ECB-ART-44697
Immunol Cell Biol
2016 Oct 01;949:861-874. doi: 10.1038/icb.2016.51.
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Perturbation of gut bacteria induces a coordinated cellular immune response in the purple sea urchin larva.
Ch Ho E
,
Buckley KM
,
Schrankel CS
,
Schuh NW
,
Hibino T
,
Solek CM
,
Bae K
,
Wang G
,
Rast JP
.
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The purple sea urchin (Strongylocentrotus purpuratus) genome sequence contains a complex repertoire of genes encoding innate immune recognition proteins and homologs of important vertebrate immune regulatory factors. To characterize how this immune system is deployed within an experimentally tractable, intact animal, we investigate the immune capability of the larval stage. Sea urchin embryos and larvae are morphologically simple and transparent, providing an organism-wide model to view immune response at cellular resolution. Here we present evidence for immune function in five mesenchymal cell types based on morphology, behavior and gene expression. Two cell types are phagocytic; the others interact at sites of microbial detection or injury. We characterize immune-associated gene markers for three cell types, including a perforin-like molecule, a scavenger receptor, a complement-like thioester-containing protein and the echinoderm-specific immune response factor 185/333. We elicit larval immune responses by (1) bacterial injection into the blastocoel and (2) seawater exposure to the marine bacterium Vibrio diazotrophicus to perturb immune state in the gut. Exposure at the epithelium induces a strong response in which pigment cells (one type of immune cell) migrate from the ectoderm to interact with the gut epithelium. Bacteria that accumulate in the gut later invade the blastocoel, where they are cleared by phagocytic and granular immune cells. The complexity of this coordinated, dynamic inflammatory program within the simple larval morphology provides a system in which to characterize processes that direct both aspects of the echinoderm-specific immune response as well as those that are shared with other deuterostomes, including vertebrates.
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???displayArticle.link??? Immunol Cell Biol
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MOP74667 CIHR
Genes referenced: LOC100887844 LOC100893907 LOC115919910 LOC115925133 LOC115929142 LOC583082 LOC586858 LOC594725 LOC763692
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Figure 1. Purple sea urchin larvae are morphologically simple yet have several immune cell types. (a) The purple sea urchin has a biphasic life history. Although many sea urchin species have similar life cycles, the times shown apply to S. purpuratus. Adult animals have life spans of several decades. Fertilized zygotes develop through a rapid cleavage stage to form a mesenchyme blastula of ~800 cells by 24 hpf. In this stage, the NSM is partitioned along the oral (O; blue)/aboral (A; red) axis. In the aboral NSM pigment cell precursors ingress into the blastocoel at ~30 hpf. Oral NSM cells are marked by expression of Gata1/2/3 and Scl homologs and differentiate later into several blastocoelar cell types as they ingress at ~42 hpf (see c–f). Larvae are characterized by a tripartite gut (foregut, midgut and hindgut) and a calcite skeleton. Pigment cells are typically apposed to the ectoderm. The blastocoel is populated with several morphologically distinct types of blastocoelar cells. (b–f) Five types of immune cells are present in sea urchin larva. (b) Pigment cells have two morphologies. A collection of pigment cells near the ectoderm (b1, b3) and a single pigment cell (b2, b4) are shown. In their resting state, pigment cells are stellate (b1, b2). In response to immune stimuli, they become rounded (b3, b4). (c–f) Morphology and behavior define four types of blastocoelar cells. These include (c) globular cells, (d) a subset of filopodial cells, (e) ovoid cells and (f) amoeboid cells. Details of these cells are found in Supplementary Table S1. Scale bar represents 20 μm. | |
