Dopamine modulates hemocyte phagocytosis via a D1-like receptor in the rice stem borer, Chilo suppressalis

Dopamine (DA) is a signal moiety bridging the nervous and immune systems. DA dysregulation is linked to serious human diseases, including addiction, schizophrenia, and Parkinson’s disease. However, DA actions in the immune system remain incompletely understood. In this study, we found that DA modulates insect hemocyte phagocytosis using hemocytes prepared from the rice stem borer (RSB), Chilo suppressalis. We investigated whether insect hemocytes are capable of de novo DA production. Here we show that exposing hemocytes to lipopolysaccharide (LPS) led to induction of DA-generating enzymes. Exogenous DA induced rapid phosphorylation of extracellular signal-regulated kinase (ERK) in naïve hemocytes. Activation of ERK was inhibited by preincubating with a DOP1 receptor antagonist. Thus, DA signaling via the DOP1 receptor may contribute to early hemocyte activation. DA synthesized and released from hemocytes may act in an autocrine mechanism to stimulate or maintain phagocytic activity. Consistent with this hypothesis, we found that inhibition of DA synthesis with α-methyl-DL-tyrosine methyl ester hydrochloride or blockage of DOP1 receptor with antagonist SCH23390 impaired hemocyte phagocytosis. Topical DA application also significantly decreased RSB mortality following challenge with the insect pathogenic fungus, Beauveria bassiana. We infer that a DA-dependent signaling system operates in hemocytes to mediate phagocytotic functions.

The central and peripheral nervous systems modulate immune function by releasing soluble factors such as neurotransmitters and neuropeptides [1][2][3] . The classical catecholamine neurotransmitter, dopamine (DA), is particularly interesting because in addition to regulating movement 4 , decision-making 5 , drug addiction 6 , pain perception 7 and endocrine functions 8 , it also modulates immune functions 9,10 . For example, DA is synthesized by several immune effector cells and its receptors are present in these cells 11,12 . Incubating human lymphocytes with DA inhibited Concanavalin A-stimulated proliferation and synthesis of IFNγ 13 . DA operates in human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV)-induced brain pathology 14,15 , and in lymphoproliferative response 16 . Gaskill et al. 17 reported that DA activated macrophages, which express DA receptors, by increasing ERK 1 phosphorylation. They also showed that DA increased HIV replication in human macrophages via activation of the DA receptor DR2 17 . Several studies have demonstrated that T cells and dendritic cells were functionally responsive to DA, suggesting it modulates immunity 12,18 . Stimulation of the DA receptor D5 on dendritic cells potentiates Th17-mediated immunity 19 .
Once released from presynaptic terminals, DA activates D1 and D2 classes of DA receptors in the target cells 20,21 . In vertebrates, the D1 class includes the D1 and D5 subtypes, which on activation increase intracellular cAMP. The D2 receptors, including D2, D3 and D4 subtypes, inhibit generation of intracellular cAMP 20,21 . Invertebrates express three distinct classes of DA receptors. One is the "DOP1" group, most closely related to the vertebrate D1-like receptors; they activate adenylate cyclase to increase intracellular cAMP [22][23][24] . Another is the invertebrate-type DA receptors, which increase intracellular cAMP levels and couple with intracellular Ca 2+ response [25][26][27] . The third group, the invertebrate D2-like receptors, share most homology with vertebrate D2 class receptors, which decrease in intracellular cAMP levels 28,29 .
Phagocytosis is one of the oldest cellular processes, serving as a feeding mechanism, a development process and also as a key defense reaction in innate immunity of all multicellular organisms 30 . Macrophages and neutrophils are the 'professional' mammalian phagocytes that internalize senescent and apoptotic cells, as well as invading pathogens 30 . Dendritic cells present processed antigens to lymphocytes, thus linking innate and adaptive immunity 31,32 . In insects, the innate immune system is divided into humoral and cellular defense responses. Cellular immune response refer to hemocyte-mediated response such as phagocytosis and encapsulation 33 . Phagocytosis is an important innate immune response against pathogens and parasites 34 . In the lepidopteran insects, the 'professional' phagocytes are granulocytes and plasmatocytes 35 . A point of interest is the potential cross-talk between the immune and nervous system may play a role in regulating phagocytosis-like response in insects during infection 33 . However, the specific actions of neurotransmitters in insect phagocytosis are less well characterized compared with vertebrate counterparts.
