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.
Precise control of the innate immune response is essential to ensure host defense against infection while avoiding inflammatory disease. Systems-level analyses of Toll-like receptor (TLR)-stimulated macrophages suggested that SHANK-associated RH domain-interacting protein (SHARPIN) might play a role in the TLR pathway. This hypothesis was supported by the observation that macrophages derived from chronic proliferative dermatitis mutation (cpdm) mice, which harbor a spontaneous null mutation in the Sharpin gene, exhibited impaired IL-12 production in response to TLR activation. Systems biology approaches were used to define the SHARPIN-regulated networks. Promoter analysis identified NF-κB and AP-1 as candidate transcription factors downstream of SHARPIN, and network analysis suggested selective attenuation of these pathways. We found that the effects of SHARPIN deficiency on the TLR2-induced transcriptome were strikingly correlated with the effects of the recently described hypomorphic L153P/panr2 point mutation in Ikbkg [NF-κB Essential Modulator (NEMO)], suggesting that SHARPIN and NEMO interact. We confirmed this interaction by co-immunoprecipitation analysis and furthermore found it to be abrogated by panr2. NEMOdependent signaling was affected by SHARPIN deficiency in a manner similar to the panr2 mutation, including impaired p105 and ERK phosphorylation and p65 nuclear localization. Interestingly, SHARPIN deficiency had no effect on IκBα degradation and on p38 and JNK phosphorylation. Taken together, these results demonstrate that SHARPIN is an essential adaptor downstream of the branch point defined by the panr2 mutation in NEMO.innate immunity | signal transduction | pattern-recognition | ubiquitylation T he innate immune system is critical for host defense but, unchecked, can cause severe inflammatory disease (1-5). Inflammatory sequelae are mitigated at a number of levels. Principal among these is the precise identification of the threat and the appropriate tailoring of the response. Infectious agents are precisely identified by a variety of pattern recognition receptors, including Toll-like receptors (TLRs), which recognize molecular motifs that are specific to the pathogen (6). Although much is known about the mechanisms through which TLRs mediate immune responses, a number of important questions remain unanswered (7). Central to these is a complete knowledge of all of the critical components within the TLR-signaling pathways and how dynamic interactions between them lead to the appropriate coordination of host defense. The precise titration of the response requires multiple levels of regulation that include crosstalk and feedback between various signaling pathways and gene regulatory networks operating on very different spatial and temporal scales. Systems biology provides a framework in which this complexity can be addressed. Systems approaches combine prior knowledge and biological insight with global measurement technologies and computational methods both to reveal regulatory interactions and to ...
We previously described the purification of a membrane-bound diacylglycerol kinase highly selective for sn-1-acyl-2-arachidonoyl diacylglycerols (Walsh, J. P., Suen, R., Lemaitre, R. N., and Glomset, J. A. (1994) J. Biol. Chem. 269, 21155-21164). This enzyme appears to be responsible for the rapid clearance of the arachidonaterich pool of diacylglycerols generated during stimulusinduced phosphoinositide turnover. We have now shown phosphatidylinositol 4,5-bisphosphate to be a potent and specific inhibitor of arachidonoyl-diacylglycerol kinase. Kinetic analyses indicated a K i for phosphatidylinositol 4,5-bisphosphate of 0.04 mol %. Phosphatidic acid also was an inhibitor with a K i of 0.7 mol %. Other phospholipids had only small effects at these concentrations. A series of multiply phosphorylated lipid analogs also inhibited the enzyme, indicating that the head group phosphomonoesters are the primary determinants of the polyphosphoinositide effect. However, these compounds were not as potent as phosphatidylinositol 4,5-bisphosphate, indicating some specificity for the polyphosphoinositide additional to its total charge. Five other diacylglycerol kinases were activated to varying degrees by phosphatidylinositol 4,5-bisphosphate and phosphatidic acid, suggesting that inhibition by acidic lipids may be specific for the arachidonoyl-DAG kinase isoform. Given the presumed role of arachidonoyl-diacylglycerol kinase in the phosphoinositide cycle, this inhibition may represent a mechanism for polyphosphoinositides to regulate their own synthesis.Diacylglycerol kinases catalyze the ATP-dependent phosphorylation of sn-1,2-diacylglycerol (DAG) 1 to phosphatidic acid (PA) (1-3). As such, they are widely regarded as attenuators of the DAG signaling and protein kinase C activation that occur during stimulus-induced PI turnover (4). The recent identification of a specific DAG kinase essential for PI-mediated invertebrate visual transduction is a striking confirmation of the involvement of DAG kinases in the PI cycle (5). It has recently become evident that DAG kinases are a diverse family of isoenzymes (2). The first of these to be purified and cloned was an 82.6-kDa isoform expressed predominantly in brain and thymus (6, 7). Several homologs of this enzyme also have been cloned, each of which has its own highly specific pattern of expression in cells and tissues (8 -12). Additional DAG kinases, which appear distinct from the cloned isoforms described above, also have been reported (13-21). However, detailed enzymologic data on these are not available at this time. Our laboratory has described and purified a membranebound DAG kinase highly selective for DAG molecular species containing arachidonate as the sn-2 fatty acyl moiety (21-24). This activity can be distinguished from other DAG kinases by a variety of enzymologic properties in addition to its substrate specificity (21-24). Arachidonoyl-DAG kinase activity varies widely between different tissues, but it is detectable in all cells and tissues we have examined (21-24). G...
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