bluuebunny/crossref_metadata_2025 huggingface dataset:
PTEN Down-Regulates IL-17 Expression in a Murine Model of Toluene Diisocyanate-Induced Airway Disease
Authors: Kim So Ri , Lee Kyung Sun , Park Seoung Ju , Min Kyung Hoon , Lee Ka Young , Choe Yeong Hun , Lee Young Rae , Kim Jong Suk , Hong Soo Jong , Lee Yong Chul
Published: April 2014

Toluene diisocyanate (TDI)-induced airway disease is a disorder characterized by chronic airway inflammation and airway remodeling. A recently discovered group of cytokines is the IL-17 family, which has been introduced as an important regulator of immune and inflammatory responses, including airway inflammation. Recently, we have reported that phosphatase and tensin homologue deleted on chromosome 10 (PTEN) plays a pivotal role in the pathogenesis of bronchial asthma. However, there are no available data for the effects of PTEN or IL-17 on TDI-induced airway disease and the relationship between PTEN and IL-17. We used a murine model to determine the role of PTEN in the pathogenesis of TDI-induced airway disease and the regulation of IL-17 production. These mice developed the typical pathophysiological features of TDI-induced airway disease and increased IL-17 expression in the lungs. Administration of phosphoinositide 3-kinase inhibitors or adenoviruses carrying PTEN cDNA (AdPTEN) reduced the pathophysiological features of TDI-induced airway disease and decreased the increased levels of IL-17 expression. Our results also showed that PI3K inhibitors or AdPTEN down-regulated a transcription factor, NF-κB activity, and BAY 11-7085 substantially reduced the increased levels of IL-17 after TDI inhalation. We also found that inhibition of IL-17 activity with an anti-IL-17 Ab reduced airway inflammation and airway hyperresponsiveness. These results suggest that PTEN plays a protective role in the pathogenesis of TDI-induced airway disease, at least in part through the regulation of IL-17 expression. Thus, PTEN may be a useful target for treating TDI-induced airway disease by modulating IL-17 expression.
Raf Signaling but not the ERK Effector SAP-1 Is Required for Regulatory T Cell Development
Authors: Willoughby Jane E. , Costello Patrick S. , Nicolas Robert H. , Robinson Nicholas J. , Stamp Gordon , Powrie Fiona , Treisman Richard
Published: April 2014

Regulatory T cells (Treg) play an important role in immune regulation. Their development in the thymus requires TCR activation and recognition of peptide-MHC, although the downstream signals controlling commitment to the lineage are unclear. To compare the requirements for positive selection and Treg development, we studied knockout and transgenic mice defective in Raf signaling and the ERK effector SRF accessory protein 1 (SAP-1), a member of the ternary complex factor family of Ets domain transcription factors. Although SAP-1 deficient mice display a severe defect in thymocyte positive selection, Treg development was unimpaired as assessed by expression of Foxp3 and the activation markers CD25, GITR, CTLA4, and CD103 in the CD4+ cell population. In contrast, inhibition of Raf signaling by the interfering dominant negative Raf derivative reduced both Foxp3+ and Foxp3− CD4+ populations. In SAP-1-deficient CD4+CD25+ Treg cells, TCR crosslinking efficiently induced ERK activation, but transcriptional induction of the immediate early gene Egr-1 was impaired. Nevertheless, neither deletion of SAP-1 nor expression of a dominant negative Raf derivative affected the ability of CD4+CD25+ Treg cells to suppress CD4+CD25− cell proliferation in vitro. Finally the suppressive activity of CD4+CD25+ Treg cells lacking SAP-1 in an in vivo colitis model was not significantly impaired. The signaling requirements for development of Treg cells in the thymus are thus distinct from those required for “conventional” T cell positive selection, and ERK signaling to SAP-1 is not required for the suppressive activity of Treg cells.
Trans-Sialidase Recombinant Protein Mixed with CpG Motif-Containing Oligodeoxynucleotide Induces Protective Mucosal and Systemic Trypanosoma cruzi Immunity Involving CD8+ CTL and B Cell-Mediated Cross-Priming
Authors: Hoft Daniel F. , Eickhoff Christopher S. , Giddings Olivia K. , Vasconcelos José R. C. , Rodrigues Maurício M.
Published: April 2014

The Trypanosoma cruzi trans-sialidase (TS) is a unique enzyme with neuraminidase and sialic acid transfer activities important for parasite infectivity. The T. cruzi genome contains a large family of TS homologous genes, and it has been suggested that TS homologues provide a mechanism of immune escape important for chronic infection. We have investigated whether the consensus TS enzymatic domain could induce immunity protective against acute and chronic, as well as mucosal and systemic, T. cruzi infection. We have shown that: 1) TS-specific immunity can protect against acute T. cruzi infection; 2) effective TS-specific immunity is maintained during chronic T. cruzi infection despite the expression of numerous related TS superfamily genes encoding altered peptide ligands that in theory could promote immune tolerization; and 3) the practical intranasal delivery of recombinant TS protein combined with a ssDNA oligodeoxynucleotide (ODN) adjuvant containing unmethylated CpG motifs can induce both mucosal and systemic protective immunity. We have further demonstrated that the intranasal delivery of soluble TS recombinant Ag combined with CpG ODN induces both TS-specific CD4+ and CD8+ T cells associated with vaccine-induced protective immunity. In addition, optimal protection induced by intranasal TS Ag combined with CpG ODN requires B cells, which, after treatment with CpG ODN, have the ability to induce TS-specific CD8+ T cell cross-priming. Our results support the development of TS vaccines for human use, suggest surrogate markers for use in future human vaccine trials, and mechanistically identify B cells as important APC targets for vaccines designed to induce CD8+ CTL responses.
CCAAT/Enhancer-Binding Protein β and δ Binding to CIITA Promoters Is Associated with the Inhibition of CIITA Expression in Response toMycobacterium tuberculosis19-kDa Lipoprotein
Authors: Pennini Meghan E. , Liu Yi , Yang Jianqi , Croniger Colleen M. , Boom W. Henry , Harding Clifford V.
Published: April 2014

