Stimulation Of Human Monocytes Research Articles

Multifaceted Proteome Analysis at Solubility, Redox, and Expression Dimensions for Target Identification.

Multifaceted interrogation of the proteome deepens the system-wide understanding of biological systems; however, mapping the redox changes in the proteome has so far been significantly more challenging than expression and solubility/stability analyses. Here, the first high-throughput redox proteomics approach integrated with expression analysis (REX) is devised and combined with the Proteome Integral Solubility Alteration (PISA) assay. The whole PISA-REX experiment with up to four biological replicates can be multiplexed into a single tandem mass tag TMTpro set. For benchmarking this compact tool, HCT116 cells treated with auranofin are analyzed, showing great improvement compared with previous studies. PISA-REX is then applied to study proteome remodeling upon stimulation of human monocytes by interferon α (IFN-α). Applying this tool to study the proteome changes in plasmacytoid dendritic cells (pDCs) isolated from wild-type versus Ncf1-mutant mice treated with interferon α, shows that NCF1 deficiency enhances the STAT1 pathway and modulates the expression, solubility, and redox state of interferon-induced proteins. Providing comprehensive multifaceted information on the proteome, the compact PISA-REX has the potential to become an industry standard in proteomics and to open new windows into the biology of health and disease.

Exploring the expression and potential function of follicle stimulating hormone receptor in extragonadal cells related to abdominal aortic aneurysm.

Follicle stimulating hormone (FSH) is identified to play a role in postmenopausal disease and hypothesized to affect abdominal aortic aneurysm (AAA) onset/progression in postmenopausal women. We aimed to detect FSHR gene expression in AAA tissue and cell types involved in AAA formation. FSH stimulation of human umbilical cord endothelial cells (HUVECs), smooth muscle cells (HUCs) and PMA-differentiated macrophages to assess gene expression of FSHR and various markers. Human macrophages activated with various stimuli were assessed for FSHR gene expression. AAA dataset, AAA tissue samples and AAA-derived smooth muscle cells (SMC) obtained from elderly female donors were assessed for FSHR gene expression. AAA-SMCs were stimulated with FSH to assess its effect on gene expression. Lastly, oxidized low-density-lipoprotein (ox-LDL) uptake and abundance of cell surface protein markers were assessed by flow cytometry after FSH stimulation of human monocytes. FSH stimulation showed similar levels of gene expression in HUVECs and HUCs. Only ACTA2 was downregulated in HUCs. In PMA-differentiated macrophages, gene expression of inflammation markers was unchanged after FSH stimulation. FSHR gene expression was found to be low in the AAA datasets. Female AAA-SMCs show occasional FSHR gene expression at a very low level, yet stimulation with FSH did not affect gene expression of SMC- or inflammation markers. FSH stimulation did not impact ox-LDL uptake or alter cell surface protein expression in monocytes. While FSHR gene expression was detected in human testis tissue, it was below quantification level in all other investigated cell types, even upon activation of macrophages with various stimuli. Despite previous reports, we did not detect FSHR gene expression in various extragonadal cell types, except in occasional female AAA-SMCs. No clear effect on cell activation was observed upon FSH stimulation in any cell type. Our data suggest that a direct effect of FSH in AAA-related extragonadal cells is unlikely to influence AAA.

The SARS-CoV-2 encoded ORF8 protein Stimulates Human Monocytes to Produce Pro-Inflammatory Cytokines

Abstract Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can result in a cytokine storm that is associated with poor outcomes in patients. The activation of a NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome following SARS-CoV-2 infection has been shown to play a major role in inflammatory immune responses. In this study, we show that open reading frame 8 (ORF8) protein is abundantly secreted as a glycoprotein in vitro. The glycosylated ORF8 stimulates human CD14+/CD16+ monocytes to cause upregulation of proinflammatory cytokine production and cell surface marker expression within 24 hours. The data suggests that stimulation of human monocytes by ORF8 is not receptor mediated. Rather, ORF8 is likely phagocytosed by human monocytes. ORF8 then stimulates the monocytes by independently binding to the NACHT and LRR domains of the NLRP3 protein. Pharmacologic inhibition of NLRP3 significantly diminished the production of pro-inflammatory cytokines from ORF8-stimulated human monocytes. Finally, this study shows that the ORF8 protein is also secreted in patients with newly diagnosed coronavirus disease 2019 (COVID-19). Levels of ORF8 in the blood of patients diagnosed with COVID-19 correlated with disease mortality and trajectory of disease. ORF8 stimulation of monocytes causes pro-inflammatory cytokine production that leads to the development of severe COVID-19.

