Synthetic glycolipid-based TLR4 antagonists negatively regulate TRIF-dependent TLR4 signalling in human macrophages

TLRs, including TLR4, play a crucial role in inflammatory-based diseases, and TLR4 has been identified as a therapeutic target for pharmacological intervention. In previous studies, we investigated the potential of FP7, a novel synthetic glycolipid active as a TLR4 antagonist, to inhibit haematopoietic and non-haematopoietic MyD88-dependent TLR4 pro-inflammatory signalling. The main aim of this study was to investigate the action of FP7 and its derivative FP12 on MyD88-independent TLR4 signalling in THP-1 derived macrophages. Western blotting, Ab array and ELISA approaches were used to explore the effect of FP7 and FP12 on TRIF-dependent TLR4 functional activity in response to LPS and other endogenous TLR4 ligands in THP-1 macrophages. A different kinetic in the inhibition of endotoxin-driven TBK1, IRF3 and STAT1 phosphorylation was observed using different LPS chemotypes. Following activation of TLR4 by LPS, data revealed that FP7 and FP12 inhibited TBK1, IRF3 and STAT1 phosphorylation which was associated with down-regulation IFN-β and IP-10. Specific blockage of the IFN type one receptor showed that these novel molecules inhibited TRIF-dependent TLR4 signalling via IFN-β pathways. These results add novel information on the mechanism of action of monosaccharide FP derivatives. The inhibition of the TRIF-dependent pathway in human macrophages suggests potential therapeutic uses for these novel TLR4 antagonists in pharmacological interventions on inflammatory diseases.     

Therapeutic implications of the TLR and VDR partnership

The innate immune system provides the host with an immediate and rapid defense against invading microbes. Detection of foreign invaders is mediated by a class of receptors that are known as the pattern recognition receptors, such as the family of Toll-like receptors (TLRs). In humans, ten functional TLRs have been identified and they respond to conserved pathogen-associated molecular patterns derived from bacteria, mycoplasma, fungi and viruses. TLR activation leads to direct antimicrobial activity against both intracellular and extracellular bacteria, and induces an antiviral gene program. Recently, it was reported that TLR2 activation leads to the use of vitamin D3 as a mechanism to combat Mycobacterium tuberculosis. Here, we focus on recent findings concerning the TLR-induced antimicrobial mechanisms in humans and the therapeutic implications of these findings. Owing to their capability to combat a wide array of pathogens, TLRs are attractive therapeutic targets. However, additional knowledge about their antimicrobial mechanisms is needed.     

Coordinate analysis of murine immune cell surface markers and intracellular phosphoproteins by flow cytometry

Recently, phosphospecific flow cytometry has emerged as a powerful tool to analyze intracellular signaling events in complex populations of cells because of its ability to simultaneously discriminate cell types based on surface marker expression and measure levels of intracellular phosphoproteins. This has provided novel insights into the cell- and pathway-specific nature of immune signaling. However, we and others have found that the fixation and permeabilization steps necessary for phosphoprotein analysis often negatively affect the resolution of cell types based on surface marker analysis and light scatter characteristics. Therefore, we performed a comprehensive profile of >35 different murine surface marker Abs to understand the effects of fixation and permeabilization on surface Ag staining. Fortuitously, approximately 80% of the Abs tested resolved cell populations of interest, although with decreased separation between positive and negative populations and at very different titers than those used on live cells. The other 20% showed either complete loss of separation between populations or loss of intermediately staining populations. We were able to rescue staining of several of these Ags by performing staining after fixation, but before permeabilization, although with limited fluorophore choices. Scatter characteristics of lymphocytes were well retained, but changed dramatically for monocyte and neutrophil populations. These results compile a comprehensive resource for researchers interested in applying phosphospecific flow cytometry to complex populations of cells while outlining steps necessary to successfully apply new surface marker Abs to this platform.     

TLR activation of Langerhans cell-like dendritic cells triggers an antiviral immune response

Langerhans cells (LC) are a unique subset of dendritic cells (DC), present in the epidermis and serving as the first line of defense against pathogens invading the skin. To investigate the role of human LCs in innate immune responses, we examined TLR expression and function of LC-like DCs derived from CD34+ progenitor cells and compared them to DCs derived from peripheral blood monocytes (monocyte-derived DC; Mo-DC). LC-like DCs and Mo-DCs expressed TLR1-10 mRNAs at comparable levels. Although many of the TLR-induced cytokine patterns were similar between the two cell types, stimulation with the TLR3 agonist poly(I:C) triggered significantly higher amounts of the IFN-inducible chemokines CXCL9 (monokine induced by IFN-gamma) and CXCL11 (IFN-gamma-inducible T cell alpha chemoattractant) in LC-like DCs as compared with Mo-DCs. Supernatants from TLR3-activated LC-like DCs reduced intracellular replication of vesicular stomatitis virus in a type I IFN-dependent manner. Finally, CXCL9 colocalized with LCs in skin biopsy specimens from viral infections. Together, our data suggest that LCs exhibit a direct antiviral activity that is dependent on type I IFN as part of the innate immune system.     

