NLRP3 activation and mitosis are mutually exclusive events coordinated by NEK7, a new inflammasome component.

The NLRP3 inflammasome responds to microbes and danger signals by processing and activating proinflammatory cytokines, including interleukin 1ß (IL-1ß) and IL-18. We found here that activation of the NLRP3 inflammasome was restricted to interphase of the cell cycle by NEK7, a serine-threonine kinase previously linked to mitosis. Activation of the NLRP3 inflammasome required NEK7, which bound to the leucine-rich repeat domain of NLRP3 in a kinase-independent manner downstream of the induction of mitochondrial reactive oxygen species (ROS). This interaction was necessary for the formation of a complex containing NLRP3 and the adaptor ASC, oligomerization of ASC and activation of caspase-1. NEK7 promoted the NLRP3-dependent cellular inflammatory response to intraperitoneal challenge with monosodium urate and the development of experimental autoimmune encephalitis in mice. Our findings suggest that NEK7 serves as a cellular switch that enforces mutual exclusivity of the inflammasome response and cell division.

Immunoediting role for major vault protein in apoptotic signaling induced by bacterial N-acyl homoserine lactones.

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

The use of small molecule probes to study spatially separated stimulus-induced signaling pathways.

Simultaneous activation of signaling pathways requires dynamic assembly of higher-order protein complexes at the cytoplasmic domains of membrane-associated receptors in a stimulus-specific manner. Here, using the paradigm of cellular activation through cytokine and innate immune receptors, we demonstrate the proof-of-principle application of small molecule probes for the dissection of receptor-proximal signaling processes, such as activation of the transcription factor NF-κB and the protein kinase p38.

An inflammasome-independent role for epithelial-expressed Nlrp3 in renal ischemia-reperfusion injury.

Cytoplasmic innate immune receptors are important therapeutic targets for diseases associated with overproduction of proinflammatory cytokines. One cytoplasmic receptor complex, the Nlrp3 inflammasome, responds to an extensive array of molecules associated with cellular stress. Under normal conditions, Nlrp3 is autorepressed, but in the presence of its ligands, it oligomerizes, recruits apoptosis-associated speck-like protein containing a caspase recruitment domain (Asc), and triggers caspase 1 activation and the maturation of proinflammatory cytokines such as IL-1ß and IL-18. Because ischemic tissue injury provides a potential source for Nlrp3 ligands, our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in components of the Nlrp3 inflammasome (Nlrp3(-/-) and Asc(-/-) mice). To examine the role of the inflammasome in renal ischemia-reperfusion injury (IRI) we also tested its downstream targets caspase 1, IL-1ß, and IL-18. Both Nlrp3 and Asc were highly expressed in renal tubular epithelium of humans and mice, and the absence of Nlrp3, but not Asc or the downstream inflammasome targets, dramatically protected from kidney IRI. We conclude that Nlrp3 contributes to renal IRI by a direct effect on renal tubular epithelium and that this effect is independent of inflammasome-induced proinflammatory cytokine production.

Nod1 and nod2 are expressed in human and murine renal tubular epithelial cells and participate in renal ischemia reperfusion injury.

Nucleotide-binding oligomerization domain (Nod) 1 and Nod2 are members of a family of intracellular innate sensors that participate in innate immune responses to pathogens and molecules released during the course of tissue injury, including injury induced by ischemia. Ischemic injury to the kidney is characterized by renal tubular epithelial apoptosis and inflammation. Among the best studied intracellular innate immune receptors known to contribute to apoptosis and inflammation are Nod1 and Nod2. Our study compared and contrasted the effects of renal ischemia in wild-type mice and mice deficient in Nod1, Nod2, Nod(1 x 2), and in their downstream signaling molecule receptor-interacting protein 2. We found that Nod1 and Nod2 were present in renal tubular epithelial cells in both mouse and human kidneys and that the absence of these receptors in mice resulted in protection from kidney ischemia reperfusion injury. Significant protection from kidney injury was seen with a deficiency of Nod2 and receptor-interacting protein 2, and the simultaneous deficiency of Nod1 and Nod2 provided even greater protection. We conclude that the intracellular sensors Nod1 and Nod2 play an important role in the pathogenesis of acute ischemic injury of the kidney, although possibly through different mechanisms.

Enhanced bacterial clearance and sepsis resistance in caspase-12-deficient mice.