TLR2 signaling by Mycobacterium tuberculosis 19-kDa lipoprotein (LpqH) inhibits IFN-γ-induced expression of CIITA by macrophages. Microarray analysis, quantitative RT-PCR, and Western blots showed that LpqH induced C/EBPβ and C/EBPδ in kinetic correlation with inhibition of CIITA expression. Of the C/EBPβ isoforms, liver inhibitory protein (LIP) was notably induced and liver-activating protein was increased by LpqH. Putative C/EBP binding sites were identified in CIITA promoters I and IV (pI and pIV). LpqH induced binding of C/EBPβ (LIP and liver-activating protein) to biotinylated oligodeoxynucleotide containing the pI or pIV binding sites, and chromatin immunoprecipitation showed that LpqH induced binding of C/EBPβ and C/EBPδ to endogenous CIITA pI and pIV. Constitutive expression of C/EBPβ LIP inhibited IFN-γ-induced CIITA expression in transfected cells. In summary, LpqH induced expression of C/EBPβ and C/EBPδ, and their binding to CIITA pI and pIV, in correlation with inhibition of IFN-γ-induced expression of CIITA in macrophages, suggesting a role for C/EBP as a novel regulator of CIITA expression.
Inflammasome-Mediated Production of IL-1β Is Required for Neutrophil Recruitment against Staphylococcus aureus In Vivo
Authors: Miller Lloyd S. , Pietras Eric M. , Uricchio Lawrence H. , Hirano Kathleen , Rao Shyam , Lin Heping , O’Connell Ryan M. , Iwakura Yoichiro , Cheung Ambrose L. , Cheng Genhong , Modlin Robert L.
Published: April 2014

IL-1R activation is required for neutrophil recruitment in an effective innate immune response against Staphylococcus aureus infection. In this study, we investigated the mechanism of IL-1R activation in vivo in a model of S. aureus infection. In response to a S. aureus cutaneous challenge, mice deficient in IL-1β, IL-1α/IL-1β, but not IL-1α, developed larger lesions with higher bacterial counts and had decreased neutrophil recruitment compared with wild-type mice. Neutrophil recruitment and bacterial clearance required IL-1β expression by bone marrow (BM)-derived cells and not by non-BM-derived resident cells. In addition, mice deficient in the inflammasome component apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) had the same defects in neutrophil recruitment and host defense as IL-1β-deficient mice, demonstrating an essential role for the inflammasome in mediating the production of active IL-1β to promote neutrophil recruitment in host defense against S. aureus. This finding was further supported by the ability of recombinant active IL-1β to control the infection and promote bacterial clearance in IL-1β-deficient mice. These studies define a key host defense circuit where inflammasome-mediated IL-1β production by BM-derived cells signals IL-1R on non-BM-derived resident cells to activate neutrophil recruitment in the innate immune response against S. aureus in vivo.
Noduler, A Novel Immune Up-Regulated Protein Mediates Nodulation Response in Insects
Authors: Gandhe Archana S. , John Serene H. , Nagaraju Javaregowda
Published: April 2014

Insect immune system comprises of both humoral and cellular defenses. Nodulation is one of the major, yet very poorly understood cellular responses against microbial infections in insects. Through screening for novel immune genes from an Indian saturniid silkmoth Antheraea mylitta, we identified a protein up-regulated in hemolymph within minutes upon bacterial challenge. We have shown here, for first time, the involvement of this novel protein in mediating nodulation response against bacteria and hence designated it as Noduler. Noduler possessed a characteristic reeler domain found in several extracellular matrix vertebrate proteins. Noduler was shown in vitro to bind a wide range of bacteria, yeast, and also insect hemocytes. Furthermore, Noduler specifically bound LPS, lipotechoic acid, and β-1, 3 glucan components of microbial cell walls. RNA-interference mediated knock-down of the Noduler resulted in significant reduction in the number of nodules and consequent increase in bacterial load in larval hemolymph. The results suggest that the Noduler is widely conserved and is involved in very early clearance of bacteria by forming nodules of hemocytes and bacterial complexes in insects. The results would promote further studies for understanding of the crucial but hitherto overlooked nodulation mechanism in insects and also provide cues for the study of similar mammalian proteins whose function is not understood.
Altered Inflammatory Responses in TLR5-Deficient Mice Infected with Legionella pneumophila
Authors: Hawn Thomas R. , Berrington William R. , Smith Ian A. , Uematsu Satoshi , Akira Shizuo , Aderem Alan , Smith Kelly D. , Skerrett Shawn J.
Published: April 2014

Legionella pneumophila (Lp), an important cause of morbidity and mortality from pneumonia, infects alveolar macrophages (AMs) and is recognized by several TLRs as well as Birc1e (NAIP5) and IL-1 converting enzyme-protease activating factor. We examined the role of TLR5 during the murine response to aerosolized Lp infection. At 4 h after infection, Tlr5−/− mice had lower numbers of polymorphonuclear neutrophils (PMNs) in their broncho-alveolar lavage fluid in comparison to wild-type (WT) mice. At 24 and 72 h, the PMN recruitment was similar. WT mice infected with a flagellin-deficient strain (LpFlaA−) also showed an impaired early PMN response at 4 h compared with those infected with the WT strain. There was no consistent difference in bacterial counts at any of the time points when comparing the Tlr5−/− and WT mice. However, at 6 days after infection, the Tlr5−/− mice had increased leukocytic infiltrates in the alveolar and peribronchial interstitial spaces that were consistent with organizing pneumonia. We also examined the role of TLR5 during macrophage infection. In contrast to bone marrow-derived macrophages, AMs secreted TNF-α after stimulation with purified flagellin. In addition, WT, but not Tlr5−/−, AMs produced TNF-α after stimulation with Lp. Live LpFlaA− did not induce TNF-α secretion in AM. These results suggested that AMs recognize Lp flagellin and that a majority of the Lp-induced TNF-α response is TLR5-mediated. Thus, TLR5 mediates recognition of Lp in AMs and performs a distinct role during the in vivo pulmonary immune response through regulation of early PMN recruitment and subsequent later development of pneumonia.
Differential Expression of Inflammatory and Fibrogenic Genes and Their Regulation by NF-κB Inhibition in a Mouse Model of Chronic Colitis
Authors: Wu Feng , Chakravarti Shukti
Published: April 2014

