Human Macrophage Cell Line THP-1 Research Articles

Dexamethasone protects against asthma via regulating Hif-1α-glycolysis-lactate axis and protein lactylation

PurposeAsthma can not be eradicated till now and its control primarily relies on the application of corticosteroids. Recently, glycolytic reprogramming has been reportedly contributed to asthma, this study aimed to reveal whether the effect of corticosteroids on asthma control is related to their regulation of glycolysis and glycolysis-dependent protein lactylation. MethodsOvalbumin (OVA) aeroallergen was used to challenge mice and stimulate human macrophage cell line THP-1 following dexamethasone (DEX) treatment. Airway hyperresponsiveness, airway inflammation, the expressions of key glycolytic enzymes and pyroptosis markers, the level of lactic acid, real-time glycolysis and oxidative phosphorylation (OXPHOS), and protein lactylation were analyzed. ResultsDEX significantly attenuated OVA-induced eosinophilic airway inflammation, including airway hyperresponsiveness, leukocyte infiltration, goblet cell hyperplasia, Th2 cytokines production and pyroptosis markers expression. Meanwhile, OVA-induced Hif-1α-glycolysis axis was substantially downregulated by DEX, which resulted in low level of lactic acid. Besides, key glycolytic enzymes in the lungs of asthmatic mice were notably co-localized with F4/80-positive macrophages, indicating metabolic shift to glycolysis in lung macrophages during asthma. This was confirmed in OVA-stimulated THP-1 cells that DEX treatment resulted in reductions in pyroptosis, glycolysis and lactic acid level. Finally, protein lactylation was found significantly increased in the lungs of asthmatic mice and OVA-stimulated THP-1 cells, which were both inhibited by DEX. ConclusionOur present study revealed that the effect of DEX on asthma control was associated with its suppressing of Hif-1α-glycolysis-lactateaxis and subsequent protein lactylation, which may open new avenues for the therapy of eosinophilic asthma.

Transcriptional Activation of a Pro-Inflammatory Response (NF-κB, AP-1, IL-1β) by the Vibrio cholerae Cytotoxin (VCC) Monomer through the MAPK Signaling Pathway in the THP-1 Human Macrophage Cell Line.

This study describes, to some extent, the VCC contribution as an early stimulation of the macrophage lineage. Regarding the onset of the innate immune response caused by infection, the β form of IL-1 is the most important interleukin involved in the onset of the inflammatory innate response. Activated macrophages treated in vitro with VCC induced the activation of the MAPK signaling pathway in a one-hour period, with the activation of transcriptional regulators for a surviving and pro-inflammatory response, suggesting an explanation inspired and supported by the inflammasome physiology. The mechanism of IL-1β production induced by VCC has been gracefully outlined in murine models, using bacterial knockdown mutants and purified molecules; nevertheless, the knowledge of this mechanism in the human immune system is still under study. This work shows the soluble form of 65 kDa of the Vibrio cholerae cytotoxin (also known as hemolysin), as it is secreted by the bacteria, inducing the production of IL-1β in the human macrophage cell line THP-1. The mechanism involves triggering the early activation of the signaling pathway MAPKs pERK and p38, with the subsequent activation of (p50) NF-κB and AP-1 (cJun and cFos), determined by real-time quantitation. The evidence shown here supports that the monomeric soluble form of the VCC in the macrophage acts as a modulator of the innate immune response, which is consistent with the assembly of the NLRP3 inflammasome actively releasing IL-1β.

Proinflammatory Responses in SARS-CoV-2 and Soluble Spike Glycoprotein S1 Subunit Activated Human Macrophages.

