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SARM regulates CCL5 production in macrophages by promoting the recruitment of transcription factors and RNA polymerase II to the Ccl5 promoter

Mon, 12/08/2014 - 5:39pm

The four Toll/IL-1R domain-containing adaptor proteins MyD88, MAL, TRIF, and TRAM are well established as essential mediators of TLR signaling and gene induction following microbial detection. In contrast, the function of the fifth, most evolutionarily conserved Toll/IL-1R adaptor, sterile alpha and HEAT/Armadillo motif-containing protein (SARM), has remained more elusive. Recent studies of Sarm(-/-) mice have highlighted a role for SARM in stress-induced neuronal cell death and immune responses in the CNS. However, whether SARM has a role in immune responses in peripheral myeloid immune cells is less clear. Thus, we characterized TLR-induced cytokine responses in SARM-deficient murine macrophages and discovered a requirement for SARM in CCL5 production, whereas gene induction of TNF, IL-1beta, CCL2, and CXCL10 were SARM-independent. SARM was not required for TLR-induced activation of MAPKs or of transcription factors implicated in CCL5 induction, namely NF-kappaB and IFN regulatory factors, nor for Ccl5 mRNA stability or splicing. However, SARM was critical for the recruitment of transcription factors and of RNA polymerase II to the Ccl5 promoter. Strikingly, the requirement of SARM for CCL5 induction was not restricted to TLR pathways, as it was also apparent in cytosolic RNA and DNA responses. Thus, this study identifies a new role for SARM in CCL5 expression in macrophages.

Bacterial RNA:DNA hybrids are activators of the NLRP3 inflammasome

Mon, 12/08/2014 - 5:39pm

Enterohemorrhagic Escherichia coli (EHEC) is an extracellular pathogen that causes hemorrhagic colitis and hemolytic uremic syndrome. The proinflammatory cytokine, interleukin-1beta, has been linked to hemolytic uremic syndrome. Here we identify the nucleotide-binding domain and leucine rich repeat containing family, pyrin domain containing 3 (NLRP3) inflammasome as an essential mediator of EHEC-induced IL-1beta. Whereas EHEC-specific virulence factors were dispensable for NLRP3 activation, bacterial nucleic acids such as RNA:DNA hybrids and RNA gained cytosolic access and mediated inflammasome-dependent responses. Consistent with a direct role for RNA:DNA hybrids in inflammasome activation, delivery of synthetic EHEC RNA:DNA hybrids into the cytosol triggered NLRP3-dependent responses, and introduction of RNase H, which degrades such hybrids, into infected cells specifically inhibited inflammasome activation. Notably, an E. coli rnhA mutant, which is incapable of producing RNase H and thus harbors increased levels of RNA:DNA hybrid, induced elevated levels of NLRP3-dependent caspase-1 activation and IL-1beta maturation. Collectively, these findings identify RNA:DNA hybrids of bacterial origin as a unique microbial trigger of the NLRP3 inflammasome.

Post-transcriptional regulation of gene expression in innate immunity

Mon, 12/08/2014 - 5:39pm

Innate immune responses combat infectious microorganisms by inducing inflammatory responses, antimicrobial pathways and adaptive immunity. Multiple genes within each of these functional categories are coordinately and temporally regulated in response to distinct external stimuli. The substantial potential of these responses to drive pathological inflammation and tissue damage highlights the need for rigorous control of these responses. Although transcriptional control of inflammatory gene expression has been studied extensively, the importance of post-transcriptional regulation of these processes is less well defined. In this Review, we discuss the regulatory mechanisms that occur at the level of mRNA splicing, mRNA polyadenylation, mRNA stability and protein translation, and that have instrumental roles in controlling both the magnitude and duration of the inflammatory response.

3-Hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor (statin)-induced 28-kDa interleukin-1beta interferes with mature IL-1beta signaling

Mon, 12/08/2014 - 5:39pm

Multiple clinical trials have shown that the 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors known as statins have anti-inflammatory effects. However, the underlying molecular mechanism remains unclear. The proinflammatory cytokine interleukin-1beta (IL-1beta) is synthesized as a non-active precursor. The 31-kDa pro-IL-1beta is processed into the 17-kDa active form by caspase-1-activating inflammasomes. Here, we report a novel signaling pathway induced by statins, which leads to processing of pro-IL-1beta into an intermediate 28-kDa form. This statin-induced IL-1beta processing is independent of caspase-1- activating inflammasomes. The 28-kDa form of IL-1beta cannot activate interleukin-1 receptor-1 (IL1R1) to signal inflammatory responses. Instead, it interferes with mature IL-1beta signaling through IL-1R1 and therefore may dampen inflammatory responses initiated by mature IL-1beta. These results may provide new clues to explain the anti-inflammatory effects of statins.

