Human THP-1 Macrophages Research Articles

Hepcidin Deficiency Protects Against Atherosclerosis.

Objective- Inflammatory stimuli enhance the progression of atherosclerotic disease. Inflammation also increases the expression of hepcidin, a hormonal regulator of iron homeostasis, which decreases intestinal iron absorption, reduces serum iron levels and traps iron within macrophages. The role of macrophage iron in the development of atherosclerosis remains incompletely understood. The objective of this study was to investigate the effects of hepcidin deficiency and decreased macrophage iron on the development of atherosclerosis. Approach and Results- Hepcidin- and LDL (low-density lipoprotein) receptor-deficient ( Hamp-/-/ Ldlr-/-) mice and Hamp+/+/ Ldlr-/- control mice were fed a high-fat diet for 21 weeks. Compared with control mice, Hamp-/-/ Ldlr-/- mice had decreased aortic macrophage activity and atherosclerosis. Because hepcidin deficiency is associated with both increased serum iron and decreased macrophage iron, the possibility that increased serum iron was responsible for decreased atherosclerosis in Hamp-/-/ Ldlr-/- mice was considered. Hamp+/+/ Ldlr-/- mice were treated with iron dextran so as to produce a 2-fold increase in serum iron. Increased serum iron did not decrease atherosclerosis in Hamp+/+/ Ldlr-/- mice. Aortic macrophages from Hamp-/-/ Ldlr-/- mice had less labile free iron and exhibited a reduced proinflammatory (M1) phenotype compared with macrophages from Hamp+/+/ Ldlr-/- mice. THP1 human macrophages treated with an iron chelator were used to model hepcidin deficiency in vitro. Treatment with an iron chelator reduced LPS (lipopolysaccharide)-induced M1 phenotypic expression and decreased uptake of oxidized LDL. Conclusions- In summary, in a hyperlipidemic mouse model, hepcidin deficiency was associated with decreased macrophage iron, a reduced aortic macrophage inflammatory phenotype and protection from atherosclerosis. The results indicate that decreasing hepcidin activity, with the resulting decrease in macrophage iron, may prove to be a novel strategy for the treatment of atherosclerosis.

Production and Characterization of Chemically Cross-Linked Collagen Scaffolds.

Chemical cross-linking of collagen-based devices is used as a means of increasing the mechanical stability and control the degradation rate upon implantation. Herein, we describe techniques to produce cross-linked with glutaraldehyde (GTA; amine terminal cross-linker), 4-arm polyethylene glycol succinimidyl glutarate (4SP; amine terminal cross-linker), diphenyl phosphoryl azide (DPPA; carboxyl terminal cross-linker), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC; carboxyl terminal cross-linker) collagen films. In addition, we provide protocols to characterize the biophysical (swelling), biomechanical (tensile), and biological (metabolic activity, proliferation and viability using human dermal fibroblasts and THP-1 macrophages) properties of the cross-linked collagen scaffolds.

In vitro exposure to 2,2′,4,4′-tetrabromodiphenyl ether (PBDE-47) impairs innate inflammatory response

Polybrominated diphenyl ethers (PBDEs) are persistent organic pollutants that are added to numerous products to prevent accidental fires. PBDEs are present in the environment and they bio-accumulate in human and animal tissues. Recently, their presence has been correlated to several pathologies but little is known about their effect on the human innate immune system activity. In this study we investigated the effect of the congener 2,2′,4,4′-Tetrabromodiphenyl ether (PBDE-47) on the functional activity of the THP-1 human macrophages cell line and on ex vivo freshly isolated human basophils. Cytotoxicity and genotoxicity studies showed that PBDE-47 was able to induce toxic effects on the THP-1 cell line viability at concentrations ≥25 μM. Immune function of THP-1 was studied after stimulation with bacterial lipopolysaccharide (LPS) and PBDE-47 exposure at concentrations granting macrophage viability. Two dimensional electrophoresis showed modification of the proteome in the 3 μM PBDE-47 treated sample and Real Time PCR and ELISA demonstrated a statistically significant reduction in the expression of IL-1β, IL-6 and TNF-α cytokines. Furthermore, PBDE-47 was able to perturbate genes involved in cell motility upregulating CDH-1 and downregulating MMP-12 expressions. Finally, basophil activation assay showed reduced CD63 activation in PBDE-47 treated samples. In conclusion, our study demonstrated that PBDE-47 may perturb the activities of cells involved in innate immunity dampening the expression of macrophage pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and genes involved in cell motility (MMP-12 and E-cadherin) and interfering with basophil activation suggesting that this compound can impair innate immune response.