Critically ill COVID-19 patients display signs of generalized hyperinflammation. Macrophages trigger inflammation to eliminate pathogens and repair tissue, but this process can also lead to hyperinflammation and resulting exaggerated disease. The role of macrophages in dysregulated inflammation during SARS-CoV-2 infection is poorly understood. We inoculated and treated human macrophage cell line THP-1 with SARS-CoV-2 and purified, glycosylated, soluble SARS-CoV-2 spike protein S1 subunit (S1) to clarify the role of macrophages in pro-inflammatory responses. Soluble S1 upregulated TNF-α and CXCL10 mRNAs, and induced secretion of TNF-α from THP-1 macrophages. While THP-1 macrophages did not support productive SARS-CoV-2 replication or viral entry, virus exposure resulted in upregulation of both TNF-α and CXCL10 genes. Our study shows that extracellular soluble S1 protein is a key viral component inducing pro-inflammatory responses in macrophages, independent of virus replication. Thus, virus- or soluble S1-activated macrophages may become sources of pro-inflammatory mediators contributing to hyperinflammation in COVID-19 patients.

Mycobacterium tuberculosis sRNA MTS2823 regulates the growth of the multidrug-resistant strain in macrophages.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (MTB), is a serious contagious disease. MTB-encoded small regulatory RNA (sRNA) MTS2823 was reported to be upregulated in the plasma of TB patients. Nevertheless, whether MTS2823 is implicated in MTB drug resistance is unclear. Human macrophage cell line THP-1 was infected with the drug-susceptible strain H37Rv or the multidrug-resistant (MDR) strain 8462. Colony-forming unit assay was implemented for evaluating intracellular growth of the MTB strains. Enzyme-linked immunosorbent assay was used for measurement of inflammatory cytokines. Real-time quantitative polymerase chain reaction was utilized to assess MTS2823 and recombinase A (recA) expression in strains 8462 and H37Rv. Nitric oxide (NO) production in the MDR strain-infected THP-1 cells was measured. In this study, MTS2823 was found to display a low level in the MDR strain. Overexpressing MTS2823 promoted intracellular growth of the MDR strain and inhibited inflammatory cytokine and NO production in infected THP-1 cells. RecA might be a target of MTS2823 in the MDR strain. Overall, MTB-encoded sRNA MTS2823 displays a low level and regulates the growth of the MDR strain in THP-1 cells by modulating recA.

Functional differences between primary monocyte-derived and THP-1 macrophages and their response to LCPUFAs

BackgroundIn immune cell models, macrophages are one of the most frequently used cell types. THP-1 cells are often used as model to study macrophage function, however they may act differently from primary human monocyte derived macrophages (MDMs). MethodsIn this study, we investigated the intrinsic baseline differences between the human macrophage cell line THP-1 and human primary MDMs. Additionally, we studied the difference in response to treatment with long-chain polyunsaturated fatty acids (LCPUFAs): well-described immunomodulators. ResultsAlthough the amount of cells that phagocytose were similar between the cell types, primary MDMs consumed significantly more E. coli bioparticles compared to THP-1 macrophages. In M1 macrophages, IL-12 secretion was almost fifty times higher by primary MDMs compared to THP-1 macrophages, thereby increasing the IL-12/IL-10 ratio. Despite this, the IL-12 secretion by THP-1 M1 macrophages was higher that the secretion of IL-10, thereby showing that it is still a suitable M1 type. Cytokine profiles differed between primary MDMs and THP-1 M1 and M2 macrophages. In response to LCPUFAs, primary M1 MDMs and THP-1 M1 macrophages were alike. Interestingly, primary M2 MDMs secreted less IL-10 and CCL22 when treated with LCPUFAs, whereas THP-1 M2 macrophages secreted more IL-10 when treated with LCPUFAs and showed no difference in CCL22 secretion. ConclusionsIn conclusion, in an M1 setting, both THP-1 and primary MDMs are suitable models. However, when interested in M2 models, the model choice highly depends on the research question.

Anti-Inflammatory Properties of Statin-Loaded Biodegradable Lecithin/Chitosan Nanoparticles: A Step Toward Nose-to-Brain Treatment of Neurodegenerative Diseases.

