Immune responses of mussel hemocyte ... - Semantic Scholar

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Immune responses of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC, and ERK kinase families.

Erick García-García1, Maria Prado-Álvarez2, Beatriz Novoa2, Antonio Figueras2, and Carlos Rosales1*

1Department of Immunology, Instituto de Investigaciones Biomédicas, Universidad Nacional Autónoma de México, Mexico City, MEXICO, and 2Instituto de Investigaciones Marinas, CSIC, Spanish National Reference Laboratory for Mollusc Diseases, 36208 Vigo, SPAIN.

* Corresponding author: Carlos Rosales, Department of Immunology, Instituto de Investigaciones Biomédicas - UNAM, Apdo. Postal 70228, Cd. Universitaria, México D.F. - 04510, MEXICO. Phone: 52 - 55 - 5622 – 8951 FAX: 52 - 55 - 5622 – 9212. Email: [email protected]

2 Immune functions of mussel hemocyte subpopulations are differentially regulated by enzymes of the PI 3-K, PKC and ERK kinases families Abstract. Various hemocyte cell types have been described in invertebrates, but for most species a functional characterization of different hemocyte cell types is still lacking. In order

to

characterize

some

immunological

properties

of

mussel

(Mytilus

galloprovincalis) hemocytes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. PI 3-K, PKC and ERK inhibitors were also used to biochemically characterize these cell responses. Four morphologically distinct subpopulations, designated R1 to R4, were detected. R1, R2, and R3 cells presented different levels of phagocytosis towards zymosan, latex beads, and two bacteria species. Similarly, R1 to R3, but not R4 cells produced ROS, while all subpopulations produced NO, in response to zymosan. Internalisation of all phagocytic targets was blocked by PI 3-K inhibition. In addition, internalisation of latex particles, but not of bacteria, was partially blocked by PKC or ERK inhibition. Interestingly, phagocytosis of zymosan was impaired by PKC, or ERK inhibitors only in R2 cells. Zymosan-induced ROS production was blocked by PI 3-K inhibition, but not by PKC, or ERK inhibition. In addition, zymosan-stimulated NO production was affected by PI 3-K inhibition in R1 and R2, but not in R3 or R4 cells. NO production in all cell types was unaffected by PKC inhibition, but ERK inhibition blocked it in R2 cells. These data reveal the existence of profound functional and biochemical differences in mussel hemocytes, and indicate that M. galloprovinciallis hemocytes are specialized cells fulfilling specific tasks in the context of host defence.

3 Key words: mollusc, signal transduction, innate immunity, phagocytosis, respiratory burst, wortmannin, staurosporine, PD98059, professional phagocytes.

1. Introduction. Despite the lack of an adaptive immune system, invertebrates are able to survive among potentially pathogenic viruses, bacteria, and fungi [1, 2], and respond to infection by activating various defence mechanisms [2, 3]. This suggests a key role for their apparently limited innate immune system, in protecting these invertebrates from infections. Among the defence mechanisms described in invertebrates are phagocytosis, activation of the phenoloxydase system, encapsulation, the respiratory burst, nitric oxide production, and the production and release of various microbicidal molecules [3]. It is however unclear which of the different cells integrating the invertebrate innate immune system, usually named hemocytes, are responsible for these cell responses. Different types of hemocytes have been described in various invertebrate species, but an accurate universal classification of invertebrate hemocytes is still lacking. A consensus hemocyte classification is not available yet, due in part to intrinsic differences in the immune systems of even closely related invertebrate species [4-6], and also to differences in the experimental methods used to study them [7-11]. Depending on the species, and the experimental methodology, hemocytes have been classified in as few as two, or up to eleven categories [7, 11, 12]. In molluscs, hemocyte classification has largely depended on cell granularity, determined either by microscopy or by flow cytometry [6, 13-16]. However, as in most invertebrate phyla, no definitive mollusc hemocyte classification exists. Extensive studies have shown that blood cells appear to have conserved roles in vertebrate organisms ranging from fish to mammals [17-19]. In contrast, few attempts

4 have been made to functionally characterize the different hemocyte subtypes of most invertebrate organisms. For example, while little doubt exists regarding the phagocytic and microbicidal abilities of macrophages [19], even in lower vertebrates such as fish [18], the information regarding the specific defence mechanisms displayed by different hemocyte subtypes is scarce, and has largely focused only on phagocytic capacity [8-10, 20-22]. In mammals, phagocytosis and the respiratory burst are fundamental defence mechanisms of professional phagocytes, that often take place simultaneously [23-26]. Phagocytosis is the physical internalisation of microbes or cellular debris [25, 26]. The respiratory burst on the other hand, is a series of biochemical reactions that produce -

highly microbicidal reactive oxygen species (ROS), such as superoxide (O2 ), hydrogen .

peroxide (H2O2), and hydroxyl radical (OH ) [23, 24]. Phagocytosis and activation of the respiratory burst have been observed in hemocytes of various mollusc species [8, 27-33]. However, the specific hemocyte subtypes that present these cell responses are still unknown. In the mammalian immune system, the production of nitric oxide (NO) is part of the defence mechanisms displayed by professional phagocytes such as macrophages [34, 35]. NO is a highly cytotoxic and microbicidal molecule [34, 35], also capable of activating other leukocytes [36]. The expression of a nitric oxide synthase, and the production of NO by hemocytes has been reported in various mollusc species [37-43]. Yet, no information is available on the capacity of specific hemocyte subtypes to produce NO. In mammalian leukocytes, the signalling pathways regulating immune responses, such as phagocytosis, differ depending on the type of phagocyte involved [44], the specific nature of the receptor involved [45], and the differentiation and

