bluuebunny/crossref_metadata_2025 huggingface dataset:
Tobacco Smoking Inhibits Expression of Proinflammatory Cytokines and Activation of IL-1R-Associated Kinase, p38, and NF-κB in Alveolar Macrophages Stimulated with TLR2 and TLR4 Agonists
Authors: Chen Haiyan , Cowan Mark J. , Hasday Jeffrey D. , Vogel Stefanie N. , Medvedev Andrei E.
Published: April 2014

Tobacco smoking has been associated with impaired pulmonary functions and increased incidence of infections; however, mechanisms that underlie these phenomena are poorly understood. In this study, we examined whether smokers’ alveolar macrophages (AM) exhibit impaired sensing of bacterial components via TLR2 and TLR4 and determined the effect of smoking on expression levels of TLR2, TLR4 and coreceptors, and activation of signaling intermediates. Smokers’ AMs exhibited reduced gene expression and secretion of proinflammatory cytokines (TNF-α, IL-1β, IL-6) and chemokines (RANTES and IL-8) upon stimulation with TLR2 and TLR4 agonists, S-[2,3-bis(palmitoyloxy)-(2-RS)-propyl]-N-palmitoyl-(R)-Cys-(S)-Ser-Lys4-OH trihydrochloride (Pam3Cys), and LPS, whereas expression of anti-inflammatory cytokines (IL-10 and IL-1 receptor antagonist) was not affected. TLR3 activation with polyinosinic-polycytidylic acid led to comparable or even higher cytokine responses in smokers’ AMs, indicating that smoking-induced suppression does not affect all TLRs. Comparable expression of cytokines and chemokines was detected in PBMC and purified monocytes obtained from smokers and nonsmokers, demonstrating that the suppressive effect of smoking is restricted to the lung. TLR2/4-inducible IL-1R-associated kinase-1 (IRAK-1) and p38 phosphorylation and NF-κB activation was suppressed in smokers’ AMs, whereas TLR2, TLR4, CD14, MD-2 mRNA levels, and TLR4 protein expression were not altered. These data suggest that changes in expression and/or activities of signaling intermediates at the postreceptor level account for smoking-induced immunosuppression. Thus, exposure of AMs to tobacco smoke induces a hyporesponsive state similar to endotoxin tolerance as manifested by inhibited TLR2/4-induced expression of proinflammatory cytokines, chemokines, and impaired activation of IRAK-1, p38, and NF-κB, resulting in suppressed expression of proinflammatory mediators.
VAP-1-Deficient Mice Display Defects in Mucosal Immunity and Antimicrobial Responses: Implications for Antiadhesive Applications
Authors: Koskinen Kaisa , Nevalainen Suvi , Karikoski Marika , Hänninen Arno , Jalkanen Sirpa , Salmi Marko
Published: April 2014

VAP-1, an ecto-enzyme expressed on the surface of endothelial cells, is involved in leukocyte trafficking between the blood and tissues under physiological and pathological conditions. In this study, we used VAP-1-deficient mice to elucidate whether absence of VAP-1 alters the immune system under normal conditions and upon immunization and microbial challenge. We found that VAP-1-deficient mice display age-dependent paucity of lymphocytes, in the Peyer’s patches of the gut. IgA concentration in serum was also found to be lower in VAP-1−/− animals than in wild-type mice. Although there were slightly less CD11a on B and T cells isolated from VAP-1-deficient mice than on those from wild-type mice, there were no differences in the expression of gut-homing-associated adhesion molecules or chemokine receptors. Because anti-VAP-1 therapies are being developed for clinical use to treat inflammation, we determined the effect of VAP-1 deletion on useful immune responses. Oral immunization with OVA showed defective T and B cell responses in VAP-1-deficient mice. Antimicrobial immune responses against Staphylococcus aureus and coxsackie B4 virus were also affected by the absence of VAP-1. Importantly, when the function of VAP-1 was acutely neutralized using small molecule enzyme inhibitors and anti-VAP-1 Abs rather than by gene deletion, no significant impairment in antimicrobial control was detected. In conclusion, VAP-1-deficient mice have mild deviations in the mucosal immune system and therapeutic targeting of VAP-1 does not appear to cause a generalized increase in the risk of infection.
The Role of Dendritic Cells in the Development of Acute Dextran Sulfate Sodium Colitis
Authors: Berndt Bradford E. , Zhang Min , Chen Gwo-Hsiao , Huffnagle Gary B. , Kao John Y.
Published: April 2014

Dendritic cells (DCs) are essential mediators of the host immune response to surrounding microbes. In this study, we investigate the role of DCs in the pathogenesis of a widely used colitis model, dextran sulfate sodium-induced colitis. The effect of dextran sulfate sodium on the production of proinflammatory cytokines and chemokines by bone marrow-derived DCs (BM-DCs) was analyzed. BM-DCs were adoptively transferred into C57BL/6 mice or DCs were ablated using transgenic CD11c-DTR/GFP mice before treatment with 5% dextran sulfate sodium in drinking water. We found that dextran sulfate sodium induced production of proinflammatory cytokines (IL-12 and TNF-α) and chemokines (KC, MIP-1α, MIP-2, and MCP-1) by DCs. Adoptive transfer of BM-DCs exacerbated dextran sulfate sodium colitis while ablation of DCs attenuated the colitis. We conclude that DCs are critical in the development of acute dextran sulfate sodium colitis and may serve a key role in immune balance of the gut mucosa.
The Physiology of Leukocyte Recruitment: An In Vivo Perspective
Authors: Petri Björn , Phillipson Mia , Kubes Paul
Published: April 2014

