Bovine milk RNases modulate pro-inflammatory responses induced by nucleic acids in cultured immune and epithelial cells

https://doi.org/10.1016/j.dci.2016.11.015Get rights and content

Highlights

  • Milk RNase4 and 5 stimulate pro-inflammatory responses against nucleic acid mimetics.

  • Both RNases affect TLR3, TLR7 and TLR9 signalling in a mouse macrophage cell line.

  • These effects were also observed with natural DNA in bovine mammary epithelial cells.

  • The RNases showed distinct effects on the responses induced by natural DNA in bovine PBMCs.

  • The results suggest the RNases assist pathogen recognition in the mammary gland.

Abstract

Activation of innate immune receptors by exogenous substances is crucial for the detection of microbial pathogens and a subsequent inflammatory response. The inflammatory response to microbial lipopolysaccharide via Toll-like receptor 4 (TLR4) is facilitated by soluble accessory proteins, but the role of such proteins in the activation of other pathogen recognition receptors for microbial nucleic acid is not well understood. Here we demonstrate that RNase4 and RNase5 purified from bovine milk bind to Salmonella typhimurium DNA and stimulate pro-inflammatory responses induced by nucleic acid mimetics and S. typhimurium DNA in an established mouse macrophage cell culture model, RAW264.7, as well as in primary bovine mammary epithelial cells. RNase4 and 5 also modulated pro-inflammatory signalling in response to nucleic acids in bovine peripheral blood mononuclear cells, although producing a distinct response. These results support a role for RNase4 and RNase5 in mediating inflammatory signals in both immune and epithelial cells, involving mechanisms that are cell-type specific.

Introduction

The cells of the innate immune system can detect a variety of exogenous substances, including molecules of microbial origin. These so-called pathogen-associated molecular patterns (PAMPs) activate specific pattern recognition receptors present in immune cells and on mucosal epithelia. The recognition of some PAMPs is known to be stimulated by extracellular factors which bind to them and facilitate their presentation to the receptors. For example, lipopolysaccharide (LPS)-binding protein (LBP) and cluster of differentiation14 (CD14) facilitate the recognition of LPS by TLR4 (Ahrén et al., 2001, Jiang et al., 2005b). The recognition of exogenous nucleic acid is thought to occur mainly through endosomally located TLR3, TLR7 and TLR9 (Kawai and Akira, 2010). The high mobility group box proteins, the human cathelicidin peptide, and human β-defensin 2 and 3 have been shown to bind nucleic acids and facilitate their recognition by TLR7 and TLR9 (Ganguly et al., 2009, Lande et al., 2007, Tewary et al., 2013, Yanai et al., 2009). The means by which exogenous nucleic acids present in the extracellular space activate the TLRs in endosomes is complex and not fully understood, leaving open the possibility of involvement of additional accessory proteins.

Milk contains many proteins that appear to be involved in pathogen recognition (Hettinga et al., 2011, Smolenski et al., 2007, Wheeler et al., 2012b). The most studied of these are the secretory immunoglobulins, which interact with exogenous molecules and activate host defence responses (Hosea Blewett et al., 2008). Other milk proteins facilitate interaction between PAMPs and receptors. For example, the above-described accessory proteins, LBP and CD14, are present in milk (Bannerman et al., 2003). Milk also contains multiple complement factors, which bind to bacteria and facilitate activation of neutrophils (Shuster et al., 1997, Tomlinson, 1993). At least two members of the Ribonuclease A (RNaseA) family, RNase4 and RNase5/angiogenin, are also present in cows’ milk (Di Liddo et al., 2010, Harris et al., 2010, Murata et al., 2013). The RNase family comprises at least 13 members that have relatively high sequence divergence among species and are considered to be rapidly evolving (Cho et al., 2005, Wheeler et al., 2012a). These are secreted from a range of tissues and are present in mucosal secretions (Gupta et al., 2016, Wheeler et al., 2012a). Some of the RNases have been suggested to function in host defence (Boix and Nogues, 2007, Gupta et al., 2013, Koczera et al., 2016, Rosenberg, 2008), and some have been shown to stimulate cytokine production (Shcheglovitova et al., 2003, Yang et al., 2008). Bovine RNase4 and RNase5 share only 45% amino acid sequence identity with each other, and the number of RNase5-like genes and their sequence varies markedly among species (Cho et al., 2005, Wheeler et al., 2012a). On the other hand, only one relatively well-conserved RNase4 gene has been identified in mammalian genomes.

