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1.  The methyltransferase Setdb2 mediates virus-induced susceptibility to bacterial superinfection 
Nature immunology  2014;16(1):67-74.
Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that SET domain bifurcated 2 (Setdb2) was the only protein lysine methyltransferase induced during influenza virus infection. Setdb2 expression depended on type-I interferon signaling and it repressed the expression of the neutrophil attractant Cxcl1 and other NF-κB target genes. This coincided with Setdb2 occupancy at the Cxcl1 promoter, which in the absence of Setdb2 displayed reduced H3K9 tri-methylation. Setdb2 hypomorphic gene-trap mice exhibited increased neutrophil infiltration in sterile lung inflammation and were less sensitive to bacterial superinfection upon influenza virus infection. This suggests that a Setdb2-mediated regulatory crosstalk between the type-I interferon and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.
doi:10.1038/ni.3046
PMCID: PMC4320687  PMID: 25419628
2.  Antigen expression determines adenoviral vaccine potency independent of IFN and STING signaling 
The Journal of Clinical Investigation  2015;125(3):1129-1146.
Recombinant adenoviral vectors (rAds) are lead vaccine candidates for protection against a variety of pathogens, including Ebola, HIV, tuberculosis, and malaria, due to their ability to potently induce T cell immunity in humans. However, the ability to induce protective cellular immunity varies among rAds. Here, we assessed the mechanisms that control the potency of CD8 T cell responses in murine models following vaccination with human-, chimpanzee-, and simian-derived rAds encoding SIV-Gag antigen (Ag). After rAd vaccination, we quantified Ag expression and performed expression profiling of innate immune response genes in the draining lymph node. Human-derived rAd5 and chimpanzee-derived chAd3 were the most potent rAds and induced high and persistent Ag expression with low innate gene activation, while less potent rAds induced less Ag expression and robustly induced innate immunity genes that were primarily associated with IFN signaling. Abrogation of type I IFN or stimulator of IFN genes (STING) signaling increased Ag expression and accelerated CD8 T cell response kinetics but did not alter memory responses or protection. These findings reveal that the magnitude of rAd-induced memory CD8 T cell immune responses correlates with Ag expression but is independent of IFN and STING and provide criteria for optimizing protective CD8 T cell immunity with rAd vaccines.
doi:10.1172/JCI78280
PMCID: PMC4362254  PMID: 25642773
3.  Systems-Level Analysis of Innate Immunity 
Annual review of immunology  2014;32:547-577.
Systems-level analysis of biological processes strives to comprehensively and quantitatively evaluate the interactions between the relevant molecular components over time, thereby enabling development of models that can be employed to ultimately predict behavior. Rapid development in measurement technologies (omics), when combined with the accessible nature of the cellular constituents themselves, is allowing the field of innate immunity to take significant strides toward this lofty goal. In this review, we survey exciting results derived from systems biology analyses of the immune system, ranging from gene regulatory networks to influenza pathogenesis and systems vaccinology.
doi:10.1146/annurev-immunol-032713-120254
PMCID: PMC4084718  PMID: 24655298
systems biology; transcriptomics; proteomics; lipidomics; influenza; systems vaccinology
4.  A Polymorphism in Toll-Interleukin 1 Receptor Domain Containing Adaptor Protein Is Associated with Susceptibility to Meningeal Tuberculosis 
The Journal of infectious diseases  2006;194(8):1127-1134.
Background
Although meningitis is the most severe form of infection caused by Mycobacterium tuberculosis, the immunopathogenesis of this disease is poorly understood. We tested the hypothesis that polymorphisms in Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP), an adaptor protein that mediates signals from Toll-like receptors activated by mycobacteria, are associated with susceptibility to tuberculosis (TB).
Methods
We used a case-population study design in Vietnam with cord-blood control samples (n = 392) and case patients (n = 358) who had either pulmonary (n = 183) or meningeal (n = 175) TB.
