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1.  B Lymphocyte intestinal homing in inflammatory bowel disease 
BMC Immunology  2011;12:71.
Inflammatory bowel disease (IBD) is thought to be due to an abnormal interaction between the host immune system and commensal microflora. Within the intestinal immune system, B cells produce physiologically natural antibodies but pathologically atypical anti-neutrophil antibodies (xANCAs) are frequently observed in patients with IBD. The objective is to investigate the localisation of immunoglobulin-producing cells (IPCs) in samples of inflamed intestinal tissue taken from patients with IBD, and their possible relationship with clinical features.
The IPCs in small intestinal, colonic and rectal biopsy specimens of patients with IBD were analysed by means of immunofluorescence using polyclonal rabbit anti-human Ig and goat anti-human IgM. The B cell phenotype of the IPC-positive samples was assessed using monoclonal antibodies specific for CD79, CD20, CD23, CD21, CD5, λ and κ chains. Statistical correlations were sought between the histological findings and clinical expression.
The study involved 96 patients (64 with ulcerative colitis and 32 with Crohn's disease). Two different patterns of B lymphocyte infiltrates were found in the intestinal tissue: one was characterised by a strong to moderate stromal localisation of small IgM+/CD79+/CD20-/CD21-/CD23-/CD5± IPCs (42.7% of cases); in the other (57.3%) no such small IPCs were detected in stromal or epithelial tissues. IPCs were significantly less frequent in the patients with Crohn's disease than in those with ulcerative colitis (p = 0.004).
Our findings suggest that different immunopathogenetic pathways underlie chronic intestinal inflammation with different clinical expressions. The presence of small B lymphocytes resembling B-1 cells also seemed to be negatively associated with Crohn's disease. It can therefore be inferred that the gut contains an alternative population of B cells that have a regulatory function.
PMCID: PMC3261110  PMID: 22208453
Inflammatory bowel disease; inflammation; mucosal immunity; lymphocytes; B1 cells; lymphocyte homing
2.  Neuroantigen-specific, tolerogenic vaccines: GM-CSF is a fusion partner that facilitates tolerance rather than immunity to dominant self-epitopes of myelin in murine models of experimental autoimmune encephalomyelitis (EAE) 
BMC Immunology  2011;12:72.
Vaccination strategies that elicit antigen-specific tolerance are needed as therapies for autoimmune disease. This study focused on whether cytokine-neuroantigen (NAg) fusion proteins could inhibit disease in chronic murine models of experimental autoimmune encephalomyelitis (EAE) and thus serve as potential therapeutic modalities for multiple sclerosis.
A fusion protein comprised of murine GM-CSF as the N-terminal domain and the encephalitogenic MOG35-55 peptide as the C-terminal domain was tested as a tolerogenic, therapeutic vaccine (TTV) in the C57BL/6 model of EAE. Administration of GMCSF-MOG before active induction of EAE, or alternatively, at the onset of EAE blocked the development and progression of EAE. Covalent linkage of the GM-CSF and MOG35-55 domains was required for tolerogenic activity. Likewise, a TTV comprised of GM-CSF and PLP139-151 was a tolerogen in the SJL model of EAE.
These data indicated that fusion proteins containing GM-CSF coupled to myelin auto-antigens elicit tolerance rather than immunity.
PMCID: PMC3261124  PMID: 22208499
3.  Selectively enhanced expression of prophenoloxidase activating enzyme 1 (PPAE1) at a bacteria clearance site in the white shrimp, Litopenaeus vannamei 
BMC Immunology  2011;12:70.
The prophenoloxidase-activating (PO activating) system plays an important role in the crustacean innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating enzyme (PPAE), which is the direct activator of prophenoloxidase (proPO). Despite their importance in crustacean PO activating system, the studies on them remain limited.
Here we report on a PPAE of white shrimp, Litopenaeus vannamei (lvPPAE1), which showed 94% similarity to PPAE1 of Penaeus monodon. We found that lvPPAE1 in fluid hemocytes was down regulated after challenge by Vibrio harveyi but was enhanced when shrimps were exposed to a bacteria-rich environment for long-term. In vivo gene silence of lvPPAE1 by RNAi can significantly reduce the phenoloxidase activity (PO) and increase the susceptibility of shrimps to V. harveyi. Although lvPPAE1 was down-regulated in fluid hemocytes by Vibrio challenge, its expression increased significantly in gill after bacteria injection, which is the primary bacteria-clearance tissue.
Suppressed expression in fluid hemocytes and enhanced expression in gill indicates selectively enhanced expression at the bacterial clearance site. This is a novel feature for PPAE expression. The results will contribute to our understanding of the PO activating system in crustaceans.
PMCID: PMC3268739  PMID: 22208405
4.  Impact of acute stress on antimicrobial polypeptides mRNA copy number in several tissues of marine sea bass (Dicentrarchus labrax) 
BMC Immunology  2011;12:69.
In comparison to higher vertebrates, fish are thought to rely heavily on innate immune system for initial protection against pathogen invasion because their acquired immune system displays a considerably poor immunological memory, and short-lived secondary response. The endogenous antimicrobial polypeptides (AMPPs) directly and rapidly killing pathogens such as bacteria, fungi, parasites, and viruses are included within the realm of innate defenses. In addition to piscidins, AMPPs that in recent years have been shown to be commonly linked to innate defense, are histones and their polypeptide fragments, and peptides derived from the respiratory protein hemoglobin. There is evidence that a number of stresses lead to significant regulation of AMPPs and thus their monitoring could be a highly sensitive measure of health status and risk of an infectious disease outbreak, which is a major impediment to the continued success of virtually all aquaculture enterprises and is often the most significant cause of economic losses.
