Toll-like receptors (TLRs) detect microorganisms and protect multicellular organisms from infection. TLRs transduce their signals through MyD88 and the serine/threonine kinase IRAK. The IRAK family consists of two active kinases, IRAK and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. IRAK-M expression is restricted to monocytes/macrophages, whereas other IRAKs are ubiquitous. We show here that IRAK-M is induced upon TLR stimulation and negatively regulates TLR signaling. IRAK-M prevented dissociation of IRAK and IRAK-4 from MyD88 and formation of IRAK-TRAF6 complexes. IRAK-M(-/-) cells exhibited increased cytokine production upon TLR/IL-1 stimulation and bacterial challenge, and IRAK-M(-/-) mice showed increased inflammatory responses to bacterial infection. Endotoxin tolerance, a protection mechanism against endotoxin shock, was significantly reduced in IRAK-M(-/-) cells. Thus, IRAK-M regulates TLR signaling and innate immune homeostasis.

Mutations in the NALP3/CIAS1/cryopyrin gene are linked to three autoinflammatory disorders: Muckle-Wells syndrome, familial cold autoinflammatory syndrome, and chronic infantile neurologic cutaneous and articular syndrome. NALP3, with the adaptor molecule ASC, has been proposed to form a caspase-1-activating "inflammasome," a complex with pro-IL1beta-processing activity. Here, we demonstrate the effect of NALP3 deficiency on caspase-1 function. NALP3 was essential for the ATP-driven activation of caspase-1 in lipopolysaccharide-stimulated macrophages and for the efficient secretion of the caspase-1-dependent cytokines IL-1alpha, IL-1beta, and IL-18. IL-1beta has been shown to play a key role in contact hypersensitivity; we show that ASC- and NALP3-deficient mice also demonstrate an impaired contact hypersensitivity response to the hapten trinitrophenylchloride. NALP3, however, was not required for caspase-1 activation by Salmonella typhimurium, and NALP3 deficiency only partially protects mice from the lethal effects of endotoxin. These data suggest that NALP3 plays a specific role in the caspase-1 activation pathway.

Bacteria that have sustained long-standing close associations with eukaryotic hosts have evolved specific adaptations to survive and replicate in this environment. Perhaps one of the most remarkable of those adaptations is the type III secretion system (T3SS)--a bacterial organelle that has specifically evolved to deliver bacterial proteins into eukaryotic cells. Although originally identified in a handful of pathogenic bacteria, T3SSs are encoded by a large number of bacterial species that are symbiotic or pathogenic for humans, other animals including insects or nematodes, and plants. The study of these systems is leading to unique insights into not only organelle assembly and protein secretion but also mechanisms of symbiosis and pathogenesis.

Invasion of the intestinal epithelium is thought to be an important step in the pathogenesis of Salmonella infections. Using an in vitro system, we have isolated a genetic locus, inv, that confers to a noninvasive strain of Salmonella typhimurium the ability to penetrate tissue culture cells. Highly virulent S. typhimurium strains carrying inv mutations were defective for entry into Henle-407 cells while remaining unaffected in their ability to attach to cultured cells. When administered perorally to BALB/c mice, inv mutants of S. typhimurium had higher 50% lethal doses (LD50) than their wild-type parent strains. To the contrary, there were no differences in the observed LD50 when strains were administered intraperitoneally. In addition, inv mutants presented decreased ability to colonize the Peyer's patches, the small intestinal wall, and the spleen when

S. typhimurium stimulates signaling pathways leading to membrane ruffling, actin cytoskeleton rearrangements, and nuclear responses. The stimulation requires a protein secretion system (type III) that translocates bacterial proteins into the host cell. We show that SopE, a substrate of this secretion system, stimulates cytoskeletal reorganization and JNK activation in a CDC42- and Rac-1-dependent manner. A lambda gt11 cDNA library screen for proteins that interact with SopE identified Rac-1 and CDC42. Furthermore, purified SopE was shown to stimulate GDP/GTP nucleotide exchange in several Rho GTPases in vitro, including Rac-1 and CDC42. These findings establish a paradigm for microbial stimulation of cellular responses in which the pathogen induces signaling events by directly engaging the signaling machinery within the host cell.

The type III secretion system of Salmonella typhimurium directs the translocation of proteins into host cells. Evolutionarily related to the flagellar assembly machinery, this system is also present in other pathogenic bacteria, but its organization is unknown. Electron microscopy revealed supramolecular structures spanning the inner and outer membranes of flagellated and nonflagellated strains; such structures were not detected in strains carrying null mutations in components of the type III apparatus. Isolated structures were found to contain at least three proteins of this secretion system. Thus, the type III apparatus of S. typhimurium, and presumably other bacteria, exists as a supramolecular structure in the bacterial envelope.

The immune system consists of two evolutionarily different but closely related responses, innate immunity and adaptive immunity. Each of these responses has characteristic receptors-Toll-like receptors (TLRs) for innate immunity and antigen-specific receptors for adaptive immunity. Here we show that the caspase recruitment domain (CARD)-containing serine/threonine kinase Rip2 (also known as RICK, CARDIAK, CCK and Ripk2) transduces signals from receptors of both immune responses. Rip2 was recruited to TLR2 signalling complexes after ligand stimulation. Moreover, cytokine production in Rip2-deficient cells was reduced on stimulation of TLRs with lipopolysaccharide, peptidoglycan and double-stranded RNA, but not with bacterial DNA, indicating that Rip2 is downstream of TLR2/3/4 but not TLR9. Rip2-deficient cells were also hyporesponsive to signalling through interleukin (IL)-1 and IL-18 receptors, and deficient for signalling through Nod proteins-molecules also implicated in the innate immune response. Furthermore, Rip2-deficient T cells showed severely reduced NF-kappaB activation, IL-2 production and proliferation on T-cell-receptor (TCR) engagement, and impaired differentiation to T-helper subtype 1 (TH1) cells, indicating that Rip2 is required for optimal TCR signalling and T-cell differentiation. Rip2 is therefore a signal transducer and integrator of signals for both the innate and adaptive immune systems.

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