5 activation state of the phagocytic cells [44, 46, 47]. Kinases of the phosphatidylinositol 3-kinase (PI 3-K), and protein kinase C (PKC) families are key regulators of phagocytosis mediated through various membrane receptors [25, 26]. In addition, the extracellular signal-regulated kinase (ERK) is an enzyme involved in phagocytosis regulation, depending on the phagocytic receptor involved [25], and also on the phagocytic cell type [44, 48, 49]. The role of these signalling molecules in the regulation of hemocyte phagocytosis is unclear. Similarly, the participation of these signalling enzymes in the regulation of the respiratory burst or NO production in invertebrate organisms is poorly defined. In order to characterize some immunological properties of the mussel M. galloprovincialis hemocyte subtypes, cells were separated by flow cytometry and their capacity for phagocytosis, production of reactive oxygen species (ROS), and production of nitric oxide (NO), was examined. PI 3-K, PKC and ERK inhibitors were used to biochemically characterize these cell functions. Four morphologically distinct subpopulations, designated Large granular (R1), Large semigranular (R2), Small semigranular (R3), and Small hyaline (R4) hemocytes, were detected in mussel hemolymph. While Large granular (R1), Large semigranular (R2), and Small semigranular (R3) cells were phagocytic, and capable of activating the respiratory burst, Small hyaline (R4) cells lacked these two capabilities. Nevertheless, all hemocyte subpopulations were capable of NO production. Biochemical characterization of hemocyte functions showed a clear role for PI 3-K in phagocytosis and the respiratory burst, and also for NO production in some cell types. PKC or ERK participated in phagocytosis regulation, depending on the hemocyte subtype and the phagocytic target, but had no role in the regulation of zymosan-induced respiratory burst. While zymosaninduced NO production was independent of PKC, this cell response required ERK, but

6 only in some cell types. These data revealed the existence of critical functional and biochemical differences in mussel hemocytes, and indicate that M. galloprovinciallis hemocytes are specialized cells fulfilling specific tasks in the context of host defence.

2. Materials and Methods. 2.1 Reagents Sea water was obtained from the Ría de Vigo (NW Spain). It was filtered sequentially through 0.45 μm and 0.22 μm porous membranes before use. Flouresceinlabelled 1μm latex beads (catalogue number F8823), flourescein-labelled zymosan bioparticles (catalogue number Z-284), flourescein-labelled E. coli bacteria (catalogue number E-2861), the reactive oxygen species (ROS)-specific intracellular indicator chloromethyl-H2DCFDA (catalogue number C6827), and the nitric oxide (NO)-specific intracellular indicator DAF-FM/ DAF-FM diacetate (catalogue number D-23844), were all from Molecular Probes (Eugene, OR). Fluorescein-labelled Vibrio alginolyticus bacteria were prepared as described previously [50]. PD98059 (catalogue number V1191) was from Promega, (Madison, WI). Wortmannin (catalogue number 19545-267), phorbol myristate acetate (PMA) (catalogue number P8139), staurosporine (catalogue number 55921), and all other chemicals were from Sigma Chemical Company (St. Louis, MO). 2.2 Mussel hemocytes Mussels (Mytilus galloprovincialis) were obtained from the Ría de Vigo (NW Spain). They were maintained in filtered sea water tanks at 15 ºC with aeration, and fed daily with the alga Tetraselmis svecica. Mussel shells were notched, and one to three ml of hemolymph per individual were withdrawn from the adductor muscle. All

7 experiments were performed with hemolymph pools from a minimum of three individuals. 2.3 Flow cytometry analysis of hemocyte subpopulations Freshly isolated hemocytes diluted two fold with cold filtered sea water, or fixed for 20 minutes with cold 2 % paraformaldehyde in sea water, were analysed in a FACScalibur flow cytometer (Becton Dickinson; Franklin Lakes, NJ) equipped with cell sorting and cell concentrator modules. Cell size (Forward light Scatter, FSC) and cellular complexity (Side light Scatter, SSC) parameters were used to distinguish different hemocyte subpopulations in dot-plots. Instrument settings were as follows: -1

FSC set in log scale at E , SSC set in log scale at 287, the primary parameter for threshold was FSC set at 217. Ten thousand events were recorded for each hemolymph sample. No differences were observed in dot-plots between fresh or 2 % paraformaldehyde-fixed hemolymph samples (data not shown). 2.4 Morphological analysis of hemocyte subpopulations. Cell sorting of paraformaldehyde-fixed cells was performed using sea water as sheath fluid. Cells were recovered from the concentrator module, centrifuged at 300 x g in Eppendorf tubes for 10 minutes, resuspended in 3.5 μl 10 % bovine serum albumin in PBS, and placed on glass slides. Cells were then either placed under glass cover-slips, and observed directly with a Nikon Optiphot phase-contrast microscope (Nikon Instruments; Melville, NY), or air-dried and stained with hemacolor (catalogue numbers: Red 1.11956.2500, Blue 1.11957.2500; Merk KGaA, Darmstadt, Germany) for observation in a Nikon Optiphot light transmission microscope (Nikon Instruments; Melville, NY). 2.5 Phagocytosis assays

