TL1A-DR3 interaction regulates Th17 cell function and Th17-mediated autoimmune disease.

T helper type 17 (Th17) cells play an important pathogenic function in autoimmune diseases; their regulation, however, is not well understood. We show that the expression of a tumor necrosis factor receptor family member, death receptor 3 (DR3; also known as TNFRSF25), is selectively elevated in Th17 cells, and that TL1A, its cognate ligand, can promote the proliferation of effector Th17 cells. To further investigate the role of the TL1A-DR3 pathway in Th17 regulation, we generated a TL1A-deficient mouse and found that TL1A(-/-) dendritic cells exhibited a reduced capacity in supporting Th17 differentiation and proliferation. Consistent with these data, TL1A(-/-) animals displayed decreased clinical severity in experimental autoimmune encephalomyelitis (EAE). Finally, we demonstrated that during EAE disease progression, TL1A was required for the optimal differentiation as well as effector function of Th17 cells. These observations thus establish an important role of the TL1A-DR3 pathway in promoting Th17 cell function and Th17-mediated autoimmune disease.

TRPM5-mediated calcium uptake regulates mucin secretion from human colon goblet cells

Mucin 5AC (MUC5AC) is secreted by goblet cells of the respiratory tract and, surprisingly, also expressed de novo in mucus secreting cancer lines. siRNA-mediated knockdown of 7343 human gene products in a human colonic cancer goblet cell line (HT29-18N2) revealed new proteins, including a Ca(2+)-activated channel TRPM5, for MUC5AC secretion. TRPM5 was required for PMA and ATP-induced secretion of MUC5AC from the post-Golgi secretory granules. Stable knockdown of TRPM5 reduced a TRPM5-like current and ATP-mediated Ca(2+) signal. ATP-induced MUC5AC secretion depended strongly on Ca(2+) influx, which was markedly reduced in TRPM5 knockdown cells. The difference in ATP-induced Ca(2+) entry between control and TRPM5 knockdown cells was abrogated in the absence of extracellular Ca(2+) and by inhibition of the Na(+)/Ca(2+) exchanger (NCX). Accordingly, MUC5AC secretion was reduced by inhibition of NCX. Thus TRPM5 activation by ATP couples TRPM5-mediated Na(+) entry to promote Ca(2+) uptake via an NCX to trigger MUC5AC secretion

Recognition of Gram-positive Intestinal Bacteria by Hybridoma- and Colostrum-derived Secretory Immunoglobulin A Is Mediated by Carbohydrates.

Humans live in symbiosis with 10(14) commensal bacteria among which >99% resides in their gastrointestinal tract. The molecular bases pertaining to the interaction between mucosal secretory IgA (SIgA) and bacteria residing in the intestine are not known. Previous studies have demonstrated that commensals are naturally coated by SIgA in the gut lumen. Thus, understanding how natural SIgA interacts with commensal bacteria can provide new clues on its multiple functions at mucosal surfaces. Using fluorescently labeled, nonspecific SIgA or secretory component (SC), we visualized by confocal microscopy the interaction with various commensal bacteria, including Lactobacillus, Bifidobacteria, Escherichia coli, and Bacteroides strains. These experiments revealed that the interaction between SIgA and commensal bacteria involves Fab- and Fc-independent structural motifs, featuring SC as a crucial partner. Removal of glycans present on free SC or bound in SIgA resulted in a drastic drop in the interaction with Gram-positive bacteria, indicating the essential role of carbohydrates in the process. In contrast, poor binding of Gram-positive bacteria by control IgG was observed. The interaction with Gram-negative bacteria was preserved whatever the molecular form of protein partner used, suggesting the involvement of different binding motifs. Purified SIgA and SC from either mouse hybridoma cells or human colostrum exhibited identical patterns of recognition for Gram-positive bacteria, emphasizing conserved plasticity between species. Thus, sugar-mediated binding of commensals by SIgA highlights the currently underappreciated role of glycans in mediating the interaction between a highly diverse microbiota and the mucosal immune system.