Nasal delivery has been indicated as one of the most interesting alternative routes for the brain delivery of neuroprotective drugs. Nanocarriers have emerged as a promising strategy for the delivery of neurotherapeutics across the nasal epithelia. In this work, hybrid lecithin/chitosan nanoparticles (LCNs) were proposed as a drug delivery platform for the nasal administration of simvastatin (SVT) for the treatment of neuroinflammatory diseases. The impact of SVT nanoencapsulation on its transport across the nasal epithelium was investigated, as well as the efficacy of SVT-LCNs in suppressing cytokines release in a cellular model of neuroinflammation. Drug release studies were performed in simulated nasal fluids to investigate SVT release from the nanoparticles under conditions mimicking the physiological environment present in the nasal cavity. It was observed that interaction of nanoparticles with a simulated nasal mucus decreased nanoparticle drug release and/or slowed drug diffusion. On the other hand, it was demonstrated that two antibacterial enzymes commonly present in the nasal secretions, lysozyme and phospholipase A2, promoted drug release from the nanocarrier. Indeed, an enzyme-triggered drug release was observed even in the presence of mucus, with a 5-fold increase in drug release from LCNs. Moreover, chitosan-coated nanoparticles enhanced SVT permeation across a human cell model of the nasal epithelium (×11). The nanoformulation pharmacological activity was assessed using an accepted model of microglia, obtained by activating the human macrophage cell line THP-1 with the Escherichia coli–derived lipopolysaccharide (LPS) as the pro-inflammatory stimulus. SVT-LCNs were demonstrated to suppress the pro-inflammatory signaling more efficiently than the simple drug solution (−75% for IL-6 and −27% for TNF-α vs. −47% and −15% at 10 µM concentration for SVT-LCNs and SVT solution, respectively). Moreover, neither cellular toxicity nor pro-inflammatory responses were evidenced for the treatment with the blank nanoparticles even after 36 h of incubation, indicating a good biocompatibility of the nanomedicine components in vitro. Due to their biocompatibility and ability to promote drug release and absorption at the biointerface, hybrid LCNs appear to be an ideal carrier for achieving nose-to-brain delivery of poorly water-soluble drugs such as SVT.

Identification of cGAS as an innate immune sensor of extracellular bacterium Pseudomonas aeruginosa.

SummaryCyclic GMP-AMP synthase (cGAS) is reported essential for detecting intracellular bacteria. However, it remains to be determined whether and how cGAS is involved in extracellular bacterial infection. Here, we report that cGAS is essential for mediating type I interferon (IFN) production in infection by multiple extracellular pathogens, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Staphylococcus aureus. In addition, the canonical cGAS-stimulator of interferon gene (STING)-IFN axis is required for protecting mice from P. aeruginosa-induced mouse acute pulmonary infection, confirmed in cGAS pathway-specific gene deficiency mouse models. cGAS−/− and STING−/− mice exhibited reduced type I IFNs production, excessive inflammatory response accompanied with decreased resistance to P. aeruginosa challenge. Unfolded protein response was also modulated by cGAS through IRF3 and type I IFNs under P. aeruginosa infection. Collectively, these findings uncover the importance of cGAS in initiating immune responses against extracellular bacterial infection.

Primary Amine Modified Gold Nanodots Regulate Macrophage Function and Antioxidant Response: Potential Therapeutics Targeting of Nrf2.

BackgroundGold nanoparticles with high biocompatibility and immunomodulatory properties have potential applications in the development of new diagnostic and therapeutic strategies for nanomedicine. Nanoparticles targeting macrophages can manipulate or control immunological diseases. This study assessed the activity of dendrimer-encapsulated gold nanodots (AuNDs) with three surface modifications [ie, outfacing groups with primary amine (AuNDs-NH2), hydroxyl (AuNDs-OH), and quaternary ammonium ions (AuNDs-CH3)] regulated macrophage function and antioxidant response through Nrf2-dependent pathway.MethodsAuNDs were prepared and characterized. Intracellular distribution of AuNDs in human macrophages was observed through confocal microscopy. The activity of AuNDs was evaluated using macrophage functions and antioxidant response in the human macrophage cell line THP-1.ResultsAuNDs-NH2 and AuNDs-CH3, but not AuNDs-OH, drove the obvious Nrf2-antioxidant response element pathway in THP-1 cells. Of the three, AuNDs-NH2 considerably increased mRNA levels and antioxidant activities of heme oxygenase 1 and NAD(P)H quinone dehydrogenase 1 in THP-1 cells. IL-6 mRNA and protein expression was mediated through Nrf2 activation in AuNDs-NH2-treated macrophages. Furthermore, Nrf2 activation by AuNDs-NH2 increased the phagocytic ability of THP-1 macrophages.ConclusionAuNDs-NH2 had immunomodulatory activities in macrophages. The findings of the present work suggested that AuNDs have potential effects against chronic inflammatory diseases via the Nrf2 pathway.

