Connexin-36 contributes to control function of insulin-producing cells.

Connexin-36 (Cx36) is a gap junction protein expressed by the insulin-producing beta-cells. We investigated the contribution of this protein in normal beta-cell function by using a viral gene transfer approach to alter Cx36 content in the insulin-producing line of INS-1E cells and rat pancreatic islets. Transcripts for Cx43, Cx45, and Cx36 were detected by reverse transcriptase-PCR in freshly isolated pancreatic islets, whereas only a transcript for Cx36 was detected in INS-1E cells. After infection with a sense viral vector, which induced de novo Cx36 expression in the Cx-defective HeLa cells we used to control the transgene expression, Western blot, immunofluorescence, and freeze-fracture analysis showed a large increase of Cx36 within INS-1E cell membranes. In contrast, after infection with an antisense vector, Cx36 content was decreased by 80%. Glucose-induced insulin release and insulin content were decreased, whether infected INS-1E cells expressed Cx36 levels that were largely higher or lower than those observed in wild-type control cells. In both cases, basal insulin secretion was unaffected. Comparable observations on basal secretion and insulin content were made in freshly isolated rat pancreatic islets. The data indicate that large changes in Cx36 alter insulin content and, at least in INS-1E cells, also affect glucose-induced insulin release.

The specificity of TRIM5 alpha-mediated restriction is influenced by its coiled-coil domain.

Retroviruses are both powerful evolutionary forces and dangerous threats to genome integrity. As such, they have imposed strong selective pressure on their hosts, notably triggering the emergence of restriction factors, such as TRIM5 alpha, that act as potent barriers to their cross-species transmission. TRIM5 alpha orthologues from different primates have distinct retroviral restriction patterns, largely dictated by the sequence of their C-terminal PRYSPRY domain, which binds the capsid protein of incoming virions. Here, by combining genetic and functional analyses of human and squirrel monkey TRIM5 alpha, we demonstrate that the coiled-coil domain of this protein, thus far essentially known for mediating oligomerization, also conditions the spectrum of antiretroviral activity. Furthermore, we identify three coiled-coil residues responsible for this effect, one of which has been under positive selection during primate evolution, notably in New World monkeys. These results indicate that the PRYSPRY and coiled-coil domains cooperate to determine the specificity of TRIM5 alpha-mediated capture of retroviral capsids, shedding new light on this complex event.

Immune regulation and function of the NOD-like receptors NLRC5 and NLRP3

AbstractThe vertebrate immune system is composed of the innate and the adaptive branches. Innate immune cells represent the first line of defense and detect pathogens through pattern recognition receptors (PRRs), detecting evolutionary conserved pathogen- and danger- associated molecular patterns. Engagement of these receptors initiates the inflammatory response, but also instructs antigen-specific adaptive immune cells. NOD-like receptors (NLRs) are an important group of PRRs, leading to the production of inflammatory mediators and favoring antigen presentation to Τ lymphocytes through the regulation of major histocompatibility complex (MHC) molecules.In this work we focused our attention on selected NOD-like receptors (NLRs) and their role at the interface between innate and adaptive immunity. First, we describe a new regulatory mechanism controlling IL-1 production. Our results indicate that type I interferons (IFNs) block NLRP1 and NLRP3 inflammasome activity and interfere with LPS-driven proIL-Ια and -β induction. As type I IFNs are produced upon viral infections, these anti-inflammatory effects of type I IFN could be relevant in the context of superinfections, but could also help explaining the efficacy of IFN-β in multiple sclerosis treatment.The second project addresses the role of a novel NLR family member, called NLRC5. The function of this NLR is still matter of debate, as it has been proposed as both an inhibitor and an activator of different inflammatory pathways. We found that the expression of this protein is restricted to immune cells and is positively regulated by IFNs. We generated Nlrc5-deficient mice and found that this NLR plays an essential role in Τ, NKT and, NK lymphocytes, in which it drives the expression of MHC class I molecules. Accordingly, we could show that CD8+ Τ cell-mediated killing of target lymphocytes lacking NLRC5 is strongly impaired. Moreover, NLRC5 expression was found to be low in many lymphoid- derived tumor cell lines, a mechanism that could be exploited by tumors to escape immunosurveillance.Finally, we found NLRC5 to be involved in the production of IL-10 by CD4+ Τ cells, as Nlrc5- deficient Τ lymphocytes produced less of this cytokine upon TCR triggering. In line with these observations, Mrc5-deficient CD4+ Τ cells expanded more than control cells when transferred into lymphopenic hosts and led to a more rapid appearance of colitis symptoms. Therefore, our work gives novel insights on the function of NLRC5 by using knockout mice, and strongly supports the idea that NLRs direct not only innate, but also adaptive immune responses.

