Inflammatory Response In Human Macrophages Research Articles

Characteristics and health risks of the inhalable fraction of metal additive manufacturing powders

AbstractMetal additive manufacturing (AM) is gaining traction but raises worker health concerns due to micron‐sized powders, including fine inhalable particles. This study explored particle and surface characteristics, electrochemical properties, metal release in artificial lysosomal fluid (ALF), and potential toxicity of virgin and sieved virgin Fe‐based powders, stainless steel (316L), Fe, and two tooling steels. Virgin particles ranged in size from 1 to 100 µm, while sieved particles were within the respirable size range (<5–10 µm). Surface oxide composition differed from bulk composition. The Fe powder showed low corrosion resistance and high metal release due to a lack of protective surface oxide. Sieved particles of 316L, Fe, and one tooling steel released more metals into ALF than virgin particles, with the opposite was observed for the other tooling steel. Sieved particles had no notable impact on cell viability or micronuclei formation in human bronchial epithelial cells. Inflammatory response in human macrophages was generally low, except for the Fe powder and one tooling steel, which induced increased interleukin‐8 (IL‐8/CXCL‐8) and monocyte chemoattractant protein‐1 (MCP‐1/CCL‐2) secretion. This study underscores distinctions between virgin and sieved Fe‐based powders and suggests relatively low acute toxicity.

SARS-CoV-2 activates the TLR4/MyD88 pathway in human macrophages: A possible correlation with strong pro-inflammatory responses in severe COVID-19

BackgroundToll-like receptors (TLRs) play a pivotal role in the immunologic response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Exaggerated inflammatory response of innate immune cells, however, may drive morbidity and death in Coronavirus disease 19 (COVID-19). ObjectiveWe investigated the engagement of SARS-CoV-2 with TLR4 in order to better understand how to tackle hyperinflammation in COVID-19. MethodsWe combined RNA-sequencing data of human lung tissue and of bronchoalveolar lavage fluid cells derived from COVID-19 patients with functional studies in human macrophages using SARS-CoV-2 spike proteins and viable SARS-CoV-2. Pharmacological inhibitors as well as gene editing with CRISPR/Cas9 were used to delineate the signalling pathways involved. ResultsWe found TLR4 to be the most abundantly upregulated TLR in human lung tissue irrespective of the underlying pathology. Accordingly, bronchoalveolar lavage fluid cells from patients with severe COVID-19 showed an NF-κB-pathway dominated immune response, whereas they were mostly defined by type I interferon signalling in moderate COVID-19. Mechanistically, we found the Spike ectodomain, but not receptor binding domain monomer to induce TLR4-dependent inflammation in human macrophages. By using pharmacological inhibitors as well as CRISPR/Cas9 deleted macrophages, we identify SARS-CoV-2 to engage canonical TLR4-MyD88 signalling. Importantly, we demonstrate that TLR4 blockage prevents exaggerated inflammatory responses in human macrophages infected with different SARS-CoV-2 variants, including immune escape variants B.1.1.7.-E484K and B.1.1.529 (omicron). ConclusionOur study critically extends the current knowledge on TLR-mediated hyperinflammatory responses to SARS-CoV-2 in human macrophages, paving the way for novel approaches to tackle severe COVID-19. Take-home messageOur study combining human lung transcriptomics with functional studies in human macrophages clearly supports the design and development of TLR4 - directed therapeutics to mitigate hyperinflammation in severe COVID-19.

Hypertonic saline induces inflammation in human macrophages through the NLRP1 inflammasome.

Nebulized hypertonic saline (3-7%) is commonly used to increase mucociliary clearance in patients with chronic airway disease and/or virus infections. However, altered salt concentrations may contribute to inflammatory responses. The aim of this study was to investigate whether 500 mM NaCl (3%) triggers inflammation in human macrophages and identify the molecular mechanisms involved. NaCl-induced pyroptosis, IL-1β, IL-18 and ASC speck release were measured in primary human monocyte-derived macrophages. Treatment with the recombinant IL-1 receptor antagonist anakinra or the NLRP3 inhibitor MCC950 did not affect NaCl-mediated inflammasome assembly. Knock-down of NLRP1 expression, but not of NLRP3 and NLRC4, reduced NaCl-induced pyroptosis, pro-inflammatory cytokine and ASC speck release from human THP-1-derived macrophages. Data from this study suggest that 3% NaCl-induced inflammatory responses in human macrophages depend on NLRP1 and inflammasome assembly. Targeting inflammation in addition to inhalation with hypertonic saline may benefit patients with inflammatory airway disease.