Results
The TIRAP single-nucleotide polymorphism (SNP) C558T was associated with increased susceptibility to TB, with a 558T allele frequency of 0.035 in control samples versus 0.074 in case patients (odds ratio [OR], 2.25; P < .001). Subgroup analysis revealed that SNP 558T was more strongly associated with susceptibility to meningeal TB (OR, 3.02; P < .001) than to pulmonary TB (OR, 1.55; P = .22). In comparison to the 558CC genotype, the 558TT genotype was associated with decreased whole-blood interleukin-6 production, which suggests that TIRAP influence disease susceptibility by modulating the inflammatory response.
Conclusions
These results provide the firs evidence of an association of a TIRAP SNP with the risk of any disease and also suggest that the Toll-like receptor pathway influence susceptibility to meningeal and pulmonary TB by different immune mechanisms.
doi:10.1086/507907
PMCID: PMC4333200  PMID: 16991088
5.  A hydrolase of trehalose dimycolate induces nutrient influx and stress sensitivity to balance intracellular growth of Mycobacterium tuberculosis 
Cell host & microbe  2014;15(2):153-163.
Summary
Chronic tuberculosis in an immunocompetent host is a consequence of the delicately balanced growth of Mycobacterium tuberculosis (Mtb) in the face of host defense mechanisms. We identify an Mtb enzyme (TdmhMtb) that hydrolyzes the mycobacterial glycolipid trehalose dimycolate and plays a critical role in balancing the intracellular growth of the pathogen. TdmhMtb is induced under nutrient limiting conditions and remodels the Mtb envelope to increase nutrient influx, but concomitantly sensitizes Mtb to stresses encountered in the host. Consistent with this, a ΔtdmhMtb mutant is more resilient to stress and grows to higher levels than wild-type in immunocompetent mice. By contrast, mutant growth is retarded in MyD88−/− mice indicating that TdmhMtb provides a growth advantage to intracellular Mtb in an immunocompromised host. Thus, the effects and counter-effects of TdmhMtb play an important role in balancing intracellular growth of Mtb in a manner that is directly responsive to host innate immunity.
doi:10.1016/j.chom.2014.01.008
PMCID: PMC3974621  PMID: 24528862
6.  Epigenome-Guided Analysis of the Transcriptome of Plaque Macrophages during Atherosclerosis Regression Reveals Activation of the Wnt Signaling Pathway 
PLoS Genetics  2014;10(12):e1004828.
We report the first systems biology investigation of regulators controlling arterial plaque macrophage transcriptional changes in response to lipid lowering in vivo in two distinct mouse models of atherosclerosis regression. Transcriptome measurements from plaque macrophages from the Reversa mouse were integrated with measurements from an aortic transplant-based mouse model of plaque regression. Functional relevance of the genes detected as differentially expressed in plaque macrophages in response to lipid lowering in vivo was assessed through analysis of gene functional annotations, overlap with in vitro foam cell studies, and overlap of associated eQTLs with human atherosclerosis/CAD risk SNPs. To identify transcription factors that control plaque macrophage responses to lipid lowering in vivo, we used an integrative strategy – leveraging macrophage epigenomic measurements – to detect enrichment of transcription factor binding sites upstream of genes that are differentially expressed in plaque macrophages during regression. The integrated analysis uncovered eight transcription factor binding site elements that were statistically overrepresented within the 5′ regulatory regions of genes that were upregulated in plaque macrophages in the Reversa model under maximal regression conditions and within the 5′ regulatory regions of genes that were upregulated in the aortic transplant model during regression. Of these, the TCF/LEF binding site was present in promoters of upregulated genes related to cell motility, suggesting that the canonical Wnt signaling pathway may be activated in plaque macrophages during regression. We validated this network-based prediction by demonstrating that β-catenin expression is higher in regressing (vs. control group) plaques in both regression models, and we further demonstrated that stimulation of canonical Wnt signaling increases macrophage migration in vitro. These results suggest involvement of canonical Wnt signaling in macrophage emigration from the plaque during lipid lowering-induced regression, and they illustrate the discovery potential of an epigenome-guided, systems approach to understanding atherosclerosis regression.