Gadolinium-based compounds induce NLRP3-dependent IL-1beta production and peritoneal inflammation

Mon, 12/08/2014 - 5:39pm

OBJECTIVE: Nephrogenic systemic fibrosis (NSF) is a progressive fibrosing disorder that may develop in patients with chronic kidney disease after administration of gadolinium (Gd)-based contrast agents (GBCAs). In the setting of impaired renal clearance of GBCAs, Gd deposits in various tissues and fibrosis subsequently develops. However, the precise mechanism by which fibrosis occurs in NSF is incompletely understood. Because other profibrotic agents, such as silica or asbestos, activate the nucleotide-binding oligomerisation domain (NOD)-like receptor protein 3 (NLRP3) inflammasome and initiate interleukin (IL)-1beta release with the subsequent development of fibrosis, we evaluated the effects of GBCAs on inflammasome activation.

METHODS: Bone marrow derived macrophages from C57BL/6, Nlrp3-/- and Asc-/- mice were incubated with three Gd-containing compounds and IL-1beta activation and secretion was detected by ELISA and western blot analysis. Inflammasome activation and regulation was investigated in IL-4- and interferon (IFN)gamma-polarised macrophages by ELISA, quantitative real time (qRT)-PCR and NanoString nCounter analysis. Furthermore, C57BL/6 and Nlrp3-/-mice were intraperitoneally injected with GBCA and recruitment of inflammatory cells to the peritoneum was analysed by fluorescence-activated cell sorting (FACS).

RESULTS: Free Gd and GBCAs activate the NLRP3 inflammasome and induce IL-1beta secretion in vitro. Gd-diethylenetriaminepentaacetic acid also induces the recruitment of neutrophils and inflammatory monocytes to the peritoneum in vivo. Gd activated IL-4-polarised macrophages more effectively than IFNgamma-polarised macrophages, which preferentially expressed genes known to downregulate inflammasome activity.

CONCLUSIONS: These data suggest that Gd released from GBCAs triggers a NLRP3 inflammasome-dependent inflammatory response that leads to fibrosis in an appropriate clinical setting. The preferential activation of IL-4-differentiated macrophages is consistent with the predominantly fibrotic presentation of NSF. already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.

A single vertebrate DNA virus protein disarms invertebrate immunity to RNA virus infection

Mon, 12/08/2014 - 5:39pm

Virus-host interactions drive a remarkable diversity of immune responses and countermeasures. We found that two RNA viruses with broad host ranges, vesicular stomatitis virus (VSV) and Sindbis virus (SINV), are completely restricted in their replication after entry into Lepidopteran cells. This restriction is overcome when cells are co-infected with vaccinia virus (VACV), a vertebrate DNA virus. Using RNAi screening, we show that Lepidopteran RNAi, Nuclear Factor-kappaB, and ubiquitin-proteasome pathways restrict RNA virus infection. Surprisingly, a highly conserved, uncharacterized VACV protein, A51R, can partially overcome this virus restriction. We show that A51R is also critical for VACV replication in vertebrate cells and for pathogenesis in mice. Interestingly, A51R colocalizes with, and stabilizes, host microtubules and also associates with ubiquitin. We show that A51R promotes viral protein stability, possibly by preventing ubiquitin-dependent targeting of viral proteins for destruction. Importantly, our studies reveal exciting new opportunities to study virus-host interactions in experimentally-tractable Lepidopteran systems.