Caspases function in both apoptosis and inflammatory cytokine processing and thereby have a role in resistance to sepsis. Here we describe a novel role for a caspase in dampening responses to bacterial infection. We show that in mice, gene-targeted deletion of caspase-12 renders animals resistant to peritonitis and septic shock. The resulting survival advantage was conferred by the ability of the caspase-12-deficient mice to clear bacterial infection more efficiently than wild-type littermates. Caspase-12 dampened the production of the pro-inflammatory cytokines interleukin (IL)-1beta, IL-18 (interferon (IFN)-gamma inducing factor) and IFN-gamma, but not tumour-necrosis factor-alpha and IL-6, in response to various bacterial components that stimulate Toll-like receptor and NOD pathways. The IFN-gamma pathway was crucial in mediating survival of septic caspase-12-deficient mice, because administration of neutralizing antibodies to IFN-gamma receptors ablated the survival advantage that otherwise occurred in these animals. Mechanistically, caspase-12 associated with caspase-1 and inhibited its activity. Notably, the protease function of caspase-12 was not necessary for this effect, as the catalytically inactive caspase-12 mutant Cys299Ala also inhibited caspase-1 and IL-1beta production to the same extent as wild-type caspase-12. In this regard, caspase-12 seems to be the cFLIP counterpart for regulating the inflammatory branch of the caspase cascade. In mice, caspase-12 deficiency confers resistance to sepsis and its presence exerts a dominant-negative suppressive effect on caspase-1, resulting in enhanced vulnerability to bacterial infection and septic mortality.

Infection control by antibody disruption of bacterial quorum sensing signaling.

Quorum sensing (QS) is the process through which bacteria communicate utilizing small diffusible molecules termed autoinducers. It has been demonstrated that QS controls a plethora of microbial processes including the expression of virulence factors. Here we report an immunopharmacotherapeutic approach for the attenuation of QS in the Gram-positive human pathogen Staphylococcus aureus. An anti-autoinducer monoclonal antibody, AP4-24H11, was elicited against a rationally designed hapten, and efficiently inhibited QS in vitro through the sequestration of the autoinducing peptide (AIP)-4 produced by S. aureus RN4850. Importantly, AP4-24H11 suppressed S. aureus pathogenicity in an abscess formation mouse model in vivo and provided complete protection against a lethal S. aureus challenge. These findings provide a strong foundation for further investigations of immunopharmacotherapy for the treatment of bacterial infections in which QS controls the expression of virulence factors.

Toll-like receptor 9 mediates CpG-DNA signaling.

Among the bacterial products known to activate the innate immune '1system is bacterial DNA. This activity resides within the nonmethylated CpG motifs of the DNA and is recapitulated using appropriate synthetic CpG containing oligodeoxynucleotides (CpG-ODN). TLR9-deficient mice were shown to exhibit a nonresponsive phenotype-to-bacterial DNA and CpG-ODN. Here, we describe a model system to further characterize CpG-ODN and TLR9 interactions using ectopically expressed TLR9 in HEK293 cells. Expression of TLR9 confers cellular responsiveness to CpG-ODN but not to the other bacterial products. Previous studies identified species-specific CpG-containing sequences; here, we show that expression of murine TLR9 favors responses to CpG-ODN motifs specific to mouse cells, and expression of human TLR9 favors CpG-ODN known to preferentially activate human cells. Response patterns to various CpG-ODN motifs were parallel when cells containing an ectopically expressed TLR9 and endogenous receptor were compared. Here, we also show that TLR9 acts at the cell surface and engages an intracellular signaling pathway that includes MyD88, IRAK, and TRAF6.

IRAK-4 kinase activity-dependent and -independent regulation of lipopolysaccharide-inducible genes.

IRAK-4 kinase inactive (IRAK-4 KD) knock-in mice display defects in TLR- and IL-1 receptor signaling and are resistant to LPS-induced shock. In the present study we examined the LPS-induced response in IRAK-4 KD mice in more detail. We show that IRAK-4 kinase activity is required for certain aspects of TLR-mediated signaling but not for others. We found that IRAK-4 KD cells displayed reduced JNK and p38 signaling, while NF-kappaB was activated to a normal level but with delayed kinetics compared to wild-type cells. TLR4-mediated IRF3 activation was intact in these cells. Comprehensive analysis of expression of LPS-inducible genes by microarray demonstrated that IRAK-4 KD cells were severely impaired in the expression of many pro-inflammatory genes, suggesting their dependence on IRAK-4 kinase activity. In contrast, the expression of a subset of LPS-induced genes of anti-viral response was not affected by IRAK-4 kinase deficiency. Additionally, we demonstrate that LPS-activated early expression and production of some cytokines, e.g., TNF-alpha, is partially induced in the absence of IRAK-4 kinase activity. This suggests that the partially unaffected TLR4-mediated signaling could still drive expression of these genes in early phases and that IRAK-4 kinase activity is important for a more sustained anti-bacterial response.