Human macrophages and monocyte-derived dendritic cells stimulate the proliferation of endothelial cells through midkine production.

The cytokine midkine (MK) is a growth factor that is involved in different physiological processes including tissue repair, inflammation, the development of different types of cancer and the proliferation of endothelial cells. The production of MK by primary human macrophages and monocyte-derived dendritic cells (MDDCs) was never described. We investigated whether MK is produced by primary human monocytes, macrophages and MDDCs and the capacity of macrophages and MDDCs to modulate the proliferation of endothelial cells through MK production. The TLR stimulation of human monocytes, macrophages and MDDCs induced an average of ≈200-fold increase in MK mRNA and the production of an average of 78.2, 62, 179 pg/ml MK by monocytes, macrophages and MDDCs respectively (p < 0.05). MK production was supported by its detection in CD11c+ cells, CLEC4C+ cells and CD68+ cells in biopsies of human tonsils showing reactive lymphoid follicular hyperplasia. JSH-23, which selectively inhibits NF-κB activity, decreased the TLR-induced production of MK in PMBCs, macrophages and MDDCs compared to the control (p < 0.05). The inhibition of MK production by macrophages and MDDCs using anti-MK siRNA decreased the capacity of their supernatants to stimulate the proliferation of endothelial cells (p = 0.01 and 0.04 respectively). This is the first study demonstrating that the cytokine MK is produced by primary human macrophages and MDDCs upon TLR triggering, and that these cells can stimulate endothelial cell proliferation through MK production. Our results also suggest that NF-κB plays a potential role in the production of MK in macrophages and MDDCs upon TLR stimulation. The production of MK by macrophages and MDDCs and the fact that these cells can enhance the proliferation of endothelial cells by producing MK are novel immunological phenomena that have potentially important therapeutic implications.

Generation and functional characterization of recombinant Porphyromonas gingivalis W83 FimA

Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1β, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.

GLUT3 as an Intersection of Glycerophospholipid Metabolism and the Innate Immune Response to Candida albicans.

Immune cells can optimize the management of metabolic resources to balance their energy requirements in order to regulate immune responses. The interconnection between immunometabolism and fungal infections is becoming increasingly apparent. Using proteome and metabolome assays, we found that stimulation of primary human monocytes by Candida albicans was accompanied by upregulation of glucose transporter 3 (GLUT3) and activation of the glycerophospholipid metabolism pathway. Upregulated GLUT3 expression has been preliminarily confirmed in monocytes from patients with C. albicans bloodstream infection. Our findings support the importance of GLUT3 in the complex network of glycerophospholipid metabolism and the innate immune responses against C. albicans. In summary, this study might contribute to decipher the regulatory mechanism between the monocyte metabolic reprogramming and innate immune response and reveal potential targets for the antifungal treatments.

TLR8 is Activated by 5’-Methylthioinosine, a &lt;i&gt;Plasmodium Falciparum-Specific&lt;/i&gt; Intermediate of the Purine Salvage Pathway

