In humans, ten Toll-like receptor (TLR) paralogues sense molecular components of microbes, initiating the production of cytokine mediators that create the inflammatory response. Using N-ethyl-N-nitrosourea, we induced a germline mutation called Lps2, which abolishes cytokine responses to double-stranded RNA and severely impairs responses to the endotoxin lipopolysaccharide (LPS), indicating that TLR3 and TLR4 might share a specific, proximal transducer. Here we identify the Lps2 mutation: a distal frameshift error in a Toll/interleukin-1 receptor/resistance (TIR) adaptor protein known as Trif or Ticam-1. Trif(Lps2) homozygotes are markedly resistant to the toxic effects of LPS, and are hypersusceptible to mouse cytomegalovirus, failing to produce type I interferons when infected. Compound homozygosity for mutations at Trif and MyD88 (a cytoplasmic TIR-domain-containing adaptor protein) loci ablates all responses to LPS, indicating that only two signalling pathways emanate from the LPS receptor. However, a Trif-independent cell population is detectable when Trif(Lps2) mutant macrophages are stimulated with LPS. This reveals that an alternative MyD88-dependent 'adaptor X' pathway is present in some, but not all, macrophages, and implies afferent immune specialization.

Several subsets of dendritic cells have been shown to produce type I IFN in response to viral infections, thereby assisting the natural killer cell-dependent response that eliminates the pathogen. Type I IFN production can be induced both by unmethylated CpG-oligodeoxynucleotide and by double-stranded RNA. Here, we describe a codominant CpG-ODN unresponsive phenotype that results from an N-ethyl-N-nitrosourea-induced missense mutation in the Tlr9 gene (Tlr9 CpG1 ). Mice homozygous for the Tlr9 CpG1 allele are highly susceptible to mouse cytomegalovirus infection and show impaired infection-induced secretion of IFN-␣͞␤ and natural killer cell activation. We also demonstrate that both the Toll-like receptor (TLR) 9 3 MyD88 and TLR3 3 Trif signaling pathways are activated in vivo on viral inoculation, and that each pathway contributes to innate defense against systemic viral infection. Whereas both pathways lead to type I IFN production, neither pathway offers full protection against mouse cytomegalovirus infection in the absence of the other. The Tlr9 CpG1 mutation alters a leucine-rich repeat motif and lies within a receptor domain that is conserved within the evolutionary cluster encompassing TLRs 7, 8, and 9. In other TLRs, including three mouse-specific TLRs described in this paper, the affected region is not represented. The phenotypic effect of the Tlr9 CpG1 allele thus points to a critical role for TLR9 in viral sensing and identifies a vulnerable amino acid within the ectodomain of three TLR proteins, essential for a ligand response.

Here we have identified 'triple D' (3d), a recessive N-ethyl-N-nitrosourea-induced mutation and phenotype in which no signaling occurs via the intracellular Toll-like receptors 3, 7 and 9 (sensors for double-stranded RNA, single-stranded RNA and unmethylated DNA, respectively). The 3d mutation also prevented cross-presentation and diminished major histocompatibility complex class II presentation of exogenous antigen; it also caused hypersusceptibility to infection by mouse cytomegalovirus and other microbes. By positional identification, we found 3d to be a missense allele of Unc93b1, which encodes the 12-membrane-spanning protein UNC-93B, a highly conserved molecule found in the endoplasmic reticulum with multiple paralogs in mammals. Innate responses to nucleic acids and exogenous antigen presentation, which both initiate in endosomes, thus seem to depend on an endoplasmic reticulum-resident protein, which suggests communication between these organellar systems.

IKKbeta-dependent NF-kappaB activation plays a key role in innate immunity and inflammation, and inhibition of IKKbeta has been considered as a likely anti-inflammatory therapy. Surprisingly, however, mice with a targeted IKKbeta deletion in myeloid cells are more susceptible to endotoxin-induced shock than control mice. Increased endotoxin susceptibility is associated with elevated plasma IL-1beta as a result of increased pro-IL-1beta processing, which was also seen upon bacterial infection. In macrophages enhanced pro-IL-1beta processing depends on caspase-1, whose activation is inhibited by NF-kappaB-dependent gene products. In neutrophils, however, IL-1beta secretion is caspase-1 independent and depends on serine proteases, whose activity is also inhibited by NF-kappaB gene products. Prolonged pharmacologic inhibition of IKKbeta also augments IL-1beta secretion upon endotoxin challenge. These results unravel an unanticipated role for IKKbeta-dependent NF-kappaB signaling in the negative control of IL-1beta production and highlight potential complications of long-term IKKbeta inhibition.