Forskolin inhibited the secretion of interleukin-1β in activated human THP-1 macrophages

Objective To investigate the effects of Forskolin on the activation of nucleotide-binding oligomerization domain like receptor family, pyrin domain-containing 3(NLRP-3) inflammasome and the secretion of interleukin-1β(IL-1β) in activated human THP-1 macrophages, which can provide evidence for clinical treatment of inflammatory diseases in children. Methods Human monocyte cell line THP-1 cells were induced to differentiate into macrophages by Phorbol-12-myristate-13-acetate(PMA), and Forskolin(10, 50, 100 μmol/L) stimulated activated macrophages by nigericin.The mRNA of the inflammasome markers NLRP-3, IL-1β and Caspase-1 were detected by real-time quantitative polymerase chain reaction(qPCR) method.The protein of NLRP-3, pro-IL-1β, pro-Caspase-1 and Caspase-1 were detected by Western blot.The secretion of inflammatory cytokines IL-1β was detected by enzyme-linked immuno-sorbent assay(ELISA). Results Nigericin activated the cells and the mRNA expression of NLRP-3, IL-1β and Caspase-1 increased by 7.59 times(P 0.05). Western blot showed that Forskolin also had no effect on the protein expression of pro-Caspase-1 and pro-IL-1β in activated THP-1 macrophages(P>0.05), but the expression of NLRP-3 and Caspase-1 decreased significantly (P<0.001). The results of ELISA showed that the IL-1β secretion increased from basal 584.0 nmol/L to activated 2 695.6 nmol/L(t=16.031 1, P<0.001) on THP-1 macrophages by nigericin, but Forskolin(10, 50, 100 μmol/L) reduced it to 1 858.4 nmol/L(t=5.365 5, P<0.001), 1 467.9 nmol/L(t=8.047 5, P<0.001) and 1 246.7 nmol/L(t=10.199 0, P<0.001) on the activated THP-1 macrophages. Conclusions Forskolin can not affect the expression of pro-cytokines at the gene transcription and protein translation levels in activated THP-1 cells, but inhibit the secretion of inflammatory cytokines IL-1β, so as to inhibit inflammatory response, which can be used to treat pediatric inflammatory diseases caused by IL-1β. Key words: Inflammation; Forskolin; Macrophage; Inflammasome; Interleukin-1β

Polarization of Human Macrophages by Interleukin-4 Does Not Require ATP-Citrate Lyase.

Macrophages exposed to the Th2 cytokines interleukin (IL) IL-4 and IL-13 exhibit a distinct transcriptional response, commonly referred to as M2 polarization. Recently, IL-4-induced polarization of murine bone marrow-derived macrophages (BMDMs) has been linked to acetyl-CoA levels through the activity of the cytosolic acetyl-CoA-generating enzyme ATP-citrate lyase (ACLY). Here, we studied how ACLY regulated IL-4-stimulated gene expression in human monocyte-derived macrophages (MDMs). Although multiple ACLY inhibitors attenuated IL-4-induced target gene expression, this effect could not be recapitulated by silencing ACLY expression. Furthermore, ACLY inhibition failed to alter cellular acetyl-CoA levels and histone acetylation. We generated ACLY knockout human THP-1 macrophages using CRISPR/Cas9 technology. While these cells exhibited reduced histone acetylation levels, IL-4-induced gene expression remained intact. Strikingly, ACLY inhibitors still suppressed induction of target genes by IL-4 in ACLY knockout cells, suggesting off-target effects of these drugs. Our findings suggest that ACLY may not be the major regulator of nucleocytoplasmic acetyl-CoA and IL-4-induced polarization in human macrophages. Furthermore, caution should be warranted in interpreting the impact of pharmacological inhibition of ACLY on gene expression.