Fibrosis is a major complication of chronic inflammation, as seen in Crohn’s disease and ulcerative colitis, two forms of inflammatory bowel diseases. To elucidate inflammatory signals that regulate fibrosis, we investigated gene expression changes underlying chronic inflammation and fibrosis in trinitrobenzene sulfonic acid-induced murine colitis. Six weekly 2,4,6-trinitrobenzene sulfonic acid enemas were given to establish colitis and temporal gene expression patterns were obtained at 6-, 8-, 10-, and 12-wk time points. The 6-wk point, TNBS-w6, was the active, chronic inflammatory stage of the model marked by macrophage, neutrophil, and CD3+ and CD4+ T cell infiltrates in the colon, consistent with the idea that this model is T cell immune response driven. Proinflammatory genes Cxcl1, Ccl2, Il1b, Lcn2, Pla2g2a, Saa3, S100a9, Nos2, Reg2, and Reg3g, and profibrogenic extracellular matrix genes Col1a1, Col1a2, Col3a1, and Lum (lumican), encoding a collagen-associated proteoglycan, were up-regulated at the active/chronic inflammatory stages. Rectal administration of the NF-κB p65 antisense oligonucleotide reduced but did not abrogate inflammation and fibrosis completely. The antisense oligonucleotide treatment reduced total NF-κB by 60% and down-regulated most proinflammatory genes. However, Ccl2, a proinflammatory chemokine known to promote fibrosis, was not down-regulated. Among extracellular matrix gene expressions Lum was suppressed while Col1a1 and Col3a1 were not. Thus, effective treatment of fibrosis in inflammatory bowel disease may require early and complete blockade of NF-κB with particular attention to specific proinflammatory and profibrogenic genes that remain active at low levels of NF-κB.
Resolvin E1 Regulates Inflammation at the Cellular and Tissue Level and Restores Tissue Homeostasis In Vivo
Authors: Hasturk Hatice , Kantarci Alpdogan , Goguet-Surmenian Emilie , Blackwood Amanda , Andry Chris , Serhan Charles N. , Van Dyke Thomas E.
Published: April 2014

Resolvin E1 (RvE1) is a potent proresolving mediator of inflammation derived from omega-3 eicosapentaenoic acid that acts locally to stop leukocyte recruitment and promote resolution. RvE1 displays potent counter-regulatory and tissue-protective actions in vitro and in vivo. Periodontal disease is a local inflammatory disease initiated by bacteria characterized by neutrophil-mediated tissue injury followed by development of a chronic immune lesion. In this study, we report the treatment of established periodontitis using RvE1 as a monotherapy in rabbits compared with structurally related lipids PGE2 and leukotriene B4. PGE2 and leukotriene B4 each enhanced development of periodontitis and worsened the severity of disease. Promotion of resolution of inflammation as a therapeutic target with RvE1 resulted in complete restoration of the local lesion, and reduction in the systemic inflammatory markers C-reactive protein and IL-1β. This report is the first to show that resolution of inflammation by a naturally occurring endogenous lipid mediator results in complete regeneration of pathologically lost tissues, including bone.
Inhibition of NF-κB Activation Reduces the Tissue Effects of Transgenic IL-13
Authors: Chapoval Svetlana P. , Al-Garawi Amal , Lora Jose M. , Strickland Ian , Ma Bing , Lee Patty J. , Homer Robert J. , Ghosh Sankar , Coyle Anthony J. , Elias Jack A.
Published: April 2014

IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Rα/IL-13Rα1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-κB. As a result, we hypothesized that NF-κB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-κB activity. Three pharmacologic approaches were used to inhibit NF-κB including 1) PS1145, a small molecule inhibitor of IκBα kinase (IKK2), 2) antennapedia-linked NF-κB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-κB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-κB is an attractive target for immunotherapy of IL-13-mediated diseases.
TNF Pretreatment Interferes with Mitochondrial Apoptosis in the Mouse Liver by A20-Mediated Down-Regulation of Bax
Authors: Sass Gabriele , Shembade Noula Dattu , Haimerl Florian , Lamoureux Nicolas , Hashemolhosseini Said , Tannapfel Andrea , Tiegs Gisa
Published: April 2014

