Trends Immunol Research Articles

Serum beta-2 microglobulin concentration predicts cardiovascular and all-cause mortality

Beta-2 microglobulin (B2M) is the light chain in the major histocompatibility complex (MHC) Class 1 molecule [ [1] Saper M.A. Bjorkman P.J. Wiley D.C. Refined structure of the human histocompatibility antigen HLA-A2 at 2.6 A resolution. J Mol Biol. 1991; 219: 277-319 Crossref PubMed Scopus (965) Google Scholar ], which is present in all nucleated cells. It has been suggested as an initiator of inflammation [ [2] Xie J. Yi Q. Beta2-microglobulin as a potential initiator of inflammatory responses. Trends Immunol. 2003; 24 ([author reply 229–230]): 228-229 Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar ], and is an early marker of diseases related to inflammation such as cardiovascular and renal disease [ 3 Winchester J.F. Novel changes in beta2-microglobulin in dialysis patients. Clin Chem. 2005; 51: 1089-1090 Crossref PubMed Scopus (4) Google Scholar , 4 Liabeuf S. Lenglet A. Desjardins L. et al. Plasma beta-2 microglobulin is associated with cardiovascular disease in uremic patients. Kidney Int. 2012; 82: 1297-1303 Crossref PubMed Scopus (104) Google Scholar ]. B2M is renally excreted and so the serum level increases in renal dysfunction. We and others have reported that serum B2M level predicts mortality in certain patient populations [ 4 Liabeuf S. Lenglet A. Desjardins L. et al. Plasma beta-2 microglobulin is associated with cardiovascular disease in uremic patients. Kidney Int. 2012; 82: 1297-1303 Crossref PubMed Scopus (104) Google Scholar , 5 Astor B.C. Shafi T. Hoogeveen R.C. et al. Novel markers of kidney function as predictors of ESRD, cardiovascular disease, and mortality in the general population. Am J Kidney Dis. 2012; 59: 653-662 Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar , 6 Cheung A.K. Greene T. Leypoldt J.K. et al. Association between serum 2-microglobulin level and infectious mortality in hemodialysis patients. Clin J Am Soc Nephrol. 2008; 3: 69-77 Crossref PubMed Scopus (80) Google Scholar , 7 Cheung A.K. Rocco M.V. Yan G. et al. Serum beta-2 microglobulin levels predict mortality in dialysis patients: results of the HEMO study. J Am Soc Nephrol. 2006; 17: 546-555 Crossref PubMed Scopus (354) Google Scholar , 8 Shinkai S. Chaves P.H. Fujiwara Y. et al. Beta2-microglobulin for risk stratification of total mortality in the elderly population: comparison with cystatin C and C-reactive protein. Arch Intern Med. 2008; 168: 200-206 Crossref PubMed Scopus (84) Google Scholar , 9 Cheung C.L. Lam K.S. Cheung B.M. Serum beta-2 microglobulin predicts mortality in people with diabetes. Eur J Endocrinol. 2013; 169: 1-7 Crossref PubMed Scopus (23) Google Scholar ]. We therefore examined whether serum B2M level predicted cardiovascular and all-cause mortality in a large nationally representative cohort.

OP0218 Regulation of auto-antibody production by auto-immune complexes on follicular dendritic cells

BackgroundFollicular dendritic cells (FDCs) are characteristic components of organized tertiary lymphoid structures (TLS) containing germinal centres (GCs) in chronically inflamed tissues. However, the role of FDCs in initiation, maintenance, and...

SP0020 Adam17 as a masterswitch in the regulation of inflammation and cancer

Cytokine receptors exist in membrane bound and soluble form. While most soluble receptors are antagonists, some soluble receptors are agonists like soluble receptors of the gp130 cytokine family. In vivo,...

FRI0358 The association between inflammatory bowel diseases and familial mediterranean fever in turkish children diagnosed as familial mediterranean fever

BackgroundCrohn’s disease and ulcerative colitis are multifactorial and heterogeneous, chronically relapsing inflammatory bowel diseases (IBD) that are thought to result from a dysregulated mucosal immune response to gut lumen bacterial...