Nobiletin reduces LPL-mediated lipid accumulation and pro-inflammatory cytokine secretion through upregulation of miR-590 expression

Nobiletin has protective effects on cardiovascular diseases, but the mechanism is not clear. In this study, we examined whether nobiletin affects the expression of miR-590/LPL and its relative effects on lipid accumulation and pro-inflammatory cytokine secretion in human THP-1 macrophages. RT-qPCR analysis showed that nobiletin increased the expression of miR-590. Western blot analysis showed that nobiletin-suppressed LPL expression was enhanced by miR-590 mimic and abrogated by miR-590 inhibitor. Oil Red O staining and high-performance liquid chromatography assays showed that nobiletin attenuated lipid accumulation in macrophages. Treatment with nobiletin and miR-590 mimic decreased cellular lipid accumulation, whereas treatment with miR-590 inhibitor increased cellular lipid accumulation. ELISA illustrated that nobiletin alleviated pro-inflammatory cytokine secretion in macrophages as measured by, which was reduced by miR-590 mimic and increased by miR-590 inhibitor. In conclusion, nobiletin may alleviate lipid accumulation and secretion of pro-inflammatory cytokines by enhancing the inhibitory effect of miR-590 on LPL expression, suggesting a promising strategy for potential drug development for atherosclerosis.

Attenuating Effects of Nortrachelogenin on IL-4 and IL-13 Induced Alternative Macrophage Activation and on Bleomycin-Induced Dermal Fibrosis.

Excessive alternative macrophage activation contributes to fibrosis. We studied the effects of nortrachelogenin, the major lignan component of Pinus sylvestris knot extract, on alternative (M2) macrophage activation. J774 murine and THP-1 human macrophages were cultured with IL-4+IL-13 to induce alternative activation, together with the extract and its components. Effects of nortrachelogenin were also studied in bleomycin-induced murine dermal fibrosis model. Knot extract significantly decreased the expression of alternative activation markers-arginase 1 in murine macrophages (97.4 ± 1.3% inhibition at 30 μg/mL) and CCL13 and PDGF in human macrophages-as did nortrachelogenin (94.9 ± 2.4% inhibition of arginase 1 at 10 μM). Nortrachelogenin also decreased PPARγ expression but had no effect on STAT6 phosphorylation. In vivo, nortrachelogenin reduced bleomycin-induced increase in skin thickness as well as the expression of collagens COL1A1, COL1A2, and COL3A1 (all by >50%). In conclusion, nortrachelogenin suppressed IL-4+IL-13-induced alternative macrophage activation and ameliorated bleomycin-induced fibrosis, indicating therapeutic potential in fibrosing conditions.

IL-6 mediates ER expansion during hyperpolarization of alternatively activated macrophages.

Although recent evidence has shown that IL‐6 is involved in enhanced alternative activation of macrophages toward a profibrotic phenotype, the mechanisms leading to their increased secretory capacity are not fully understood. Here, we investigated the effect of IL‐6 on endoplasmic reticulum (ER) expansion and alternative activation of macrophages in vitro. An essential mediator in this ER expansion process is the IRE1 pathway, which possesses a kinase and endoribonuclease domain to cleave XBP1 into a spliced bioactive molecule. To investigate the IRE1‐XBP1 expansion pathway, IL‐4/IL‐13 and IL‐4/IL‐13/IL‐6‐mediated alternative programming of murine bone marrow‐derived and human THP1 macrophages were assessed by arginase activity in cell lysates, CD206 and arginase‐1 expression by flow cytometry, and secreted CCL18 by ELISA, respectively. Ultrastructural intracellular morphology and ER biogenesis were examined by transmission electron microscopy and immunofluorescence. Transcription profiling of 128 genes were assessed by NanoString and Pharmacological inhibition of the IRE1‐XBP1 arm was achieved using STF‐083010 and was verified by RT‐PCR. The addition of IL‐6 to the conventional alternative programming cocktail IL‐4/IL‐13 resulted in increased ER and mitochondrial expansion, profibrotic profiles and unfolded protein response‐mediated induction of molecular chaperones. IRE1‐XBP1 inhibition substantially reduced the IL‐6‐mediated hyperpolarization and normalized the above effects. In conclusion, the addition of IL‐6 enhances ER expansion and the profibrotic capacity of IL‐4/IL‐13‐mediated activation of macrophages. Therapeutic strategies targeting IL‐6 or the IRE1‐XBP1 axis may be beneficial to prevent the profibrotic capacity of macrophages.

