fed with CT or PBS. The impact of CT on DC subsets in the mesenteric lymph node (MLN) was assessed by flow cytometry. Ag presentation assays were performed with DCs isolated from the MLN of PBS- or CT-fed mice, using OVA-specific CD4+ T cells as responder cells. Gene expression in MLN DCs was determined by real-time PCR, and neutralizing Abs were used to test the function of OX40 ligand (OX40L) in Th2 skewing. Oral administration of CT induced an increase in the total CD11c+ population in the MLN. CT induced a selective increase in migration of the CD11c+CD11b−CD8α− DC subset and the maturation of all DC subsets. Maturation of DCs in vivo enhanced T cell proliferation and cytokine secretion. Oral CT induced up-regulation of Jagged-2 and OX40L by MLN DCs. Neutralizing anti-OX40L Abs completely abrogated the CT-induced Th2 cytokine response. We show that oral CT induces selective DC migration, maturation, and T cell priming activity in the MLN. Th2 skewing is mediated by OX40L, and we speculate that this molecule may be an important inducer of allergic sensitization to food allergens.
De Novo Production of K-α1 Tubulin-Specific Antibodies: Role in Chronic Lung Allograft Rejection
Authors: Goers Trudie A. , Ramachandran Sabarinathan , Aloush Aviva , Trulock Elbert , Patterson G. Alexander , Mohanakumar Thalachallour
Published: April 2014

Lung transplantation is the treatment option for a variety of end-stage pulmonary diseases. Posttransplant development of Abs against donor HLA and non-HLA Ags have been associated with acute and chronic rejection of transplanted organs. Development of bronchiolitis obliterans syndrome (BOS) following lung transplantation has been correlated with de novo production of anti-donor-HLA Abs. However, only a portion of the patients with BOS demonstrate detectable anti-donor-HLA Abs. Airway epithelium is considered as a major target for lung allograft rejection. In this study we demonstrate that many BOS+ patients (12 of 36) develop Abs reactive to epithelial cell Ag that are distinct from HLA. Furthermore, de novo production of antiepithelial cell Ab precedes clinical onset of BOS. N-terminal sequencing and blastx analysis as well as blocking with K-α1 tubulin-specific Ab identified the epithelial Ag as K-α1 tubulin. Binding of the de novo-produced anti-K-α1 tubulin Abs to the airway epithelial cells resulted in the increased expression of transcription factors (TCF5 and c-Myc), leading to increased expression of fibrogenic growth factors, activation of cell cycle signaling, and fibroproliferation, the central events in immunopathogenesis of BOS following human lung transplantation.
Aberrant Genetic Control of Invariant TCR-Bearing NKT Cell Function in New Zealand Mouse Strains: Possible Involvement in Systemic Lupus Erythematosus Pathogenesis
Authors: Tsukamoto Kazuyuki , Ohtsuji Mareki , Shiroiwa Wakana , Lin Qingshun , Nakamura Kazuhiro , Tsurui Hiromichi , Jiang Yi , Sudo Katsuko , Nishimura Hiroyuki , Shirai Toshikazu , Hirose Sachiko
Published: April 2014

Both suppressive and promoting roles of NKT cells have been reported in the pathogenesis of systemic lupus erythematosus (SLE). Herein, we found that although New Zealand mice have normal frequencies of NKT cells, their in vitro potential to produce IL-4 and IFN-γ in response to α-galactosylceramide was remarkably impaired in New Zealand Black (NZB) mice prone to mild SLE, while production was highly up-regulated in nonautoimmune New Zealand White (NZW) mice and at intermediate levels in (NZB × NZW)F1 mice, which are prone to severe SLE. Because this aberration is evident in young mice before disease onset, genetic mechanisms are thought to be involved. Genome-wide quantitative trait locus analysis and association studies revealed that a locus linked to D11Mit14 on chromosome 11 may be involved in the difference in cytokine-producing potential between NZB and NZW NKT cells. Additionally, (NZB × NZW)F1 × NZB backcross progeny with the NZW genotype for D11Mit14 showed significantly increased frequencies of age-associated SLE phenotypes, such as high serum levels of IgG, IgG anti-DNA Abs, and lupus nephritis. In coculture studies, α-galactosylceramide-stimulated NKT cells from NZW and (NZB × NZW)F1 mice, but not from NZB mice, showed significantly enhanced Ig synthesis by B cells. These findings suggest that the D11Mit14-linked NZW locus may contribute to the development of SLE in (NZB × NZW)F1 mice through a mechanism that up-regulates NKT cell function. Thus, this NZW allele may be a candidate of the NZW modifiers that act to promote (NZB × NZW)F1 disease.
Mast Cell Migration from the Skin to the Draining Lymph Nodes upon Ultraviolet Irradiation Represents a Key Step in the Induction of Immune Suppression
Authors: Byrne Scott N. , Limón-Flores Alberto Y. , Ullrich Stephen E.
Published: April 2014