Author Summary
Atherosclerosis, a progressive accumulation of lipid-rich plaque within arteries, is an inflammatory disease in which the response of macrophages (a key cell type of the innate immune system) to plasma lipoproteins plays a central role. In humans, the goal of significantly reducing already-established plaque through drug treatments, including statins, remains elusive. In mice, atherosclerosis can be reversed by experimental manipulations that lower circulating lipid levels. A common feature of many regression models is that macrophages transition to a less inflammatory state and emigrate from the plaque. While the molecular regulators that control these responses are largely unknown, we hypothesized that by integrating global measurements of macrophage gene expression in regressing plaques with measurements of the macrophage chromatin landscape, we could identify key molecules that control macrophage responses to the lowering of circulating lipid levels. Our systems biology analysis of plaque macrophages yielded a network in which the Wnt signaling pathway emerged as a candidate upstream regulator. Wnt signaling is known to affect both inflammation and the ability of macrophages to migrate from one location to another, and our targeted validation studies provide evidence that Wnt signaling is increased in plaque macrophages during regression. Our findings both demonstrate the power of a systems approach to uncover candidate regulators of regression and to identify a potential new therapeutic target.
doi:10.1371/journal.pgen.1004828
PMCID: PMC4256277  PMID: 25474352
7.  Multiscale Representation of Genomic Signals 
Nature methods  2014;11(6):689-694.
Genomic information is encoded on a wide range of distance scales, ranging from tens of base pairs to megabases. We developed a multiscale framework to analyze and visualize the information content of genomic signals. Different types of signals, such as GC content or DNA methylation, are characterized by distinct patterns of signal enrichment or depletion across scales spanning several orders of magnitude. These patterns are associated with a variety of genomic annotations, including genes, nuclear lamina associated domains, and repeat elements. By integrating the information across all scales, as compared to using any single scale, we demonstrate improved prediction of gene expression from Polymerase II chromatin immunoprecipitation sequencing (ChIP-seq) measurements and we observed that gene expression differences in colorectal cancer are not most strongly related to gene body methylation, but rather to methylation patterns that extend beyond the single-gene scale.
doi:10.1038/nmeth.2924
PMCID: PMC4040162  PMID: 24727652
8.  A comprehensive collection of systems biology data characterizing the host response to viral infection 
Scientific Data  2014;1:140033.
The Systems Biology for Infectious Diseases Research program was established by the U.S. National Institute of Allergy and Infectious Diseases to investigate host-pathogen interactions at a systems level. This program generated 47 transcriptomic and proteomic datasets from 30 studies that investigate in vivo and in vitro host responses to viral infections. Human pathogens in the Orthomyxoviridae and Coronaviridae families, especially pandemic H1N1 and avian H5N1 influenza A viruses and severe acute respiratory syndrome coronavirus (SARS-CoV), were investigated. Study validation was demonstrated via experimental quality control measures and meta-analysis of independent experiments performed under similar conditions. Primary assay results are archived at the GEO and PeptideAtlas public repositories, while processed statistical results together with standardized metadata are publically available at the Influenza Research Database (www.fludb.org) and the Virus Pathogen Resource (www.viprbrc.org). By comparing data from mutant versus wild-type virus and host strains, RNA versus protein differential expression, and infection with genetically similar strains, these data can be used to further investigate genetic and physiological determinants of host responses to viral infection.
doi:10.1038/sdata.2014.33
PMCID: PMC4410982  PMID: 25977790
9.  Pan-viral specificity of IFN-induced genes reveals new roles for cGAS in innate immunity 
Nature  2013;505(7485):691-695.
The type I interferon (IFN) response protects cells from viral infection by inducing hundreds of interferon-stimulated genes (ISGs), some of which encode direct antiviral effectors1–3. Recent screening studies have begun to catalogue ISGs with antiviral activity against several RNA and DNA viruses4–13. However, antiviral ISG specificity across multiple distinct classes of viruses remains largely unexplored. Here we used an ectopic expression assay to screen a library of more than 350 human ISGs for effects on 14 viruses representing 7 families and 11 genera. We show that 47 genes inhibit one or more viruses, and 25 genes enhance virus infectivity. Comparative analysis reveals that the screened ISGs target positive-sense single-stranded RNA viruses more effectively than negative-sense single-stranded RNA viruses. Gene clustering highlights the cytosolic DNA sensor cyclic GMP-AMP synthase (cGAS, also known as MB21D1) as a gene whose expression also broadly inhibits several RNA viruses. In vitro, lentiviral delivery of enzymatically active cGAS triggers a STING-dependent, IRF3-mediated antiviral program that functions independently of canonical IFN/STAT1 signalling. In vivo, genetic ablation of murine cGAS reveals its requirement in the antiviral response to two DNA viruses, and an unappreciated contribution to the innate control of an RNA virus. These studies uncover new paradigms for the preferential specificity of IFN-mediated antiviral pathways spanning several virus families.