Insights into Streptococcus agalactiae PI-2b pilus biosynthesis and role in adherence to host cells

The core PI-2b pilus present in “hypervirulent” ST-17 Streptococcus agalactiae strains consists of three pilin subunits (Spb1, Ap1 and Ap2) assembled by sortase SrtC1 and cell-wall anchored by Srt2. Spb1 was shown to be the major pilin and Ap2 the anchor pilin. Ap1 is a putative adhesin. Two additional genes, orf and lep, are part of this operon. The contribution of Lep and Ap1 to the biogenesis of the PI-2b pilus was investigated. Concerning the role of PI-2b, we found that higher PI-2b expression resulted in higher adherence to human brain endothelial cells and higher phagocytosis by human THP1 macrophages.

Recovery From a Myocardial Infarction Is Impaired in Male C57bl/6 N Mice Acutely Exposed to the Bisphenols and Phthalates That Escape From Medical Devices Used in Cardiac Surgery.

Bisphenols and phthalates leach from medical devices, and this exposure is likely to increase in postcardiac surgery patients. Previous studies suggest that such chemical exposure may impact recovery and wound healing, yet the direct effects of bisphenols and phthalates are unknown in this context. To study the direct effect of clinically based chemical exposures, we measured the metabolites representative of 6 bisphenols and 10 phthalates in men before and after cardiac surgery and then replicated this exposure in a mouse model of cardiac surgery and assessed survival, cardiac function and inflammation. Bisphenol A (BPA), di-ethyl hexyl phthalate (DEHP), butylbenzyl phthalate, di-isodecyl phthalate, and di-n-butyl phthalate metabolites were increased after surgery. DEHP exposure predominated, was positively correlated with duration on the cardiopulmonary bypass machine and exceeded its tolerable daily intake limit by 37-fold. In vivo, C57bl/6 N male mice treated with BPA+phthalates during recovery from surgery-induced myocardial infarction had reduced survival, greater cardiac dilation, reduced cardiac function and increased infiltration of neutrophils, monocytes and macrophages suggesting impaired recovery. Of interest, genetic ablation or estrogen receptor beta (ERβ) antagonism did not improve recovery and replacement of DEHP with tri-octyl trimellitate or removal of BPA from the mixture did not ameliorate these effects. To examine the direct effects on inflammation, treatment of human THP-1 macrophages with BPA and phthalates induced a dysfunctional proinflammatory macrophage phenotype with increased expression of M1-type macrophage polarization markers and MMP9 secretion, yet reduced phagocytic activity. These results suggest that chemicals escape from medical devices and may impair patient recovery.

Shiga Toxin/Lipopolysaccharide Activates Caspase-4 and Gasdermin D to Trigger Mitochondrial Reactive Oxygen Species Upstream of the NLRP3 Inflammasome

The non-canonical caspase-4 and canonical NLRP3 inflammasomes are both activated by intracellular lipopolysaccharide (LPS), but the crosstalk between these two pathways remains unclear. Shiga toxin 2 (Stx2)/LPS complex, from pathogenic enterohemorrhagic Escherichia coli, activates caspase-4, gasdermin D (GSDMD), and the NLRP3 inflammasome in human THP-1 macrophages, but not mouse macrophages that lack the Stx receptor CD77. Stx2/LPS-mediated IL-1β secretion and pyroptosis are dependent on mitochondrial reactive oxygen species (ROS) downstream of the non-canonical caspase-4 inflammasome and cleaved GSDMD, which is enriched at the mitochondria. Blockade of caspase-4 activation and ROS generation as well as GSDMD deficiency significantly reduces Stx2/LPS-induced IL-1β production and pyroptosis. The NLRP3 inflammasome plays a significant role in amplifying Stx2/LPS-induced GSDMD cleavage and pyroptosis, with significant reduction of these responses in NLRP3-deficientTHP-1 cells. Together, these data show that Stx2/LPS complex activates the non-canonical inflammasome and mitochondrial ROS upstream of the NLRP3 inflammasome to promote cytokine maturation and pyroptosis.

PGE2 Augments Inflammasome Activation and M1 Polarization in Macrophages Infected With Salmonella Typhimurium and Yersinia enterocolitica.