Immune and inflammatory responses of human macrophages, dendritic cells, and T-cells in presence of micro- and nanoplastic of different types and sizes

Environmental pollution by microplastics (MPs) is a growing concern regarding their impact on aquatic and terrestrial systems and human health. Typical exposure routes of MPs are dermal contact, digestion, and inhalation. Recent in vitro and in vivo studies observed alterations in immunity after MPs exposure, but systemic studies using primary human immune cells are scarce. In our investigation, we addressed the effect of polystyrene (PS) and poly methyl methacrylate (PMMA) in three different sizes (50–1100 nm) as well as amino-modified PS (PS-NH2; 50 nm) on cells of the adaptive and innate immune system. T-cells isolated from human peripheral blood mononuclear cells (PBMCs) were least affected regarding the cytotoxicity but displayed increased activation marker expression after 72 h, and strongly modulated cytokine secretion patterns. Conversely, phagocytic dendritic cells and macrophages derived from isolated monocytes were highly sensitive to pristine MPs. Their marker expression suggested a downregulation of the inflammatory phenotypes indicative of M2 macrophage induction after MPs exposure for 24 h. Our results showed that even pristine MPs affected immune cell function and inflammatory phenotype dependent on MPs polymers, size, and immune cell type.

Human metapneumovirus driven IFN-β production antagonizes macrophage transcriptional induction of IL1-β in response to bacterial pathogens.

Human metapneumovirus (HMPV) is a pneumovirus that may cause severe respiratory disease in humans. HMPV infection has been found to increase susceptibility to bacterial superinfections leading to increased morbidity and mortality. The molecular mechanisms underlying HMPV-mediated increase in bacterial susceptibility are poorly understood and largely understudied. Type I interferons (IFNs), while critical for antiviral defenses, may often have detrimental effects by skewing the host immune response and cytokine output of immune cells. It is currently unknown if HMPV skews the inflammatory response in human macrophages triggered by bacterial stimuli. Here we report that HMPV pre-infection impacts production of specific cytokines. HMPV strongly suppresses IL-1β transcription in response to LPS or heat-killed Pseudomonas aeruginosa and Streptococcus pneumonia, while enhancing mRNA levels of IL-6, TNF-α and IFN-β. We demonstrate that in human macrophages the HMPV-mediated suppression of IL-1β transcription requires TANK-binding kinase 1 (TBK1) and signaling via the IFN-β-IFNAR axis. Interestingly, our results show that HMPV pre-infection did not impair the LPS-stimulated activation of NF-κB and HIF-1α, transcription factors that stimulate IL-1β mRNA synthesis in human cells. Furthermore, we determined that sequential HMPV-LPS treatment resulted in accumulation of the repressive epigenetic mark H3K27me3 at the IL1B promoter. Thus, for the first time we present data revealing the molecular mechanisms by which HMPV shapes the cytokine output of human macrophages exposed to bacterial pathogens/LPS, which appears to be dependent on epigenetic reprogramming at the IL1B promoter leading to reduced synthesis of IL-1β. These results may improve current understanding of the role of type I IFNs in respiratory disease mediated not only by HMPV, but also by other respiratory viruses that are associated with superinfections.

1107 TRPV4 activation inhibits inflammatory responses in human macrophages

1107 TRPV4 activation inhibits inflammatory responses in human macrophages

Protective Effect of Glycomacropeptide on the Inflammatory Response of U937 Macrophages.

Macrophages play crucial roles in inflammation and oxidative stress associated with noncommunicable diseases, such as cardiovascular diseases, diabetes, and cancer. Glycomacropeptide (GMP) is a bioactive peptide derived from milk κ-casein that contains abundant sialic acid and has shown anti-inflammatory, antioxidative, anti-obesity, and anti-diabetic properties when is orally administered. The aim of this study was to evaluate the effect of GMP on the regulation of the inflammatory response in human macrophages and the participation of sialic acid in this activity. GMP pretreatment decreased by 35%, 35%, and 49% the production of nitrites, interleukin (IL)-1β, and tumor necrosis factor (TNF)-α, respectively, in activated human macrophages U937. The same effect was obtained when cells were pretreated with asialo GMP, and no change on the gene expression of the lectins associated with the recognition of sialic acids, SIGLEC5, 7, and 9, was induced by GMP on macrophages, which suggests that sialic acid might not be involved in this immunoregulatory effect. Interestingly, GMP increased 8.9- and 3.5-fold the gene expression of the canonical anti-inflammatory protein SOCS3 and the antioxidant enzyme HMOX1, respectively, in U937 cells. Thus, GMP exerts anti-inflammatory and antioxidative activities on activated macrophages in a sialic acid-independent manner, which might be related to its in vivo reported bioactivity.