Regulation of mass and function of pancreatic β-cells: identification of anti-apoptotic peptides and role of GLP-1

Résumé La masse de cellules β sécrétrices d'insuline est un tissu dynamique qui s'adapte aux variations de la demande métabolique pour assurer une normoglycémie. Cette adaptation se fait par un changement de sécrétion d'insuline et de la masse totale des cellules β. Une perte complète ou partielle des cellules β conduit respectivement à un diabète de type 1 et de type 2. Les mécanismes qui régulent la masse de cellules β et maintiennent leur phénotype differencié sont encore peu connus. Leur identification est nécessaire pour comprendre le développement du diabète et développer des stratégies de traitement. La greffe d'îlots est une approche thérapeutique prometteuse pour le diabète de type 1, mais est limitée par une perte précoce des cellules β due à une apoptose induite par des cytokines. Afin d'améliorer la survie des cellules β lors de la greffe d'îlots, le premier but était de trouver des peptides pouvant bloquer l'apoptose induite par FasL et TNF-α. Pour ce faire, deux librairies de phages ont été criblées pour sélectionner des peptides se liant au Fas DD ou au TNFRl DD. Nous avons identifié six peptides différents. Cependant, aucun d'entre eux n'était capable de protéger les cellules de l'apoptose induite par FasL ou TNF-α. Deuxièmement, le GLP-1 est une hormone qui stimule la sécrétion d'insuline, et est impliquée dans la prolifération des cellules β, la différentiation, et inhibe l'apoptose. Nous avons fait l'hypothèse que le GLP-1 joue un rôle crucial dans le contrôle de la masse et de la fonction des cellules β. Afin de l'évaluer, une analyse par puce à ADN a été réalisée en comparant des cellules βTC-Tet traitées avec du GLP-1 à des cellules non-traitées. 376 gènes régulés ont été identifiés, dont RGS2, CREM, ICERI et DUSP14, augmentés significativement par le GLP-1. Nous avons confirmé que le GLP-1 augmente l'expression de ces gènes, aussi bien au niveau des transcripts que des protéines. De plus, nous avons montré que le GLP-1 induit leur expression par activation de la voie cAMP/PKA, et nécessite l'entrée de calcium extracellulaire. D'après leur fonction biologique, nous avons ensuite supposé que ces gènes pourraient agir comme régulateurs négatifs de la signalisation du GLP-l, et donc freiner son effet proliférateur. Pour vérifier notre hypothèse, des siRNAs contre ces gènes ont été développés, et leurs effets sur la prolifération des cellules β seront évalués ultérieurement. Abstract The pancreatic β-cell mass is a dynamic tissue which adapts to variations in metabolic demand in order to ensure normoglycemia. This adaptation occurs through a change in both insulin secretion and the total mass of ,β-cells. An absolute or relative loss of β-cells leads to type 1 and type 2 diabetes, respectively. The mechanisms that regulate the pancreatic β-cell mass and maintain the fully differentiated phenotype of the insulin-secreting β-cells are only poorly defined. Their identification is required to understand the progression of diabetes, but also to design strategies for the treatment of diabetes. Islet transplantation is a promising therapeutic approach for type 1 diabetes, but it is still limited by an early graft loss due to cytokine-induced apoptosis. In order to improve β-cell survival during islet transplantation, our first goal was to find novel blockers of FasL- and TNF-α-mediated cell death in the form of peptides. To that end, we screened two phage display libraries to select Fas DD- or TNFR1 DD-binding peptides. We identified six different small peptides. However, none of these peptides was able to prevent cells from FasL- or TNF-α-mediated apoptosis. Secondly, GLP-1 is a hormone that has been shown to stimulate insulin secretion and to be involved in β-cell proliferation, differentiation and inhibition of apoptosis. We hypothesized that GLP-1 plays a crucial role to control mass and function of β-cells. To evaluate this hypothesis, we performed a cDNA microarray analysis with GLP-1-treated βTC-Tet cells compared to untreated cells. We found 376 regulated genes, among these, RGS2, CREM, ICERI and DUSP14, which were significantly upregulated by GLP-1. We confirmed that both their mRNA and protein levels were strongly and rapidly increased after GLP-1 treatment. Moreover, we found that GLP-1 activates their expression mainly through the activation of the cAMP/PKA signaling pathway, and requires extracellular calcium entry. According to their biological function, we then hypothesized that these genes might act as negative regulators of the GLP-1 signaling. In particular, they might brake the effects of GLP-1 on β-cell proliferation. To verify this hypothesis, siRNAs against these genes were developed. The effect of these siRNAs on GLP-1-induced β-cell proliferation will be evaluated later.