The UV radiation in sunlight is the primary cause of skin cancer. UV is also immunosuppressive and numerous studies have shown that UV-induced immune suppression is a major risk factor for skin cancer induction. Previous studies demonstrated that dermal mast cells play a critical role in the induction of immune suppression. Mast cell-deficient mice are resistant to the immunosuppressive effects of UV radiation, and UV-induced immune suppression can be restored by injecting bone marrow-derived mast cells into the skin of mast cell- deficient mice. The exact process however, by which mast cells contribute to immune suppression, is not known. In this study, we show that one of the first steps in the induction of immune suppression is mast cell migration from the skin to the draining lymph nodes. UV exposure, in a dose-dependent manner, causes a significant increase in lymph node mast cell numbers. When GFP+ skin was grafted onto mast cell-deficient mice, we found that GFP+ mast cells preferentially migrated into the lymph nodes draining the skin. The mast cells migrated primarily to the B cell areas of the draining nodes. Mast cells express CXCR4+ and UV exposure up-regulated the expression of its ligand CXCL12 by lymph node B cells. Treating UV-irradiated mice with a CXCR4 antagonist blocked mast cell migration and abrogated UV-induced immune suppression. Our findings indicate that UV-induced mast cell migration to draining lymph nodes, mediated by CXCR4 interacting with CXCL12, represents a key early step in UV-induced immune suppression.
Combined CXCR3/CCR5 Blockade Attenuates Acute and Chronic Rejection
Authors: Schnickel Gabriel T. , Bastani Sam , Hsieh George R. , Shefizadeh Ali , Bhatia Rubina , Fishbein Michael C. , Belperio John , Ardehali Abbas
Published: April 2014

Chemokine-chemokine receptor interactions orchestrate mononuclear cells recruitment to the allograft, leading to acute and chronic rejection. Despite biologic redundancy, several experimental studies have demonstrated the importance of CXCR3 and CCR5 in acute rejection of allografts. In these studies, deficiency or blockade of CXCR3 or CCR5 led to prolongation of allograft survival, yet allografts were ultimately lost to acute rejection. Given the above findings and the specificity of mononuclear cells bearing CXCR3 and CCR5, we hypothesized that combined blockade of CXCR3 and CCR5 will lead to indefinite (>100 days) graft survival in a full MHC-mismatched murine cardiac allograft model. The donor hearts in the control group were rejected in 6 ± 1 days after transplantation. Combined blockade of CXCR3 and CCR5 prolonged allograft survival >15-fold vs the control group; all allografts survived for >100 days. More importantly, the donor hearts did not display any intimal lesions characteristic of chronic rejection. Further analysis of the donor hearts in the CXCR3/CCR5 blockade group demonstrated graft infiltration with CD4+CD25+ T cells expressing the Foxp3 gene. Depletion of CD25+ cells in the combined CXCR3 and CCR5 blockade group resulted in acute rejection of the allografts in 22 ± 2 days. Combined CXCR3 and CCR5 blockade also reduced alloantigen-specific T lymphocyte proliferation. Combined CXCR3 and CCR5 blockade is effective in preventing acute and chronic rejection in a robust murine model. This effect is mediated, in part, by CD25+ regulatory T cell recruitment and control of T lymphocyte proliferation.
Isoform-Specific Inhibition of RORα-Mediated Transcriptional Activation by Human FOXP3
Authors: Du Jianguang , Huang Chunjian , Zhou Baohua , Ziegler Steven F.
Published: April 2014

FOXP3 is a forkhead family transcriptional repressor important for the development and function of CD4+CD25+ regulatory T cells. In humans, FOXP3 is expressed as two isoforms, a full-length form and a smaller form lacking exon 2. These two isoforms are expressed in approximately equal amounts in circulating regulatory T cells, and are induced equally in freshly activated CD4+CD25− T cells. Herein, we show that FOXP3 interacts with retinoic acid receptor-related orphan receptor (ROR)α, and that this interaction inhibits transcriptional activation mediated by RORα. Full-length FOXP3, but not the isoform lacking exon 2, interacts with RORα, and the region of FOXP3 involved in the interaction is encoded by exon 2. Mutation of the LxxLL motif in FOXP3, located in exon 2, abolished interaction and repression by FOXP3. Additionally, the inhibition of RORα by FOXP3 does not require an intact forkhead domain, demonstrating a mode of FOXP3 function that is independent of DNA binding. Interestingly, expression of RORα in T cells leads to the expression of genes that define Th17 cells, and the expression of each of these gene was inhibited by coexpression of full-length, but not ΔEx2, FOXP3. These data expand the possible targets of FOXP3-mediated repression and demonstrate functional differences between FOXP3 isoforms.
Intercellular Adhesion Molecule-3 Binding of Integrin αLβ2 Requires Both Extension and Opening of the Integrin Headpiece
Authors: Tang Xiao-Yan , Li Yan-Feng , Tan Suet-Mien
Published: April 2014

The leukocyte-restricted integrin αLβ2 is required in immune processes such as leukocyte adhesion, migration, and immune synapse formation. Activation of αLβ2 by conformational changes promotes αLβ2 binding to its ligands, ICAMs. It was reported that different affinity states of αLβ2 are required for binding ICAM-1 and ICAM-3. Recently, the bent, extended with a closed headpiece, and extended with open headpiece conformations of αLβ2, was reported. To address the overall conformational requirements of αLβ2 that allow selective binding of these ICAMs, we examined the adhesion properties of these αLβ2 conformers. αLβ2 with different conformations were generated by mutations, and verified by using a panel of reporter mAbs that detect αLβ2 extension, hybrid domain movement, or I-like domain activation. We report a marked difference between extended αLβ2 with closed and open headpieces in their adhesive properties to ICAM-1 and ICAM-3. Our data show that the extension of αLβ2 alone is sufficient to mediate ICAM-1 adhesion. By contrast, an extended αLβ2 with an open headpiece is required for ICAM-3 adhesion.
Leishmania major-Specific B Cells Are Necessary for Th2 Cell Development and Susceptibility toL. majorLV39 in BALB/c Mice
Authors: Ronet Catherine , Voigt Heike , Himmelrich Hayo , Doucey Marie-Agnès , Hauyon-La Torre Yazmin , Revaz-Breton Mélanie , Tacchini-Cottier Fabienne , Bron Claude , Louis Jacques , Launois Pascal
Published: April 2014