The mechanisms of leukocyte recruitment have been studied extensively in vitro and have shed light on the basic molecular structure-function relationship of adhesion and signaling molecules involved in this essential immune response. This review will summarize how these in vitro observations extend to leukocyte behavior in inflamed blood vessels in the microcirculation. We highlight physiological results that might not have been predicted from in vitro systems. Special attention is placed on the physiology of rolling, adhesion, and intralumenal crawling in blood vessels. The importance of the glycocalyx, secondary tethers, shear, and the microenvironment are discussed. Docking structures forming rings of adhesion molecules together with a novel endothelial dome-like structure in vivo during transmigration are highlighted. Transcellular and paracellular emigration out of inflamed blood vessels is also discussed. The last section highlights leukocyte recruitment in some organs that do not always follow the accepted paradigm of leukocyte recruitment.
Cutting Edge: MHC Class I–Ly49 Interaction Regulates Neuronal Function
Authors: Zohar Ofer , Reiter Yoram , Bennink Jack R. , Lev Avital , Cavallaro Sebastiano , Paratore Sabrina , Pick Chaim G. , Brooker Gary , Yewdell Jonathan W.
Published: April 2014

MHC class I molecules (MHC-I) have been implicated in nervous system development in the mouse. In this study we present evidence for the interaction of MHC-I with the NK cell receptor Ly49 in primary cortical neuronal cultures. We show that MHC-I and Ly49 are expressed on neuronal soma and axon surfaces, with Ly49 also present on dendrites. Anti-MHC-I Abs reduce synapsin-I expression and enhance neurite outgrowth and neuronal death. Conversely, anti-Ly49 mAbs increase synapsin-I expression, reduce neurite outgrowth, and promote neuron viability. Because we show that Ly49 genes are selectively expressed in the adult brain, these findings suggest an unsuspected role for the MHC-I-Ly49 interaction in the development and function of the brain.
Functional Consequences of Interactions between Human NKR-P1A and Its Ligand LLT1 Expressed on Activated Dendritic Cells and B Cells
Authors: Rosen David B. , Cao Wei , Avery Danielle T. , Tangye Stuart G. , Liu Yong-Jun , Houchins J. P. , Lanier Lewis L.
Published: April 2014

Lectin-like transcript-1 (LLT1) (also named osteoclast inhibitory lectin or CLEC2D) is a ligand for the human NKR-P1A (CD161) receptor, present on NK cells and T cells. To further understand the physiological relevance of this interaction, we developed mAbs against LLT1, characterized the expression pattern of LLT1, and explored the functional consequence of LLT1 engagement of the NKR-P1A receptor on NK cells and T cells. LLT1 is expressed on TLR-activated plasmacytoid dendritic, TLR-activated monocyte-derived dendritic cells, and on B cells stimulated through TLR9, surface Ig, or CD40. Interactions between NKR-P1A on NK cells and LLT1 on target cells inhibit NK cell-mediated cytotoxicity and cytokine production and can inhibit TNF-α production by TCR-activated NKR-P1A+ CD8+ T cells. In contrast, NKR-P1A failed to inhibit or augment the TCR-dependent activation of NKR-P1A-bearing CD4+ T cells. Expression of LLT1 on activated dendritic cells and B cells suggests that it might regulate the cross-talk between NK cells and APCs.
Activation of a TGF-β-Specific Multistep Gene Expression Program in Mature Macrophages Requires Glucocorticoid-Mediated Surface Expression of TGF-β Receptor II
Authors: Gratchev Alexei , Kzhyshkowska Julia , Kannookadan Sheila , Ochsenreiter Miriam , Popova Anna , Yu Xiaolei , Mamidi Srinivas , Stonehouse-Usselmann Eugenia , Muller-Molinet Isabelle , Gooi LiMing , Goerdt Sergij
Published: April 2014

Alternatively activated (M2) macrophages regulate steady state-, cancer-, and inflammation-related tissue remodeling. They are induced by Th2-cytokines and glucocorticoids (GC). The responsiveness of mature macrophages to TGF-β, a cytokine involved in inflammation, cancer, and atherosclerosis, is currently controversial. Recently, we demonstrated that IL-17 receptor B is up-regulated in human monocyte-derived macrophages differentiated in the presence of Th2 cytokines IL-4 and TGF-β1. In this study, we show that mature human macrophages differentiated in the presence of IL-4, and dexamethasone (M2IL-4/GC) but not M2IL-4 responds to TGF-β1 which induced a gene expression program comprising 111 genes including transcriptional/signaling regulators (ID3 and RGS1), immune modulators (ALOX5AP and IL-17 receptor B) and atherosclerosis-related genes (ALOX5AP, ORL1, APOC1, APOC2, and APOE). Analysis of molecular mechanism underlying GC/TGF-β cooperation revealed that surface expression of TGF-βRII was high in M2GC and M2IL-4/GC, but absent from M2IL-4, whereas the expression of TGF-βRI/II mRNA, TGF-βRII total protein, and surface expression of TGF-βRIII were unchanged. GC dexamethasone was essential for increased surface expression of functional TGF-βRII because its effect was observed also in combination with IL-13, M-CSF, and GM-CSF. Prolonged Smad2-mediated signaling observed in TGF-β1-treated M2IL-4/GC was due to insufficient activity of negative feedback mechanism what can be explained by up-regulation of SIRT1, a negative regulator of Smad7, and the retention of TGF-βRII complex on the cell surface. In summary, mature human M2 macrophages made permissive to TGF-β by GC-induced surface expression of TGF-βRII activate in response to TGF-β1, a multistep gene expression program featuring traits of macrophages found within an atherosclerotic lesion.
Human Cytomegalovirus Latent Infection of Myeloid Cells Directs Monocyte Migration by Up-Regulating Monocyte Chemotactic Protein-1
Authors: Stern J. Lewis , Slobedman Barry
Published: April 2014

