Expression In Human Macrophages Research Articles

Cannflavin A inhibits TLR4-induced chemokine and cytokine expression in human macrophages

Cannflavin A (CFL-A), a flavonoid present in the hemp plant Cannabis sativa L. (C. sativa), has anti-inflammatory and neuroprotective capacity. Research continues to elucidate the anti-inflammatory effects of components of C. sativa, with evidence that plant-derived cannabinoids and terpenes can mediate anti-inflammatory activity by targeting toll-like receptor (TLR) signalling, the sensors of pathogen-associated molecules. This study set out to determine if TLR-mediated inflammatory signalling is a CFL-A target using the endotoxin lipopolysaccharide (LPS) to induce TLR4 signalling in human THP-1-derived macrophages. TLR4 activation promoted the production of the chemokine CXCL10 and cytokines IL-1β and TNFα. Treatment with CFL-A dose-dependently attenuated TLR4-induced CXCL10 and IL-1β secretion, with our findings also indicating that the inhibitory effects of CFL-A on chemokine/cytokine secretion are in line with an NF-κB inhibitor. This study highlights TLR4 signalling as a cannflavin target in macrophages.

Assessment of the alpha 7 nicotinic acetylcholine receptor as an imaging marker of cardiac repair-associated processes using NS14490

BackgroundCardiac repair and remodeling following myocardial infarction (MI) is a multifactorial process involving pro-reparative inflammation, angiogenesis and fibrosis. Noninvasive imaging using a radiotracer targeting these processes could be used to elucidate cardiac wound healing mechanisms. The alpha7 nicotinic acetylcholine receptor (ɑ7nAChR) stimulates pro-reparative macrophage activity and angiogenesis, making it a potential imaging biomarker in this context. We investigated this by assessing in vitro cellular expression of ɑ7nAChR, and by using a tritiated version of the PET radiotracer [18F]NS14490 in tissue autoradiography studies.Resultsɑ7nAChR expression in human monocyte-derived macrophages and vascular cells showed the highest relative expression was within macrophages, but only endothelial cells exhibited a proliferation and hypoxia-driven increase in expression. Using a mouse model of inflammatory angiogenesis following sponge implantation, specific binding of [3H]NS14490 increased from 3.6 ± 0.2 µCi/g at day 3 post-implantation to 4.9 ± 0.2 µCi/g at day 7 (n = 4, P < 0.01), followed by a reduction at days 14 and 21. This peak matched the onset of vessel formation, macrophage infiltration and sponge fibrovascular encapsulation. In a rat MI model, specific binding of [3H]NS14490 was low in sham and remote MI myocardium. Specific binding within the infarct increased from day 14 post-MI (33.8 ± 14.1 µCi/g, P ≤ 0.01 versus sham), peaking at day 28 (48.9 ± 5.1 µCi/g, P ≤ 0.0001 versus sham). Histological and proteomic profiling of ɑ7nAChR positive tissue revealed strong associations between ɑ7nAChR and extracellular matrix deposition, and rat cardiac fibroblasts expressed ɑ7nAChR protein under normoxic and hypoxic conditions.Conclusionɑ7nAChR is highly expressed in human macrophages and showed proliferation and hypoxia-driven expression in human endothelial cells. While NS14490 imaging displays a pattern that coincides with vessel formation, macrophage infiltration and fibrovascular encapsulation in the sponge model, this is not the case in the MI model where the ɑ7nAChR imaging signal was strongly associated with extracellular matrix deposition which could be explained by ɑ7nAChR expression in fibroblasts. Overall, these findings support the involvement of ɑ7nAChR across several processes central to cardiac repair, with fibrosis most closely associated with ɑ7nAChR following MI.

Tim-3 Is Differentially Expressed during Cell Activation and Interacts with the LSP-1 Protein in Human Macrophages.