Modulation of gene expression via disruption of NF-kappaB signaling by a bacterial small molecule.

The control of innate immune responses through activation of the nuclear transcription factor NF-kappaB is essential for the elimination of invading microbial pathogens. We showed that the bacterial N-(3-oxo-dodecanoyl) homoserine lactone (C12) selectively impairs the regulation of NF-kappaB functions in activated mammalian cells. The consequence is specific repression of stimulus-mediated induction of NF-kappaB-responsive genes encoding inflammatory cytokines and other immune regulators. These findings uncover a strategy by which C12-producing opportunistic pathogens, such as Pseudomonas aeruginosa, attenuate the innate immune system to establish and maintain local persistent infection in humans, for example, in cystic fibrosis patients.

IRAK-4 kinase activity is required for interleukin-1 (IL-1) receptor- and toll-like receptor 7-mediated signaling and gene expression.

IRAK-4 is an essential component of the signal transduction complex downstream of the IL-1- and Toll-like receptors. Although regarded as the first kinase in the signaling cascade, the role of IRAK-4 kinase activity versus its scaffold function is still controversial. To investigate the role of IRAK-4 kinase function in vivo, "knock-in" mice were generated by replacing the wild type IRAK-4 gene with a mutant gene encoding kinase-deficient IRAK-4 protein (IRAK-4 KD). IRAK-4 kinase was rendered inactive by mutating the conserved lysine residues in the ATP pocket essential for coordinating ATP. Analyses of embryonic fibroblasts and macrophages obtained from IRAK-4 KD mice demonstrate lack of cellular responsiveness to stimulation with IL-1beta or a Toll-like receptor 7 (TLR7) agonist. IRAK-4 kinase deficiency prevents the recruitment of IRAK-1 to the IL-1 receptor complex and its subsequent phosphorylation and degradation. IRAK-4 KD cells are severely impaired in NFkappaB, JNK, and p38 activation in response to IL-1beta or TLR7 ligand. As a consequence, IL-1 receptor/TLR7-mediated production of cytokines and chemokines is largely absent in these cells. Additionally, microarray analysis identified IL-1beta response genes and revealed that the induction of IL-1beta-responsive mRNAs is largely ablated in IRAK-4 KD cells. In summary, our results suggest that IRAK-4 kinase activity plays a critical role in IL-1 receptor (IL-1R)/TLR7-mediated induction of inflammatory responses.

Triad3A regulates ubiquitination and proteasomal degradation of RIP1 following disruption of Hsp90 binding.

Toll-like receptors (TLRs) play a crucial role in innate immunity by recognizing microbial pathogens. Triad3A is an E3 ubiquitin-protein ligase that interacts with the Toll/interleukin-1 receptor domain of TLRs and promotes their proteolytic degradation. In the present study, we further investigated its activity on signaling molecules downstream of TLRs and tumor necrosis factor (TNF) receptor 1. Triad3A promoted down-regulation of two TIR domain-containing adapter proteins, TIRAP and TRIF, as well as a RIP1 but had no effect on other adapter molecules in either the TLRs or TNF-alpha signaling pathways. Multiple sequence alignment analysis suggested that RIP1 contains a TIR homologous domain, and mutation of amino acid residues in this domain identified three residues critical for its interaction with Triad3A. Moreover, Triad3A acted as a negative regulator in TNF-alpha signaling. Reduction of Triad3A expression by small interference RNAs rendered cells hyperresponsive to TNF-alpha stimulation. Conversely, overexpression of Triad3A in cells blocked TNF-alpha-induced cell activation. This negative regulation was effected independently of changes in the cellular protein level of RIP1. Further studies indicated that RIP1 formed a complex with Triad3A and heat shock protein 90 (Hsp90), which is a chaperone protein capable of maintaining the stability of its client proteins. Treatment of cells with geldanamycin to disrupt the Hsp90 complex led to proteasomal degradation of RIP1. Depletion of Triad3A by small interference RNA treatment inhibited geldanamycin-activated ubiquitination and proteolytic degradation of RIP1. These results suggest that Triad3A is an E3 ubiquitin-protein ligase to RIP1 and that Hsp90 and Triad3A cooperatively maintain the homeostasis of RIP1.

NF-kappa B-inducing kinase regulates selected gene expression in the Nod2 signaling pathway.