Following primary infection, human cytomegalovirus (HCMV) establishes a latent infection in hematopoietic cells from which it reactivates to cause serious disease in immunosuppressed patients such as allograft recipients. HCMV is a common cause of disease in newborns and transplant patients and has also been linked with vascular diseases such as primary and post-transplant arteriosclerosis. A major factor in the pathogenesis of vascular disease is the CC chemokine MCP-1. In this study, we demonstrate that granulocyte macrophage progenitors (GMPs) latently infected with HCMV significantly increased expression of MCP-1 and that this phenotype was dependent on infection with viable virus. Inhibitors of a subset of Gα proteins and PI3K inhibited the up-regulation of MCP-1 in latently infected cultures, suggesting that the mechanism underlying this phenotype involves signaling through a G-protein coupled receptor. In GMPs infected with the low passage viral strain Toledo, up-regulated MCP-1 was restricted to a subset of myeloid progenitor cells expressing CD33, HLA-DR, and CD14 but not CD1a, CD15, or CD16, and the increase in MCP-1 was sufficient to enhance migration of CD14+ monocytes to latently infected cells. Latent HCMV-mediated up-regulation of MCP-1 provides a mechanism by which HCMV may contribute to vascular disease during the latent phase of infection or facilitate dissemination of virus upon reactivation from latency.
Stabilized β-Catenin Potentiates Fas-Mediated T Cell Apoptosis
Authors: Huang Zhaofeng , Wang Ruiqing , Xie Huimin , Shang Weirong , Manicassamy Santhakumar , Sun Zuoming
Published: April 2014

In response to Ag stimulation, Ag-specific T cells proliferate and accumulate in the peripheral lymphoid tissues. To avoid excessive T cell accumulation, the immune system has developed mechanisms to delete clonally expanded T cells. Fas/FasL-mediated apoptosis plays a critical role in the deletion of activated peripheral T cells, which is clearly demonstrated by superantigen (staphylococcal enterotoxin B)-induced deletion of Vβ8+ T cells. Using transgenic mice expressing a stabilized β-catenin (β-catTg), we show here that β-catenin was able to enhance apoptosis of activated T cells by up-regulating Fas. In response to staphylococcal enterotoxin B stimulation, β-catTg mice exhibited accelerated deletion of CD4+Vβ8+ T cells compared with wild type mice. Surface Fas levels were significantly higher on activated T cells obtained from β-catTg mice than that from wild type mice. Additionally, T cells from β-catTg mice were more sensitive to apoptosis induced by crosslinking Fas, activation-induced cell death, and to apoptosis induced by cytokine withdrawal. Lastly, β-catenin bound to and stimulated the Fas promoter. Therefore, our data demonstrated that the β-catenin pathway was able to promote the apoptosis of activated T cells in part via up-regulation of Fas.
A Novel B Cell-Mediated Transport of IgE-Immune Complexes to the Follicle of the Spleen
Authors: Hjelm Fredrik , Karlsson Mikael C. I. , Heyman Birgitta
Published: April 2014

Ag administered i.v. to mice along with specific IgE or IgG2a induces higher Ab- and CD4+ T cell responses than Ag administered alone. The IgE effect is completely dependent on the low-affinity receptor for IgE, CD23, whereas the IgG2a effect depends on activating FcγRs. In vitro studies suggest that IgE/Ag is presented more efficiently than Ag alone to CD4+ T cells by CD23+ B cells and that IgG2a/Ag is presented by FcγR+ dendritic cells (DCs). In this study, we investigate in vivo the early events leading to IgE- and IgG2a-mediated enhancement of immune responses. OVA administered i.v. in PBS in combination with specific IgE binds circulating B cells after 5 min and is found in B cell follicles bound to follicular B cells (CD23high) after 30 min. This novel B cell-dependent route of entry is specific for IgE because IgG2a-Ag complexes were trapped in the marginal zone. OVA-specific CD4+ T cells were found at the T-B border in the T cell zones 12 h after immunization both with IgE/OVA or IgG2a/OVA and proliferated vigorously after 3 days. The findings suggest that IgE- and IgG2a-immune complexes are efficient stimulators of early CD4+ T cell responses and that Ag bound to IgE has a specific route for transportation into follicles.
IL-12-Impaired and IL-12-Secreting Dendritic Cells Produce IL-23 upon CD154 Restimulation
Authors: Jasny Edith , Eisenblätter Martin , Mätz-Rensing Kerstin , Tenner-Racz Klara , Tenbusch Matthias , Schrod Annette , Stahl-Hennig Christiane , Moos Verena , Schneider Thomas , Racz Paul , Überla Klaus , Kaup Franz-Josef , Ignatius Ralf
Published: April 2014

Experimental studies in monkeys on the basis of ex vivo-generated, reinjected dendritic cells (DCs) allow investigations of primate DC biology in vivo. To study in vitro and in vivo properties of DCs with a reduced capacity to produce IL-12, we adapted findings obtained in vitro with human cells to the rhesus macaque model. Following exposure of immature monocyte-derived monkey DCs to the immunomodulating synthetic polypeptide glatiramer acetate (GA) and to dibutyryl-cAMP (d-cAMP; i.e., a cAMP enhancer that activates DCs but inhibits the induction of Th1 immune responses), the resulting DCs displayed a mature phenotype with enhanced Ag-specific T cell stimulatory function, notably also for memory Th1 cells. Phosphorylation of p38 MAPK was not induced in GA/d-cAMP-activated DCs. Accordingly, these cells secreted significantly less IL-12p40 (p ≤ 0.001) than did cytokine-activated cells. However, upon restimulation with rhesus macaque CD154, GA/d-cAMP-activated DCs produced IL-12p40/IL-23. Additionally, DCs activated by proinflammatory cytokines following protocols for the generation of cells used in clinical studies secreted significantly more IL-23 upon CD154 restimulation than following prior activation. Two days after intradermal injection, GA/d-cAMP-activated fluorescence-labeled DCs were detected in the T cell areas of draining lymph nodes. When similarly injected, GA/d-cAMP as well as cytokine-activated protein-loaded DCs induced comparable Th immune responses characterized by secretion of IFN-γ, TNF, and IL-17, and transiently expanded FOXP3+ regulatory T cells. Reactivation of primate DCs through CD154 considerably influences their immmunostimulatory properties. This may have a substantial impact on the development of innovative vaccine approaches.
Location and Time-Dependent Control of Rejection by Regulatory T Cells Culminates in a Failure to Generate Memory T Cells
Authors: Carvalho-Gaspar Manuela , Jones Nick D. , Luo Shiqiao , Martin Laurent , Brook Matthew O. , Wood Kathryn J.
Published: April 2014