Mitigating effects of hUCB-MSCs on the hematopoietic syndrome resulting from total body irradiation

This study evaluated the clinical and pathologic effects of human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs) in the recovery from total body irradiation by comparing it with the effects of granulocyte-colony stimulating factor (G-CSF), an efficacious drug in the treatment of acute bone marrow radiation syndrome. BALB/c mice were treated with G-CSF or hUCB-MSCs after they were irradiated with 7 Gy cobalt-60 γ-rays. Circulating blood counts, histopathologic changes in the bone marrow, and plasma level of Flt-3L and transforming growth factor (TGF-β1) were monitored in the postirradiation period. Hematologic analysis revealed that the peripheral leukocyte counts were markedly increased in the hUCB-MSCs-treated group, whereas G-CSF-treated mice did not recover significantly. Moreover, differential counts showed that hUCB-MSC treatment has regenerative effects on white blood cells, lymphocytes, and monocytes compared with the irradiated group. Treatment with hUCB-MSCs or G-CSF significantly increased immunoreactivity of Ki-67 until 3 weeks after total body irradiation. However, at 3 weeks, the number of Ki-67 immunoreactive cells significantly increased in the hUCB-MSCs-treated group compared with the G-CSF-treated group. Furthermore, hUCB-MSC treatment significantly modulated plasma levels of the hematopoietic cytokines Flt-3L and TGF-β1, whereas G-CSF treatment failed to decrease the plasma Flt-3L levels at 2 weeks after irradiation. Based on the differences in circulating blood cell reconstitution and cell density of bone marrow, the authors suggest that MSC treatment is superior to G-CSF treatment for hematopoietic reconstitution following sublethal dose radiation exposure.

Phenotypic evaluation of CD28 negative T cells in primary sclerosing cholangitis

Event Abstract Back to Event Phenotypic evaluation of CD28 negative T cells in primary sclerosing cholangitis Evaggelia Liaskou1*, Louisa Jeffery2, Shankar Suresh1, Palak Trivedi1, Tony Bruns1, 3, David Adams1 and David Sansom2 1 University of Birmingham, Centre for Liver Research and NIHR Biomedical Research Unit in Liver Disease, Institute of Biomedical Research, United Kingdom 2 Medical Research Council Centre for Immune Regulation, School of Immunity & Infection, Institute of Biomedical Research,, United Kingdom 3 Friedrich Schiller University of Jena, Germany Background/Aims: Genetic, functional and epidemiologic studies implicate CD28 and vitamin D (vitD) as relevant to autoimmune diseases including primary sclerosing cholangitis (PSC). Here we studied the role of CD28-ve T cells in PSC and the effects of vitD on their phenotype. Methods: Liver-infiltrating (LIMCs) and peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors or patients with PSC. The frequency and phenotype of CD28-ve cells [expression of activation (CD69, CD25) and exhaustion (PD-1) markers, perforin and granzyme B] were studied by flow cytometry. PSC LIMCs and PBMCs were activated and the release of IFNγ was examined. CD28-ve and CD28+ve cells from PSC PBMCs were stimulated with anti-CD3, autologous CD14+monocytes (5:1 ratio) with/without vitD for 4 days. Results: CD4+CD28-ve and CD8+CD28-ve T cells were highly expanded in PSC peripheral blood (3.9- and 2.1-fold increase respectively versus normal blood) and in PSC liver (17.8- and 2.45-fold increase respectively versus normal liver). The frequency of CD4+CD28-ve T cells in PSC liver was significantly higher than PSC peripheral blood (p<0.01). Liver-infiltrating CD4+CD28-ve T cells showed an activated CD25+CD69+PD-1- and cytotoxic phenotype granzymeB+perforin+. In-vitro stimulation of PSC liver-infiltrating CD4+CD28-ve cells induced the production of IFNγ (41%), which was much higher compared to their CD28+ve counterparts (12%). Stimulation of PSC peripheral blood CD28-ve and CD28+ve cells with vitD induced CD28 expression on CD28-ve (+52%) and CD28+ve (+65%) cells and suppressed IFNγ production (-43% and 67%, respectively). Conclusion: In PSC, CD28 negative T cells are pro-inflammatory, an effect that can be reduced by vitamin D supplementation. References Krones E., et al, Liver Int. 2012; 32(3):352-269 Ludwig J., et al, Hepatology 1981 Bo X., et al., Gut 2001; 49: 131-141 Weng NP., et al, Trends Immunol 2009; 30(7):306-312 Keywords: Vitamin D, CD28null, primary sclerosing cholangitis, biliary epithelial cells, Cytotoxicity Conference: 15th International Congress of Immunology (ICI), Milan, Italy, 22 Aug - 27 Aug, 2013. Presentation Type: Abstract Topic: Immune-mediated disease pathogenesis Citation: Liaskou E, Jeffery L, Suresh S, Trivedi P, Bruns T, Adams D and Sansom D (2013). Phenotypic evaluation of CD28 negative T cells in primary sclerosing cholangitis. Front. Immunol. Conference Abstract: 15th International Congress of Immunology (ICI). doi: 10.3389/conf.fimmu.2013.02.00085 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 08 Mar 2013; Published Online: 22 Aug 2013. * Correspondence: Dr. Evaggelia Liaskou, University of Birmingham, Centre for Liver Research and NIHR Biomedical Research Unit in Liver Disease, Institute of Biomedical Research, Birmingham, United Kingdom, e.liaskou@bham.ac.uk Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Evaggelia Liaskou Louisa Jeffery Shankar Suresh Palak Trivedi Tony Bruns David Adams David Sansom Google Evaggelia Liaskou Louisa Jeffery Shankar Suresh Palak Trivedi Tony Bruns David Adams David Sansom Google Scholar Evaggelia Liaskou Louisa Jeffery Shankar Suresh Palak Trivedi Tony Bruns David Adams David Sansom PubMed Evaggelia Liaskou Louisa Jeffery Shankar Suresh Palak Trivedi Tony Bruns David Adams David Sansom Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Lipid profile analysis in spinal trauma patients shows severe distortion of AA/DHA after injury