T-cell Immunoglobulin and Mucin Domain 3 (TIM-3) is an immune checkpoint receptor known to regulate T-cell activation and has been targeted for immunotherapy in cancer and other diseases. However, its expression and function in other cell types, such as macrophages, are poorly understood. This study investigated TIM-3 expression in human macrophages polarized to M1 (stimulated with IFN-γ and LPS) and M2 (stimulated with IL-4 and IL-13) phenotypes using an in vitro model. Our results show that M1 macrophages have a lower frequency of TIM-3+ cells compared to M2 macrophages at 48 and 72 hr poststimulation. Additionally, we observed differential levels of soluble ADAM 10, an enzyme responsible for TIM-3 release, in the supernatants of M1 and M2 macrophages at 72 hr. We also found that the TIM-3 intracellular tail might associate with lymphocyte-specific protein 1 (LSP-1), a protein implicated in cell motility and podosome formation. These findings enhance our understanding of TIM-3 function in myeloid cells such as macrophages and may inform the development of immunotherapies with reduced immune-related adverse effects.

Extracellular vesicles secreted by adipose tissue during obesity and type 2 diabetes mellitus influence cholesterol metabolism gene expression in human macrophages

Extracellular vesicles secreted by adipose tissue during obesity and type 2 diabetes mellitus influence cholesterol metabolism gene expression in human macrophages

TNF promotes DECTIN2 family C-type lectin receptor expression in human macrophages.

TNF blockade constitutes an effective therapy for inflammatory bowel disease, yet increases the risk for infection, including active tuberculosis. The DECTIN2 family C-type lectin receptors MINCLE, MCL, and DECTIN2 sense mycobacterial ligands and activate myeloid cells. In mice, upregulation of DECTIN2 family C-type lectin receptor after stimulation with Mycobacterium bovis Bacille Calmette-Guérin requires TNF. Here, we investigated whether TNF controls inducible C-type lectin receptor expression in human myeloid cells. Monocyte-derived macrophages were stimulated with Bacille Calmette-Guérin and the TLR4 ligand lipopolysaccharide, and expression of C-type lectin receptor was analyzed. Bacille Calmette-Guérin and lipopolysaccharide strongly upregulated messenger RNA expression of DECTIN2 family C-type lectin receptor but not of DECTIN1. Bacille Calmette-Guérin and lipopolysaccharide also induced robust production of TNF. Recombinant TNF was sufficient to upregulate expression of DECTIN2 family C-type lectin receptor. Blocking TNF with the TNFR2-Fc fusion protein etanercept abrogated, as expected, the effect of recombinant TNF and impaired induction of DECTIN2 family C-type lectin receptor by Bacille Calmette-Guérin and lipopolysaccharide. Flow cytometry confirmed upregulation of MCL at the protein level by recombinant TNF and showed inhibition of Bacille Calmette-Guérin-induced MCL by etanercept. To investigate the impact of TNF on C-type lectin receptor expression in vivo, we analyzed peripheral blood mononuclear cells of patients with inflammatory bowel disease and observed downregulation of MINCLE and MCL expression after therapeutic TNF blockade. Together, TNF is sufficient to upregulate DECTIN2 family C-type lectin receptor in human myeloid cells and contributes to this process after encounter with Bacille Calmette-Guérin or lipopolysaccharide. Impaired C-type lectin receptor expression in patients receiving TNF blockade may dampen the sensing of microbes and defense against infection.

Gene expression profiling of human macrophages after graphene oxide and graphene nanoplatelets treatment reveals particle-specific regulation of pathways

Graphene and its derivatives are attractive materials envisaged to enable a wealth of novel applications in many fields including energy, electronics, composite materials or health. A comprehensive understanding of the potential adverse effects of graphene-related materials (GRM) in humans is a prerequisite to the safe use of these promising materials. Here, we exploited gene expression profiling to identify transcriptional responses and toxicity pathways induced by graphene oxide (GO) and graphene nanoplatelets (GNP) in human macrophages. Primary human monocyte-derived macrophages (MDM) and a human macrophage cell line, i.e. differentiated THP-1 cells, were exposed to 5 or 20 μg/mL GO and GNP for 6 and 24 h to capture early and more persistent acute responses at realistic or slightly overdose concentrations. GO and GNP induced time-, dose- and macrophage type-specific differential expression of a substantial number of genes with some overlap between the two GRM types (up to 384 genes (9.6%) or 447 genes (20.4%) in THP-1 or MDM, respectively) but also a high number of genes exclusively deregulated from each material type. Furthermore, GRM responses on gene expression were highly different from those induced by inflammogenic material crystalline quartz (maximum of 64 (2.3%) or 318 (11.3%) common genes for MDM treated with 20 μg/mL GO and GNP, respectively). Further bioinformatics analysis revealed that GNP predominantly activated genes controlling inflammatory and apoptotic pathways whereas GO showed only limited inflammatory responses. Interestingly, both GRM affected the expression of genes related to antigen processing and presentation and in addition, GO activated pathways of neutrophil activation, degranulation and immunity in MDM. Overall, this study provides an extensive resource of potential toxicity mechanisms for future safety assessment of GRM in more advanced model systems to verify if the observed changes in gene expression in human macrophages could lead to long-term consequences on human health.