doi:10.1038/nature12862
PMCID: PMC4077721  PMID: 24284630
10.  Lipidomic Profiling of Influenza Infection Identifies Mediators that Induce and Resolve Inflammation 
Cell  2013;154(1):213-227.
Summary
Bioactive lipid mediators play a crucial role in the induction and resolution of inflammation. To elucidate their involvement during influenza infection, LC/MS lipidomic profiling of 141 lipid species was performed on a mouse influenza model using two viruses of significantly different pathogenicity. Infection by the low pathogenicity strain, X31/H3N2, induced a pro-inflammatory response followed by a distinct anti-inflammatory response; infection by the high pathogenicity strain, PR8/H1N1, resulted in overlapping pro- and anti-inflammatory states. Integration of the large-scale lipid measurements with targeted gene expression data demonstrated that 5 lipoxygenase metabolites correlated with the pathogenic phase of the infection whereas 12/15-lipoxygenase metabolites were associated with the resolution phase. Hydroxylated linoleic acid, specifically the ratio of 13- to 9-HODE, was identified as a potential biomarker for immune status during an active infection. Importantly, some of the findings from the animal model were recapitulated in studies of human nasopharyngeal lavages obtained during the 2009–2011 influenza seasons.
doi:10.1016/j.cell.2013.05.052
PMCID: PMC3753192  PMID: 23827684
11.  Systems biology approach predicts immunogenicity of the yellow fever vaccine in humans 
Nature immunology  2008;10(1):116-125.
A major challenge in vaccinology is to prospectively determine vaccine efficacy. Here we have used a systems biology approach to identify early gene ‘signatures’ that predicted immune responses in humans vaccinated with yellow fever vaccine YF-17D. Vaccination induced genes that regulate virus innate sensing and type I interferon production. Computational analyses identified a gene signature, including complement protein C1qB and eukaryotic translation initiation factor 2 alpha kinase 4—an orchestrator of the integrated stress response—that correlated with and predicted YF-17D CD8+ T cell responses with up to 90% accuracy in an independent, blinded trial. A distinct signature, including B cell growth factor TNFRS17, predicted the neutralizing antibody response with up to 100% accuracy. These data highlight the utility of systems biology approaches in predicting vaccine efficacy.
doi:10.1038/ni.1688
PMCID: PMC4049462  PMID: 19029902
12.  Caspase-11 protects against bacteria that escape the vacuole 
Science (New York, N.Y.)  2013;339(6122):975-978.
Caspases are either apoptotic or inflammatory. The inflammatory Caspases-1 and -11 trigger pyroptosis, a form of programmed cell death. Whereas both can be detrimental in inflammatory disease, only Caspase-1 has an established protective role during infection. Herein, we report that Caspase-11 is required for innate immunity to cytosolic, but not vacuolar, bacteria. While Salmonella typhimurium and Legionella pneumophila normally reside in the vacuole, specific mutants (sifA and sdhA, respectively) that aberrantly enter the cytosol triggered Caspase-11, enhancing clearance of S. typhimurium sifA in vivo. This response did not require NLRP3, NLRC4, or ASC inflammasome pathways. Burkholderia species that naturally invade the cytosol also triggered Caspase-11, protecting mice from lethal challenge with B. thailandensis and B. pseudomallei. Thus, Caspase-11 is critical for surviving exposure to ubiquitous environmental pathogens.