The caspase-8 homolog Dredd cleaves Imd and Relish but is not inhibited by p35

Mon, 12/08/2014 - 5:39pm

In Drosophila, the Imd pathway is activated by diaminopimelic acid-type peptidoglycan and triggers the humoral innate immune response, including the robust induction of antimicrobial peptide gene expression. Imd and Relish, two essential components of this pathway, are both endoproteolytically cleaved upon immune stimulation. Genetic analyses have shown that these cleavage events are dependent on the caspase-8 like Dredd, suggesting that Imd and Relish are direct substrates of Dredd. Among the seven Drosophila caspases, we find that Dredd uniquely promotes Imd and Relish processing, and purified recombinant Dredd cleaves Imd and Relish in vitro. In addition, interdomain cleavage of Dredd is not required for Imd or Relish processing and is not observed during immune stimulation. Baculovirus p35, a suicide substrate of executioner caspases, is not cleaved by purified Dredd in vitro. Consistent with this biochemistry but contrary to earlier reports, p35 does not interfere with Imd signaling in S2* cells or in vivo.

TRIL is involved in cytokine production in the brain following Escherichia coli infection

Mon, 12/08/2014 - 5:39pm

TLR4 interactor with leucine-rich repeats (TRIL) is a brain-enriched accessory protein that is important in TLR3 and TLR4 signaling. In this study, we generated Tril(-/-) mice and examined TLR responses in vitro and in vivo. We found a role for TRIL in both TLR4 and TLR3 signaling in mixed glial cells, consistent with the high level of expression of TRIL in these cells. We also found that TRIL is a modulator of the innate immune response to LPS challenge and Escherichia coli infection in vivo. Tril(-/-) mice produce lower levels of multiple proinflammatory cytokines and chemokines specifically within the brain after E. coli and LPS challenge. Collectively, these data uncover TRIL as a mediator of innate immune responses within the brain, where it enhances neuronal cytokine responses to infection.

Interferon gamma-inducible protein (IFI) 16 transcriptionally regulates type i interferons and other interferon-stimulated genes and controls the interferon response to both DNA and RNA viruses

Mon, 12/08/2014 - 5:39pm

The interferon gamma-inducible protein 16 (IFI16) has recently been linked to the detection of nuclear and cytosolic DNA during infection with herpes simplex virus-1 and HIV. IFI16 binds dsDNA via HIN200 domains and activates stimulator of interferon genes (STING), leading to TANK (TRAF family member-associated NF-kappaB activator)-binding kinase-1 (TBK1)-dependent phosphorylation of interferon regulatory factor (IRF) 3 and transcription of type I interferons (IFNs) and related genes. To better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as cyclic dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to DNA ligands and viruses. In contrast, expression of the NF-kappaB-regulated cytokines IL-6 and IL-1beta was unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of IFN-alpha and the IFN-stimulated gene retinoic acid-inducible gene I (RIG-I) in response to cyclic GMP-AMP, a second messenger produced by cyclic GMP-AMP synthase (cGAS) as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-alpha promoter in these cells, suggesting that transcription of IFN-stimulated genes is dependent on IFI16. These results indicate a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in antiviral immunity.

Genetic rescue of functional senescence in synaptic and behavioral plasticity

Mon, 12/08/2014 - 5:39pm

STUDY OBJECTIVES: Aging has been linked with decreased neural plasticity and memory formation in humans and in laboratory model species such as the fruit fly, Drosophila melanogaster. Here, we examine plastic responses following social experience in Drosophila as a high-throughput method to identify interventions that prevent these impairments.

PATIENTS OR PARTICIPANTS: Wild-type and transgenic Drosophila melanogaster.

DESIGN AND INTERVENTIONS: Young (5-day old) or aged (20-day old) adult female Drosophila were housed in socially enriched (n = 35-40) or isolated environments, then assayed for changes in sleep and for structural markers of synaptic terminal growth in the ventral lateral neurons (LNVs) of the circadian clock.

MEASUREMENTS AND RESULTS: When young flies are housed in a socially enriched environment, they exhibit synaptic elaboration within a component of the circadian circuitry, the LNVs, which is followed by increased sleep. Aged flies, however, no longer exhibit either of these plastic changes. Because of the tight correlation between neural plasticity and ensuing increases in sleep, we use sleep after enrichment as a high-throughput marker for neural plasticity to identify interventions that prolong youthful plasticity in aged flies. To validate this strategy, we find three independent genetic manipulations that delay age-related losses in plasticity: (1) elevation of dopaminergic signaling, (2) over-expression of the transcription factor blistered (bs) in the LNVs, and (3) reduction of the Imd immune signaling pathway. These findings provide proof-of-principle evidence that measuring changes in sleep in flies after social enrichment may provide a highly scalable assay for the study of age-related deficits in synaptic plasticity.