P-039 COMPOSITE PP/PLLA MONOFILAMENT 4DVENTRAL® PROMOTES AN ANTI-INFLAMMATORY RESPONSE FROM HUMAN MACROPHAGES IN VITRO

Abstract The repair of abdominal hernias with prosthetic mesh can significantly reduce the risk of hernia recurrence. A new type of hernia implants combines monofilaments, Polypropylene (PP) and a biobased and natural slowly bioabsorbable polymer, Poly-L-Lactic Acid (PLLA). The partially (60%) resorbable PLLA/PP and PP meshes manufactured by COUSIN Surgery are 4DVentral® and Biomesh P1®. The contribution of PLLA in regulated inflammatory response and in enhancing abdominal repair remains unknown. Inflammation has a functional role during tissue regeneration process. An abnormal macrophage response characterized by an uncontrolled release of pro-inflammatory cytokines, chemokines, and tissue-degrading enzymes has been shown to be responsible for postoperative complications. In contrast, the angiogenic and tissue remodeling activities of the alternative anti-inflammatory M2 macrophages have potential use in tissue regenerative. Here, the characterization of the inflammatory response of human macrophages in vitro regarding 4DVentral® compared to Biomesh P1® was performed. Human monocytic THP-1 cells were differentiated into macrophages and polarized in M1 macrophages by incubation with IFN-γ and LPS or not (M0 macrophages). We found that 4DVentral® induced higher secretion of anti-inflammatory cytokines (IL-10 and Il1-RA) compared to PP Mesh in M0 macrophages. Interestingly, the secretion of pro-inflammatory cytokine IL-6 in M1 macrophages was dramatically reduced in the presence of 4DVentral® mesh compared to PP Biomesh P1®. However, the secretion of IL-10 was significantly increased with 4DVentral®. The composite PLLA/PP monofilament 4DVentral® positively drives human macrophages to an anti-inflammatory profile, resolving inflammation and promoting tissue repair.

Immunolipidomics Reveals a Globoside Network During the Resolution of Pro-Inflammatory Response in Human Macrophages.

Toll-like receptor 4 (TLR4)-mediated changes in macrophages reshape intracellular lipid pools to coordinate an effective innate immune response. Although this has been previously well-studied in different model systems, it remains incompletely understood in primary human macrophages. Here we report time-dependent lipidomic and transcriptomic responses to lipopolysaccharide (LPS) in primary human macrophages from healthy donors. We grouped the variation of ~200 individual lipid species measured by LC-MS/MS into eight temporal clusters. Among all other lipids, glycosphingolipids (glycoSP) and cholesteryl esters (CE) showed a sharp increase during the resolution phase (between 8h or 16h post LPS). GlycoSP, belonging to the globoside family (Gb3 and Gb4), showed the greatest inter-individual variability among all lipids quantified. Integrative network analysis between GlycoSP/CE levels and genome-wide transcripts, identified Gb4 d18:1/16:0 and CE 20:4 association with subnetworks enriched for T cell receptor signaling (PDCD1, CD86, PTPRC, CD247, IFNG) and DC-SIGN signaling (RAF1, CD209), respectively. Our findings reveal Gb3 and Gb4 globosides as sphingolipids associated with the resolution phase of inflammatory response in human macrophages.

WS12.06 Hypertonic saline triggers inflammatory responses in human macrophages

WS12.06 Hypertonic saline triggers inflammatory responses in human macrophages

Recombinant Lloviu virus as a tool to study viral replication and host responses.

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.

Hyperglycemia Induces Inflammatory Response of Human Macrophages to CD163-Mediated Scavenging of Hemoglobin-Haptoglobin Complexes.