The major insect cellular immune reactions to infection and invasion are phagocytosis, nodulation and encapsulation, all mediated by a substantial range of compounds, some of which exert similar actions in vertebrate and invertebrate immunity. The similarity provoked us to pose the hypothesis that DA is one the signal moieties responsible for mediating phagocytosis by insect hemocytes. Here we present the outcomes of experiments with the rice stem borer (RSB), Chilo suppressalis, designed to test our hypothesis.

Results
Bacterial LPS challenge stimulates hemocyte DA synthesis. Figure 1A outlines insect DA biosynthesis, in which the tyrosine hydroxylase step is rate-limiting 35 . Figure 1B shows DA granules labeled with anti-DA antibody in the cytosol of hemocytes ( untreated with LPS). To determine whether hemocytes generate and release DA, cells were incubated with bacterial LPS and amounts of DA in cell supernatants were determined by HPLC-MS. Hemocytic DA amounts increased significantly from about 5 ng/ml to over 25 ng/ml in hemocyte supernatants 4 h after exposure to LPS (Fig. 1C). On the basis of these findings, we evaluated the expression of genes encoding tyrosine hydroxylase and dopa decarboxylase by qPCR. Hemocytes were incubated for 15 min and 4 h with 100 ng/ml LPS. To confirm the hemocytes recognized the LPS challenge, we used qPCR to determine expression levels of the genes encoding the anti-fungal peptide defensin and the pattern recognition receptor PGRP-S2 ( Figure S1A and B); expression of both genes was substantially induced by LPS. Figure 1D,E show that expression of tyrosine hydroxylase increased by about 2.5-fold and expression of dopa decarboxylase increased by about 1.5-fold at 4 h post LPS challenge. We confirmed our qPCR results by Western blotting; at 4 h post LPS challenge the expression of both proteins was markedly increased (Fig. 1F,G).
Hemocytes express CsDOP1. We tested DA receptor gene expression in hemocytes. Although CsDOP2 and CsDOP3 was also detected by qPCR, the Ct values were larger (Table S2). We analyzed control hemocyte preparations immediately following isolation; and experimental preparations after 4 h exposure to 100 ng/ml LPS. As shown in Fig. 2A,B, control and experimental hemocyte preparations express the CsDOP1 receptor, and levels of the transcript and protein remained constant following LPS-activation. We confirmed the presence of CsDOP1 using immunofluorescence to directly visualize CsDOP1 within hemocytes (Fig. 2C). The figure also indicates that CsDOP1 was expressed on the plasmatocyte and granulocyte membranes. CsDOP1 receptors couple with a G s protein and increase intracellular cAMP concentrations ([cAMP] i ) in a DA dose-dependent manner, from about 15 pmol/ well in controls to about 50 pmol/well after DA treatment at 10 −7 M and higher concentrations (Fig. 2D).
We ruled out the possibility that the functional of CsDOP2 (increase intracellular Ca 2+ ) or a CsDOP3 (decrease cAMP] i ) operates in RSB hemocytes 28 . We stimulated in vitro hemocyte preparations with DA and monitored intracellular Ca 2+ and, separately, [cAMP] i concentrations. Figure 2E shows that DA treatments (10 μ M) did not induce a Ca 2+ response; Fig. 2F shows that DA treatments did not reduce forskolin-activated [cAMP] i . We infer that hemocytes express a functional CsDOP1, but not a CsDOP2 or CsDOP3 receptor.