Fluorescent Labeling of Helminth Extracellular Vesicles Using an In Vivo Whole Organism Approach

In the last two decades, extracellular vesicles (EVs) from the three domains of life, Archaea, Bacteria and Eukaryotes, have gained increasing scientific attention. As such, the role of EVs in host-pathogen communication and immune modulation are being intensely investigated. Pivotal to EV research is the determination of how and where EVs are taken up by recipient cells and organs in vivo, which requires suitable tracking strategies including labelling. Labelling of EVs is often performed post-isolation which increases risks of non-specific labelling and the introduction of labelling artefacts. Here we exploited the inability of helminths to de novo synthesise fatty acids to enable labelling of EVs by whole organism uptake of fluorescent lipid analogues and the subsequent incorporation in EVs. We showed uptake of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DOPE-Rho) in Anisakis spp. and Trichuris suis larvae. EVs isolated from the supernatant of Anisakis spp. labelled with DOPE-Rho were characterised to assess the effects of labelling on size, structure and fluorescence of EVs. Fluorescent EVs were successfully taken up by the human macrophage cell line THP-1. This study, therefore, presents a novel staining method that can be utilized by the EV field in parasitology and potentially across multiple species.

Effects of Cigarette Smoke and Opium on the Expression of CD9, CD36, and CD68 at mRNA and Protein Levels in Human Macrophage Cell Line THP-1.

Cigarette smoking and opium use are risk factors for coronary artery disease (CAD). It has been known that scavenger receptors such as CD36 and CD68 play critical roles in the pathogenesis of CAD. CD9, as a member of the tetraspanin, has been shown to interact with scavenger receptors. The aim of this study was to investigate the effects of these risk factors on expression levels of CD9, CD36, and CD68 on the THP-1 cell line. The THP-1 cell line treated with cigarette smoke extract (CSE( and opium, both individually and combinatory, in 24 h incubation. The protein and mRNA levels of CD9, CD36, and CD68 were evaluated by flow cytometry and quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR) techniques, respectively. CD36 and CD68 mRNA and protein expression levels were significantly increased in the cells treated with cigarette smoke extract compared to the control (p<0.001 in mRNA expression levels and p=0.016 and p=0.012 in protein expression levels, respectively). The CSE increased the level of CD9 protein expression compared to the control group (p=0.041) on the human macrophage cell line THP-1. No significant differences were observed in the CD9, CD36, and CD68 gene expression and at the protein levels between opium-treated THP-1 cells and controls. In conclusion, cigarettes by increasing the levels of CD36, CD68, and CD9 can be a risk factor in the development of many inflammatory diseases, including cardiovascular diseases, chronic obstructive pulmonary disease (COPD) and lung carcinoma.

Mechanistic profiling of the release kinetics of siRNA from lipidoid-polymer hybrid nanoparticles in vitro and in vivo after pulmonary administration

