FoxO and Stress Responses in the Cnidarian Hydra vulgaris

Background: In the face of changing environmental conditions, the mechanisms underlying stress responses in diverse organisms are of increasing interest. In vertebrates, Drosophila, and Caenorhabditis elegans, FoxO transcription factors mediate cellular responses to stress, including oxidative stress and dietary restriction. Although FoxO genes have been identified in early-arising animal lineages including sponges and cnidarians, little is known about their roles in these organisms. Methods/Principal Findings: We have examined the regulation of FoxO activity in members of the well-studied cnidarian genus Hydra. We find that Hydra FoxO is expressed at high levels in cells of the interstitial lineage, a cell lineage that includes multipotent stem cells that give rise to neurons, stinging cells, secretory cells and gametes. Using transgenic Hydra that express a FoxO-GFP fusion protein in cells of the interstitial lineage, we have determined that heat shock causes localization of the fusion protein to the nucleus. Our results also provide evidence that, as in bilaterian animals, Hydra FoxO activity is regulated by both Akt and JNK kinases. Conclusions: These findings imply that basic mechanisms of FoxO regulation arose before the evolution of bilaterians an

Human Dectin-1 Deficiency Impairs Macrophage-Mediated Defense Against Phaeohyphomycosis

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, β-glucan-binding receptor, Dectin-1. The patient\u27s PBMCs failed to produce TNF-α and IL-1β in response to β-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1β and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1β-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi

FoxO and stress responses in the cnidarian Hydra vulgaris

Background: In the face of changing environmental conditions, the mechanisms underlying stress responses in diverse organisms are of increasing interest. In vertebrates, Drosophila, and Caenorhabditis elegans, FoxO transcription factors mediate cellular responses to stress, including oxidative stress and dietary restriction. Although FoxO genes have been identified in early-arising animal lineages including sponges and cnidarians, little is known about their roles in these organisms. Methods/Principal Findings: We have examined the regulation of FoxO activity in members of the well-studied cnidarian genus Hydra. We find that Hydra FoxO is expressed at high levels in cells of the interstitial lineage, a cell lineage that includes multipotent stem cells that give rise to neurons, stinging cells, secretory cells and gametes. Using transgenic Hydra that express a FoxO-GFP fusion protein in cells of the interstitial lineage, we have determined that heat shock causes localization of the fusion protein to the nucleus. Our results also provide evidence that, as in bilaterian animals, Hydra FoxO activity is regulated by both Akt and JNK kinases. Conclusions: These findings imply that basic mechanisms of FoxO regulation arose before the evolution of bilaterians and raise the possibility that FoxO is involved in stress responses of other cnidarian species, including corals. © 2010 Bridge et al

FoxO and Stress Responses in the Cnidarian Hydra vulgaris

BackgroundIn the face of changing environmental conditions, the mechanisms underlying stress responses in diverse organisms are of increasing interest. In vertebrates, Drosophila, and Caenorhabditis elegans, FoxO transcription factors mediate cellular responses to stress, including oxidative stress and dietary restriction. Although FoxO genes have been identified in early-arising animal lineages including sponges and cnidarians, little is known about their roles in these organisms.Methods/Principal FindingsWe have examined the regulation of FoxO activity in members of the well-studied cnidarian genus Hydra. We find that Hydra FoxO is expressed at high levels in cells of the interstitial lineage, a cell lineage that includes multipotent stem cells that give rise to neurons, stinging cells, secretory cells and gametes. Using transgenic Hydra that express a FoxO-GFP fusion protein in cells of the interstitial lineage, we have determined that heat shock causes localization of the fusion protein to the nucleus. Our results also provide evidence that, as in bilaterian animals, Hydra FoxO activity is regulated by both Akt and JNK kinases.ConclusionsThese findings imply that basic mechanisms of FoxO regulation arose before the evolution of bilaterians and raise the possibility that FoxO is involved in stress responses of other cnidarian species, including corals

Effects of experimental treatments on FoxO-GFP localization.