Adaptive CD25+CD4+ regulatory T cells (Treg) can be induced following exposure to alloantigen and may function alongside naturally occurring Treg to suppress allograft rejection when present in sufficient numbers. However, the location of the Treg as they function in vivo and the mechanisms used to control donor-reactive T cells remains ill-defined. In this study, we used a CD8+ TCR transgenic model of skin allograft rejection to characterize in vivo activity of donor-reactive Treg cells during induction of transplantation tolerance. We demonstrate that, initially after skin transplantation, Treg attenuate the priming of donor-reactive naive CD8+ T cells in the lymphoid tissue draining the graft site. However, with time, peripheral suppression is overcome despite the continued presence of Treg, resulting in the priming of donor-reactive CD8+ T cells and graft infiltration by the resultant effector T cells and induction of a “Tc1-like” intragraft gene expression profile. These intragraft effector CD8+ T cells are then prevented from eliciting rejection by Treg that simultaneously infiltrate the skin allografts, resulting in a failure to generate donor-reactive memory CD8+ T cells. Overall, these data demonstrate for the first time that donor-reactive Treg can suppress allograft rejection using distinct mechanisms at different sites in vivo with the overall outcome of preventing the generation of donor-reactive memory T cells.
Overlapping and Distinct Roles of STAT4 and T-bet in the Regulation of T Cell Differentiation and Allergic Airway Inflammation
Authors: Furuta Shunsuke , Kagami Shin-ichiro , Tamachi Tomohiro , Ikeda Kei , Fujiwara Michio , Suto Akira , Hirose Koichi , Watanabe Norihiko , Saito Yasushi , Iwamoto Itsuo , Nakajima Hiroshi
Published: April 2014

T-bet and STAT4 play critical roles in helper T cell differentiation, especially for Th1 cells. However, it is still unknown about the relative importance and redundancy of T-bet and STAT4 for Th1 differentiation. It is also unknown about their independent role of T-bet and STAT4 in the regulation of allergic airway inflammation. In this study, we addressed these issues by comparing T-bet-deficient (T-bet−/−) mice, STAT4−/− mice, and T-bet- and STAT4-double-deficient (T-bet−/−STAT4−/−) mice on the same genetic background. Th1 differentiation was severely decreased in T-bet−/− mice and STAT4−/− mice as compared with that in wild-type mice, but Th1 differentiation was still observed in T-bet−/− mice and STAT4−/− mice. However, Th1 cells were hardly detected in T-bet−/−STAT4−/− mice. In contrast, the maintenance of Th17 cells was enhanced in T-bet−/− mice but was reduced in STAT4−/− mice and T-bet−/−STAT4−/− mice. In vivo, Ag-induced eosinophil and neutrophil recruitment into the airways was enhanced in T-bet−/− mice but was attenuated in STAT4−/− mice and T-bet−/−STAT4−/− mice. Ag-induced IL-17 production in the airways was also diminished in STAT4−/− mice and T-bet−/−STAT4−/− mice. These results indicate that STAT4 not only plays an indispensable role in T-bet-independent Th1 differentiation but also is involved in the maintenance of Th17 cells and the enhancement of allergic airway inflammation.
DNA Microarray Gene Expression Profile of Marginal Zone versus Follicular B Cells and Idiotype Positive Marginal Zone B Cells before and after Immunization with Streptococcus pneumoniae
Authors: Kin Nicholas W. , Crawford Dianna M. , Liu Jiabin , Behrens Timothy W. , Kearney John F.
Published: April 2014

Marginal zone (MZ) B cells play an important role in the clearance of blood-borne bacterial infections via rapid T-independent IgM responses. We have previously demonstrated that MZ B cells respond rapidly and robustly to bacterial particulates. To determine the MZ-specific genes that are expressed to allow for this response, MZ and follicular (FO) B cells were sort purified and analyzed via DNA microarray analysis. We identified 181 genes that were significantly different between the two B cell populations. Ninety-nine genes were more highly expressed in MZ B cells while 82 genes were more highly expressed in FO B cells. To further understand the molecular mechanisms by which MZ B cells respond so rapidly to bacterial challenge, Id-positive and -negative MZ B cells were sort purified before (0 h) or after (1 h) i.v. immunization with heat-killed Streptococcus pneumoniae, R36A, and analyzed via DNA microarray analysis. We identified genes specifically up-regulated or down-regulated at 1 h following immunization in the Id-positive MZ B cells. These results give insight into the gene expression pattern in resting MZ vs FO B cells and the specific regulation of gene expression in Ag-specific MZ B cells following interaction with Ag.
Signaling Mechanism of HIV-1 gp120 and Virion-Induced IL-1β Release in Primary Human Macrophages
Authors: Cheung Ricky , Ravyn Vipa , Wang Lingshu , Ptasznik Andrzej , Collman Ronald G.
Published: April 2014

HIV-1 envelope glycoprotein gp120 induces, independently of infection, the release of proinflammatory cytokines, including IL-1β from macrophages, that are implicated in the pathogenesis of HIV-associated dementia. However, the signal transduction pathways involved have not been fully defined. Previously, our laboratory reported that soluble gp120 activates multiple protein kinases in primary human monocyte-derived macrophages, including the Src family kinase Lyn, PI3K, and the focal adhesion-related proline-rich tyrosine kinase Pyk2. In this study we showed that gp120 induces IL-1β release from macrophages in a time- and concentration-dependent manner through binding to the chemokine receptor CCR5 and coupling to Giα protein. Using pharmacological inhibitors and small interfering RNA gene knockdown, we demonstrated that concomitant activation of Lyn, Pyk2, and class IA PI3K are required for gp120-induced IL-1β production. By coimmunoprecipitation and immunofluorescence confocal microscopy, we showed that CCR5 activation by gp120 triggered the assembly of a signaling complex involving endogenous Lyn, PI3K, and Pyk2 and is associated with PI3K and Pyk2 translocation from the cytoplasm to the membrane where they colocalized with Lyn. Finally, we demonstrated that virion-associated gp120 induced similar response, as structurally intact whole virions also triggered IL-1β release and re-localization of PI3K and Pyk2. This study identifies a novel signaling mechanism for HIV-1-induced IL-1β production by primary human macrophages that may be involved in the neuropathogenesis of HIV-associated dementia.
The Downstream Transcriptional Enhancer, Ed, Positively Regulates Mouse Igκ Gene Expression and Somatic Hypermutation
Authors: Xiang Yougui , Garrard William T.
Published: April 2014

