Nitric oxide production by Biomphalaria glabrata haemocytes: effects of Schistosoma mansoni ESPs and regulation through the extracellular signal-regulated kinase pathway

Abstract

Background: Schistosoma mansoni uses Biomphalaria glabrata as an intermediate host during its complex life cycle. In the snail, the parasite initially transforms from a miracidium into a mother sporocyst and during this process excretory-secretory products (ESPs) are released. Nitric oxide (NO) and its reactive intermediates play an important role in host defence responses against pathogens. This study therefore aimed to determine the effects of S. mansoni ESPs on NO production in defence cells (haemocytes) from schistosome-susceptible and schistosome-resistant B. glabrata strains. As S. mansoni ESPs have previously been shown to inhibit extracellular signal-regulated kinase (ERK) phosphorylation (activation) in haemocytes from susceptible, but not resistant, B. glabrata the regulation of NO output by ERK in these cells was also investigated.

Results: Haemocytes from resistant snails challenged with S. mansoni ESPs (20 mug/ml) over 5 h displayed an increase in NO production that was 3.3 times greater than that observed for unchallenged haemocytes; lower concentrations of ESPs (0.1-10 mug/ml) did not significantly increase NO output. In contrast, haemocytes from susceptible snails showed no significant change in NO output following challenge with ESPs at any concentration used (0.1-20 mug/ml). Western blotting revealed that U0126 (1 muM or 10 muM) blocked the phosphorylation (activation) status of ERK in haemocytes from both snail strains. Inhibition of ERK signalling by U0126 attenuated considerably intracellular NO production in haemocytes from both susceptible and resistant B. glabrata strains, identifying ERK as a key regulator of NO output in these cells.

Conclusion: S. mansoni ESPs differentially influence intracellular NO levels in susceptible and resistant B. glabrata haemocytes, possibly through modulation of the ERK signalling pathway. Such effects might facilitate survival of S. mansoni in its intermediate host.

Figure 1

Relative NO produced in haemocytes from schistosome-susceptible (S) and schistosome-resistant (R) B. glabrata strains in the presence and absence of 20 μg/ml ESPs over time. Equal volumes (100 μl per well) of diluted haemolymph (A), or an equal number of haemocytes (3 × 10³ cells per well) (B) from each snail strain were used in NO assays. Mean relative fluorescence from a single pool of haemolymph (± SEM, n = 4) is shown for each time point as a proportion of background fluorescence (no DAF-FM diacetate, indicated by the dotted line). Data shown are from one assay and are representative of three different assays conducted on separate days. *P ≤ 0.05 and **P ≤ 0.01 for differences in mean fluorescence values between treatments at 2 h.

Figure 2

Relative NO produced in haemocytes from schistosome-susceptible (A) and schistosome-resistant (B) B. glabrata strains challenged with different concentrations of ESPs (0, 0.1, 1, 10 and 20 μg/ml) over time. Equal numbers of haemocytes (3 × 10³ cells per well) from each snail strain were used in each experiment. Mean relative fluorescence from a single pool of haemolymph (± SEM, n = 3) is shown for each time point as a proportion of background fluorescence (no DAF-FM diacetate, indicated by the dotted line). Data shown are from one assay and are representative of three different assays conducted on separate days. *P ≤ 0.05 for differences in mean fluorescence values at 2 h between unexposed haemocytes and haemocytes exposed to ESPs.

Figure 3

Western blot showing phosphorylated ERK (p-ERK) levels in haemocytes from schistosome-susceptible and schistosome-resistant B. glabrata strains. Haemocytes were exposed to the MEK1/2 inhibitor, U0126 (1 μM or 10 μM) for 20 min before cell lysis. Unexposed haemocytes (C) and a mammalian (M) cell line (HC60) were used as controls. Results are representative of two independent experiments.

Figure 4

Relative NO levels in haemocytes from (A) schistosome-susceptible and (B) schistosome-resistant B. glabrata following exposure to ESPs (20 μg/ml), U0126 (1 μM or 10 μÌ) or both. Equal numbers of haemocytes (3 × 10³ cells per well) from susceptible or resistant snails were exposed to U0126 (1 μM or 10 μM), ESPs (20 μg/ml), or both. Mean relative fluorescence from a single pool of haemolymph (± SEM, n = 3) is shown for each time point as a proportion of background fluorescence (no DAF-FM diacetate indicated by a dotted line). Data shown are from one assay and are representative of three different assays conducted on separate days. *P ≤ 0.05 for differences in mean fluorescence values between treatments at 2 h.