Phagocytosis of different target particles by goldfish and lamprey primary leukocytes.

<p>(A) Goldfish primary kidney leukocytes (PKL) or lamprey primary typhlosole leukocytes (PTL) were incubated with 3 µm YG latex beads, <i>E. coli</i> DH5α-GFP, or zymosan-FITC at the indicated concentrations for the specified times. Cells were then fixed and phagocytosis was quantified by flow cytometry. Grey bars represent percent internalized. Hatched white bars represent percent surface bound. For all n = 4, over 2 examined over a minimum of two independent experiments. * p<0.05 for % internalized,+p<0.05 for % surface bound- between 10∶1 and 5∶1 particle to cell ratios in each graph. (B) Representative images of no internalization (No), surface bound (B), and internalized beads (Int.) from ImageStream MkII flow cytometer (Amnis).</p

Effect of pre-incubation with zymosan and apoptotic cells on respiratory burst responses of individual phagocytes.

<p>Goldfish PKL and lamprey PTL were incubated with both zymosan and apoptotic cells (5∶1 ratio for each) for 2 h and 6 h, respectively. (A) To investigate the effects of pre-incubation with apoptotic cells, apoptotic cells were added 2 h prior to zymosan. Respiratory burst (measured as % DHR positive) was then analyzed based on phagocytic capacity across the four resulting sub-populations: non-phagocytic cells, phagocytes containing only zymosan, phagocytes containing only apoptotic cells, and phagocytes that contain both. (B) The percent of total population found in each of the four sub-populations of (A); no internalization, zymosan only, apoptotic cells only, zymosan and apoptotic cells. (C) The phagocytic index of the Zym+AC group in (A). For all n = 4, examined over a minimum of two independent experiments. * p<0.05 and ** p<0.01 compared to No;+p<0.05 and++p<0.01 compared to Zym.</p

Divergent pro-inflammatory and homeostatic responses of lamprey and goldfish phagocytes.

<p>Goldfish PKL and lamprey PTL were incubated with both zymosan and apoptotic cells (5∶1 ratio for each) for 2 h and 6 h, respectively. (A) Respiratory burst (measured as % DHR positive) was then analyzed based on phagocytic capacity across the four resulting sub-populations: non-phagocytic cells, phagocytes containing only zymosan, phagocytes containing only apoptotic cells, and phagocytes that contain both. (B) The percent of total population found in each of the four sub-populations of (A); no internalization, zymosan only, apoptotic cells only, zymosan and apoptotic cells. * p<0.05 and ** p<0.01 compared to No;+p<0.05 and++p<0.01 compared to Zym. (C) The phagocytic index of the Zym+AC group in (A). (D) Respiratory burst analyzed according to the number of zymosan particles internalized in the Zym+AC group. * p<0.05 and ** p<0.01 compared to goldfish. No- no internalized particle; AC- apoptotic cells; Zym- zymosan. For all n = 4, examined over a minimum of two independent experiments.</p

Fish and Mammalian Phagocytes Differentially Regulate Pro-Inflammatory and Homeostatic Responses <em>In Vivo</em>

<div><p>Phagocytosis is a cellular mechanism that is important to the early induction of antimicrobial responses and the regulation of adaptive immunity. At an inflammatory site, phagocytes serve as central regulators for both pro-inflammatory and homeostatic anti-inflammatory processes. However, it remains unclear if this is a recent evolutionary development or whether the capacity to balance between these two seemingly contradictory processes is a feature already displayed in lower vertebrates. In this study, we used murine (C57BL/6) and teleost fish (<em>C. auratus</em>) <em>in vitro</em> and <em>in vivo</em> models to assess the evolutionary conservation of this dichotomy at a site of inflammation. At the level of the macrophage, we found that teleost fish already displayed divergent pro-inflammatory and homeostatic responses following internalization of zymosan or apoptotic bodies, respectively, and that these were consistent with those of mice. However, fish and mice displayed significant differences <em>in vivo</em> with regards to the level of responsiveness to zymosan and apoptotic bodies, the identity of infiltrating leukocytes, their rate of infiltration, and the kinetics and strength of resulting antimicrobial responses. Unlike macrophages, significant differences were identified between teleost and murine neutrophilic responses. We report for the first time that activated murine, but not teleost neutrophils, possess the capacity to internalize apoptotic bodies. This internalization translates into reduction of neutrophil ROS production. This may play an important part in the recently identified anti-inflammatory activity that mammalian neutrophils display during the resolution phase of inflammation. Our observations are consistent with continued honing of inflammatory control mechanisms from fish to mammals, and provide added insights into the evolutionary path that has resulted in the integrated, multilayered responses that are characteristic of higher vertebrates.</p> </div

Pro-inflammatory (zymosan) and homeostatic (apoptotic bodies) stimuli differentially impact leukocyte infiltration profiles in goldfish and mice.

