Attenuation of pattern recognition receptor signaling is mediated by a MAP kinase kinase kinase

Pattern recognition receptors (PRRs) play a key role in plant and animal innate immunity. PRR binding of their cognate ligand triggers a signaling network and activates an immune response. Activation of PRR signaling must be controlled prior to ligand binding to prevent spurious signaling and immune activation. Flagellin perception in Arabidopsis through FLAGELLIN‐SENSITIVE 2 (FLS2) induces the activation of mitogen‐activated protein kinases (MAPKs) and immunity. However, the precise molecular mechanism that connects activated FLS2 to downstream MAPK cascades remains unknown. Here, we report the identification of a differentially phosphorylated MAP kinase kinase kinase that also interacts with FLS2. Using targeted proteomics and functional analysis, we show that MKKK7 negatively regulates flagellin‐triggered signaling and basal immunity and this requires phosphorylation of MKKK7 on specific serine residues. MKKK7 attenuates MPK6 activity and defense gene expression. Moreover, MKKK7 suppresses the reactive oxygen species burst downstream of FLS2, suggesting that MKKK7‐mediated attenuation of FLS2 signaling occurs through direct modulation of the FLS2 complex.     

A phase 1/2 clinical trial of enzyme replacement in fabry disease: pharmacokinetic, substrate clearance, and safety studies

Fabry disease results from deficient alpha-galactosidase A (alpha-Gal A) activity and the pathologic accumulation of the globotriaosylceramide (GL-3) and related glycosphingolipids, primarily in vascular endothelial lysosomes. Treatment is currently palliative, and affected patients generally die in their 40s or 50s. Preclinical studies of recombinant human alpha-Gal A (r-halphaGalA) infusions in knockout mice demonstrated reduction of GL-3 in tissues and plasma, providing rationale for a phase 1/2 clinical trial. Here, we report a single-center, open-label, dose-ranging study of r-halphaGalA treatment in 15 patients, each of whom received five infusions at one of five dose regimens. Intravenously administered r-halphaGalA was cleared from the circulation in a dose-dependent manner, via both saturable and non-saturable pathways. Rapid and marked reductions in plasma and tissue GL-3 were observed biochemically, histologically, and/or ultrastructurally. Clearance of plasma GL-3 was dose-dependent. In patients with pre- and posttreatment biopsies, mean GL-3 content decreased 84% in liver (n=13), was markedly reduced in kidney in four of five patients, and after five doses was modestly lowered in the endomyocardium of four of seven patients. GL-3 deposits were cleared to near normal or were markedly reduced in the vascular endothelium of liver, skin, heart, and kidney, on the basis of light- and electron-microscopic evaluation. In addition, patients reported less pain, increased ability to sweat, and improved quality-of-life measures. Infusions were well tolerated; four patients experienced mild-to-moderate reactions, suggestive of hypersensitivity, that were managed conservatively. Of 15 patients, 8 (53%) developed IgG antibodies to r-halphaGalA; however, the antibodies were not neutralizing, as indicated by unchanged pharmacokinetic values for infusions 1 and 5. This study provides the basis for a phase 3 trial of enzyme-replacement therapy for Fabry disease.     

Tolerization of mice to Schistosoma mansoni egg antigens causes elevated type 1 and diminished type 2 cytokine responses and increased mortality in acute infection

The granuloma that surrounds the Schistosoma mansoni egg is the cause of pathology in murine schistosomiasis, and its formation is driven by egg Ag-stimulated type 1 and type 2 cytokines. To determine the role of egg-driven immune responses during schistosome infection we rendered CBA/Ca mice unresponsive to schistosome eggs by combined cyclophosphamide treatment and thymectomy. In the early acute stages of schistosome infection, egg-tolerized mice suffered high mortalities. Granuloma size and deposition of collagen in the liver were significantly reduced in egg-tolerized mice. Similarly, limited granuloma responses were detected in the intestines of these mice, and this was associated with a >90% reduction in egg excretion. Histologically, egg-tolerized mice had exacerbated hepatocyte damage, with extensive microvesicular steatosis. Elevated plasma transaminase levels confirmed the damage to hepatocytes. Infected egg-tolerized mice had impaired proliferation responses to egg Ag but intact responses to worm Ag. Tolerized mice had diminished Ab responses to egg Ag and had a type 1 cytokine isotype pattern to worm Ag, with elevated IgG2a and diminished IgG1 and IgE. Egg-tolerized mice failed to down-regulate type 1 cytokines that are normally elicited during early schistosome infection. Hepatic granuloma cells from egg-tolerized mice were also type 1 cytokine dominated, with elevated frequencies of Tc1/Th1 and reduced Tc2/Th2 cells. This study demonstrates that mice tolerized to schistosome eggs have elevated type 1 cytokine responses with diminished type 2 responses and reduced anti-egg Ab during schistosome infection, and these effects are detrimental to the host.     