Under lean conditions, the adipose-derived hormone leptin maintains energy balance in part through CNS-mediated increases in sympathetic outflow that enhance fat burning 1,2. Triggering of beta adrenergic receptors in adipocytes stimulates energy expenditure via cAMP-dependent increases in lipolysis and fatty acid oxidation 3. Although the underlying mechanism is unclear, catecholamine signaling in fat cells is thought to be disrupted in obesity 4, where it may contribute to the ectopic accumulation of lipid in liver and to the development of insulin resistance 5,6. Here we show that the cAMP responsive CREB coactivator CRTC3 promotes obesity by attenuating beta adrenergic receptor signaling in adipose; mice with a knockout of the CRTC3 gene have increased energy expenditure, are resistant to diet induced obesity, and are protected from the development of hepatic steatosis under high fat diet feeding conditions. CRTC3 was activated in response to catecholamine signals, when it reduced adenyl cyclase activity by upregulating the expression of RGS2 7–9, a metabolic syndrome susceptibility gene 10, which we show here is also a direct target of CREB and CRTC3. RGS2 expression was down-regulated in adipocytes from CRTC3−/− mice, leading to increases in insulin and catecholamine signaling that enhanced glucose and fatty acid oxidation. As a common human CRTC3 variant (Ser72Asn), with increased transcriptional activity, is associated with several anthropometric indices of adiposity in two distinct Mexican-American cohorts, our results suggest that adipocyte CRTC3 may play a role in the development of obesity in this population.

In the fasted state, increases in circulating glucagon promote hepatic glucose production through induction of the gluconeogenic program. Triggering of the cAMP pathway increases gluconeogenic gene expression via the de-phosphorylation of the CREB coactivator CRTC2 1. Glucagon promotes CRTC2 dephosphorylation in part through the PKA-mediated inhibition of the CRTC2 kinase SIK2. A number of Ser/Thr phosphatases appear capable of dephosphorylating CRTC2 2,3, but the mechanisms by which hormonal cues regulate these enzymes remain unclear. Here we show that glucagon stimulates CRTC2 dephosphorylation in hepatocytes by mobilizing intracellular calcium stores and activating the calcium/calmodulin dependent Ser/Thr phosphatase calcineurin/PP2B. Glucagon increased cytosolic calcium through the PKA-mediated phosphorylation of inositol 1,4,5-trisphosphate receptors (InsP3Rs), which we show here associate with CRTC2. Following their activation, InsP3Rs enhanced gluconeogenic gene expression by promoting the calcineurin-mediated dephosphorylation of CRTC2. During feeding, increases in insulin signaling reduced CRTC2 activity via the AKT-mediated inactivation of InsP3Rs. InsP3R activity was increased in diabetes, leading to upregulation of the gluconeogenic program. As hepatic down-regulation of InsP3Rs and calcineurin improved circulating glucose levels in insulin resistance, these results demonstrate how cross-talk between cAMP and calcium pathways at the level of the InsP3 receptor modulates hepatic glucose production under fasting conditions and in diabetes.

In fasted mammals, glucose homeostasis is maintained through induction of the cAMP response element-binding protein (CREB) coactivator transducer of regulated CREB activity 2 (TORC2), which stimulates the gluconeogenic program in concert with the forkhead factor FOXO1. Here we show that starvation also triggers TORC activation in Drosophila, where it maintains energy balance through induction of CREB target genes in the brain. TORC mutant flies have reduced glycogen and lipid stores and are sensitive to starvation and oxidative stress. Neuronal TORC expression rescued stress sensitivity as well as CREB target gene expression in TORC mutants. During refeeding, increases in insulin signaling inhibited TORC activity through the salt-inducible kinase 2 (SIK2)-mediated phosphorylation and subsequent degradation of TORC. Depletion of neuronal SIK2 increased TORC activity and enhanced stress resistance. As disruption of insulin signaling also augmented TORC activity in adult flies, our results illustrate the importance of an insulin-regulated pathway that functions in the brain to maintain energy balance.

Both forward and reverse genetic techniques have been used to define components of the mammalian lipopolysaccharide (LPS) receptor. TLR4, identified by a forward genetic approach as the product of the classical Lps locus, is the only known transmembrane component of the mammalian LPS receptor. Gene knockout work has also established that LPS signal transduction requires the integrity of CD14, MD-2, and, in part, MyD88, IRAK4, and TRAF-6. However, there is no reason to believe that these are the only proteins that make up the receptor/transducer apparatus. To examine the possibility that other proteins may be involved, we initiated a mutagenesis program, in which germline mutations are induced in mice using N-ethyl-N-nitrosourea (ENU), and macrophages from individual animals are screened for their competence to respond to LPS. We now report the existence of a new locus, Lps2, which is required for TNF production in response to LPS. The Lps2 mutation that we have identified is co-dominant, is similar in phenotypic effect to Lpsd, and does not represent a novel allele of any of the genes that are known to encode the 'core' LPS signaling apparatus. The Lps2 mutation does not preclude signaling initiated by peptidoglycan or unmethylated DNA. Hence, genetic data suggest that there is at least one 'missing' component of the LPS receptor complex that has yet to be found.