Lipid profile analysis in spinal trauma patients shows severe distortion of AA/DHA after injury

Caspase-8 Blocks Kinase RIPK3-Mediated Activation of the NLRP3 Inflammasome

Caspase-8 deficiency in certain cells prompts chronic inflammation. One mechanism suggested to account for this inflammation is enhanced signaling for necrotic cell death, mediated by the protein kinases RIPK1 and RIPK3 that caspase-8 can cleave. We describe an activity of caspase-8 in dendritic cells that controls the initiation of inflammation in another way. Caspase-8 deficiency in thesecells facilitated lipopolysaccharide-induced assembly and function of the NLRP3 inflammasome. This effect depended on the functions of RIPK1 and RIPK3, as well as of MLKL and PGAM5, two signaling proteins recently shown to contribute to RIPK3-mediated induction of necrosis. However, although enhancement of inflammasome assembly in the caspase-8-deficient cells shares proximal signaling events with the induction of necrosis, it occurred independently of cell death. These findings provide new insight into potentially pathological inflammatory processes to which RIPK1- and RIPK3-mediated signaling contributes.

Abstract SY32-01: The immunologic constant of rejection and cancer

Abstract The advent of high-throughput technology challenges the traditional histopathological classification of cancer, and proposes new taxonomies derived from global transcriptional patterns. Although most of these molecular re-classifications did not endure the test of time, they provided bulk of new information that can reframe our understanding of human cancer biology. Together with Galon's (1,2) and other groups, we propose an immunologic interpretation of cancer that segregates oncogenic processes independent from their tissue derivation into at least two categories of which one bears the footprints of immune activation. Several observations describe a cancer phenotype where the expression of interferon stimulated genes and immune effector mechanisms reflect patterns commonly observed during the inflammatory response against pathogens, which leads to elimination of infected cells (3). As these signatures are observed in growing cancers, they are not sufficient to entirely clear the organism of neoplastic cells but they sustain, as in chronic infections, a self-perpetuating inflammatory process(4). Yet, several studies determined an association between this inflammatory status and a favorable natural history of the disease or a better responsiveness to cancer immune therapy. Indeed, these signatures that define a good prognostic cancer phenotype are similar to those associated with the broader phenomenon of immune-mediated, tissue-specific destruction (TSD) in other immune pathologies such as regression of cancer during immunotherapy, allograft rejection, graft versus host disease, flares of autoimmunity or destruction of virally infected cells to clear intra-cellular pathogens during acute infection. Direct human observations collected by studying tissues during the acute phases of TSD demonstrated a convergence toward a common final mechanism including the activation of interferon stimulated genes, genes associated with immune effector function and the presence of a restricted group of chemokines targeting activated T and natural killer cells. We defined this set of genes “the immunologic constant of rejection” (ICR)(5,4). In cancer, it appears that the ICR genes are present not only during the acute phase of rejection, but also are expressed though with lower intensity in tumors that bear better prognosis and in tumors most likely to response to immune therapy. In particular, we recently followed a limited set of melanoma metastases undergoing immunotherapy with systemic high-dose interleukin-2. As previously observed (6), metastases that underwent complete regression following therapy differed in their transcriptional profile from non responding ones even before treatment (7) and expressed a subset of ICR genes. By serially biopsying the same metastases using minimally invasive techniques, we observed that during treatment, the responsive metastases underwent a further activation of ICR genes that was both qualitative and quantitative while the non-responding ones remained silent. This observation suggests that immune rejection of cancer is part of the intrinsic biology of individual cancers which may be predisposed to immune responsiveness and their natural history can be modulated with interventions that can enhance a naturally occurring phenomenon. It remains, however, unknown why it occurs in immune competent hosts. It also remains uncertain whether a genetically determined response of the host to its own cancer, the genetic makeup of the neoplastic process or a combination of both drives the inflammatory process. Recent work from our group suggests that a combination of the two could be responsible for this multifactorial event. Analysis of two cases in which patients with melanoma experienced a mixed response during therapy suggests that the activation of ICR genes and consequently immune rejection of cancer is genetically determined by the tumor phenotypes as distinct tumors responded differently in the same patient at the same time, therefore, eliminating the genetic background of the host and/or environmental factors as determinants in these cases (8). On the other hand, recent work also from our group identified a polymorphism of the interferon-regulatory factor-5 (IRF-5) as a potential predictor of non-responsiveness to immunotherapy (9). Thus, we propose a hierarchical model of immune responsiveness in which the likelihood of a tumor to respond to immunotherapy is first dependent upon a check-point determined by the genetic background of the patient. As a second check point, in patients genetically predisposed to respond to therapy, is the genetic makeup of individual cancers that can further determine their responsiveness. Finally, as a third check point, environmental factors such as quality, intensity and type of therapy may modulate, within each cancer the likelihood of response. As several of these determinants are observed in other forms of TSD such for instance systemic lupus erythematosus, we propose, that cancer rejection in response to immunotherapy, it an aspect of auto-immunity that targets cancer-specific biological processes and it occurs only when a full-fledged acute inflammatory status is reached with complete activation of ICR genes.

Phospholipase Cγ2 (PLCγ2) Is Key Component in Dectin-2 Signaling Pathway, Mediating Anti-fungal Innate Immune Responses

C-type lectin receptors (CLRs) such as Dectin-2 function as pattern recognition receptors to sense fungal infection. However, the signaling pathways induced by these receptors remain largely unknown. Previous studies suggest that the CLR-induced signaling pathway may utilize similar signaling components as the B cell receptor-induced signaling pathway. Phospholipase Cγ2 (PLCγ2) is a key component in B cell receptor signaling, but its role in other signaling pathways has not been fully characterized. Here, we show that PLCγ2 functions downstream of Dectin-2 in response to the stimulation by the hyphal form of Candida albicans, an opportunistic pathogenic fungus. Using PLCγ2- and PLCγ1-deficient macrophages, we found that the lack of PLCγ2, but not PLCγ1, impairs cytokine production in response to infection with C. albicans. PLCγ2 deficiency results in the defective activation of NF-κB and MAPK and a significantly reduced production of reactive oxygen species following fungal challenge. In addition, PLCγ2-deficient mice are defective in clearing C. albicans infection in vivo. Together, these findings demonstrate that PLCγ2 plays a critical role in CLR-induced signaling pathways, governing antifungal innate immune responses.