doi:10.1126/science.1230751
PMCID: PMC3697099  PMID: 23348507
13.  miR-451 regulates dendritic cell cytokine responses to influenza infection1 
MicroRNAs are important post-transcriptional regulators in immune cells, but how viral infection regulates microRNA expression to shape dendritic cell responses has not been well characterized. We identified 20 miRNAs that were differentially expressed in primary murine dendritic cells in response to the double-stranded RNA agonist poly(I:C), a subset of which were modestly regulated by influenza infection. miR-451 was unique because it was induced more strongly in primary splenic and lung dendritic cells by live viral infection than by purified agonists of pattern recognition receptors. We determined that miR-451 regulates a subset of pro-inflammatory cytokine responses. Three types of primary dendritic cells treated with anti-sense RNA antagomirs directed against miR-451 secreted elevated levels of IL-6, TNF, CCL5/RANTES, and CCL3/MIP1α, and these results were confirmed using miR-451null cells. miR-451 negatively regulates YWHAZ/14-3-3ζ protein levels in various cell types, and we measured a similar inhibition of YWHAZ levels in dendritic cells. It is known that YWHAZ can control the activity of two negative regulators of cytokine production: FOXO3, which is an inhibitory transcription factor, and ZFP36/Tristetraprolin, which binds to AU-rich elements within 3′-UTRs to destabilize cytokine mRNAs. Inhibition of miR-451 expression correlated with increased YWHAZ protein expression and decreased ZFP36 expression, providing a possible mechanism for the elevated secretion of IL-6, TNF, CCL5/RANTES, and CCL3/MIP1α. miR-451 levels are themselves increased by IL-6 and type I interferon, potentially forming a regulatory loop. These data suggest that viral infection specifically induces a miRNA that directs a negative regulatory cascade to tune dendritic cell cytokine production.
doi:10.4049/jimmunol.1201437
PMCID: PMC3528339  PMID: 23169590
14.  Differential Host Response, Rather Than Early Viral Replication Efficiency, Correlates with Pathogenicity Caused by Influenza Viruses 
PLoS ONE  2013;8(9):e74863.
Influenza viruses exhibit large, strain-dependent differences in pathogenicity in mammalian hosts. Although the characteristics of severe disease, including uncontrolled viral replication, infection of the lower airway, and highly inflammatory cytokine responses have been extensively documented, the specific virulence mechanisms that distinguish highly pathogenic strains remain elusive. In this study, we focused on the early events in influenza infection, measuring the growth rate of three strains of varying pathogenicity in the mouse airway epithelium and simultaneously examining the global host transcriptional response over the first 24 hours. Although all strains replicated equally rapidly over the first viral life-cycle, their growth rates in both lung and tracheal tissue strongly diverged at later times, resulting in nearly 10-fold differences in viral load by 24 hours following infection. We identified separate networks of genes in both the lung and tracheal tissues whose rapid up-regulation at early time points by specific strains correlated with a reduced viral replication rate of those strains. The set of early-induced genes in the lung that led to viral growth restriction is enriched for both NF-κB binding site motifs and members of the TREM1 and IL-17 signaling pathways, suggesting that rapid, NF-κB –mediated activation of these pathways may contribute to control of viral replication. Because influenza infection extending into the lung generally results in severe disease, early activation of these pathways may be one factor distinguishing high- and low-pathogenicity strains.
doi:10.1371/journal.pone.0074863
PMCID: PMC3779241  PMID: 24073225
15.  miRNA regulation of macrophage fusion into multinucleated giant cells 
Cellular fusion of macrophages into multinucleated giant cells is a distinguishing feature of the granulomatous response to inflammation, infection and foreign bodies (1). We observed a marked increase in fusion of macrophages genetically deficient in Dicer, an enzyme required for canonical miRNA biogenesis. Gene expression profiling of miRNA deficient macrophages revealed an up-regulation of the IL4 responsive fusion protein Tm7sf4, analyses identify miR-7a-1 as a negative regulator of macrophage fusion, functioning by directly targeting Tm7sf4 mRNA. miR-7a-1 is itself an IL4 responsive gene in macrophages, suggesting feedback control of cellular fusion. Collectively these data indicate that miR-7a-1 functions to regulate IL4 directed multinucleated giant cell formation.
doi:10.4049/jimmunol.1102477
PMCID: PMC3381877  PMID: 22661094
16.  A FOXO3/IRF7 gene regulatory circuit limits inflammatory sequelae of antiviral responses 
Nature  2012;490(7420):421-425.
