Barela Hudgell MA et al. (2022), Coelomocyte populations in the sea urchin, Stro...

ECB-ART-50893

Front Immunol 2022 Jan 01;13:940852. doi: 10.3389/fimmu.2022.940852.

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Coelomocyte populations in the sea urchin, Strongylocentrotus purpuratus, undergo dynamic changes in response to immune challenge.

Barela Hudgell MA , Grayfer L , Smith LC .

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The sea urchin, Strongylocentrotus purpuratus has seven described populations of distinct coelomocytes in the coelomic fluid that are defined by morphology, size, and for some types, by known functions. Of these subtypes, the large phagocytes are thought to be key to the sea urchin cellular innate immune response. The concentration of total coelomocytes in the coelomic fluid increases in response to pathogen challenge. However, there is no quantitative analysis of how the respective coelomocyte populations change over time in response to immune challenge. Accordingly, coelomocytes collected from immunoquiescent, healthy sea urchins were evaluated by flow cytometry for responses to injury and to challenge with either heat-killed Vibrio diazotrophicus, zymosan A, or artificial coelomic fluid, which served as the vehicle control. Responses to the initial injury of coelomic fluid collection or to injection of V. diazotrophicus show significant increases in the concentration of large phagocytes, small phagocytes, and red spherule cells after one day. Responses to zymosan A show decreases in the concentration of large phagocytes and increases in the concentration of small phagocytes. In contrast, responses to injections of vehicle result in decreased concentration of large phagocytes. When these changes in coelomocytes are evaluated based on proportions rather than concentration, the respective coelomocyte proportions are generally maintained in response to injection with V. diazotrophicus and vehicle. However, this is not observed in response to zymosan A and this lack of correspondence between proportions and concentrations may be an outcome of clearing these large particles by the large phagocytes. Variations in coelomocyte populations are also noted for individual sea urchins evaluated at different times for their responses to immune challenge compared to the vehicle. Together, these results demonstrate that the cell populations in sea urchin immune cell populations undergo dynamic changes in vivo in response to distinct immune stimuli and to injury and that these changes are driven by the responses of the large phagocyte populations.