Mitochondrial ferritin expression in human macrophages is facilitated by thrombin-mediated cleavage under hypoxia.

Ferritins are iron storage proteins, which maintain cellular iron homeostasis. Among these proteins, the ferritin heavy chain is well characterized, but the regulatory principles of mitochondrial ferritin (FTMT) remain elusive. FTMT appears to be cleaved from a 27 kDa to a 22 kDa form. In human macrophages, FTMT increased under hypoxia in a hypoxia-inducible factor 2-dependent manner. Occurrence of FTMT resulted from cleavage by thrombin, which was supplied by serum. Inhibition of thrombin as well as serum removal decreased FTMT, while supplementation of thrombin under serum-deprived conditions restored its expression. Besides hypoxia, thrombin facilitated FTMT expression after treatment with the ferroptosis inducer RSL3 and the pro-inflammatory stimulus lipopolysaccharide. This study provides insights into the regulation of FTMT under hypoxia and identifies thrombin as a FTMT maturation-associated peptidase.

POP1 inhibits MSU-induced inflammasome activation and ameliorates gout.

Canonical inflammasomes are innate immune protein scaffolds that enable the activation of inflammatory caspase-1, and subsequently the processing and release of interleukin (IL)-1β, IL-18, and danger signals, as well as the induction of pyroptotic cell death. Inflammasome assembly and activation occurs in response to sensing of infectious, sterile and self-derived molecular patterns by cytosolic pattern recognition receptors, including the Nod-like receptor NLRP3. While these responses are essential for host defense, excessive and uncontrolled NLRP3 inflammasome responses cause and contribute to a wide spectrum of inflammatory diseases, including gout. A key step in NLRP3 inflammasome assembly is the sequentially nucleated polymerization of Pyrin domain (PYD)- and caspase recruitment domain (CARD)-containing inflammasome components. NLRP3 triggers polymerization of the adaptor protein ASC through PYD-PYD interactions, but ASC polymerization then proceeds in a self-perpetuating manner and represents a point of no return, which culminates in the activation of caspase-1 by induced proximity. In humans, small PYD-only proteins (POPs) lacking an effector domain regulate this key process through competitive binding, but limited information exists on their physiological role during health and disease. Here we demonstrate that POP1 expression in macrophages is sufficient to dampen MSU crystal-mediated inflammatory responses in animal models of gout. Whether MSU crystals are administered into a subcutaneous airpouch or into the ankle joint, the presence of POP1 significantly reduces neutrophil infiltration. Also, airpouch exudates have much reduced IL-1β and ASC, which are typical pro-inflammatory indicators that can also be detected in synovial fluids of gout patients. Exogenous expression of POP1 in mouse and human macrophages also blocks MSU crystal-induced NLRP3 inflammasome assembly, resulting in reduced IL-1β and IL-18 secretion. Conversely, reduced POP1 expression in human macrophages enhances IL-1β secretion. We further determined that the mechanism for the POP1-mediated inhibition of NLRP3 inflammasome activation is through its interference with the crucial NLRP3 and ASC interaction within the inflammasome complex. Strikingly, administration of an engineered cell permeable version of POP1 was able to ameliorate MSU crystal-mediated inflammation in vivo, as measured by neutrophil infiltration. Overall, we demonstrate that POP1 may play a crucial role in regulating inflammatory responses in gout.