<p>Goldfish and C57BL/6 mice were injected intraperitoneally with saline, apoptotic bodies (5×10⁶) or zymosan (2.5 mg). Apoptotic bodies were also pre-injected 4 h before zymosan injections. Goldfish leukocyte populations were defined by imaging flow cytometry (area, internal complexity, and morphology) and staining patterns with Sudan Black and an anti-CSF-1R antibody (Figure S3). Murine cells were defined based on surface markers for neutrophils (F4/80⁻/Gr1⁺/CD11b⁺), monocytes (F4/80^lo/Gr1^+/−/CD11b⁺), macrophages (F4/80^hi/Gr1^+/−/CD11b⁺) and lymphocytes (F4/80⁻/Gr1⁻; CD3, B220, NK1.1; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047070#pone.0047070.s004" target="_blank">Figure S4A</a>). n = 4; * p<0.05 and ** p<0.01 compared to control; + p<0.05 and ++ p<0.01 compared to zymosan. No- no internalized particle; AB- apoptotic body; Zy- zymosan.</p

Apoptotic bodies downregulate murine neutrophil ROS production in a contact dependent manner.

<p>Goldfish (left) and C57BL/6 mice (right) were injected intraperitoneally with zymosan (2.5 mg). Activated peritoneal cells from were harvested by peritoneal lavage and subpopulations were isolated by density centrifugation. (A) Separated neutrophil or mononuclear populations were incubated with labeled apoptotic bodies for 2 h and internalization was analyzed. n = 4; * p<0.05 and ** p<0.01; Mφ/M  =  macrophage/monocyte. (B) Isolated populations were cultured for 2 h in the presence of the indicated stimuli. Conditions denoted within brackets were contained within a 4 μm transwell. After 2 h, responder cells outside the transwells were harvested and respiratory burst was assayed using DHR.</p

<i>In vivo</i> administration of apoptotic bodies leads to a more dramatic reduction of pro-inflammatory respiratory burst responses in teleost fish compared to mice.

<p>Goldfish and C57BL/6 mice were injected intraperitoneally with saline, apoptotic bodies (5×10⁶) or zymosan (2.5 mg) and incubated for 24 h. Apoptotic bodies were also pre-injected 0, 2, or 4 h before zymosan injections to assess the contributions of kinetics to these responses. Cells from injected animals were harvested by peritoneal lavage and respiratory burst was assayed with DHR. n = 4; * p<0.05 and ** p<0.01 compared to PBS (saline) control; + p<0.05 and ++ p<0.01 compared to zymosan. No- no internalized particle; A.B.− apoptotic body; Zy- zymosan.</p

Murine neutrophil respiratory burst antimicrobial responses are most greatly affected by the presence of apoptotic bodies.

<p>(A) C57BL/6 mice were injected intraperitoneally with saline, apoptotic bodies (5×10⁶) or zymosan (2.5 mg). Apoptotic bodies were also pre-injected 0, 2, or 4 h before zymosan injections. Cells from injected mice were harvested by peritoneal lavage and respiratory burst was assayed with DHR in peritoneal cell subpopulations based on forward scatter and side scatter profiles (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047070#pone.0047070.s004" target="_blank">Figure S4B</a>). n = 4; * p<0.05 and ** p<0.01 compared to control; + p<0.05 and ++ p<0.01 compared to zymosan. No- no internalized particle; A.B.- apoptotic body; Zy- zymosan. (B) Histograms show representative DHR responses for total leukocytes, neutrophils and monocytes/macrophages. We found that the high responders were predominantly neutrophils (>90%). Pre-incubation with apoptotic bodies resulted in a preferential switch in the neutrophil population from high responders to mid/low responders.</p

<i>In vivo</i> administration of zymosan induces a marked infiltration of leukocytes that is linked to high levels of respiratory burst.

<p>Goldfish (left) and C57BL/6 mice (right) were injected intraperitoneally with 2.5 mg of zymosan. Cells were harvested by peritoneal lavage at 0 h (saline alone), 8, 24 and 48 h and counted (top row). Injection of zymosan resulted in a marked increase in cell numbers isolated from the peritoneum that peaked at 48 h for mice and 24 h for goldfish. Respiratory burst in isolated cells at these time points was determined with DHR (bottom row). n = 4; * p<0.05; ** p<0.01.</p

DP2 agonist-induced Ca²⁺ flux in human mast cells.

<p>After measuring baseline fluorescence of Fluo-4 AM loaded MC (1.25×10⁵ cells in 50 µL/well), (A, C) DK-PGD₂ or (B, D) 15R-15-methyl PGD₂ was given to the MC and intracellular Ca²⁺ flux was assessed by measuring fluorescence change. (A, B) Cytosolic free Ca²⁺ changes induced by DP2 agonists are presented as ΔFluorescence ratio (fluorescence ratio of agonist treatment – fluorescence ratio of sham treatment), where fluorescence ratio is fluorescence unit at each time point/baseline fluorescence unit. Arrow indicates the time when agonist was given. (C, D) Cytosolic free Ca²⁺ changes induced by DP2 agonist treatment are presented as ΔIntegral for 3 min from ΔFluorescent ratio curves shown in A and B. Results are expressed as mean ± SEM for three separate experiments. *p<0.05, **p<0.01 compared with 100 nM agonist treatment by repeated measures ANOVA followed by the Bonferroni post-test.</p