Figure 2. Mesenchymal immune cells specifically express immune genes during development and in response to bacterial challenge. (a–d) The tecp2 transcript is expressed in pigment cells. (a) WMISH localizes tecp2 expression to the secondary mesenchyme cells ingressing into the blastocoel at 27 hpf and (b) within cells populating the blastocoel and at the ectoderm at 48 hpf. A GFP reporter construct localizes tecp2 expression to pigment cells. (c, d) A larva (4 DPF) expressing GFPtecp2 is shown (c; fluorescence channel c′); the box indicates the inset in (d; fluorescence channel d′). See Supplementary Figure S2 for a detailed time course of WMISH and GFP expression. (e, f) Fluorescent WMISH colocalizes SRCR142 to some tecp2+ pigment cells. tecp2 (green) and srcr142 (pink) (g, indicates diffuse gut autofluorescence; e, e′, f, f′: single fluorescence channels; e′′, f′′ both channels). (g–j) The macpfA.2 transcript localizes to globular cells. WMISH indicates that macpfA2 expression is present in several cells at 48 hpf (g). In larvae (10 DPF), macpfA2+cells are evident throughout the blastocoel as well as the larval arms and apex (h). In embryos transgenic for a macpfA2 GFP reporter, GFP fluorescence is restricted to globular cells (i′, j′). The inset shown in (j) is indicated by the white box in (i) (i, j, DIC; i′, j′, DIC/fluorescence overlay). (k, l) The srcr142 and 185/333 transcripts are expressed in distinct cell populations. Fluorescent WMISH was performed using probes specific for srcr142 (pink) and 185/333 (green) on larvae (10 DPF) exposed to V. diazotrophicus for 24 h. No colocalization was evident between these two transcripts. The location of the 185/333+ cells within the blastocoel as well as their morphology suggests that these cells are filopodial cells. (k, k′, l, l′) Single fluorescence channels; (k′′, l′′) channel overlay. (l) blowup from white box in (k). White scale bars indicate 50 μm; yellow scale bars indicate 20 μm. | |
Figure 3. Larval blastocoelar cells respond to foreign particles in the blastocoel. (a) Zymosan A is phagocytosed by ovoid cells in an injected larva. Three time points extracted from time-lapse images are shown (a1–a3). One of the Zymosan A particles is indicated by a white arrow. (b, c) Injection of two larvae with E. coli DH10B strain expressing GFP. The insets (b2, c2) are enlargements of the dashed boxes in (b1, c1) showing phagocytosis by filopodial cells. (d1–d6) Injection of V. diazotrophicus. A cluster of V. diazotrophicus is indicated with a yellow circle (d1–d3). The dotted lines in (d3) indicate phagocytosis of this cluster. Times are indicated in yellow (h:min). Images in (a) are stills from Supplementary Video S1. (e) Larvae phagocytose the majority of V. diazotrophicus within 2 h of injection. Larvae were injected with V. diazotrophicus and subjected to time-lapse imaging for up to 2 h. Bacteria were enumerated in 10 min intervals and plotted as the proportion of the initial bacterial count. FC, filopodial cell; hg, hindgut; mg, midgut; OC, ovoid cell. All scale bars are 20 μm. | |
Figure 4. Seawater exposure to V. diazotrophicus induces a system-wide larval cellular response. (a–d) The larval immune response includes migration of immune cells to the gut epithelium and changes in gut morphology. Before challenge the pigment cells are at the aboral ectoderm and the gut is extended (a, c). After 24 h of exposure to V. diazotrophicus, the larval gut is reduced in size and the gut epithelium thickens (b, d). (e) Pigment cell migration is dependent on bacterial concentration. Larvae exposed to increasing concentrations of V. diazotrophicus were monitored for pigment cell migration over the course of 72 h. Each point represents the number of pigment cells not associated with the ectoderm within an individual larva (including cells within the blastocoel and those associated with the gut). Inset shows the corresponding average cell migration over time (dots) and the proportion of larvae that have migratory pigment cells (bars). (f) Immune cell velocity is affected by exposure to V. diazotrophicus. Larvae were subjected to time-lapse imaging after 0, 6, 12 and 24 h of exposure to V. diazotrophicus. Each point represents the velocity of an individual cell (all cells were tracked in n=3 larvae for each time point). Cells were classified as either amoeboid (blue), globular (gray) or pigment cells located in either the ectoderm (dark red), larval arms or apex (red) or within the blastocoel (pink). Pigment cells were absent from the blastocoels of uninfected larvae (*). Error bars indicate the mean±s.e.m. Black bars indicate comparisons of a single cell type across time points; gray bars indicate