Inflammasome activation by adenylate cyclase toxin directs Th17 responses and protection against Bordetella pertussis.

Inflammasome-mediated IL-1beta production is central to the innate immune defects that give rise to certain autoinflammatory diseases and may also be associated with the generation of IL-17-producing CD4(+) T (Th17) cells that mediate autoimmunity. However, the role of the inflammasome in driving adaptive immunity to infection has not been addressed. In this article, we demonstrate that inflammasome-mediated IL-1beta plays a critical role in promoting Ag-specific Th17 cells and in generating protective immunity against Bordetella pertussis infection. Using a murine respiratory challenge model, we demonstrated that the course of B. pertussis infection was significantly exacerbated in IL-1R type I-defective (IL-1RI(-/-)) mice. We found that adenylate cyclase toxin (CyaA), a key virulence factor secreted by B. pertussis, induced robust IL-1beta production by dendritic cells through activation of caspase-1 and the NALP3-containing inflammasome complex. Using mutant toxins, we demonstrate that CyaA-mediated activation of caspase-1 was not dependent on adenylate cyclase enzyme activity but was dependent on the pore-forming capacity of CyaA. In addition, CyaA promoted the induction of Ag-specific Th17 cells in wild-type but not IL-1RI(-/-) mice. Furthermore, the bacterial load was enhanced in IL-17-defective mice. Our findings demonstrate that CyaA, a virulence factor from B. pertussis, promotes innate IL-1beta production via activation of the NALP3 inflammasome and, thereby, polarizes T cell responses toward the Th17 subtype. In addition to its known role in subverting host immunity, our findings suggest that CyaA can promote IL-1beta-mediated Th17 cells, which promote clearance of the bacteria from the respiratory tract.

Impact of biocontrol strain Pseudomonas fluorescens CHA0 on rhizosphere bacteria isolated from barley (Hordeum vulgare L.) with special reference to Cytophaga-like bacteria.

AIMS: To assess the impact of the biocontrol strain Pseudomonas fluorescens CHA0 on a collection of barley rhizosphere bacteria using an agar plate inhibition assay and a plant microcosm, focusing on a CHA0-sensitive member of the Cytophaga-like bacteria (CLB). METHODS AND RESULTS: The effect of strain CHA0 on a collection of barley rhizosphere bacteria, in particular CLB and fluorescent pseudomonads sampled during a growth season, was assessed by a growth inhibition assay. On average, 85% of the bacteria were sensitive in the May sample, while the effect was reduced to around 68% in the July and August samples. In the May sample, around 95% of the CLB and around 45% of the fluorescent pseudomonads were sensitive to strain CHA0. The proportion of CHA0-sensitive CLB and fluorescent pseudomonad isolates decreased during the plant growth season, i.e. in the July and August samples. A particularly sensitive CLB isolate, CLB23, was selected, exposed to strain CHA0 (wild type) and its genetically modified derivatives in the rhizosphere of barley grown in gnotobiotic soil microcosms. Two dry-stress periods were imposed during the experiment. Derivatives of strain CHA0 included antibiotic or exopolysaccharide (EPS) overproducing strains and a dry-stress-sensitive mutant. Despite their inhibitory activity against CLB23 in vitro, neither wild-type strain CHA0, nor any of its derivatives, had a major effect on culturable and total cell numbers of CLB23 during the 23-day microcosm experiment. Populations of all inoculants declined during the two dry-stress periods, with soil water contents below 5% and plants reaching the wilting point, but they recovered after re-wetting the soil. Survival of the dry-stress-sensitive mutant of CHA0 was most affected by the dry periods; however, this did not result in an increased population density of CLB23. CONCLUSIONS: CLB comprise a large fraction of barley rhizosphere bacteria that are sensitive to the biocontrol pseudomonad CHA0 in vitro. However, in plant microcosm experiments with varying soil humidity conditions, CHA0 or its derivatives had no major impact on the survival of the highly sensitive CLB strain, CLB23, during two dry-stress periods and a re-wetting period; all co-existed well in the rhizosphere of barley plants. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate a lack of interaction between the biocontrol pseudomonad CHA0 and a sensitive CLB when the complexity increases from agar plate assays to plant microcosm experiments. This suggests the occurrence of low levels of antibiotic production and/or that the two bacterial genera occupy different niches in the rhizosphere.

