技术资料

BACKGROUND: The cellular sulfonation pathway modulates key steps of virus replication. This pathway comprises two main families of sulfonate-conjugating enzymes: Golgi sulfotransferases,which sulfonate proteins,glycoproteins,glycolipids and proteoglycans; and cytosolic sulfotransferases (SULTs),which sulfonate various small molecules including hormones,neurotransmitters,and xenobiotics. Sulfonation controls the functions of numerous cellular factors such as those involved in cell-cell interactions,cell signaling,and small molecule detoxification. We previously showed that the cellular sulfonation pathway regulates HIV-1 gene expression and reactivation from latency. Here we show that a specific cellular sulfotransferase can regulate HIV-1 replication in primary human monocyte-derived macrophages (MDMs) by yet another mechanism,namely reverse transcription. METHODS: MDMs were derived from monocytes isolated from donor peripheral blood mononuclear cells (PBMCs) obtained from the San Diego Blood Bank. After one week in vitro cell culture under macrophage-polarizing conditions,MDMs were transfected with sulfotranserase-specific or control siRNAs and infected with HIV-1 or SIV constructs expressing a luciferase reporter. Infection levels were subsequently monitored by luminescence. Western blotting was used to assay siRNA knockdown and viral protein levels,and qPCR was used to measure viral RNA and DNA products. RESULTS: We demonstrate that the cytosolic sulfotransferase SULT1A1 is highly expressed in primary human MDMs,and through siRNA knockdown experiments,we show that this enzyme promotes infection of MDMs by single cycle VSV-G pseudotyped human HIV-1 and simian immunodeficiency virus vectors and by replication-competent HIV-1. Quantitative PCR analysis revealed that SULT1A1 affects HIV-1 replication in MDMs by modulating the kinetics of minus-strand DNA elongation during reverse transcription. CONCLUSIONS: These studies have identified SULT1A1 as a cellular regulator of HIV-1 reverse transcription in primary human MDMs. The normal substrates of this enzyme are small phenolic-like molecules,raising the possibility that one or more of these substrates may be involved. Targeting SULT1A1 and/or its substrate(s) may offer a novel host-directed strategy to improve HIV-1 therapeutics. View Publication

The high risk of neonatal death from sepsis is thought to result from impaired responses by innate immune cells; however,the clinical observation of hyperinflammatory courses of neonatal sepsis contradicts this concept. Using transcriptomic,epigenetic and immunological approaches,we demonstrated that high amounts of the perinatal alarmins S100A8 and S100A9 specifically altered MyD88-dependent proinflammatory gene programs. S100 programming prevented hyperinflammatory responses without impairing pathogen defense. TRIF-adaptor-dependent regulatory genes remained unaffected by perinatal S100 programming and responded strongly to lipopolysaccharide,but were barely expressed. Steady-state expression of TRIF-dependent genes increased only gradually during the first year of life in human neonates,shifting immune regulation toward the adult phenotype. Disruption of this critical sequence of transient alarmin programming and subsequent reprogramming of regulatory pathways increased the risk of hyperinflammation and sepsis. Collectively these data suggest that neonates are characterized by a selective,transient microbial unresponsiveness that prevents harmful hyperinflammation in the delicate neonate while allowing for sufficient immunological protection. View Publication

Low-grade,chronic inflammation has been associated with many diseases of aging,but the mechanisms responsible for producing this inflammation remain unclear. Inflammasomes can drive chronic inflammation in the context of an infectious disease or cellular stress,and they trigger the maturation of interleukin-1β (IL-1β). Here we find that the expression of specific inflammasome gene modules stratifies older individuals into two extremes: those with constitutive expression of IL-1β,nucleotide metabolism dysfunction,elevated oxidative stress,high rates of hypertension and arterial stiffness; and those without constitutive expression of IL-1β,who lack these characteristics. Adenine and N(4)-acetylcytidine,nucleotide-derived metabolites that are detectable in the blood of the former group,prime and activate the NLRC4 inflammasome,induce the production of IL-1β,activate platelets and neutrophils and elevate blood pressure in mice. In individuals over 85 years of age,the elevated expression of inflammasome gene modules was associated with all-cause mortality. Thus,targeting inflammasome components may ameliorate chronic inflammation and various other age-associated conditions. View Publication