Pretreatment with low doses of the proinflammatory cytokine TNF has been shown to prevent hepatocellular apoptosis and liver damage in inflammatory as well as in ischemia/reperfusion-induced liver injury. The underlying mechanisms of protection have not been elucidated so far. In this study, these mechanisms were investigated in murine hepatocyte cultures as well as in a mouse model of TNF-dependent apoptotic liver damage (galactosamine/TNF model). Our results show that pretreatment with TNF, or application of small-interfering RNA directed against the proapoptotic Bcl2 family member Bax, interfered with the onset of mitochondrial apoptosis in vivo. Knockdown of TNF-α-induced-protein 3 (A20) restored mitochondrial apoptosis, Bax expression, and liver damage. The underlying mechanism of protection seems to involve a cascade of events, where TNF induces the expression of A20 in hepatocytes, A20 down-modulates Bax expression by interference with transcriptional activation, and the reduced availability of Bax interferes with the onset of mitochondrial apoptosis and the ensuing apoptotic liver damage. In conclusion, we identified Bax and A20 as key players in TNF-induced protection from apoptotic liver damage. Because treatment with TNF itself might be a risk factor for patients, we propose that overexpression of A20 might represent an alternative approach for protection from inflammation related apoptotic liver damage, as well as for TNF preconditioning during transplantation.
Resolution of Der p1-Induced Allergic Airway Inflammation Is Dependent on CD4+CD25+Foxp3+ Regulatory Cells
Authors: Leech Melanie D. , Benson Robert A. , deVries Annick , Fitch Paul M. , Howie Sarah E. M.
Published: April 2014

Allergic airway inflammation (AAI) is characterized by airway hyperreactivity, eosinophilia, goblet cell hyperplasia, and elevated serum IgE, however, it is unclear what mediates natural resolution after cessation of allergen exposure. This is important because the outcome of subsequent allergen challenge may depend on the concurrent inflammatory milieu of the lung. Using a murine AAI model, we demonstrate that after exposure to a defined natural protein allergen, Der p1, the response in lungs and draining mediastinal lymph nodes (dMLN) peaks between 4 and 6 days then declines until resolution by 21 days. Der p1-specific serum IgE follows the same pattern while IgG1 continues to increase. Resolution of AAI is mediated by CD4+CD25+Foxp3+ regulatory T cells (Tregs), which appear in lungs and dMLN following airway challenge. Treg depletion exacerbated lung eosinophilia, increased dMLN IL-5 and IL-13 but not IL-10 secretion, and increased allergic Ab responses. Most convincingly, transfer of CD4+CD25+Foxp3+ T cells from Ag naive mice (natural Tregs) abolished AAI, decreased dMLN IL-5 and IL-13 secretion, increased dMLN IL-10 secretion, abolished IgE, and decreased IgG1 Abs. Blocking IL-10 receptor function in vivo did not block the anti-inflammatory function of transferred natural Tregs but did restore dMLN IL-5 and IL-13 secretion. Thus natural Tregs can control AAI in an IL-10 independent manner.
FcεRI- and Fcγ Receptor-Mediated Production of Reactive Oxygen Species by Mast Cells Is Lipoxygenase- and Cyclooxygenase-Dependent and NADPH Oxidase-Independent
Authors: Swindle Emily J. , Coleman John W. , DeLeo Frank R. , Metcalfe Dean D.
Published: April 2014

We investigated the enzymes responsible for FcεRI-dependent production of reactive oxygen species (ROS) and the influence of ROS on mast cell secretory responses. 5-Lipoxygenase (5-LO) was the primary enzyme involved in ROS production by human mast cells (huMC) and mouse bone marrow-derived mast cells (mBMMC) following FcεRI aggregation because incubation with 5-LO inhibitors (AA861, nordihydroguaiaretic acid, zileuton) but not a flavoenzyme inhibitor (diphenyleneiodonium) completely abrogated Ag-induced dichlorodihydrofluorescein (DCF) fluorescence. Furthermore, 5-LO-deficient mBMMC had greatly reduced FcεRI-dependent DCF fluorescence compared with wild type mBMMC or those lacking a functional NADPH oxidase (i.e., gp91phox- or p47phox-deficient cells). A minor role for cyclooxygenase (COX)-1 in FcεRI-dependent ROS production was demonstrated by inhibition of Ag-mediated DCF fluorescence by a COX-1 inhibitor (FR122047) and reduced DCF fluorescence in COX-1-deficient mBMMC. Complete abrogation of FcεRI-dependent ROS production in mast cells had no effect on degranulation or cytokine secretion. In response to the NADPH oxidase-stimulating agents including PMA, mBMMC and huMC produced negligible ROS. IgG-coated latex beads did stimulate ROS production in huMC, and in this experiment 5-LO and COX again appeared to be the enzymatic sources of ROS. In contrast, IgG-coated latex bead-induced ROS production in human polymorphonuclear leukocytes occurred by the NADPH oxidase pathway. Thus mBMMC and huMC generate ROS by 5-LO and COX-1 in response to FcεRI aggregation; huMC generate ROS upon exposure to IgG-coated latex beads by 5-LO and COX; and ROS appear to have no significant role in FcεRI-dependent degranulation and cytokine production.
Catalytically Inactive Cyclooxygenase 2 and Absence of Prostaglandin E2 Biosynthesis in Murine Peritoneal Macrophages following In Vivo Phagocytosis of Heat-Killed Mycobacterium bovis Bacillus Calmette-Guérin
Authors: Yamashita Makiko , Shinohara Tsutomu , Tsuji Shoutaro , Myrvik Quentin N. , Nishiyama Akihito , Henriksen Ruth Ann , Shibata Yoshimi
Published: April 2014