Oligomerization of the alpha 1a- and alpha 1b-adrenergic receptor subtypes. Potential implications in receptor internalization.

We combined biophysical, biochemical, and pharmacological approaches to investigate the ability of the alpha 1a- and alpha 1b-adrenergic receptor (AR) subtypes to form homo- and hetero-oligomers. Receptors tagged with different epitopes (hemagglutinin and Myc) or fluorescent proteins (cyan and green fluorescent proteins) were transiently expressed in HEK-293 cells either individually or in different combinations. Fluorescence resonance energy transfer measurements provided evidence that both the alpha 1a- and alpha 1b-AR can form homo-oligomers with similar transfer efficiency of approximately 0.10. Hetero-oligomers could also be observed between the alpha 1b- and the alpha 1a-AR subtypes but not between the alpha 1b-AR and the beta2-AR, the NK1 tachykinin, or the CCR5 chemokine receptors. Oligomerization of the alpha 1b-AR did not require the integrity of its C-tail, of two glycophorin motifs, or of the N-linked glycosylation sites at its N terminus. In contrast, helix I and, to a lesser extent, helix VII were found to play a role in the alpha 1b-AR homo-oligomerization. Receptor oligomerization was not influenced by the agonist epinephrine or by the inverse agonist prazosin. A constitutively active (A293E) as well as a signaling-deficient (R143E) mutant displayed oligomerization features similar to those of the wild type alpha 1b-AR. Confocal imaging revealed that oligomerization of the alpha1-AR subtypes correlated with their ability to co-internalize upon exposure to the agonist. The alpha 1a-selective agonist oxymetazoline induced the co-internalization of the alpha 1a- and alpha 1b-AR, whereas the alpha 1b-AR could not co-internalize with the NK1 tachykinin or CCR5 chemokine receptors. Oligomerization might therefore represent an additional mechanism regulating the physiological responses mediated by the alpha 1a- and alpha 1b-AR subtypes.

Mutagenesis and modelling of the alpha(1b)-adrenergic receptor highlight the role of the helix 3/helix 6 interface in receptor activation.

Computer simulations on a new model of the alpha1b-adrenergic receptor based on the crystal structure of rhodopsin have been combined with experimental mutagenesis to investigate the role of residues in the cytosolic half of helix 6 in receptor activation. Our results support the hypothesis that a salt bridge between the highly conserved arginine (R143(3.50)) of the E/DRY motif of helix 3 and a conserved glutamate (E289(6.30)) on helix 6 constrains the alpha1b-AR in the inactive state. In fact, mutations of E289(6.30) that weakened the R143(3.50)-E289(6.30) interaction constitutively activated the receptor. The functional effect of mutating other amino acids on helix 6 (F286(6.27), A292(6.33), L296(6.37), V299(6.40,) V300(6.41), and F303(6.44)) correlates with the extent of their interaction with helix 3 and in particular with R143(3.50) of the E/DRY sequence.

Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that can be activated by various xenobiotics and natural fatty acids. These transcription factors primarily regulate genes involved in lipid metabolism and also play a role in adipocyte differentiation. We present the expression patterns of the PPAR subtypes in the adult rat, determined by in situ hybridization using specific probes for PPAR-alpha, -beta and -gamma, and by immunohistochemistry using a polyclonal antibody that recognizes the three rat PPAR subtypes. In numerous cell types from either ectodermal, mesodermal, or endodermal origin, PPARs are coexpressed, with relative levels varying between them from one cell type to the other. PPAR-alpha is highly expressed in hepatocytes, cardiomyocytes, enterocytes, and the proximal tubule cells of kidney. PPAR-beta is expressed ubiquitously and often at higher levels than PPAR-alpha and -gamma. PPAR-gamma is expressed predominantly in adipose tissue and the immune system. Our results suggest new potential directions to investigate the functions of the different PPAR subtypes.