comparisons of different cell types (solid lines, P<0.001; dashed lines, P<0.05). (g) Activation of larval cellular response requires live bacteria. Larvae (7 DPF) were exposed to V. diazotrophicus neutralized by three different methods (as well as live bacteria). Pigment cell migration was quantified after 24 h of exposure. ***Significant difference relative to the corresponding unexposed set within exposure types; Letter groupings (a, b) indicate significant differences within time groups (all P<0.0001). (h) Larvae return to pre-exposure state following removal of bacteria. Pigment cell migration was assessed after 24 h of exposure to V. diazotrophicus, and at 24 and 48 h following removal of bacteria. Significantly different groupings are indicated (a, b; P<0.0001). (i–l) Bacteria infiltrate the blastocoel during V. diazotrophicus exposure. Larvae were exposed to V. diazotrophicus and analyzed in fluorescent in situ hybridization (FISH) experiments using a Cy5-conjugated 16S RNA probe (EUB338, pink). Nuclei are counterstained with Hoechst (blue). The fluorescent signal in the gut (i, j) is the result of autofluorescence. Results are representative of six experiments. White squares indicate the regions shown in more detail (h′, i′). The gut is outlined with a dashed line in (i′). White arrows highlight bacteria in the blastocoel. (k, k') Bacteria are evident within 185/333+ cells in the blastocoel. Larvae exposed to V. diazotrophicus were used in WMISH using probes specific for 185/333 (green) and 16S rRNA from V. diazotrophicus (pink). Enlargements from two representative larvae are shown. (l) The number of bacteria within the larval blastocoel increase over time in response to exposure to V. diazotrophicus. Each data point represents the number of bacteria counted within the blastocoel of an individual larva. Results are pooled from two sets of larval samples in six experiments. Statistical groupings are indicated (a, b, c; P<0.0001). Inset shows the frequency of larvae with any bacteria in their gut (dark gray) or blastocoel (light gray). Sample size (n) is indicated under the abscissa in (e–h). All error bars indicate s.e.m. Black scale bars indicate 100 μm; white scale bars indicate 20 μm; yellow bars indicate 10 μm. Significance in all cases was determined using an unpaired, two-tailed Student's t-test. | |
Figure 5. Immune cells interact with the gut epithelium and other cell types. (a) Pigment cells and amoeboid cells interact. The amoeboid cell (labeled AC) approaches the pigment cell (PC; a1, a2) that briefly extends a single granule-filled pseudopod to interact with the amoeboid cell (a3, duration of contact is ∼1 min), and then retreats (a4, see Supplementary Video S3). (b) Several immune cell types interact after microbial disturbance. A pigment cell (PC) interacts with a filopodial cell (FC) in the blastocoel, whereas a globular cell (GC) interacts with the gut epithelium in a larva exposed to V. diazotrophicus. (c) Pigment cells and an amoeboid cell interact with the gut epithelium. Two pigment cells (PC, and 1) and an amoeboid cell (AC4). The pigment cells extend and retract pseudopodia to interact with the gut epithelium in an immune-challenged larva (24 h of V. diazotrophicus exposure). Images shown are captured from a 6 h time-lapse microscopy video of a larva infected with V. diazotrophicus after 24 h exposure (Supplementary Video S4). (d1–d8) Pigment cells and amoeboid cells form a long-lasting tight cluster near the ectoderm. Three pigment cells (1–3) interact near the ectoderm. Cells 1 and 4 are the same cells shown in (c) that have traveled from the gut back to the ectoderm. These cells form a close association, and are shortly joined by two amoeboid cells (cells 4 and 5; d2 and d4). These five cells interact dynamically for ~2 h before dispersing (d8). The times (shown in yellow) indicate the time (h:mm) from the start of the imaging. Scale bars indicate 20 μm. | |
Figure 6. Larvae respond to bacteria by altering gene expression levels in immune cells. Larvae were infected with V. diazotrophicus and used in quantitative PCR (qPCR) analyses to assess transcript prevalence (a, c, e) and WMISH to count number of cells (b, d, f) expressing the globular cell marker macpfa2 (a, b), pigment cell markers srcr142, tecp2 and mif7 (c, d) and filopodial cell marker 185/333 (e, f). Proportions of larvae that have 185/333+ cells are indicated in parentheses in (f). Error bars are assessed based on four qPCR replicates. Student's t-test indicates that the numbers of cells that express macpfA2 are statistically different at the time points of exposure (thick line, P<0.005; dashed line, P<0.05). Sample size of larvae counted (n) is indicated. | |