We firstly isolated and deposited in Genbank database the cDNA sequences encoding for hemoglobin-β-like protein (Hb-LP) [GeneBank: JN410659], H2B histone-like protein 1 (HLP1) GenBank: JN410660], and HLP2 [GenBank: JN410661]. The "de novo" prediction of the three-dimensional structures for each protein is presented. Phylogenetic trees were constructed on Hb-LP, HLP1, and HLP2 sequences of sea bass and those of other teleost, avian, reptiles, amphibian and mammalian species. We then used real time RT-PCR technology to monitor for the first time in sea bass, dynamic changes in mRNA copy number of Hb-LP, HLP1, HLP2, and dicentracin in gills, skin, eyes, stomach and proximal intestine in response to acute crowding/confinement stress. We showed that acute crowding stress induces an increase in the expression levels of the aforementioned genes, in gills and skin of sea bass, but not in other tissues, and that this expression patterns are not always rapidly reversed upon re-exposure to normal conditions.
The higher expression of the four target genes in gills and skin of sea bass suggests that this AMPP represents a first and immediate line of defense in combating pathogens and stressors since these tissues constitute the first physiological barriers of the animal.
PMCID: PMC3274460  PMID: 22204309
5.  The effect of conditional EFNB1 deletion in the T cell compartment on T cell development and function 
BMC Immunology  2011;12:68.
Eph kinases are the largest family of cell surface receptor tyrosine kinases. The ligands of Ephs, ephrins (EFNs), are also cell surface molecules. Ephs interact with EFNs transmitting signals in both directions, i.e., from Ephs to EFNs and from EFNs to Ephs. EFNB1 is known to be able to co-stimulate T cells in vitro and to modulate thymocyte development in a model of foetal thymus organ culture. To further understand the role of EFNB1 in T cell immunity, we generated T-cell-specific EFNB1 gene knockout mice to assess T cell development and function in these mice.
The mice were of normal size and cellularity in the thymus and spleen and had normal T cell subpopulations in these organs. The bone marrow progenitors from KO mice and WT control mice repopulated host spleen T cell pool to similar extents. The activation and proliferation of KO T cells was comparable to that of control mice. Naïve KO CD4 cells showed an ability to differentiate into Th1, Th2, Th17 and Treg cells similar to control CD4 cells.
Our results suggest that the function of EFNB1 in the T cell compartment could be compensated by other members of the EFN family, and that such redundancy safeguards the pivotal roles of EFNB1 in T cell development and function.
PMCID: PMC3287259  PMID: 22182253
6.  Indispensable roles of OX40L-derived signal and epistatic genetic effect in immune-mediated pathogenesis of spontaneous pulmonary hypertension 
BMC Immunology  2011;12:67.
Pulmonary hypertension (PH) refers to a spectrum of diseases with elevated pulmonary artery pressure. Pulmonary arterial hypertension (PAH) is a disease category that clinically presents with severe PH and that is histopathologically characterized by the occlusion of pulmonary arterioles, medial muscular hypertrophy, and/or intimal fibrosis. PAH occurs with a secondary as well as a primary onset. Secondary PAH is known to be complicated with immunological disorders. The aim of the present study is to histopathologically and genetically characterize a new animal model of PAH and clarify the role of OX40 ligand in the pathogenesis of PAH.
Spontaneous onset of PAH was stably identified in mice with immune abnormality because of overexpression of the tumor necrosis factor (TNF) family molecule OX40 ligand (OX40L). Histopathological and physical examinations revealed the onset of PAH-like disorders in the C57BL/6 (B6) strain of OX40L transgenic mice (B6.TgL). Comparative analysis performed using different strains of transgenic mice showed that this onset depends on the presence of OX40L in the B6 genetic background. Genetic analyses demonstrated a susceptibility locus of a B6 allele to this onset on chromosome 5. Immunological analyses revealed that the excessive OX40 signals in TgL mice attenuates expansion of regulatory T cells the B6 genetic background, suggesting an impact of the B6 genetic background on the differentiation of regulatory T cells.
Present findings suggest a role for the OX40L-derived immune response and epistatic genetic effect in immune-mediated pathogenesis of PAH.
PMCID: PMC3269997  PMID: 22171643
7.  Vasoactive Intestinal Peptide Knockout (VIP KO) mouse model of sulfite-sensitive asthma: up-regulation of novel lung carbonyl reductase 
BMC Immunology  2011;12:66.
We earlier reported spontaneous features of asthma in Vasoactive Intestinal Peptide knockout mice (VIP KO): 1) peribronchiolar airway inflammation, with accumulation of lymphocytes and eosinophils, 2) pro-inflammatory cytokine production of IL-5, IL-6, with IFN-γ, and 3) airway hyper-responsiveness to inhaled methacholine. In human asthma, a phenotype with sulfite sensitivity leads to airway inflammation and hyper-responsiveness to inhaled sulfites, and is associated with upregulation of anti-oxidant protein lung carbonyl reductase. For the present experiments, we examined the role of VIP in modulating anti-oxidant genes and their proteins, including lung carbonyl reductase.