B lymphocytes are considered to play a minimal role in host defense against Leishmania major. In this study, the contribution of B cells to susceptibility to infection with different strains of L. major was investigated in BALB/c mice lacking mature B cells due to the disruption of the IgM transmembrane domain (μMT). Whereas BALB/c μMT remained susceptible to infection with L. major IR173 and IR75, they were partially resistant to infection with L. major LV39. Adoptive transfer of naive B cells into BALB/c μMT mice before infection restored susceptibility to infection with L. major LV39, demonstrating a role for B cells in susceptibility to infection with this parasite. In contrast, adoptive transfer of B cells that express an IgM/IgD specific for hen egg lysozyme (HEL), an irrelevant Ag, did not restore disease progression in BALB/c μMT mice infected with L. major LV39. This finding was likely due to the inability of HEL Tg B cells to internalize and present Leishmania Ags to specific T cells. Furthermore, specific Ig did not contribute to disease progression as assessed by transfer of immune serum in BALB/c μMT mice. These data suggest that direct Ag presentation by specific B cells and not Ig effector functions is involved in susceptibility of BALB/c mice to infection with L. major LV39.
CD209+ Macrophages Mediate Host Defense against Propionibacterium acnes
Authors: Liu Philip T. , Phan Jenny , Tang Dominic , Kanchanapoomi Melissa , Hall Brian , Krutzik Stephan R. , Kim Jenny
Published: April 2014

Propionibacterium acnes is a major etiological factor of acne, triggering an inflammatory response in part through the activation of TLR2. In this study, we demonstrate that activation of peripheral blood monocytes with P. acnes in vitro induced their differentiation into two distinct innate immune cell subsets, CD209+ macrophages and CD1b+ dendritic cells. Furthermore, P. acnes induced expression of mRNA for the cytokines IL-15 and GM-CSF, which differentiate CD209+ and CD1b+ cells, respectively. The CD209+ cells were more effective in uptake of P. acnes, compared with the CD1b+ cells, and demonstrated a 2-fold greater antimicrobial activity against the phagocytosed bacteria. Although CD1b+ cells secreted inflammatory cytokines in response to both P. acnes and a TLR2 ligand control, the CD209+ cells responded only to P. acnes. The addition of all-trans retinoic acid, a commonly used agent for the treatment of acne, directly induced differentiation of monocytes into CD209+ macrophages and enhanced the P. acnes-mediated differentiation of the CD209+ subset. Therefore, the differentiation of monocytes into CD209+ macrophages and CD1b+ dendritic cells distinctly mediate the innate immune response to P. acnes.
IL-13 Attenuates Gastrointestinal Candidiasis in Normal and Immunodeficient RAG-2−/− Mice via Peroxisome Proliferator-Activated Receptor-γ Activation
Authors: Coste Agnès , Lagane Céline , Filipe Cédric , Authier Hélène , Galès Amandine , Bernad José , Douin-Echinard Victorine , Lepert Jean-Claude , Balard Patricia , Linas Marie-Denise , Arnal Jean-François , Auwerx Johan , Pipy Bernard
Published: April 2014

We recently demonstrated that in vitro peroxisome proliferator-activated receptor-γ (PPARγ) activation of mouse peritoneal macrophages by IL-13 or PPARγ ligands promotes uptake and killing of Candida albicans through mannose receptor overexpression. In this study, we demonstrate that i.p. treatment of immunocompetent and immunodeficient (RAG-2−/−) mice with natural and synthetic PPARγ-specific ligands or with IL-13 decreases C. albicans colonization of the gastrointestinal (GI) tract 8 days following oral infection with the yeast. We also showed that Candida GI infection triggers macrophage recruitment in cecum mucosa. These mucosal macrophages, as well as peritoneal macrophages, overexpress the mannose receptor after IL-13 and rosiglitazone treatments. The treatments promote macrophage activation against C. albicans as suggested by the increased ability of peritoneal macrophages to phagocyte C. albicans and to produce reactive oxygen intermediates after yeast challenge. These effects on C. albicans GI infection and on macrophage activation are suppressed by treatment of mice with GW9662, a selective PPARγ antagonist, and are reduced in PPARγ+/− mice. Overall, these data demonstrate that IL-13 or PPARγ ligands attenuate C. albicans infection of the GI tract through PPARγ activation and hence suggest that PPARγ ligands may be of therapeutic value in esophageal and GI candidiasis in immunocompromised patients.
Suppression of Proteoglycan-Induced Arthritis by Anti-CD20 B Cell Depletion Therapy Is Mediated by Reduction in Autoantibodies and CD4+ T Cell Reactivity
Authors: Hamel Keith , Doodes Paul , Cao Yanxia , Wang Yumei , Martinson Jeffrey , Dunn Robert , Kehry Marilyn R. , Farkas Balint , Finnegan Alison
Published: April 2014

