Ch Ho E et al. (2016), Perturbation of gut bacteria induces a coordina...

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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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.

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