Four male VIP KO mice and four wild-type age- and gender matched mice had lungs examined for whole genome microarray and a proteomics approach using mass spectrometry. The proteomics analysis revealed that a novel variant of anti-oxidant protein lung carbonyl reductase (car3) was uniquely and markedly elevated in the VIP KO mice. RT-PCR indicated that carbonic anhydrase 3, which is an anti-oxidant protein, was elevated in the VIP KO mice.
These data support the concept that VIP influences the endogenous oxidant/antioxidant balance. One potential implication is that VIP and its analogues may be used to treat inflammatory diseases, including asthma.
PMCID: PMC3277465  PMID: 22103391
8.  Cytokine expression during early and late phase of acute Puumala hantavirus infection 
BMC Immunology  2011;12:65.
Hantaviruses of the family Bunyaviridae are emerging zoonotic pathogens which cause hemorrhagic fever with renal syndrome (HFRS) in the Old World and hantavirus pulmonary syndrome (HPS) in the New World. An immune-mediated pathogenesis is discussed for both syndromes. The aim of our study was to investigate cytokine expression during the course of acute Puumala hantavirus infection.
We retrospectively studied 64 patients hospitalised with acute Puumala hantavirus infection in 2010 during a hantavirus epidemic in Germany. Hantavirus infection was confirmed by positive anti-hantavirus IgG/IgM. Cytokine expression of IL-2, IL-5, IL-6, IL-8, IL-10, IFN-γ, TNF-α and TGF-β1 was analysed by ELISA during the early and late phase of acute hantavirus infection (average 6 and 12 days after onset of symptoms, respectively). A detailed description of the demographic and clinical presentation of severe hantavirus infection requiring hospitalization during the 2010 hantavirus epidemic in Germany is given. Acute hantavirus infection was characterized by significantly elevated levels of IL-2, IL-6, IL-8, TGF-β1 and TNF-α in both early and late phase compared to healthy controls. From early to late phase of disease, IL-6, IL-10 and TNF-α significantly decreased whereas TGF-β1 levels increased. Disease severity characterized by elevated creatinine and low platelet counts was correlated with high pro-inflammatory IL-6 and TNF-α but low immunosuppressive TGF-β1 levels and vice versa .
High expression of cytokines activating T-lymphocytes, monocytes and macrophages in the early phase of disease supports the hypothesis of an immune-mediated pathogenesis. In the late phase of disease, immunosuppressive TGF-β1 level increase significantly. We suggest that delayed induction of a protective immune mechanism to downregulate a massive early pro-inflammatory immune response might contribute to the pathologies characteristic of human hantavirus infection.
PMCID: PMC3259039  PMID: 22085404
9.  Blockade of leukocyte haptokinesis and haptotaxis by ketoprofen, diclofenac and SC-560 
BMC Immunology  2011;12:64.
Nonsteroidal anti-inflammatory drugs (NSAID) represent a one of the most widely used anti-inflammatory substances. Their anti-inflammatory effects are mainly based on inhibition of cyclooxygenase. The potential direct effect of NSAID on leukocyte migration was poorly investigated. Using time-lapse microscopy and 96-well fluorescence-based assay, we studied the effect of three different NSAID, ketoprofen, diclofenac and SC-560, on leukocyte haptokinesis and haptotaxis in vivo and in vitro.
NSAID induced an immediate inhibiting effect on leukocyte migration both in vitro and in vivo. This effect was dose-dependent and was not restricted to a specific type of leukocytes. The inhibition of leukocyte migration by NSAID was partially re-stored after removal of inhibiting agent. Only complete blockade of leukocyte migration was accompanied by a strong reduction of [Ca2+]i.
NSAID strongly supress leukocyte migration. The results of the present study may have important clinical implications since blockade of leukocyte migration can be achieved after topical application of NSAID.
PMCID: PMC3247092  PMID: 22078067
10.  The absence of MyD88 has no effect on the induction of alternatively activated macrophage during Fasciola hepatica infection 
BMC Immunology  2011;12:63.
Alternatively activated macrophages (AAMϕ) play important roles in allergies and responses to parasitic infections. However, whether signaling through toll-like receptors (TLRs) plays any role in AAMϕ induction when young Fasciola hepatica penetrates the liver capsule and migrates through the liver tissue is still unclear.
The data show that the lack of myeloid differentiation factor 88 (MyD88) has no effect on the AAMϕ derived from the bone marrow (BMMϕ) in vitro and does not impair the mRNA expression of arginase-1, resistin-like molecule (RELMα), and Ym1 in BMMϕs. The Th2 cytokine production bias in splenocytes was not significantly altered in F. hepatica-infected mice in the absence of MyD88 in vitro and in the pleural cavity lavage in vivo. In addition, MyD88-deficiency has no effect on the arginase production of the F. hepatica elicited macrophages (Fe Mϕs), production of RELMα and Ym1 proteins and mRNA expression of Ym1 and RELMα of macrophages in the peritoneal cavity 6 weeks post F. hepatica infection.
The absence of MyD88 has no effect on presence of AAMϕ 6 weeks post F. hepatica infection.
PMCID: PMC3226545  PMID: 22074389
11.  Differential expression of CD300a/c on human TH1 and TH17 cells 
BMC Immunology  2011;12:62.