RNase4 and 5 appear to have distinct biological activities. RNase5 has a distinct substrate preference for its ribonucleolytic activity and this activity is much weaker than that of RNase4 and RNaseA (Gupta, 2013, Russo et al., 1996). Digestion of RNA by RNase5 results in oligonucleotides (Shapiro et al., 1986), whereas digestion by RNase4 or RNaseA results in single nucleotides (Harris et al., 2010). RNase5 has been reported to have antimicrobial activity (Hooper et al., 2003), and is significantly more potent than RNase4 in suppressing the growth of the fungal pathogen, Candida albicans (Harris et al., 2010). Both RNase4 and RNase5 also have been reported to bind DNA (Harris et al., 2010, Rybak and Vallee, 1988, Xu et al., 2003), a property that is not a known universal characteristic of the RNase family. The presence of RNase5 has been shown to stimulate cytokine production in leukocytes (Shcheglovitova et al., 2003).

The physiological role of RNase4 and RNase5 in milk has not been very well characterised. We hypothesised that one of the functions of RNase4 and RNase5 in milk is to bind to microbial nucleic acid and facilitate its recognition by pattern recognition receptors on immune cells, which are present in milk in high numbers during mastitis, or by mammary epithelial cells, and thereby to contribute to the host's response to infection of the mammary gland. As a first step in addressing this we investigated the ability of RNase4 and RNase5 purified from cows' milk to facilitate the recognition of synthetic and natural microbial nucleic acid ligands using three in vitro cell culture models; the mouse monocyte/macrophage derived cell line, RAW264.7, peripheral blood mononuclear cells (PBMCs) isolated from bovine blood, and primary cultures of bovine mammary epithelial cells (pbMEC). We found that RNase4 and RNase5 stimulated responses to synthetic as well as microbial DNA in RAW264.7 cells and pbMEC, but suppressed the nucleic acid responses in bovine PBMCs. These findings suggest that RNase4 and 5 could facilitate the local host response to mammary infection in vivo.

Section snippets

Reagents

All reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. The TLR7/8 agonist R-848 (imidazoquinoline), an adenine analogue with anti-viral activity, was purchased from Invivogen (Cat No. tlr-r848, San Diego, CA). Mouse-specific unmethylated CpG-ODN 1826 (TCCATGACGTTCCTGACGTT) and bovine-specific unmethylated CpG-ODN 2007 (TCGTCGTTGTCGTTTTGTCGTT) were obtained from Invitrogen (Auckland, New Zealand) as high performance liquid chromatography purified preparations.

Interaction of RNase4 and RNase5 with synthetic nucleic acid mimetics and natural DNA

One key requirement for an immune recognition accessory protein is that it interacts with the exogenous substance that is being recognised. To address whether this occurs for RNase4 and RNase5, the interaction between bovine milk-derived RNase4 and RNase5 with synthetic nucleic acid mimetics as well as native DNA purified from biological sources was evaluated using an agarose gel shift assay. The synthetic double-stranded viral RNA analogue, polyinosinic–polycytidylic acid (polyI:C), which

Discussion

The results presented here establish that RNase4 and RNase5 derived from cows’ milk are capable of modulating innate immune responses elicited by synthetic nucleic acid mimetics as well as DNA of pathogenic origin in multiple cell culture models of the inflammatory response. A physiological level of RNase5 produced as much as a 7.5-fold increase or up to a 5-fold decrease in the response to an activating ligand, depending on the model used. This level of modulation in the immune response would

Conclusions

This study establishes that bovine milk-derived RNase4 and RNase5 modulate pro-inflammatory responses induced by at least one species of microbial nucleic acid in multiple cell culture models. These findings contribute to a more sophisticated understanding of the physiological role of the RNases and help to advance understanding of how these RNases contribute to host defence. The findings reported here also provide the impetus to establish the significance of the immunomodulatory activity of

Competing interests

All authors declare they have no competing interests.

Acknowledgements

This work was supported by a New Zealand Ministry of Science and Innovation research contract (C10X0707) ‘Host Defence Proteins in Milk’ awarded to TTW, and Post Graduate Research Scholarship from the University of Otago and AgResearch awarded to SKG.

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