8 Five hundred μl of hemolymph were placed into individual wells of tissueculture 24-well plates. Hemocyte number in each sample was estimated by counting cells in a hemocytometer. Hemocytes were allowed to adhere to the well bottom by incubating them for 30 minutes at 15 ºC in the dark. Hemolymph supernatant was then removed by blotting onto paper towels. Five hundred μl of filtered sea water were then added to each well. Flourescein-labelled 1μm latex beads, flourescein-labelled zymosan bioparticles, flourescein-labelled E. coli bacteria, or fluorescein-labelled Vibrio alginolyticus bacteria were added at a 10:1 target:hemocyte ratio. After a two hour incubation at 15 ºC in the dark, uninternalised particles were removed by washing wells twice with 500 μl PBS. Cells were finally resuspended in 500 μl PBS by gently detaching them from the well bottom, using a rubber cell scraper. Fifty μl of 0.8 % trypan blue in PBS were added to each sample to quench external fluorescence. Ten thousand cells were then analysed by flow cytometry. Cell fluorescence was analysed through the FL-1 channel set at log scale. FL-1 was adjusted for each phagocytic target, so that positive cells (cells that internalised al least one particle) fell within the same fluorescence range, independently of the phagocytic target used. Phagocytosis was expressed as the percentage of cells that internalised at least one fluorescent particle (positive cells). In selected experiments PI 3-K, PKC, or ERK inhibitors, or only the DMSO solvent, were added to the cells and incubated at 15 ºC in the dark for 30 minutes before mixing them with the fluorescent particles. 2.5 Analysis of the respiratory burst and nitric oxide production Two hundred and fifty μl of hemolymph pools were placed into individual wells of tissue-culture U-bottom 96-well plates. After 30 minutes at 15 ºC in the dark, the hemolymph supernatant was removed, and 200 μl filtered sea water, containing 0.4 % DMSO and 5 μg/ml of chloromethyl-H2DCFDA, or DAF-FM/ DAF-FM diacetate were

9 added per well. In some experiments PI 3-K, PKC, or ERK inhibitors were added together with the ROS or NO indicators. After 30 minutes at 15 ºC in the dark, the supernatant was removed, and wells washed twice with filtered sea water. Zymosan (1 mg/ml), or PMA (1-1000 ng/ml) were then added in 200 μl sea water. Cells were incubated for 1 hour at 15 ºC in the dark, washed twice with PBS, and finally resuspended in 200 μl PBS. Cell fluorescence was analysed by flow cytometry through the FL-1 channel, set at log scale. The mean fluorescence intensity (MFI) values of the different hemocyte subpopulations in the resting state, was considered to be 100%. The change in MFI value, induced by zymosan or PMA stimulation, was then plotted as a percentage of the resting state value for each hemocyte subpopulation.. 2.6 Statistical analysis Data were analysed by paired-samples T-tests; or by one-way ANOVA with post-hoc multiple-comparisons tests (Dunnett), using the Statistical Package for the Social Sciences v8.0 software (SPSS Inc, Chicago, IL). A significance level of P < 0.05 was used. The maximum p values for the statistically different points in each data series are given in the figure legends.

3. Results 3.1 Characterization of hemocyte subpopulations. M. galloprovincialis hemolymph, withdrawn from the adductor muscle, presented a mixed population of adherent cells differing in size and morphology (Fig. 1A). Flow cytometry analysis was used to characterize the cell types present in mussel hemolymph. Based on measurements of cell size (Forward light Scatter, FSC) and complexity (Side light Scatter, SSC), four morphologically distinct hemocyte subpopulations could be separated. These hemocyte subpopulations were designated R1

10 to R4 (Fig. 1B). R1 are large granular cells with high SSC values, sizing between 15 and 20 μm in diameter (Fig. 1B and 1C), and possessing a relatively small nucleus located eccentrically (Fig. 1C). R2 are large semigranular cells with intermediate SSC values, sizing between 15 and 20 μm in diameter, and presenting a larger nucleus (Fig. 1B and 1D). R3 cells are smaller semigranular cells with intermediate SSC values (Fig. 1B and 1E), presenting a small nucleus and several membrane extensions (Fig. 1E). R4 cells are small agranular (hyaline) cells, with the lowest SSC values (Fig. 1B and 1F), and a prominent nucleus that covers most of the cytoplasm. Small hyaline cells were around 10 to 15 μm in diameter (Fig. 1F). 3.2 Large granular, Large semigranular, and Small semigranular hemocyte subpopulations are phagocytic. To assess the phagocytosis potential of the various mussel hemocyte subtypes, cells were mixed with several fluorescein-labelled phagocytic targets, allowed to ingest these targets, and then analysed by flow cytometry. Hemocytes in regions R1 (Large granular hemocytes), R2 (Large semigranular hemocytes), and R3 (Small semigranular hemocytes) were all capable of phagocytosis, although at different levels (Fig. 2). Zymosan (Fig. 2B), latex beads (Fig. 2C), and the bacteria V. alginolyticus (Fig. 2D) were ingested efficiently by all Large granular (R1), Large semigranular (R2), and Small semigranular (R3) hemocytes. In contrast, the bacteria E. coli, which appears to be non-pathogenic for molluscs [50, 51], was poorly phagocytosed (Fig. 2E). Small hyaline (R4) hemocytes were not phagocytic at all (Fig. 2). Large granular (R1) hemocytes were the most efficient phagocytic cells, followed by Large semigranular (R2) hemocytes, and then Small semigranular (R3) hemocytes (Fig. 3). Zymosan was ingested by about 40 % of the total of Large granular (R1) cells, and by about 25 % of the total of Large semigranular (R2) or Small semigranular (R3) cells (Fig. 3A). Latex