Inadequate T follicular cell help impairs B cell immunity during HIV infection.

The majority of HIV-infected individuals fail to produce protective antibodies and have diminished responses to new immunizations. We report here that even though there is an expansion of follicular helper T (TFH) cells in HIV-infected individuals, the cells are unable to provide adequate B cell help. We found a higher frequency of programmed cell death ligand 1 (PD-L1)(+) germinal center B cells from lymph nodes of HIV-infected individuals suggesting a potential role for PD-1-PD-L1 interaction in regulating TFH cell function. In fact, we show that engagement of PD-1 on TFH cells leads to a reduction in cell proliferation, activation, inducible T-cell co-stimulator (ICOS) expression and interleukin-21 (IL-21) cytokine secretion. Blocking PD-1 signaling enhances HIV-specific immunoglobulin production in vitro. We further show that at least part of this defect involves IL-21, as addition of this cytokine rescues antibody responses and plasma cell generation in vitro. Our results suggest that deregulation of TFH cell-mediated B cell help diminishes B cell responses during HIV infection and may be related to PD-1 triggering on TFH cells. These results demonstrate a role for TFH cell impairment in HIV pathogenesis and suggest that enhancing their function could have a major impact on the outcome and control of HIV infection, preventing future infections and improving immune responses to vaccinations.

Gene transfer of a soluble IL-1 type 2 receptor-Ig fusion protein improves cardiac allograft survival in rats.

OBJECTIVE: Interleukin-1 (IL-1) mediates ischemia-reperfusion injury and graft inflammation after heart transplantation. IL-1 affects target cells through two distinct types of transmembrane receptors, type-1 receptor (IL-1R1), which transduces the signal, and the non-signaling type-2 receptor (IL-1R2), which acts as a ligand sink that subtracts IL-1beta from IL-1R1. We analyzed the efficacy of adenovirus (Ad)-mediated gene transfer of a soluble IL-1R2-Ig fusion protein in delaying cardiac allograft rejection and the mechanisms underlying the protective effect. METHODS: IL-1 inhibition by IL-1R2-Ig was tested using an in vitro functional assay whereby endothelial cells preincubated with AdIL-1R2-Ig or control virus were stimulated with recombinant IL-1beta or tumor necrosis factor-alpha (TNF-alpha), and urokinase-type plasminogen activator (u-PA) induction was measured by zymography. AdIL-1R2-Ig was delivered to F344 rat donor hearts ex vivo, which were placed in the abdominal position in LEW hosts. Intragraft inflammatory cell infiltrates and proinflammatory cytokine expression were analyzed by immunohistochemistry and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. RESULTS: IL-1R2-Ig specifically inhibited IL-1beta-induced u-PA responses in vitro. IL-1R2-Ig gene transfer reduced intragraft monocytes/macrophages and CD4(+) cell infiltrates (p<0.05), TNF-alpha and transforming growth factor-beta (TGF-beta) expression (p<0.05), and prolonged graft survival (15.6+/-5.7 vs 10.3+/-2.5 days with control vector and 10.1+/-2.1 days with buffer alone; p<0.01). AdIL-1R2-Ig combined with a subtherapeutic regimen of cyclosporin A (CsA) was superior to CsA alone (19.4+/-3.0 vs 15.9+/-1.8 days; p<0.05). CONCLUSIONS: Soluble IL-1 type-2 receptor gene transfer attenuates cardiac allograft rejection in a rat model. IL-1 inhibition may be useful as an adjuvant therapy in heart transplantation.

A PiggyBac-mediated approach for muscle gene transfer or cell therapy.

An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies.

Induction of cardiac Angptl4 by dietary fatty acids is mediated by peroxisome proliferator-activated receptor beta/delta and protects against fatty acid-induced oxidative stress.

