Mannan binding lectin attenuates double-stranded RNA-mediated TLR3 activation and innate immunity

Mannan binding lectin (MBL) functions as a pattern recognition molecule (PRM) which is able to initiate complement activation. Here, we characterize a previously unrecognized attribute of MBL as a double-stranded RNA (dsRNA) binding protein capable of modifying Toll like receptor 3 (TLR3) activation. MBL interacts with poly(I:C) and suppresses poly(I:C)-induced activation of TLR3 pathways and subsequent cytokine production. In addition, MBL binds to TLR3 directly. Surprisingly, disrupting the interaction between MBL and complement receptor 1 (CR1) or restraining the traffic of MBL to phagosome reversed the MBL limited TLR3 activation. We demonstrate the importance of MBL guided ligands intracellular localization, emphasizing the significance of understanding the dynamics of TLR agonists complexed with MBL or other PRMs inside the cell in immune defense.     

A protein-kinase, IFN-inducible double-stranded RNA dependent inhibitor and repressor of p58 (PRKRIR) enhances type I IFN-mediated antiviral response through the stability control of RIG-I protein

The cellular RIG-I-like receptor (RLR) senses pathogenic RNA molecular patterns and transmits signals for type I interferon (IFN) production. It acts as a center for antiviral responses, and large numbers of RIG-I (retinoic acid inducible gene-I) interacting proteins are identified as signaling regulators. In the present study, we report PRKRIR, a negative regulator of PKR inhibitor, as a novel RIG-I interacting protein. In HEK293FT cells, PRKRIR synergistically enhances type I IFN production mediated by a signal activated- or constitutively active form of RIG-I. The C-terminal domain of the PRKRIR was required for physical interaction and the signal augmentation. The PRKRIR blocks poly-ubiquitination and protein degradation of RIG-I, thereby increasing cellular levels of RIG-I proteins. Furthermore, overexpression of PRKRIR, along with a signal activated- or constitutively active form of RIG-I, efficiently inhibits virus replication in the infected host. In conclusion, PRKRIR provides a novel positive regulator controlling the RIG-I-IFN production system through protein stability control.     

A novel binding protein of single immunoglobulin IL-1 receptor-related molecule: Paralemmin-3

Previous studies have shown that single immunoglobulin IL-1 receptor-related molecule (SIGIRR) is a negative regulator of Toll-Interleukin-1 receptor signaling. Nevertheless, the molecular mechanism of the negatively regulatory effect of SIGIRR remains unknown. Using a yeast two-hybrid screen, we identified paralemmin-3 (PALM3) as a novel binding protein of SIGIRR. This interaction of SIGIRR with PALM3 was confirmed by coimmunoprecipitation in mammalian cells. In addition, the PALM3 mRNA expression was upregulated by lipopolysaccharide (LPS)-stimulation in a human alveolar epithelial cell line (A549 cells). Furthermore, silencing PALM3 by RNA interference inhibited the release of inflammatory cytokines in A549 cells after LPS-stimulation. These results suggest that PALM3 may function as an adaptor in the LPS- Toll-like receptor 4 signaling and the interaction of SIGIRR with PALM3 may partly account for the mechanism of the negatively regulatory effect of SIGIRR.     

The role of phagocytosis in IL-8 production by human monocytes in response to lipoproteins on Staphylococcus aureus

Cytokine responses to microbes are triggered by pattern recognition receptors, such as Toll-like receptors (TLRs), which sense pathogen-associated molecular patterns. Cell wall-associated triacylated lipoproteins in Staphylococcus aureus are known to be native TLR2 ligands that mediate host inflammatory responses against S. aureus. However, the mechanism by which these lipidated lipoproteins, which are buried under the thick S. aureus cell wall, work to stimulate TLR2 remains unclear. Heat-killed wild type S. aureus cells activated human monocytic THP-1 cells to produce proinflammatory cytokines, including interleukin (IL)-8, whereas the lipoprotein lipidation-deficient lgt mutant induced less than an eighth of the amount of IL-8 induced by the wild type. IL-8 induction in response to heat-killed S. aureus cells in THP-1 cells was not inhibited by a blocking antibody against cell surface TLR2, suggesting that intracellular TLR2 might be involved in the induction of IL-8 by S. aureus lipoprotein. The relationship between phagocytosis and IL-8 production in THP-1 cells was analyzed on a single-cell level by flow cytometry using fluorescein-labeled S. aureus cells and phycoerythrin-labeled anti-IL-8 antibody. Production of intracellular IL-8 was correlated with phagocytosis of S. aureus cells in THP-1 cells and in human peripheral blood mononuclear cells. Opsonization of S. aureus cells enhanced both the phagocytosis of S. aureus cells and the production of intracellular IL-8 in THP-1 cells. These results suggest that lipidated lipoproteins on S. aureus cells stimulate human monocytes after phagocytosis.