Exogenous DA leads to rapid phosphorylation of ERK. We investigated the time course of ERK phosphorylation in DA-treated hemocytes. ERK phosphorylation increased above basal levels within 5 min and peaked between 10 and 20 min after DA treatment (at 10 μ M; Fig. 4A). We confirmed the action of CsDOP1 in ERK activation by pretreating hemocytes with the most potent CsDOP1 receptor antagonist, SCH23390 (at 1 nM and 10 μ M). Figure 4B shows that 10 μ M, but not 1 nM, SCH23390 fully inhibited DA-induced ERK phosphorylation.
Inhibition of DA signaling impairs hemocyte phagocytosis. We treated hemocyte preparations with α -methyl-DL-tyrosine methyl ester hydrochloride (AMPT), a tyrosine hydroxylase inhibitor 41 and recorded substantial reductions in the basal DA synthesis compared with controls. We saw in Fig. 1C that LPS treatments led to increased DA concentrations. Figure S2 shows that LPS did not lead to increased DA in AMPT-treated hemocyte preparations. DA enhanced the phagocytosis in untreated, but not in AMPT-treated hemocyte preparations (Fig. 5). The rescue experiment showed that DA plus AMPT did not influence phagocytosis relative to untreated control hemocytes (Fig. 5D,E).

DA rescued mortality following fungal challenge. Fungal inoculation (Beauveria bassiana)
was performed by immersing the insect body into a spore solution. DA was applied topically on the middle-abdomenal notum to avoid septic injury. The unchallenged, control RSBs received DA at 1025 ng/ larva and were then treated with PBS; all control insects survived (Fig. 7). Survivorship was reduced to about 70% at 9 days post challenge in larvae without DA rescue treatment; fungal challenge with a subsequent DA rescue treatment increased survival to about 88% from day 5 to day 10 (χ 2 = 10.87, P < 0.01; Kaplan-Meier analysis)

Discussion
The results reported in this paper firmly support our hypothesis that DA is one of the signal moieties responsible for mediating phagocytosis by insect hemocytes. Several lines of data form a complete argument. First, challenging RSBs with a fungal pathogen led to substantial larval mortality, which was reduced in larvae treated with DA. Second, DA is present in RSB plasmatocytes and granulocytes, the lepidopteran immune-conferring hemocytes. Third, in vitro challenge with bacterial LPS stimulated DA production in experimental hemocytes and led to increased expression of genes encoding TH and DDC, the two enzyme responsible for DA biosynthesis, shown by increased levels of the cognate mRNA transcripts and protein. The hemocytes were immunocompetent because LPS challenge led to expression of genes encoding two immune defense proteins, defensin and PGRP-S2. Fourth, hemocytes constitutively express a gene encoding the DA receptor, CsDOP1, again shown by the presence of the receptor transcripts and protein. Our immunohistochemical protocol shows the receptor is located in the hemocyte plasmamembranes, as expected for GPCRs. Fifth, the DA-activated CsDOP1 couples with a G s protein, which leads to increased [cAMP] i s in a dose-dependent manner. We also show that DA treatments did   Eighth, DA stimulated phagocytosis in in vitro hemocyte preparations, which was inhibited in preparations treated with the TH inhibitor AMPT. The elements in this summary form a powerful array of evidence supporting our view that DA signals phagocytosis in hemocytes of microbially challenged RSBs.
Our mortality data show that the fungal challenges in untreated larvae led to decreased survivorship, however, a considerable number of larvae, about 70%, survived. We infer that the cellular immune functions operated during the fungal challenges and that cellular immune signaling effectively protected the infected larvae. The DA treatments enhanced survivorship. This result gives additional credence to our view that DA is produced in hemocytes and acts in an autocrine, or perhaps paracrine mechanism to increase cellular defense reactions to infection. Our view of an auto-or paracrine mechanism is supported also by presence of DA in hemocytes. We detected DA in unchallenged hemocytes and registered infection-driven increases in expression of genes encoding TH and DDC and increases in DA synthesis. Hemocytes constitutively express a plasmamembrane DA receptor, CsDOP1, indicating that individual hemocytes are equipped to respond to DA released from themselves or neighboring hemocytes. It was also reported that prostaglandins (PGs) act in auto-and paracrine mechanisms 42 and they influence insect hemocyte phagocytosis, likely in a similar auto-and paracrine manner 1,43 .