Regulation of Cathelicidin Antimicrobial Peptide Expression by an Endoplasmic Reticulum (ER) Stress Signaling, Vitamin D Receptor-independent Pathway

Vitamin D receptor (VDR)-dependent mechanisms regulate human cathelicidin antimicrobial peptide (CAMP)/LL-37 in various cell types, but CAMP expression also increases after external perturbations (such as infection, injuries, UV irradiation, and permeability barrier disruption) in parallel with induction of endoplasmic reticulum (ER) stress. We demonstrate that CAMP mRNA and protein expression increase in epithelial cells (human primary keratinocytes, HaCaT keratinocytes, and HeLa cells), but not in myeloid (U937 and HL-60) cells, following ER stress generated by two mechanistically different, pharmacological stressors, thapsigargin or tunicamycin. The mechanism for increased CAMP following exposure to ER stress involves NF-κB activation leading to CCAAT/enhancer-binding protein α (C/EBPα) activation via MAP kinase-mediated phosphorylation. Furthermore, both increased CAMP secretion and its proteolytic processing to LL-37 are required for antimicrobial activities occur following ER stress. In addition, topical thapsigargin also increases production of the murine homologue of CAMP in mouse epidermis. Finally and paradoxically, ER stress instead suppresses the 1,25(OH)(2) vitamin D(3)-induced activation of VDR, but blockade of VDR activity does not alter ER stress-induced CAMP up-regulation. Hence, ER stress increases CAMP expression via NF-κB-C/EBPα activation, independent of VDR, illuminating a novel VDR-independent role for ER stress in stimulating innate immunity.

Homologous Elements hs3a and hs3b in the 3′ Regulatory Region of the Murine Immunoglobulin Heavy Chain (Igh) Locus Are Both Dispensable for Class-switch Recombination

Immunoglobulin heavy chain (IgH) genes are formed, tested, and modified to yield diverse, specific, and high affinity antibody responses to antigen. The processes involved must be regulated, however, to avoid unintended damage to chromosomes. The 3' regulatory region of the Igh locus plays a major role in regulating class-switch recombination (CSR), the process by which antibody effector functions are modified during an immune response. Loss of all known enhancer-like elements in this region dramatically impairs CSR, but individual element deletions have no effect on this process. In the present study, we explored the hypothesis that an underlying functional redundancy in the homologous elements hs3a and hs3b was masking the importance of either element to CSR. Several transgenic mouse lines were generated, each carrying a bacterial artificial chromosome transgene that mimicked Igh locus structure but in which hs3a was missing and hs3b was flanked by loxP sites. Matings to Cyclization Recombination Enzyme-expressing mice established "pairs" of lines that differed only in the presence or absence of hs3b. Remarkably, CSR remained robust in the absence of both hs3a and hs3b, suggesting that the remaining two elements of the 3' regulatory region, hs1.2 and hs4, although individually dispensable for CSR, are, together, sufficient to support CSR.

Leukocyte rolling and adhesion via ICAM-1 signals to endothelial permeability. Focus on “Leukocyte rolling and adhesion both contribute to regulation of microvascular permeability to albumin via ligation of ICAM-1”