The innate immune recognition of the malaria causing pathogen Plasmodium falciparum (Pf) is not yet fully explored. Utilizing genetic complementation assays, Toll-like receptor (TLR)-deficient immune cells, inhibitor studies and molecular modelling, we identified the nucleoside 5’-methylthioinosine (MTI) as a new natural human TLR8 agonist. MTI is a Plasmodium-specific intermediate of the purine salvage pathway and activates TLR8 to induce IL-6 and IL-1β secretion, respectively. Co-incubation of synthetic MTI with the TLR8 enhancer poly(dT) as well as synthetic single-stranded RNA or RNA purified from P. falciparum strongly increased its stimulatory activity. Importantly, MTI generated from methylthioadenosine (MTA) in vitro by P. falciparum lysates under conditions where MTI metabolization through purine nucleoside phosphorylase ( Pf PNP) is blocked by immucillins, produced inflammatory cytokines. Similarly, phagocytosed Pf infected red blood cells incubated with MTI led to increased immune stimulation of human monocytes. In summary, the pathogen-derived nucleoside MTI is a new natural human TLR8 ligand underlining the in vivo relevance for MTI in immune response to P. falciparum. The use of immucillins may boost TLR8-mediated immunity against Plasmodium by accumulation of MTI. These findings enhance our understanding how the innate immune system senses Plasmodium and offer new avenues for treatment.

Neutral Lipids Are Not a Source of Arachidonic Acid for Lipid Mediator Signaling in Human Foamy Monocytes

Human monocytes exposed to free arachidonic acid (AA), a secretory product of endothelial cells, acquire a foamy phenotype which is due to the accumulation of cytoplasmic lipid droplets with high AA content. Recruitment of foamy monocytes to the inflamed endothelium contributes to the development of atherosclerotic lesions. In this work, we investigated the potential role of AA stored in the neutral lipids of foamy monocytes to be cleaved by lipases and contribute to lipid mediator signaling. To this end, we used mass spectrometry-based lipidomic approaches combined with strategies to generate monocytes with different concentrations of AA. Results from our experiments indicate that the phospholipid AA pool in monocytes is stable and does not change upon exposure of the cells to the external AA. On the contrary, the AA pool in triacylglycerol is expandable and can accommodate relatively large amounts of fatty acid. Stimulation of the cells with opsonized zymosan results in the expected decreases of cellular AA. Under all conditions examined, all of the AA decreases observed in stimulated cells were accounted for by decreases in the phospholipid pool; we failed to detect any contribution of the triacylglycerol pool to the response. Experiments utilizing selective inhibitors of phospholipid or triacylglyerol hydrolysis confirmed that the phospholipid pool is the sole contributor of the AA liberated by stimulated cells. Thus, the AA in the triacylglycerol is not a source of free AA for the lipid mediator signaling during stimulation of human foamy monocytes and may be used for other cellular functions.

The impact of sex hormones on BCG-induced trained immunity.

The anti-tuberculosis vaccine Bacillus Calmette-Guérin (BCG) is a well-known immune modulator that induces nonspecific protective effects against heterologous infections through induction of innate immune memory, also termed "trained immunity." In randomized trials in low weight newborns, BCG vaccination reduced neonatal mortality due to decreased incidence of sepsis and respiratory infections. In many studies, sex-differential nonspecific effects of vaccines have been observed, but the mechanisms behind these differential effects are unknown. We investigated whether the important sex hormones estrogen and dihydrotestosterone (DHT) influence BCG-induced trained immunity in human primary monocytes. Although addition of estradiol and DHT to BCG inhibited the production of proinflammatory cytokines after direct stimulation of human monocytes, they did not influence the induction of trained immunity by BCG. In addition, estradiol or DHT did not induce training or tolerance in monocytes themselves. We conclude that these important sex hormones are unlikely to explain the sex-differential effects after BCG vaccination. Future studies should focus on the investigation of alternative mechanisms as an explanation for sex-differential nonspecific effects of BCG vaccination.

Suppression of CYP1 members of the AHR response by pathogen-associated molecular patterns.