11 beads were also internalised by about 40 % of Large granular (r1) cells, and by about 30 % of Large semigranular (R2) cells, but only by about 10 % of Small semigranular (R3) cells (Fig. 4B). In addition, the bacteria V. alginolyticus, was efficiently ingested by about 60 % of the total of Large granular (R1) cells, by about 45 % of Large semigranular (R2) cells, and by almost 30 % of Small semigranular (R3) cells (Fig. 3C). In contrast, the bacteria E. coli, was poorly phagocytosed by all hemocyte subtypes (Fig. 3 D). These results indicated that Large granular (R1) and Large semigranular (R2) mussel hemocytes are efficient phagocytes, with different phagocytic abilities depending on the target they are presented. 3.3 Phagocytosis regulation by PI 3-K, PKC, or ERK. To investigate the participation of PI 3-K, PKC, and ERK enzymes in phagocytosis, mussel hemocytes were treated with specific inhibitors for these enzymes before allowing them to phagocytose fluorescein-labelled particles. Phagocytosis of zymosan was inhibited by wortmannin (the PI 3-K inhibitor) in the three phagocytic hemocyte subpopulations (Fig. 4 A). Phagocytosis by Large granular cells was the most sensitive to PI 3-K inhibition. Inhibition of PKC, and ERK (by staurosporine, and PD98059, respectively) did not affect zymosan phagocytosis in either Large granular or Small semigranular hemocytes (Fig. 4B and 4C). In contrast, zymosan phagocytosis by Large semigranular hemocytes was significantly reduced after treatment with staurosporine or PD98059 (Fig. 4B and 4C). None of the inhibitors affected cell viability at the highest concentrations used: 85.2 ± 5.5 % hemocytes were viable after DMSO treatment, 86 ± 2.6 % after wortmannin treatment, 87.6 ± 1.9 % after staurosporine treatment, and 83.6 ± 4.2 % after PD98059 treatment. These data showed that all types of hemocytes require PI 3-K activity for phagocytosis of zymosan, and that only Large semigranular hemocytes also needed PKC and ERK for zymosan

12 internalisation. Phagocytosis of latex beads was also inhibited by wortmannin in the three phagocytic hemocyte subpopulations (Fig. 5A). All cells seemed to be similarly sensitive to PI 3-K inhibition (Fig. 5A). In contrast, inhibition of PKC and ERK caused only a minute decrease in the level of phagocytosis by all phagocytic hemocytes (Fig. 5B and 5C). Because the phagocytic activity of mussel hemocytes seemed to vary depending on the type of bacteria being ingested (Fig. 4), the biochemical requirements for these forms of phagocytosis were evaluated using the inhibitors for PI 3-K, PKC, and ERK. Phagocytosis of the bacteria V. alginolyticus was inhibited, about 50 %, by wortmannin in the three phagocytic hemocyte subpopulations (Fig. 6A). In contrast, inhibition of PKC and ERK did not affect V. alginolyticus phagocytosis by any type of hemocytes (Fig. 6B and 6C). Interestingly, even though phagocytosis of the bacteria E. coli was very poor in all types of hemocytes (Fig. 4), it was also inhibited about 50 % by wortmannin in the three phagocytic hemocyte subpopulations (Fig. 7A). Similarly to V. alginolyticus, inhibition of PKC and ERK did not affect E. coli phagocytosis by any type of hemocytes (Fig. 7B and 7C). These data suggested that all types of hemocytes require PI 3-K activity for phagocytosis of bacteria, and that this type of phagocytosis is independent of PKC and ERK activities. Data also suggested that efficient phagocytosis of different bacterial species probably involves other mechanisms besides the use of PI 3-K. 3.4 Large granular, Large semigranular, and Small semigranular hemocyte subpopulations produce reactive oxygen species. Zymosan was used to determine which of the different hemocyte subpopulations presented was capable of producing reactive oxygen species (ROS). Large granular (R1), Large semigranular (R2), and Small semigranular (R3) hemocytes were all

13 capable of ROS production at comparable levels (Fig. 8). Small hyaline (R4) hemocytes showed a modest increase in ROS, but this increment was not statistically different from control (unstimulated) cells (Fig.8). 3.5 Regulation of reactive oxygen species production by PI 3-K or PKC. Similarly to phagocytosis, the participation of certain enzymes, such as PI 3-K, PKC, and ERK, in ROS production was investigated with the use of pharmacological inhibitors. Zymosan-induced ROS production was inhibited by wortmannin in the three phagocytic hemocyte subpopulations (Fig. 9A). Large semigranular cells were the most sensitive to PI 3-K inhibition. Inhibition of PKC, and ERK did not affect ROS production by any of the hemocyte subpopulations (Fig. 9B and 9C). Although, PKC inhibition by staurosporine did not affect zymosan-induced ROS production, it has been reported in other cell types that PKC is in fact a good activator of the respiratory burst [52, 53]. Treatment of mussel hemocytes with the phorbol ester PMA, a well known activator of PKC, indeed resulted in strong ROS production independently of any other stimulus. This increase in ROS production was three to four-fold in the phagocytic hemocyte subpopulations (Fig. 9D). In Small hyaline (R4) cells, PMA-induced activation of PKC caused only a small increment in ROS production (not shown). These data showed that Large granular (R1), Large semigranular (R2), and Small semigranular (R3) hemocyte subtypes require PI 3-K activity for zymosan-induced ROS production, and that neither PKC, nor ERK, participate in this cell response. However, PKC seems to participate in other pathways that lead to an even stronger activation of the respiratory burst. 3.6 All hemocyte subpopulations produce nitric oxide. All hemocyte subpopulations were capable of producing nitric oxide (NO) in response to zymosan (Fig. 10). Large granular (R1), and Large semigranular (R2)