Eicosanoids are cellular metabolites, which shape the immune response, including inflammatory processes in macrophages. The effects of these lipid mediators on inflammation and bacterial pathogenesis are not clearly understood. Certain eicosanoids are suspected to act as molecular sensors for the recruitment of neutrophils, while others regulate bacterial uptake. In this study, gene expression analyses indicated that genes involved in eicosanoid biosynthesis including COX-1, COX-2, DAGL, and PLA-2 are differentially regulated in THP-1 human macrophages infected with Salmonella enterica Typhimurium or Yersinia enterocolitica. By using targeted metabolomics approach, we found that the eicosanoid precursor, arachidonic acid (AA) as well as its derivatives, including prostaglandins (PGs) PGF2α or PGE2/PGD2, and thromboxane TxB2, are rapidly secreted from macrophages infected with these Gram-negative pathogenic bacteria. The magnitude of eicosanoid biosynthesis in infected host cells depends on the presence of virulence factors of Y. enterocolitica and S. Typhimurium strains, albeit in an opposite way in Y. enterocolitica compared to S. Typhimurium infection. Trials with combinations of EP2/EP4 PGE2 receptor agonists and antagonists suggest that PGE2 signaling in these infection models works primarily through the EP4 receptor. Downstream of EP4 activation, PGE2 enhances inflammasome activation and represses M2 macrophage polarization while inducing key M1-type markers. PGE2 also led to a decreased numbers of Y. enterocolitica within macrophages. To summarize, PGE2 is a potent autocrine/paracrine activator of inflammation during infection in Gram-negative bacteria, and it affects macrophage polarization, likely controlling bacterial clearance by macrophages.

PACAP deficiency aggravates atherosclerosis in ApoE deficient mice

Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) plays an important role in cytoprotection, inflammation and cardiovascular regulation. Thus, we studied the involvement of PACAP in atherogenesis. Differentiated human THP-1 macrophages (MΦ) were stimulated with oxidized low-density lipoproteins (oxLDL) and the influence of PACAP38 treatment on lipid content and TNF release was determined. To test the effect of PACAP deficiency (PACAP−/−) on the development of atherosclerosis under standard chow (SC) or cholesterol-enriched diet (CED) in vivo, PACAP−/− mice were crossbred with ApoE−/− to generate PACAP−/−/ApoE−/− mice. Blood cholesterol and triglyceride levels were quantified. Lumen stenosis in the brachiocephalic trunk, cellularity and amounts of pro-inflammatory as well as autophagy-, apoptosis- and necroptosis-relevant proteins were analysed in atherosclerotic plaques by quantitative immunohistochemistry. In vitro, PACAP38 inhibited oxLDL-induced intracellular lipid storage as well as TNF release in MФ. In vivo, after SC, but not under CED, PACAP−/−/ApoE−/− mice showed an increased lumen stenosis compared to ApoE−/− mice. In atherosclerotic plaques of PACAP−/−/ApoE−/− mice, the immunoreactive areas of TNF+, IL-1β+, autophagic, apoptotic and necroptotic cells were increased. In contrast, the overall cell density was decreased compared to ApoE−/− under SC, while no differences were seen under CED. Similar plasma cholesterol levels were observed in PACAP−/−/ApoE−/− and ApoE−/− mice under the respective feeding regime. Thus, PACAP−/-/ApoE−/− mice represent a novel mouse model of accelerated atherosclerosis where CED is not required. Our data indicate that PACAP acts as an endogenous atheroprotective neuropeptide. Thus, stable PACAP agonists may have potential as anti-atherosclerotic therapeutics. The specific PACAP receptor(s) mediating atheroprotection remain(s) to be identified.

Blockade of NEAT1 represses inflammation response and lipid uptake via modulating miR-342-3p in human macrophages THP-1 cells.