THE VASCULAR ENDOTHELIUM plays a vital role in the inflammatory response by induction and surface expression of adhesion molecules and chemokines. At sites of acute inflammation, blood flow is increased and postcapillary venules exhibit increased permeability (leakiness) and support the influx of blood leukocytes, primarily neutrophils. The relationship between neutrophil influx and increased vessel permeability has been a topic of investigation for decades. Many investigations have reported a direct neutrophil-dependent increase in vessel permeability during inflammation using in vivo and in vitro models (reviewed in Ref. 4). One aspect that has received increased attention is whether endothelial cell adhesion molecules contribute to the regulation of vessel permeability, in addition to their well-characterized role in neutrophil rolling and adhesion. Intercellular adhesion molecule-1 (ICAM-1, also known as CD54) is an immunoglobulin superfamily member that is abundantly expressed on the endothelial cell surface and is enriched at endothelial cell borders in vivo and in vitro (2, 12, 14, 17). ICAM-1 surface expression is upregulated in endothelial cells by proinflammatory cytokines like TNF-, IL-1, IFN-, or bacterial endotoxins, and ICAM-1 serves as a receptor for leukocyte 2-integrins (LFA-1 and Mac-1) (reviewed in Ref. 15). In an experiment of nature, patients with leukocyte adhesion deficiency-1 (LAD-1) have a severe primary immune deficiency in which blood neutrophils fail to localize to sites of inflammation or injury. This defect occurs because patients’ neutrophils either lack 2-integrins or contain mutations in these molecules. Studies using neutrophils from LAD-1 patients or 2-integrin-deficient mice clearly demonstrate that these cells have normal selectin-dependent rolling on activated endothelium but fail to stably arrest and transmigrate. Additional studies have demonstrated that endothelial-expressed ICAM-1 contributes to both kinetics of leukocyte rolling and arrest in vivo as determined by intravital microscopy of the cremaster muscle microcirculation in ICAM-1 / mice (16). In this issue of American Journal of Physiology-Cell Physiology, Sumagin and colleagues (18) use elegant in vivo studies to demonstrate that microvascular permeability in the cremaster muscle model is regulated by leukocyte engagement of ICAM-1. The authors monitored permeability (Ps) in cremaster microcirculation by efflux of luminal fluorescent-tagged albumin by fluorescence confocal intravital microscopy. Prior studies by Sarelius and colleagues (19) in the same model demonstrated that ICAM-1 engagement induced a localized increase in vessel permeability in unstimulated arterioles and in TNF-stimulated venules. In agreement with this finding ,4ho f TNF- stimulation of arterioles and venules in ICAM-1 knockout (KO) animals did not result in increased vessel permeability as compared with wild-type (WT) mice under identical conditions. Similar results were seen with 2-integrin KO mice. In the current study, the authors provide further evidence linking ICAM-1 expression to regulation of endothelial permeability. Comparisons between unstimulated venules and TNF--treated venules reveal that neutrophil rolling regulates venule permeability in unstimulated venules while leukocyte adhesion regulates venule permeability in TNF--stimulated venules. The 2-integrin-blocking antibodies, which inhibit neutrophil arrest on endothelium, reduced the vessel permeability, and this was further reduced by treatment with a combination of 2-integrin and P-selectin blocking antibodies that inhibits both rolling and arrest. Interestingly, the reduction in venule permeability observed by blockade of P-selectin and 2-integrin function were suggested to be due to insufficient ICAM-1 engagement due to the absence of rolling leukocytes. To examine the role that neutrophils play, circulating neutrophils were depleted by antineutrophil GR-1 antibody treatment. Notably, depletion of neutrophils caused a marked decrease in