Porphyromonas gingivalis outer membrane vesicles modulate cytokine and chemokine production by gingipain-dependent mechanisms in human macrophages

ObjectiveThe aim was to determine the changes of inflammatory mediator expression in human macrophages stimulated with outer membrane vesicles purified from Porphyromonas gingivalis. Designouter membrane vesicles purified by ultracentrifugation from ATCC 33277 and W83 P. gingivalis strains were used for stimulating human macrophages and determine their inflammatory mediator expression changes. U937 monocyte cells line were differentiated into macrophages and stimulated with outer membrane vesicles for 30 min and six hours. In Independent experiments, the outer membrane vesicles and viable bacteria control were pre-treated with the gingipain inhibitors KYT-1 and KYT-36 (Arg-gingipain and Lys-gingipain, respectively) or Polymyxin-B to block the lipopolysaccharide activity to evaluate the secretion changes of immune mediators IL-1β, IL-6, TNF-α, IL-8, MCP-1, MIP-1α and RANTES by flow cytometry. A factorial ANOVA was used to analyze the data. ResultsThe outer membrane vesicles of P. gingivalis ATCC 33277 displayed higher Arg-gingipain activity than those obtained from the P. gingivalis W83 strain (0.6 U/μg vs. 0.46 U/μg). Although the outer membrane vesicles of P. gingivalis stimulated the production of cytokines and chemokines, specific Arg-gingipain and Lys-gingipain inhibition induced significant increases in IL-1β, IL-6, IL-8, MCP-1, and RANTES levels, and this induction was significantly greater at 6 h compared to 30 min (*p < 0.05). On the contrary, TNF-α secretion decreased when gingipains were blocked. Conclusionsouter membrane vesicles may play a dual role during P. gingivalis infection based on their ability to induce changes in the immune responses of human macrophages, probably via gingipain-dependent events.

The long noncoding RNA LUCAT1 promotes immune gene expression in human macrophages

Abstract Long noncoding RNAs (lncRNA) are transcripts larger than 200 nucleotides that mediate their molecular function in an RNA-dependent manner by regulating gene expression at either the transcriptional or post-transcriptional level. lncRNAs play vital roles in many biological processes; however, their functions in immune cells are largely unexplored. Here we describe the expression, function and the mechanism of action of LUCAT1 (Lung cancer associated transcript 1; SCAL1) in human macrophages. While elevated LUCAT1 expression is observed across many cancers, its cellular function in physiological contexts remains unknown. We show that Toll-like receptor 4 (TLR4) and PMA upregulates LUCAT1 expression in human monocyte-derived macrophages and THP1 cells through ERK1/2 signaling. Using 3’-RACE, long-range PCR and RNA FISH, we demonstrate that the LUCAT1 gene transcribes an abundant, 2562-nucleotides long nuclear-localized RNA. Transient perturbation of the nuclear LUCAT1 using antisense oligonucleotides followed by RNA sequencing and protein assays revealed that LUCAT1 promotes the expression of cytokines and chemokines (CSF3, IL24 and CXCL5), transcription factors (NR4A1 and NR4A3), matrix metalloproteinases (MMP1, MMP3 and MMP19) and others in human macrophages. Using siRNA-mediated knockdown of LUCAT1-interacting nuclear proteins, we show that RBMX contributes to the molecular function of LUCAT1, whereas SFPQ controls the biogenesis of the mature LUCAT1. Further, the nuclear LUCAT1 is chromatin-associated, and acts at the level of gene transcription. Collectively, in this study we have uncovered the function of LUCAT1 in macrophages, underscoring the critical roles of lncRNAs in the immune system.

TAAR1 Expression in Human Macrophages and Brain Tissue: A Potential Novel Facet of MS Neuroinflammation.