Retinal degeneration progression changes lentiviral vector cell targeting in the retina.

In normal mice, the lentiviral vector (LV) is very efficient to target the RPE cells, but transduces retinal neurons well only during development. In the present study, the tropism of LV has been investigated in the degenerating retina of mice, knowing that the retina structure changes during degeneration. We postulated that the viral transduction would be increased by the alteration of the outer limiting membrane (OLM). Two different LV pseudotypes were tested using the VSVG and the Mokola envelopes, as well as two animal models of retinal degeneration: light-damaged Balb-C and Rhodopsin knockout (Rho-/-) mice. After light damage, the OLM is altered and no significant increase of the number of transduced photoreceptors can be obtained with a LV-VSVG-Rhop-GFP vector. In the Rho-/- mice, an alteration of the OLM was also observed, but the possibility of transducing photoreceptors was decreased, probably by ongoing gliosis. The use of a ubiquitous promoter allows better photoreceptor transduction, suggesting that photoreceptor-specific promoter activity changes during late stages of photoreceptor degeneration. However, the number of targeted photoreceptors remains low. In contrast, LV pseudotyped with the Mokola envelope allows a wide dispersion of the vector into the retina (corresponding to the injection bleb) with preferential targeting of Müller cells, a situation which does not occur in the wild-type retina. Mokola-pseudotyped lentiviral vectors may serve to engineer these glial cells to deliver secreted therapeutic factors to a diseased area of the retina.

Long-term swimming exercise does not modulate the Akt-dependent endothelial nitric oxide synthase phosphorylation in healthy mice.

Molecular mechanisms by which exercise exerts cardiovascular benefits are poorly understood. Exercise-induced increase of endothelial NO synthase (eNOS) phosphorylation through the protein kinase Akt has been shown to be a key mechanism underlying the beneficial effect of exercise in coronary artery disease patients. We examined whether this protective pathway might also be activated in long-term-exercised healthy mice. C57BL/6 wild-type mice swam for 24 weeks. A group of sedentary animals were used as controls. Aortic levels of total protein kinase Akt (protein kinase B), phosphorylated Akt at ser473 (p-Akt), total eNOS, phosphorylated eNOS at Ser1177 (p-eNOS), and PECAM-1 (platelet endothelial cell adhesion molecule-1) were assessed by Western blotting. Protein expressions of Akt, p-Akt, eNOS, p-eNOS, and PECAM-1 were not modulated by 24 weeks of exercise. The Akt-dependent eNOS phosphorylation did not seem to be a primary molecular adaptation in response to long-term exercise in healthy mice.

Regulatory B cells shape the development of Th2 immune responses in BALB/c mice infected with Leishmania major through IL-10 production.

Recent evidence indicates that B cells are required for susceptibility to infection with Leishmania major in BALB/c mice. In this study, we analyzed the role of the IL-10 produced by B cells in this process. We showed that B cells purified from the spleen of BALB/c mice produced IL-10 in response to stimulation with L. major in vitro. In vivo, early IL-10 mRNA expression is detected after L. major infection in B cells from draining lymph nodes of susceptible BALB/c, but not of resistant C57BL/6 mice. Although adoptive transfer of naive wild-type B cells prior to infection in B cell-deficient BALB/c mice restored Th2 cell development and susceptibility to infection with L. major of these otherwise resistant mice, adoptive transfer of IL-10(-/-) B cells mice did not. B cells stimulated by L. major, following in vitro or in vivo encounter, express the CD1d and CD5 molecules and the IL-10 produced by these cells downregulate IL-12 production by L. major-stimulated dendritic cells. These observations indicate that IL-10 secreting B cells are phenotypically and functionally regulatory B cells. Altogether these results demonstrate that the IL-10 produced by regulatory CD1d+ CD5+ B cells in response to L. major is critical for Th2 cell development in BALB/c mice.

An eceriferum locus, cer-zv, is associated with a defect in cutin responsible for water retention in barley (Hordeum vulgare) leaves.