The aryl hydrocarbon receptor (AHR) is a ligand-activated transcription factor that triggers a broad response, which includes the regulation of proinflammatory cytokine production by monocytes and macrophages. AHR is negatively regulated by a set of genes that it transcriptionally activates, including the AHR repressor (Ahrr) and the cytochrome P450 1 (Cyp1) family, which are critical for preventing exacerbated AHR activity. An imbalance in these regulatory mechanisms has been shown to cause severe defects in lymphoid cells. Therefore, we wanted to assess how AHR activation is regulated in monocytes and macrophages in the context of innate immune responses induced by pathogen-associated molecular patterns (PAMPs). We found that concomitant stimulation of primary human monocytes with PAMPs and the AHR agonist 6-formylindolo(3,2-b)carbazole (FICZ) led to a selective dose-dependent inhibition of Cyp1 family members induction. Two other AHR-dependent genes [Ahrr and NADPH quinone dehydrogenase 1 (Nqo1)] were not affected under these conditions, suggesting a split in the AHR regulation by PAMPs. This down-regulation of Cyp1 family members did not require de novo protein production nor signaling through p38, ERK, or PI3K-Akt-mammalian target of rapamycin (mTOR) pathways. Furthermore, such a split regulation of the AHR response was more apparent in GM-CSF-derived macrophages, a finding corroborated at the functional level by decreased CYP1 activity and decreased proinflammatory cytokine production in response to FICZ and LPS. Collectively, our findings identify a role for pattern recognition receptor (PRR) signaling in regulating the AHR response through selective down-regulation of Cyp1 expression in human monocytes and macrophages.

A System Dynamics Model to Predict the Human Monocyte Response to Endotoxins.

System dynamics is a powerful tool that allows modeling of complex and highly networked systems such as those found in the human immune system. We have developed a model that reproduces how the exposure of human monocytes to lipopolysaccharides (LPSs) induces an inflammatory state characterized by high production of tumor necrosis factor alpha (TNFα), which is rapidly modulated to enter into a tolerant state, known as endotoxin tolerance (ET). The model contains two subsystems with a total of six states, seven flows, two auxiliary variables, and 14 parameters that interact through six differential and nine algebraic equations. The parameters were estimated and optimized to obtain a model that fits the experimental data obtained from human monocytes treated with various LPS doses. In contrast to publications on other animal models, stimulation of human monocytes with super-low-dose LPSs did not alter the response to a second LPSs challenge, neither inducing ET, nor enhancing the inflammatory response. Moreover, the model confirms the low production of TNFα and increased levels of C–C motif ligand 2 when monocytes exhibit a tolerant state similar to that of patients with sepsis. At present, the model can help us better understand the ET response and might offer new insights on sepsis diagnostics and prognosis by examining the monocyte response to endotoxins in patients with sepsis.

Abstract 260: Monocyte to Macrophage Conversion is Guided by Isoform Switching of the Actin Capping Protein Y-Adducin

Background: Modulation of cellular function necessitates a versatile utilization of the transcriptome, thereby altering the proteome. These functional adaptations are post-transcriptionally guided by RNA-binding proteins (RBP), which confer cells with the capacity to rapidly respond to various stimuli and stressors. We recently discovered a pivotal role for the RBP Quaking (QKI) in directing the conversion of monocytes into macrophages, with in particular alternative splicing of pre-mRNAs critically impacting the acquisition of a pro-inflammatory phenotype. Computational analysis indicated that this differentiation process was dependent on dynamic mobilization of the actin cytoskeletal network, which prompted us to focus our attention on an exclusion event of exon 13 at the C-terminus of the actin-capping protein gamma-Adducin (ADD3). At present, the functional consequences of ADD3 isoform switching are unknown. Methods and Results: Stimulation of primary human monocytes with GM-CSF initially led to a decrease in ADD3 mRNA expression while ADD3 exon 13 usage was preserved. However, mRNA expression was restored upon differentiation towards the pro-inflammatory macrophage phenotype (t=3 days), while exon 13 inclusion strikingly dropped from 47.2% ± 10.5 to 7.7% ± 2.6 (p=0.0387, n=3). Having previously shown that QKI protein expression increases upon conversion to a macrophage, we sought to pinpoint whether QKI specifically regulates this ADD3 splicing event. Therefore, we mutated the QKI binding site (ACUAA → ACGAA) proximal to exon 13 using an ADD3 minigene. These studies revealed that disrupting the capacity for QKI to bind at this intronic sequence almost completely abolished exon 13 splicing (ACGAA: 94.9% ± 0.0025 inclusion vs. ACUAA: 2.0% ± 0.0009 inclusion; p&lt;0.0001, n=3). Using predictive bioinformatics tools, exclusion of exon 13 was found to induce conformational changes in ADD3 protein structure. We subsequently utilized atomic force microscopy to study actomyosin dynamics and cell stiffness, pressure and tension in monocytes overexpressing the distinct ADD3 isoforms. Conclusions: QKI-mediated splicing of ADD3 triggers an isoform switch that impacts monocyte and macrophage function by altering the actin cytoskeleton.