Over 25 years ago, it was observed that peritoneal macrophages (Mφ) isolated from mice given heat-killed Mycobacterium bovis bacillus Calmette-Guérin (HK-BCG) i.p. did not release PGE2. However, when peritoneal Mφ from untreated mice are treated with HK-BCG in vitro, cyclooxygenase 2 (COX-2), a rate-limiting enzyme for PGE2 biosynthesis, is expressed and the release of PGE2 is increased. The present study of peritoneal Mφ obtained from C57BL/6 mice and treated either in vitro or in vivo with HK-BCG was undertaken to further characterize the cellular responses that result in suppression of PGE2 release. The results indicate that Mφ treated with HK-BCG in vivo express constitutive COX-1 and inducible COX-2 that are catalytically inactive, are localized subcellularly in the cytoplasm, and are not associated with the nuclear envelope (NE). In contrast, Mφ treated in vitro express catalytically active COX-1 and COX-2 that are localized in the NE and diffusely in the cytoplasm. Thus, for local Mφ activated in vivo by HK-BCG, the results indicate that COX-1 and COX-2 dissociated from the NE are catalytically inactive, which accounts for the lack of PGE2 production by local Mφ activated in vivo with HK-BCG. Our studies further indicate that the formation of catalytically inactive COX-2 is associated with in vivo phagocytosis of HK-BCG, and is not dependent on extracellular mediators produced by in vivo HK-BCG treatment. This attenuation of PGE2 production may enhance Mφ-mediated innate and Th1-acquired immune responses against intracellular infections which are suppressed by PGE2.
Overexpression of BclXL in B Cells Promotes Th1 Response and Exacerbates Collagen-Induced Arthritis
Authors: Zheng Biao , Marinova Ekaterina , Switzer Kirsten , Wansley Daniel , He Hongxia , Bheekha-Escura Roy , Behrens Timothy W. , Han Shuhua
Published: April 2014

B cells play a pathogenic or regulatory role in many autoimmune diseases through production of autoantibodies, cytokine production, and Ag presentation. However, the mechanisms that regulate these B cell functions under different autoimmune settings remain unclear. In the current study, we found that when B cells overexpress an antiapoptotic gene, BclXL, they significantly increased production of IFN-γ and enhanced Th1 response. Consistently, Bcl-xL transgenic mice developed more severe and sustained collagen-induced arthritis due to the enhanced Th1 response. The production of autoantibodies in BclXL transgenic mice was comparable to that in wild-type mice. Thus, our results indicate a novel role of BclXL in regulating B cell functions and immune responses. In patients with rheumatoid arthritis, arthritogenic B cells often up-regulate BclXL expression, which may not only render B cells resistant to apoptosis but also alter the ability of the autoreactive B cells to produce cytokines and modulate the inflammatory response. This may have therapeutic implications if BclXL expression can be down-regulated in autoreactive B cells.
Mast Cells and Basophils Are Selectively Activated In Vitro and In Vivo through CD200R3 in an IgE-Independent Manner
Authors: Kojima Toshiyuki , Obata Kazushige , Mukai Kaori , Sato Shingo , Takai Toshiyuki , Minegishi Yoshiyuki , Karasuyama Hajime
Published: April 2014

Mast cells and basophils have been implicated in the host defense system against pathogens and in the development of allergic disorders. Although IgE-dependent responses via FcεRI on these cells have been extensively studied, little is known about cell surface molecules that are selectively expressed by these cells and engaged in their activation via an IgE-independent mechanism. We have recently established two mAbs that reacted specifically with murine mast cells and basophils, and one of them selectively depleted basophils when administered in vivo. Biochemical and flow cytometric analyses revealed that both mAbs specifically recognized a CD200R-like protein, CD200R3, but not other CD200R family members. CD200R3 existed as a disulfide-linked dimer, unlike other CD200Rs, and was expressed on mast cells and basophils primarily in association with an ITAM-bearing adaptor DAP12. Cross-linking of CD200R3 with the mAbs induced degranulation in mast cells and production of the cytokine IL-4 in basophils in vitro. Administration of the nondepleting mAb in vivo elicited systemic and local anaphylaxis in a CD200R3-dependent manner. These results suggest that CD200R3 functions as an activating receptor on mast cells and basophils to regulate IgE-independent immune responses in cooperation with an inhibitory receptor CD200R, similar to the paired receptors expressed on NK cells.
MAPK p38 Regulates Transcriptional Activity of NF-κB in Primary Human Astrocytes via Acetylation of p65
Authors: Saha Ramendra N. , Jana Malabendu , Pahan Kalipada
Published: April 2014

MAPK-p38 plays an important role in inflammation. Several studies have shown that blocking p38 activity attenuates the transcriptional activity of the proinflammatory transcription factor NF-κB without altering its DNA-binding activity. We have also observed that blocking p38 in human primary astrocytes suppresses the transcriptional but not the DNA-binding activity of NF-κB and down-regulates the expression of an NF-κB-dependent gene, inducible NO synthase. However, the molecular mechanism of p38-mediated regulation of NF-κB remains largely unknown. In this study, we delineate that p38 controls the transcriptional activity of NF-κB by regulating acetylation of p65, but not its phosphorylation. The combination of IL-1β and IFN-γ, previously shown to strongly induce inducible NO synthase in human primary astrocytes, induced p38-dependent phosphorylation of acetyltransferase coactivator p300, but not p65, and subsequent association of p300 with p65. Furthermore, immunocomplex-histone acetyltransferase assays demonstrated that cytokine-induced association of p65 with biologically active immunocomplex-histone acetyltransferase assay was dependent on p38. It has been previously reported that acetylation of p65 at K310 residue is important for transcriptional activity of NF-κB. Accordingly, we found that cytokine-induced association of p65 with p300 led to acetylation of p65 at K310. Because p38 regulated the association between p65 and p300, blocking p38 activity also led to attenuation of p65-K310 acetylation in cytokine-stimulated astrocytes. Taken together, this study illuminates a novel regulatory role of p38 during neuroinflammation where this MAP kinase controls acetylation of NF-κB p65 by regulating acetyltransferase activity of coactivator p300.
Induction of IRAK-M Is Associated with Lipopolysaccharide Tolerance in a Human Endotoxemia Model
Authors: van ‘t Veer Cornelis , van den Pangaart Petra S. , van Zoelen Marieke A. D. , de Kruif Martijn , Birjmohun Rakesh S. , Stroes Eric S. , de Vos Alex F. , van der Poll Tom
Published: April 2014