Drought limits plant growth and threatens crop productivity. A barley (Hordeum vulgare) ethylene imine-induced monogenic recessive mutant cer-zv, which is sensitive to drought, was characterized and genetically mapped in the present study. Detached leaves of cer-zv lost 34.2 % of their initial weight after 1 h of dehydration. The transpiration was much higher in cer-zv leaves than in wild-type leaves under both light and dark conditions. The stomata of cer-zv leaves functioned normally, but the cuticle of cer-zv leaves showed increased permeability to ethanol and toluidine blue dye. There was a 50-90 % reduction in four major cutin monomers, but no reduction in wax loads was found in the cer-zv mutant as compared with the wild type. Two F(2) mapping populations were established by the crosses of 23-19 × cer-zv and cer-zv × OUH602. More polymorphisms were found in EST sequences between cer-zv and OUH602 than between cer-zv and 23-19. cer-zv was located in a pericentromeric region on chromosome 4H in a 10.8 cM interval in the 23-19 × cer-zv map based on 186 gametes tested and a 1.7 cM interval in the cer-zv × OUH602 map based on 176 gametes tested. It co-segregated with EST marker AK251484 in both maps. The results indicated that the cer-zv mutant is defective in cutin, which might be responsible for the increased transpiration rate and drought sensitivity, and that the F(2) of cer-zv × OUH602 might better facilitate high resolution mapping of cer-zv.

Secreted glutamic protease rescues aspartic protease Pep deficiency in Aspergillus fumigatus during growth in acidic protein medium.

In an acidic protein medium Aspergillus fumigatus secretes an aspartic endoprotease (Pep) as well as tripeptidyl-peptidases, a prolyl-peptidase and carboxypeptidases. In addition, LC-MS/MS revealed a novel glutamic protease, AfuGprA, homologous to Aspergillus niger aspergillopepsin II. The importance of AfuGprA in protein digestion was evaluated by deletion of its encoding gene in A. fumigatus wild-type D141 and in a pepΔ mutant. Either A. fumigatus Pep or AfuGprA was shown to be necessary for fungal growth in protein medium at low pH. Exoproteolytic activity is therefore not sufficient for complete protein hydrolysis and fungal growth in a medium containing proteins as the sole nitrogen source. Pep and AfuGprA constitute a pair of endoproteases active at low pH, in analogy to A. fumigatus alkaline protease (Alp) and metalloprotease I (Mep), where at least one of these enzymes is necessary for fungal growth in protein medium at neutral pH. Heterologous expression of AfuGprA in Pichia pastoris showed that the enzyme is synthesized as a preproprotein and that the propeptide is removed through an autoproteolytic reaction at low pH to generate the mature protease. In contrast to A. niger aspergillopepsin II, AfuGprA is a single-chain protein and is structurally more similar to G1 proteases characterized in other non-Aspergillus fungi.

The serine-rich N-terminal region of Arabidopsis phytochrome A is required for protein stability.

Deletion or substitution of the serine-rich N-terminal stretch of grass phytochrome A (phyA) has repeatedly been shown to yield a hyperactive photoreceptor when expressed under the control of a constitutive promoter in transgenic tobacco or Arabidopsis seedlings retaining their native phyA. These observations have lead to the proposal that the serine-rich region is involved in negative regulation of phyA signaling. To re-evaluate this conclusion in a more physiological context we produced transgenic Arabidopsis seedlings of the phyA-null background expressing Arabidopsis PHYA deleted in the sequence corresponding to amino acids 6-12, under the control of the native PHYA promoter. Compared to the transgenic seedlings expressing wild-type phyA, the seedlings bearing the mutated phyA showed normal responses to pulses of far-red (FR) light and impaired responses to continuous FR light. In yeast two-hybrid experiments, deleted phyA interacted normally with FHY1 and FHL, which are required for phyA accumulation in the nucleus. Immunoblot analysis showed reduced stability of deleted phyA under continuous red or FR light. The reduced physiological activity can therefore be accounted for by the enhanced destruction of the mutated phyA. These findings do not support the involvement of the serine-rich region in negative regulation but they are consistent with a recent report suggesting that phyA turnover is regulated by phosphorylation.

A member of the PLEIOTROPIC DRUG RESISTANCE family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis.

