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Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors.

Qi YX, Huang J, Li MQ, Wu YS, Xia RY, Ye GY - Elife (2016)

Bottom Line: Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors.The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis.Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Rice Biology, Institute of Insect Sciences, Zhejiang University, Hangzhou, China.

ABSTRACT
Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B- Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution.

No MeSH data available.


Related in: MedlinePlus

Knockdown of 5-HT1B in hemocytes affects Drosophila phagocytosis and survival.(A) Quantification of in vivo phagocytosis of E. coli in WT/UAS-1B RNAi, hml> UAS-1B RNAi and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice, (B) Quantification of in vivo phagocytosis of E. coli in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice. (C) Quantification of in vivo phagocytosis of S. aureus in WT/UAS-1B RNAi, hml> UAS-1B RNAi, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice, (D) Quantification of in vivo phagocytosis of S. aureus in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice. (E) Quantification of in vivo phagocytosis of red fluorescently labeled latex beads in WT/UAS-1B RNAi, hml>UAS-1B RNAi, and hml/WT flies after a 30 min preinjection of either PBS or 1μg/μl 5-HT. Approximately six flies per genotype were used in each experiment. Experiments were done twice, (F) Representative survival curves of WT/UAS-1B RNAi, hml>UAS-1B RNAi, and hml/WT male flies after injection of S. aureus. n=19–22 flies. Data are representatives of three independent experiments. Each experiment was performed in triplicate. (G) Representative survival curves of WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT male flies after injection of S. aureus. n=19–21 flies. Data are representatives of two independent experiments. Each experiment was performed in triplicate. (H) Comparison of the S. aureus (OD 0.4) recovered in WT/UAS-1B RNAi, hml>UAS-1B RNAi and hml/WT flies 0, and 18 hr post infection. Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment. Experiments were performed in triplicate. (I) Comparison of the S. aureus (OD 0.4) recovered in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies 0 and 18 hr post infection. Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment. Experiments were performed in triplicate. One-way ANOVA followed by Tukey’s multiple comparison test for A, B, C, D, E, H, and I. Error bars indicate ± s.e.m., ***p<0.001, **p<0.01, *p<0.05.DOI:http://dx.doi.org/10.7554/eLife.12241.013
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fig7: Knockdown of 5-HT1B in hemocytes affects Drosophila phagocytosis and survival.(A) Quantification of in vivo phagocytosis of E. coli in WT/UAS-1B RNAi, hml> UAS-1B RNAi and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice, (B) Quantification of in vivo phagocytosis of E. coli in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice. (C) Quantification of in vivo phagocytosis of S. aureus in WT/UAS-1B RNAi, hml> UAS-1B RNAi, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice, (D) Quantification of in vivo phagocytosis of S. aureus in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice. (E) Quantification of in vivo phagocytosis of red fluorescently labeled latex beads in WT/UAS-1B RNAi, hml>UAS-1B RNAi, and hml/WT flies after a 30 min preinjection of either PBS or 1μg/μl 5-HT. Approximately six flies per genotype were used in each experiment. Experiments were done twice, (F) Representative survival curves of WT/UAS-1B RNAi, hml>UAS-1B RNAi, and hml/WT male flies after injection of S. aureus. n=19–22 flies. Data are representatives of three independent experiments. Each experiment was performed in triplicate. (G) Representative survival curves of WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT male flies after injection of S. aureus. n=19–21 flies. Data are representatives of two independent experiments. Each experiment was performed in triplicate. (H) Comparison of the S. aureus (OD 0.4) recovered in WT/UAS-1B RNAi, hml>UAS-1B RNAi and hml/WT flies 0, and 18 hr post infection. Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment. Experiments were performed in triplicate. (I) Comparison of the S. aureus (OD 0.4) recovered in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies 0 and 18 hr post infection. Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment. Experiments were performed in triplicate. One-way ANOVA followed by Tukey’s multiple comparison test for A, B, C, D, E, H, and I. Error bars indicate ± s.e.m., ***p<0.001, **p<0.01, *p<0.05.DOI:http://dx.doi.org/10.7554/eLife.12241.013