Antiviral responses must be tightly regulated to rapidly defend against infection while minimizing inflammatory damage. Type 1 interferons (IFN-I) are crucial mediators of antiviral responses1 and their transcription is regulated by a variety of transcription factors2; principal amongst these is the family of interferon regulatory factors (IRFs)3. The IRF gene regulatory networks are complex and contain multiple feedback loops. The tools of systems biology are well suited to elucidate the complex interactions that give rise to precise coordination of the interferon response. Here we have used an unbiased systems approach to predict that a member of the forkhead family of transcription factors, FOXO3, is a negative regulator of a subset of antiviral genes. This prediction was validated using macrophages isolated from Foxo3-null mice. Genome-wide location analysis combined with gene deletion studies identified the Irf7 gene as a critical target of FOXO3. FOXO3 was identified as a negative regulator of Irf7 transcription and we have further demonstrated that FOXO3, IRF7 and IFN-I form a coherent feed-forward regulatory circuit. Our data suggest that the FOXO3-IRF7 regulatory circuit represents a novel mechanism for establishing the requisite set points in the interferon pathway that balances the beneficial effects and deleterious sequelae of the antiviral response.
doi:10.1038/nature11428
PMCID: PMC3556990  PMID: 22982991
17.  Caspase-1 induced pyroptotic cell death 
Immunological reviews  2011;243(1):206-214.
Summary
Programmed cell death is a necessary part of development and tissue homeostasis enabling the removal of unwanted cells. In the setting of infectious disease, cells that have been commandeered by microbial pathogens become detrimental to the host. When macrophages and dendritic cells are compromised in this way, they can be lysed by pyroptosis, a cell death mechanism that is distinct from apoptosis and oncosis/necrosis. Pyroptosis is triggered by Caspase-1 after its activation by various inflammasomes, and results in lysis of the affected cell. Both pyroptosis and apoptosis are programmed cell death mechanisms, but are dependent on different caspases, unlike oncosis. Similar to oncosis, and unlike apoptosis, pyroptosis results in cellular lysis and release of the cytosolic contents to the extracellular space. This event is predicted to be inherently inflammatory, and additionally coincides with IL-1β and IL-18 secretion. We discuss the role of distinct inflammasomes, including NLRC4, NLRP3 and AIM2, as well as the role of the ASC focus in Caspase-1 signaling. We further review the importance of pyroptosis in vivo as a potent mechanism to clear intracellular pathogens.
doi:10.1111/j.1600-065X.2011.01044.x
PMCID: PMC3609431  PMID: 21884178
monocytes/macrophages; Toll-like Receptors/Pattern recognition receptors; Apoptosis/Autophagy; Pyroptosis; Caspase-1
18.  ATF3 protects against atherosclerosis by suppressing 25-hydroxycholesterol–induced lipid body formation 
The transcription factor ATF3 inhibits lipid body formation in macrophages during atherosclerosis in part by dampening the expression of cholesterol 25-hydroxylase.
Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of lipid-loaded macrophages in the arterial wall. We demonstrate that macrophage lipid body formation can be induced by modified lipoproteins or by inflammatory Toll-like receptor agonists. We used an unbiased approach to study the overlap in these pathways to identify regulators that control foam cell formation and atherogenesis. An analysis method integrating epigenomic and transcriptomic datasets with a transcription factor (TF) binding site prediction algorithm suggested that the TF ATF3 may regulate macrophage foam cell formation. Indeed, we found that deletion of this TF results in increased lipid body accumulation, and that ATF3 directly regulates transcription of the gene encoding cholesterol 25-hydroxylase. We further showed that production of 25-hydroxycholesterol (25-HC) promotes macrophage foam cell formation. Finally, deletion of ATF3 in Apoe−/− mice led to in vivo increases in foam cell formation, aortic 25-HC levels, and disease progression. These results define a previously unknown role for ATF3 in controlling macrophage lipid metabolism and demonstrate that ATF3 is a key intersection point for lipid metabolic and inflammatory pathways in these cells.
doi:10.1084/jem.20111202
PMCID: PMC3328364  PMID: 22473958
19.  Generation of a Listeria vaccine strain by enhanced Caspase-1 activation 
European Journal of Immunology  2011;41(7):1934-1940.