Understanding the release kinetics of siRNA from nanocarriers, their cellular uptake, their in vivo biodistribution and pharmacokinetics is a fundamental prerequisite for efficient optimisation of the design of nanocarriers for siRNA-based therapeutics. Thus, we investigated the influence of composition on the siRNA release from lipid-polymer hybrid nanoparticles (LPNs) consisting of cationic lipidoid 5 (L5) and poly(DL-lactic-co-glycolic acid) (PLGA) intended for pulmonary administration. An array of siRNA-loaded LPNs was prepared by systematic variation of: (i) the L5 content (10–20%, w/w), and (ii) the L5:siRNA ratio (10,1–30:1, w/w). For comparative purposes, L5-based lipoplexes, L5-based stable nucleic acid lipid nanoparticles (SNALPs). and dioleoyltrimethylammoniumpropane (DOTAP)-modified LPNs loaded with siRNA were also prepared. Release studies in buffer and lung surfactant-containing medium showed that siRNA release is dependent on the presence of both surfactant and heparin (a displacing agent) in the release medium, since these interact with the lipid shell structure thereby facilitating decomplexation of L5 and siRNA, as evident from the retarded siRNA release when the L5 content and the L5:siRNA ratio were increased. This confirms the hypothesis that siRNA loaded in LPNs is predominantly present as complexes with the cationic lipid and primarily is located near the particle surface. Cellular uptake and tolerability studies in the human macrophage cell line THP-1 and the type I-like human alveolar epithelial cell line hAELVi, which together represents a monolayer-based barrier model of lung epithelium, indicated that uptake of LPNs was much higher in THP-1 cells in agreement with their primary clearance role. In vivo biodistributions of formulations loaded with Alexa Fluor® 750-labelled siRNA after pulmonary administration in mice were compared by using quantitative fluorescence imaging tomography. The L5-modified LPNs, SNALPs and DOTAP-modified LPNs displayed significantly increased lung retention of siRNA as compared to L5-based lipoplexes, which had a biodistribution profile comparable to that of non-loaded siRNA, for which >50% of the siRNA dose permeated the air-blood barrier within 6 h and subsequently was excreted via the kidneys. Hence, the enhanced lung retention upon pulmonary administration of siRNA-loaded LPNs represents a promising characteristic that can be used to control the delivery of the siRNA cargo to lung tissue for local management of disease.

Cyclic Stretch Force Induces Periodontal Ligament Cells to Secrete Exosomes That Suppress IL-1β Production Through the Inhibition of the NF-κB Signaling Pathway in Macrophages.

In the oral mechanical environment, periodontal ligament cells (PDL cells) contribute to maintaining periodontal tissue homeostasis. Recent studies showed that exosomes, which are small vesicles secreted by various types of cells, play a pivotal role in cell-to-cell communication in biological processes. We examined the secretion of exosomes from PDL cells stimulated with cyclic stretch and their role in the inflammatory response of macrophages using the human macrophage cell line THP-1 and human primary monocytes/macrophages. We prepared supernatants from human PDL cells (PDL-sup) stimulated with cyclic stretch. The treatment of macrophages with PDL-sup, but not PDL-sup from unstimulated PDL cells, inhibited the production of IL-1β in LPS/nigericin-stimulated macrophages. The pretreatment of PDL cells with GW4869, an inhibitor of exosome secretion, or siRNA for Rab27B, which controls exosome secretion, abrogated the inhibitory effects of PDL-sup. A transmission electron microscopy analysis demonstrated the existence of exosomes with diameters ranging between 30 and 100 nm in PDL-sup, suggesting that exosomes in PDL-sup contribute to this inhibition. An immunofluorescence microscopy analysis revealed that exosomes labeled with PKH67, a fluorescent dye, were incorporated by macrophages as early as 2 h after the addition of exosomes. Purified exosomes inhibited IL-1β production in LPS/nigericin-stimulated macrophages and the nuclear translocation of NF-κB as well as NF-κB p65 DNA-binding activity in LPS-stimulated macrophages, suggesting that exosomes suppress IL-1β production by inhibiting the NF-κB signaling pathway. Our results indicate that PDL cells in mechanical environments contribute to the maintenance of periodontal immune/inflammatory homeostasis by releasing exosomes.

IL-18 induced IL-23/IL-17 expression impairs Aβ clearance in cultured THP-1 and BV2 cells