Dual function of eosinophils in pathogenesis and protective immunity against parasites

The functional duality of eosinophils, involved in a protective response or in pathogenesis is illustrated in various parasitic infections. In schistosomiasis, eosinophils have been shown to mediate schistosomula killing, in the presence of antibodies. The association of eosinophil-dependent cytotoxic antibody isotypes with resistance of reinfection (IgE and IgA antibodies), whereas in vitro blocking antibody isotypes (IgG4, IgM) were detected in susceptible subjects, suggested a participation of eosinophils in antibody-dependent protective response. However eosinophils could participate to granuloma formation and consequently to the pathological reactions during schistosomiasis. Activation of eosinophils by antibodies, leading to release of granule proteins have been studied in patients with filariasis. Eosinophil peroxidase, EPO was released safter IgE-dependent activation whereas Eosinophil Cationic Protein, ECP, was released after IgG- and IgA-dependent activation of eosinophils, results suggesting a process of differential release mediators. Interactions between eosinophils and interleukins, and specially IL-5 are discussed. Whereas a receptor for IL-5 has been characterized on human eosinophils, recent studies have shown that eosinophils, expressed the messenger RNA encoding IL-5. These results associated to data showing the synthesis of other cytokines indicate that eosinophils are not only the source of cytotoxic mediators involved in the effector phase of immunity but also of growth and regualtory factors, participating to immunoregulation.

Determination of human plasma levels of levo-alpha-acetylmethadol and its metabolites by gas chromatography-mass spectrometry.

A gas chromatography-mass spectrometry (GC-MS) method is presented which allows the simultaneous determination of the plasma concentrations of the levo-alpha-acetylmethadol (LAAM) and of its active metabolites (NorLAAM and DiNorLAAM), after derivatization with the reagent trifluoroacetic anhydride (TFAA). No interferences from endogenous compounds were observed following the extraction of plasma samples from 11 different human subjects. The standard curves were linear over a working range of 5-200ng/ml for the three compounds. Recoveries measured at three concentrations ranged from 47 to 67% for LAAM, from 50 to 69% for NorLAAM and from 28 to 50% for DiNorLAAM. Intra- and interday coefficients of variation determined at three concentrations ranged from 5 to 13% for LAAM, from 3 to 9% for NorLAAM and from 5 to 13% for DiNorLAAM. The limits of quantitation of the method were found to be 4ng/ml for the three compounds. No interference was noted from methadone. This sensitive and specific analytical method could be useful for assessing the in vivo relationship between LAAM's blood levels, clinical efficacy and/or cardiotoxicity

A new function of the Fas-FasL pathway in macrophage activation.

Upon infection with the protozoan parasite Leishmania major, susceptible BALB/c mice develop unhealing lesions associated with the maturation of CD4(+)Th2 cells secreting IL-4. In contrast, resistant C57BL/6 mice heal their lesions, because of expansion and secretion of IFN-gamma of CD4(+) Th1 cells. The Fas-FasL pathway, although not involved in Th cell differentiation, was reported to be necessary for complete resolution of lesions. We investigate here the role of IFN-gamma and IL-4 on Fas-FasL nonapoptotic signaling events leading to the modulation of macrophage activation. We show that addition of FasL and IFN-gamma to BMMø led to their increased activation, as reflected by enhanced secretion of TNF, IL-6, NO, and the induction of their microbicidal activity, resulting in the killing of intracellular L. major. In contrast, the presence of IL-4 decreased the synergy of IFN-gamma/FasL significantly on macrophage activation and the killing of intracellular L. major. These results show that FasL synergizes with IFN-gamma to activate macrophages and that the tight regulation by IFN-gamma and/or IL-4 of the nonapoptotic signaling events triggered by the Fas-FasL pathway affects significantly the activation of macrophages to a microbicidal state and may thus contribute to the pathogenesis of L. major infection.

Time-Course In Vivo Trafficking Pattern and Effector Function of Donor-Specific Regulatory T Cells in Response to an Allograft.

