Macrophage Cell Responses Research Articles

Macrophage cell responses in cutaneous leishmaniasis caused by Leishmania donovani

Macrophage cell responses in cutaneous leishmaniasis caused by Leishmania donovani

Immunomodulation activity of alkali extract polysaccharide from Plantago asiatic L. seeds

The MAPK signaling pathway is greatly involved in PLP-induced macrophage cell response.

Mouse aorta-derived mesenchymal progenitor cells contribute to and enhance the immune response of macrophage cells under inflammatory conditions.

IntroductionMesenchymal progenitor cells interact with immune cells and modulate inflammatory responses. The cellular characteristics required for this modulation are under fervent investigation. Upon interaction with macrophage cells, they can contribute to or suppress an inflammatory response. Current studies have focused on mesenchymal progenitors derived from bone marrow, adipose, and placenta. However, the arterial wall contains many mesenchymal progenitor cells, which during vascular disease progression have the potential to interact with macrophage cells. To examine the consequence of vascular-tissue progenitor cell-macrophage cell interactions in an inflammatory environment, we used a recently established mesenchymal progenitor cell line derived from the mouse aorta.MethodsMouse bone marrow-derived macrophage (MΦ) cells and mouse aorta-derived mesenchymal progenitor (mAo) cells were cultured alone or co-cultured directly and indirectly. Cells were treated with oxidized low-density lipoprotein (ox-LDL) or exposed to the inflammatory mediators lipopolysaccharide (LPS) and interferon-gamma (IFNγ) or both. A Toll-like receptor-4 (TLR4)-deficient macrophage cell line was used to determine the role of the mAo cells. To monitor inflammation, nitric oxide (NO), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNFα) secretions were measured.ResultsMesenchymal progenitor cells isolated from aorta and cloned by high proliferative capacity (mAo) can differentiate into multiple mesenchymal lineages and are positive for several commonly used mouse mesenchymal stem cell markers (that is, CD29, CD44, CD105, CD106, and Sca-1) but are negative for CD73 and ecto-5′-nucleotidase. In co-culture with MΦ cells, they increase MΦ oxidized-LDL uptake by 52.2%. In an inflammatory environment, they synergistically and additively contribute to local production of both NO and IL-6. After exposure to ox-LDL, the inflammatory response of MΦ cells to LPS and LPS/IFNγ is muted. However, when lipid-laden MΦ cells are co-cultured with mAo cell progenitors, the muted response is recovered and the contribution by the mAo cell progenitor is dependent upon cell contact.ConclusionsThe resident mesenchymal progenitor cell is a potential contributor to vascular inflammation when in contact with inflamed and lipid-laden MΦ cells. This interaction represents an additional target in vascular disease treatment. The potential for resident cells to contribute to the local immune response should be considered when designing therapeutics targeting inflammatory vascular disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0071-8) contains supplementary material, which is available to authorized users.

Involvement of redox balance in in vitro osteoclast formation of RAW 264.7 macrophage cells in response to LPS

Here we report that LPS induces osteoclast (OC) formation in murine RAW 264.7 macrophage cells in RPMI-1640 medium but not in α-minimum essential medium (α-MEM) as the original culture medium. LPS-induced OC formation in both media was examined to clarify the differential response. Receptor activator of NF-κB ligand induced OC formation in either α-MEM or RPMI-1640 medium. However, LPS-induced OC formation in RAW 264.7 cells maintained in RPMI-1640 medium, but not α-MEM, which was also supported by mouse bone marrow-derived macrophages, although they were less sensitive to LPS than RAW 264.7 cells. LPS augmented the expression of nuclear factor of activated T-cells (NFATc1) as a key transcription factor of osteoclastogenesis in cells maintained in RPMI-1640 medium, but reduced it in cells maintained in α-MEM. A high concentration of LPS was cytotoxic against cells maintained in α-MEM. Glutathione exclusively present in RPMI-1640 medium prevented LPS-induced cell death in α-MEM and augmented LPS-induced NFATc1 expression, followed by enhanced LPS-induced OC formation. LPS induced higher generation of reactive oxygen species in α-MEM than RPMI-1640 medium. An antioxidant enhanced LPS-induced OC formation, whereas a pro-oxidant reduced it. Taken together, redox balance in the culture condition was suggested to regulate in vitro LPS-induced OC formation.