Our data indicated that when the hemocytes were challenged with LPS. The unknown receptor which located on the membrane of hemocyte will be activated. It might enhance the expression of dopamine-producing enzymes (TH and DDC). The generated DA might be transported from intracellular into extracellular by vesicular monoamine transporter (VMAT). Finally, the DA signaling pathway is immediately launched . DA binds with its hemocyte receptor, CsDOP1 and then the intracellular component couples with a Gs protein that activates adenylyl cyclase to increase [cAMP] i s was also initiated. We find that hemocyte-expressed CsDOP1 receptor are functional. Signaling through the CsDOP1 receptor might induces the seconder messenger phospho-ERK and activates the transcription of immune-related genes (Fig. 8). It might be thought that other DA receptors act in phagocytosis, however, we deem that an unlikely possibility because we found that DA did not reduce forskolin-driven high cAMP concentrations in in vitro hemocyte preparations, nor did DA treatments lead to release of Ca 2+ from intracellular stores. We infer a single DA receptor, CsDOP1, but not other DA receptors, acts in hemocyte phagocytosis. Our pharmacological data similarly indicates a single DA receptor type. While DA stimulated ERK phosphorylation, we found the DA receptor antagonist, SCH23390 cancelled the DA effect. SCH23390 is a highly selective vertebrate D1 receptor antagonist 44 . The selectivity of SCH23390 and high sensitivity for one agonist over others indicates a single DA receptor type in RSB hemocytes and likely most lepidopteran hemocytes. The idea that DA acts in insect immunity has been recognized. Sideri et al. (2008) reported on the presence of DDC associated with medfly, Ceratitis capitata hemocytes 45 . They found that siRNA silencing a gene encoding DDC blocked phagocytosis and suggested that phagocytosis and nodulation depend on DA and DA-derived compounds. Similarly, in their work on the Cotesia kariyai/Pseudaletia separata host/parasitoid relationship, Noguchi et al. (2003) found increased concentrations of DA are associated with increases in the insect cytokine, growth-blocking peptide (GBP) 46 . They found that a gene encoding DDC (converts dopa to dopamine, Fig. 1) is expressed at low levels in epidermis, brain and hemocytes; GBP injections led to increased expression of DDC in all three tissues. Although they did not report on the influence of DA on hemocyte behavior, the authors indicate that DA acts in host/parasitoid relationships. Similarly, Gorman et al. (2007) reported that TH activity was up-regulated in tobacco hornworm Manduca sexta hemocytes and fat body following immune challenge 35 . This is in concert with our supplementary data showing that treating RSB hemocyte preparations with the TH inhibitor, AMPT, effectively blocked DA production. In their model, DA produced after infection is incorporated into melanin. The melanin can be used in melanization of nodules and other defense actions. We infer DA has at least two general roles in insect immune reactions, one as a signal moiety in hemocytic phagocytosis and another as a structural component of melanin.
DA-mediated phagocytosis is likely a general insect cellular defense mechanism because DA treatments mitigated the lethality of a fungal pathogen B. bassiana and challenging hemocytes with LPS prepared from the bacterium, E. coli, led to increased expression of genes encoding TH and DDC, increased DA production and increased phagocytosis. Several other signal moieties also mediate insect cellular immune functions. The biogenic amines, octopamine and serotonin, signal phagocytosis and nodulation in cockroach, Periplaneta americana hemocytes 47 ; the insect cytokine, plasmatocyte spreading peptide (PSP) stimulates hemocyte spreading on surfaces. Several groups have reported that prostaglandins (PGs) and other eicosanoids mediate a number of hemocyte actions, including phagocytosis, nodulation, cell spreading and cell migration 48 . These findings prompt the issue of how are these several mediators integrated to produce the observable hemocytic defense behaviors. Kim and his colleagues 48,49 proposed a model in which cytokines, PGs, biogenic amines and possibly other signal molecules act via specific GPCRs, all of which stimulate the small G-protein, Rac1. Rac1 activates the first step in biosynthesis of prostaglandins and other eicosanoids, phospholipase A 2 (PLA2), leading to PG biosynthesis and hemocytic responses. We speculate the CsDOP1 similarly acts via Rac1 in RSB hemocytes.

