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Viral sensing in myeloid cells involves inflammasome activation leading to gasdermin pore formation, cytokine release, and cell death. However, less is known about viral sensing in barrier epithelial cells, which are critical to the innate immune response to RNA viruses. Here, we show that poly(I:C), a mimic of viral dsRNA, is sensed by NLRP1 in human bronchial epithelial cells, leading to inflammasome-dependent gasdermin D (GSDMD) pore formation via caspase-1. DsRNA also stimulated a parallel sensing pathway via PKR which activated caspase-3 to cleave gasdermin E (GSDME) to form active pores. Influenza A virus (IAV) infection of cells caused GSDME activation, cytokine release, and cell death, in a PKR-dependent but NLRP1-independent manner, involving caspase-8 and caspase-3. Suppression of GSDMD and GSDME expression increased IAV replication. These data clarify mechanisms of gasdermin cleavage in response to viral sensing and reveal that gasdermin pore formation is intrinsically antiviral in human epithelial cells.

Recognition of DNA by the innate immune system is central to antiviral and antibacterial defenses, as well as an important contributor to autoimmune diseases involving self DNA. AIM2 (absent in melanoma 2) and IFI16 (interferon-inducible protein 16) have been identified as DNA receptors that induce inflammasome formation and interferon production, respectively. Here we present the crystal structures of their HIN domains in complex with double-stranded (ds) DNA. Non-sequence-specific DNA recognition is accomplished through electrostatic attraction between the positively charged HIN domain residues and the dsDNA sugar-phosphate backbone. An intramolecular complex of the AIM2 Pyrin and HIN domains in an autoinhibited state is liberated by DNA binding, which may facilitate the assembly of inflammasomes along the DNA staircase. These findings provide mechanistic insights into dsDNA as the activation trigger and oligomerization platform for the assembly of large innate signaling complexes such as the inflammasomes.

It has been assumed that cells distinguish viral from cellular DNA due to the former's presence in the cytosol. However, in this issue, Kerur et al. (2011) propose that the DNA genome of Kaposi's sarcoma-associated herpesvirus (KSHV) is recognized inside the nucleus by the DNA sensor IFI16, leading inflammasome activation.

It has been assumed that cells distinguish viral from cellular DNA due to the former's presence in the cytosol. However, in this issue, Kerur et al. (2011) propose that the DNA genome of Kaposi's sarcoma-associated herpesvirus (KSHV) is recognized inside the nucleus by the DNA sensor IFI16, leading inflammasome activation.

The immediate response to viral infection relies on pattern-recognition receptors (PRRs), most prominently the Toll-like receptors (TLRs) and the RNA helicases RIG-I and MDA5, as well as double stranded RNA-dependent protein kinase (PKR) and the DNA receptor, DAI. These PRRs recognize pathogen-associated molecular patterns (PAMPs) such as viral proteins and nucleic acids. The engagement of these receptors then initiates intracellular signaling cascades which ultimately cause the activation of transcription factors and the expression of type I interferons and pro-inflammatory cytokines. This innate response establishes an anti-viral state in the infected cell and its neighbours and alerts immune cells to the danger. In order to establish a productive infection, viruses need to overcome this initial anti-viral response. Evasion of innate immune defences is achieved by means of viral proteins that inhibit the signaling cascades emanating from the PRRs. The same innate signal transduction pathways have been implicated in conditions of sterile inflammation, such as rheumatoid arthritis and multiple sclerosis, and in autoimmunity. Because viral proteins target crucial host proteins involved in these pathways, they can point the way to key drug targets. Further, the viral proteins themselves or derivatives of them may be of use therapeutically to curtail inflammation and autoimmunity.

The expression of pattern-recognition receptors (PRRs) by immune and tissue cells provides the host with the ability to detect and respond to infection by viruses and other microorganisms. Significant progress has been made from studying this area, including the identification of PRRs, such as Toll-like receptors and RIG-I-like receptors, and the description of the molecular basis of their signalling pathways, which lead to the production of interferons and other cytokines. In parallel, common mechanisms used by viruses to evade PRR-mediated responses or to actively subvert these pathways for their own benefit are emerging. Accumulating evidence on how viral infection and PRR signalling pathways intersect is providing further insights into the function of the pathways involved, their constituent proteins and ways in which they could be manipulated therapeutically.

The expression of pattern-recognition receptors (PRRs) by immune and tissue cells provides the host with the ability to detect and respond to infection by viruses and other microorganisms. Significant progress has been made from studying this area, including the identification of PRRs, such as Toll-like receptors and RIG-I-like receptors, and the description of the molecular basis of their signalling pathways, which lead to the production of interferons and other cytokines. In parallel, common mechanisms used by viruses to evade PRR-mediated responses or to actively subvert these pathways for their own benefit are emerging. Accumulating evidence on how viral infection and PRR signalling pathways intersect is providing further insights into the function of the pathways involved, their constituent proteins and ways in which they could be manipulated therapeutically.

Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll–interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction ‘downstream’ of TRIF but not of MyD88. SARM associated with TRIF, and ‘knockdown’ of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling.

Toll-like receptors discriminate between different pathogen-associated molecules and activate signaling cascades that lead to immune responses. The specificity of Toll-like receptor signaling occurs by means of adaptor proteins containing Toll–interleukin 1 receptor (TIR) domains. Activating functions have been assigned to four TIR adaptors: MyD88, Mal, TRIF and TRAM. Here we characterize a fifth TIR adaptor, SARM, as a negative regulator of TRIF-dependent Toll-like receptor signaling. Expression of SARM blocked gene induction ‘downstream’ of TRIF but not of MyD88. SARM associated with TRIF, and ‘knockdown’ of endogenous SARM expression by interfering RNA led to enhanced TRIF-dependent cytokine and chemokine induction. Thus, the fifth mammalian TIR adaptor SARM is a negative regulator of Toll-like receptor signaling.

Toll-like receptor 3 (TLR3), which recognizes double-stranded (ds)RNA, was the first identified antiviral TLR and, because dsRNA is a universal viral molecular pattern, TLR3 has been assumed to have a central role in the host response to viruses. However, this role has recently been questioned by in vivo studies and the discovery of several other antiviral pattern-recognition receptors. In this review, the function of TLR3 in the context of these other receptors, namely TLR7, 8 and 9 and the newly identified dsRNA-receptor retinoic-acid inducible gene-I (RIG-I) is discussed. Also, recent research concerning the expression profile of TLR3, its evasion by viruses and a potential role in crosspriming is addressed, which reveals a clearer appreciation of the contribution of TLR3 to antiviral immunity.

Toll-like receptor 3 (TLR3), which recognizes double-stranded (ds)RNA, was the first identified antiviral TLR and, because dsRNA is a universal viral molecular pattern, TLR3 has been assumed to have a central role in the host response to viruses. However, this role has recently been questioned by in vivo studies and the discovery of several other antiviral pattern-recognition receptors. In this review, the function of TLR3 in the context of these other receptors, namely TLR7, 8 and 9 and the newly identified dsRNA-receptor retinoic-acid inducible gene-I (RIG-I) is discussed. Also, recent research concerning the expression profile of TLR3, its evasion by viruses and a potential role in crosspriming is addressed, which reveals a clearer appreciation of the contribution of TLR3 to antiviral immunity.

It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-kB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-kB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-kB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-kB and stimulation of IkBa degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive to Nacetyl- L-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-kB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-kB activation and IkBa degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6,6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-kB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-kB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-kB activation b

It has been proposed that reactive oxygen species, and in particular H2O2, may be involved in the activation of NF-kB by diverse stimuli in different cell types. Here we have investigated the effect of a range of putative antioxidants on NF-kB activation by interleukin-1 and tumor necrosis factor as well as the ability of H2O2 to activate NF-kB in primary human umbilical vein endothelial cells and the transformed human endothelial cell line ECV304. Activation of NF-kB and stimulation of IkBa degradation by H2O2 was only evident in the transformed cells and required much longer contact times than that observed with interleukin-1 or tumor necrosis factor. Furthermore, only H2O2 was sensitive to Nacetyl- L-cysteine, and no increase in H2O2 was detected in response to either cytokine. Pyrrolidine dithiocarbamate has been purported to be a specific antioxidant inhibitor of NF-kB that acts independently of activating agent or cell type. However, we found that tumor necrosis factor- but not interleukin-1-driven NF-kB activation and IkBa degradation were sensitive to pyrrolidine dithiocarbamate in transformed cells, while neither pathway was inhibited in primary cells. Phorbol ester-mediated activation was sensitive in both transformed and primary cells. Other antioxidants failed to inhibit either cytokine, while the iron chelators desferrioxamine and 2,2,6,6-tetramethylpiperidine-1-oxyl mimicked the pattern of inhibition seen for the dithiocarbamate. This suggested that pyrrolidine dithiocarbamate was inhibiting NF-kB activation in endothelial cells primarily through its iron-chelating properties. Tumor necrosis factor, but not interleukin-1, was found to induce lipid peroxidation in ECV304 cells. This was inhibited by pyrrolidine dithiocarbamate and desferrioxamine. t-Butyl hydroperoxide, which induces lipid peroxidation, activated NF-kB. Finally, butylated hydroxyanisole, which inhibits lipid peroxidation but has no iron-chelating properties, inhibited NF-kB activation b