Hyperglycemia, a hallmark of diabetes, can induce inflammatory programming of macrophages. The macrophage scavenger receptor CD163 internalizes and degrades hemoglobin-haptoglobin (Hb-Hp) complexes built due to intravascular hemolysis. Clinical studies have demonstrated a correlation between impaired scavenging of Hb-Hp complexes via CD163 and diabetic vascular complications. Our aim was to identify whether hyperglycemia is able to amplify inflammation via Hb-Hp complex interactions with the immune system. M(IFNγ), M(IL-4), and control M0 macrophages were differentiated out of primary human monocytes in normo- (5 mM) and hyperglycemic (25 mM) conditions. CD163 gene expression was decreased 5.53 times in M(IFNγ) with a further decrease of 1.99 times in hyperglycemia. Hyperglycemia suppressed CD163 surface expression in M(IFNγ) (1.43 times). Flow cytometry demonstrated no impairment of Hb-Hp uptake in hyperglycemia. However, hyperglycemia induced an inflammatory response of M(IFNγ) to Hb-Hp1-1 and Hb-Hp2-2 uptake with different dynamics. Hb-Hp1-1 uptake stimulated IL-6 release (3.03 times) after 6 h but suppressed secretion (5.78 times) after 24 h. Contrarily, Hb-Hp2-2 uptake did not affect IL-6 release after 6h but increased secretion after 24 h (3.06 times). Our data show that hyperglycemia induces an inflammatory response of innate immune cells to Hb-Hp1-1 and Hb-Hp2-2 uptake, converting the silent Hb-Hp complex clearance that prevents vascular damage into an inflammatory process, hereby increasing the susceptibility of diabetic patients to vascular complications.

In Vitro Effect of Replicated Porous Polymeric Nano-MicroStructured Biointerfaces Characteristics on Macrophages Behavior.

In the last decades, optimizing implant properties in terms of materials and biointerface characteristics represents one of the main quests in biomedical research. Modifying and engineering polyvinylidene fluoride (PVDF) as scaffolds becomes more and more attractive to multiples areas of bio-applications (e.g., bone or cochlear implants). Nevertheless, the acceptance of an implant is affected by its inflammatory potency caused by surface-induced modification. Therefore, in this work, three types of nano-micro squared wells like PVDF structures (i.e., reversed pyramidal shape with depths from 0.8 to 2.5 microns) were obtained by replication, and the influence of their characteristics on the inflammatory response of human macrophages was investigated in vitro. FTIR and X-ray photoelectron spectroscopy analysis confirmed the maintaining chemical structures of the replicated surfaces, while the topographical surface characteristics were evaluated by AFM and SEM analysis. Contact angle and surface energy analysis indicated a modification from superhydrophobicity of casted materials to moderate hydrophobicity based on the structure’s depth change. The effects induced by PVDF casted and micron-sized reversed pyramidal replicas on macrophages behavior were evaluated in normal and inflammatory conditions (lipopolysaccharide treatment) using colorimetric, microscopy, and ELISA methods. Our results demonstrate that the depth of the microstructured surface affects the activity of macrophages and that the modification of topography could influence both the hydrophobicity of the surface and the inflammatory response.

Metformin Regulates the Inflammatory Response of Human Macrophages to Mycobacterium tuberculosis Through a Reduction of M1 Polarization

Metformin Regulates the Inflammatory Response of Human Macrophages to Mycobacterium tuberculosis Through a Reduction of M1 Polarization

Bioactive hyaluronic acid fragments inhibit lipopolysaccharide-induced inflammatory responses via the Toll-like receptor 4 signaling pathway