Recent in vitro and murine in vivo studies have identified several potential LPS tolerance factors. In this study, we describe the expression kinetics of these LPS tolerance factors in standardized human endotoxemia models using i.v. LPS bolus administration. Responsiveness to LPS as well as the expression of potential regulators of LPS signaling were determined in peripheral whole blood. Intravenous LPS administration (4 ng/kg) resulted in peak plasma levels of TNF-α at 1.5 h followed by subsequent peaks of the classic negative feedback inhibitors A20 and IL-10 at 2 and 3 h, respectively. Circulating blood monocyte counts decimated during the initial inflammatory response, but normalized in the period between 4 and 8 h post-LPS. The LPS response as determined by ex vivo TNF release per monocyte in whole blood was profoundly decreased at 6–8 h post-LPS injection despite cessation of A20 and IL-10 expression after 4 h. Analysis of MyD88short, IL-1R-associated kinase (IRAK)-1, IRAK-M, ST2, suppressor of cytokine signaling-1 and -3, SHIP-1, and MAP kinase phosphatase-1 expression indicated that the observed LPS tolerance was associated with decreased IRAK-1 and elevated IRAK-M expression in this human model. Interestingly, a lower dose of LPS (1 ng/kg) induced LPS tolerance accompanied with IRAK-M up-regulation but without depletion of IRAK-1. In vitro studies in whole blood showed that IRAK-M up-regulation by LPS is largely dependent on TNF-α. The observed rise of IRAK-M transcription in the human endotoxemia model appeared much greater compared with in vitro-stimulated whole blood. In conclusion, LPS tolerance in human endotoxemia models is associated with IRAK-M up-regulation.
Apigenin Blocks Lipopolysaccharide-Induced Lethality In Vivo and Proinflammatory Cytokines Expression by Inactivating NF-κB through the Suppression of p65 Phosphorylation
Authors: Nicholas Courtney , Batra Sanjay , Vargo Melissa A. , Voss Oliver H. , Gavrilin Mikhail A. , Wewers Mark D. , Guttridge Denis C. , Grotewold Erich , Doseff Andrea I.
Published: April 2014

LPS stimulates monocytes/macrophages through the activation of signaling events that modulate the production of inflammatory cytokines. Apigenin, a flavonoid abundantly found in fruits and vegetables, exhibits anti-proliferative and anti-inflammatory activities through poorly defined mechanisms. In this study, we demonstrate that apigenin inhibits the production of proinflammatory cytokines IL-1β, IL-8, and TNF in LPS-stimulated human monocytes and mouse macrophages. The inhibitory effect on proinflammatory cytokine production persists even when apigenin is administered after LPS stimulation. Transient transfection experiments using NF-κB reporter constructs indicated that apigenin inhibits the transcriptional activity of NF-κB in LPS-stimulated mouse macrophages. The classical proteasome-dependent degradation of the NF-κB inhibitor IκBα was observed in apigenin LPS-stimulated human monocytes. Using EMSA, we found that apigenin does not alter NF-κB-DNA binding activity in human monocytes. Instead we show that apigenin, as part of a non-canonical pathway, regulates NF-κB activity through hypophosphorylation of Ser536 in the p65 subunit and the inactivation of the IKK complex stimulated by LPS. The decreased phosphorylation on Ser536 observed in LPS-stimulated mouse macrophages treated with apigenin was overcome by the over-expression of IKKβ. In addition, our studies indicate that apigenin inhibits in vivo LPS-induced TNF and the mortality induced by lethal doses of LPS. Collectively, these findings suggest a molecular mechanism by which apigenin suppresses inflammation and modulates the immune response in vivo.
Extracellular Branched-Chain Amino Acids, Especially Valine, Regulate Maturation and Function of Monocyte-Derived Dendritic Cells
Authors: Kakazu Eiji , Kanno Noriatsu , Ueno Yoshiyuki , Shimosegawa Tooru
Published: April 2014

The functions of dendritic cells (DCs) are impaired in patients with liver cirrhosis. It is well-known that cirrhotic patients show decreased levels of plasma branched-chain amino acids (BCAA). Although amino acids are associated with maintaining the cell structure and function in many organs, limited data are available regarding the role of amino acids including BCAA in the immune system. We aimed to investigate the roles of BCAA in the function of human monocyte-derived DCs (MoDC). CD14-positive monocytes (CD14 +) were isolated from PBMC from healthy volunteers and hepatitis C virus (HCV) cirrhotic patients. In medium deprived of BCAA or valine, monocytes were able to differentiate into immature, but not into mature, DCs and showed weak expression of CD83. The deprivation of leucine or isoleucine did not affect this process. The MoDC allostimulatory capacity was significantly decreased in medium deprived of BCAA or valine (p = 0.017, p = 0.012, Bonferroni’s analysis, respectively). Annexin VFITC/propidium iodide staining showed that the DC yield and viability were not significantly different under any medium. Immunoblotting demonstrated that depletion of valine or leucine decreased phospho-S6 kinase expression. Valine increased dose-dependently the allostimulatory capacity and IL-12 production of MoDC from both healthy volunteers and HCV cirrhotic patients. An elevated extracellular concentration of valine could improve the DC function in cirrhotic patients. These data provide a rationale for nutrition therapy that could be beneficial to patients with cirrhosis.
Jak2 Dampens the Induction by IL-1β of Prostaglandin Endoperoxide H Synthase 2 Expression in Human Orbital Fibroblasts: Evidence for Divergent Influence on the Prostaglandin E2 Biosynthetic Pathway
Authors: Han Rui , Chen Beiling , Smith Terry J.
Published: April 2014

