We report here the identification and characterization of a protein, ERIS, an endoplasmic reticulum (ER) IFN stimulator, which is a strong type I IFN stimulator and plays a pivotal role in response to both non-self-cytosolic RNA and dsDNA. ERIS (also known as STING or MITA) resided exclusively on ER membrane. The ER retention/ retrieval sequence RIR was found to be critical to retain the protein on ER membrane and to maintain its integrity. ERIS was dimerized on innate immune challenges. Coumermycin-induced ERIS dimerization led to strong and fast IFN induction, suggesting that dimerization of ERIS was critical for self-activation and subsequent downstream signaling.innate immunity ͉ type I IFN ͉ functional cDNA library screening ͉ cytosolic RNA and dsDNA ͉ ER retention signal M icrobial infection-induced host immune responses are initiated by the germline-encoded pattern recognition receptors, which recognize components specific to microorganisms. There are 3 major classes of such receptors: Toll-like receptors (TLRs), RIG-I-like helicases (RLHs) and NOD-like receptors (1). During infection, nucleic acids derived from microbes are recognized by TLRs and RLHs, which then trigger a series of signaling events leading to the production of type I IFNs and proinflammatory cytokines.RLHs have recently been identified to sense the invading viruses in the cytoplasm. Unlike TLRs, which are expressed in specific cells like macrophages and dendritic cells, RLHs are found in most cell types (2). They contain caspase recruitment domain (CARD) and DExD/H helicase domain. RLHs interact with microbial nucleotides through their helicase domain. The N-terminal CARDs are responsible for activating downstream signaling pathways that mediate type I IFN production. Genetic analyses demonstrate that RIG-I and MDA5 sense distinct types of viruses (3-5). RIG-I and MDA5 use a common adaptor molecule, IPS-1 (also known as Cardif, MAVS, or VISA) (6-9). IPS-1 is found to reside on the mitochondrial membrane by its C-terminal transmembrane (TM) domain. It also contains a CARD-like domain at its N-terminus, which mediates the interaction with MDA5 or RIG-I. IPS-1 transmits the signal to TANK-binding kinase-1 (TBK1)/I B kinase i (IKKi; also known as IKK ) and the IKK complex to activate interferon regulatory factor (IRF)-3/IRF-7 and NF-B, respectively, collectively eliciting innate antiviral immune responses, including type I IFN production.On the other hand, dsDNA in the cytosol, for example, genomic DNA from intracellular bacteria (e.g., Listeria, Legionella), also causes a strong host immune response independent of TLRs, leading to the induction of type I IFN. A recent report has indicated that the molecule DAI (also known as ZBP1) might serve as a cytosolic dsDNA sensor (10). However, ZBP1 Ϫ/Ϫ cells showed normal type I IFN production in response to dsDNA stimulation (11). Meanwhile, reports showed that IPS-1/Cardif/MAVS/VISA was not required for dsDNA-caused innate immune activation (12).The signaling induced by cytoplasmic dsDNA leading ...

The recessive mutation 'Heedless' (hdl) was detected in third-generation N-ethyl-N-nitrosourea-mutated mice that showed defective responses to microbial inducers. Macrophages from Heedless homozygotes signaled by the MyD88-dependent pathway in response to rough lipopolysaccharide (LPS) and lipid A, but not in response to smooth LPS. In addition, the Heedless mutation prevented TRAM-TRIF-dependent signaling in response to all LPS chemotypes. Heedless also abolished macrophage responses to vesicular stomatitis virus and substantially inhibited responses to specific ligands for the Toll-like receptor 2 (TLR2)-TLR6 heterodimer. The Heedless phenotype was positionally ascribed to a premature stop codon in Cd14. Our data suggest that the TLR4-MD-2 complex distinguishes LPS chemotypes, but CD14 nullifies this distinction. Thus, the TLR4-MD-2 complex receptor can function in two separate modes: one in which full signaling occurs and one limited to MyD88-dependent signaling.

Classical genetic methods, driven by phenotype rather than hypotheses, generally permit the identification of all proteins that serve nonredundant functions in a defined biological process. Long before this goal is achieved, and sometimes at the very outset, genetics may cut to the heart of a biological puzzle. So it was in the field of mammalian innate immunity. The positional cloning of a spontaneous mutation that caused lipopolysaccharide resistance and susceptibility to Gram-negative infection led directly to the understanding that Toll-like receptors (TLRs) are essential sensors of microbial infection. Other mutations, induced by the random germ line mutagen ENU (N-ethyl-N-nitrosourea), have disclosed key molecules in the TLR signaling pathways and helped us to construct a reasonably sophisticated portrait of the afferent innate immune response. A still broader genetic screen--one that detects all mutations that compromise survival during infection--is permitting fresh insight into the number and types of proteins that mammals use to defend themselves against microbes.

Manganese (Mn) is essential for many physiological processes, but its functions in innate immunity remain undefined. Here, we found that Mn was required for the host defense against DNA viruses by increasing the sensitivity of the DNA sensor cGAS and its downstream adaptor protein STING. Mn was released from membrane-enclosed organelles upon viral infection and accumulated in the cytosol where it bound directly to cGAS. Mn enhanced the sensitivity of cGAS to double-stranded DNA (dsDNA) and its enzymatic activity, enabling cGAS to produce secondary messenger cGAMP in the presence of low concentrations of dsDNA that would otherwise be non-stimulatory. Mn also enhanced STING activity by augmenting cGAMP-STING binding affinity. Mn-deficient mice showed diminished cytokine production and were more vulnerable to DNA viruses, and Mn-deficient STING-deficient mice showed no increased susceptibility. These findings indicate that Mn is critically involved and required for the host defense against DNA viruses.