Toll-like receptors (TLRs) are activated by pathogen-associated molecular patterns to induce innate immune responses and production of pro-inflammatory cytokines, interferons and anti-inflammatory cytokines¹. TLRs activate downstream effectors through adaptors that contain Toll/interleukin-1 receptor (TIR) domains², but the mechanisms accounting for diversification of TLR effector functions are unclear. To dissect biochemically TLR signalling, we established a system for isolating signalling complexes assembled by dimerized adaptors. Using MyD88 as a prototypical adaptor, we identified TNF receptor-associated factor 3 (TRAF3) as a new component of TIR signalling complexes that is recruited along with TRAF6. Using myeloid cells from TRAF3- and TRAF6-deficient mice, we show that TRAF3 is essential for the induction of type I interferons (IFN) and the anti-inflammatory cytokine interleukin-10 (IL-10), but is dispensable for expression of pro-inflammatory cytokines. In fact, TRAF3-deficient cells overproduce pro-inflammatory cytokines owing to defective IL-10 production. Despite their structural similarity, the functions of TRAF3 and TRAF6 are largely distinct. TRAF3 is also recruited to the adaptor TRIF (Toll/IL-1 receptor domain-containing adaptor-inducing IFN-β) and is required for marshalling the protein kinase TBK1 (also called NAK) into TIR signalling complexes, thereby explaining its unique role in activation of the IFN response.

Variants of NOD2, an intracellular sensor of bacteria-derived muramyl dipeptide (MDP), increase susceptibility to Crohn's disease (CD). These variants are thought to be defective in activation of nuclear factor κB (NF-κB) and antibacterial defenses, but CD clinical specimens display elevated NF-κB activity. To illuminate the pathophysiological function of NOD2, we introduced such a variant to the mouse Nod2 locus. Mutant mice exhibited elevated NF-κB activation in response to MDP and more efficient processing and secretion of the cytokine interleukin-1β (IL-1β). These effects are linked to increased susceptibility to bacterial-induced intestinal inflammation and identify NOD2 as a positive regulator of NF-κB activation and IL-1β secretion.

NOD2, a NOD-like receptor (NLR), is an intracellular sensor of bacterial muramyl dipeptide (MDP) that was suggested to promote secretion of the proinflammatory cytokine IL-1β. Yet, the molecular mechanism by which NOD2 can stimulate IL-1β secretion, and its biological significance were heretofore unknown. We found that NOD2 through its N-terminal caspase recruitment domain directly binds and activates caspase-1 to trigger IL-1β processing and secretion in MDP-stimulated macrophages, whereas the C-terminal leucine-rich repeats of NOD2 prevent caspase-1 activation in nonstimulated cells. MDP challenge induces the association of NOD2 with another NLR protein, NALP1, and gel filtration analysis revealed the formation of a complex consisting of NOD2, NALP1, and caspase-1. Importantly, Bacillus anthracis infection induces IL-1β secretion in a manner that depended on caspase-1 and NOD2. In vitro , Anthrax lethal toxin strongly potentiated IL-1β secretion, and that response was NOD2 and caspase-1-dependent. Thus, NOD2 plays a key role in the B. anthracis -induced inflammatory response by being a critical mediator of IL-1β secretion.

Macrophages are pivotal constituents of the innate immune system, vital for recognition and elimination of microbial pathogens¹. Macrophages use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns—including bacterial cell wall components, such as lipopolysaccharide or lipoteichoic acid, and viral nucleic acids, such as double-stranded (ds)RNA—and in turn activate effector functions, including anti-apoptotic signalling pathways². Certain pathogens, however, such as Salmonella spp., Shigellae spp. and Yersiniae spp., use specialized virulence factors to overcome these protective responses and induce macrophage apoptosis³. We found that the anthrax bacterium, Bacillus anthracis, selectively induces apoptosis of activated macrophages⁴ through its lethal toxin, which prevents activation of the anti-apoptotic p38 mitogen-activated protein kinase⁴. We now demonstrate that macrophage apoptosis by three different bacterial pathogens depends on activation of TLR4. Dissection of anti- and pro-apoptotic signalling events triggered by TLR4 identified the dsRNA responsive protein kinase PKR as a critical mediator of pathogen-induced macrophage apoptosis. The pro-apoptotic actions of PKR are mediated both through inhibition of protein synthesis and activation of interferon response factor 3.