pH Dependence of structural stability of interleukin-2 and granulocyte colony-stimulating factor

After a cytokine binds to its receptor on the cell surface (pH approximately 7), the complex is internalized into acidic endosomal compartments (pH approximately 5-6), where partially unfolded intermediates can form. The nature of these structural transitions was studied for wild-type interleukin-2 (IL-2) and wild-type granulocyte colony-stimulating factor (G-CSF). A noncoincidence of denaturation transitions in the secondary and tertiary structure of IL-2 and tertiary structural perturbations in G-CSF suggest the presence of an intermediate state for each, a common feature of this structural family of four-helical bundle proteins. Unexpectedly, both IL-2 and G-CSF display monotonic increases in stability as the pH is decreased from 7 to 4. We hypothesize that such cytokines with cell-based clearance mechanisms in vivo may have evolved to help stabilize endosomal complexes for sorting to lysosomal degradation. We show that mutants of both IL-2 and G-CSF have differential stabilities to their wild-type counterparts as a function of pH, and that these differences may explain the differences in ligand trafficking and depletion. Further understanding of the structural changes accompanying unfolding may help guide cytokine design with respect to ligand binding, endocytic trafficking, and, consequently, therapeutic efficacy.     

A closed compact structure of native Ca(2+)-calmodulin

Calmodulin has been a subject of intense scrutiny since its discovery because of its unusual properties in regulating the functions of about 100 diverse target enzymes and structural proteins. The original and to date only crystal conformation of native eukaryotic Ca(2+)-calmodulin (Ca(2+)-CaM) is a very extended molecule with two widely separated globular domains linked by an exposed long helix. Here we report the 1.7 A X-ray structure of a new native Ca(2+)-CaM that is in a compact ellipsoidal conformation and shows a sharp bend in the linker helix and a more contracted N-terminal domain. This conformation may offer advantages for recognition of kinase-type calmodulin targets or small organic molecule drugs.     

Detection of lysozyme-like enzymatic activity secreted by an immune-responsive mosquito cell line

Using molecular approaches, we have recently shown that the C7-10 mosquito cell line from Aedes albopictus, and the Aag-2 line from Aedes aegypti, secrete a variety of immune peptides into the culture medium, including cecropins, defensins, transferrin, and lysozyme. The diversity of these peptides makes it difficult to quantify the relative activities of each molecule, because possible synergistic interactions may occur. Using a microtiter plate assay with live bacteria, we now show that C7-10 cells secrete an activity that is more potent against the Gram-positive bacterium, Micrococcus luteus, than against Gram-negative Escherichia coli. This lysozyme-like activity is accompanied by production of a lytic zone in an agarose plate assay containing commercially available, lyophilized M. luteus. Properties of the lysozyme-like activity from C7-10 cells included a broad pH optimum from 5.5 to 6.5, and heat-sensitivity above 42 degrees C. Amounts of secreted activity increased during the initial 24h of incubation with heat-killed bacteria. During this induction, lysozyme-like activity was found primarily in the cell culture supernatant.     

Citrate oscillates in liver and pancreatic beta cell mitochondria and in INS-1 insulinoma cells

Oscillations in citric acid cycle intermediates have never been previously reported in any type of cell. Here we show that adding pyruvate to isolated mitochondria from liver, pancreatic islets, and INS-1 insulinoma cells or adding glucose to intact INS-1 cells causes sustained oscillations in citrate levels. Other citric acid cycle intermediates measured either did not oscillate or possibly oscillated with a low amplitude. In INS-1 mitochondria citrate oscillations are in phase with NAD(P) oscillations, and in intact INS-1 cells citrate oscillations parallel oscillations in ATP, suggesting that these processes are co-regulated. Oscillations have been extensively studied in the pancreatic beta cell where oscillations in glycolysis, NAD(P)/NAD(P)H and ATP/ADP ratios, plasma membrane electrical activity, calcium levels, and insulin secretion have been well documented. Because the mitochondrion is the major site of ATP synthesis and NADH oxidation and the only site of citrate synthesis, mitochondria need to be synchronized for these factors to oscillate. In suspensions of mitochondria from various organs, most of the citrate is exported from the mitochondria. In addition, citrate inhibits its own synthesis. We propose that this enables citrate itself to act as one of the cellular messengers that synchronizes mitochondria. Furthermore, because citrate is a potent inhibitor of the glycolytic enzyme phosphofructokinase, the pacemaker of glycolytic oscillations, citrate may act as a metabolic link between mitochondria and glycolysis. Citrate oscillations may coordinate oscillations in mitochondrial energy production and anaplerosis with glycolytic oscillations, which in the beta cell are known to parallel oscillations in insulin secretion.     