High-content single-cell drug screening with phosphospecific flow cytometry

Drug screening is often limited to cell-free assays involving purified enzymes, but it is arguably best applied against systems that represent disease states or complex physiological cellular networks. Here, we describe a high-content, cell-based drug discovery platform based on phosphospecific flow cytometry, or phosphoflow, that enabled screening for inhibitors against multiple endogenous kinase signaling pathways in heterogeneous primary cell populations at the single-cell level. From a library of small-molecule natural products, we identified pathway-selective inhibitors of Jak-Stat and MAP kinase signaling. Dose-response experiments in primary cells confirmed pathway selectivity, but importantly also revealed differential inhibition of cell types and new druggability trends across multiple compounds. Lead compound selectivity was confirmed in vivo in mice. Phosphoflow therefore provides a unique platform that can be applied throughout the drug discovery process, from early compound screening to in vivo testing and clinical monitoring of drug efficacy.     

Resistance of tomato infected with cucumber mosaic virus satellite RNA to potato spindle tuber viroid

Tomato (Lycopersicon esculentum cvs Rutgers and Lichun) plants were firstly pre-inoculated either with a cucumber mosaic virus (CMV) isolate containing satellite RNA (CMV-S52) or with a CMV isolate without satellite RNA, and then challenged 14 days later with a severe strain of potato spindle tuber viroid (PSTVd). Also, tomato plants transformed with CMV satellite cDNA and non-transgenic control plants were directly inoculated with PSTVd. Protection effects were assessed by the observation of symptoms and by assay of PSTVd accumulation in tomato plants using return polyacrylamide gel electrophoresis and silver staining. The results indicated that the satellite-transgenic plants and plants pre-inoculated with CMV-S52 showed much milder symptoms of PSTVd infection than the respective control plants. The concentration of PSTVd RNA in the satellite-transgenic plants and CMV-S52 pre-inoculated plants was reduced to about 0.02-0.03 of the controls. PSTVd infection did not increase the amount of satellite ds-RNA in plants. It is concluded that the plant resistance to PSTVd is induced by the presence of satellite RNA rather than the CMV infection. It is suggested that as there is considerable sequence similarity between satellite RNA and PSTVd, base pairings may be a cause of reduction of both symptoms and the accumulation of PSTVd.     

情前期与间情期大鼠内侧视前区-下丘脑前区神经元的电活动

在氯化筒箭毒制动的情前期及间情期大鼠上进行实验。用玻璃微电极记录内侧视前区-下丘脑前区(mPOA-AHA)的单位放电。mPOA-AHA 的大多数单位表现连续性或周期性自发放电。情前期大鼠周期性放电单位所占的百分比显著地多于间情期大鼠(P<0.01)。刺激子宫颈部可以使单位放电发生两种反应。一种单位对宫颈刺激发生放电频率增加的反应(宫颈兴奋神经元,CE 神经元),另一种单位则发生放电频率降低的反应(宫颈抑制神经元,CI神经元)。情前期大鼠 CE 与 CI 神经元所占的百分比与间情期大鼠没有明显的差异(P>0.05)。但情前期大鼠的 CE和 CI 神经元多呈现周期性放电,而间情期大鼠的 CE 和 CI 神经元则多呈现连续性放电。这说明 mPOA-AHA 神经元的电活动随生殖周期发生明显的变化。脑室注射去甲肾上腺素(NE)能使 mPOA-AHA 内大多数 CE 神经元对宫颈刺激的兴奋反应暂时受到抑制,而注射 NE 却使大多数 CI 神经元发生抑制解除。这些结果提示 NE 可能有抑制 CE 神经元和刺激 CI 神经元的作用。     

Macrophages acquire neutrophil granules for antimicrobial activity against intracellular pathogens

A key target of many intracellular pathogens is the macrophage. Although macrophages can generate antimicrobial activity, neutrophils have been shown to have a key role in host defense, presumably by their preformed granules containing antimicrobial agents. Yet the mechanism by which neutrophils can mediate antimicrobial activity against intracellular pathogens such as Mycobacterium tuberculosis has been a long-standing enigma. We demonstrate that apoptotic neutrophils and purified granules inhibit the growth of extracellular mycobacteria. Phagocytosis of apoptotic neutrophils by macrophages results in decreased viability of intracellular M. tuberculosis. Concomitant with uptake of apoptotic neutrophils, granule contents traffic to early endosomes, and colocalize with mycobacteria. Uptake of purified granules alone decreased growth of intracellular mycobacteria. Therefore, the transfer of antimicrobial peptides from neutrophils to macrophages provides a cooperative defense strategy between innate immune cells against intracellular pathogens and may complement other pathways that involve delivery of antimicrobial peptides to macrophages.