The immunostimulatory properties conferred by vaccine adjuvants require Caspase-1 for processing of IL-1β and IL-18. Caspase-1 is activated in response to a breach of the cytosolic compartment by microbes and the process is initiated by intracellular pattern recognition receptors within inflammasomes. Listeria monocytogenes is detected in the cytosol by the NLRC4, NLRP3 and AIM2 inflammasomes. NLRC4 is activated by flagellin, and L. monocytogenes evades this detector by repressing flagellin expression. We generated an L. monocytogenes strain that was forced to express flagellin in the host cell cytosol. This strain hyperactivated Caspase-1 and was preferentially cleared via NLRC4 detection in an IL-1β/IL-18 independent manner. We also created a strain of L. monocytogenes with forced expression of another NLRC4 agonist, PrgJ from the Type III secretion system of S. typhimurium. Forced expression of flagellin or PrgJ resulted in attenuation, yet both strains conferred protective immunity in mice against lethal challenge with L. monocytogenes. This work is the first demonstration of specific targeting of the Caspase-1 activation pathway to generate a safe and potent L. monocytogenes based vaccine. Moreover, the attenuated strains with embedded flagellin or PrgJ adjuvants, represent attractive vectors for vaccines aimed at eliciting T cell responses.
doi:10.1002/eji.201041214
PMCID: PMC3375905  PMID: 21538346
20.  Leishmania promotes its own virulence by inducing expression of the host immune inhibitory ligand CD200 
Cell host & microbe  2011;9(6):463-471.
Summary
Leishmania parasites infect macrophages, cells normally involved in innate defense against pathogens. L. amazonensis and L. major cause severe or mild disease, respectively, consistent with each parasite’s ability to survive within activated macrophages. The mechanisms underlying increased virulence of L. amazonensis are mostly unknown. We show that L. amazonensis promotes its own survival by inducing expression of CD200, an immunoregulatory molecule that inhibits macrophage activation. L. amazonensis does not form typical non-healing lesions in CD200−/− mice and cannot replicate in CD200−/− macrophages, an effect reversed by exogenous administration of soluble CD200-Fc. The less virulent L. major does not induce CD200 expression and forms small, self-healing lesions in both wild type and CD200−/− mice. Notably, CD200-Fc injection transforms the course of L. major infection to one resembling L. amazonensis, with large, non-healing lesions. CD200-dependent iNOS inhibition allows parasite growth in macrophages, identifying a mechanism for the increased virulence of L. amazonensis.
doi:10.1016/j.chom.2011.04.014
PMCID: PMC3118640  PMID: 21669395
21.  Systems Biology of Seasonal Influenza Vaccination in Humans 
Nature immunology  2011;12(8):786-795.
We used a systems biological approach to study innate and adaptive responses to influenza vaccination in humans, during 3 consecutive influenza seasons. Healthy adults were vaccinated with inactivated (TIV) or live attenuated (LAIV) influenza vaccines. TIV induced greater antibody titers and enhanced numbers of plasmablasts than LAIV. In TIV vaccinees, early molecular signatures correlated with, and accurately predicted, later antibody titers in two independent trials. Interestingly, the expression of Calcium/calmodulin-dependent kinase IV (CamkIV) at day 3 was inversely correlated with later antibody titers. Vaccination of CamkIV −/− mice with TIV induced enhanced antigen-specific antibody titers, demonstrating an unappreciated role for CaMKIV in the regulation of antibody responses. Thus systems approaches can predict immunogenicity, and reveal new mechanistic insights about vaccines.
doi:10.1038/ni.2067
PMCID: PMC3140559  PMID: 21743478
22.  Activation of the NLRP3 inflammasome by intracellular poly I:C 
FEBS letters  2010;584(22):4627-4632.