The innate immune system surveys the extra- and intracellular environment for the presence of microbes. Among the intracellular sensors is a protein known as Nod2, a cytosolic protein containing a leucine-rich repeat domain. Nod2 is believed to play a role in determining host responses to invasive bacteria. A key element in upregulating host defense involves activation of the NF-kappaB pathway. It has been suggested through indirect studies that NF-kappaB-inducing kinase, or NIK, may be involved in Nod2 signaling. Here we have used macrophages derived from primary explants of bone marrow from wild-type mice and mice that either bear a mutation in NIK, rendering it inactive, or are derived from NIK-/- mice, in which the NIK gene has been deleted. We show that NIK binds to Nod2 and mediates induction of specific changes induced by the specific Nod2 activator, muramyl dipeptide, and that the role of NIK occurs in settings where both the Nod2 and TLR4 pathways are activated by their respective agonists. Specifically, we have linked NIK to the induction of the B-cell chemoattractant known as BLC and suggest that this chemokine may play a role in processes initiated by Nod2 activation that lead to improved host defense.

N-(3-oxo-acyl)homoserine lactones signal cell activation through a mechanism distinct from the canonical pathogen-associated molecular pattern recognition receptor pathways.

Innate immune system receptors function as sensors of infection and trigger the immune responses through ligand-specific signaling pathways. These ligands are pathogen-associated products, such as components of bacterial walls and viral nuclear acids. A common response to such ligands is the activation of mitogen-activated protein kinase p38, whereas double-stranded viral RNA additionally induces the phosphorylation of eukaryotic translation initiation factor 2alpha (eIF2alpha). Here we have shown that p38 and eIF2alpha phosphorylation represent two biochemical markers of the effects induced by N-(3-oxo-acyl)homoserine lactones, the secreted products of a number of Gram-negative bacteria, including the human opportunistic pathogen Pseudomonas aeruginosa. Furthermore, N-(3-oxo-dodecanoyl)homoserine lactone induced distension of mitochondria and the endoplasmic reticulum as well as c-jun gene transcription. These effects occurred in a wide variety of cell types including alveolar macrophages and bronchial epithelial cells, requiring the structural integrity of the lactone ring motif and its natural stereochemistry. These findings suggest that N-(3-oxo-acyl)homoserine lactones might be recognized by receptors of the innate immune system. However, we provide evidence that N-(3-oxo-dodecanoyl)homoserine lactone-mediated signaling does not require the presence of the canonical innate immune system receptors, Toll-like receptors, or two members of the NLR/Nod/Caterpillar family, Nod1 and Nod2. These data offer a new understanding of the effects of N-(3-oxo-dodecanoyl)homoserine lactone on host cells and its role in persistent airway infections caused by P. aeruginosa.

Innate immune responses during infection.

The innate immune system senses bacteria in the environment and defends against infection. Here we will discuss two types of sensor protein families. The plasma membrane receptors that comprise the Toll-like receptor (TLRs) family and the intracellular proteins termed NOD1 and NOD2. These proteins directly bind bacterial products such as lipopolysaccharides (LPS), peptidoglycan fragments, bacterial DNA, and receptor binding leads to intracellular signaling and gene expression. TLR signaling involves members of the MyD88 family of adaptor proteins. In contrast NOD1 or NOD2 utilize pathways that do not depend on the MyD88 family members.

IKKi/IKKepsilon plays a key role in integrating signals induced by pro-inflammatory stimuli.

We report that the product of the inducible gene encoding the kinase known as IKKi/IKKepsilon (IKKi) is required for expression of a group of genes up-regulated by pro-inflammatory stimuli such as bacterial endotoxin (lipopolysaccharide (LPS)). Here, using murine embryonic fibroblasts obtained from mice bearing deletions in IKK2, p65, and IKKi genes, we provide evidence to support a link between signaling through the NF-kappaB and CCAAA/enhancer-binding protein (C/EBP) pathways. This link includes an NF-kappaB-dependent regulation of C/EBPbeta and C/EBPdelta gene transcription and IKKi-mediated activation of C/EBP. Disruption of the NF-kappaB pathway results in the blockade of the inducible up-regulation of C/EBPbeta, C/EBPdelta, and IKKi genes. Cells lacking IKKi are normal in activation of the canonical NF-kappaB pathway but fail to induce C/EBPdelta activity and transcription of C/EBP and C/EBP-NF-kappaB target genes in response to LPS. In addition we show that, in response to LPS or tumor necrosis factor alpha, both beta and delta subunits of C/EBP interact with IKKi promoter, suggesting a feedback mechanism in the regulation of IKKi-dependent cellular processes. These data are among the first to provide insights into the biological function of IKKi.