West Nile virus (WNV) infection is a mosquito-borne zoonosis with increasing prevalence in the United States. WNV infection begins in the skin,and the virus replicates initially in keratinocytes and dendritic cells (DCs). In the skin and cutaneous lymph nodes,infected DCs are likely to interact with invariant natural killer T cells (iNKTs). Bidirectional interactions between DCs and iNKTs amplify the innate immune response to viral infections,thus controlling viral load and regulating adaptive immunity. iNKTs are stimulated by CD1d-bound lipid antigens or activated indirectly by inflammatory cytokines. We exposed human monocyte-derived DCs to WNV Kunjin and determined their ability to activate isolated blood iNKTs. DCs became infected as judged by synthesis of viral mRNA and Envelope and NS-1 proteins,but did not undergo significant apoptosis. Infected DCs up-regulated the co-stimulatory molecules CD86 and CD40,but showed decreased expression of CD1d. WNV infection induced DC secretion of type I interferon (IFN),but no or minimal interleukin (IL)-12,IL-23,IL-18 or IL-10. Unexpectedly,we found that the WNV-infected DCs stimulated human iNKTs to up-regulate CD69 and produce low amounts of IL-10,but not proinflammatory cytokines such as IFN-γ or tumour necrosis factor (TNF)-α. Both CD1d and IFNAR blockade partially abrogated this iNKT response,suggesting involvement of a T cell receptor (TCR)-CD1d interaction and type I interferon receptor (IFNAR) signalling. Thus,WNV infection interferes with DC-iNKT interactions by preventing the production of proinflammatory cytokines. iNKTs may be a source of IL-10 observed in human flavivirus infections and initiate an anti-inflammatory innate response that limits adaptive immunity and immune pathology upon WNV infection. View Publication

In this study,we examined whether bacterial pathogen-associated molecular patterns recognized by toll-like receptors (TLRs) can modify the CCR7-dependent migration of human monocytes. MonoMac-1 (MM-1) cells and freshly isolated human monocytes were cultivated in the presence of agonists for TLR4 (which senses lipopolysaccharides from gram-negative bacteria),TLR1/2 (which senses peptidoglycan from gram-positive bacteria),and TLR9 (which recognizes bacterial DNA rich in unmethylated CpG DNA). CCR7 mRNA transcription was measured using quantitative reverse transcription polymerase chain reaction and protein expression was examined using flow cytometry. CCR7 function was monitored using migration and transmigration assays in response to CCL19/CCL21,which are natural ligands for CCR7. Our results show that TLR4 strongly increases monocyte migratory capacity in response to CCL19 in chemotaxis and transmigration assays in a model that mimics the human blood-brain barrier,whereas TLR1/2 and 9 have no effect. Examination of monocyte migration in response to TLRs that are activated by bacterial components would contribute to understanding the excessive monocyte migration that characterizes the pathogenesis of bacterial infections and/or neuroinflammatory diseases. View Publication

Proof of drug-target engagement in physiologically-relevant contexts is a key pillar of successful therapeutic target validation. We developed two orthogonal technologies,the cellular thermal shift assay (CETSA) and a covalent chemical probe reporter approach (harnessing sulfonyl fluoride tyrosine labeling and subsequent click chemistry) to measure the occupancy of the mRNA-decapping scavenger enzyme DcpS by a small molecule inhibitor in live cells. Enzyme affinity determined using isothermal dose response fingerprinting (ITDRFCETSA) and the concentration required to occupy 50% of the enzyme (OC50) using the chemical probe reporter assay were very similar. In this case,the chemical probe method worked well due to the long offset kinetics of the reversible inhibitor (determined using a fluorescent dye-tagged probe). This work suggests that CETSA could become the first choice assay to determine in-cell target engagement due to its simplicity. View Publication