Transcriptome profile of the murine macrophage cell response to Candida parapsilosis

Candida parapsilosis is a human fungal pathogen with increasing global significance. Understanding how macrophages respond to C. parapsilosis at the molecular level will facilitate the development of novel therapeutic paradigms. The complex response of murine macrophages to infection with C. parapsilosis was investigated at the level of gene expression using an Agilent mouse microarray. We identified 155 and 511 differentially regulated genes at 3 and 8h post-infection, respectively. Most of the upregulated genes encoded molecules involved in immune response and inflammation, transcription, signaling, apoptosis, cell cycle, electron transport and cell adhesion. Typical of the classically activated macrophages, there was significant upregulation of genes coordinating the production of inflammatory cytokines such as TNF, IL-1 and IL-15. Further, we used both primary murine macrophages and macrophages differentiated from human peripheral mononuclear cells to confirm the upregulation of the TNF-receptor family member TNFRSF9 that is associated with Th1 T-helper cell responses. Additionally, the microarray data indicate significant differences between the response to C. parapsilosis infection and that of C. albicans.

Tunicamycin inhibits Toll-like receptor-activated inflammation in RAW264.7 cells by suppression of NF-κB and c-Jun activity via a mechanism that is independent of ER-stress and N-glycosylation

In this study, we investigated the effect of tunicamycin on the production of pro-inflammatory molecules in RAW264.7 macrophage cells in response to lipopolysaccharide (LPS) and Toll-like receptor (TLR) agonists. Tunicamycin caused a reduction in LPS-induced nitric oxide (NO) production and expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin-1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α). In contrast, other ER stress-inducing chemicals, such as A23187 and thapsigargin (TG), increased LPS-induced COX-2 expression and had no effect on LPS-induced iNOS, TNF-α or IL-1β expression. Furthermore, the inhibitory effect of tunicamycin on LPS-induced inflammation was not influenced by salubrinal, an ER stress inhibitor, suggesting that the anti-inflammatory effect of tunicamycin is independent of ER stress. Tunicamycin also inhibited the expression of inflammatory molecule mRNAs induced by stimulation of TLR2 (with lipoteichoic acid) or TLR3 (with polyinosinic:polycytidylic acid), which do not require myeloid differentiation protein-2 (MD2) for their activation. Moreover, inhibition of LPS-induced iNOS expression was not inhibited by castanospermine, another N-glycosylation inhibitor, suggesting that the inhibitory effect of tunicamycin on LPS-induced iNOS induction is likely independent of MD2 N-glycosylation. Tunicamycin inhibited nuclear factor-kappaB (NF-κB) activity by suppressing LPS-induced nuclear translocation of p50 and subsequent DNA binding of p50 and p65 to the NF-κB site of the iNOS promoter. Tunicamycin also inhibited the transcriptional activity of a cAMP-response element (CRE) reporter, possibly by inhibiting c-Jun activation. Therefore, we conclude that tunicamycin represses TLR-induced inflammation through suppression of NF-κB and CRE activity via a mechanism that is independent of ER-stress and N-glycosylation.

MiR-146a Regulates IL-6 Production in Lipopolysaccharide-Induced RAW264.7 Macrophage Cells by Inhibiting Notch1

Inflammatory cells, macrophages induced by lipopolysaccharide (LPS) stimulation, lead to the production of inflammatory cytokines, which are crucial to host defense. MicroRNAs are short noncoding RNAs that regulate key biological processes via suppression of gene expression at posttranscriptional levels. Recently, miR-146a has been shown to be involved in the regulation of immune and inflammatory responses. However, the role of miR-146a in LPS-induced RAW264.7 macrophage cells remains unclear. In this study, we found that the expression of miR-146a was upregulated in RAW264.7 macrophage cells in response to LPS stimulation in a dose- and time-dependent manner by one-step real-time quantitative PCR. In addition, miR-146a mimics decreased, while miR-146a inhibitor increased, the expression of inflammatory cytokine interleukin-6, but did not affect tumor necrosis factor-α expression in LPS-stimulated RAW264.7 macrophage cells. Bioinformatics analyses predict that Notch1 is a potential target of miR-146a. Moreover, miR-146a overexpression in LPS-treated RAW264.7 macrophage cells did significantly decrease Notch1 mRNA and protein levels. These results suggested that miR-146a may function as a novel feedback negative regulator to LPS-induced production of inflammatory cytokines, at least in part, via inhibiting the expression of Notch1.

Effect of Surface-Modified Gold Nanorods on the Inflammatory Cytokine Response in Macrophage Cells

Inflammatory cytokine responses in macrophage cells (Raw264.7) exposed to gold nanorods (GNRs) depend on surface coating materials and on the concentration of the GNRs. At a high concentrations of GNRs, methoxy(polyethylene glycol)-thiol coated GNR (PEG-GNR) can be a good candidate for use in biological systems.