The high- and the low-molecular weight hyaluronic acids (HMW-HA and LMW-HA, respectively) showed different biological activities in inflammation. However, the role of LMW-HA in inflammatory response is controversial. In this study, we aimed to investigate the effect of bioactive hyaluronan (B-HA) on lipopolysaccharide (LPS)-induced inflammatory responses in human macrophages and mice. B-HA was produced from HA treated with glycosylated recombinant human hyaluronidase PH20. Human THP-1 cells were induced to differentiate into macrophages. THP-1-derived macrophages were treated with B-HA, LPS, or B-HA + LPS. The mRNA expression and the production of inflammatory cytokines were determined using quantitative real-time PCR and enzyme-linked immunosorbent assay. The phosphorylation levels of proteins in the nuclear factor-κB (NF-κB), mitogen-activated protein kinase (MAPK), and IRF-3 signaling pathways were measured using Western blot. The in vivo efficacy of B-HA was assessed in a mouse model of LPS-induced inflammation. Results showed that B-HA inhibited the expression of TNF-α, IL-6, IL-1, and IFN-β, and enhanced the expression of the antiinflammatory cytokine IL-10 in LPS-induced inflammatory responses in THP-1-derived macrophages and in vivo. B-HA significantly suppressed the phosphorylation of the TLR4 signaling pathway proteins p65, IKKα/β, IκBα, JNK1/2, ERK1/2, p38, and IRF-3. In conclusion, our results demonstrated that the B-HA attenuated the LPS-stimulated inflammatory response by inhibiting the activation of the TLR4 signaling pathway. B-HA could be a potential anti-inflammatory drug in the treatment of inflammatory disease.

Investigating the Cellular Transcriptomic Response Induced by the Makona Variant of Ebola Virus in Differentiated THP-1 Cells.

Recent studies have shown that transcriptomic analysis of blood samples taken from patients with acute Ebola virus disease (EVD) during the 2013–2016 West African outbreak was suggestive that a severe inflammatory response took place in acutely ill patients. The significant knowledge gained from studying the Makona variant, a cause of the largest known EVD outbreak, may be applicable to other species of ebolavirus, and other variants of the Ebola virus (EBOV) species. To investigate the ability of Makona to initiate an inflammatory response in human macrophages and characterise the host response in a similar manner to previously characterised EBOV variants, the human monocytic cell line THP-1 was differentiated into macrophage-like cells and infected with Makona. RNA-Seq and quantitative proteomics were used to identify and quantify host mRNA and protein abundance during infection. Data from infection with Reston virus (RESTV) were used as comparators to investigate changes that may be specific to, or enhanced in, Makona infection in relation to a less pathogenic species of ebolavirus.. This study found demonstrable induction of the inflammatory response, and increase in the activation state of THP-1 macrophages infected with Makona. NFκB and inflammation-associated transcripts displayed significant changes in abundance, reflective of what was observed in human patients during the 2013–2016 EBOV outbreak in West Africa, and demonstrated that transcriptomic changes found in Makona-infected cells were similar to that observed in Reston virus infection and that have been described in previous studies of other variants of EBOV.

Vitamin D modulates human macrophage response to Mycobacterium tuberculosis DNA

Mycobacterium tuberculosis (Mtb) is a facultative intracellular pathogen that infects macrophages where it avoids elimination by interfering with host defense mechanisms, including phago-lysosome fusion. Endosomal Toll-like receptors (TLRs) generate Type I Interferons (IFNs), which are associated with active tuberculosis (TB).We aimed to explore if DNA from different Mtb lineages lead to differences in the inflammatory response of human monocytic/macrophage cells. THP-1 cells which express two inducible reporter constructs for interferons (IFNs) as well as for NF-κB, were stimulated via endosomal delivery of Mtb DNA as a nanocomplex with PEI. DNA from different Mtb phylogenetic lineages elicited differential inflammatory responses in human macrophages. An initial relatively weak IRF-mediated response to DNA from HN878 and H37Rv increased if the cells were pre-treated with Vitamin D (Vit D) for 72 h. RNAseq of THP-1 under different transformation conditions showed that pre-treatment with Vit D upregulated several TLR9 variants, as well as genes involved in inflammatory immune response to infection, immune cell activation, Type I IFN regulation, and regulation of inflammation.Vit D appears to be important in increasing low IRF responses to DNA from certain lineages of Mtb. Variations in the IRF-mediated response to DNA derived from different Mtb genotypes are potentially important in the pathogenesis of tuberculosis since Type I IFN responses are associated with active disease. The role of Vit D in these responses could also translate into future therapeutic approaches.