TAAR1 is a neuroregulator with emerging evidence suggesting a role in immunomodulation. Multiple sclerosis (MS) is an immune-mediated demyelinating disease of the central nervous system. Here, we investigate TAAR1 expression in human primary monocytes, peripherally-derived macrophages, and MS brain tissue. RT-qPCR was used to assess TAAR1 levels in MS monocytes. Using a previously validated anti-human TAAR1 antibody and fluorescence microscopy, TAAR1 protein was visualized in lipopolysaccharide-stimulated or basal human macrophages, as well as macrophage/microglia populations surrounding, bordering, and within a mixed active/inactive MS lesion. In vivo, TAAR1 mRNA expression was significantly lower in MS monocytes compared to age- and sex-matched healthy controls. In vitro, TAAR1 protein showed a predominant nuclear localization in quiescent/control macrophages with a shift to a diffuse intracellular distribution following lipopolysaccharide-induced activation. In brain tissue, TAAR1 protein was predominantly expressed in macrophages/microglia within the border region of mixed active/inactive MS lesions. Considering that TAAR1-mediated anti-inflammatory effects have been previously reported, decreased mRNA in MS patients suggests possible pathophysiologic relevance. A shift in TAAR1 localization following pro-inflammatory activation suggests its function is altered in pro-inflammatory states, while TAAR1-expressing macrophages/microglia bordering an MS lesion supports TAAR1 as a novel pharmacological target in cells directly implicated in MS neuroinflammation.

Regulation of Apolipoprotein A-I Gene Expression in Human Macrophages by Oxidized Low-Density Lipoprotein.

Apolipoprotein A-I (ApoA-I) is a key component of reverse cholesterol transport in humans. In the previous studies, we demonstrated expression of the apoA-I gene in human monocytes and macrophages; however, little is known on the regulation of the apoA-I expression in macrophages during the uptake of modified low-density lipoprotein (LDL), which is one of the key processes in the early stages of atherogenesis leading to formation of foam cells. Here, we demonstrate a complex nature of the apoA-I regulation in human macrophages during the uptake of oxidized LDL (oxLDL). Incubation of macrophages with oxLDL induced expression of the apoA-I gene within the first 24 hours, but suppressed it after 48 h. Both effects depended on the interaction of oxLDL with the TLR4 receptor, rather than on the oxLDL uptake by the macrophages. The oxLDL-mediated downregulation of the apoA-I gene depended on the ERK1/2 and JNK cascades, as well as on the NF-κB cascade.

β-Arrestin 2 Regulates Inflammatory Responses against Mycobacterium tuberculosis Infection through ERK1/2 Signaling.

Mycobacterium tuberculosis, the pathogen that causes tuberculosis, exhibits complex host-pathogen interactions. Pattern recognition receptors and their downstream signaling pathways play crucial roles in determining the outcome of infection. In particular, the scaffold protein β-arrestin 2 mediates downstream signaling of G protein-coupled receptors. However, the role of β-arrestin 2 in conferring immunity against M. tuberculosis has not yet been explored. We found that β-arrestin 2 was upregulated in the lesioned regions of lung tissues in patients with tuberculosis. M. tuberculosis infection upregulated β-arrestin 2 expression in human macrophages, and silencing of β-arrestin 2 significantly enhanced bactericidal activity by enhancing the expression of proinflammatory cytokines such as TNF-α. β-Arrestin 2 was shown to inhibit the activation of the TLR2/ERK1/2 pathway and its transcriptional regulation activity upon M. tuberculosis infection. Furthermore, β-arrestin 2 transcriptionally regulates TNF-α by binding to CREB1. These observations revealed that the upregulation of β-arrestin 2 is critical for M. tuberculosis to escape immune surveillance through an unknown mechanism. Our research offers a novel interference modality to enhance the immune response against tuberculosis by targeting β-arrestin 2 to modulate the TLR2-β-arrestin 2-ERK1/2-CREB1-TNF-α regulatory axis.

PU.1 and IRF8 Modulate Activation of NLRP3 Inflammasome via Regulating Its Expression in Human Macrophages.