B cells have been implicated in the pathogenesis of rheumatoid arthritis (RA) since the discovery of RA as an autoimmune disease. There is renewed interest in B cells in RA based on the clinical efficacy of B cell depletion therapy in RA patients. Although, reduced titers of rheumatoid factor and anti-cyclic citrullinated peptide Abs are recorded, the mechanisms that convey clinical improvement are incompletely understood. In the proteoglycan-induced arthritis (PGIA) mouse model of RA, we reported that Ag-specific B cells have two important functions in the development of arthritis. PG-specific B cells are required as autoantibody-producing cells as well as Ag-specific APCs. Herein we report on the effects of anti-CD20 mAb B cell depletion therapy in PGIA. Mice were sensitized to PG and treated with anti-CD20 Ab at a time when PG-specific autoantibodies and T cell activation were evident but before acute arthritis. In mice treated with anti-CD20 mAb, development of arthritis was significantly reduced in comparison to control mAb-treated mice. B cell depletion reduced the PG-specific autoantibody response. Furthermore, there was a significant reduction in the PG-specific CD4+ T cell recall response as well as significantly fewer PG-specific CD4+ T cells producing IFN-γ and IL-17, but not IL-4. The reduction in PG-specific T cells was confirmed by the inability of CD4+ T cells from B cell-depleted mice to adoptively transfer disease into SCID mice. Overall, B cell depletion during PGIA significantly reduced disease and inhibited both autoreactive B cell and T cell function.
The Synthetic Peroxisome Proliferator-Activated Receptor-γ Agonist Ciglitazone Attenuates Neuroinflammation and Accelerates Encapsulation in Bacterial Brain Abscesses
Authors: Kielian Tammy , Syed Mohsin Md. , Liu Shuliang , Phulwani Nirmal K. , Phillips Napoleon , Wagoner Gail , Drew Paul D. , Esen Nilufer
Published: April 2014

Brain abscesses result from a pyogenic parenchymal infection commonly initiated by Gram-positive bacteria such as Staphylococcus aureus. Although the host immune response elicited following infection is essential for effective bacterial containment, this response also contributes to the significant loss of brain parenchyma by necrosis that may be reduced by modulating the inflammatory response. Ciglitazone, a PPAR-γ agonist with anti-inflammatory properties, was evaluated for its ability to influence the course of brain abscess development when treatment was initiated 3 days following infection. Interestingly, abscess-associated bacterial burdens were significantly lower following ciglitazone administration, which could be explained, in part, by the finding that ciglitazone enhanced S. aureus phagocytosis by microglia. In addition, ciglitazone attenuated the expression of select inflammatory mediators during brain abscess development including inducible NO synthase, TNF-α, IL-1β, CXCL2, and CCL3. Unexpectedly, ciglitazone also accelerated brain abscess encapsulation, which was typified by the heightened expression of fibronectin and α-smooth muscle actin-positive myofibroblasts. Collectively, through its ability to attenuate excessive inflammation and accelerate abscess encapsulation, ciglitazone may effectively sequester brain abscesses and limit bacterial dissemination.
Macrophages Produce TGF-β-Induced (β-ig-h3) following Ingestion of Apoptotic Cells and Regulate MMP14 Levels and Collagen Turnover in Fibroblasts
Authors: Nacu Natalia , Luzina Irina G. , Highsmith Kendrick , Lockatell Virginia , Pochetuhen Kerill , Cooper Zachary A. , Gillmeister Michael P. , Todd Nevins W. , Atamas Sergei P.
Published: April 2014

Phagocytic clearance of apoptotic cells by macrophages is an essential part in the resolution of inflammation. It coincides with activation of repair mechanisms, including accumulation of extracellular matrix. A possible link between clearance of apoptotic debris and accumulation of extracellular matrix has not been investigated. Production of collagen was measured in primary fibroblasts cocultured with macrophages. Ingestion of apoptotic cells by monocyte-derived macrophages led to up-regulation of collagen. Direct contact between macrophages and fibroblasts was not required for collagen up-regulation. Macrophages produced TGF-β following ingestion of apoptotic cells, but the levels of this cytokine were lower than those required for a significant up-regulation of collagen. Simultaneously, the levels of TGF-β-induced (TGFBI), or keratoepithelin/BIGH3, mRNA and protein were increased. In contrast, primary alveolar macrophages stimulated collagen production without exposure to apoptotic cells; there was no further increase in the levels of TGFBI, mRNA or protein, or collagen after ingestion of apoptotic cells. Stimulation of fibroblasts with TGFBI down-regulated MMP14 levels, decreased DNA binding by p53, increased DNA binding by PU.1, and up-regulated collagen protein but not mRNA levels. Overexpression of MMP14 or p53, or small interfering RNA-mediated inhibition of PU.1 led to an increase in MMP14 and a decline in collagen levels, whereas small interfering RNA-mediated inhibition of MMP14 led to elevation of collagen levels. In conclusion, monocyte-derived but not alveolar macrophages produce TGFBI following ingestion of apoptotic cells, leading to the down-regulation of MMP14 levels in fibroblasts through a mechanism involving p53 and PU.1, and to subsequent accumulation of collagen.
Human CD4+ T Cells Recognize an Epitope within α-Fetoprotein Sequence and Develop into TGF-β-Producing CD4+ T Cells
Authors: Alisa Akeel , Boswell Sandra , Pathan Ansar A. , Ayaru Lakshmana , Williams Roger , Behboudi Shahriar
Published: April 2014