Recent studies have provided overwhelming evidence of the involvement of microglia-related molecular networks in the pathogenesis of Alzheimer’s diseases (AD). The potential involvement of pro-inflammatory cytokines interleukin (IL)-18, IL-23 and IL-17 on amyloid (Aβ) clearance is still unclear. In this study, we addressed that there might be a net relationship among IL-18, IL-23, and IL-17 and they can affect Aβ clearance in cultured macrophage/microglia cells. In human macrophage cell line THP-1, Aβ42 incubation could increase the expression of IL-18, IL-23 and IL-17 in a concentration dependent manner. THP-1 cell could clear Aβ42 in the culture medium time-dependently, but its capacity of Aβ clearance was impaired by IL-18, IL-23 or IL-17 treatment. Similarly, the capacity of the microglia cell line BV2 to clear Aβ42 was impaired by IL-18, IL-23 or IL-17 treatment. In co-cultures of BV2 with APP/PS1 neuron, Aβ was efficiently cleared by BV2 cell, but Aβ clearance was impaired by IL-18, IL-23 or IL-17 treatment. The effects of IL-18, IL-23 and IL-17 could be blocked by their corresponding neutralizing antibodies. In addition, the inhibitory effects of IL-18 were blocked by IL-23 or IL-17 neutralizing antibodies while the inhibitory effects of IL-23 were blocked by IL-17 neutralizing antibodies. Our study provides evidences showing that amyloid induced IL-18/IL-23/IL-17 axis could impair macrophage and microglia-mediated Aβ clearance. Thus, IL-18/IL-23/IL-17 axis might be a therapeutic target in AD.

Facilitating THP-1 macrophage studies by differentiating and investigating cell functions in polystyrene test tubes

Macrophage cell lines are a useful model to explore the properties of primary macrophages. However, a major limitation in the use of these cells is that when they are differentiated, they become adherent and hence present with the same limitation as natural macrophages. The cells need to be detached and are often subjected to detachment techniques such as detachment buffers containing proteolytic enzymes or scraping with a rubber ‘policeman’. These steps are time-consuming, reduce cell yields as well as cell viability and function. We have therefore investigated the possibility of differentiating the human macrophage THP-1 cell line in polystyrene FACS tubes to enable cells to be directly used for investigations by flow cytometry. Here we demonstrate that when the human macrophage cell line THP-1 are cultured in FACS tubes with phorbol myristate acetate added, they undergo differentiation into macrophages, assessed morphologically and by autofluorescence expression, in a similar manner to those cultured in tissue culture dishes. The cells can be readily washed and adjusted in concentration by centrifugation in the same tubes and can be directly tested for expression of cell surface markers and function by flow cytometry. This avoids the use of either detachment reagents or physical cell scraping. Consequently, we showed that the tube culture method results in increased cell yield and viability compared to those subjected to detachment procedures. The tube method generated functional macrophages which expressed the complement receptors, CR3 and CR4, and effectively phagocytosed complement opsonised Staphylococcus aureus via these receptors.

The microRNA-1908 up-regulation in the peripheral blood cells impairs amyloid clearance by targeting ApoE.

To give a new insight into the mechanism of ApoE dysregulation and microRNA-1908 in Alzheimer's disease (AD). Plasma ApoE levels were measured in 20 AD patients and 20 healthy controls. THP-1 was maintained in RPMI1640 with 10% fetal bovine serum. Quantitative real-time polymerase chain reaction was performed to detect 13-microRNA and ApoE mRNA in cultured cell lines. Enzyme-linked immunosorbent assay was used to measure human ApoE in the plasma or culture medium of cell lines and also used to quantify the human Aβ42 in the culture medium of cell lines. We found plasma ApoE level reduced in AD patients (2.28 vs 3.78μg/mL, P<.001), and microRNA-1908 was up-regulated in AD patients and was negatively associated with plasma ApoE (r=-0.32, P=.012). In human macrophage cell line THP-1 and astrocytoma cell line U87, microRNA-1908 could inhibit the mRNA and protein levels of ApoE by targeting its 3'untranslated region. Consistently, microRNA-1908 inhibits the ApoE-mediated Aβ clearance. Our study provides new insight into the mechanism of ApoE dysregulation in AD patients, and microRNA-1908 might be a therapeutic target for AD treatment.

