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Nucleotide-binding oligomerization domain-like receptors (NLRs) detect pathogens and danger-associated signals within the cell. Salmonella enterica serovar Typhimurium, an intracellular pathogen, activates caspase-1 required for the processing of the proinflammatory cytokines, pro-IL-1β and pro-IL-18, and pyroptosis. In this study, we show that Salmonella infection induces the formation of an apoptosis-associated specklike protein containing a CARD (ASC)-Caspase-8-Caspase-1 inflammasome in macrophages. Caspase-8 and caspase-1 are recruited to the ASC focus independently of one other. Salmonella infection initiates caspase-8 proteolysis in a manner dependent on NLRC4 and ASC, but not NLRP3, caspase-1 or caspase-11. Caspase-8 primarily mediates the synthesis of pro-IL-1β, but is dispensable for Salmonella-induced cell death. Overall, our findings highlight that the ASC inflammasome can recruit different members of the caspase family to induce distinct effector functions in response to Salmonella infection. [ABSTRACT FROM AUTHOR]

Allergic responses can be triggered by structurally diverse allergens. Most allergens are proteins, yet extensive research has not revealed how they initiate the allergic response and why the myriad of other inhaled proteins do not. Among these allergens, the cat secretoglobulin protein Fel d 1 is a major allergen and is responsible for severe allergic responses. In this study, we show that similar to the mite dust allergen Der p 2, Fel d 1 substantially enhances signaling through the innate receptors TLR4 and TLR2. In contrast to Der p 2, however, Fel d 1 does not act by mimicking the TLR4 coreceptor MD2 and is not able to bind stably to the TLR4/MD2 complex in vitro. Fel d 1 does, however, bind to the TLR4 agonist LPS, suggesting that a lipid transfer mechanism may be involved in the Fel d 1 enhancement of TLR signaling. We also show that the dog allergen Can f 6, a member of a distinct class of lipocalin allergens, has very similar properties to Fel d 1. We propose that Fel d 1 and Can f 6 belong to a group of allergen immunomodulatory proteins that enhance innate immune signaling and promote airway hypersensitivity reactions in diseases such as asthma. [ABSTRACT FROM AUTHOR]

Nucleotide-binding oligomerization domain-like receptors (NLRs) detect pathogens and danger-associated signals within the cell. Salmonella enterica serovar Typhimurium, an intracellular pathogen, activates caspase-1 required for the processing of the proinflammatory cytokines, pro-IL-1β and pro-IL-18, and pyroptosis. In this study, we show that Salmonella infection induces the formation of an apoptosis-associated specklike protein containing a CARD (ASC)-Caspase-8-Caspase-1 inflammasome in macrophages. Caspase-8 and caspase-1 are recruited to the ASC focus independently of one other. Salmonella infection initiates caspase-8 proteolysis in a manner dependent on NLRC4 and ASC, but not NLRP3, caspase-1 or caspase-11. Caspase-8 primarily mediates the synthesis of pro-IL-1β, but is dispensable for Salmonella-induced cell death. Overall, our findings highlight that the ASC inflammasome can recruit different members of the caspase family to induce distinct effector functions in response to Salmonella infection.

TLRs are critical pattern recognition receptors that recognize bacterial and viral pathogen-associated molecular patterns leading to innate and adaptive immune responses. TLRs signal via homotypic interactions between their cytoplasmic Toll/IL-1R (TIR) domains and TIR domain-containing adaptor proteins. Over the course of evolution, viruses have developed various immune evasion strategies, one of which involves inhibiting TLR signaling pathways to avoid immune detection. Thus, vaccinia virus encodes the A46 protein, which binds to multiple TIR-domain containing proteins, ultimately preventing TLRs from signaling. We have identified an 11-aa-long peptide from A46 (termed viral inhibitor peptide of TLR4, or VIPER), which, when fused to a cell-penetrating delivery sequence, potently inhibits TLR4-mediated responses. VIPER was TLR4 specific, being inert toward other TLR pathways, and was active in murine and human cells and in vivo, where it inhibited LPS-induced IL-12p40 secretion. VIPER also prevented TLR4-mediated MAPK and transcription factor activation, suggesting it acted close to the TLR4 complex. Indeed, VIPER directly interacted with the TLR4 adaptor proteins MyD88 adaptor-like (Mal) and TRIF-related adaptor molecule (TRAM). Viral proteins target host proteins using evolutionary optimized binding surfaces. Thus, VIPER possibly represents a surface domain of A46 that specifically inhibits TLR4 by masking critical binding sites on Mal and TRAM. Apart from its potential therapeutic and experimental use in suppressing TLR4 function, identification of VIPER's specific binding sites on TRAM and Mal may reveal novel therapeutic target sites. Overall, we demonstrate for the first time disruption of a specific TLR signaling pathway by a short virally derived peptide.

TLR4 is the primary sensor of LPS. In this study, we describe for the first time TLR4 interactor with leucine-rich repeats (TRIL), which is a novel component of the TLR4 complex. TRIL is expressed in a number of tissues, most prominently in the brain but also in the spinal cord, lung, kidney, and ovary. TRIL is composed of a signal sequence, 13 leucine-rich repeats, a fibronectin domain, and a single transmembrane spanning region. TRIL is induced by LPS in the human astrocytoma cell line U373, in murine brain following i.p. injection, and in human PBMC. Endogenous TRIL interacts with TLR4 and this interaction is greatly enhanced following LPS stimulation. TRIL also interacts with the TLR4 ligand LPS. Furthermore, U373 cells stably overexpressing TRIL display enhanced cytokine production in response to LPS. Finally, knockdown of TRIL using small interfering RNA attenuates LPS signaling and cytokine production in cell lines, human PBMC, and primary murine mixed glial cells. These results demonstrate that TRIL is a novel component of the TLR4 complex which may have particular relevance for the functional role of TLR4 in the brain.

LPS signals through a membrane bound-complex of the lipid binding protein MD-2 and the receptor TLR4. In this study we identify discrete regions in both MD-2 and TLR4 that are required for signaling by lipid IVa, an LPS derivative that is an agonist in horse but an antagonist in humans. We show that changes in the electrostatic surface potential of both MD-2 and TLR4 are required in order that lipid IVa can induce signaling. In MD-2, replacing horse residues 57-66 and 82-89 with the equivalent human residues confers a level of constitutive activity on horse MD-2, suggesting that conformational switching in this protein is likely to be important in ligand-induced activation of MD-2/TLR4. We identify leucine-rich repeat 14 in the C terminus of TLR4 as essential for lipid IVa activation of MD-2/TLR4. Remarkably, we identify a single residue in the glycan-free flank of the horse TLR4 solenoid that confers the ability to signal in response to lipid IVa. These results suggest a mechanism of signaling that involves crosslinking mediated by both MD-2-receptor and receptor-receptor contacts in a model that shows striking similarities to the recently published structure (Cell 130: 1071-1082) of the ligand-bound TLR1/2 ectodomain heterodimer.