Lipids derived from Camel milk regulate NLRP3 inflammasome-dependent inflammatory responses in human macrophages

Background: Camel milk is widely used for its reported anti-diabetic and health promoting effects. Lipids derived from the milk have also been shown to exhibit potent anti-inflammatory effects. The mechanism through which these lipids and constituent fatty acids exert these effects remains elusive. The aim of this study was to investigate the effect of camel milk on glycated protein-mediated macrophage inflammation.Methods: To determine the effect of Total Lipids (TL) and Total Fatty Acids (TFA) derived from camel milk on an in vitro model of diabetic inflammation, differentiated THP-1 (dTHP-1) cells stimulated with glycated serum albumin (gBSA) was employed. Cells were pre-treated with TL or TFA before challenging cells with gBSA.Results: Gas Chromatography-Mass Spectrometry (GC-MS) analysis found that TL was 96% triacylglycerol (TAG) while the TFA comprised 65% saturated and 35% unsaturated fatty acids. Both TL and TFA significantly (p<0.05) decreased gBSA-induced secretion of pro-inflammatory cytokines (Tumour necrosis factor-(TNF)-α, Interleukin-(IL)-1β/18). TL also demonstrated the ability to regulate the expression of p50/p65 sub-units of Nuclear Factor-kappa B (NF-κB), while concomitantly increasing the expression of regulatory cytokines IL-10, IL-1 Receptor Antagonist (IL-1Ra) and Cluster of Differentiation 163 (CD163)-shifting cells towards an M2 macrophage phenotype. Additionally, we found that TL significantly regulated the expression of Nucleotide-binding oligomerization domain-like receptor family pyrin domain containing-3 (NLRP3) inflammasome subunit and its regulator; Ten-Eleven Translocation-2 (TET-2).Conclusion: This paper demonstrates the ability of camel milk lipids to regulate gBSA-induced macrophage inflammation in vitro, by modulating the expression of key inflammatory regulators such NF-kB and NLRP3 inflammasome subunit. Keywords: Camel milk lipids, Macrophages, NF-kB, NLRP3-inflammasome, TET-2

Dietary Advanced Glycation Endproducts Induce an Inflammatory Response in Human Macrophages in Vitro.

Advanced glycation endproducts (AGEs) can be found in protein- and sugar-rich food products processed at high temperatures, which make up a vast amount of the Western diet. The effect of AGE-rich food products on human health is not yet clear and controversy still exists due to possible contamination of samples with endotoxin and the use of endogenous formed AGEs. AGEs occur in food products, both as protein-bound and individual molecules. Which form exactly induces a pro-inflammatory effect is also unknown. In this study, we exposed human macrophage-like cells to dietary AGEs, both in a protein matrix and individual AGEs. It was ensured that all samples did not contain endotoxin concentrations > 0.06 EU/mL. The dietary AGEs induced TNF-alpha secretion of human macrophage-like cells. This effect was decreased by the addition of N(ε)-carboxymethyllysine (CML)-antibodies or a receptor for advanced glycation endproducts (RAGE) antagonist. None of the individual AGEs induce any TNF-alpha, indicating that AGEs should be bound to proteins to exert an inflammatory reaction. These findings show that dietary AGEs directly stimulate the inflammatory response of human innate immune cells and help us define the risk of regular consumption of AGE-rich food products on human health.

AICAR inhibits NF\u03baB DNA binding independently of AMPK to attenuate LPS-triggered inflammatory responses in human macrophages

5-aminoimidazole-4-carboxamide-1-β-D-ribofuranoside (AICAR) is an established pharmacological activator of AMP-activated protein kinase (AMPK). Both, AICAR and AMPK were reported to attenuate inflammation. However, AICAR is known for many AMPK-independent effects, although the mechanisms remain incompletely understood. Here we report a potent suppression of lipopolysaccharide (LPS)-induced inflammatory gene expression by AICAR in primary human macrophages, which occurred independently of its conversion to AMPK-activating 5-aminoimidazole-4-carboxamide-1-β-D-ribofuranosyl monophosphate. Although AICAR did not interfere with activation of cytosolic signalling cascades and nuclear translocation of nuclear factor - κB (NFκB) by LPS, it prevented the recruitment of NFκB and RNA polymerase II to target gene promoters. AICAR also inhibited signal transducer and activator of transcription 3 (STAT3)-dependent induction of interleukin (IL) IL-6 and IL-10 targets, while leaving STAT6 and HIF1α-dependent gene expression in IL-4 and dimethyloxalylgylcine-treated macrophages intact. This points to a transcription factor-specific mode of action. Attenuated gene expression correlated with impaired NFκB and STAT3, but not HIF-binding in electrophoretic mobility shift assays in vitro. Conclusively, AICAR interferes with DNA binding of NFκB and STAT3 to modulate inflammatory responses.