Background: Experimental data have suggested that adoptive transfer of CD4+CD25+Foxp3+ regulatory T cells (Tregs), capable of controlling immune responses to specifi c auto- or alloantigens, could be used as a therapeutic strategy to promote specifi c tolerance in T-cell mediated diseases and in organ transplantation (Tx). However, before advocating the application of immunotherapy with Tregs in Tx, we need to improve our understanding of their in vivo homeostasis, traffi cking pattern and effector function in response to alloantigens. Methods : Donor-antigen specifi c murine Tregs were generated and characterized in vitro following our described protocols. Using an adoptive transfer and skin allotransplantation model, we have analyzed the in vivo expansion and homing of fl uorescent-labeled effector T cells (Teff) and Tregs, at different time-points after Tx, using fl ow-cytometry as well as fl uorescence microscopy techniques. Results: Tregs expressed CD62L, CCR7 and CD103 allowing their homing into lymphoid and non-lymphoid tissues (gut, skin) after intravenous injection. While hyporesponsive to TCR stimulation in vitro, transferred Tregs survived, migrated to secondary lymphoid organs and preferentially expanded within the allograft draining lymph nodes. Furthermore, Foxp3+ cells could be detected inside the allograft as early as day 3-5 after Tx. At a much later time-point (day 60 after Tx), graft-infi ltrating Foxp3+ cells were also detectable in tolerant recipients. When transferred alone, CD4+CD25- Teff cells expanded within secondary lymphoid organs and infi ltrated the allograft by day 3-5 after Tx. The co-transfer of Tregs limited the expansion of alloreactive Teff cells as well as their recruitment into the allograft. The promotion of graft survival observed in the presence of Tregs was in part mediated by the inhibition of the production of effector cytokines by CD4+CD25- T cells. Conclusion: Taken together, our results suggest that the suppression of allograft rejection and the induction of Tx tolerance are in part dependant on the alloantigendriven homing and expansion of Tregs. Thus, the appropriate localization of Tregs may be critical for their suppressive function in vivo.

TIE-2 and VEGFR Kinase Activities Drive Immunosuppressive Function of TIE-2-Expressing Monocytes in Human Breast Tumors.

PURPOSE: Tumor-associated TIE-2-expressing monocytes (TEM) are highly proangiogenic cells critical for tumor vascularization. We previously showed that, in human breast cancer, TIE-2 and VEGFR pathways control proangiogenic activity of TEMs. Here, we examine the contribution of these pathways to immunosuppressive activity of TEMs. EXPERIMENTAL DESIGN: We investigated the changes in immunosuppressive activity of TEMs and gene expression in response to specific kinase inhibitors of TIE-2 and VEGFR. The ability of tumor TEMs to suppress tumor-specific T-cell response mediated by tumor dendritic cells (DC) was measured in vitro. Characterization of TEM and DC phenotype in addition to their interaction with T cells was done using confocal microscopic images analysis of breast carcinomas. RESULTS: TEMs from breast tumors are able to suppress tumor-specific immune responses. Importantly, proangiogenic and suppressive functions of TEMs are similarly driven by TIE-2 and VEGFR kinase activity. Furthermore, we show that tumor TEMs can function as antigen-presenting cells and elicit a weak proliferation of T cells. Blocking TIE-2 and VEGFR kinase activity induced TEMs to change their phenotype into cells with features of myeloid dendritic cells. We show that immunosuppressive activity of TEMs is associated with high CD86 surface expression and extensive engagement of T regulatory cells in breast tumors. TIE-2 and VEGFR kinase activity was also necessary to maintain high CD86 surface expression levels and to convert T cells into regulatory cells. CONCLUSIONS: These results suggest that TEMs are plastic cells that can be reverted from suppressive, proangiogenic cells into cells that are able to mediate an antitumoral immune response. Clin Cancer Res; 19(13); 3439-49. ©2013 AACR.

Maintenance of hepatic nuclear factor 6 in postnatal islets impairs terminal differentiation and function of beta-cells.