Cholesterol crystals enhance TLR2- and TLR4-mediated pro-inflammatory cytokine responses of monocytes to the proatherogenic oral bacterium Porphyromonas gingivalis.

Cholesterol deposits and pro-inflammatory cytokines play an essential role in the pathogenesis of atherosclerosis, a predominant cause of cardiovascular disease (CVD). Epidemiological evidence has linked periodontal disease (PD) with atherosclerotic CVD. Accordingly, viable periodontal pathogens, including Porphyromonas gingivalis, have been found in atherosclerotic plaques in humans and mice. We aimed to determine whether cholesterol crystals (CHCs) and oral bacteria synergize in the stimulation of human monocytes. Incubation of human monocytes with CHCs induced secretion of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, and IL-8. Moreover, CHCs markedly enhanced secretion of IL-1β by monocytes stimulated with the toll-like receptor (TLR) 4 agonist Escherichia coli lipopolysaccharide (LPS), and the TLR2 agonist Staphylococcus aureus lipoteichoic acid. Notably, CHCs also enhanced IL-1β secretion induced by P. gingivalis LPS and IL-1β secretion induced by whole P. gingivalis bacteria. This enhancement was abrogated by the NLRP3 inflammasome inhibitors Z-YVAD-FMK and glibenclamide. CHCs had no effect on cytokine production induced by P. gingivalis gingipains. Taken together, our findings support that CHCs, via stimulation of NLRP3 inflammasomes, act in synergy with the periodontal pathogen P. gingivalis to promote monocyte secretion of pro-atherogenic cytokines.

IL12, IL10, IFNγ and TNFα Expression in Human Primary Monocytes Stimulated with Bacterial Heat Shock GroEL (Hsp64) Protein.

Actinobacillus (Aggregatibacter) actinomycetemicomitans (Aa) is a bacterium that lives in the oral cavity and plays an important role in periodontal diseases. The effect of A.actinomycetemcomitans’s heat shock family protein GroEL on host or immune cells including monocytes is quite limited. In this study, the recombinant A.actinomycetemcomitans’s GroEL protein (rAaGroEL) was used as an antigen and its effects on monocytes of peripheral blood mononuclear cells (PBMCs) was investigated. To do that, PBMCs were stimulated with rAaGroEL protein at different time points (16h to 96h) and the cytokines of CD14+ monocytes were detected with intracellular cytokine staining by Flow cytometry. Data showed that AaGroEL protein has an antigenic effect on human primary monocytes. AaGroEL protein responsive CD14 monocytes stimulates the expression of IL12, IL10, IFNγ and TNFα cytokines with different kinetics and expression profile. Therefore, A. actinomycetemcomitans’s heat shock GroEL protein can modulate innate and adaptive immune responses and contribute to an inflammatory diseases pathology.

Redox Stimulation of Human THP-1 Monocytes in Response to Cold Physical Plasma.

In plasma medicine, cold physical plasma delivers a delicate mixture of reactive components to cells and tissues. Recent studies suggested a beneficial role of cold plasma in wound healing. Yet, the biological processes related to the redox modulation via plasma are not fully understood. We here used the monocytic cell line THP-1 as a model to test their response to cold plasma in vitro. Intriguingly, short term plasma treatment stimulated cell growth. Longer exposure only modestly compromised cell viability but apparently supported the growth of cells that were enlarged in size and that showed enhanced metabolic activity. A significantly increased mitochondrial content in plasma treated cells supported this notion. On THP-1 cell proteome level, we identified an increase of protein translation with key regulatory proteins being involved in redox regulation (hypoxia inducible factor 2α), differentiation (retinoic acid signaling and interferon inducible factors), and cell growth (Yin Yang 1). Regulation of inflammation is a key element in many chronic diseases, and we found a significantly increased expression of the anti-inflammatory heme oxygenase 1 (HMOX1) and of the neutrophil attractant chemokine interleukin-8 (IL-8). Together, these results foster the view that cold physical plasma modulates the redox balance and inflammatory processes in wound related cells.