14 hemocytes presented NO production that doubled the basal NO levels of unstimulated cells (Fig. 10A and 10B). Small semigranular (R3) and Small hyaline (R4) hemocytes showed a smaller increase in NO production, but this increment was statistically different from control (unstimulated) cells (Fig. 10C and 10D). 3.7 Regulation of NO production by PI 3-K, PKC, or ERK. Similarly to ROS production, the participation of PI 3-K, PKC, and ERK, in NO production was investigated with the use of pharmacological inhibitors. Zymosaninduced NO production was inhibited by wortmannin in the Large granular and Large semigranular hemocyte subpopulations (Fig. 11A). Large semigranular cells were also the most sensitive to PI 3-K inhibition. The small zymosan-induced increase in NO production by Small semigranular and Small hyaline hemocytes was not affected by wortmannin (Fig. 11A). Inhibition of PKC, did not affect NO production in any of the hemocyte subpopulations (Fig. 11B), suggesting that PKC is not used for zymosaninduced NO production. Similarly to phagocytosis (Fig. 4C) NO production was only affected by EKR inhibition in Large semigranular hemocytes (Fig. 11C). Interestingly, in absence of any other stimulus, PKC activation by PMA resulted in diminished levels of NO in phagocytic hemocytes (Fig. 11D). These data suggest that efficient NO production requires PI 3-K (in Large granular and Large semigranular cells), and possibly ERK (in Large semigranular cells) activities.

15 4. Discussion. The information regarding cell functions in invertebrate hemolymph has increased substantially over the last decade, but still hemocyte classification remains controversial. This is partially due to dissimilar classification criteria adopted for each species. Classifications have been based on morphology and cytochemical properties for the most part, and to a lesser extent on cell functions. Using flow cytometry techniques, we have identified four morphologically distinct hemocyte subpopulations in the mussel M. galloprovincialis: Large granular cells (R1), Large semigranular cells (R2), Small semigranular cells (R3), and Small Hyaline cells (R4). Through the use of flow cytometry we were able to show, for the first time, the capacity for phagocytosis, ROS production, and NO production, of clearly defined hemocyte subpopulations in a mollusc species. From the four M. galloprovincialis hemocyte subpopulations, we identified two professional phagocytes: Large granular cells (R1), and Large semigranular cells (R2). As in mammals [19, 23, 24, 34, 35], the two M. galloprovincialis professional phagocytes present high phagocytic capacity, and produce high levels of ROS and NO upon activation. M. galloprovincialis Small semigranular (R3) hemocytes appear to play a minor role in phagocytosis, and ROS and NO production, and it is possible that they participate in other immune functions, such as coagulation, encapsulation, or defence against viruses. M. galloprovincialis Small hyaline hemocytes (R4) present no phagocytic capacity, and is very likely that these cells are specialized for coagulation or encapsulation, as has been suggested for hyalinocytes of other invertebrates [10]. It is thus clear that M. galloprovinciallis hemocytes are specialized cells fulfilling specific tasks in the context of host defence. In agreement with our finding that M. galloprovincialis Large granular and Large semigranular hemocytes are highly phagocytic, others have reported in various

16 invertebrates that granulocytes are efficient phagocytes [9, 20, 21, 30, 32]. M. galloprovincialis Small hyaline M. galloprovincialis hemocytes were, on the other hand, non-phagocytic. Contradictory information exists regarding the phagocytic capacity of hyaline hemocytes. While some reports indicate a lack of phagocytic potential in these cells [9, 32], other reports indicate a low phagocytic potential in hyaline hemocytes [21, 30]. Considering that hyaline hemocytes are reported to posses, at the best, a low level of phagocytosis, we are inclined to believe that these cells perform defence mechanisms other than phagocytosis. Many marine molluscs posses great economical importance as a source of nourishment for humans; and also as possible sources of microbicidal molecules that could be used to treat human diseases [54]. Therefore, the interest in understanding the basic mechanisms that govern invertebrate immune systems has risen in the last few years. In mammalian leukocytes, enzymes of the PI 3-K, PKC, and ERK families appear to be central in the regulation of host defence mechanism including phagocytosis, the respiratory burst, and NO production [25, 44, 52, 53, 55-61]. The participation of PI 3-K, PKC, and ERK families of kinases in invertebrate defence mechanisms is just starting to be explored. The existence of enzymes of these families in mollusc species is mostly inferred from experiments in which cell responses are blocked by pharmacological inhibitors [33, 62-65]. In a few species, including M. galloprovincialis, the existence of enzymes of these families has been demonstrated by Western-blotting techniques, using antibodies directed against enzymes of mammalian origin [64, 66-68]. Thus it appears that PI 3-K, PCK, and ERK enzymes have been conserved through evolution. Here we show for the first time that, as in vertebrates [25, 44], the regulation of hemocyte phagocytosis by enzymes of the PI 3-K, PKC, and ERK families is subjected

17 to the nature of the cell subtype, and to the nature of the particle triggering the phagocytic process. In M. galloprovincialis PI 3-K appears to have a conserved role in phagocytosis regulation, independently of the hemocyte subtype, or the nature of the phagocytic target. In mammals, PI 3-K also participates in the regulation of phagocytosis mediated by various membrane receptors [25]. The role of PI 3-K in the regulation of phagocytosis thus appears to be evolutionarily conserved. The role of ERK and PKC in phagocytosis regulation, on the other hand, is less generalized, and is possibly restricted to specific targets (e.g. yeast, or bacteria), or to specific hemocyte subtypes (e.g. Large semigranular cells). It is possible that, as has been suggested in mammals [25], these differences in the utilisation of PKC, or ERK for phagocytosis are due to differential stimulation of phagocytic receptors. However, phagocytic receptors in most invertebrates, including molluscs, are yet to be characterised. PI 3-K played an important role in role in the regulation of ROS production by different M. galloprovincialis hemocyte subtypes. This enzyme has also been reported to regulate ROS production in vertebrates [57, 58], which suggests that regulation of the respiratory burst by PI 3-K is an evolutionarily-conserved mechanism. Zymosaninduced ROS production by M. galloprovincialis hemocytes was, on the other hand, independent of PKC, or ERK. In agreement with our data, other reports suggest that, in mussels, ERK is not required for bacterial killing mechanisms possibly related to ROS production [63, 67]. So, it seems that ERK is not used by any type of mussel hemocytes for ROS production. These observations also suggest the existence of alternative ERKand PKC-independent signalling pathways regulating the respiratory burst in M. galloprovincialis hemocytes. In contrast, it has been reported that in snails, ERK is required for the respiratory burst [65], and for regulation of ROS production [69]. One possible explanation for this is that differences in ERK and PKC utilisation for the