The mouse Igκ locus has three known transcriptional enhancers: the matrix association region/intronic enhancer, the 3′ enhancer (E3′), and the further downstream enhancer (Ed). Previous studies have shown that both matrix association region/intronic and E3′ enhancers are required for maximal gene rearrangement of the locus, and that E3′ is also required for maximal expression and somatic hypermutation (SHM). To functionally elucidate Ed in vivo, we generated knockout mice with a targeted germline deletion of Ed. Ed deleted homozygous mice (Ed−/−) have moderately reduced numbers of Igκ expressing B cells and correspondingly increased numbers of Igλ expressing B cells in spleen. Ed−/− mice also have decreased Igκ mRNA expression in resting and T cell-dependent activated splenic B cells and reduced Igκ chains in sera. However, our analysis indicates that Igκ gene rearrangement is normal in Ed−/− mice. In addition, our results show that Ed−/− mice exhibit reduced SHM in the Igκ gene J-C intronic region in germinal center B cells from Peyer’s patches. We conclude that Ed positively regulates Igκ gene expression and SHM, but not gene rearrangement.
The Ewing Sarcoma Protein (EWS) Binds Directly to the Proximal Elements of the Macrophage-Specific Promoter of the CSF-1 Receptor (csf1r) Gene
Authors: Hume David A. , Sasmono Tedjo , Himes S. Roy , Sharma Sudarshana M. , Bronisz Agnieszka , Constantin Myrna , Ostrowski Michael C. , Ross Ian L.
Published: April 2014

Many macrophage-specific promoters lack classical transcriptional start site elements such as TATA boxes and Sp1 sites. One example is the CSF-1 receptor (CSF-1R, CD115, c-fms), which is used as a model of the transcriptional regulation of macrophage genes. To understand the molecular basis of start site recognition in this gene, we identified cellular proteins binding specifically to the transcriptional start site (TSS) region. The mouse and human csf1r TSS were identified using cap analysis gene expression (CAGE) data. Conserved elements flanking the TSS cluster were analyzed using EMSAs to identify discrete DNA-binding factors in primary bone marrow macrophages as candidate transcriptional regulators. Two complexes were identified that bind in a highly sequence-specific manner to the mouse and human TSS proximal region and also to high-affinity sites recognized by myeloid zinc finger protein 1 (Mzf1). The murine proteins were purified by DNA affinity isolation from the RAW264.7 macrophage cell line and identified by mass spectrometry as EWS and FUS/TLS, closely related DNA and RNA-binding proteins. Chromatin immunoprecipitation experiments in bone marrow macrophages confirmed that EWS, but not FUS/TLS, was present in vivo on the CSF-1R proximal promoter in unstimulated primary macrophages. Transfection assays suggest that EWS does not act as a conventional transcriptional activator or repressor. We hypothesize that EWS contributes to start site recognition in TATA-less mammalian promoters.
Dynamics of Immune Cell Recruitment during West Nile Encephalitis and Identification of a New CD19+B220−BST-2+ Leukocyte Population
Authors: Bréhin Anne-Claire , Mouriès Juliette , Frenkiel Marie-Pascale , Dadaglio Gilles , Desprès Philippe , Lafon Monique , Couderc Thérèse
Published: April 2014

West Nile virus (WNV) is an emerging neurotropic flavivirus. We investigated the dynamics of immune cell recruitment in peripheral tissues and in the CNS during WNV encephalitis in an immunocompetent mouse model. In the periphery, immune cell expansion can successfully limit viremia and lymphoid tissue infection. However, viral clearance in the periphery is too late to prevent viral invasion of the CNS. In the CNS, innate immune cells, including microglia/macrophages, NK cells, and plasmacytoid dendritic cells, greatly expand as the virus invades the brain, whereas B and T cells are recruited after viral invasion, and fail to control the spread of the virus. Thus, the onset of WNV encephalitis was correlated both with CNS viral infection and with a large local increase of innate immune cells. Interestingly, we identify a new immune cell type: CD19+B220− BST-2+, which we name G8-ICs. These cells appear during peripheral infection and enter the CNS. G8-ICs express high levels of MHC class II, stain for viral Ag, and are localized in the paracortical zone of lymph nodes, strongly suggesting they are previously unidentified APCs that appear in response to viral infection.
Additive Roles for MCP-1 and MCP-3 in CCR2-Mediated Recruitment of Inflammatory Monocytes during Listeria monocytogenes Infection
Authors: Jia Ting , Serbina Natalya V. , Brandl Katharina , Zhong Maggie X. , Leiner Ingrid M. , Charo Israel F. , Pamer Eric G.
Published: April 2014