Elevated numbers and frequencies of CD56- CD3- NK cells with high perforin occur in healthy donors (160.14)

Abstract Routinely, human natural killer (NK) lymphocytes are identified as CD3eneg CD56bright or dim. CD3neg CD56neg cells with low perforin are found in viremic patients infected with human immunodeficiency virus and hepatitis C virus (reviewed, N.K. Bjorkstorm et al., Trends Immunol. 31:401, 2010). To our surprise, we found by flow cytometry that CD3neg perforin(PRF1) positive NK cells included as many as 50% CD56neg cells within the blood of 97 healthy subjects. Among these subjects (aged 21 to 62 years), the CD3eneg PRF1positive cells included at least 10% CD56neg cells in 24 of 97 (~25%) subjects. CD56neg perforin high CD3neg cells were over 50 cells/mm3 of blood in 14% of the healthy donors. As anticipated, the CD56dim cells had ~7 fold higher perforin MFI than the CD56bright cells. Notably in the healthy donors with high frequencies of CD56neg cells, these cells had bimodal perforin levels comparable to the levels in the CD56bright and CD56dim subsets. The perforin levels indicate that many CD56neg NK cells may have strong cytotoxicity in healthy donors in contrast to the low perforin and cytotoxicity observed in CD56neg NK cells of the viremic patients. We conclude that CD56neg NK cells now warrant attention as potential cytotoxic cells after receiving considerable attention as high chemokine-secreting cells.

Variable Antibody-dependent Activation of Complement by Functionalized Phospholipid Nanoparticle Surfaces

A wide variety of nanomaterials are currently being developed for use in the detection and treatment of human diseases. However, there is no systematic way to measure and predict the action of such materials in biological contexts. Lipid-encapsulated nanoparticles (NPs) are a class of nanomaterials that includes the liposomes, the most widely used and clinically proven type of NPs. Liposomes can, however, activate the complement system, an important branch of innate immunity, resulting in undesirable consequences. Here, we describe the complement response to lipid-encapsulated NPs that are functionalized on the surface with various lipid-anchored gadolinium chelates. We developed a quantitative approach to examine the interaction of NPs with the complement system using in vitro assays and correlating these results with those obtained in an in vivo mouse model. Our results indicate that surface functionalization of NPs with certain chemical structures elicits swift complement activation that is initiated by a natural IgM antibody and propagated via the classical pathway. The intensity of the response is dependent on the chemical structures of the lipid-anchored chelates and not zeta potential effects alone. Moreover, the extent of complement activation may be tempered by complement inhibiting regulatory proteins that bind to the surface of NPs. These findings represent a step forward in the understanding of the interactions between nanomaterials and the host innate immune response and provide the basis for a systematic structure-activity relationship study to establish guidelines that are critical to the future development of biocompatible nanotherapeutics.