Human memory CD4+ T cells can be either CD300a/c+ or CD300a/c- and subsequent analyses showed that CD4+ effector memory T (TEM) cells are mostly CD300a/c+, whereas CD4+ central memory T (TCM) cells have similar frequencies of CD300a/c+ and CD300a/c- cells.
Extensive phenotypical and functional characterization showed that in both TCM and TEM cells, the CD300a/c+ subset contained a higher number of TH1 (IFN-γ producing) cells. Alternatively, TH17 (IL-17a producing) cells tend to be CD300a/c-, especially in the TEM subset. Further characterization of the IL-17a+ cells showed that cells that produce only this cytokine are mostly CD300a/c-, while cells that produce IL-17a in combination with other cytokines, especially IFN-γ, are mostly CD300a/c+, indicating that the expression of this receptor is associated with cells that produce IFN-γ. Co-ligation of the TCR and CD300a/c in CD4+ T cells inhibited Ca2+ mobilization evoked by TCR ligation alone and modulated IFN-γ production on TH1 polarized cells.
We conclude that the CD300a/c receptors are differentially expressed on human TH1 and TH17 cells and that their ligation is capable of modulating TCR mediated signals.
PMCID: PMC3219710  PMID: 22046970
12.  Immune enhancement by novel vaccine adjuvants in autoimmune-prone NZB/W F1 mice: relative efficacy and safety 
BMC Immunology  2011;12:61.
Vaccines have profoundly impacted global health although concerns persist about their potential role in autoimmune or other adverse reactions. To address these concerns, vaccine components like immunogens and adjuvants require critical evaluation not only in healthy subjects but also in those genetically averse to vaccine constituents. Evaluation in autoimmune-prone animal models of adjuvants is therefore important in vaccine development. The objective here was to assess the effectiveness of experimental adjuvants: two phytol-derived immunostimulants PHIS-01 (phytanol) and PHIS-03 (phytanyl mannose), and a new commercial adjuvant from porcine small intestinal submucosa (SIS-H), relative to a standard adjuvant alum. Phytol derivatives are hydrophobic, oil-in water diterpenoids, while alum is hydrophilic, and SIS is essentially a biodegradable and collagenous protein cocktail derived from extracellular matrices.
We studied phthalate -specific and cross-reactive anti-DNA antibody responses, and parameters associated with the onset of autoimmune disorders. We determined antibody isotype and cytokine/chemokine milieu induced by the above experimental adjuvants relative to alum. Our results indicated that the phytol-derived adjuvant PHIS-01 exceeded alum in enhancing anti-phthalate antibody without much cross reactivity with ds-DNA. Relatively, SIS and PHIS-03 proved less robust, but they were also less inflammatory. Interestingly, these adjuvants facilitated isotype switching of anti-hapten, but not of anti-DNA response. The current study reaffirms our earlier reports on adjuvanticity of phytol compounds and SIS-H in non autoimmune-prone BALB/c and C57BL/6 mice. These adjuvants are as effective as alum also in autoimmune-prone NZB/WF1 mice, and they have little deleterious effects.
Although all adjuvants tested impacted cytokine/chemokine milieu in favor of Th1/Th2 balance, the phytol compounds fared better in reducing the onset of autoimmune syndromes. However, SIS is least inflammatory among the adjuvants evaluated.
PMCID: PMC3214834  PMID: 22024358
13.  Transfer of in vivo primed transgenic T cells supports allergic lung inflammation and FIZZ1 and Ym1 production in an IL-4Rα and STAT6 dependent manner 
BMC Immunology  2011;12:60.
CD4+ T helper type 2 (TH2) cells, their cytokines IL-4, IL-5 and IL-13 and the transcription factor STAT6 are known to regulate various features of asthma including lung inflammation, mucus production and airway hyperreactivity and also drive alternative activation of macrophages (AAM). However, the precise roles played by the IL-4/IL-13 receptors and STAT6 in inducing AAM protein expression and modulating specific features of airway inflammation are still unclear. Since TH2 differentiation and activation plays a pivotal role in this disease, we explored the possibility of developing an asthma model in mice using T cells that were differentiated in vivo.
In this study, we monitored the activation and proliferation status of adoptively transferred allergen-specific naïve or in vivo primed CD4+ T cells. We found that both the naïve and in vivo primed T cells expressed similar levels of CD44 and IL-4. However, in vivo primed T cells underwent reduced proliferation in a lymphopenic environment when compared to naïve T cells. We then used these in vivo generated effector T cells in an asthma model. Although there was reduced inflammation in mice lacking IL-4Rα or STAT6, significant amounts of eosinophils were still present in the BAL and lung tissue. Moreover, specific AAM proteins YM1 and FIZZ1 were expressed by epithelial cells, while macrophages expressed only YM1 in RAG2-/- mice. We further show that FIZZ1 and YM1 protein expression in the lung was completely dependent on signaling through the IL-4Rα and STAT6. Consistent with the enhanced inflammation and AAM protein expression, there was a significant increase in collagen deposition and smooth muscle thickening in RAG2-/- mice compared to mice deficient in IL-4Rα or STAT6.
These results establish that transfer of in vivo primed CD4+ T cells can induce allergic lung inflammation. Furthermore, while IL-4/IL-13 signaling through IL-4Rα and STAT6 is essential for AAM protein expression, lung inflammation and eosinophilia are only partially dependent on this pathway. Further studies are required to identify other proteins and signaling pathways involved in airway inflammation.