Anti-inflammatory effect of multistrain probiotic formulation ( L. rhamnosus, B. lactis, and B. longum )

In recent years, a great number of studies have been directed toward the evaluation of gastrointestinal microbiota modulation through the introduction of beneficial microorganisms, also known as probiotics. Many studies have highlighted how this category of bacteria is very important for the good development, functioning, and maintenance of our immune system. There is a delicate balance between the immune system, located under the gut epithelial barrier, and the microbiota, but many factors can induce a disequilibrium that leads to an inflammatory state and dysbiosis. The aim of this work is to verify the anti-inflammatory effects of a probiotic formulation of Lactobacillus rhamnosus, Bifidobacterium lactis, and Bifidobacterium longum (Serobioma). To mimic the natural host compartmentalization between probiotics and immune cells through the intestinal epithelial barrier in vitro, the transwell model was used. We focused on a particular subset of immune cells that play a key role in the mucosal immune system. The immunomodulatory effects of probiotic formulation were investigated in the human macrophage cell line THP1 and macrophages derived from ex vivo human peripheral blood mononuclear cells. Probiotic formulation induced a significant increase in anti-inflammatory cytokine interleukin-10 (IL-10) production and was able to decrease the secretion of the major proinflammatory cytokines IL-1β and IL-6 by 70% and 80%, respectively. Finally, for the first time, the ability of probiotic formulation to favor the macrophage M2 phenotype has been identified. The transwell model is an intriguing toll approach to studying the human epithelial barrier.

Functional and Structural Analysis of a Highly-Expressed Yersinia pestis Small RNA following Infection of Cultured Macrophages.

Non-coding small RNAs (sRNAs) are found in practically all bacterial genomes and play important roles in regulating gene expression to impact bacterial metabolism, growth, and virulence. We performed transcriptomics analysis to identify sRNAs that are differentially expressed in Yersinia pestis that invaded the human macrophage cell line THP-1, compared to pathogens that remained extracellular in the presence of host. Using ultra high-throughput sequencing, we identified 37 novel and 143 previously known sRNAs in Y. pestis. In particular, the sRNA Ysr170 was highly expressed in intracellular Yersinia and exhibited a log2 fold change ~3.6 higher levels compared to extracellular bacteria. We found that knock-down of Ysr170 expression attenuated infection efficiency in cell culture and growth rate in response to different stressors. In addition, we applied selective 2’-hydroxyl acylation analyzed by primer extension (SHAPE) analysis to determine the secondary structure of Ysr170 and observed structural changes resulting from interactions with the aminoglycoside antibiotic gentamycin and the RNA chaperone Hfq. Interestingly, gentamicin stabilized helix 4 of Ysr170, which structurally resembles the native gentamicin 16S ribosomal binding site. Finally, we modeled the tertiary structure of Ysr170 binding to gentamycin using RNA motif modeling. Integration of these experimental and structural methods can provide further insight into the design of small molecules that can inhibit function of sRNAs required for pathogen virulence.

Transcriptional Profiling of Mycobacterium tuberculosis Exposed to In Vitro Lysosomal Stress.

Increasing experimental evidence supports the idea that Mycobacterium tuberculosis has evolved strategies to survive within lysosomes of activated macrophages. To further our knowledge of M. tuberculosis response to the hostile lysosomal environment, we profiled the global transcriptional activity of M. tuberculosis when exposed to the lysosomal soluble fraction (SF) prepared from activated macrophages. Transcriptome sequencing (RNA-seq) analysis was performed using various incubation conditions, ranging from noninhibitory to cidal based on the mycobacterial replication or killing profile. Under inhibitory conditions that led to the absence of apparent mycobacterial replication, M. tuberculosis expressed a unique transcriptome with modulation of genes involved in general stress response, metabolic reprogramming, respiration, oxidative stress, dormancy response, and virulence. The transcription pattern also indicates characteristic cell wall remodeling with the possible outcomes of increased infectivity, intrinsic resistance to antibiotics, and subversion of the host immune system. Among the lysosome-specific responses, we identified the glgE-mediated 1,4 α-glucan synthesis pathway and a defined group of VapBC toxin/anti-toxin systems, both of which represent toxicity mechanisms that potentially can be exploited for killing intracellular mycobacteria. A meta-analysis including previously reported transcriptomic studies in macrophage infection and in vitro stress models was conducted to identify overlapping and nonoverlapping pathways. Finally, the Tap efflux pump-encoding gene Rv1258c was selected for validation. An M. tuberculosis ΔRv1258c mutant was constructed and displayed increased susceptibility to killing by lysosomal SF and the antimicrobial peptide LL-37, as well as attenuated survival in primary murine macrophages and human macrophage cell line THP-1.