Chemokine receptor-mediated recruitment of inflammatory cells is essential for innate immune defense against microbial infection. Recruitment of Ly6Chigh inflammatory monocytes from bone marrow to sites of microbial infection is dependent on CCR2, a chemokine receptor that responds to MCP-1 and MCP-3. Although CCR2−/− mice are markedly more susceptible to Listeria monocytogenes infection than are wild-type mice, MCP-1−/− mice have an intermediate phenotype, suggesting that other CCR2 ligands contribute to antimicrobial defense. Herein, we show that L. monocytogenes infection rapidly induces MCP-3 in tissue culture macrophages and in serum, spleen, liver, and kidney following in vivo infection. Only cytosol invasive L. monocytogenes induce MCP-3, suggesting that cytosolic innate immune detection mechanisms trigger chemokine production. MCP-3−/− mice clear bacteria less effectively from the spleen than do wild-type mice, a defect that correlates with diminished inflammatory monocyte recruitment. MCP-3−/− mice have significantly fewer Ly6Chigh monocytes in the spleen and bloodstream, and increased monocyte numbers in bone marrow. MCP-3−/− mice, like MCP-1−/− mice, have fewer TNF- and inducible NO synthase-producing dendritic cells (Tip-DCs) in the spleen following L. monocytogenes infection. Our data demonstrate that MCP-3 and MCP-1 provide parallel contributions to CCR2-mediated inflammatory monocyte recruitment and that both chemokines are required for optimal innate immune defense against L. monocytogenes infection.
Functional Th1 Cells Are Required for Surgical Adhesion Formation in a Murine Model
Authors: Tzianabos Arthur O. , Holsti Matthew A. , Zheng Xin-Xiao , Stucchi Arthur F. , Kuchroo Vijay K. , Strom Terry B. , Glimcher Laurie H. , Cruikshank William W.
Published: April 2014

Tissue trauma in the peritoneal and pelvic cavities following surgery or bacterial infection results in adhesions that are a debilitating cause of intestinal obstruction, chronic pelvic pain, and infertility in women. We recently demonstrated that CD4+ αβ T cells are essential for development of this process. Using a murine model of experimental adhesion formation, we now demonstrate that adhesion formation is characterized by the selective recruitment of Tim-3+, CCR5+, CXCR3+, IFN-γ+ cells, indicating the presence of a Th1 phenotype. We further demonstrate that adhesion formation is critically dependent on the function of Th1 cells because mice genetically deficient for IFN-γ, T-bet, or treated with Abs to the Th1-selective chemoattractant IL-16 show significantly less adhesion formation than wild-type mice. In addition, disrupting the interaction of the Th1-specific regulatory molecule Tim-3, with its ligand, significantly exacerbates adhesion formation. This enhanced response is associated with increases in the level of neutrophil-attracting chemokines KC and MIP-2, known to play a role in adhesiogenesis. These data demonstrate that the CD4+ T cells orchestrating adhesion formation are of the Th1 phenotype and delineate the central role of T-bet, Tim-3, IFN-γ, and IL-16 in mediating this pathogenic tissue response.
Simvastatin Inhibits IL-17 Secretion by Targeting Multiple IL-17-Regulatory Cytokines and by Inhibiting the Expression of IL-17 Transcription Factor RORC in CD4+ Lymphocytes
Authors: Zhang Xin , Jin Jianping , Peng Xueyan , Ramgolam Vinod S. , Markovic-Plese Silva
Published: April 2014

Statins, extensively used as cholesterol-lowering agents, have recently been identified as immunomodulatory agents. This study investigated the statins’ mechanisms that target the autoimmune response in humans, and evaluated their therapeutic potential in multiple sclerosis. Our results demonstrated statin-mediated increases in suppressor of cytokine secretion (SOCS) 3 and suppressor of cytokine secretion 7, which negatively regulate the STAT/JAK signal transduction pathway and IL-6 and IL-23 gene expression in monocytes. Simvastatin also induced IFN-γ, IL-4, and IL-27 production in monocytes, which together inhibited IL-17 transcription and secretion in CD4+ T cells. IL-17-producing CD4+ cells, referred to as Th17 cells, have recently been found to play a central role in the development of autoimmune diseases. Furthermore, simvastatin directly inhibited the expression of retinoic acid-related orphan nuclear hormone receptor C, a transcription factor that controls IL-17 production in CD4+ T cells. This effect was reversed by mevalonic acid, a downstream metabolite of 3-hydroxy-3-methylglutaryl CoA reductase, confirming that simvastatin’s specific effect is through the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase. These results provide evidence for the novel immunomodulatory mechanisms of statins, which selectively target the regulation of cytokine transcription involved in the development of the human autoimmune response. Based on the described immunomodulatory mechanisms, good safety profile and oral bioavailability, statins represent a promising therapeutic approach for multiple sclerosis and other chronic inflammatory diseases.
Long-Term Engraftment and Expansion of Tumor-Derived Memory T Cells Following the Implantation of Non-Disrupted Pieces of Human Lung Tumor into NOD-scid IL2Rγnull Mice
Authors: Simpson-Abelson Michelle R. , Sonnenberg Gregory F. , Takita Hiroshi , Yokota Sandra J. , Conway Thomas F. , Kelleher Raymond J. , Shultz Leonard D. , Barcos Maurice , Bankert Richard B.
Published: April 2014

Non-disrupted pieces of primary human lung tumor implanted into NOD-scid IL2Rγnull mice consistently result in successful xenografts in which tissue architecture, including tumor-associated leukocytes, stromal fibroblasts, and tumor cells are preserved for prolonged periods with limited host-vs-graft interference. Human CD45+ tumor-associated leukocytes within the xenograft are predominantly CD3+ T cells with fewer CD138+ plasma cells. The effector memory T cells that had been shown to be quiescent in human lung tumor microenvironments can be activated in situ as determined by the production of human IFN-γ in response to exogenous IL-12. Plasma cells remain functional as evidenced by production of human Ig. Significant levels of human IFN-γ and Ig were detected in sera from xenograft-bearing mice for up to 9 wk postengraftment. Tumor-associated T cells were found to migrate from the microenvironment of the xenograft to the lung, liver, and primarily the spleen. At 8 wk postengraftment, a significant portion of cells isolated from the mouse spleens were found to be human CD45+ cells. The majority of CD45+ cells were CD3+ and expressed a phenotype consistent with an effector memory T cell, consisting of CD4+ or CD8+ T cells that were CD45RO+, CD44+, CD62L−, and CD25−. Following adoptive transfer into non-tumor bearing NOD-scid IL2Rγnull mice, these human T cells were found to expand in the spleen, produce IFN-γ, and maintain an effector memory phenotype. We conclude that the NOD-scid IL2Rγnull tumor xenograft model provides an opportunity to study tumor and tumor-stromal cell interactions in situ for prolonged periods.
Cutting Edge: IL-21 Is Not Essential for Th17 Differentiation or Experimental Autoimmune Encephalomyelitis
Authors: Coquet Jonathan M. , Chakravarti Sumone , Smyth Mark J. , Godfrey Dale I.
Published: April 2014