Methods
Organisms and drugs. The larvae of the rice stem borer, Chilo suppressalis were collected from fields in Fuyang, Zhejiang Province, China. They were reared using rice seedling method reported by Shang et al. 50  Hemocyte isolation and stimulation. The fifth-instar larvae of C. suppressalis were surface sterilized with 70% ethanol. The proleg was cut with a pair of scissors and the hemolymph was collected in Grace's medium (1:10, v/v; Invitrogen, Carlsbad, CA). Hemocytes were treated with 50 or 100 ng of lipopolysaccharides (LPS) (Escherichia coli 0111:B4; Sigma Aldrich). Supernatants were obtained and stored in liquid nitrogen. DA detection. The hemolymph from five fifth-instar larvae was collected and mixed. The combined hemolymph (30 μ L) was mixed with 170 μ L Grace's Insect Medium (as described above) containing 50 μ g/ mL tetracycline and 2 μ L saturated 2-phenylthiourea (PTU). The diluted hemolymph was added to each well of an 8-well chambered coverglasses (Lab-Tek ™ , Nunc, Thermo Fisher Scientific, Rochester, USA) and hemocytes were allowed to adhere to the slide for 20 min at 25 °C to form monolayers. Then, hemocytes were fixed with 4% paraformaldehyde. Dopamine (DA) was labeled with anti-dopamine antibodies (ab6427; Abcam, Cambridge, U.K.) for 72 hours at room temperature, followed by biotinylated anti-rabbit Ig for 24 hours at 4 °C and SA-Alexa Fluor 546 (Invitrogen) for 1 hour at room temperature. The nuclei of hemocytes were stained with 1 μ g/mL of 4′ -6-diamidino-2-phenylindole (DAPI, Beyotime Biotech, Jiangsu, China) for 5 min and hemocytes were observed by fluorescent microscope (Zeiss, Göttingen, Germany).
Dopamine in the supernatants of hemocytes was quantified with reverse-phase high performance liquid chromatography (HPLC) and mass spectrometry (MS) analysis. Briefly, the supernatants were filtered by 0.45 μ m filter, transferred to 1.5 ml Eppendorf tubes and stored at − 80 °C until MS analysis. RNA isolation and qPCR. Total RNA was extracted from hemocytes with Trizol reagent (Life Technologies) in accordance with the manufacturer's instructions. Reverse transcription was performed with 1 μ g of RNA by using a ReverTra Ace ® qPCR RT kit (Toyobo, Osaka, Japan). Real-time quantitative PCR was then performed on cDNA preparations using the SsoFast Eva Green Supermix with Low Rox (Bio-Rad, Hercules, CA) and Applied Biosystems 7500 Real-Time PCR System (Applied Biosystems by Life Technologies, Carlsbad, CA) following the manufacturer's instructions. The quantification of transcript levels of different genes was conducted according to the 2 −ΔΔCT method 51 . The primers used in this study are shown in Table S1.
Western blot. After isolation and exposure to LPS, hemocytes were lysed in Laemmli buffer (1% Triton X-100, 150 mM NaCl, 10 mM Tris-HCl pH 7.5, 5 mM EDTA, 1 mM sodiumo-vanadate) containing 5% mercaptoethanol. Debris were sedimented by centrifugation (15,500 g, 10 minutes, 4 °C), supernatants were collected, and their protein content was determined (Bio-Rad Laboratories). Samples were then separated in a denaturing polyacrylamide gel and transferred to a nitrocellulose membrane. After blocking (5% Tris-buffered saline pH 7.0 containing 0.1% Tween 20) and washing, membranes were then incubated overnight with primary antibodies against TH, DDC, DOP1 and COMT (1:1,000). Membranes were then incubated with secondary antibody horseradish peroxidase-conjugated goat anti-rabbit IgG diluted 1:5,000 in TBS-T. Membranes were rinsed 3 times with wash buffer and then incubated with with the ECL SuperSignal System (Pierce, USA). Membranes were stripped and reprobed with antibodies against Actin (Abcam) for 3 hours at room temperature, followed by incubation with secondary antibody. Immunoblot signals were quantified using a Bio-Rad GS-700 Image densitometer and analyzed using Molecular Analyst (v2.1.2; Bio-Rad). Densitometry units for each band are expressed as OD adjusted by surface (OD × mm 2 ).
