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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

Effect of siPr5-HT2B and siPr5-HT7 on hemocyte phagocytosis.(A) Confirmation of knock-down effect of Pr5-HT2B by real-time PCR. The P. rapae 18s rRNA gene was used as an internal reference gene (n = 3). (B) Western blot analysis of knock-down effect of Pr5-HT2B. β-actin was used to show equal protein loading. (C-D) Effect of siPr5-HT2B on hemocyte phagocytosis was visualized by florescence microscope. (E) Quantification of siPr5-HT2B effect on hemocyte phagocytosis (n = 3). (F) Confirmation of knock-down effect of Pr5-HT7 by real-time PCR. The P. rapae 18s rRNA gene was used as an internal reference gene (n= 3). (G) Quantification of siPr5-HT7 effect on hemocyte phagocytosis (n = 3). Two-tailed t-test for A, E, F and G. Error bars indicate ± s.e.m., ***p<0.001, **p<0.01, *p<0.05, and NS means no significant difference.DOI:http://dx.doi.org/10.7554/eLife.12241.010
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fig4s1: Effect of siPr5-HT2B and siPr5-HT7 on hemocyte phagocytosis.(A) Confirmation of knock-down effect of Pr5-HT2B by real-time PCR. The P. rapae 18s rRNA gene was used as an internal reference gene (n = 3). (B) Western blot analysis of knock-down effect of Pr5-HT2B. β-actin was used to show equal protein loading. (C-D) Effect of siPr5-HT2B on hemocyte phagocytosis was visualized by florescence microscope. (E) Quantification of siPr5-HT2B effect on hemocyte phagocytosis (n = 3). (F) Confirmation of knock-down effect of Pr5-HT7 by real-time PCR. The P. rapae 18s rRNA gene was used as an internal reference gene (n= 3). (G) Quantification of siPr5-HT7 effect on hemocyte phagocytosis (n = 3). Two-tailed t-test for A, E, F and G. Error bars indicate ± s.e.m., ***p<0.001, **p<0.01, *p<0.05, and NS means no significant difference.DOI:http://dx.doi.org/10.7554/eLife.12241.010

Mentions: We further found that the 5-HT1B blocker SB 216641 affected hemocyte phagocytosis in a dose-dependent manner (Figure 4A). To confirm the above results, we performed siRNA-mediated interference to knock-down each receptor in hemocytes. The results showed that siPr5-HT1Bdecreased Pr5-HT1B expression significantly at both mRNA and protein levels (Figures 4B–C) after 24 hr and 48 hr, respectively. As expected, the siPr5-HT1B treated hemocytes phagocytose E. coli poorly compared with control (Figure 4D–F). Significantly knock-down of Pr5-HT2B was also observed at both transcript and protein levels (Figure 4—figure supplement 1A–B) at 48 hr, which promoted hemocyte phagocytosis (Figure 4—figure supplement 1C–E). However, knock-down of Pr5-HT7have no significant effect on hemocyte phagocytosis (Figure 4—figure supplement 1F–G). The results demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors but that these two receptors act in opposite ways.10.7554/eLife.12241.009Figure 4.Pr5-HT1B mediates hemocyte phagocytosis.


Serotonin modulates insect hemocyte phagocytosis via two different serotonin receptors.

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

Effect of siPr5-HT2B and siPr5-HT7 on hemocyte phagocytosis.(A) Confirmation of knock-down effect of Pr5-HT2B by real-time PCR. The P. rapae 18s rRNA gene was used as an internal reference gene (n = 3). (B) Western blot analysis of knock-down effect of Pr5-HT2B. β-actin was used to show equal protein loading. (C-D) Effect of siPr5-HT2B on hemocyte phagocytosis was visualized by florescence microscope. (E) Quantification of siPr5-HT2B effect on hemocyte phagocytosis (n = 3). (F) Confirmation of knock-down effect of Pr5-HT7 by real-time PCR. The P. rapae 18s rRNA gene was used as an internal reference gene (n= 3). (G) Quantification of siPr5-HT7 effect on hemocyte phagocytosis (n = 3). Two-tailed t-test for A, E, F and G. Error bars indicate ± s.e.m., ***p<0.001, **p<0.01, *p<0.05, and NS means no significant difference.DOI:http://dx.doi.org/10.7554/eLife.12241.010
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fig4s1: Effect of siPr5-HT2B and siPr5-HT7 on hemocyte phagocytosis.(A) Confirmation of knock-down effect of Pr5-HT2B by real-time PCR. The P. rapae 18s rRNA gene was used as an internal reference gene (n = 3). (B) Western blot analysis of knock-down effect of Pr5-HT2B. β-actin was used to show equal protein loading. (C-D) Effect of siPr5-HT2B on hemocyte phagocytosis was visualized by florescence microscope. (E) Quantification of siPr5-HT2B effect on hemocyte phagocytosis (n = 3). (F) Confirmation of knock-down effect of Pr5-HT7 by real-time PCR. The P. rapae 18s rRNA gene was used as an internal reference gene (n= 3). (G) Quantification of siPr5-HT7 effect on hemocyte phagocytosis (n = 3). Two-tailed t-test for A, E, F and G. Error bars indicate ± s.e.m., ***p<0.001, **p<0.01, *p<0.05, and NS means no significant difference.DOI:http://dx.doi.org/10.7554/eLife.12241.010
Mentions: We further found that the 5-HT1B blocker SB 216641 affected hemocyte phagocytosis in a dose-dependent manner (Figure 4A). To confirm the above results, we performed siRNA-mediated interference to knock-down each receptor in hemocytes. The results showed that siPr5-HT1Bdecreased Pr5-HT1B expression significantly at both mRNA and protein levels (Figures 4B–C) after 24 hr and 48 hr, respectively. As expected, the siPr5-HT1B treated hemocytes phagocytose E. coli poorly compared with control (Figure 4D–F). Significantly knock-down of Pr5-HT2B was also observed at both transcript and protein levels (Figure 4—figure supplement 1A–B) at 48 hr, which promoted hemocyte phagocytosis (Figure 4—figure supplement 1C–E). However, knock-down of Pr5-HT7have no significant effect on hemocyte phagocytosis (Figure 4—figure supplement 1F–G). The results demonstrate that 5-HT mediates hemocyte phagocytosis through 5-HT1B and 5-HT2B receptors but that these two receptors act in opposite ways.10.7554/eLife.12241.009Figure 4.Pr5-HT1B mediates hemocyte phagocytosis.

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