Fibroblast growth factor (FGF)-4 can induce proliferation of cardiac cushion mesenchymal cells during early valve leaflet formation

While much has been learned about how endothelial cells transform to mesenchyme during cardiac cushion formation, there remain fundamental questions about the developmental fate of cushions. In the present work, we focus on the growth and development of cushion mesenchyme. We hypothesize that proliferative expansion and distal elongation of cushion mesenchyme mediated by growth factors are the basis of early valve leaflet formation. As a first step to test this hypothesis, we have localized fibroblast growth factor (FGF)-4 protein in cushion mesenchymal cells at the onset of prevalve leaflet formation in chick embryos (Hamburger and Hamilton stage 20-25). Ligand distribution was correlated with FGF receptor (FGFR) expression. In situ hybridization data indicated that FGFR3 mRNA was confined to the endocardial rim of the atrioventricular (AV) cushion pads, whereas FGFR2 was expressed exclusively in cushion mesenchymal cells. FGFR1 expression was detected in both endocardium and cushion mesenchyme as well as in myocardium. To determine whether the FGF pathways play regulatory roles in cushion mesenchymal cell proliferation and elongation into prevalvular structure, FGF-4 protein was added to the cushion mesenchymal cells explanted from stage 24-25 chick embryos. A significant increase in proliferative ability was strongly suggested in FGF-4-treated mesenchymal cells as judged by the incorporation of 5'-bromodeoxyuridine (BrdU). To determine whether cushion cells responded similarly in vivo, a replication-defective retrovirus encoding FGF-4 with the reporter, bacterial beta-galactosidase was microinjected into stage 18 chick cardiac cushion mesenchyme along the inner curvature where AV and outflow cushions converge. As compared with vector controls, overexpression of FGF-4 clearly induced expansion of cushion mesenchyme toward the lumen. To further test the proliferative effect of FGF-4 in cardiac cushion expansion in vivo (ovo), FGF-4 protein was microinjected into stage 18 chick inner curvature. An assay for BrdU incorporation indicated a significant increase in proliferative ability in FGF-4 microinjected cardiac cushion mesenchyme as compared with BSA-microinjected controls. Together, these results suggest a role of FGF-4 for cardiac valve leaflet formation through proliferative expansion of cushion mesenchyme.     

Island and taxon effects in parasitism revisited: avian malaria in the Lesser Antilles

We identify and describe the distribution of 12 genetically distinct malaria parasite lineages over islands and hosts in four common passerine birds in the Lesser Antilles. Combined parasite prevalence demonstrates strong host effects, little or no island effect, and a significant host-times-island interaction, indicating independent outcomes of host-parasite infections among island populations of the same host species. Host- and/or island-specific parasite lineages do not explain these host-parasite associations; rather, individual lineages themselves demonstrate the same type of independent interactions. Unlike overall prevalence, individual parasite lineages show considerable geographic structure (i.e., island effects) as well as species effects indicating that parasite lineages are constrained in their ability to move between hosts and locations. Together, our results suggest an upper limit to the number of host individuals that malaria parasites, as a community, can infect. Within this limit, however, the relative frequency of the different lineages varies reflecting fine scale interactions between host and parasite populations. Patterns of host-parasite associations within this system suggest both historical co-evolution and ecologically dynamic and independent host-parasite interactions.     

Exploration of mosquito immunity using cells in culture

The propagation of immune-responsive cells in vitro has provided the basis for substantial contributions to our understanding of many aspects of the mammalian immune response. In contrast, the potential for exploring the innate immune response of insects using cultured cells is only beginning to be developed, particularly with various mosquito cell lines from the genera Aedes and Anopheles. Immune-reactive mosquito cell lines express various defensive factors, including transferrin, lysozyme, cecropin, defensin, and prophenoloxidase activities. In this review, we discuss insect immunity in the context of key concepts that have emerged in the study of the mammalian immune system, with emphasis on the properties of the cells that participate in the immune response. The nature of established cell lines and their contributions to our understanding of immune functions in humans and insects is described, with emphasis on our own work with the C7-10 and Aag-2 mosquito cell lines from Aedes albopictus and Aedes aegypti, respectively. Finally, we offer some speculation on further advances in insect immunology that may be facilitated by work with cells in culture.