Immunofluorescence. For immunofluorescence of CsDOP1, it is the same with DA detection except using the anti-CsDOP1 as the primary antibody.
Intracellular Ca 2+ and cAMP assays. Ca 2+ imaging and cAMP assays were conducted as previously described 52 .
ERK phosphorylation. Hemocytes (2.5 × 10 4 cells/μ L) were stimulated with DA (10 μ M) for 5, 15, or 30 minutes at 25 °C. In some experiments DOP1 receptor signaling was blocked by preincubation with the antagonist, SCH 23390 (Sigma; S7389) at the concentration of 1 nM and 10 μ M for 60 minutes at 25 °C. Cell lysates were prepared, and proteins were resolved by SDS-PAGE. Blots were immunolabeled overnight at 4 °C using 1 μ g/mL polyclonal rabbit Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody (Cell Signaling Technology, Beverly, MA) diluted in TBS-T. Secondary labeling, detection, and analysis were performed as previously described. Membranes were stripped and reprobed with polyclonal rabbit p44/42 MAPK (ERK1/2) antibody (1 μ g/mL; Cell Signaling Technology). Phagocytosis assay. The assay for phagocytosis was performed according to the method described by Huang et al. 52 and Wu et al. 53 with minor modifications. Briefly, larval hemocytes (2 × 10 5 cells) were incubated in 45 μ l Grace's medium containing FITC-labeled E. coli (Invitrogen) with 10 bacteria per hemocyte, in the presence of drugs solution or control solution (5 μ l) for 30 min at 26 °C in a 96 well tissue culture plate (Nunc, Roskilde, Denmark). After incubation in darkness in a moist chamber, internalized FITC-labeled E. coli was measured by quenching attached FITC-labeled E. coli with 200 μ l of PBS containing Trypan blue (0.4%). After washing each well three times with PBS, hemocytes were treated with 4% formaldehyde for 15 min to fix the monolayer. The nuclei of hemocytes were also stained with 1 μ g/mL of DAPI (Beyotime Biotech). The proportion of cells that had phagocytosed labeled E. coli was determined under a Florescence microscope (TS100, Nikon, Tokyo, Japan) in fluorescence mode at 200 × magnification in five different fields.

Survival analysis.
To dilute DA, we used acetone and PBS buffer (pH 7.2) with 1:1 (v/v) mixture as solvent. Fourth-instar larvae of rice stem borer (8-9 mg) were topically treated with 1 μ l of DA solution (1025 ng/μ l) onto the middle-abdomen notum of the larvae with a hand micro-applicator (Institute of Plant Physiology and Ecology, Chinese Academy of Sciences, Shanghai, China). After twenty minutes, the rice stem borers were immersed in a spore suspension (1 × 10 6 spores/ml) of the Beauveria bassiana for ten seconds. Rice stem borer that received DA at 1025 ng/larva and then PBS served as the unchallenged controls. There were three treatments and each treatments was conducted with 30 larvae. The experiment lasted 10 days.

Statistical analysis.
All values are expressed as means ± standard error (s.e.m.). Data were analyzed using the analysis of variance (ANOVA) with Tukey-Kramer post-hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). Before analysis, the percentage data were normalized using an arcsine transformation. All curve fitting and statistical calculations were performed with Origin 8.0 (Origin Lab, Northampton, MA, USA). EC 50 is the agonist concentration that evoked the half-maximal response.