There is limited information on the influence of tumor growth on the expansion of tumor-specific TGF-β-producing CD4+ T cells in humans. α-Fetoprotein (AFP) is an oncofetal Ag and has intrinsic immunoregulatory properties. In this study, we report the identification and characterization of subsets of CD4+ T cells that recognize an epitope within the AFP sequence (AFP46–55) and develop into TGF-β-producing CD4+ T cells. In a peptide-specific and dose-dependent manner, AFP46–55 CD4+ T cells produce TGF-β, GM-CSF, and IL-2 but not Th1-, Th2-, Th17-, or Tr1-type cytokines. These cells express CTLA-4 and glucocorticoid-induced TNR receptor and inhibit T cell proliferation in a contact-dependent manner. In this study, we show that the frequency of AFP46–55 CD4+ T cells is significantly higher (p = 001) in patients with hepatocellular carcinoma than in healthy donors, suggesting that these cells are expanded in response to tumor Ag. In contrast, tumor necrosis-inducing treatments that are shown to improve survival rate can shift the Th1/TGF-β-producing CD4+ T cell balance in favor of Th1 responses. Our data demonstrate that tumor Ags may contain epitopes which activate the expansion of inducible regulatory T cells, leading to evasion of tumor control.
Zebrafish TRIF, a Golgi-Localized Protein, Participates in IFN Induction and NF-κB Activation
Authors: Fan Shan , Chen Shangwu , Liu Yanhui , Lin Yiqun , Liu Hui , Guo Lei , Lin Bin , Huang Shengfeng , Xu Anlong
Published: April 2014

The antiviral immune responses were triggered by the innate immune recognition of viral infection. The type I IFNs (IFN-β and IFN-α) are the key cytokines produced upon viral infection and consequently link innate immunity with adaptive immunity. A main antiviral system in mammals is TRIF-dependent TLRs pathway, but the TRIF-independent RIG-I pathway, has also been discovered recently. In this manuscript, our study focuses on the functional characterization of zebrafish TRIF based on the comparison of its sequence and functional evolution from zebrafish to mammals. Our experimental results show that the full length cDNA of zebrafish TRIF cloned by RACE-PCR approach encodes a protein of 556 amino acids. Luciferase reporter assay confirms that zebrafish TRIF is able to induce the IFN promoter as well as activate NF-κB response promoter. The IFN induction function of zebrafish TRIF is abolished when Ala359 is mutated to Pro or His. Laser confocal microscopy shows that zebrafish TRIF is colocalized with a Golgi apparatus marker, implying its unique subcellular localization in Golgi apparatus. In zebrafish, the mRNA expression of molecules participating in RIG-I pathway are much more sensitive and specific to polyinosine-polycytidylic acid induction compared with those in TRIF-dependent antiviral pathway. The TRIF-dependent TLR4 IFN induction signaling appears not to be functional in zebrafish, since IFN expression cannot be up-regulated by LPS. These two striking findings from de novo ligand induction experiments suggest a novel antiviral mechanism in zebrafish.
Unique Effects of KIT D816V in BaF3 Cells: Induction of Cluster Formation, Histamine Synthesis, and Early Mast Cell Differentiation Antigens
Authors: Mayerhofer Matthias , Gleixner Karoline V. , Hoelbl Andrea , Florian Stefan , Hoermann Gregor , Aichberger Karl J. , Bilban Martin , Esterbauer Harald , Krauth Maria-Theresa , Sperr Wolfgang R. , Longley Jack B. , Kralovics Robert , Moriggl Richard , Zappulla Jacques , Liblau Roland S. , Schwarzinger Ilse , Sexl Veronika , Sillaber Christian , Valent Peter
Published: April 2014

Oncogenic tyrosine kinases (TK) usually convert growth factor-dependent cells to factor independence with autonomous proliferation. However, TK-driven neoplasms often are indolent and characterized by cell differentiation rather than proliferation. A prototype of an indolent TK-driven neoplasm is indolent systemic mastocytosis. We found that the D816V-mutated variant of KIT, a TK detectable in most patients with systemic mastocytosis, induces cluster formation and expression of several mast cell differentiation and adhesion Ags, including microphthalmia transcription factor, IL-4 receptor, histamine, CD63, and ICAM-1 in IL-3-dependent BaF3 cells. By contrast, wild-type KIT did not induce cluster formation or mast cell differentiation Ags. Additionally, KIT D816V, but not wild-type KIT, induced STAT5 activation in BaF3 cells. However, despite these intriguing effects, KIT D816V did not convert BaF3 cells to factor-independent proliferation. Correspondingly, BaF3 cells with conditional expression of KIT D816V did not form tumors in nude mice. Together, the biologic effects of KIT D816V in BaF3 cells match strikingly with the clinical course of indolent systemic mastocytosis and with our recently established transgenic mouse model, in which KIT D816V induces indolent mast cell accumulations but usually does not induce a malignant mast cell disease. Based on all these results, it is hypothesized that KIT D816V as a single hit may be sufficient to cause indolent systemic mastocytosis, whereas additional defects may be required to induce aggressive mast cell disorders.
Homeostatic Intracellular-Free Ca2+ Is Permissive for Rap1-Mediated Constitutive Activation of α4 Integrins on Eosinophils
Authors: Ulfman Laurien H. , Kamp Vera M. , van Aalst Corneli W. , Verhagen Liesbeth P. , Sanders Marjolein E. , Reedquist Kris A. , Buitenhuis Miranda , Koenderman Leo
Published: April 2014