PMCID: PMC3212823  PMID: 22014099
14.  Endogenous annexin A1 counter-regulates bleomycin-induced lung fibrosis 
BMC Immunology  2011;12:59.
The balancing functions of pro/anti-inflammatory mediators of the complex innate responses have been investigated in a variety of experimental inflammatory settings. Annexin-A1 (AnxA1) is one mediator of endogenous anti-inflammation, affording regulation of leukocyte trafficking and activation in many contexts, yet its role in lung pathologies has been scarcely investigated, despite being highly expressed in lung cells. Here we have applied the bleomycin lung fibrosis model to AnxA1 null mice over a 21-day time-course, to monitor potential impact of this mediator on the control of the inflammatory and fibrotic phases.
Analyses in wild-type mice revealed strict spatial and temporal regulation of the Anxa1 gene, e.g. up-regulation in epithelial cells and infiltrated granulocytes at day 7, followed by augmented protein levels in alveolar macrophages by day 21. Absence of AnxA1 caused increases in: i) the degree of inflammation at day 7; and ii) indexes of fibrosis (assessed by deposition of hydroxyproline in the lung) at day 7 and 21. These alterations in AnxA1 null mice were paralleled by augmented TGF-β1, IFN-γ and TNF-α generation compared to wild-type mice. Finally, treatment of wild type animals with an AnxA1 peptido-mimetic, given prophylactically (from day 0 to 21) or therapeutically (from day 14 onward), ameliorated both signs of inflammation and fibrosis.
Collectively these data reveal a pathophysiological relevance for endogenous AnxA1 in lung inflammation and, more importantly, fibrosis, and may open new insights for the pharmacological treatment of lung fibrosis.
PMCID: PMC3212807  PMID: 22011168
anti-inflammation; fibrosis; lung inflammation; macrophage; neutrophil; transforming growth factor (TGF-β)
15.  Comparison of static immersion and intravenous injection systems for exposure of zebrafish embryos to the natural pathogen Edwardsiella tarda 
BMC Immunology  2011;12:58.
The zebrafish embryo is an important in vivo model to study the host innate immune response towards microbial infection. In most zebrafish infectious disease models, infection is achieved by micro-injection of bacteria into the embryo. Alternatively, Edwardsiella tarda, a natural fish pathogen, has been used to treat embryos by static immersion. In this study we used transcriptome profiling and quantitative RT-PCR to analyze the immune response induced by E. tarda immersion and injection.
Mortality rates after static immersion of embryos in E. tarda suspension varied between 25-75%, while intravenous injection of bacteria resulted in 100% mortality. Quantitative RT-PCR analysis on the level of single embryos showed that expression of the proinflammatory marker genes il1b and mmp9 was induced only in some embryos that were exposed to E. tarda in the immersion system, whereas intravenous injection of E. tarda led to il1b and mmp9 induction in all embryos. In addition, microarray expression profiles of embryos subjected to immersion or injection showed little overlap. E. tarda-injected embryos displayed strong induction of inflammatory and defense genes and of regulatory genes of the immune response. E. tarda-immersed embryos showed transient induction of the cytochrome P450 gene cyp1a. This gene was also induced after immersion in Escherichia coli and Pseudomonas aeruginosa suspensions, but, in contrast, was not induced upon intravenous E. tarda injection. One of the rare common responses in the immersion and injection systems was induction of irg1l, a homolog of a murine immunoresponsive gene of unknown function.
Based on the differences in mortality rates between experiments and gene expression profiles of individual embryos we conclude that zebrafish embryos cannot be reproducibly infected by exposure to E. tarda in the immersion system. Induction of il1b and mmp9 was consistently observed in embryos that had been systemically infected by intravenous injection, while the early transcriptional induction of cyp1a and irg1l in the immersion system may reflect an epithelial or other tissue response towards cell membrane or other molecules that are shed or released by bacteria. Our microarray expression data provide a useful reference for future analysis of signal transduction pathways underlying the systemic innate immune response versus those underlying responses to external bacteria and secreted virulence factors and toxins.
PMCID: PMC3206475  PMID: 22003892
16.  DC-STAMP knock-down deregulates cytokine production and T-cell stimulatory capacity of LPS-matured dendritic cells 
BMC Immunology  2011;12:57.
Dendritic cells (DCs) are the highly specialized antigen presenting cells of the immune system that play a key role in regulating immune responses. DCs can efficiently initiate immune responses or induce tolerance. Due to this dual function, DCs are studied in the context of immunotherapy for both cancer and autoimmune diseases. Characterization of DC-specific genes, leading to better understanding of DC immunobiology, will help to guide their use in clinical settings. We previously identified DC-STAMP, a multi-membrane spanning protein preferentially expressed by DCs. DC-STAMP resides in the endoplasmic reticulum (ER) of immature DCs and translocates towards the Golgi compartment upon maturation. In this study we knocked down DC-STAMP in mouse bone marrow-derived DCs (mBMDCs) to determine its function.
We demonstrate that DC-STAMP knock-down mBMDCs secrete less IL-6, IL-12, TNF-α and IL-10 while IL-1 production is enhanced. Moreover, LPS-matured DC-STAMP knock-down mBMDCs show impaired T cell activation potential and induction of Th1 responses in an alloreaction.