Although the multilayered structure of the plant cuticle was discovered many years ago, the molecular basis of its formation and the functional relevance of the layers are not understood. Here, we present the permeable cuticle1 (pec1) mutant of Arabidopsis thaliana, which displays features associated with a highly permeable cuticle in several organs. In pec1 flowers, typical cutin monomers, such as ω-hydroxylated fatty acids and 10,16-dihydroxypalmitate, are reduced to 40% of wild-type levels and are accompanied by the appearance of lipidic inclusions within the epidermal cell. The cuticular layer of the cell wall, rather than the cuticle proper, is structurally altered in pec1 petals. Therefore, a significant role for the formation of the diffusion barrier in petals can be attributed to this layer. Thus, pec1 defines a new class of mutants. The phenotypes of the pec1 mutant are caused by the knockout of ATP BINDING CASSETTEG32 (ABCG32), an ABC transporter from the PLEIOTROPIC DRUG RESISTANCE family that is localized at the plasma membrane of epidermal cells in a polar manner toward the surface of the organs. Our results suggest that ABCG32 is involved in the formation of the cuticular layer of the cell wall, most likely by exporting particular cutin precursors from the epidermal cell.

Engineered aprotinin for improved stability of fibrin biomaterials.

Fibrin has been long used clinically for hemostasis and sealing, yet extension of use in other applications has been limited due to its relatively rapid resorption in vivo, even with addition of aprotinin or other protease inhibitors. We report an engineered aprotinin variant that can be immobilized within fibrin and thus provide extended longevity. When recombinantly fused to a transglutaminase substrate domain from α(2)-plasmin inhibitor (α(2)PI(1-8)), the resulting variant, aprotinin-α(2)PI(1-8), was covalently crosslinked into fibrin matrices during normal thrombin/factor XIIIa-mediated polymerization. Challenge with physiological plasmin concentrations revealed that aprotinin-α(2)PI(1-8)-containing matrices retained 78% of their mass after 3 wk, whereas matrices containing wild type (WT) aprotinin degraded completely within 1 wk. Plasmin challenge of commercial sealants Omrixil and Tisseel, supplemented with aprotinin-α(2)PI(1-8) or WT aprotinin, showed extended longevity as well. When seeded with human dermal fibroblasts, aprotinin-α(2)PI(1-8)-supplemented matrices supported cell growth for at least 33% longer than those containing WT aprotinin. Subcutaneously implanted matrices containing aprotinin-α(2)PI(1-8) were detectable in mice for more than twice as long as those containing WT aprotinin. We conclude that our engineered recombinant aprotinin variant can confer extended longevity to fibrin matrices more effectively than WT aprotinin in vitro and in vivo.

Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.

Prolonged deprivation of food induces dramatic changes in mammalian metabolism, including the release of large amounts of fatty acids from the adipose tissue, followed by their oxidation in the liver. The nuclear receptor known as peroxisome proliferator-activated receptor alpha (PPARalpha) was found to play a role in regulating mitochondrial and peroxisomal fatty acid oxidation, suggesting that PPARalpha may be involved in the transcriptional response to fasting. To investigate this possibility, PPARalpha-null mice were subjected to a high fat diet or to fasting, and their responses were compared with those of wild-type mice. PPARalpha-null mice chronically fed a high fat diet showed a massive accumulation of lipid in their livers. A similar phenotype was noted in PPARalpha-null mice fasted for 24 hours, who also displayed severe hypoglycemia, hypoketonemia, hypothermia, and elevated plasma free fatty acid levels, indicating a dramatic inhibition of fatty acid uptake and oxidation. It is shown that to accommodate the increased requirement for hepatic fatty acid oxidation, PPARalpha mRNA is induced during fasting in wild-type mice. The data indicate that PPARalpha plays a pivotal role in the management of energy stores during fasting. By modulating gene expression, PPARalpha stimulates hepatic fatty acid oxidation to supply substrates that can be metabolized by other tissues.

Protection from lethal gram-negative bacterial sepsis by targeting Toll-like receptor 4.

Toll-like receptor 4 (TLR4), the signal-transducing molecule of the LPS receptor complex, plays a fundamental role in the sensing of LPS from gram-negative bacteria. Activation of TLR4 signaling pathways by LPS is a critical upstream event in the pathogenesis of gram-negative sepsis, making TLR4 an attractive target for novel antisepsis therapy. To validate the concept of TLR4-targeted treatment strategies in gram-negative sepsis, we first showed that TLR4(-/-) and myeloid differentiation primary response gene 88 (MyD88)(-/-) mice were fully resistant to Escherichia coli-induced septic shock, whereas TLR2(-/-) and wild-type mice rapidly died of fulminant sepsis. Neutralizing anti-TLR4 antibodies were then generated using a soluble chimeric fusion protein composed of the N-terminal domain of mouse TLR4 (amino acids 1-334) and the Fc portion of human IgG1. Anti-TLR4 antibodies inhibited intracellular signaling, markedly reduced cytokine production, and protected mice from lethal endotoxic shock and E. coli sepsis when administered in a prophylactic and therapeutic manner up to 13 h after the onset of bacterial sepsis. These experimental data provide strong support for the concept of TLR4-targeted therapy for gram-negative sepsis.