Several RNA viruses can be detected by the inflammasome, which promotes IL-1β and IL-18 secretion, but the underlying mechanisms of detection remain unclear. Cytosolic dsRNA is a replication intermediate of many RNA viruses. We show here that transfection of the dsRNA analogue poly I:C activates the NLRP3 inflammasome via a pathway requiring endosomal acidification. This detection is independent of the other poly I:C sensors: TLR3 and MDA5. These results suggest a mechanism by which cytosolic dsRNA produced during viral infection could activate the NLRP3 inflammasome.
doi:10.1016/j.febslet.2010.10.036
PMCID: PMC3005299  PMID: 20971108
Inflammasome; poly I:C; RNA virus; IL-1β
23.  The NLRP3 Inflammasome Detects Encephalomyocarditis Virus and Vesicular Stomatitis Virus Infection▿ 
Journal of Virology  2011;85(9):4167-4172.
Inflammasomes are cytosolic protein complexes that regulate caspase-1 activation and the secretion of interleukin-1β (IL-1β) and IL-18. Several different inflammasome complexes have been identified, but the NLRP3 inflammasome is particularly notable because of its central role in diseases of inflammation. Recent work has demonstrated an essential role for the NLRP3 inflammasome in host defense against influenza virus. We show here that two other RNA viruses, encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV), activate the NLRP3 inflammasome in dendritic cells and macrophages through a mechanism requiring viral replication. Inflammasome activation in response to both viruses does not require MDA5 or RIG-I signaling. Despite the ability of the NLRP3 inflammasome to detect EMCV and VSV, wild-type and caspase-1-deficient mice were equally susceptible to infection with both viruses. These findings indicate that the NLRP3 inflammasome may be a common pathway for RNA virus detection, but its precise role in the host response may be variable.
doi:10.1128/JVI.01687-10
PMCID: PMC3126243  PMID: 21289120
24.  Cutting Edge: Cytosolic Bacterial DNA Activates the Inflammasome via Aim2 
Pathogens are detected by pattern recognition receptors that, upon activation, orchestrate an appropriate immune response. The TLRs and the nucleotide-binding oligomerization domain-like receptors (NLRs) are prototypic pattern recognition receptors that detect extracellular and cytosolic pathogens, respectively. Listeria monocytogenes has both extracellular and cytosolic phases and is detected in the cytosol by members of the NLR family. These include two NLR members, NLRC4 and NLRP3, that, upon detection of cytosolic L. monocytogenes, induce the assembly of the inflammasome. Inflammasomes serve as platforms for the activation of the protease caspase 1, which mediates the processing and secretion of pro–IL-1β and pro–IL-18. We previously provided evidence that L. monocytogenes is also detected by a third inflammasome. We now use biochemical and genetic approaches to demonstrate that the third detector senses bacterial DNA and identify it as Aim2, a receptor that has previously been shown to detect viral DNA.
doi:10.4049/jimmunol.1000724
PMCID: PMC2993756  PMID: 20562263
25.  Caspase-1-induced pyroptosis is an innate immune effector mechanism against intracellular bacteria 
Nature immunology  2010;11(12):1136-1142.
Summary
Macrophages mediate crucial innate immune responses via caspase-1-dependent processing and secretion of IL-1β and IL-18. While wild type Salmonella typhimurium infection is lethal to mice, a strain that persistently expresses flagellin was cleared by the cytosolic flagellin detection pathway via NLRC4 activation of caspase-1; however, this clearance was independent of IL-1β and IL-18. Instead, caspase-1 induced pyroptotic cell death, released bacteria from macrophages and exposed them to uptake and killing by reactive oxygen species in neutrophils. Similarly, caspase-1 cleared unmanipulated Legionella and Burkholderia by cytokine-independent mechanisms. This demonstrates for the first time that caspase-1 clears intracellular bacteria in vivo independent of IL-1β and IL-18, and establishes pyroptosis as an efficient mechanism of bacterial clearance by the innate immune system.
doi:10.1038/ni.1960
PMCID: PMC3058225  PMID: 21057511
Caspase-1; pyroptosis; IL-1β Salmonella; cell death

Results 1-25 (67)