NLRP3 inflammasomes play crucial roles in the initiation of host defense by converting pro-Caspase-1 to mature Caspase-1, which in turn processes immature IL-1β and IL-18 into their biologically active forms. Although NLRP3 expression is restricted to monocytic lineages such as monocytes, macrophages, and dendritic cells, the mechanisms determining the lineage-specific expression of NLRP3 remain largely unknown. In this study, we investigated the transcription factors involved in cell-type-specific transcription of NLRP3. We found that a distal, rather than a proximal, promoter of human NLRP3 was predominantly used in the human monocytic cell lines and macrophages. Reporter analysis showed that an Ets/IRF composite element (EICE) at -309/-300 and an Ets motif at +5/+8 were critical for transcriptional activity of the distal promoter. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays demonstrated that two transcription factors, PU.1 and IRF8, both of which play essential roles in development and gene expression of the monocytic lineage, were bound to the EICE site, whereas PU.1 alone was bound to the Ets site. Knockdown of PU.1 and/or IRF8 mediated by small interfering RNA downregulated expression of NLRP3 and related molecules and markedly diminished the LPS-induced release of IL-1β in THP-1, suggesting that activity of the NLRP3 inflammasome was suppressed by knockdown of PU.1 and IRF8. Taken together, these results indicate that PU.1 and IRF8 are involved in the monocytic lineage-specific expression of NLRP3 by binding to regulatory elements within its promoter and that PU.1 and IRF8 are potential targets for regulating the activity of the NLRP3 inflammasome.

Sensing soluble uric acid by Naip1-Nlrp3 platform

Uric acid (UA), a product of purine nucleotide degradation able to initiate an immune response, represents a breakpoint in the evolutionary history of humans, when uricase, the enzyme required for UA cleavage, was lost. Despite being inert in human cells, UA in its soluble form (sUA) can increase the level of interleukin-1β (IL-1β) in murine macrophages. We, therefore, hypothesized that the recognition of sUA is achieved by the Naip1-Nlrp3 inflammasome platform. Through structural modelling predictions and transcriptome and functional analyses, we found that murine Naip1 expression in human macrophages induces IL-1β expression, fatty acid production and an inflammation-related response upon sUA stimulation, a process reversed by the pharmacological and genetic inhibition of Nlrp3. Moreover, molecular interaction experiments showed that Naip1 directly recognizes sUA. Accordingly, Naip may be the sUA receptor lost through the human evolutionary process, and a better understanding of its recognition may lead to novel anti-hyperuricaemia therapies.

Saturated Fatty Acids Promote GDF15 Expression in Human Macrophages through the PERK/eIF2/CHOP Signaling Pathway.

Growth differentiation factor-15 (GDF-15) and its receptor GFRAL are both involved in the development of obesity and insulin resistance. Plasmatic GDF-15 level increases with obesity and is positively associated with disease progression. Despite macrophages have been recently suggested as a key source of GDF-15 in obesity, little is known about the regulation of GDF-15 in these cells. In the present work, we sought for potential pathophysiological activators of GDF15 expression in human macrophages and identified saturated fatty acids (SFAs) as strong inducers of GDF15 expression and secretion. SFAs increase GDF15 expression through the induction of an ER stress and the activation of the PERK/eIF2/CHOP signaling pathway in both PMA-differentiated THP-1 cells and in primary monocyte-derived macrophages. The transcription factor CHOP directly binds to the GDF15 promoter region and regulates GDF15 expression. Unlike SFAs, unsaturated fatty acids do not promote GDF15 expression and rather inhibit both SFA-induced GDF15 expression and ER stress. These results suggest that free fatty acids may be involved in the control of GDF-15 and provide new molecular insights about how diet and lipid metabolism may regulate the development of obesity and T2D.

Regulation of IL12B Expression in Human Macrophages by TALEN-mediated Epigenome Editing.

Although the exact etiology of inflammatory bowel disease (IBD) remains unclear, exaggerated immune response in genetically predisposed individuals has been reported. Th1 and Th17 cells mediate IBD development. Macrophages produce IL-12 and IL-23 that share p40 subunit encoded by IL12B gene as heteromer partner to drive Th1 and Th17 differentiation. The available animal and human data strongly support the pathogenic role of IL-12/IL-23 in IBD development and suggest that blocking p40 might be the potential strategy for IBD treatment. Furthermore, aberrant alteration of some cytokines expression via epigenetic mechanisms is involved in pathogenesis of IBD. In this study, we analyzed core promoter region of IL12B gene and investigated whether IL12B expression could be regulated through targeted epigenetic modification with gene editing technology. Transcription activator-like effectors (TALEs) are widely used in the field of genome editing and can specifically target DNA sequence in the host genome. We synthesized the TALE DNA-binding domains that target the promoter of human IL12B gene and fused it with the functional catalytic domains of epigenetic enzymes. Transient expression of these engineered enzymes demonstrated that the TALE-DNMT3A targeted the selected IL12B promoter region, induced loci-specific DNA methylation, and down-regulated IL-12B expression in various human cell lines. Collectively, our data suggested that epigenetic editing of IL12B through methylating DNA on its promoter might be developed as a potential therapeutic strategy for IBD treatment.