Mentions: 5-HT1B function in the regulation of hemocyte phagocytosis was further confirmed by RNAi. We used the HmlΔ-Gal4 (Sinenko et al., 2004) driver to specifically express UAS-1B RNAi (Yuan et al. 2005) and UAS-5-HT1B RNAi25833 respectively, both of which led to decreased phagocytosis of E. coli and S. aureus (Figure 7A–D). To test whether the hemocyte-specific knockdown of 5-HT1B dampened phagocytosis in general, or whether there was a lack of inducibility after immune challenge, we injected flies with PBS or 5-HT and then latex beads. When flies were injected with PBS, all flies showed similar phagocytic capacity. However, when flies were injected with 5-HT, knocking down 5-HT1B failed to enhance latex beads phagocytosis as controls (Figure 7E). After injection with S. aureus, the flies expressing 5-HT1B RNAi in their blood cells were much weaker than the control flies (Figure 7F–I). Our RNAi data indicate that the increased susceptibility to bacteria in 5-HT1B mutants is due to 5-HT1B malfunction in hemocytes.10.7554/eLife.12241.013Figure 7.Knockdown of 5-HT1B in hemocytes affects Drosophila phagocytosis and survival.


Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors.

Qi YX, Huang J, Li MQ, Wu YS, Xia RY, Ye GY - Elife (2016)

Knockdown of 5-HT1B in hemocytes affects Drosophila phagocytosis and survival.(A) Quantification of in vivo phagocytosis of E. coli in WT/UAS-1B RNAi, hml> UAS-1B RNAi and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice, (B) Quantification of in vivo phagocytosis of E. coli in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice. (C) Quantification of in vivo phagocytosis of S. aureus in WT/UAS-1B RNAi, hml> UAS-1B RNAi, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice, (D) Quantification of in vivo phagocytosis of S. aureus in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice. (E) Quantification of in vivo phagocytosis of red fluorescently labeled latex beads in WT/UAS-1B RNAi, hml>UAS-1B RNAi, and hml/WT flies after a 30 min preinjection of either PBS or 1μg/μl 5-HT. Approximately six flies per genotype were used in each experiment. Experiments were done twice, (F) Representative survival curves of WT/UAS-1B RNAi, hml>UAS-1B RNAi, and hml/WT male flies after injection of S. aureus. n=19–22 flies. Data are representatives of three independent experiments. Each experiment was performed in triplicate. (G) Representative survival curves of WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT male flies after injection of S. aureus. n=19–21 flies. Data are representatives of two independent experiments. Each experiment was performed in triplicate. (H) Comparison of the S. aureus (OD 0.4) recovered in WT/UAS-1B RNAi, hml>UAS-1B RNAi and hml/WT flies 0, and 18 hr post infection. Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment. Experiments were performed in triplicate. (I) Comparison of the S. aureus (OD 0.4) recovered in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies 0 and 18 hr post infection. Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment. Experiments were performed in triplicate. One-way ANOVA followed by Tukey’s multiple comparison test for A, B, C, D, E, H, and I. Error bars indicate ± s.e.m., ***p<0.001, **p<0.01, *p<0.05.DOI:http://dx.doi.org/10.7554/eLife.12241.013
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4829436&req=5