Targeted Proteomics of the Eicosanoid Biosynthetic Pathway Completes an Integrated Genomics-Proteomics-Metabolomics Picture of Cellular Metabolism

Eicosanoids constitute a diverse class of bioactive lipid mediators that are produced from arachidonic acid and play critical roles in cell signaling and inflammatory aspects of numerous diseases. We have previously quantified eicosanoid metabolite production in RAW264.7 macrophage cells in response to Toll-like receptor 4 signaling and analyzed the levels of transcripts coding for the enzymes involved in the eicosanoid metabolite biosynthetic pathways. We now report the quantification of changes in protein levels under similar experimental conditions in RAW264.7 macrophages by multiple reaction monitoring mass spectrometry, an accurate targeted protein quantification method. The data complete the first fully integrated genomic, proteomic, and metabolomic analysis of the eicosanoid biochemical pathway.

Nodakenin Suppresses Lipopolysaccharide-Induced Inflammatory Responses in Macrophage Cells by Inhibiting Tumor Necrosis Factor Receptor-Associated Factor 6 and Nuclear Factor-κB Pathways and Protects Mice from Lethal Endotoxin Shock

Nodakenin, a coumarin isolated from the roots of Angelicae gigas, has been reported to possess neuroprotective, antiaggregatory, antibacterial, and memory-enhancing effects. In the present study, we investigated the anti-inflammatory effects of nodakenin by examining its in vitro inhibitory effects on inducible nitric-oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and proinflammatory cytokines in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages and mouse peritoneal macrophages and its in vivo effects on LPS-induced septic shock in mice. Our results indicate that nodakenin concentration-dependently inhibits iNOS and COX-2 at the protein, mRNA, and promoter binding levels, and these inhibitions cause attendant decreases in the production of nitric oxide (NO) and prostaglandin E₂ (PGE₂). Furthermore, we found that nodakenin inhibits the production and mRNA expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β induced by LPS. Molecular data revealed that nodakenin suppressed the transcriptional activity and translocation of nuclear factor-κB (NF-κB) by inhibiting inhibitory κB-α degradation and IκB kinase-α/β phosphorylation. In addition, nodakenin was found to significantly inhibit the LPS-induced binding of transforming growth factor-β-activated kinase 1 to tumor necrosis factor receptor-associated factor 6 (TRAF6) by reducing TRAF6 ubiquitination. Pretreatment with nodakenin reduced the serum levels of NO, PGE₂, and proinflammatory cytokines and increased the survival rate of mice with LPS-induced endotoxemia. Taken together, our data suggest that nodakenin down-regulates the expression of the proinflammatory iNOS, COX-2, TNF-α, IL-6, and IL-1β genes in macrophages by interfering with the activation of TRAF6, thus preventing NF-κB activation.

Consequences of Alteration in Leucine Zipper Sequence of Melittin in Its Neutralization of Lipopolysaccharide-induced Proinflammatory Response in Macrophage Cells and Interaction with Lipopolysaccharide

The bee venom antimicrobial peptide, melittin, besides showing versatile activity against microorganisms also neutralizes lipopolysaccharide (LPS)-induced proinflammatory responses in macrophage cells. However, how the amino acid sequence of melittin contributes in its anti-inflammatory properties is mostly unknown. To determine the importance of the leucine zipper sequence of melittin in its neutralization of LPS-induced inflammatory responses in macrophages and interaction with LPS, anti-inflammatory properties of melittin and its three analogues and their interactions with LPS were studied in detail. Two of these analogues, namely melittin Mut-1 (MM-1) and melittin Mut-2 (MM-2), possess leucine to alanine substitutions in the single and double heptadic leucine residue(s) of melittin, respectively, whereas the third analogue is a scrambled peptide (Mel-SCR) that contains the amino acid composition of melittin with minor rearrangement in its leucine zipper sequence. Although MM-1 partly inhibited the production of proinflammatory cytokines in RAW 264.7 and rat primary macrophage cells in the presence of LPS, MM-2 and Mel-SCR were negligibly active. A progressive decrease in interaction of melittin with LPS, aggregation in LPS, and dissociation of LPS aggregates with alteration in the leucine zipper sequence of melittin was observed. Furthermore, with alteration in the leucine zipper sequence of melittin, these analogues failed to exhibit cellular responses associated with neutralization of LPS-induced inflammatory responses in macrophage cells by melittin. The data indicated a probable important role of the leucine zipper sequence of melittin in neutralizing LPS-induced proinflammatory responses in macrophage cells as well as in its interaction with LPS.