Figure 7. Larvae differentially respond to immune challenge with several bacterial isolates. Larvae were exposed to varying concentrations of five of the isolated bacterial strains (see Supplementary Table S2 for details). The numbers of pigment cells within the blastocoel (including those associated with the gut) were counted after 24 h of exposure. Experiments at 108 bacteria per ml were not determined (n.d.) for isolates 58 and 59 because of bacterial clumping. Asterisks indicate significant differences in pigment cell migration compared with the corresponding concentration of V. diazotrophicus according to the two-tailed unpaired Student's t-test (*P⩽0.05; **P⩽0.005). Sample size (n) is indicated under the abscissa. | |
Figure 8. The larval response to gut-associated bacterial perturbation occurs in cells and tissues throughout the organism. A graphic timeline of the larval response to pathogenic bacteria in the sea water is shown including changes in the gut morphology, cell behavior and alterations in gene expression levels. Under uninfected conditions, stellate pigment cells are apposed to the ectoderm, low levels of 185/333 expression are evident in a subset of filopodial cells and some cell/cell interactions are rare. Exposure to V. diazotrophicus induces a rapid thickening of the gut epithelium, pigment cell and amoeboid migration to the gut, and upregulation of 185/333. As bacterial cells penetrate the gut epithelium and enter the blastocoel, they are phagocytosed by 185/333+ filopodial cells. AC, amoeboid cell; GC, globular cell. |
References [+] :
Amann,
Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations.
1990, Pubmed
Amann, Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. 1990, Pubmed
Bosch, Uncovering the evolutionary history of innate immunity: the simple metazoan Hydra uses epithelial cells for host defence. 2009, Pubmed
Buckley, Diversity of animal immune receptors and the origins of recognition complexity in the deuterostomes. 2015, Pubmed , Echinobase
Buckley, Dynamic evolution of toll-like receptor multigene families in echinoderms. 2012, Pubmed , Echinobase
Calestani, Isolation of pigment cell specific genes in the sea urchin embryo by differential macroarray screening. 2003, Pubmed , Echinobase
Canton, Scavenger receptors in homeostasis and immunity. 2013, Pubmed
Clow, The sea urchin complement homologue, SpC3, functions as an opsonin. 2004, Pubmed , Echinobase
Croce, Dynamics of Delta/Notch signaling on endomesoderm segregation in the sea urchin embryo. 2010, Pubmed , Echinobase
Davidson, A genomic regulatory network for development. 2002, Pubmed , Echinobase
Dishaw, Gut immunity in a protochordate involves a secreted immunoglobulin-type mediator binding host chitin and bacteria. 2016, Pubmed
Duboc, Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo. 2004, Pubmed , Echinobase
Ernst, Offerings from an urchin. 2011, Pubmed , Echinobase
Fugmann, An ancient evolutionary origin of the Rag1/2 gene locus. 2006, Pubmed , Echinobase
Furukawa, Characterization of a scavenger receptor cysteine-rich-domain-containing protein of the starfish, Asterina pectinifera: ApSRCR1 acts as an opsonin in the larval and adult innate immune systems. 2012, Pubmed , Echinobase
Furukawa, Defense system by mesenchyme cells in bipinnaria larvae of the starfish, Asterina pectinifera. 2009, Pubmed , Echinobase
Ghosh, Sp185/333: a novel family of genes and proteins involved in the purple sea urchin immune response. 2010, Pubmed , Echinobase
Gibson, Migratory and invasive behavior of pigment cells in normal and animalized sea urchin embryos. 1987, Pubmed , Echinobase
Gibson, The origin of pigment cells in embryos of the sea urchin Strongylocentrotus purpuratus. 1985, Pubmed , Echinobase
Gross, SpC3, the complement homologue from the purple sea urchin, Strongylocentrotus purpuratus, is expressed in two subpopulations of the phagocytic coelomocytes. 2000, Pubmed , Echinobase
Hakim, An abundance of Epsilonproteobacteria revealed in the gut microbiome of the laboratory cultured sea urchin, Lytechinus variegatus. 2015, Pubmed , Echinobase