The Interleukin 13 (IL-13) Pathway in Human Macrophages Is Modulated by MicroRNA-155 via Direct Targeting of Interleukin 13 Receptor α1 (IL13Rα1)

Macrophages play a central role in the balance and efficiency of the immune response and are at the interface between innate and adaptive immunity. Their phenotype is a delicate equilibrium between the M1 (classical, pro-Th(1)) and M2 (alternative, pro-Th(2)) profiles. This balance is regulated by cytokines such as interleukin 13 (IL-13), a typical pro-M2-Th(2) cytokine that has been related to allergic disease and asthma. IL-13 binds to IL-13 receptor α1 (IL13Rα1), a component of the Type II IL-4 receptor, and exerts its effects by activating the transcription factor signal transducer and activator of transcription 6 (STAT6) through phosphorylation. MicroRNAs are short (∼22 nucleotide) inhibitory non-coding RNAs that block the translation or promote the degradation of their specific mRNA targets. By bioinformatics analysis, we found that microRNA-155 (miR-155) is predicted to target IL13Rα1. This suggested that miR-155 might be involved in the regulation of the M1/M2 balance in macrophages by modulating IL-13 effects. miR-155 has been implicated in the development of a healthy immune system and function as well as in the inflammatory pro-Th(1)/M1 immune profile. Here we have shown that in human macrophages, miR-155 directly targets IL13Rα1 and reduces the levels of IL13Rα1 protein, leading to diminished activation of STAT6. Finally we also demonstrate that miR-155 affects the IL-13-dependent regulation of several genes (SOCS1, DC-SIGN, CCL18, CD23, and SERPINE) involved in the establishment of an M2/pro-Th(2) phenotype in macrophages. Our work shows a central role for miR-155 in determining the M2 phenotype in human macrophages.

Tumor-secreted PGE2 Inhibits CCL5 Production in Activated Macrophages through cAMP/PKA Signaling Pathway

One of the major characteristics of tumors is their ability to evade immunosurveillance through altering the properties and functions of host stromal and/or immune cells. CCL5 has been shown to play important roles in T cell proliferation, IFN-γ, and IL-2 production, which promotes the differentiation and proliferation of Th1 cells important for immune defense against intracellular infection. In this study we found that tumor-bearing mice were more susceptible to bacterial infection and showed reduced CCL5 levels in serum during endotoxic shock. Our data further demonstrated that the soluble factors secreted by mammary gland tumor cells but not normal mammary gland epithelial cells inhibited CCL5 expression in macrophages in response to LPS, but not to TNF-α stimulation. The inhibitory effect of tumor-secreted molecules on LPS-induced CCL5 expression was regulated at the post-transcriptional level. Blocking PGE(2) synthesis by NS398 or through the use of PGE(2) receptor antagonists AH-6809 (EP2 antagonist) and AH-23848 (EP4 antagonist) completely reversed the inhibitory effect of tumor-conditioned medium (TCM) on LPS-induced CCL5 expression. Moreover, PGE(2) and the cAMP analog forskolin could mimic tumor-mediated CCL5 inhibition, and the inhibitory effects of TCM, PGE(2), and cAMP analog on LPS-induced CCL5 expression could be completely reversed by the PKA inhibitor H89. Furthermore, blocking PGE(2) synthesis in vivo led to partial recovery of CCL5 production during endotoxic shock. Taken together, our data indicate that PGE(2) secreted from breast cancer cells suppresses CCL5 secretion in LPS-activated macrophages through a cAMP/PKA signaling pathway, which may result in suppression of host immune responses against subsequent bacterial infection.