18 regulation of ROS production are species-specific. The analysis of ROS production among closely-related species will probably help to clarify this issue. Participation of PI 3-K in the regulation of NO production was restricted to M. galloprovincialis hemocytes presenting the highest levels of NO production: the professional phagocytes Large granular, and Large semigranular hemocytes. It is thus very likely that, in M. galloprovincialis professional phagocytes, PI 3-K is central in the regulation of pathogen killing, by coordinating efficient phagocytosis with efficient ROS and NO production. ERK, on the other hand, regulated NO production only in Large semigranular hemocytes. In snails ERK was also reported to be required for NO production [43], but the methodology used did not allow for the identification of the hemocyte subtypes producing NO. Our data support the idea that ERK utilisation for the regulation of NO production is hemocyte subtype-specific, since only one type of hemocytes showed ERK participation in this cell response. However, ERK utilization for NO production might still exist for other types of stimuli. This is to say, that ERK participation may be just the result of differential membrane receptor stimulation. We only tested NO production in response to zymosan. Other stimuli need to be tested in order to decide whether ERK is used for NO production by only one type of hemocyte as we show here, or whether ERK is needed depending on the receptor engaged on the hemocyte plasma membrane. Both possibilities seem at the moment equally possible. PKC played no role in the regulation of zymosan-induced NO production in any of the M. galloprovincialis hemocyte subtypes, suggesting the existence of PKC independent mechanisms for NO production. By measuring NO metabolites, rather than NO itself, others have suggested that PKC indeed participates in the regulation of NO production [38, 41-43]. Interestingly, we observed that direct activation of PKC with PMA actually reduces the basal levels of NO in the ROS-producing Large granular,

19 Large semigranular, and Small semigranular hemocytes. This effect of PMA was more marked in the Large granular and Large semigranular hemocytes. the cell types that also presented the strongest respiratory burst. The formation of peroxynitrite, from respiratory burst-derived superoxide anions and NO, has been recorded in M. galloprovincialis hemocytes after PMA stimulation [38]. It is thus possible that the PMA-induced reduction in NO levels is due to peroxynitrite formation; which would consume the available NO required for the oxidation of the NO-specific indicator. If this is the case, hemocyte-pathogen interactions that resulted in PKC activation would be predicted to promote the production of large amounts peroxynitrite, and rapid pathogen killing. Lack of a consensus criterion for mollusc hemocyte classification makes it difficult to integrate the information on the basic mechanisms of invertebrate host defence, obtained by different research groups. See for example: [6, 14, 70, 71]. Unifying criteria for hemocyte classification is thus urgent, and should involve a standardised methodology for the separation of hemocyte subtypes, as well as for their morphological and functional characterization. Due to its fast and powerful analytic capacity, allowing for accurate cell separation, we propose that flow cytometry could be used as standard methodology for the classification of invertebrate hemocytes. Given the availability of a wide variety of fluorescent phagocytic particles, and a wide array of cellular probes tailored for the measurement of various cellular functions, flow cytometry easily allows for the functional characterization of hemocyte subtypes. The strong potential of flow cytometry for hemocyte classification is therefore illustrated by our results, as we were able to specifically record phagocytosis, ROS production, and NO production, in clearly defined hemocyte subpopulations.

20 It is important to bear in mind that the gating and separation of complex cell mixtures requires careful selection of FSC and SSC amplification, and threshold settings [72]. Failure to accurately adjust these critical flow cytometry parameters may preclude the visualization of discernible cell subpopulations in dot-plots. For example, a previous attempt to study M. galloprovincialis hemocytes by flow cytometry reported only one hemocyte population visualized in dot-plots [73]. This is clearly at odds with the large differences in hemocyte size and shape observed in mussel hemolymph preparations under the microscope. Contradictory results, when using flow cytometry, have also been reported in the American oyster Crassostrea virginica [71, 74]. The selection of FSC and SSC amplification, and threshold settings is thus critical for the identification of cell subpopulations. Fortunately, most flow cytometers are now equipped with software containing tools that aid in the identification and gating of separate cell populations. It is therefore very likely that flow cytometry can easily be applied to the morphological and functional characterization of hemocytes of many invertebrate species. In conclusion, the immune system of M. galloprovinciallis is composed of at least four hemocyte subtypes, specialized to fulfil specific tasks in the context of host defence. Two out of these four hemocyte subtypes, Large granular and Large semigranular cells, are professional phagocytes, that share features with mammalian professional phagocytes. PI 3-K appears to have an evolutionarily-conserved role in the regulation of phagocytosis, and in ROS and NO production. Differential utilisation of PKC, or ERK for the regulation of phagocytosis of diverse targets, and for the regulation of ROS and NO production, reveals hemocyte subtype-specific variations in signalling mechanisms, which could be due to the differential expression of membrane

21 receptors. The search for these invertebrate “immune receptors” shall be an exciting quest in the near future.