Prostaglandin endoperoxide H synthase 2 (PGHS-2) catalyzes the rate-limiting steps in the synthesis of PGE2. It is substantially but transiently induced in human orbital fibroblasts treated with IL-1β. In this study, we report that the induction of PGHS-2 by IL-1β is dramatically enhanced and prolonged when Jak2 signaling is abrogated, either with the specific inhibitor AG490 or by transiently transfecting fibroblasts with a dominant negative mutant Jak2. Attenuating Jak2 increases PGHS-2 steady-state mRNA levels, a consequence of increased gene transcription and mRNA survival in IL-1β-treated cultures. Surprisingly, interrupting Jak2 function also blocked the expected increase in PGE2 synthesis usually provoked by IL-1β. This resulted from the rapid loss of IL-1β-dependent arachidonate release and by attenuation of group IIA secreted PLA2 (sPLA2) gene induction. Supplying Jak2-compromised cultures with exogenous arachidonate failed to increase PGE2 production in response to IL-1β until cells were mechanically disrupted. However, transiently transfecting them with wild-type sPLA2 fully restored prostanoid production to anticipated levels. sPLA2 expression following transfection resulted in increased IL-1β-dependent PGHS-2 and microsomal PGE2 synthase levels. Thus, sPLA2 plays important roles in PGE2 synthesis in addition to its release of arachidonate. Our findings suggest that Jak2 ordinarily dampens and limits the duration of the PGHS-2 induction by IL-1β. Moreover, it is required for IL-1β-dependent signaling to sPLA2, the expression and activity of which are necessary for up-regulating PGE2 synthesis in orbital fibroblasts.
Elevated Urinary VCAM-1, P-Selectin, Soluble TNF Receptor-1, and CXC Chemokine Ligand 16 in Multiple Murine Lupus Strains and Human Lupus Nephritis
Authors: Wu Tianfu , Xie Chun , Wang Hong W. , Zhou Xin J. , Schwartz Noa , Calixto Sergio , Mackay Meggan , Aranow Cynthia , Putterman Chaim , Mohan Chandra
Published: April 2014

In an effort to identify potential biomarkers in lupus nephritis, urine from mice with spontaneous lupus nephritis was screened for the presence of VCAM-1, P-selectin, TNFR-1, and CXCL16, four molecules that had previously been shown to be elevated in experimental immune nephritis, particularly at the peak of disease. Interestingly, all four molecules were elevated ∼2- to 4-fold in the urine of several strains of mice with spontaneous lupus nephritis, including the MRL/lpr, NZM2410, and B6.Sle1.lpr strains, correlating well with proteinuria. VCAM-1, P-selectin, TNFR-1, and CXCL16 were enriched in the urine compared with the serum particularly in active disease, and were shown to be expressed within the diseased kidneys. Finally, all four molecules were also elevated in the urine of patients with lupus nephritis, correlating well with urine protein levels and systemic lupus erythematosus disease activity index scores. In particular, urinary VCAM-1 and CXCL16 showed superior specificity and sensitivity in distinguishing subjects with active renal disease from the other systemic lupus erythematosus patients. These studies uncover VCAM-1, P-selectin, TNFR-1, and CXCL16 as a quartet of molecules that may have potential diagnostic significance in lupus nephritis. Longitudinal studies are warranted to establish the clinical use of these potential biomarkers.
Cutting Edge: A Transcriptional Repressor and Corepressor Induced by the STAT3-Regulated Anti-Inflammatory Signaling Pathway
Authors: El Kasmi Karim C. , Smith Amber M. , Williams Lynn , Neale Geoffrey , Panopolous Athanasia , Watowich Stephanie S. , Häcker Hans , Foxwell Brian M. J. , Murray Peter J.
Published: April 2014

IL-10 regulates anti-inflammatory signaling via the activation of STAT3, which in turn controls the induction of a gene expression program whose products execute inhibitory effects on proinflammatory mediator production. In this study we show that IL-10 induces the expression of an ETS family transcriptional repressor, ETV3, and a helicase family corepressor, Strawberry notch homologue 2 (SBNO2), in mouse and human macrophages. IL-10-mediated induction of ETV3 and SBNO2 expression was dependent upon both STAT3 and a stimulus through the TLR pathway. We also observed that ETV3 expression was strongly induced by the STAT3 pathway regulated by IL-10 but not by STAT3 signaling activated by IL-6, which cannot activate the anti-inflammatory signaling pathway. ETV3 and SBNO2 repressed NF-κB- but not IFN regulatory factor 7 (IRF7)-activated transcriptional reporters. Collectively our data suggest that ETV3 and SBNO2 are components of the pathways that contribute to the downstream anti-inflammatory effects of IL-10.
Cutting Edge: Tissue-Resident Memory CTL Down-Regulate Cytolytic Molecule Expression following Virus Clearance
Authors: Mintern Justine D. , Guillonneau Carole , Carbone Francis R. , Doherty Peter C. , Turner Stephen J.
Published: April 2014

CTL express lytic proteins that mediate the cytolysis of virus-infected cells. In this study, cytolytic transcriptional profiles were determined for individual CTL responding to influenza A virus and HSV-1. During acute infection, influenza-specific CTL in the spleen and respiratory airways displayed highly activated cytolytic profiles, as did HSV-1-specific CTL localized in the spleen, skin, and dorsal root ganglia (DRG). In contrast, memory CTL dramatically down-regulated cytolytic molecule transcription. This occurred for both lymphoid (spleen) and tissue-resident (skin and/or lung) memory CTL. In contrast, HSV-1-specific CTL localized in the dorsal root ganglia in the presence latent HSV-1 Ag did not down-regulate cytolytic molecule transcription. Therefore, both lymphoid and tissue-resident memory CTL down-regulate cytolytic molecule transcription following virus clearance unless localized Ag is present.
Specific Immunogenicity of Heat Shock Protein gp96 Derives from Chaperoned Antigenic Peptides and Not from Contaminating Proteins
Authors: Binder Robert J. , Kelly John B. , Vatner Ralph E. , Srivastava Pramod K.
Published: April 2014