Although much progress has been made in understanding the molecular mechanisms underlying agonist-induced “inside-out” activation of integrins, little is known about how basal levels of integrin function are maintained. This is particularly important for nonactivated eosinophils, where intermediate activation of α4β1 integrin supports recruitment to endothelial cells under flow conditions. Depletion of intracellular Ca2+ and pharmacological inhibition of phospholipase C (but not other intracellular signaling molecules, including PI3K, ERK1/2, p38 MAPK, and tyrosine kinase activity) abrogated basal α4 integrin activity in nonactivated eosinophils. Basal α4 integrin activation was associated with activation of the small GTPase Rap1, a known regulator of agonist-induced integrin function. Basal Rap activation was dependent upon phospholipase C, but not intracellular Ca2+. However, depletion of intracellular Ca2+ in CD34+ hematopoietic progenitor cells abolished RapV12-mediated induction of α4 integrin activity. Thus, residual Rap activity or constitutively active Rap activity in Ca2+-depleted cells is not sufficient to induce α4 integrin activation. These data suggest that activation of functional α4 integrin activity in resting eosinophils is mediated by Rap1 provided that the intracellular-free Ca2+ is at a normal homeostatic concentration.
Utilization of Fc Receptors as a Mucosal Vaccine Strategy against an Intracellular Bacterium, Francisella tularensis
Authors: Rawool Deepak B. , Bitsaktsis Constantine , Li Ying , Gosselin Diane R. , Lin Yili , Kurkure Nitin V. , Metzger Dennis W. , Gosselin Edmund J.
Published: April 2014

Numerous studies have demonstrated that targeting Ag to Fc receptors (FcR) on APCs can enhance humoral and cellular immunity. However, studies are lacking that examine both the use of FcR-targeting in generating immune protection against infectious agents and the use of FcRs in the induction of mucosal immunity. Francisella tularensis is a category A intracellular mucosal pathogen. Thus, intense efforts are underway to develop a vaccine against this organism. We hypothesized that protection against mucosal infection with F. tularensis would be significantly enhanced by targeting inactivated F. tularensis live vaccine strain (iFt) to FcRs at mucosal sites, via intranasal immunization with mAb-iFt complexes. These studies demonstrate for the first time that: 1) FcR-targeted immunogen enhances immunogen-specific IgA production and protection against subsequent infection in an IgA-dependent manner, 2) FcγR and neonatal FcR are crucial to this protection, and 3) inactivated F. tularensis, when targeted to FcRs, enhances protection against the highly virulent SchuS4 strain of F. tularensis, a category A biothreat agent. In summary, these studies show for the first time the use of FcRs as a highly effective vaccination strategy against a highly virulent mucosal intracellular pathogen.
NK Cells Play a Critical Protective Role in Host Defense against Acute ExtracellularStaphylococcus aureusBacterial Infection in the Lung
Authors: Small Cherrie-Lee , McCormick Sarah , Gill Navkiran , Kugathasan Kapilan , Santosuosso Michael , Donaldson Nickett , Heinrichs David E. , Ashkar Ali , Xing Zhou
Published: April 2014

Staphylococcus aureus remains a common cause of nosocomial bacterial infections and are often antibiotic resistant. The role of NK cells and IL-15 and their relationship in host defense against extracellular bacterial pathogens including S. aureus remain unclear. We have undertaken several approaches to address this issue using wild type (WT), IL-15 gene knock-out (KO), and NK cell-depleted mouse models. Upon pulmonary staphylococcal infection WT mice had markedly increased activated NK cells, but not NKT or γδ T cells, in the airway lumen that correlated with IL-15 production in the airway and with alveolar macrophages. In vitro exposure to staphylococcal products and/or coculture with lung macrophages directly activated NK cells. In contrast, lung macrophages better phagocytosed S. aureus in the presence of NK cells. In sharp contrast to WT controls, IL-15 KO mice deficient in NK cells were found to be highly susceptible to pulmonary staphylococcal infection despite markedly increased neutrophils and macrophages in the lung. In further support of these findings, WT mice depleted of NK cells were similarly susceptible to staphylococcal infection while they remained fully capable of IL-15 production in the lung at levels similar to those of NK-competent WT hosts. Our study thus identifies a critical role for NK cells in host defense against pulmonary extracellular bacterial infection and suggests that IL-15 is involved in this process via its indispensable effect on NK cells, but not other innate cells. These findings hold implication for the development of therapeutics in treating antibiotic-resistant S. aureus infection.
The Tyrosine Kinase Pyk2 Mediates Lipopolysaccharide-Induced IL-8 Expression in Human Endothelial Cells
Authors: Anand Appakkudal R. , Cucchiarini Magali , Terwilliger Ernest F. , Ganju Ramesh K.
Published: April 2014