22 5. Figure Legends. Figure 1. Four hemocyte subpopulations are present in mussel hemolymph. (A) Mussel hemolymph was observed directly by phase-contrast microscopy at 40 X magnification. (B) Dot-plots of hemolymph from four individual mussels, analysed by size (FSC) vs. cellular complexity (SSC), reveal four distinct cellular subpopulations, desiganted R1 through R4. Particles detected in the lower left corner of the dot-plots were cell debris and unidentified material (Deb). C-F) Mussel hemocyte subpopulations R1 to R4 were physically separated by flow cytometry. (C) R1 cells, (D) R2 cells, (E) R3 cells, (F) R4 cells. Cells in each region were observed directly by phase-contrast microscopy (upper panels), or stained with Hemacolor and observed by light transmission microscopy (lower panels). Figure 2. Mussel Large granular (R1), Large semigranular (R2), and Small semigranular (R3) hemocyte subpopulations present phagocytic capacity. Mussel hemocytes were incubated at 15 ºC for two hours with nothing (A), or with different fluorescein-labelled phagocytic targets: (B) zymosan, (C) latex beads, (D) V. alginolyticus, (E) E. coli. Left panels show fluorescence histograms of hemolymph samples after phagocytosis. Right panels show dot-plots of cells within the fluorescence gate (R5) in the corresponding left-hand histogram. Figure 3. Mussel hemocyte subpopulations have different phagocytic efficiency. Mussel hemocytes were incubated at 15 ºC for two hours with different fluorescein-labelled phagocytic targets: (A) zymosan, (B) latex beads, (C) V. alginolyticus, (D) E. coli. Phagocytosis is expressed as % of positive cells (cells internalising at least one fluorescent particle) from the total number of cells within each hemocyte region (R1, R2, or R3). Data are mean + S.D. of over 10 determinations.

23 Figure 4. Effect of PI 3-K, PCK, or ERK inhibitors on phagocytosis of zymosan. Mussel hemocytes were incubated at 15 ºC for 30 minutes with various concentrations of wortmannin (A), staurosporine (B), or PD98059 (C) before allowing them to phagocytose fluorescein-labelled zymosan. Phagocytosis for the different hemocyte subpopulations, Large granular (R1), Large semigranular (R2), and Small semigranular (R3) is expressed as a percentage of control samples (inhibitor concentration = 0), considered 100 %. Open symbols represent data points with no statistical difference from controls. Filled symbols represent data points that are statistically different from controls. Data are mean + s.e.m of 12-16 determinations. Maximum p values, for statistically different data points are: A) p < 0.04, n=12. B) p < 0.03, n=16. C) p < 0.012, n=16. Figure 5. Effect of PI 3-K, PCK, or ERK inhibitors on phagocytosis of latex beads. Mussel hemocytes were incubated at 15 ºC for 30 minutes with various concentrations of wortmannin (A), staurosporine (B), or PD98059 (C) before allowing them to phagocytose fluorescein-labelled latex beads. Phagocytosis for the different hemocyte subpopulations Large granular (R1), Large semigranular (R2), and Small semigranular (R3) is expressed as a percentage of control samples (inhibitor concentration = 0), considered 100 %. Open symbols represent data points with no statistical difference from controls. Filled symbols represent data points that are statistically different from controls. Data are mean + s.e.m of 12-20 determinations. Maximum p values, for statistically different data points are: A) p < 0.04, n=16. B) p < 0.03, n=20. C) p < 0.03, n=12. Figure 6. Effect of PI 3-K, PCK, or ERK inhibitors on phagocytosis of the bacteria Vibrio alginolyticus. Mussel hemocytes were incubated at 15 ºC for 30 minutes with various concentrations of wortmannin (A), staurosporine (B), or PD98059

24 (C) before allowing them to phagocytose fluorescein-labelled V. alginolyticus. Phagocytosis for the different hemocyte subpopulations Large granular (R1), Large semigranular (R2), and Small semigranular (R3) is expressed as a percentage of control samples (inhibitor concentration = 0), considered 100 %. Open symbols represent data points with no statistical difference from controls. Filled symbols represent data points that are statistically different from controls. Data are mean + s.e.m of 10 determinations. Maximum p values, for statistically different data points are: A) p < 0.016, n=10. Figure 7. Effect of PI 3-K, PCK, or ERK inhibitors on phagocytosis of the bacteria E. coli. Mussel hemocytes were incubated at 15 ºC for 30 minutes with various concentrations of wortmannin (A), staurosporine (B), or PD98059 (C) before allowing them to phagocytose fluorescein-labelled E. coli. Phagocytosis for the different hemocyte subpopulations Large granular (R1), Large semigranular (R2), and Small semigranular (R3) is expressed as a percentage of control samples (inhibitor concentration = 0), considered 100 %. Open symbols represent data points with no statistical difference from controls. Filled symbols represent data points that are statistically different from controls. Data are mean + s.e.m of 12-20 determinations. Maximum p values, for statistically different data points are: A) p < 0.02, n=12. Figure 8. Mussel hemocytes produce reactive oxide species (ROS) in response to zymosan. Mussel hemocytes were incubated at 15 °C for 30 minutes with a reactive oxygen species (ROS)-sensitive intracellular indicator, and then stimulated with 1 mg/ml zymosan. After one hour, ROS production was quantified by flow cytometry in each hemocyte subpopulation: Large granular (R1), Large semigranular (R2), Small semigranular (R3), and Small hyaline (R4). Zymosan-stimulated ROS production was plotted as a percentage of the resting level, considered 100 %. Data are