Atherosclerosis has been recognized as a chronic inflammation process induced by lipid of the vessel wall. Oxidized low-density lipoprotein (ox-LDL) can drive atherosclerosis progression involving macrophages. Recently, long noncoding RNAs (lncRNAs) have been reported to play critical roles in atherosclerosis development. In our current study, we focused on the biological roles of lncRNA NEAT1 in atherosclerosis progress. Here, we found that ox-LDL was able to trigger human macrophages THP-1 cells, a human monocytic cell line, apoptosis in a dose-dependent and time-dependent course. In addition, we observed that NEAT1 was significantly increased in THP-1 cells incubated with ox-LDL and meanwhile miR-342-3p was greatly decreased. Then, NEAT1 was silenced by transfection of small interfering RNA (siRNA) of NEAT1 into THP-1 cells. As exhibited, CD36, oil-red staining levels, total cholesterol (TC), total cholesterol (TG) levels and THP-1 cell apoptosis were obviously repressed by knockdown of NEAT1. Furthermore, inhibition of NEAT1 contributed to the repression of inflammation in vitro. Interleukin 6 (IL-6), IL-1β, cyclooxygenase-2 (COX-2) and tumour necrosis factor-alpha (TNF-α) protein levels were remarkably depressed by NEAT1 siRNA in THP-1 cells. By using bioinformatics analysis, miR-342-3p was predicted as a downstream target of NEAT1 and the correlation between them was confirmed in our study. Moreover, overexpression of miR-342-3p could also greatly suppress inflammation response and lipid uptake in THP-1 cells. Knockdown of NEAT1 and miR-342-3p mimics inhibited lipid uptake in THP-1 cells. In conclusion, we implied that blockade of NEAT1 repressed inflammation response through modulating miR-342-3p in human macrophages THP-1 cells and NEAT1 may offer a promising strategy to treat atherosclerotic cardiovascular diseases.

Silence of long intergenic noncoding RNA HOTAIR ameliorates oxidative stress and inflammation response in ox‐LDL‐treated human macrophages by upregulating miR‐330‐5p

Evidence of the involvement of long noncoding RNAs (lncRNAs) in atherosclerosis is growing but still not well characterized. Here, we concentrated on the biological roles of lncRNA HOX transcription antisense RNA (HOTAIR) in atherosclerosis. In our study, we found that oxidized low-density lipoprotein (ox-LDL) induced human macrophages THP-1 cells apoptosis dose dependently and time dependently. Meanwhile, HOTAIR was significantly increased in THP-1 cells treated with ox-LDL. Then, HOTAIR was modulated by infection of LV-short hairpin RNA (shRNA) and LV-HOTAIR into THP-1 cells. As displayed, CD36, Oil Red O staining levels, total cholesterol, triglyceride levels and dil-ox-LDL uptake rate were greatly repressed by the silence of HOTAIR while triggered by overexpression of HOTAIR. Moreover, knockdown of HOTAIR suppressedreactive oxygen species, malondialdehyde levels, increased superoxide dismutase activity and cell apoptosis were also restrained. Reversely, overexpression of HOTAIR exhibited an opposite phenomenon. In addition, interleukin 6 (IL-6), IL-1β, cyclo-oxygenase 2, and tumor necrosis factor α protein levels were significantly depressed by LV-shRNA) of HOTAIR while increased by upregulation of HOTAIR in THP-1 cells. By carrying out bioinformatics analysis, miR-330-5p was predicted as a target of HOTAIR and the correlation between them was validated in our current study. MiR-330-5p was greatly decreased in THP-1 cells incubated with ox-LDL and overexpression of miR-330-5p was able to inhibit oxidative stress and inflammation process. Taken together, it was implied that HOTAIR contributed to atherosclerosis development by downregulating miR-330-5p in human macrophages.

High glucose induces a priming effect in macrophages and exacerbates the production of pro-inflammatory cytokines after a challenge.

IntroductionPainful diabetic neuropathy is associated with chronic inflammation, in which macrophages are the key effectors. We utilized an in vitro approach to determine the effects of high glucose on macrophage phenotype.Materials and methodsWe exposed human THP-1 macrophages to normal glucose (5 mM) and a clinically relevant high glucose environment (15 mM) and measured the expression and concentration of molecules associated with a diabetic cellular phenotype.ResultsWe found that THP-1 macrophages in high glucose conditions did not influence the basal expression of cyclooxygenase-2, Toll-like receptor-4, or class A scavenger receptor mRNA, or the concentrations of the cytokines interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and IL-10, but induced a priming effect on tumor necrosis factor (TNF)-α. Then, we stimulated THP-1 macrophages with a strong pro-inflammatory stimulus lipopolysaccharide (LPS; 5 µg/mL). After stimulation with LPS, we observed an exacerbated increase in TNF-α, IL-6, and MCP-1 concentration in the high glucose condition compared to the normal glucose environment. THP-1 macrophages in high glucose conditions developed tolerance to IL-10 anti-inflammatory effects (TNF-α production) when challenged with LPS.ConclusionOur in vitro approach allows the study of macrophages as potential targets for therapeutic purposes since it compares them to primary human macrophages exposed to high glucose and macrophages from patients with diabetes or complications of painful diabetic neuropathy (i.e. ulcers, adipocytes, and pancreas).