Classical NF-κB Activation Negatively Regulates Noncanonical NF-κB-dependent CXCL12 Expression

Ligation of the lymphotoxin-β receptor (LTβR) by LIGHT (lymphotoxin-related inducible ligand that competes for glycoprotein D binding to herpes virus entry mediator on T cells (TNFSF14)) activates the noncanonical (NC) NF-κB (nuclear factor-κB) pathway and up-regulates CXCL12 gene expression by human umbilical vein endothelial cells (HUVEC). In contrast, TNF only activates classical NF-κB signaling and does not up-regulate CXCL12. To determine whether cross-talk between the classical and NC pathways affects CXCL12 expression, we investigated the effects of TNF on LIGHT signaling in HUVEC. We show here that TNF inhibits both basal and LIGHT-induced CXCL12 expression. Negative regulation by TNF requires the classical NF-κB pathway as inhibition of basal and induced CXCL12 was reversed in HUVEC-expressing dominant negative IκB (inhibitor of NF-κB) kinase (IKK)β (IKKβ(K44M)). TNF did not inhibit the NC NF-κB pathway activation as LIGHT-induced p100 processing to p52 was intact; however, TNF either alone or together with LIGHT up-regulated p100 and RelB expression and induced the nuclear localization of p100-RelB complexes. Enhanced p100 and RelB expression was inhibited by IKKβ(K44M), which led us to question whether the IκB function of elevated p100 mediates the inhibition of CXCL12 expression by TNF. We retrovirally transduced HUVEC to express p100 at a level similar to that up-regulated by TNF; however, basal and LIGHT-induced CXCL12 expression was normal in the transduced cells. In contrast, ectopic RelB expression recapitulated the effects of TNF on NC signaling and inhibited basal and LIGHT-induced CXCL12 expression by HUVEC. Our findings therefore demonstrate that TNF-induced classical NF-κB signaling up-regulates RelB expression that inhibits both basal and NC NF-κB-dependent CXCL12 expression.

Mapping Surface Accessibility of the C1r/C1s Tetramer by Chemical Modification and Mass Spectrometry Provides New Insights into Assembly of the Human C1 Complex

C1, the complex that triggers the classic pathway of complement, is a 790-kDa assembly resulting from association of a recognition protein C1q with a Ca(2+)-dependent tetramer comprising two copies of the proteases C1r and C1s. Early structural investigations have shown that the extended C1s-C1r-C1r-C1s tetramer folds into a compact conformation in C1. Recent site-directed mutagenesis studies have identified the C1q-binding sites in C1r and C1s and led to a three-dimensional model of the C1 complex (Bally, I., Rossi, V., Lunardi, T., Thielens, N. M., Gaboriaud, C., and Arlaud, G. J. (2009) J. Biol. Chem. 284, 19340-19348). In this study, we have used a mass spectrometry-based strategy involving a label-free semi-quantitative analysis of protein samples to gain new structural insights into C1 assembly. Using a stable chemical modification, we have compared the accessibility of the lysine residues in the isolated tetramer and in C1. The labeling data account for 51 of the 73 lysine residues of C1r and C1s. They strongly support the hypothesis that both C1s CUB(1)-EGF-CUB(2) interaction domains, which are distant in the free tetramer, associate with each other in the C1 complex. This analysis also provides the first experimental evidence that, in the proenzyme form of C1, the C1s serine protease domain is partly positioned inside the C1q cone and yields precise information about its orientation in the complex. These results provide further structural insights into the architecture of the C1 complex, allowing significant improvement of our current C1 model.

Human Transferrin Confers Serum Resistance against Bacillus anthracis

The innate immune system in humans consists of both cellular and humoral components that collaborate to eradicate invading bacteria from the body. Here, we discover that the gram-positive bacterium Bacillus anthracis, the causative agent of anthrax, does not grow in human serum. Fractionation of serum by gel filtration chromatography led to the identification of human transferrin as the inhibiting factor. Purified transferrin blocks growth of both the fully virulent encapsulated B. anthracis Ames and the non-encapsulated Sterne strain. Growth inhibition was also observed in serum of wild-type mice but not of hypotransferrinemic mice that only have approximately 1% circulating transferrin levels. We were able to definitely assign the bacteriostatic activity of transferrin to its iron-binding function: neither iron-saturated transferrin nor a recombinant transferrin mutant unable to bind iron could inhibit growth of B. anthracis. Additional iron could restore bacterial growth in human serum. The observation that other important gram-positive pathogens are not inhibited by transferrin suggests they have evolved effective mechanisms to circumvent serum iron deprivation. These findings provide a better understanding of human host defense mechanisms against anthrax and provide a mechanistic basis for the antimicrobial activity of human transferrin.