β-glucan supplemented diets induce high and broad expression levels of TLR3 what explains protection conferred by these additives against viral infections in fish

Abstract

We have previously observed that in common carp (Cyprinus carpio), administration of β-glucan (MacroGard™) as feed additive leads to a lower expression of pro-inflammatory cytokines suggesting that this immunostimulant may be preventing an acute and potentially dangerous response to infection. However, in general, mechanisms to detect and eliminate pathogens must also be induced in order to achieve an efficient clearance of the infection. Protection against viral diseases acquired through β-glucan-supplemented feed has been extensively reported for several experimental models in fish but the underlining mechanisms are still unknown. Thus, in order to better characterize the antiviral action induced by β-glucans in fish, MacroGardTM was administered daily to common carp in the form of supplemented commercial food pellets. Carp were fed for a period of 25 days prior to intra-peritoneal injection with polyinosinic:polycytidylic acid (poly I:C), a well-known double-stranded RNA mimic that triggers a type-I interferon (IFN) response and a set of immune related genes, including Mx, were analysed by real-time PCR in liver, spleen, head-kidney and mid-gut. Results obtained confirmed that treatment with β-glucan alone generally down-regulated the mRNA expression of pro-inflammatory cytokines when compared to untreated fish, while Mx gene expression remained stable. A similar expression pattern was observed for cytokines in samples obtained from β-glucan fed fish 24 h after injection with poly I:C. However, poly I:C injection markedly increased Mx gene expression but mainly in the group fed with β-glucan. Toll-like receptor 3 (TLR3) is the candidate pattern recognition receptor possibly responsible also in fish for the binding of viral double-stranded RNA and triggering of a type-I IFN response. Through a carp genome data mining, two sequences for carp TLR3 were retrieved (ccTLR3.1 and ccTLR3.2) and characterized. Constitutive gene expression of both genes was detected by real-time PCR in cDNA of all analysed carp organs. Strikingly, 25 days after β-glucan supplementation, very high levels of ccTLR3.2 gene expression were observed in all analysed organs, with the exception of liver. This suggests that β-glucan-mediated protection against viral diseases could be the result of a general induction of ccTLR3.2 gene expression in several organs in carp.