Fanconi-Bickel syndrome and autosomal recessive proximal tubulopathy with hypercalciuria (ARPTH) are allelic variants caused by GLUT2 mutations.

CONTEXT: Many inherited disorders of calcium and phosphate homeostasis are unexplained at the molecular level. OBJECTIVE: The objective of the study was to identify the molecular basis of phosphate and calcium abnormalities in two unrelated, consanguineous families. PATIENTS: The affected members in family 1 presented with rickets due to profound urinary phosphate-wasting and hypophosphatemic rickets. In the previously reported family 2, patients presented with proximal renal tubulopathy and hypercalciuria yet normal or only mildly increased urinary phosphate excretion. METHODS: Genome-wide linkage scans and direct nucleotide sequence analyses of candidate genes were performed. Transport of glucose and phosphate by glucose transporter 2 (GLUT2) was assessed using Xenopus oocytes. Renal sodium-phosphate cotransporter 2a and 2c (Npt2a and Npt2c) expressions were evaluated in transgenically rescued Glut2-null mice (tgGlut2-/-). RESULTS: In both families, genetic mapping and sequence analysis of candidate genes led to the identification of two novel homozygous mutations (IVS4-2A>G and R124S, respectively) in GLUT2, the gene mutated in Fanconi-Bickel syndrome, a rare disease usually characterized by renal tubulopathy, impaired glucose homeostasis, and hepatomegaly. Xenopus oocytes expressing the [R124S]GLUT2 mutant showed a significant reduction in glucose transport, but neither wild-type nor mutant GLUT2 facilitated phosphate import or export; tgGlut2-/- mice demonstrated a profound reduction of Npt2c expression in the proximal renal tubules. CONCLUSIONS: Homozygous mutations in the facilitative glucose transporter GLUT2, which cause Fanconi-Bickel syndrome, can lead to very different clinical and biochemical findings that are not limited to mild proximal renal tubulopathy but can include significant hypercalciuria and highly variable degrees of urinary phosphate-wasting and hypophosphatemia, possibly because of the impaired proximal tubular expression of Npt2c.

Identification of an agrin mutation that causes congenital myasthenia and affects synapse function.

We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.

The role of SGLT1 and GLUT2 in intestinal glucose transport and sensing.

Intestinal glucose absorption is mediated by SGLT1 whereas GLUT2 is considered to provide basolateral exit. Recently, it was proposed that GLUT2 can be recruited into the apical membrane after a high luminal glucose bolus allowing bulk absorption of glucose by facilitated diffusion. Moreover, SGLT1 and GLUT2 are suggested to play an important role in intestinal glucose sensing and incretin secretion. In mice that lack either SGLT1 or GLUT2 we re-assessed the role of these transporters in intestinal glucose uptake after radiotracer glucose gavage and performed Western blot analysis for transporter abundance in apical membrane fractions in a comparative approach. Moreover, we examined the contribution of these transporters to glucose-induced changes in plasma GIP, GLP-1 and insulin levels. In mice lacking SGLT1, tissue retention of tracer glucose was drastically reduced throughout the entire small intestine whereas GLUT2-deficient animals exhibited higher tracer contents in tissue samples than wild type animals. Deletion of SGLT1 resulted also in reduced blood glucose elevations and abolished GIP and GLP-1 secretion in response to glucose. In mice lacking GLUT2, glucose-induced insulin but not incretin secretion was impaired. Western blot analysis revealed unchanged protein levels of SGLT1 after glucose gavage. GLUT2 detected in apical membrane fractions mainly resulted from contamination with basolateral membranes but did not change in density after glucose administration. SGLT1 is unequivocally the prime intestinal glucose transporter even at high luminal glucose concentrations. Moreover, SGLT1 mediates glucose-induced incretin secretion. Our studies do not provide evidence for GLUT2 playing any role in either apical glucose influx or incretin secretion.