Secretion of proinflammatory cytokines by LPS activated endothelial cells contributes substantially to the pathogenesis of sepsis. However, the mechanism involved in this process is not well understood. In the present study, we determined the role of a nonreceptor proline-rich tyrosine kinase, Pyk2, in LPS-induced IL-8 (CXCL8) production in endothelial cells. First, we observed a marked activation of Pyk2 in response to LPS. Furthermore, inhibition of Pyk2 activity in these cells by transduction with the catalytically inactive Pyk2 mutant, transfection with Pyk2-specific small interfering RNA, or treatment with Tyrphostin A9 significantly blocked LPS-induced IL-8 production. The supernatants of LPS-stimulated cells exhibiting attenuated Pyk2 activity blocked transendothelial neutrophil migration in comparison to the supernatants of LPS-treated controls, thus confirming the inhibition of functional IL-8 production. Investigations into the molecular mechanism of this pathway revealed that LPS activates Pyk2 leading to IL-8 production through the TLR4. In addition, we identified the p38 MAPK pathway to be a critical step downstream of Pyk2 during LPS-induced IL-8 production. Taken together, these results demonstrate a novel role for Pyk2 in LPS-induced IL-8 production in endothelial cells.
CRTH2 Plays an Essential Role in the Pathophysiology of Cry j 1-Induced Pollinosis in Mice
Authors: Nomiya Rie , Okano Mitsuhiro , Fujiwara Tazuko , Maeda Megumi , Kimura Yoshinobu , Kino Kosuke , Yokoyama Minehiko , Hirai Hiroyuki , Nagata Kinya , Hara Toshifumi , Nishizaki Kazunori , Nakamura Masataka
Published: April 2014

PGD2 is the major prostanoid produced during the acute phase of allergic reactions. Two PGD2 receptors have been isolated, DP and CRTH2 (chemoattractant receptor-homologous molecule expressed on Th2 cells), but whether they participate in the pathophysiology of allergic diseases remains unclear. We investigated the role of CRTH2 in the initiation of allergic rhinitis in mice. First, we developed a novel murine model of pollinosis, a type of seasonal allergic rhinitis. Additionally, pathophysiological differences in the pollinosis were compared between wild-type and CRTH2 gene-deficient mice. An effect of treatment with ramatroban, a CRTH2/T-prostanoid receptor dual antagonist, was also determined. Repeated intranasal sensitization with Cry j 1, the major allergen of Cryptomeria japonica pollen, in the absence of adjuvants significantly exacerbated nasal hyperresponsive symptoms, Cry j 1-specific IgE and IgG1 production, nasal eosinophilia, and Cry j 1-induced in vitro production of IL-4 and IL-5 by submandibular lymph node cells. Additionally, CRTH2 mRNA in nasal mucosa was significantly elevated in Cry j 1-sensitized mice. Following repeated intranasal sensitization with Cry j 1, CRTH2 gene-deficient mice had significantly weaker Cry j 1-specific IgE/IgG1 production, nasal eosinophilia, and IL-4 production by submandibular lymph node cells than did wild-type mice. Similar results were found in mice treated with ramatroban. These results suggest that the PGD2-CRTH2 interaction is elevated following sensitization and plays a proinflammatory role in the pathophysiology of allergic rhinitis, especially pollinosis in mice.
Production of Type VI Collagen by Human Macrophages: A New Dimension in Macrophage Functional Heterogeneity
Authors: Schnoor Michael , Cullen Paul , Lorkowski Julia , Stolle Katrin , Robenek Horst , Troyer David , Rauterberg Jürgen , Lorkowski Stefan
Published: April 2014

Macrophages derived from human blood monocytes perform many tasks related to tissue injury and repair. The main effect of macrophages on the extracellular matrix is considered to be destructive in nature, because macrophages secrete metalloproteinases and ingest foreign material as part of the remodeling process that occurs in wound healing and other pathological conditions. However, macrophages also contribute to the extracellular matrix and hence to tissue stabilization both indirectly, by inducing other cells to proliferate and to release matrix components, and directly, by secreting components of the extracellular matrix such as fibronectin and type VIII collagen, as we have recently shown. We now report that monocytes and macrophages express virtually all known collagen and collagen-related mRNAs. Furthermore, macrophages secrete type VI collagen protein abundantly, depending upon their mode of activation, stage of differentiation, and cell density. The primary function of type VI collagen secreted by macrophages appears to be modulation of cell-cell and cell-matrix interactions. We suggest that the production of type VI collagen is a marker for a nondestructive, matrix-conserving macrophage phenotype that could profoundly influence physiological and pathophysiological conditions in vivo.
In Vitro and In Vivo Down-Regulation of Regulatory T Cell Activity with a Peptide Inhibitor of TGF-β1
Authors: Gil-Guerrero Lucía , Dotor Javier , Huibregtse Inge Louise , Casares Noelia , López-Vázquez Ana Belén , Rudilla Francesc , Riezu-Boj José Ignacio , López-Sagaseta Jacinto , Hermida José , Van Deventer Sander , Bezunartea Jaione , Llopiz Diana , Sarobe Pablo , Prieto Jesús , Borrás-Cuesta Francisco , Lasarte Juan José
Published: April 2014

