The role of cows’ milk RNases in host defence

Abstract

The ribonucleaseA (RNaseA) superfamily is large, and comprises distinct and divergent proteins among various mammalian species. Although some of the family members possibly participate in host defence in humans and mice, little is known about their host defence-associated functions in cattle. The present study therefore set out to examine the role of two members of the RNaseA superfamily in host defence and their presence in various host defence-related tissues as well as secretions in cattle. The abundance of messenger RNA (mRNA) transcript of all other members of the RNaseA superfamily was also assessed in a range of host defence-associated tissues to determine whether these RNases have any role in host defence. RNase4 and RNase5 are present in cows’ milk and possess the ability to degrade and to bind to cow nucleic acids. Based on this ability, it was hypothesised that these proteins may bind to microbial nucleic acids to facilitate its recognition by various nucleic acid sensing receptors. To evaluate this, the responses of a mouse macrophage cell line (RAW264.7 cells) from two different sources were examined against a range of nucleic acid ligands, including polyinosinic–polycytidylic acid (polyI:C), R-848, unmethylated 2′-deoxyribo cytidine-phosphate-guanosine oligodeoxynucleotide (CpG-ODN), Salmonella typhimurium DNA or S. typhimurium RNA in the absence or presence of cows’ milk RNase4 or RNase5. A substantial difference was observed in the responsiveness of these two cell lines when stimulated with the same nucleic acid ligand. This study shows that the milk RNase-mediated recognition of nucleic acid ligands is a complex process and the RNases influence the recognition of natural nucleic acid ligands in a different manner compared with their synthetic mimetic. RNase5 showed a bimodal effect on polyI:C-induced responses that appeared to be concentration-dependent. The milk RNases showed an immunostimulatory effect on pro-inflammatory responses induced by R-848, which is a synthetic ligand for toll-like receptor (TLR7). On the other hand, the milk RNases suppressed the pro-inflammatory responses induced by the natural ligand of TLR7, S. typhimurium RNA. RNase4 suppressed the pro-inflammatory responses induced by CpG-ODN, a synthetic ligand for TLR9, while RNase5 showed an immunostimulatory effect. Both RNase4 and RNase5 were also enhanced the pro-inflammatory responses induced by S. typhimurium DNA, a natural ligand for TLR9. Experiments with bovine pancreatic RNaseA suggest that this immunomodulatory effect of the milk RNases is not shared by at least one member of the RNaseA superfamily. Further investigations using an endosomal acidification inhibitor and human embryonic kidney293 cell system suggested that the bacterial DNA in the absence or presence of the milk RNases is predominantly recognised by TLR9. These experiments also suggested the involvement of DNA sensing receptors other than TLR9. The ability of milk RNases to facilitate bacterial DNA recognition was also evaluated in primary bovine mammary epithelial cells. Milk RNase4 and RNase5 marginally enhanced the mRNA transcript levels of several immune effector genes that have been suggested to play a vital role in protecting the mammary gland from infection. Collectively the data indicate that cows’ milk RNase4 and RNase5 are capable of facilitating the recognition of microbial nucleic acid in both macrophages and epithelial cells, both of which form the first line of defence at the mucosal surface. Moreover, their presence at various mucosal surfaces and secretions further supports the idea that these proteins may participate in the mucosal host defence.