RATIONALE: Although dietary fatty acids are a major fuel for the heart, little is known about the direct effects of dietary fatty acids on gene regulation in the intact heart. OBJECTIVE: To study the effect of dietary fatty acids on cardiac gene expression and explore the functional consequences. METHODS AND RESULTS: Oral administration of synthetic triglycerides composed of one single fatty acid altered cardiac expression of numerous genes, many of which are involved in the oxidative stress response. The gene most significantly and consistently upregulated by dietary fatty acids encoded Angiopoietin-like protein (Angptl)4, a circulating inhibitor of lipoprotein lipase expressed by cardiomyocytes. Induction of Angptl4 by the fatty acid linolenic acid was specifically abolished in peroxisome proliferator-activated receptor (PPAR)beta/delta(-/-) and not PPARalpha(-/-) mice and was blunted on siRNA-mediated PPARbeta/delta knockdown in cultured cardiomyocytes. Consistent with these data, linolenic acid stimulated binding of PPARbeta/delta but not PPARalpha to the Angptl4 gene. Upregulation of Angptl4 resulted in decreased cardiac uptake of plasma triglyceride-derived fatty acids and decreased fatty acid-induced oxidative stress and lipid peroxidation. In contrast, Angptl4 deletion led to enhanced oxidative stress in the heart, both after an acute oral fat load and after prolonged high fat feeding. CONCLUSIONS: Stimulation of cardiac Angptl4 gene expression by dietary fatty acids and via PPARbeta/delta is part of a feedback mechanism aimed at protecting the heart against lipid overload and consequently fatty acid-induced oxidative stress.

Intracellular thiol-mediated modulation of epithelial sodium channel activity.

The epithelial sodium channel ENaC is physiologically important in the kidney for the regulation of the extracellular fluid volume, and in the lungs for the maintenance of the appropriate airway surface liquid volume that lines the pulmonary epithelium. Besides the regulation of ENaC by hormones, intracellular factors such as Na(+) ions, pH, or Ca(2+) are responsible for fast adaptive responses of ENaC activity to changes in the intracellular milieu. In this study, we show that ENaC is rapidly and reversibly inhibited by internal sulfhydryl-reactive molecules such as methanethiosulfonate derivatives of different sizes, the metal cations Cd(2+) and Zn(2+), or copper(II) phenanthroline, a mild oxidizing agent that promotes the formation of disulfide bonds. At the single channel level, these agents applied intracellularly induce the appearance of long channel closures, suggesting an effect on ENaC gating. The intracellular reducing agent dithiothreitol fully reverses the rundown of ENaC activity in inside-out patches. Our observations suggest that changes in intracellular redox potential modulate ENaC activity and may regulate ENaC-mediated Na(+) transport in epithelia. Finally, substitution experiments reveal that multiple cysteine residues in the amino and carboxyl termini of ENaC subunits are responsible for this thiol-mediated inhibition of ENaC.

Auxin-mediated plant architectural changes in response to shade and high temperature.

The remarkable plasticity of their architecture allows plants to adjust growth to the environment and to overcome adverse conditions. Two examples of environmental stresses that drastically affect shoot development are imminent shade and high temperature. Plants in crowded environments and plants in elevated ambient temperature display very similar phenotypic adaptations of elongated hypocotyls in seedlings and elevated and elongated leaves at later developmental stages. The comparable growth responses to shade and high temperature are partly regulated through shared signaling pathways, of which the phytohormone auxin and the phytochrome interacting factors (PIFs) are important components. During both shade- and temperature-induced elongation growth auxin biosynthesis and signaling are upregulated in a PIF-dependent manner. In this review we will discuss recent progress in our understanding of how auxin mediates architectural adaptations to shade and high temperature.

CD4+CD3- cells induce Peyer's patch development: role of alpha4beta1 integrin activation by CXCR5.