<p>Localization was examined in stenotele nematocytes, nematoblasts which are precursors to stenoteles, and ganglionic neurons. A) Effects of PI3K inhibitor treatment—1 hour incubation in 40 µM LY294002. N≥69 cells examined per animal. B) Effects of heat shock—90 minutes at 33°C. N≥73 cells examined per animal. C) Effects of heat shock on animals treated with JNK inhibitor. Control and treatment animals were subject to heat shock. Treatment involved 24 hour incubation in 2.5 µM SP60025 prior to heat shock in 2.5 µM SP60025. N≥55 cells examined per animal. D) Effects of heat shock on animals treated with JNK inhibitor. Control and treated animals were subjected to heat shock. Treatment involved 24 hour incubation in 2 µM AS601245 prior to heat shock in 2 µM AS601245. N≥56 cells examined per animal.</p

<i>Hydra</i> FoxO-GFP localization in cells of the interstitial lineage.

<p>(A, C) Nuclear localization. (B, D) Cytoplasmic localization. DAPI-stained nuclei are false-colored red, FoxO-GFP green, areas of overlap yellow. A, B) Late stage nematoblasts. The nucleus is crescent-shaped as a result of being pushed to the side of the cell by the developing nematocyst capsule. C, D) Neurons. Arrows indicate nuclei.</p

Conserved portions of the predicted <i>H. magnipapillata</i> FoxO protein aligned with other FoxO protein sequences.

<p>Akt/SGK phosphorylation motifs are enclosed in boxes, with asterisks above phosphorylated residues. The arrow indicates the location of the intron present in <i>H. magnipapillata</i> FoxO and the other sequences shown. Lines above and below the sequence indicate the forkhead domain. Basic amino acids characteristic of the nuclear localization domain are highlighted in black. Amino acids identical in <i>H. magnipapillata</i> FoxO and another sequence are shaded.</p

Results of whole mount <i>in situ</i> hybridization.

<p>A) Expression of <i>FoxO</i> mRNA in adult <i>H. magnipapillata</i>. The arrow indicates the border between ectoderm and endoderm. B) Adult <i>H. magnipapillata</i> with longer staining reaction, showing punctuate staining in the tentacles. C) Body column of control <i>H. magnipapillata</i>. D) Body column of HU-treated <i>H. magnipapillata</i>. E) <i>H. vulgaris</i> with testes, indicated by arrows. F) <i>H. vulgaris</i> with developing egg, indicated by an arrow. G) <i>H. vulgaris</i> following egg extrusion.</p

Results of phylogenetic analyses.

<p>Maximum parsimony phylogram of selected FoxO proteins rooted using <i>Mus musculus</i> FoxA1. Numbers at nodes are bootstrap support values calculated by 1000 replicates of Maximum Parsimony/Maximum Likelihood/Neighbor Joining. Bootstrap values under 50 are not shown. Asterisks at nodes indicate Bayesian PP greater than 95%. Species name abbreviations: <i>Aa: Aedes aegypti</i>; <i>Aq: Amphimedon queenslandica</i>; <i>Bf: Branchiostoma floridae</i>; <i>Ce: Caenorhabditis elegans</i>; <i>Ch: Clytia hemisphaerica</i>; <i>Ci: Ciona intestinalis</i>; <i>Dm: Drosophila melanogaster</i>; <i>Dr: Danio rerio</i>; <i>Gg: Gallus gallus</i>; <i>Hm: Hydra magnipapillata</i>; <i>Hs: Homo sapiens</i>; <i>Hv: Hydra vulgaris</i>; <i>Mm: Mus musculus</i>; <i>Ms: Metridium senile</i>; <i>Nv: Nematostella vectensis</i>; <i>Sp: Strongylocentrotus purpuratus</i>; <i>Ta: Trichoplax adhaerens</i>; <i>Xl: Xenopus laevis</i>; and <i>Xm: Xiphophorus maculatus</i>.</p