Down-regulation of CD4+CD25+ regulatory T (Treg) cell function might be beneficial to enhance the immunogenicity of viral and tumor vaccines or to induce breakdown of immunotolerance. Although the mechanism of suppression used by Treg cells remains controversial, it has been postulated that TGF-β1 mediates their immunosuppressive activity. In this study, we show that P17, a short synthetic peptide that inhibits TGF-β1 and TGF-β2 developed in our laboratory, is able to inhibit Treg activity in vitro and in vivo. In vitro studies demonstrate that P17 inhibits murine and human Treg-induced unresponsiveness of effector T cells to anti-CD3 stimulation, in an MLR or to a specific Ag. Moreover, administration of P17 to mice immunized with peptide vaccines containing tumor or viral Ags enhanced anti-vaccine immune responses and improved protective immunogenicity against tumor growth or viral infection or replication. When CD4+ T cells purified from OT-II transgenic mice were transferred into C57BL/6 mice bearing s.c. EG.7-OVA tumors, administration of P17 improved their proliferation, reduced the number of CD4+Foxp3+ T cells, and inhibited tumor growth. Also, P17 prevented development of immunotolerance induced by oral administration of OVA by genetically modified Lactococcus lactis in DO11.10 transgenic mice sensitized by s.c. injection of OVA. These findings demonstrate that peptide inhibitors of TGF-β may be a valuable tool to enhance vaccination efficacy and to break tolerance against pathogens or tumor Ags.
Coligation of the Hepatitis C Virus Receptor CD81 with CD28 Primes Naive T Lymphocytes to Acquire Type 2 Effector Function
Authors: Serra Alessandro , Nuti Sandra , Tavarini Simona , Sammicheli Chiara , Rosa Domenico , Saletti Giulietta , Soldaini Elisabetta , Abrignani Sergio , Wack Andreas
Published: April 2014

Costimuli provide supplementary signals required by naive T cells to become fully activated upon Ag encounter. Tetraspanins are a large family of transmembrane proteins that can costimulate T cells when engaged in vitro. In this study, we describe for the first time that coligation of the tetraspanins CD81, CD82, or CD9 with the costimulatory molecule CD28 in vitro leads to proliferation of naive T cells. When activated through this pathway, both CD4+ and CD8+ naive T cells differentiate into type 2 effector cells, which produce IL-4, IL-5, IL-13, and IL-10, together with IL-2 and TNF-α, but little to no IFN-γ. These effector cells descend from precursors that display early and strong production of IL-4, STAT6 phosphorylation, and up-regulation of the transcription factor GATA-3, suggesting a direct skewing toward Th2 differentiation without a Th0 intermediate. The hepatitis C virus envelope protein E2 is the only ligand known for CD81. Therefore, we propose that this new type of Ag-independent T cell activation may occur in hepatitis C virus-infected individuals, contributing to liver inflammation, impaired type 1 immune responses, and recurrent flares of type 2 immunity associated with chronic infection.
Dual Functions of Bruton’s Tyrosine Kinase and Tec Kinase during Fcγ Receptor-Induced Signaling and Phagocytosis
Authors: Jongstra-Bilen Jenny , Puig Cano Adrianet , Hasija Manvi , Xiao Haiyan , Smith C. I. Edvard , Cybulsky Myron I.
Published: April 2014

Tec family nonreceptor tyrosine kinases are expressed by hematopoietic cells, activate phospholipase C (PLC)γ, and regulate cytoskeletal rearrangement, yet their role in FcγR-induced signaling and phagocytosis remains unknown. We demonstrate in this study that Bruton’s tyrosine kinase (Btk) and Tec, the only Tec kinases expressed by RAW 264.7 cells, are activated throughout phagocytosis. Activated Btk and Tec kinase accumulate at an early stage at the base of phagocytic cups and inhibition of their activity by the specific inhibitor LFM-A13 or expression by small interfering RNA significantly inhibited FcγR-induced phagocytosis. Similarly, a significant role for these kinases in phagocytosis was found in primary macrophages. FcγR-induced activation of Mac-1, which is required for optimal phagocytosis, was markedly inhibited and our findings suggest that the roles of kinases Btk and Tec in Mac-1 activation account for their functions in the early stages of phagocytosis. Initial activation of PLCγ2, the predominant PLC isoform in RAW 264.7 cells, is dependent on Syk. In contrast, a late and prolonged activation of PLCγ2 was dependent on Btk and Tec. We found accumulation of diacylglycerol (DAG), a PLCγ product, in phagosome membranes, and activated Btk, but not Tec, colocalized with phagosomal DAG. Inhibition of Tec family kinase activity increased the level of DAG in phagosomes, suggesting a negative regulatory role for Btk. Tec, in contrast, clustered at sites near phagosome formation. In summary, we elucidated that Tec family kinases participate in at least two stages of FcγR-mediated phagocytosis: activation of Mac-1 during ingestion, and after phagosome formation, during which Btk and Tec potentially have distinct roles.
Autoreactive T Cells Escape Clonal Deletion in the Thymus by a CD24-Dependent Pathway
Authors: Carl Joseph W. , Liu Jin-Qing , Joshi Pramod S. , El-Omrani Hani Y. , Yin Lijie , Zheng Xincheng , Whitacre Caroline C. , Liu Yang , Bai Xue-Feng
Published: April 2014

Despite negative selection in the thymus, significant numbers of autoreactive T cells still escape to the periphery and cause autoimmune diseases when immune regulation goes awry. It is largely unknown how these T cells escape clonal deletion. In this study, we report that CD24 deficiency caused deletion of autoreactive T cells that normally escape negative selection. Restoration of CD24 expression on T cells alone did not prevent autoreactive T cells from deletion; bone marrow chimera experiments suggest that CD24 on radio-resistant stromal cells is necessary for preventing deletion of autoreactive T cells. CD24 deficiency abrogated the development of experimental autoimmune encephalomyelitis in transgenic mice with a TCR specific for a pathogenic autoantigen. The role of CD24 in negative selection provides a novel explanation for its control of genetic susceptibility to autoimmune diseases in mice and humans.