We show that DC-STAMP plays an important role in cytokine production by mBMDCs following LPS exposure. Our results reveal a novel function of DC-STAMP in regulating DC-initiated immune responses.
PMCID: PMC3199277  PMID: 21978263
17.  RAGE and ICAM-1 differentially control leukocyte recruitment during acute inflammation in a stimulus-dependent manner 
BMC Immunology  2011;12:56.
The receptor for advanced glycation endproducts, RAGE, is involved in the pathogenesis of many inflammatory conditions, which is mostly related to its strong activation of NF-κB but also due to its function as ligand for the β2-integrin Mac-1. To further dissect the stimulus-dependent role of RAGE on leukocyte recruitment during inflammation, we investigated β2-integrin-dependent leukocyte adhesion in RAGE-/- and Icam1-/- mice in different cremaster muscle models of inflammation using intravital microscopy.
We demonstrate that RAGE, but not ICAM-1 substantially contributes to N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced leukocyte adhesion in TNF-α-pretreated cremaster muscle venules in a Mac-1-dependent manner. In contrast, fMLP-stimulated leukocyte adhesion in unstimulated cremaster muscle venules is independent of RAGE, but dependent on ICAM-1 and its interaction with LFA-1. Furthermore, chemokine CXCL1-stimulated leukocyte adhesion in surgically prepared cremaster muscle venules was independent of RAGE but strongly dependent on ICAM-1 and LFA-1 suggesting a differential and stimulus-dependent regulation of leukocyte adhesion during inflammation in vivo.
Our results demonstrate that RAGE and ICAM-1 differentially regulate leukocyte adhesion in vivo in a stimulus-dependent manner.
PMCID: PMC3203087  PMID: 21970746
18.  Inhibition of TLR8- and TLR4-induced Type I IFN induction by alcohol is different from its effects on inflammatory cytokine production in monocytes 
BMC Immunology  2011;12:55.
Prolonged alcohol consumption is a significant co-factor in the progression of chronic viral infections including hepatitis C and HIV, which are both single-stranded RNA viruses. Toll like receptor 8 (TLR8), a pattern recognition receptor expressed in monocytes, senses viral single stranded RNA as a danger signal and leads to the induction of Type I interferon (IFN) as well as the pro-inflammatory cytokine, tumor necrosis factor alpha (TNF alpha). Lipopolysaccharide (LPS), a Toll like receptor 4 (TLR4) ligand, was shown to affect inflammatory cell activation after alcohol consumption and in HIV and HCV infections. Here we hypothesized that alcohol exposure modulates TLR8- and TLR4-ligand-induced monocyte activation and affects both type I IFN and inflammatory cytokine induction.
The TLR8 ligand, CL075, as well as the TLR4 ligand, LPS, resulted in a significant induction of TNF alpha both at the mRNA and protein levels in human monocytes. We found that both acute and prolonged alcohol treatment resulted in inhibition of type I IFN induction by either TLR8 or TLR4 ligands in human monocytes at the protein and mRNA levels. In contrast to Type I IFN production, the effects of acute and prolonged alcohol were different on inflammatory cytokine activation after TLR8 or TLR4 ligand stimulation. Acute alcohol inhibited TLR8- or TLR4-induced TNF alpha protein and mRNA induction while it augmented IL-10 production in monocytes. In contrast, prolonged alcohol treatment augmented TNF alpha without affecting IL-10 production significantly in response to either TLR8 or TLR4 ligand stimulation.
These novel results suggest first, that alcohol has a profound inhibitory effect on Type I IFN induction regardless of intracellular (TLR8) or cell surface-derived (TLR4) danger signals. Second, both acute and prolonged alcohol exposure can inhibit antiviral Type I IFN pathway activation. Third, the opposite effects of acute (inhibitory) and prolonged alcohol (augmentation) treatment on pro-inflammatory cytokine activation extend to TLR8-induced signals beyond the previously shown TLR4/LPS pathway.
PMCID: PMC3203086  PMID: 21962237
Acute; chronic; alcohol; IFNβ; IL-10; TNF alpha
19.  Regulation of IL-2 gene expression by Siva and FOXP3 in human T cells 
BMC Immunology  2011;12:54.
Severe autoinflammatory diseases are associated with mutations in the Foxp3 locus in both mice and humans. Foxp3 is required for the development, function, and maintenance of regulatory T cells (Tregs), a subset of CD4 cells that suppress T cell activation and inflammatory processes. Siva is a pro-apoptotic gene that is expressed across a range of tissues, including CD4 T cells. Siva interacts with three tumor necrosis factor receptor (TNFR) family members that are constitutively expressed on Treg cells: CD27, GITR, and OX40.
Here we report a biophysical interaction between FOXP3 and Siva. We mapped the interaction domains to Siva's C-terminus and to a central region of FOXP3. We showed that Siva repressed IL-2 induction by suppressing IL-2 promoter activity during T cell activation. Siva-1's repressive effect on IL-2 gene expression appears to be mediated by inhibition of NFkappaB, whereas FOXP3 repressed both NFkappaB and NFAT activity.
In summary, our data suggest that both FOXP3 and Siva function as negative regulators of IL-2 gene expression in Treg cells, via suppression of NFAT by FOXP3 and of NFkappaB by both FOXP3 and Siva. Our work contributes evidence for Siva's role as a T cell signalling mediator in addition to its known pro-apoptotic function. Though further investigations are needed, evidence for the biophysical interaction between FOXP3 and Siva invites the possibility that Siva may be important for proper Treg cell function.