Spleen tyrosine kinase (Syk) is an attractive drug target in autoimmune,inflammatory,and oncology disease indications. The most advanced Syk inhibitor,R406,1 (or its prodrug form fostamatinib,2),has shown efficacy in multiple therapeutic indications,but its clinical progress has been hampered by dose-limiting adverse effects that have been attributed,at least in part,to the off-target activities of 1. It is expected that a more selective Syk inhibitor would provide a greater therapeutic window. Herein we report the discovery and optimization of a novel series of imidazo[1,2-a]pyrazine Syk inhibitors. This work culminated in the identification of GS-9973,68,a highly selective and orally efficacious Syk inhibitor which is currently undergoing clinical evaluation for autoimmune and oncology indications. View Publication

Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mphi). In the present study,we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mphi that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of approximately 6.4-6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2,4-dinitroanilo)-3'-amino-N-methyldipropylamine reagent. Mphi that had ingested Saccharomyces cerevisae,a nonpathogenic yeast that is rapidly killed and degraded by Mphi,also maintained an intraphagosomal pH of approximately 6.5 over a period of several hours. Stimulation of human Mphi fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin,an inhibitor of the vacuolar ATPase. Human Mphi cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts,whereas mouse peritoneal Mphi digestion of yeasts was completely reversed in the presence of bafilomycin. However,bafilomycin did not inhibit mouse Mphi fungistatic activity induced by IFN-gamma. Thus,human Mphi do not require phagosomal acidification to kill and degrade Hc yeasts,whereas mouse Mphi do require acidification for fungicidal but not fungistatic activity. View Publication

Increasing evidence suggests that the deposition of amyloid plaques,composed primarily of the amyloid-beta protein (Abeta),within the cerebrovasculature is a frequent occurrence in Alzheimer's disease and may play a significant role in disease progression. Accordingly,the pathogenic mechanisms by which Abeta can alter vascular function may have therapeutic implications. Despite observations that Abeta elicits a number of physiological responses in endothelial cells,ranging from alteration of protein expression to cell death,the Abeta species accountable for these responses remains unexplored. In the current study,we show that isolated soluble Abeta aggregation intermediates activate human brain microvascular endothelial cells for both adhesion and subsequent transmigration of monocyte cells in the absence of endothelial cell death and monolayer disruption. In contrast,unaggregated Abeta monomer and mature Abeta fibril fail to induce any change in endothelial adhesion or transmigration. Correlations between average Abeta aggregate size and observed increases in adhesion illustrate that smaller soluble aggregates are more potent activators of endothelium. These results support previous studies demonstrating heightened neuronal activity of soluble Abeta aggregates,including Abeta-derived diffusible ligands,oligomers,and protofibrils,and further show that soluble aggregates also selectively exhibit activity in a vascular cell model. View Publication

IkappaB kinase (IKK) complex plays a key regulatory role in macrophages for NF-kappaB activation during both innate and adaptive immune responses. Because IKK1-/- mice died at birth,we differentiated functional macrophages from embryonic day 15.5 IKK1 mutant embryonic liver. The embryonic liver-derived macrophage (ELDM) showed enhanced phagocytotic clearance of bacteria,more efficient antigen-presenting capacity,elevated secretion of several key proinflammatory cytokines and chemokines,and known NFkappaB target genes. Increased NFkappaB activity in IKK1 mutant ELDM was the result of prolonged degradation of IkappaBalpha in response to infectious pathogens. The delayed restoration of IkappaBalpha in pathogen-activated IKK1-/- ELDM was a direct consequence of uncontrolled IKK2 kinase activity. We hypothesize that IKK1 plays a checkpoint role in the proper control of IkappaBalpha kinase activity in innate and adaptive immunity. View Publication