25 mean + S.D. of 28-60 determinations. Asterisks (*) indicate that differences from controls were statistically significant, p ≤ 0.0001. Figure 9. Effect of PI 3-K, PCK, or ERK inhibitors on the production of reactive oxygen (ROS) species in response to zymosan. Mussel hemocytes were incubated at 15 °C for 30 minutes with a ROS indicator and various concentrations of wortmannin (A), staurosporine (B), or PD98059 (C), and then stimulated with 1 mg/ml zymosan (A-C). After one hour, ROS production was quantified by flow cytometry in each hemocyte subpopulation: Large granular (R1), Large semigranular (R2), and Small semigranular (R3). ROS production was plotted as a percentage of the control samples (inhibitor concentration = 0), considered 100%. In (D) cells were stimulated with various concentrations of PMA in the absence of zymosan, and ROS production was plotted as a percentage of the resting level (PMA concentration = 0), considered 100 %. Open symbols represent data points with no statistical difference from controls. Filled symbols represent data points that are statistically different from controls. Data are mean + s.e.m of 8-12 determinations. Maximum p values, for statistically different data points are: A) p < 0.03, n=12. D) p < 0.038, n=8 Figure 10. Mussel hemocytes present nitric oxide production in response to zymosan. Mussel hemocytes were incubated at 15 °C for 30 minutes with a nitric oxide (NO)-sensitive intracellular indicator, and then stimulated with 1 mg/ml zymosan. After one hour, NO production was quantified by flow cytometry in each hemocyte subpopulation: Large granular (R1), Large semigranular (R2), Small semigranular (R3), and Small hyaline (R4). Zymosan-stimulated NO production was plotted as a percentage of the resting level, considered 100 %. Data are mean + S.D. of 36 determinations. Asterisks (*) indicate that differences from control were statistically significant, p ≤ 0.04.

26 Figure 11. Effect of PI 3-K, PCK, or ERK inhibitors on nitric oxide (NO) production by mussel hemocytes. Mussel hemocytes were incubated at 15 °C for 30 minutes with a NO indicator and various concentrations of wortmannin (A), staurosporine (B), or PD98059 (C), and then stimulated with 1 mg/ml zymosan (A-C). After one hour, NO production was quantified by flow cytometry in each hemocyte subpopulation: Large granular (R1), Large semigranular (R2), Small semigranular (R3), and Small hyaline (R4). NO production was plotted as a percentage of the control samples (inhibitor concentration = 0), considered 100%. In (D) cells were stimulated with various concentrations of PMA in the absence of zymosan, and NO production was plotted as a percentage of the resting level (PMA concentration = 0), considered 100 %. Open symbols represent data points with no statistical difference from controls. Filled symbols represent data points that are statistically different from controls. Data are mean + s.e.m. of 12 determinations. Maximum p values, for statistically different data points are: A) p < 0.001, n=12. C) p < 0.03, n=12. D) p < 0.009, n=8.

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110

R2 R3

100 90 80 70 60 50 40 30

0

0.5

1

1.5

2

2.5 3.5

5

10

20

30

40

Staurosporine [nM]

* R1

C


120

R2 R3

110 100 90 80 70 60 50 40 30

0

Figure 5

1

5

7.5

10

PD98059 [uM]

15

20

50

A


110

* R1

100

R2 R3

90 80 70 60 50 40 30

0

1

5

10

20

50

100

200

Wortmannin [nM]

B


110

* R1

100

R2 R3

90 80 70 60 50 40 30

0

1

2

2.5

5

10

20

40

Staurosporine [nM]


C

* R1

110

R2 R3

100 90 80 70 60 50 40 30

0

Figure 6

1

5

7,5

10

PD98059 [uM]

15

20

50

A


100

* R1

90

R2 R3

80 70 60 50 40 30

0

1

5

10

20

50

100

200

Wortmannin [nM]

B


120

* R1

110

R2 R3

100 90 80 70 60 50 40 30

0

0.5

1

1.5

2

2.5 3.5

5

10

20

30

40

Staurosporine [nM]

* R1

C


110

R2 R3

100 90 80 70 60 50 40 30

0

Figure 7

1

5

7.5

10

PD98059 [uM]

15

20

50

0

50

100

150

200

250

300

0

50

100

150

200

250

R3

Control

Control

R1

Zymosan

Zymosan

*

ROS Production (% of control)

Figure 8

C

A

D

B ROS Production (% of control) 0

50

100

150

200

250

300

0

50

100

150

200

250

300


300

*


R4

R2

Control

Control

Zymosan

Zymosan

*

10

20

50

30

40

50

60

70

80

90

110

0

1

5

10

PD98059 [uM]

7,5

15

100

50

200

R2 R3 R4

20

100

* R1 D

Wortmannin [nM]

5

B

120

30

40

50

60

70

80

90

100

1

R2 R3 R4

110

0

* R1

120

130

Figure 9

C


ROS fluorescence (% of control)

A ROS Production (% of control) ROS fluorescence (% of control) 100

150

200

250

300

350

400

450

30

40

50

60

70

80

90

100

110

120

0

0

1

2.5

5

10

1

PMA [ng/ml]

10

100

Staurosporine [nM]

2

40

1000

20

R2 R3 R4

* R1

R2 R3 R4

* R1

Figure 10

C

A

R3

R1

D

B

R4

R2

30

40

50

60

70

80

90

100

110

30

40

50

60

70

80

90

100

Figure 11

C

A

NO Production (% of control)

NO Production (% of control)

110

0

0

1

1

10

20

50

5

10

PD98059 [uM]

7.5

15

Wortmannin [nM]

5

20

100

50

200

R2 R3 R4

* R1 D

R2 R3 R4

* R1 B NO Production (% of control) NO Production (% of control) 30

40

50

60

70

80

90

100

30

40

50

60

70

80

90

100

110

120

0

0

1

2.5

5

10

1

PMA [ng/ml]

10

100

Staurosporine [nM]

2

20

1000

40

R2 R3 R4

* R1

R2 R3 R4

* R1