Heyland, A detailed staging scheme for late larval development in Strongylocentrotus purpuratus focused on readily-visible juvenile structures within the rudiment. 2014, Pubmed , Echinobase
Hibino, The immune gene repertoire encoded in the purple sea urchin genome. 2006, Pubmed , Echinobase
Johnson, The coelomic elements of sea urchins (Strongylocentrotus). I. The normal coelomocytes; their morphology and dynamics in hanging drops. 1969, Pubmed , Echinobase
Materna, Diversification of oral and aboral mesodermal regulatory states in pregastrular sea urchin embryos. 2013, Pubmed , Echinobase
McClay, Evolutionary crossroads in developmental biology: sea urchins. 2011, Pubmed , Echinobase
McCormack, Perforin-2/Mpeg1 and other pore-forming proteins throughout evolution. 2015, Pubmed
Meijering, Methods for cell and particle tracking. 2012, Pubmed
Minokawa, Expression patterns of four different regulatory genes that function during sea urchin development. 2004, Pubmed , Echinobase
Nakhamchik, Identification of a Wzy polymerase required for group IV capsular polysaccharide and lipopolysaccharide biosynthesis in Vibrio vulnificus. 2007, Pubmed
Neiman, Chitin-induced carbotype conversion in Vibrio vulnificus. 2011, Pubmed
Oliveri, Global regulatory logic for specification of an embryonic cell lineage. 2008, Pubmed , Echinobase
Pancer, Individual-specific repertoires of immune cells SRCR receptors in the purple sea urchin (S. Purpuratus). 2001, Pubmed , Echinobase
Perry, Ca2+-stimulated production of H2O2 from naphthoquinone oxidation in Arbacia eggs. 1981, Pubmed , Echinobase
Pespeni, Genome-wide polymorphisms show unexpected targets of natural selection. 2012, Pubmed , Echinobase
Pimanda, Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early hematopoietic development. 2007, Pubmed
Pruesse, SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. 2007, Pubmed
Ransick, Cis-regulatory logic driving glial cells missing: self-sustaining circuitry in later embryogenesis. 2012, Pubmed , Echinobase
Ransick, New early zygotic regulators expressed in endomesoderm of sea urchin embryos discovered by differential array hybridization. 2002, Pubmed , Echinobase
Rast, Genomic insights into the immune system of the sea urchin. 2006, Pubmed , Echinobase
Rast, brachyury Target genes in the early sea urchin embryo isolated by differential macroarray screening. 2002, Pubmed , Echinobase
Round, The gut microbiota shapes intestinal immune responses during health and disease. 2009, Pubmed
Ruffins, A clonal analysis of secondary mesenchyme cell fates in the sea urchin embryo. 1993, Pubmed , Echinobase
Schindelin, Fiji: an open-source platform for biological-image analysis. 2012, Pubmed
Schrankel, A conserved alternative form of the purple sea urchin HEB/E2-2/E2A transcription factor mediates a switch in E-protein regulatory state in differentiating immune cells. 2016, Pubmed , Echinobase
Secombes, The interleukins of fish. 2011, Pubmed
Shah, The gene encoding the sea urchin complement protein, SpC3, is expressed in embryos and can be upregulated by bacteria. 2003, Pubmed , Echinobase
Shoguchi, In situ screening for genes expressed preferentially in secondary mesenchyme cells of sea urchin embryos. 2002, Pubmed , Echinobase
Silva, The onset of phagocytosis and identity in the embryo of Lytechinus variegatus. 2000, Pubmed , Echinobase
Smith, Echinoderm immunity. 2010, Pubmed , Echinobase
Smith, The larval stages of the sea urchin, Strongylocentrotus purpuratus. 2008, Pubmed , Echinobase
Sodergren, The genome of the sea urchin Strongylocentrotus purpuratus. 2006, Pubmed , Echinobase
Solek, An ancient role for Gata-1/2/3 and Scl transcription factor homologs in the development of immunocytes. 2013, Pubmed , Echinobase
Tamboline, Secondary mesenchyme of the sea urchin embryo: ontogeny of blastocoelar cells. 1992, Pubmed , Echinobase
Tauber, Metchnikoff and the phagocytosis theory. 2003, Pubmed
Tschopp, Structural/functional similarity between proteins involved in complement- and cytotoxic T-lymphocyte-mediated cytolysis. 1986, Pubmed
Tu, Gene structure in the sea urchin Strongylocentrotus purpuratus based on transcriptome analysis. 2012, Pubmed , Echinobase
Tupin, Resistance to rifampicin: at the crossroads between ecological, genomic and medical concerns. 2010, Pubmed
Wang, Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy. 2007, Pubmed