Silencing LAIR-1 in human THP-1 macrophage increases foam cell formation by modulating PPARγ and M2 polarization

Formation of macrophage-derived foam cells may mark the initial stages of atherosclerosis. We investigated the association between the expression of the leukocyte-associated immunoglobulin-like receptor 1 (LAIR-1) in macrophages and foam cell formation. A foam cell model was established by incubating THP-1-derived macrophages and bone marrow macrophages (BMMs) with oxidized low-density lipoprotein (ox-LDL). The role of LAIR-1 in foam cell formation was evaluated via Oil Red O staining and Dil-ox-LDL fluorescence intensities. Peroxisome proliferator-activated receptor gamma (PPARγ), cholesterol metabolism-related genes, and the role of LAIR-1 in activating classically activated (M1) and alternatively activated (M2) macrophages were evaluated by qPCR. Additionally, activation of protein-tyrosine phosphatase-1 (SHP-1) and cAMP-response element binding protein (CREB) were detected by western blotting. Results indicated that silencing LAIR-1 in macrophages modulated the SHP-1/CREB/PPARγ pathway, thereby promoting M2 macrophage polarization and increasing foam cell formation. Therefore, Inhibition of LAIR-1 in macrophages may promote foam cell formation and atherosclerosis.

Anti-mycobacterial activity of some medicinal plants used traditionally by tribes from Madhya Pradesh, India for treating tuberculosis related symptoms

Ethnopharmacological relevanceTuberculosis (TB) is one of the highly infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb). Several medicinal plants are traditionally used by the tribal healers of some tribal pockets of Madhya Pradesh (M.P.), India in the treatment of various ailments including TB related symptoms. The information of traditional knowledge of plants is empirical lacking systematic scientific investigation. Aim of the studyTo provide a scientific rationale for the traditional uses of some medicinal plants in treating prolonged cough, chest complaints and TB by ethnic groups in four districts of the state of M.P., India. The anti-mycobacterial activity of these plants was also investigated against multidrug-resistant (MDR) strains of Mtb. The active plant extracts were also assessed for general cytotoxicity by human monocytic leukemia cell line, THP-1 macrophages. Materials and methodsSemi structured interviews and guided field-walk methods were used to gather information on medicinal plants used by the tribal traditional healers. The study was carried out in the tribal pockets of Anuppur, Mandla, Umariya and Dindori districts of M.P., India. A total of 35 plant species distributed in 22 families were selected based on folklore uses suggested by the tribal traditional healer. The ethanol extracts of plants were evaluated for anti-mycobacterial activity against Mtb H37Rv and six MDR clinical isolates of Mtb. The anti-mycobacterial activity of plants was determined in terms of minimum inhibitory concentration (MIC) using standard resazurin microtitre plate assay (REMA). The plant extracts found to be active against Mtb were further evaluated for general cytotoxicity against human THP-1 macrophages using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. The selectivity index (SI) of active plant extracts were also calculated on the basis of MIC and cytotoxicity. ResultsOut of the 35 plants, 11 plant species showed anti-mycobacterial activity with MIC ranging from 500 to 31.25 µg/mL against Mtb H37Rv. The plant extracts also exhibited anti-mycobacterial activity against six multi-drug resistant (MDR) clinical strains of Mtb isolated from sputum samples of pulmonary TB patients. In vitro cytotoxicity of active plant extracts was also assessed in human THP-1 macrophages. The IC50 (50% inhibitory concentration) values of most of the plant extracts on THP-1 was found to be higher than MIC values against Mtb, indicating that the THP-1 cells are not adversely affected at concentrations that are effective against Mtb. Significant anti-mycobacterial activity was observed for eleven plants viz., Alstonia scholaris (L.) R. Br., Glycyrrhiza glabra L., Holorrhena antidysentrica (Roth) Wall. exA.DC., Mallotus philippensis (Lam.) Müll. Arg., Eulophia nuda Lindl., Cocculus hirsutus (L.) Diels, Pueraria tuberosa (Willd.) DC., Cyperus rotundus L., Curcuma caesia Roxb., Sphaeranthus indicus L. and Plumbago zeylanica L. which lends support to their traditional uses. ConclusionThe present investigation supports the potential role of plants used by tribal healers as our results have shown that these plants exhibit anti-mycobacterial activity in the acceptable range against Mtb. Our study clearly lends support to the traditional uses of some plants in TB related symptoms as we have found them to exhibit significant in vitro anti-TB activity. To the best of our knowledge, anti-mycobacterial activity in plants against Mtb is being reported for the first time from tribal areas of M.P., India by the current study.