Generation and characterization of U937-TR: a platform cell line for inducible gene expression in human macrophages.

Monocytes and macrophages are involved in a wide range of biological processes and parasitic diseases. The characterization of the molecular mechanisms governing such processes usually requires precise control of the expression of genes of interest. We implemented a tetracycline-controlled gene expression system in the U937 cell line, one of the most used in vitro models for the research of human monocytes and macrophages. Here we characterized U937-derived cell lines in terms of phenotypic (morphology and marker expression) and functional (capacity for phagocytosis and for Leishmania parasite hosting) changes induced by phorbol-12-myristate-13-acetate (PMA). Finally, we provide evidence of tetracycline-inducible and reversible Lamin-A gene silencing of the PMA-differentiated U937-derived cells.

Effect of the insulin on the apolipoprotein a-i gene expression in human macrophages

The aim of the article to study the effect of insulin on apolipoprotein A-I gene expression level in human macrophages and to reveal the main signal cascades which take part in the insulin-mediated regulation of apolipoprotein A-I gene.&#x0D; Materials and methods. The experiments were carried out on the macrophages differentiated from acute monocytic leukemia cell line THP-1 and on the macrophages differentiated from the monocytes isolated from peripheral human blood. The analysis of apoA-I gene expression was performed by RealTime RT-PCR (on the mRNA level) and by flow cytofluorometry. To study the signalling cascades which take part in the insulin-mediated regulation of apoA-I gene the inhibitory analysis was used.&#x0D; Results. Insulin induces the human apoA-I gene transcription in macrophages, but decreases the level of the ApoA-I protein which binds to outer cytoplasmic membrane of macrophages. The insulin-mediated transcription of apoA-I gene depends on PI3K-AKT signal cascade and transcription factors NF-B and LXRs.&#x0D; Conclusions. Taking into account our previous data it is plausible to conclude that the elevation of ApoA-I mRNA in human macrophages after insulin treatment leads to an increase of the amplitude of macrophages anti-inflammatory response, which consists in a sharp rise in the level of surface ApoA-I in macrophages under the some proinflammatory stimuli (TNF, LPS).

Matrix Metalloproteinase 9 is Regulated by LOX-1 and erk1/2 Pathway in Dental Peri-Implantitis.

Dental peri-implantitis, which can be caused by several different microbial factors, is characterized by inflammatory lesions of the surrounding hard and soft tissues of an oral implant. Matrix Metalloproteinase 9 (MMP9) is thought to be involved in the pathogenesis of peri-implantitis. However, the regulatory mechanism of MMP9 in peri-implantitis has not been fully elucidated. In this study, we tried to evaluate the regulatory mechanism of MMP9 in peri-implantitis. We collected Peri-Implant Crevicular Fluid (PICF) from ten healthy implants and ten periimplantitis patients and compared their expression level of MMP9. We also cultured macrophages from the peripheral blood of healthy volunteers infected by Porphyromonas gingivalis to reveal the regulatory mechanism of MMP9 in peri-implantitis. Western blot, immunofluorescence staining and quantitative Polymerase Chain Reaction (RT-PCR) were used to better characterize the mechanism of MMP9. The expression of MMP9 was up-regulated in peri-implantitis patient PICF and P. gingivalis infected human macrophages. LOX-1, not dectin-1, was found to mediate MMP9 expression in human macrophages with P. gingivalis infection. Expression of Erk1/2 was responsible for infection-induced MMP9 expression. Finally, use of a broad-spectrum metalloproteinase inhibitor impaired LOX-1 expression in infected macrophages. Our results demonstrate that MMP9 is involved in dental peri-implantitis and is regulated by LOX-1 and Erk1/2. This LOX-1/MMP9 signaling pathway may represent a potential drug target for peri-implantitis.