The peptide-binding property of MHC is central to adaptive immunological functions. A similar property of heat shock proteins (HSPs) hsp70 and hsp90 has been implicated in Ag presentation by MHC and in cross-priming. The peptide-binding pocket of hsp70 has been characterized structurally and functionally and a peptide-binding site in gp96 (of hsp90 family) has been defined. Nonetheless, questions persist whether the specific immunogenicity of HSP preparations derives from the peptides chaperoned by the HSPs or by proteins contaminating the HSP preparations. Because absolute purity of a protein preparation is a metaphysical concept, other approaches are necessary to address the question. In this study, we demonstrate that the specific immunogenicity of gp96 preparations isolated from cells expressing β-galactosidase derives from the MHC I epitope precursors associated with the gp96 and not from contaminating β-galactosidase protein nor unassociated fragments derived from it. Although the observations here are limited to a single HSP and antigenic peptides chaperoned by it, they can be extended broadly.
A Novel Approach to Specific Allergy Treatment: The Recombinant Allergen-S-Layer Fusion Protein rSbsC-Bet v 1 Matures Dendritic Cells That Prime Th0/Th1 and IL-10-Producing Regulatory T Cells
Authors: Gerstmayr Marianne , Ilk Nicola , Schabussova Irma , Jahn-Schmid Beatrice , Egelseer Eva M. , Sleytr Uwe B. , Ebner Christof , Bohle Barbara
Published: April 2014

An ideal vaccine for allergen-specific immunotherapy of type I allergies should display reduced mediator-releasing capacity, induce maturation of APC, and modify the disease-eliciting Th2-dominated allergen-specific response to a more physiological response. We have previously shown that rSbsC-Bet v 1, the recombinant fusion protein of a bacterial surface (S-layer) protein of Geobacillus stearothermophilus ATCC 12980 and the major birch pollen allergen Bet v 1, exhibited reduced allergenicity and induced IFN-γ and IL-10 synthesis in Bet v 1-specific Th2 clones. In this study, we characterized the effects of rSbsC-Bet v 1 on immature monocyte-derived dendritic cells (mdDC) and the consequences for the polarization of naive CD4+ T lymphocytes isolated from the blood of birch pollen-allergic patients. mdDC responded to rSbsC-Bet v 1 with a significant up-regulation of costimulatory molecules, functional maturation, and the synthesis of IL-10 and IL-12. mdDC matured with rSbsC-Bet v 1 induced the differentiation of naive T cells into IFN-γ-producing cells. This effect was IL-12 dependent. In parallel, a substantial number of naive T cells developed into IL-10-producing CD25+Foxp3+CLTA-4+ cells capable of active suppression. Thus, rSbsC-Bet v 1 showed immune stimulatory capacity on DC, which then promoted the simultaneous differentiation of Th0/Th1 cells and regulatory T cells. These data further support that the concept of conjugating allergens to bacterial agents is a promising approach to improve vaccines for specific immunotherapy of atopic allergies.
Regulated Expression of BAFF-Binding Receptors during Human B Cell Differentiation
Authors: Darce Jaime R. , Arendt Bonnie K. , Wu Xiaosheng , Jelinek Diane F.
Published: April 2014

BAFF plays a central role in B-lineage cell biology; however, the regulation of BAFF-binding receptor (BBR) expression during B cell activation and differentiation is not completely understood. In this study, we provide a comprehensive ex vivo analysis of BBRs in human B-lineage cells at various stages of maturation, as well as describe the events that drive and regulate receptor expression. Our data reveal that B-lineage cells ranging from naive to plasma cells (PCs), excluding bone marrow PCs, express BAFF-R uniformly. In contrast, only tonsillar memory B cells (MB) and PCs, from both tonsil and bone marrow tissues, express BCMA. Furthermore, we show that TACI is expressed by MB cells and PCs, as well as a subpopulation of activated CD27neg B cells. In this regard, we demonstrate that TACI is inducible early upon B cell activation and this is independent of B cell turnover. In addition, we found that TACI expression requires activation of the ERK1/2 pathway, since its expression was blocked by ERK1/2-specific inhibitors. Expression of BAFF-R and B cell maturation Ag (BCMA) is also highly regulated and we demonstrate that BCMA expression is only acquired in MB cells and in a manner accompanied by loss of BAFF-R expression. This inverse expression coincides with MB cell differentiation into Ig-secreting cells (ISC), since blocking differentiation inhibited both induction of BCMA expression and loss of BAFF-R. Collectively, our data suggest that the BBR profile may serve as a footprint of the activation history and stage of differentiation of normal human B cells.
Murine Gammaherpesvirus-68 Infection Alters Self-Antigen Presentation and Type 1 Diabetes Onset in NOD Mice
Authors: Smith Katherine A. , Efstathiou Stacey , Cooke Anne
Published: April 2014

Recent research in line with the “hygiene hypothesis” has implicated virus infection in the delay or prevention of autoimmunity in murine models of type 1 diabetes such as the NOD mouse. We found that intraperitoneal or intranasal infection of NOD mice with the murine gammaherpesvirus-68 (MHV-68) significantly delayed diabetes onset in an age-dependent manner. The acute phase following intraperitoneal infection was associated with significantly reduced trafficking of autoreactive BDC2.5NOD CD4+ T cells to the pancreas but not the pancreatic lymph node (PLN); this was not as a result of MHV-68 M3 pan-chemokine binding protein expression. Autoreactive BDC2.5NOD CD4+ T cells within the PLN of MHV-68 infected mice were significantly more naive and proliferated to a lesser extent than those cells within the PLN of uninfected mice. These changes in autoreactive CD4+ T cell activation were associated with reduced dendritic cell endocytosis and soluble Ag presentation but were not as a result of virally induced IL-10 or changes in Ag-specific regulatory T cell populations.