PMCID: PMC3208582  PMID: 21955384
20.  Phenotype, effector function, and tissue localization of PD-1-expressing human follicular helper T cell subsets 
BMC Immunology  2011;12:53.
It is well established that PD-1 is expressed by follicular T cells but its function in regulation of human T helper cells has been unclear. We investigated the expression modality and function of PD-1 expressed by human T cells specialized in helping B cells.
We found that PD-1-expressing T cells are heterogeneous in PD-1 expression. We identified three different PD-1-expressing memory T cell subsets (i.e. PD-1low (+), PD-1medium (++), and PD-1high (+++) cells). PD-1+++ T cells expressed CXCR5 and CXCR4 and were localized in the rim of germinal centers. PD-1+ or PD-1++ cells expressed CCR7 and were present mainly in the T cell area or other parts of the B cell follicles. Utilizing a novel antigen density-dependent magnetic sorting (ADD-MS) method, we isolated the three T cell subsets for functional characterization. The germinal center-located PD-1+++ T cells were most efficient in helping B cells and in producing IL-21 and CXCL13. Other PD-1-expressing T cells, enriched with Th1 and Th17 cells, were less efficient than PD-1+++ T cells in these capacities. PD-1+++ T cells highly expressed Ki-67 and therefore appear active in cell activation and proliferation in vivo. IL-2 is a cytokine important for proliferation and survival of the PD-1+++ T cells. In contrast, IL-21, while a major effector cytokine produced by the PD-1-expressing T helper cells, had no function in generation, survival, or proliferation of the PD-1-expressing helper T cells at least in vitro. PD-1 triggering has a suppressive effect on the proliferation and B cell-helping function of PD-1+++ germinal center T cells.
Our results revealed the phenotype and effector function of PD-1-expressing T helper cell subsets and indicate that PD-1 restrains the B cell-helping function of germinal center-localized T cells to prevent excessive antibody response.
PMCID: PMC3184275  PMID: 21914188
21.  Efficient activation of T cells by human monocyte-derived dendritic cells (HMDCs) pulsed with Coxiella burnetii outer membrane protein Com1 but not by HspB-pulsed HMDCs 
BMC Immunology  2011;12:52.
Coxiella burnetii is an obligate intracellular bacterium and the etiologic agent of Q fever; both coxiella outer membrane protein 1 (Com1) and heat shock protein B (HspB) are its major immunodominant antigens. It is not clear whether Com1 and HspB have the ability to mount immune responses against C. burnetii infection.
The recombinant proteins Com1 and HspB were applied to pulse human monocyte-derived dendritic cells (HMDCs), and the pulsed HMDCs were used to stimulate isogenic T cells. Com1-pulsed HMDCs expressed substantially higher levels of surface molecules (CD83, CD40, CD80, CD86, CD54, and CD58) and a higher level of interleukin-12 than HspB-pulsed HMDCs. Moreover, Com1-pulsed HMDCs induced high-level proliferation and activation of CD4+ and CD8+ cells, which expressed high levels of T-cell activation marker CD69 and inflammatory cytokines IFN-γ and TNF-α. In contrast, HspB-pulsed HMDCs were unable to induce efficient T-cell proliferation and activation.
Our results demonstrate that Com1-pulsed HMDCs are able to induce efficient T-cell proliferation and drive T cells toward Th1 and Tc1 polarization; however, HspB-pulsed HMDCs are unable to do so. Unlike HspB, Com1 is a protective antigen, which was demonstrated by the adoptive transfer of Com1-pulsed bone marrow dendritic cells into naive BALB/c mice.
PMCID: PMC3179940  PMID: 21888659
22.  Inhibitory effects of the JAK inhibitor CP690,550 on human CD4+ T lymphocyte cytokine production 
BMC Immunology  2011;12:51.
The new JAK3 inhibitor, CP690,550, has shown efficacy in the treatment of rheumatoid arthritis. The present study was undertaken to assess the effects of CP690,550 on cytokine production and cellular signaling in human CD4+ T cells.
CD4+ T cells produced IL-2, IL-4, IL-17, IL-22 and IFN-γ in following stimulation with a CD3 antibody. At the optimal concentration, CP690,550 almost completely inhibited the production of IL-4, IL-17, IL-22 and IFN-γ from these activated CD4+ T cells, but only had marginal effects on IL-2 production. Moreover CP690,550 inhibited anti-CD3-induced phosphorylation of STAT1, STAT3, STAT4, STAT5, and STAT6, but not the TCR-associated phosphorylation of ZAP-70.
Therefore, CP690,550-mediated modification of the JAK/STAT pathway may be a new immunosuppressive strategy in the treatment of autoimmune diseases.
PMCID: PMC3179939  PMID: 21884580
CP690; 550; cytokines; Janus kinase; signal transducers and activators of transcription; T lymphocytes
23.  Classification of dendritic cell phenotypes from gene expression data 
BMC Immunology  2011;12:50.
The selection of relevant genes for sample classification is a common task in many gene expression studies. Although a number of tools have been developed to identify optimal gene expression signatures, they often generate gene lists that are too long to be exploited clinically. Consequently, researchers in the field try to identify the smallest set of genes that provide good sample classification. We investigated the genome-wide expression of the inflammatory phenotype in dendritic cells. Dendritic cells are a complex group of cells that play a critical role in vertebrate immunity. Therefore, the prediction of the inflammatory phenotype in these cells may help with the selection of immune-modulating compounds.