CONCLUSIONS: These studies demonstrate that Drosophila provides a promising model for the study of age-related loss of neural plasticity and begin to identify genes that might be manipulated to delay the onset of functional senescence.

Citrobacter rodentium: infection, inflammation and the microbiota

Mon, 12/08/2014 - 5:39pm

Citrobacter rodentium is a mucosal pathogen of mice that shares several pathogenic mechanisms with enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC), which are two clinically important human gastrointestinal pathogens. Thus, C. rodentium has long been used as a model to understand the molecular basis of EPEC and EHEC infection in vivo. In this Review, we discuss recent studies in which C. rodentium has been used to study mucosal immunology, including the deregulation of intestinal inflammatory responses during bacteria-induced colitis and the role of the intestinal microbiota in mediating resistance to colonization by enteric pathogens. These insights should help to elucidate the roles of mucosal inflammatory responses and the microbiota in the virulence of enteric pathogens.

TRIM13 is a negative regulator of MDA5-mediated type I interferon production

Mon, 12/08/2014 - 5:39pm

Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are essential intracellular detectors of viral RNA. They contribute to the type I interferon (IFN) response that is crucial for host defense against viral infections. Given the potent antiviral and proinflammatory activities elicited by the type I IFNs, induction of the type I IFN response is tightly regulated. Members of the tripartite motif (TRIM) family of proteins have recently emerged as key regulators of antiviral immunity. We show that TRIM13, an E3 ubiquitin ligase, is expressed in immune cells and is upregulated in bone marrow-derived macrophages upon stimulation with inducers of type I IFN. TRIM13 interacts with MDA5 and negatively regulates MDA5-mediated type I IFN production in vitro, acting upstream of IFN regulatory factor 3. We generated Trim13(-/-) mice and show that upon lethal challenge with encephalomyocarditis virus (EMCV), which is sensed by MDA5, Trim13(-/-) mice produce increased amounts of type I IFNs and survive longer than wild-type mice. Trim13(-/-) murine embryonic fibroblasts (MEFs) challenged with EMCV or poly(I . C) also show a significant increase in beta IFN (IFN-beta) levels, but, in contrast, IFN-beta responses to the RIG-I-detected Sendai virus were diminished, suggesting that TRIM13 may play a role in positively regulating RIG-I function. Together, these results demonstrate that TRIM13 regulates the type I IFN response through inhibition of MDA5 activity and that it functions nonredundantly to modulate MDA5 during EMCV infection. IMPORTANCE: The type I interferon (IFN) response is crucial for host defense against viral infections, and proper regulation of this pathway contributes to maintaining immune homeostasis. Retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5) are intracellular detectors of viral RNA that induce the type I IFN response. In this study, we show that expression of the gene tripartite motif 13 (Trim13) is upregulated in response to inducers of type I IFN and that TRIM13 interacts with both MDA5 and RIG-I in vitro. Through the use of multiple in vitro and in vivo model systems, we show that TRIM13 is a negative regulator of MDA5-mediated type I IFN production and may also impact RIG-I-mediated type I IFN production by enhancing RIG-I activity. This places TRIM13 at a key junction within the viral response pathway and identifies it as one of the few known modulators of MDA5 activity.

Caspase-8 modulates dectin-1 and complement receptor 3-driven IL-1beta production in response to beta-glucans and the fungal pathogen, Candida albicans