Reconfigurable microfluidic device with integrated antibody arrays for capture, multiplexed stimulation, and cytokine profiling of human monocytes.

Monocytes represent a class of immune cells that play a key role in the innate and adaptive immune response against infections. One mechanism employed by monocytes for sensing foreign antigens is via toll-like receptors (TLRs)-transmembrane proteins that distinguish classes of foreign pathogens, for example, bacteria (TLR4, 5, and 9) vs. fungi (TLR2) vs. viruses (TLR3, 7, and 8). Binding of antigens activates a signaling cascade through TLR receptors that culminate in secretion of inflammatory cytokines. Detection of these cytokines can provide valuable clinical data for drug developers and disease investigations, but this usually requires a large sample volume and can be technically inefficient with traditional techniques such as flow cytometry, enzyme-linked immunosorbent assay, or luminex. This paper describes an approach whereby antibody arrays for capturing cells and secreted cytokines are encapsulated within a microfluidic device that can be reconfigured to operate in serial or parallel mode. In serial mode, the device represents one long channel that may be perfused with a small volume of minimally processed blood. Once monocytes are captured onto antibody spots imprinted into the floor of the device, the straight channel is reconfigured to form nine individually perfusable chambers. To prove this concept, the microfluidic platform was used to capture monocytes from minimally processed human blood in serial mode and then to stimulate monocytes with different TLR agonists in parallel mode. Three cytokines, tumor necrosis factor-α, interleukin (IL)-6, and IL-10, were detected using anti-cytokine antibody arrays integrated into each of the six chambers. We foresee further use of this device in applications such as pediatric immunology or drug/vaccine testing where it is important to balance small sample volume with the need for high information content.

A Novel, Highly Selective RT-QPCR Method for Quantification of MSRV Using PNA Clamping Syncytin-1 (ERVWE1).

HERV-W is a multi-locus family of human endogenous retroviruses (HERVs) that has been found to play an important role in human physiology and pathology. Two particular members of HERV-W family are of special interests: ERVWE1 (coding syncytin-1, which is a glycoprotein essential in the formation of the placenta) and MSRV (multiple sclerosis-associated retrovirus that is thought to play a significant role in human pathology as a result of its increased expression in the brain tissue and blood cells derived from patients with multiple sclerosis (MS)). Both ERVWE1 and MSRV mRNA share high level of similarity and hence a method that allows to exclusively quantify the MSRV expression in clinical samples would be desirable. We developed a quantitative polymerase chain reaction (QPCR) technique for the detection and quantification of the multiple sclerosis-associated retrovirus. The assay utilises fluorescently labelled oligonucleotide probe, which is complementary to the conservative fragment of MSRV env gene and a peptide nucleic acid (PNA) probe, fully complementary to the ERVWE1 sequence fragment that efficiently blocks the polymerase action on ERVWE1 templates. The PNA molecule, if used parallel with hydrolysis probe in QPCR analysis, greatly facilitates the detection efficiency of MSRV even if ERVWE1 is present abundantly in respect to MSRV in the analysed sample. We achieved a wide and measurable range from 1 × 10 e2 to 1 × 10 e8 copies/reaction; the linearity of the technique was maintained even at the low MSRV level of 1 % in respect to ERVWE1. Using our newly developed method we confirmed that the expression of MSRV takes place in normal human astrocytes and in human umbilical vein endothelial cells in vitro. We also found that the stimulation of human monocytes did not influence the specific expression of MSRV but it caused changes in mRNA level of distinct HERV-W templates.

Toll-like receptor 2 agonist Pam3CSK4 up-regulates FcεRI receptor expression on monocytes from patients with severe extrinsic atopic dermatitis.