fig7: Knockdown of 5-HT1B in hemocytes affects Drosophila phagocytosis and survival.(A) Quantification of in vivo phagocytosis of E. coli in WT/UAS-1B RNAi, hml> UAS-1B RNAi and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice, (B) Quantification of in vivo phagocytosis of E. coli in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice. (C) Quantification of in vivo phagocytosis of S. aureus in WT/UAS-1B RNAi, hml> UAS-1B RNAi, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice, (D) Quantification of in vivo phagocytosis of S. aureus in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies. Approximately six flies per genotype were used in each experiment. Experiments were performed twice. (E) Quantification of in vivo phagocytosis of red fluorescently labeled latex beads in WT/UAS-1B RNAi, hml>UAS-1B RNAi, and hml/WT flies after a 30 min preinjection of either PBS or 1μg/μl 5-HT. Approximately six flies per genotype were used in each experiment. Experiments were done twice, (F) Representative survival curves of WT/UAS-1B RNAi, hml>UAS-1B RNAi, and hml/WT male flies after injection of S. aureus. n=19–22 flies. Data are representatives of three independent experiments. Each experiment was performed in triplicate. (G) Representative survival curves of WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT male flies after injection of S. aureus. n=19–21 flies. Data are representatives of two independent experiments. Each experiment was performed in triplicate. (H) Comparison of the S. aureus (OD 0.4) recovered in WT/UAS-1B RNAi, hml>UAS-1B RNAi and hml/WT flies 0, and 18 hr post infection. Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment. Experiments were performed in triplicate. (I) Comparison of the S. aureus (OD 0.4) recovered in WT/UAS-5-HT1B RNAi25833, hml>UAS-5-HT1B RNAi25833, and hml/WT flies 0 and 18 hr post infection. Bacterial load was measured in eight individual male flies per genotype at each time point in each experiment. Experiments were performed in triplicate. One-way ANOVA followed by Tukey’s multiple comparison test for A, B, C, D, E, H, and I. Error bars indicate ± s.e.m., ***p<0.001, **p<0.01, *p<0.05.DOI:http://dx.doi.org/10.7554/eLife.12241.013
Mentions: 5-HT1B function in the regulation of hemocyte phagocytosis was further confirmed by RNAi. We used the HmlΔ-Gal4 (Sinenko et al., 2004) driver to specifically express UAS-1B RNAi (Yuan et al. 2005) and UAS-5-HT1B RNAi25833 respectively, both of which led to decreased phagocytosis of E. coli and S. aureus (Figure 7A–D). To test whether the hemocyte-specific knockdown of 5-HT1B dampened phagocytosis in general, or whether there was a lack of inducibility after immune challenge, we injected flies with PBS or 5-HT and then latex beads. When flies were injected with PBS, all flies showed similar phagocytic capacity. However, when flies were injected with 5-HT, knocking down 5-HT1B failed to enhance latex beads phagocytosis as controls (Figure 7E). After injection with S. aureus, the flies expressing 5-HT1B RNAi in their blood cells were much weaker than the control flies (Figure 7F–I). Our RNAi data indicate that the increased susceptibility to bacteria in 5-HT1B mutants is due to 5-HT1B malfunction in hemocytes.10.7554/eLife.12241.013Figure 7.Knockdown of 5-HT1B in hemocytes affects Drosophila phagocytosis and survival.

Bottom Line: Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors.The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis.Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Rice Biology, Institute of Insect Sciences, Zhejiang University, Hangzhou, China.

ABSTRACT
Serotonin (5-HT) modulates both neural and immune responses in vertebrates, but its role in insect immunity remains uncertain. We report that hemocytes in the caterpillar, Pieris rapae are able to synthesize 5-HT following activation by lipopolysaccharide. The inhibition of a serotonin-generating enzyme with either pharmacological blockade or RNAi knock-down impaired hemocyte phagocytosis. Biochemical and functional experiments showed that naive hemocytes primarily express 5-HT1B and 5-HT2B receptors. The blockade of 5-HT1B significantly reduced phagocytic ability; however, the blockade of 5-HT2B increased hemocyte phagocytosis. The 5-HT1B- Drosophila melanogaster mutants showed higher mortality than controls when infected with bacteria, due to their decreased phagocytotic ability. Flies expressing 5-HT1B or 5-HT2B RNAi in hemocytes also showed similar sensitivity to infection. Combined, these data demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors and serotonergic signaling performs critical modulatory functions in immune systems of animals separated by 500 million years of evolution.

No MeSH data available.


Related in: MedlinePlus