Mon, 12/08/2014 - 5:39pm

Inflammasomes are central mediators of host defense to a wide range of microbial pathogens. The nucleotide-binding domain and leucine-rich repeat containing family (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a key role in triggering caspase-1-dependent IL-1beta maturation and resistance to fungal dissemination in Candida albicans infection. beta-Glucans are major components of fungal cell walls that trigger IL-1beta secretion in both murine and human immune cells. In this study, we sought to determine the contribution of beta-glucans to C. albicans-induced inflammasome responses in mouse dendritic cells. We show that the NLRP3-apoptosis-associated speck-like protein containing caspase recruitment domain protein-caspase-1 inflammasome is absolutely critical for IL-1beta production in response to beta-glucans. Interestingly, we also found that both complement receptor 3 (CR3) and dectin-1 play a crucial role in coordinating beta-glucan-induced IL-1beta processing as well as a cell death response. In addition to the essential role of caspase-1, we identify an important role for the proapoptotic protease caspase-8 in promoting beta-glucan-induced cell death and NLRP3 inflammasome-dependent IL-1beta maturation. A strong requirement for CR3 and caspase-8 also was found for NLRP3-dependent IL-1beta production in response to heat-killed C. albicans. Taken together, these results define the importance of dectin-1, CR3, and caspase-8, in addition to the canonical NLRP3 inflammasome, in mediating beta-glucan- and C. albicans-induced innate responses in dendritic cells. Collectively, these findings establish a novel link between beta-glucan recognition receptors and the inflammatory proteases caspase-8 and caspase-1 in coordinating cytokine secretion and cell death in response to immunostimulatory fungal components.

Long non-coding RNAs and control of gene expression in the immune system

Mon, 12/08/2014 - 5:39pm

All cells of the immune system rely on a highly integrated and dynamic gene expression program that is controlled by both transcriptional and post-transcriptional mechanisms. Recently, non-coding RNAs, including long non-coding RNAs (lncRNAs), have emerged as important regulators of gene expression in diverse biological contexts. lncRNAs control gene expression in the nucleus by modulating transcription or via post-transcriptional mechanisms targeting the splicing, stability, or translation of mRNAs. Our knowledge of lncRNA biogenesis, their cell type-specific expression, and their versatile molecular functions is rapidly progressing in all areas of biology. We discuss here these exciting new regulators and highlight an emerging paradigm of lncRNA-mediated control of gene expression in the immune system.

Transcription of Inflammatory Genes: Long Noncoding RNA and Beyond

Mon, 12/08/2014 - 5:39pm

The innate immune system must coordinate elaborate signaling pathways to turn on expression of hundreds of genes to provide protection against pathogens and resolve acute inflammation. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional on and off switches in response to distinct external stimuli. Three classes of transcription factors act together with transcriptional coregulators and chromatin-modifying complexes to control these programs. In addition, newer studies implicate long noncoding RNA (lncRNA) as additional regulators of these responses. LncRNAs promote, fine-tune, and restrain the inflammatory program. In this study, we provide an overview of gene regulation and the emerging importance of lncRNAs in the immune system.

IKKalpha negatively regulates ASC-dependent inflammasome activation

Mon, 12/08/2014 - 5:39pm

The inflammasomes are multiprotein complexes that activate caspase-1 in response to infections and stress, resulting in the secretion of pro-inflammatory cytokines. Here we report that IkappaB kinase alpha (IKKalpha) is a critical negative regulator of apoptosis-associated specklike protein containing a C-terminal caspase-activation-andrecruitment (CARD) domain (ASC)-dependent inflammasomes. IKKalpha controls the inflammasome at the level of the adaptor ASC, which interacts with IKKalpha in the nucleus of resting macrophages in an IKKalpha kinase-dependent manner. Loss of IKKalpha kinase activity results in inflammasome hyperactivation. Mechanistically, the downstream nuclear effector IKK-related kinase (IKKi) facilitates translocation of ASC from the nucleus to the perinuclear area during inflammasome activation. ASC remains under the control of IKKalpha in the perinuclear area following translocation of the ASC/IKKalpha complex. Signal 2 of NLRP3 activation leads to inhibition of IKKalpha kinase activity through the recruitment of PP2A, allowing ASC to participate in NLRP3 inflammasome assembly. Taken together, these findings reveal a IKKi-IKKalpha-ASC axis that serves as a common regulatory mechanism for ASC-dependent inflammasomes.

Innate sensing of malaria parasites

Mon, 12/08/2014 - 5:39pm

Innate immune receptors have a key role in immune surveillance by sensing microorganisms and initiating protective immune responses. However, the innate immune system is a classic 'double-edged sword' that can overreact to pathogens, which can have deleterious effects and lead to clinical manifestations. Recent studies have unveiled the complexity of innate immune receptors that function as sensors of Plasmodium spp. in the vertebrate host. This Review highlights the cellular and molecular mechanisms by which Plasmodium infection is sensed by different families of innate immune receptors. We also discuss how these events mediate both host resistance to infection and the pathogenesis of malaria.