The Onecut homeodomain transcription factor hepatic nuclear factor 6 (Hnf6) is necessary for proper development of islet beta-cells. Hnf6 is initially expressed throughout the pancreatic epithelium but is downregulated in endocrine cells at late gestation and is not expressed in postnatal islets. Transgenic mice in which Hnf6 expression is maintained in postnatal islets (pdx1(PB)Hnf6) show overt diabetes and impaired glucose-stimulated insulin secretion (GSIS) at weaning. We now define the mechanism whereby maintenance of Hnf6 expression postnatally leads to beta-cell dysfunction. We provide evidence that continued expression of Hnf6 impairs GSIS by altering insulin granule biosynthesis, resulting in a reduced response to secretagogues. Sustained expression of Hnf6 also results in downregulation of the beta-cell-specific transcription factor MafA and a decrease in total pancreatic insulin. These results suggest that downregulation of Hnf6 expression in beta-cells during development is essential to achieve a mature, glucose-responsive beta-cell.

The debate over the effector function of eosinophils in helminth infection: new evidence from studies on the regulation of vaccine immunity by IL-12

The production of Th1-type cytokines is associated with strong cell-mediated immunity while Th2-type cytokines are typically involved in the generation of humoral immune responses. In mice vaccinated a single time (1X) with attenuated cercariae of Schistosoma mansoni, the immunity induced is highly dependent on CD4+ T cells and IFN-gamma. In contrast, mice vaccinated multiple times (3X) have decreased IFN-gamma expression, develop a more dominant Th2-type cytokine response as well as protective antibodies which can passively transfer immunity to naive recipients. Previously, we demonstrated the ability of IL-12, a potent IFN-gamma-inducing cytokine to enhance (1X) schistosome cell-mediated immunity when administered during the period of immunization. More recently, we asked what effects IL-12 would have on the development humoral-based immunity. While multiply-immunized/saline-treated mice demonstrated a 70-80% reduction in parasite burden, 3X/IL-12-vaccinated animals displayed an even more striking >90% reduction in challenge infection, with many mice in the later group demonstrating complete protection. Analysis of pulmonary cytokine mRNA responses demonstrated that control challenged mice elicited a dominant Th2-type response, 3X/saline-vaccinated produced a mixed Th1/Th2-type cytokine response, while 3X/IL-12-immunized animals displayed a dominant Th1-type response. The IL-12-treated group also showed a marked reduction in total serum IgE and tissue eosinophilia while SWAP-specific IgG2a and IgG2b Abs were elevated. Interestingly, animals vaccinated with IL-12 also showed a highly significant increase in total Ig titers specific for IrV-5, a known protective antigen. More importantly, 3X/IL-12 serum alone, when transferred to naive mice reduced worm burdens by over 60% while 3X/saline serum transferred significantly less protection. Nevertheless, animals vaccinated in the presence of IL-12 also develop macrophages with enhanced nitric oxide dependent killing activity against the parasites. Together, these observations suggest that IL-12, initially described as an adjuvant for cell-mediated immunity, may also be used as an adjuvant for promoting both humoral and cell-mediated protective responses.

Mechanisms of formation and function of eosinophil lipid bodies: inducible intracellular sites involved in arachidonic acid metabolism

Lipid bodies, inducible lipid-rich cytoplasmic inclusions, are characteristically abundant in cells associated with inflammation, including eosinophils. Here we reviewed the formation and function of lipid bodies in human eosinophils. We now have evidence that the formation of lipid bodies is not attributable to adverse mechanisms, but is centrally mediated by specific signal transduction pathways. Arachidonic acid and other cis fatty acids by an NSAID-inhibitable process, diglycerides, and PAF by a 5-lipoxygenase dependent pathway are potent stimulators of lipid body induction. Lipid body formation develops rapidly by processes that involve PKC, PLC, and de novo mRNA and protein synthesis. These structures clearly serve as repositoires of arachidonyl-phospholipids and are more than inert depots. Specific enzymes, including cytosolic phospholipase A2, MAP kinases, lipoxygenases and cyclooxygenases, associate with lipid bodies. Lipid bodies appear to be dynamic, organelle-like structures involved in intracellular pathways of lipid mobilization and metabolism. Indeed, increases in lipid body numbers correlated with enhanced production of both lipoxygenase- and cyclooxygenase-derived eicosanoids. We hypothesize that lipid bodies are distinct inducible sites for generating eicosanoids as paracrine mediators with varied activities in inflammation. The capacity of lipid body formation to be specifically and rapidly induced in leukocytes enhances eicosanoid mediator formation, and conversely pharmacologic inhibition of lipid body induction represents a potential novel and specific target for anti-inflammatory therapy.