A data mining protocol was applied to microarray data for murine cell lines treated with various inflammatory stimuli. The learning and validation data sets consisted of 155 and 49 samples, respectively. The data mining protocol reduced the number of probe sets from 5,802 to 10, then from 10 to 6 and finally from 6 to 3. The performances of a set of supervised classification models were compared. The best accuracy, when using the six following genes --Il12b, Cd40, Socs3, Irgm1, Plin2 and Lgals3bp-- was obtained by Tree Augmented Naïve Bayes and Nearest Neighbour (91.8%). Using the smallest set of three genes --Il12b, Cd40 and Socs3-- the performance remained satisfactory and the best accuracy was with Support Vector Machine (95.9%). These data mining models, using data for the genes Il12b, Cd40 and Socs3, were validated with a human data set consisting of 27 samples. Support Vector Machines (71.4%) and Nearest Neighbour (92.6%) gave the worst performances, but the remaining models correctly classified all the 27 samples.
The genes selected by the data mining protocol proposed were shown to be informative for discriminating between inflammatory and steady-state phenotypes in dendritic cells. The robustness of the data mining protocol was confirmed by the accuracy for a human data set, when using only the following three genes: Il12b, Cd40 and Socs3. In summary, we analysed the longitudinal pattern of expression in dendritic cells stimulated with activating agents with the aim of identifying signatures that would predict or explain the dentritic cell response to an inflammatory agent.
PMCID: PMC3179938  PMID: 21875438
24.  Ontology-based Brucella vaccine literature indexing and systematic analysis of gene-vaccine association network 
BMC Immunology  2011;12:49.
Vaccine literature indexing is poorly performed in PubMed due to limited hierarchy of Medical Subject Headings (MeSH) annotation in the vaccine field. Vaccine Ontology (VO) is a community-based biomedical ontology that represents various vaccines and their relations. SciMiner is an in-house literature mining system that supports literature indexing and gene name tagging. We hypothesize that application of VO in SciMiner will aid vaccine literature indexing and mining of vaccine-gene interaction networks. As a test case, we have examined vaccines for Brucella, the causative agent of brucellosis in humans and animals.
The VO-based SciMiner (VO-SciMiner) was developed to incorporate a total of 67 Brucella vaccine terms. A set of rules for term expansion of VO terms were learned from training data, consisting of 90 biomedical articles related to Brucella vaccine terms. VO-SciMiner demonstrated high recall (91%) and precision (99%) from testing a separate set of 100 manually selected biomedical articles. VO-SciMiner indexing exhibited superior performance in retrieving Brucella vaccine-related papers over that obtained with MeSH-based PubMed literature search. For example, a VO-SciMiner search of "live attenuated Brucella vaccine" returned 922 hits as of April 20, 2011, while a PubMed search of the same query resulted in only 74 hits. Using the abstracts of 14,947 Brucella-related papers, VO-SciMiner identified 140 Brucella genes associated with Brucella vaccines. These genes included known protective antigens, virulence factors, and genes closely related to Brucella vaccines. These VO-interacting Brucella genes were significantly over-represented in biological functional categories, including metabolite transport and metabolism, replication and repair, cell wall biogenesis, intracellular trafficking and secretion, posttranslational modification, and chaperones. Furthermore, a comprehensive interaction network of Brucella vaccines and genes were identified. The asserted and inferred VO hierarchies provide semantic support for inferring novel knowledge of association of vaccines and genes from the retrieved data. New hypotheses were generated based on this analysis approach.
VO-SciMiner can be used to improve the efficiency for PubMed searching in the vaccine domain.
PMCID: PMC3180695  PMID: 21871085
25.  Immunome database for marsupials and monotremes 
BMC Immunology  2011;12:48.
To understand the evolutionary origins of our own immune system, we need to characterise the immune system of our distant relatives, the marsupials and monotremes. The recent sequencing of the genomes of two marsupials (opossum and tammar wallaby) and a monotreme (platypus) provides an opportunity to characterise the immune gene repertoires of these model organisms. This was required as many genes involved in immunity evolve rapidly and fail to be detected by automated gene annotation pipelines.
We have developed a database of immune genes from the tammar wallaby, red-necked wallaby, northern brown bandicoot, brush-tail possum, opossum, echidna and platypus. The resource contains 2,235 newly identified sequences and 3,197 sequences which had been described previously. This comprehensive dataset was built from a variety of sources, including EST projects and expert-curated gene predictions generated through a variety of methods including chained-BLAST and sensitive HMMER searches. To facilitate systems-based research we have grouped sequences based on broad Gene Ontology categories as well as by specific functional immune groups. Sequences can be extracted by keyword, gene name, protein domain and organism name. Users can also search the database using BLAST.
The Immunome Database for Marsupials and Monotremes (IDMM) is a comprehensive database of all known marsupial and monotreme immune genes. It provides a single point of reference for genomic and transcriptomic datasets. Data from other marsupial and monotreme species will be added to the database as it become available. This resource will be utilized by marsupial and monotreme immunologists as well as researchers interested in the evolution of mammalian immunity.
PMCID: PMC3173380  PMID: 21854560

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