Insulin-like growth factor-I serum levels and their biological effects on Leishmania isolates from different clinical forms of American tegumentary leishmaniasis.

sBackgroundAmerican tegumentary leishmaniasis (ATL) in Brazil is mostly caused by Leishmania (Viannia) braziliensis, with known forms of the disease being cutaneous (CL), mucosal (ML) and disseminated (DL) leishmaniasis. The development of the lesion in ATL is related both to the persistence of the Leishmania in the skin and to the parasite-triggered immune and inflammatory responses that ensue lesions. In this context one factor with expected role in the pathogenesis is insulin-like growth factor (IGF)-I with known effects on parasite growth and healing and inflammatory processes. In the present study, we addressed the effect of IGF-I on intracellular amastigote isolates from CL, ML and DL patients within human macrophage and we evaluated the IGF-I and IGF-binding protein-3 (IGFBP3) serum levels in patients presenting different clinical forms and controls from the endemic area.MethodsWe evaluated biological variability in the responses of intracellular amastigotes of Leishmania isolates derived from CL, ML, and DL patients from an area for ATL in response to IGF-I. Intracellular amastigote growth was evaluated using the human macrophage cell line THP-1. Arginase activity in infected cells was evaluated quantifying the generated urea concentration. Serum samples from patients and controls were assayed using chemiluminescent immunometric assay to determine IGF-I and IGFBP3 levels.ResultsWe observed an increase in intracellular parasitism upon IGF-I stimulus in 62.5 % of isolates from CL, in 85.7 % from ML and only 42.8 % from DL cases. In DL, the basal arginase activity was lower than that of CL. We then evaluated the IGF-I and IGFBP3 serum levels in patients, and we observed significantly lower levels in ML and DL than in CL and control samples.ConclusionsThe data suggest that IGF-I is modulated distinctly in different clinical forms of tegumentary leishmaniasis. IGF-I seemingly exerts effect on parasite growth likely contributing to its persistence in the skin in earlier phase. In addition the decreased IGF-I serum levels may affect the modulation of inflammation and lesion healing in chronic phase. In view of potential role of IGF-I in the pathogenesis of ATL we can speculate on therapeutic procedures taking into account the local IGF-I level.

G protein-coupled receptor160 regulates mycobacteria entry into macrophages by activating ERK

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, invades and replicates within susceptible hosts by disturbing host antimicrobial mechanisms. Although G protein-coupled receptors (GPCRs) are involved in most physiological and pathological activities of mammalian cells, the roles of GPCRs in Mtb invasion into host cell remain elusive. Here, we report that GPR160 expression is elevated at both mRNA and protein level in macrophages in response to BCG infection. Both the PiggyBac (PB) transposon-mediated mutation of gpr160 gene in mouse primary macrophages and siRNA-mediated knockdown of GPR160 in the human macrophage cell line THP-1 markedly reduced the entry of green fluorescent protein (GFP) expressing BCG (BCG-GFP), also operative in vivo. BCG infection-induced phosphorylation of ERK1/2 was significantly reduced in gpr160 mutated (gpr160−/−) macrophages relative to levels observed in wild type macrophages, while inhibition of ERK by specific inhibitor or knockdown ERK1/2 by specific siRNA markedly reduced entry of BCG. Finally, lower bacteria burdens and attenuated pathological impairments were observed in the lungs of BCG-infected gpr160−/− mice. Furthermore, gpr160−/− macrophages also exhibits reduced uptake of Escherichia coli and Francisella tularensis. Taken together, these findings suggest an important role of GPR160 in regulating the entry of BCG into macrophages by targeting the ERK signaling pathway. As GPCRs have proven to be successful drug targets in pharmaceutical industry, it's tempting to speculate that compounds targeting GPR160, a G protein-coupled receptor, could intervene in Mtb infection.