Both microbial antigens and allergens are important factors that can trigger atopic dermatitis (AD). Monocytes from patients with AD have been found to express increased and sustained levels of high-affinity IgE receptor (FcεRI) and Toll-like receptor 2 (TLR2). We hypothesized that putative interactions exist between TLR2 and FcεRI on monocytes in the pathogenesis of AD. This study aimed to understand whether activation of TLR2 by Pam3CSK4 would influence the expression of FcεRI, and whether mitogen-activated protein kinase (MAPK) signalling pathways were involved in such regulation. Peripheral blood monocytes from patients with severe extrinsic AD or healthy control patients were treated with the TLR2 agonist Pam3CSK4. The expression of FcεRI, intracellular TNF-α and MAPK family members were analysed by real-time quantitative PCR, flow cytometry and western blotting. Monocytes from patients with severe extrinsic AD expressed higher levels of surface FcεRIα than were found in monocytes from healthy controls. Stimulation of human monocytes from patients with Pam3CSK4, but not lipopolysaccharide (LPS), resulted in the up-regulation of surface FcεRI expression by inducing p38 phosphorylation. Pretreatment with a specific inhibitor of p38 kinase inhibited the Pam3CSK4-induced up-regulation of FcεRIα, suggesting the involvement of the p38 pathway in the regulation of this process. Our findings indicated interactions between TLR2 and FcεRI occurred via the activation of p38 in patients with severe extrinsic AD, which might indicate insights into understanding the mechanisms of how bacterial infection can exacerbate the clinical features of AD.

Inflammasome activation: A form of autocrine purinergic signaling in monocytes

Previously, we have demonstrated that autocrine purinergic signaling plays an important role in host defense. ATP is released from immune cells including neutrophils and T cells upon pathogen recognition. Released ATP triggers autocrine feedback signaling via purinergic receptors that regulate cell functions such as chemotaxis and T cell proliferation. Here, we studied whether inflammasome activation in monocytes involves similar autocrine purinergic signaling mechanisms. We found that toll-like receptor (TLR) stimulation of human monocytes causes the release of ATP, which could be blocked with the gap junction inhibitor carbenoxolone (CBX) (Fig. 1A). Inhibition of ATP release with CBX or interruption of autocrine purinergic feedback with suramin, a non-specific P2 receptor antagonist, dose-dependently blocked TLR-induced IL-1β expression by lipopolysaccharide (LPS) stimulated monocytes (Fig. 1B). We conclude that inflammasome activation in response to TLR stimulation involves autocrine purinergic signaling systems similar to those previously defined in neutrophils and T cells. ­­This study was supported by NIH grants R01 GM51477, R01 GM60475, R01 AI080582, and T32 GM103702.­­ Fig. 1 A. Human monocytes were stimulated with LPS (500 pg/ml), in the presence or absence of CBX (100 µM) and ATP release was measured with a luciferase assay. B. Monocytes were pre-treated for 20 min with suramin, stimulated with LPS (500 pg/ml), and IL-1β expression measured by ELISA after 24 h. Student's t-test, * p<0.05; 蠅3 independent experiments.

Synthesis and biological evaluation of pyridazino[1′,6′:1,2]pyrido[3,4-b]indolinium and pyridazino[1,6-a]benzimidazolium salts as anti-inflammatory agents

Condensed polycyclic heteroaromatic cations bearing a bridgehead nitrogen with pyridazino[1′,6′:1,2]pyrido[3,4-b]indolinium and pyridazino[1,6-a]benzimidazolium structures were assayed as inhibitors of LPS-induced TNF-α production by THP-1 cells. The hit compound 1e, which had the best IC50 value (4.49 μM) and low toxicity, was further assayed on human PMBCs (IC50 3.91 μM) and monocytes (IC50 1.82 μM). This compound also inhibited TNF-α production following poly I:C stimulation of human monocytes and monocyte-derived dendritic cells; in the latter case, inhibition of IL-12 production was also observed. Compound 1e was also able to inhibit TNF-α expression at the transcriptional level and proved to be effective in vivo. Compound 1e is an interesting potential therapeutic agent in IMIDs.