Recent studies have suggested that IL-21 is a key factor in the development of IL-17-producing CD4 T cells (Th17) and that the induction of experimental autoimmune encephalomyelitis, which depends on mounting an efficient Th17 response, is reportedly impaired in the absence of IL-21 signaling. In this study, we provide supportive in vitro evidence that IL-21 can drive Th17 responses in conjunction with TGF-β. However, more importantly we also demonstrate, using IL-21- and IL-21R-deficient mice, that IL-21 is not essential for the differentiation of Th17 cells in vitro and in vivo. Moreover, we show that IL-21- and IL-21R-deficient mice are highly susceptible to experimental autoimmune encephalomyelitis with disease scores that were comparable, or even higher at the peak of disease, to those of control mice. Thus, our results challenge the notion that IL-21 is a key factor in driving Th17 immunity and disease.
Small Interfering RNAs Induce Macrophage Migration Inhibitory Factor Production and Proliferation in Breast Cancer Cells via a Double-Stranded RNA-Dependent Protein Kinase-Dependent Mechanism
Authors: Armstrong Michelle E. , Gantier Michael , Li Lili , Chung Wen Y. , McCann Amanda , Baugh John A. , Donnelly Seamas C.
Published: April 2014

Small interfering RNAs (siRNAs) represent a novel tool to induce gene silencing in mammalian cells and clinical trials are currently ongoing to assess the therapeutic efficacy of siRNAs in various human diseases, including age-related macular degeneration and respiratory syncytial virus infection. However, previously reported off-target, nonspecific effects of siRNAs, including activation of type I IFNs and proinflammatory cytokines, remain an outstanding concern regarding use of these agents in vivo. Macrophage-migration inhibitory factor (MIF) is a pleiotropic cytokine with well-described roles in cell proliferation, tumorigenesis, and angiogenesis and represents a target gene for siRNA-based therapy in the treatment of breast cancer. However, in this study we describe an increase in MIF production from mammary adenocarcinoma (MCF-7) cells following transfection with MIF siRNA and various control siRNAs. This effect was shown to be dose-dependent and was attenuated in the presence of a double-stranded RNA-dependent protein kinase inhibitor, 2-aminopurine. Furthermore, treatment of MCF-7 cells with poly(I:C) also stimulated a PKR-dependent increase in MIF production from MCF-7 cells. The biological consequence of the siRNA-induced increase in MIF production from MCF-7 cells was a PKR-dependent increase in proliferation of breast cancer cells. Furthermore, in cDNAs prepared from a primary human breast cancer cohort, we demonstrated a significant correlation (Spearman rank correlation coefficient, r = 0.50, p < 0.0001, n = 63) between PKR- and MIF-mRNA expression. In conclusion, this study highlights the potential biological consequences of off-target, nonspecific effects of siRNAs and underlines the safety concerns regarding the use of siRNAs in the treatment of human diseases, such as cancer.
P2X7 Receptor Differentially Couples to Distinct Release Pathways for IL-1β in Mouse Macrophage
Authors: Pelegrin Pablo , Barroso-Gutierrez Consuelo , Surprenant Annmarie
Published: April 2014

The proinflammatory IL-1 cytokines IL-1α, IL-1β, and IL-18 are key mediators of the acute immune response to injury and infection. Mechanisms underlying their cellular release remain unclear. Activation of purinergic P2X7 receptors (P2X7R) by extracellular ATP is a key physiological inducer of rapid IL-1β release from LPS-primed macrophage. We investigated patterns of ATP-mediated release of IL-1 cytokines from three macrophage types in attempts to provide direct evidence for or against distinct release mechanisms. We used peritoneal macrophage from P2X7R−/− mice and found that release of IL-1α, IL-18, as well as IL-1β, by ATP resulted exclusively from activation of P2X7R, release of all these IL-1 cytokines involved pannexin-1 (panx1), and that there was both a panx1-dependent and -independent component to IL-1β release. We compared IL-1-release patterns from LPS-primed peritoneal macrophage, RAW264.7 macrophage, and J774A.1 macrophage. We found RAW264.7 macrophage readily release pro-IL-1β independently of panx1 but do not release mature IL-1β because they do not express apoptotic speck-like protein with a caspase-activating recruiting domain and so have no caspase-1 inflammasome activity. We delineated two distinct release pathways: the well-known caspase-1 cascade mediating release of processed IL-1β that was selectively blocked by inhibition of caspase-1 or panx1, and a calcium-independent, caspase-1/panx1-independent release of pro-IL-1β that was selectively blocked by glycine. None of these release responses were associated with cell damage or cytolytic effects. This provides the first direct demonstration of a distinct signaling mechanism responsible for ATP-induced release of pro-IL-1β.
Differential Effects of the Type I Interferons α4, β, and ε on Antiviral Activity and Vaccine Efficacy
Authors: Day Stephanie L. , Ramshaw Ian A. , Ramsay Alistair J. , Ranasinghe Charani
Published: April 2014

