Activation of innate immunity by rotavirus NSP4

Abstract

Introduction: Rotavirus infection is the leading cause of life-threatening disease among children in many countries. Rotavirus nonstructural protein 4 (NSP4), a trans-ER glycoprotein, mediates the maturation of intermediate vial particles to infectious virions within the infected cells. NSP4 is also the first described viral enterotoxin which mimics some of the pathogenic effects of rotavirus. It is observed that NSP4, purified from the medium of rotavirus-infected cells, binds to a wide range of cells of distinct lineage, including immune cells. Furthermore, the adjuvant-like property of NSP4 reported by others suggests a specific effect of NSP4 on immune cells. Aims: This research aims to explore the potential interactions of NSP4 with host’s innate immune system, and to identify the receptors of NSP4 on innate immune cells. Methods: PMA-treated THP-1, RAW264.7, human monocyte-derived macrophages and dendritic cells (DCs) were used as cell models for NSP4 stimulation. Cytokines produced by activated innate immune cells were measured in multiplexed bead-based immunoassays. HEK293 cells transfected with Toll-like receptors (TLRs) and macrophages derived from TLR-knockout mice were used to identify the receptor mediating NSP4 signalling. Additionally, liquid chromatography-mass spectrometry (LC-MS) was employed to identify any post-translational modification(s) on NSP4. Results and conclusions: Macrophages and DCs were activated by NSP4 to secrete various proinflammatory mediators including TNF-α and IL-6. The production of cytokines by innate immune cells is an immunological response secondary to recognition of NSP4 by TLR2 that is suggested by the TLR knockin and knockout approaches. Specifically, NSP4 induced the secretion of interleukin-8 (IL-8) from human epithelial cells transfected with TLR2 but not TLR4, and NSP4 only triggered the secretion of inflammatory cytokines from murine macrophages derived from wild-type but not MyD88 knock-out or TLR2 knock-out mice. Since TLR2 ligands are mostly lipoproteins/lipopeptides that rely on their lipid components to interact with TLR2, detergent and lipase treatment and mass spectrometric analysis of NSP4 failed to reveal a lipid motif that accounts for the immunostimulatory activity. Instead, heat-denatured and enzyme-digested NSP4 lose the ability to stimulate TLR2, suggesting an intact protein is required for its activity. Collectively, these studies suggest NSP4 may interact with TLR2 in a different manner from bacteria-derived TLR2 ligands. Taken together, this research identified that NSP4 acts as a pathogen-associated molecular pattern (PAMP) encoded by rotavirus to stimulate TLR2, and proposed a mechanism for the production of proinflammatory cytokines associated with the clinical symptoms of infection in humans and animals. Alternatively, recognition of a secreted toxin from active viral infections may represent a host adaption to detect and control viral replication, to counteract the pathological effects of enterotoxins, and to mediate tissue repair and recovery.