Combinatorial Synthesis of New Pyrimidine- and Purine-β-d-Ribonucleoside Nucleolipids: Their Distribution Between Aqueous and Organic Phases and Their In Vitro Activity Against Human- and Rat Glioblastoma Cells In Vitro.

Two series of nucleolipids, O-2',3'-heptanylidene- as well as O-2',3'-undecanylidene ketals of six β-d-ribonucleosides (type A) and partly N-farnesyl derivatives thereof (type B) were prepared in a combinatorial manner. All novel compounds were characterized by elemental analysis and/or ESI mass spectrometry and by UV-, 1 H-, and 13 C-NMR spectroscopy. Conformational parameters of the nucleosides and nucleolipids were calculated from various 3 J(H,H), 3 J(1 H,13 C), and 5 J(F,H) coupling constants. For a drug profiling, the parent nucleosides and their lipophilic derivatives were studied with respect to their distribution (log P) between water and n-octanol as well as water and cyclohexane. From these data, qualitative conclusions were drawn concerning their possible blood-brain barrier passage efficiency. Moreover, nucleolipids were characterized by their molecular descriptor amphiphilic ratio (a.r.), which describes the balance between the hydrophilicity of the nucleoside headgroup and the lipophilicity of the lipid tail. All compounds were investigated invitro with respect to their cytostatic/cytotoxic activity toward human glioblastoma (GOS 3) as well as rat malignant neuroectodermal BT4Ca cell lines invitro. In order to differentiate between anticancer and side-effects of the novel nucleolipids, they were also studied on their activity on differentiated human THP-1 macrophages.

Leucodin attenuates inflammatory response in macrophages and lipid accumulation in steatotic hepatocytes via P2x7 receptor pathway: A potential role in alcoholic liver disease

The current study was aimed to reveal that leucodin, a sesquiterpene lactone from Artemisia capillaris could inhibit the inflammatory response in macrophages and the lipid accumulation in hepatocytes via P2x7R-NLRP3 inflammasome activation. Several types of macrophages including mouse peritoneal macrophages, mouse bone marrow-derived macrophages and human macrophages THP-1 cells were pretreated with different concentrations of leucodin for 1 h and then stimulated with LPS and ATP. LPS plus ATP initiated IL-1β cleavage and release in mouse peritoneal macrophages and peaked at 4 h. Leucodin did not show significant toxicity within 200 μM and effectively inhibited pro-IL-1β cleavage and release of mature-IL-1β in macrophages. Also, P2x7R antagonist and caspase-1 inhibitor also decreased IL-1β release and cleavage. Additionally, leucodin suppressed P2x7R, TLR4 and NLRP3 expression in LPS/ATP-stimulated macrophages. HepG2 cells were pretreated with different concentrations of leucodin for 1 h and then exposed to ethanol for 24 h. Leucodin suppressed lipid accumulation and enhanced phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells exposed to ethanol. In addition, leucodin inhibited the expression of sterol regulatory element binding protein-1 (SREBP1) and ACC in ethanol-treated HepG2 cells. Leucodin possessed the capacity for inhibiting inflammatory response in macrophages and suppressing lipid accumulation in hepatocytes, suggesting a promising therapeutic potential targeting inflammation and lipid metabolism in alcoholic liver disease.