Extracellular nicotinate phosphoribosyltransferase binds Toll like receptor 4 and mediates inflammation

Antonella Managò,Alice Ianniello,Silvia Deaglio,Antonio Varriale,Nadia Raffaelli,Federica Gaudino,Katiuscia Gizzi,Francesca Mazzola,Gabriele Minazzato,Salvatore Oliviero,Giulio Mengozzi,Leonardo Sorci,Sabato D’Auria,Valentina Audrito,Nicoletta Vitale,Danny Incarnato,Gianfranco Politano

doi:10.1038/s41467-019-12055-2

Abstract

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. Here we show that nicotinate phosphoribosyltransferase (NAPRT), detected by antibody-mediated assays and mass spectrometry, is an extracellular ligand for Toll-like receptor 4 (TLR4) and a critical mediator of inflammation, acting as a DAMP. Exposure of human and mouse macrophages to NAPRT activates the inflammasome and NF-κB for secretion of inflammatory cytokines. Furthermore, NAPRT enhances monocyte differentiation into macrophages by inducing macrophage colony-stimulating factor. These NAPRT-induced effects are independent of NAD-biosynthetic activity, but rely on NAPRT binding to TLR4. In line with our finding that NAPRT mediates endotoxin tolerance in vitro and in vivo, sera from patients with sepsis contain the highest levels of NAPRT, compared to patients with other chronic inflammatory conditions. Together, these data identify NAPRT as a endogenous ligand for TLR4 and a mediator of inflammation.

Highlights

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system
Antibody binding to recombinant nicotinate phosphoribosyltransferase (NAPRT) and lack of crossreaction between rNAPRT and recombinant nicotinamide phosphoribosyltransferase (NAMPT) confirmed the specificity of the assay (Supplementary Fig. 1a, b and Supplementary Table 1). eNAPRT levels were determined in a validation cohort of 96 HD, including children, indicating median eNAPRT levels of 1.3 ± 0.08 ng/ml (Fig. 1b), with no differences according to gender or age (Supplementary Fig. 1c, d)
By using a surface coated with an anti-NAPRT antibody, we showed that a pre-mixed solution of rNAPRT and recombinant extracellular domain of TLR4 (rTLR4) resulted in increased binding when compared to rNAPRT alone, indicating that a direct molecular interaction is occurring between the proteins (Fig. 4c and Supplementary Fig. 4d)

Summary

Introduction

Damage-associated molecular patterns (DAMPs) are molecules that can be actively or passively released by injured tissues and that activate the immune system. NAPRT enhances monocyte differentiation into macrophages by inducing macrophage colony-stimulating factor These NAPRT-induced effects are independent of NAD-biosynthetic activity, but rely on NAPRT binding to TLR4. In line with our finding that NAPRT mediates endotoxin tolerance in vitro and in vivo, sera from patients with sepsis contain the highest levels of NAPRT, compared to patients with other chronic inflammatory conditions Together, these data identify NAPRT as a endogenous ligand for TLR4 and a mediator of inflammation. Intrinsic factors produced by the host can modulate this complex network of extracellular signals, thereby contributing to inflammation[3] Many of these factors have a well-characterized intracellular function and were serendipitously identified in the extracellular space, where they bind and activate pattern recognition receptors[4,5]. Sera from patients with sepsis and septic shock contain high levels of NAPRT, underling its potential use as a marker for this critical condition

Methods

Results

Conclusion