CD4+CD3- cells are the predominant hematopoietic cells found in mouse fetal intestine. We prove their role as Peyer's patch (PP)-inducing cells by transfer into neonatal PP-deficient mice. To test the requirement of chemokines and adhesion molecules in induction of PP, we studied mice deficient in CXCR5 and/or alpha4beta1 integrin-mediated adhesion. CXCR5-/- mice have CD4+CD3- cells, which are inefficient in inducing PP formation. We show here that CXCR5/CXCL13 signaling activates alpha4beta1 integrin on CD4+CD3- cells. Blocking of beta1 integrin or VCAM-1, the ligand of alpha4beta1 integrin, inhibits PP formation. This study demonstrates the link between chemokine receptors and adhesion molecules that regulates stromal/hematopoietic cell interaction leading to PP formation.

Immunopathogenesis of Asthma and Atopic Diseases – The specific Role of a selected Panel of Genes in human T helper Cell Differentiation

T helper cell (Th) functions are crucial for proper immune defence against various intra- and extracellular pathogens. According to the specific immune responses, Th cells can be classified into subtypes, Th1 and Th2 cells being the most frequently characterized classes. Th1 and Th2 cells interact with other immune cells by regulating their functions with specific cytokine production. IFN, IL-2 and TNF- are the cytokines predominantly produced by Th1 cells whereas Th2 cells produce Th2-type cytokines, such as IL-4, IL-5 and IL-13. Upon TCR activation and in the presence of polarizing cytokines, Th cells differentiate into effector subtypes from a common precursor cell. IFN and IL-12 are the predominant Th1 polarizing cytokines whereas IL-4 directs Th2 polarization. The cytokines mediate their effects through specific receptor signalling. The differentiation process is complex, involving various signalling molecules and routes, as well as functions of the specific transcription factors. The functions of the Th1/Th2 cells are tightly regulated; however, knowledge on human Th cell differentiation is, as yet, fairly poor. The susceptibility for many immune-mediated disorders often originates from disturbed Th cell responses. Thus, research is needed for defining the molecular mechanisms involved in the differentiation and balanced functions of the Th cells. Importantly, the new information obtained will be crucial for a better understanding of the pathogenesis of immune-mediated disorders, such as asthma or autoimmune diseases. In the first subproject of this thesis, the role of genetic polymorphisms in the human STAT6, GATA3 and STAT4 genes were investigated for asthma or atopy susceptibility in Finnish asthma families by association analysis. These genes code for key transcription factors regulating Th cell differentiation. The study resulted in the identification of a GATA3 haplotype that associated with asthma and related traits (high serum IgE level). In the second subproject, an optimized method for human primary T cell transfection and enrichment was established. The method can be utilized for functional studies for the selected genes of interest. The method was also utilized in the third subproject, which aimed at the identification of novel genes involved in early human Th cell polarization (0-48h) using genome-wide oligonucleotide arrays. As a result, numerous genes and ESTs with known or unknown functions were identified in the study. Using an shRNA knockdown approach, a panel of novel IL-4/STAT6 regulated genes were identified in the functional studies of the genes. Moreover, one of the genes, NDFIP2, with a previously uncharacterized role in the human Th differentiation, was observed to promote IFN production of the differentiated Th1 cells. Taken together, the results obtained have revealed potential new relevant candidate genes serving as a basis for further studies characterizing the detailed networks involved in the human Th cell differentiation as well as in the genetic susceptibility of Th-mediated immune disorders.

Perception, signaling and molecular basis of oviposition-mediated plant responses.

Eggs deposited on plants by herbivorous insects represent a threat as they develop into feeding larvae. Plants are not a passive substrate and have evolved sophisticated mechanisms to detect eggs and induce direct and indirect defenses. Recent years have seen exciting development in molecular aspects of egg-induced responses. Some egg-associated elicitors have been identified, and signaling pathways and egg-induced expression profiles are being uncovered. Depending on the mode of oviposition, both the jasmonic acid and salicylic acid pathways seem to play a role in the induction of defense responses. An emerging concept is that eggs are recognized like microbial pathogens and innate immune responses are triggered. In addition, some eggs contain elicitors that induce highly specific defenses in plants. Examples of egg-induced suppression of defense or, on the contrary, egg-induced resistance highlight the complexity of plant-egg interactions in an on-going arms race between herbivores and their hosts. A major challenge is to identify plant receptors for egg-associated elicitors, to assess the specificity of these elicitors and to identify molecular components that underlie various responses to oviposition.