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	Figure 1. Baseline coelomocyte concentrations in sea urchins prior to challenge are used as the reference for subsequent treatments. CF samples collected on day 0 (n = 18 samples) were evaluated for the different types of coelomocytes at baseline, prior to any experimental manipulations, and were used for comparison to all other time points. Each dot represents the result for an individual sea urchin. (A) Total coelomocyte concentrations are determined by cell counts with a TC20 automatic cell counter (BioRad). (B–E) Different types of coelomocytes are evaluated by flow cytometry (Accuri C6 Flow Cytometer, BD Biosciences) that identifies and quantifies concentrations of different coelomocyte populations at baseline. (F) Injury and CF withdrawal induces a fold decrease in large phagocytes compared to baseline (day 0). (G–J) Different types of coelomocytes from sea urchins (n = 11) are re-evaluated on day 1 to characterize the response to the puncture injury and CF withdrawal carried out on day 0. To calculate fold changes on day 1, baseline data shown in panels (A–E) are set to 1 in panels (F–J). The average ± SD is shown to the right of each data set. The black horizontal bars indicate significant differences (unpaired t-test; p ≤ 0.5). Note that the Y axes are not the same in the different panels.
	Figure 2. Injections of heat-killed Vibrio diazotrophicus, zymosan A, or vehicle (aCF) induce fold changes in total coelomocytes and in populations of different coelomocytes. Sea urchins were injected on days 2 and 5 (arrows along the x-axis) with either Vibrio diazotrophicus (n = 8; red), zymosan A (n = 3; purple), or vehicle as the injury control (n = 7; blue) and samples for analysis of fold changes were collected on days 3 and 5. Total coelomocytes (A) along with populations of large phagocytes (B), small phagocytes (C), and RSCs (D) were analyzed by flow cytometry and tracked over time. Responses to challenges are compared to both baseline (day 0; set to 1) and to injury and CF withdrawal (day 1). Black vertical lines indicate the ± SD for each time point for the three experimental groups. See Figures S2-S4 for graphs showing the responses to the two challenges and the vehicle control for total coelomocytes and for the different coelomocyte populations from individual sea urchins over time. Black horizontal bars with diamond ends indicate significant differences between days (unpaired t-test; significance = p ≤ 0.05). Black horizontal bars with circle ends indicate significant differences between experimental groups vs. the vehicle group (ANOVA; significance set = p ≤ 0.05.).
	Figure 3. Flow cytometry illustrates diversity of coelomocyte composition among sea urchins responding to immune challenge. Scatter plots of selected sea urchins (A–C) from each experimental group shows responses to Vibrio diazotrophicus (red), zymosan A (purple), or vehicle (blue). The plots show the gate for total coelomocytes to illustrate differences and changes in coelomocyte populations among animals. Coelomocytes were evaluated by flow cytometry at baseline (day 0), after injury and CF withdrawal (day 1), and 24 hours after the first and second response to immune challenge or vehicle (days 3, 6). Different coelomocyte populations are indicated by arrows: large phagocytes (blue), RSCs (red), vibratile and CSCs (yellow), and small phagocytes (green). See Figure S1 that shows different populations of coelomocytes as identified by flow cytometry.
	Figure 4. Injection of foreign particles shifts the proportions of coelomocyte types over time. Pie charts indicate the average proportions, shown as percentages, for each coelomocyte type calculated from the coelomocyte concentrations displayed in Figure 1 . The size of the pie charts are scaled according to the average concentration of coelomocytes at each time point, with baseline (day 0) standardized to 100%. Percent changes at each time point can be inferred from the different sizes of the pies, which are indicated by the size of each pie and by the y axes. Results are shown for all sea urchins in all groups at base line (A) and after initial injury and withdrawal of CF (B). The average coelomocyte proportions are shown for each experimental group that received injections of either Vibrio diazotrophicus (C), zymosan A (D), or vehicle (E).
	Figure 5. Coelomocyte responses to injection with Vibrio diazotrophicus is variable among sea urchins. Coelomocytes from sea urchins [SU-V2 (E–H), SU-V3 (I–L), SU-V4 (M–P)] were evaluated initially for fold changes after two injections of vehicle (blue lines). After recovery, they were injected twice with Vibrio diazotrophicus and fold changes in coelomocytes were evaluated again (red lines). Colored arrows at the x axes indicate days 2 and 5 when sea urchins were injected with either Vibrio diazotrophicus (red arrows) or vehicle (blue arrows). SU-V1 (A–D) was treated in the opposite order; V. diazotrophicus first, and then vehicle after recovery. All fold changes are relative to baseline (day 0, set to 1). Fold changes in coelomocytes are compared across days for each animal. See Figures S5A-D for fold changes in the mixed population of vibratile cells and CSCs in individual sea urchins responding to V. diazotrophicus compared to vehicle.
	Figure 6. Coelomocyte responses to injection with zymosan A are variable among sea urchins. Different types of coelomocytes from sea urchins [n = 3; SU-Z1 (A, D, G, J), SU-Z2 (B, E, H, K), SU-Z3 (C, F, I, L)] were evaluated initially after two injections of vehicle (blue lines). After a month of recovery, sea urchins were injected twice with zymosan A and fold changes in coelomocyte types were evaluated again (purple lines). Colored arrows at the x axes indicate days 2 and 5 when sea urchins were injected with either zymosan A (purple arrows) or vehicle (blue arrows). All fold changes are relative to baseline (day 0, set to 1). Fold changes in the different types of coelomocytes are compared across days for each animal. See Figures S5E-G for fold changes in the mixed population of vibratile cells and CSCs in individual sea urchins responding to zymosan A compared to vehicle.

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