The type I IFNs exert a range of activities that include antiviral, antiproliferative, and immunomodulatory effects. To study this further, we have constructed recombinant vaccinia viruses expressing HIV or hemagglutinin (HA) Ags along with murine type I IFNs, IFN-α4 (HA-VV-IFN-α4), IFN-β (HA-VV-IFN-β), or IFN-ε (HIV-VV-IFN-ε), a recently discovered member of this family. Our aims were to characterize IFN-ε functionality as a type I IFN and also to study the biological properties of these factors toward the development of safer and more effective vector-based vaccines. HIV-VV-IFN-ε and HA-VV-IFN-β grew to lower titers than did their parental controls in murine cell lines. In vivo, however, HIV-VV-IFN-ε growth was not attenuated, while IFN-β demonstrated potent local antiviral activity with no replication of HA-VV-IFN-β detected. Flow cytofluorometric analysis of B lymphocytes incubated with virally encoded IFN-ε showed up-regulation of activation markers CD69 and CD86, while RT-PCR of IFN-ε-treated cells revealed that gene expression levels of antiviral proteins were elevated, indicating the induction of an antiviral state. The use of these constructs in a poxvirus prime-boost immunization regime led to robust humoral and cellular immune responses against the encoded Ags, despite the lack of replication in the case of HA-VV-IFN-β. Thus, coexpression of these factors may be beneficial in the design of safer vector-based vaccines. Our data also indicate that while IFN-ε exhibits certain biological traits similar to other type I IFNs, it may also have a specific role in mucosal immune regulation that is quite distinct.
Tumor-Secreted Lactic Acid Promotes IL-23/IL-17 Proinflammatory Pathway
Authors: Shime Hiroaki , Yabu Masahiko , Akazawa Takashi , Kodama Ken , Matsumoto Misako , Seya Tsukasa , Inoue Norimitsu
Published: April 2014

IL-23 is a proinflammatory cytokine consisting of a p19 subunit and a p40 subunit that is shared with IL-12. IL-23 is overexpressed in and around tumor tissues, where it induces local inflammation and promotes tumor development. Many tumor cells produce large amounts of lactic acid by altering their glucose metabolism. In this study, we show that lactic acid secreted by tumor cells enhances the transcription of IL-23p19 and IL-23 production in monocytes/macrophages and in tumor-infiltrating immune cells that are stimulated with TLR2 and 4 ligands. DNA elements responsible for this enhancing activity of lactic acid were detected in a 2.7-kb 5′-flanking region of the human IL-23p19 gene. The effect of lactic acid was strictly regulated by extracellular pH. Furthermore, by inducing IL-23 overproduction, lactic acid facilitated the Ag-dependent secretion of proinflammatory cytokine IL-17 but not IFN-γ by TLR ligand-stimulated mouse splenocytes. Interestingly, this effect was observed even in the absence of TLR ligand stimulation. These results suggest that rather than just being a terminal metabolite, lactic acid is a proinflammatory mediator that is secreted by tumor cells to activate the IL-23/IL-17 proinflammatory pathway but not the Th1 pathway. Targeting the lactic acid-induced proinflammatory response may be a useful approach for treating cancer.
Effect of A2B Adenosine Receptor Gene Ablation on Proinflammatory Adenosine Signaling in Mast Cells
Authors: Ryzhov Sergey , Zaynagetdinov Rinat , Goldstein Anna E. , Novitskiy Sergey V. , Dikov Mikhail M. , Blackburn Michael R. , Biaggioni Italo , Feoktistov Igor
Published: April 2014

Pharmacological studies suggest that A2B adenosine receptors mediate proinflammatory effects of adenosine in human mast cells in part by up-regulating production of Th2 cytokines and angiogenic factors. This concept has been recently challenged by the finding that mast cells cultured from bone marrow-derived mast cells (BMMCs) of A2B knockout mice display an enhanced degranulation in response to FcεRI stimulation. This finding was interpreted as evidence of anti-inflammatory functions of A2B receptors and it was suggested that antagonists with inverse agonist activity could promote activation of mast cells. In this report, we demonstrate that genetic ablation of the A2B receptor protein has two distinct effects on BMMCs, one is the previously reported enhancement of Ag-induced degranulation, which is unrelated to adenosine signaling; the other is the loss of adenosine signaling via this receptor subtype that up-regulates IL-13 and vascular endothelial growth factor secretion. Genetic ablation of A2B receptors had no effect on A3 adenosine receptor-dependent potentiation of Ag-induced degranulation in mouse BMMCs, but abrogated A2B adenosine receptor-dependent stimulation of IL-13 and vascular endothelial growth factor secretion. Adenosine receptor antagonists MRS1706 and DPCPX with known inverse agonist activity at the A2B subtype inhibited IL-13 secretion induced by the adenosine analog NECA, but did not mimic the enhanced Ag-induced degranulation observed in A2B knockout BMMCs. Thus, our study confirmed the proinflammatory role of adenosine signaling via A2B receptors and the anti-inflammatory actions of A2B antagonists in mouse BMMCs.
Activation of NF-κB1 by OX40 Contributes to Antigen-Driven T Cell Expansion and Survival
Authors: Song Jianxun , So Takanori , Croft Michael
Published: April 2014

The costimulatory molecule OX40 (CD134) is required in many instances for effective T cell-mediated immunity, controlling proliferation, and survival of T cells after encountering specific Ag. We previously found that the functional targets of OX40 are survivin and aurora B that regulate proliferation and Bcl-2 antiapoptotic family members that regulate survival. However, the intracellular pathways from OX40 that mediate these effects are unclear. In this study, we show that OX40 signaling can target the canonical NF-κB (NF-κB1) pathway in peripheral Ag-responding CD4 T cells. Phosphorylation of IκBα, nuclear translocation of NF-κB1/p50 and RelA, and NF-κB1 activity, are impaired in OX40-deficient T cells. Retroviral transduction of active IκB kinase that constitutively activates NF-κB1 rescues the poor expansion and survival of OX40-deficient T cells, directly correlating with increased expression and activity of survivin, aurora B, and Bcl-2 family members. Moreover, active IκB kinase expression alone is sufficient to restore the defective expansion and survival of OX40-deficient T cells in vivo when responding to Ag. Thus, OX40 signals regulate T cell number and viability through the NF-κB1 pathway that controls expression and activity of intracellular targets for proliferation and survival.