Mycobacterial Secretion Systems ESX-1 and ESX-5 Play Distinct Roles in Host Cell Death and Inflammasome Activation

During infection of humans and animals, pathogenic mycobacteria manipulate the host cell causing severe diseases such as tuberculosis and leprosy. To understand the basis of mycobacterial pathogenicity, it is crucial to identify the molecular virulence mechanisms. In this study, we address the contribution of ESX-1 and ESX-5--two homologous type VII secretion systems of mycobacteria that secrete distinct sets of immune modulators--during the macrophage infection cycle. Using wild-type, ESX-1- and ESX-5-deficient mycobacterial strains, we demonstrate that these secretion systems differentially affect subcellular localization and macrophage cell responses. We show that in contrast to ESX-1, the effector proteins secreted by ESX-5 are not required for the translocation of Mycobacterium tuberculosis or Mycobacterium marinum to the cytosol of host cells. However, the M. marinum ESX-5 mutant does not induce inflammasome activation and IL-1β activation. The ESX-5 system also induces a caspase-independent cell death after translocation has taken place. Importantly, by means of inhibitory agents and small interfering RNA experiments, we reveal that cathepsin B is involved in both the induction of cell death and inflammasome activation upon infection with wild-type mycobacteria. These results reveal distinct roles for two different type VII secretion systems during infection and shed light on how virulent mycobacteria manipulate the host cell in various ways to replicate and spread.

The response of macrophage cells to sense and antisense double-stranded RNA

The response of macrophage cells to sense and antisense double-stranded RNA

Macrophage presence is essential for the regeneration of ascending afferent fibres following a conditioning sciatic nerve lesion in adult rats

BackgroundInjury to the peripheral branch of dorsal root ganglia (DRG) neurons prior to injury to the central nervous system (CNS) DRG branch results in the regeneration of the central branch. The exact mechanism mediating this regenerative trigger is not fully understood. It has been proposed that following peripheral injury, the intraganglionic inflammatory response by macrophage cells plays an important role in the pre-conditioning of injured CNS neurons to regenerate. In this study, we investigated whether the presence of macrophage cells is crucial for this type of regeneration to occur. We used a clodronate liposome technique to selectively and temporarily deplete these cells during the conditioning phase of DRG neurons.ResultsRetrograde and anterograde tracing results indicated that in macrophage-depleted animals, the regenerative trigger characteristic of pre-conditioned DRG neurons was abolished as compared to injury matched-control animals. In addition, depletion of macrophage cells led to: (i) a reduction in macrophage infiltration into the CNS compartment even after cellular repopulation, (ii) astrocyte up-regulation at rostral regions and down-regulation in brain derived neurotrophic factor (BDNF) concentration in the serum.ConclusionActivation of macrophage cells in response to the peripheral nerve injury is essential for the enhanced regeneration of ascending sensory neurons.

Anti-inflammatory and anti-nociceptive activities of compounds from Tinospora sagittata (Oliv.) Gagnep.

Radix Tinosporae is a herb widely used in traditional Chinese medicine for the treatment of various inflammatory diseases. In the present study, its anti-inflammatory and antinociceptive activities were investigated. The ethanol extract of Radix Tinosporae exhibited significant inhibitory effects on xylene-induced ear edema and acetic acid-induced writhing in mice. Using bioassay-guided fractionation, the n-butanol fraction was determined as the active fraction. Further purification of the most active n-butanol fraction led to the isolation of three compounds, palmatine, columbamine and columbinyl glucoside. All three compounds showed inhibitory activities on xylene-induced ear edema, but only palmatine and columbamine exhibited significant inhibitory effects on acetic acid-induced writhing. In addition, palmatine and columbamine markedly inhibited in vitro production of nitric oxide and nuclear factor-kappaB activation in RAW264.7 macrophage cells in response to lipopolysaccharide or tumor necrosis factor alpha stimulation. These results provide justification for the utilization of Radix Tinosporae in Chinese folk medicine for the treatment of inflammatory diseases.

Adipocyte Fatty Acid-binding Protein Modulates Inflammatory Responses in Macrophages through a Positive Feedback Loop Involving c-Jun NH2-terminal Kinases and Activator Protein-1

Adipocyte fatty acid-binding protein (A-FABP) has emerged as an important mediator of inflammation in macrophages. Macrophage-selective ablation of A-FABP alone is sufficient to prevent the development of high cholesterol diet-induced atherosclerosis in apoE-deficient mice. However, the precise mechanisms whereby A-FABP modulates inflammation remain elusive. Here, we report that A-FABP forms a finely tuned positive loop between JNK and activator protein-1 (AP-1) to exacerbate lipopolysaccharide (LPS)-induced inflammatory responses in macrophages. Real time PCR and luciferase reporter analysis showed that LPS induced A-FABP expression through transcriptional activation. This effect was mediated by JNK, which promoted the recruitment of c-Jun to a highly conserved AP-1 consensus binding motif located within the proximal region of the A-FABP promoter. LPS-induced transactivation of the A-FABP gene was diminished by either pharmacological inhibition of JNK or knocking down c-Jun or by mutating the AP-1 recognition site within the proximal region (-122 to -116 bp) of the A-FABP promoter. Conversely, the LPS-evoked phosphorylation of JNK, activation of AP-1, and production of pro-inflammatory cytokines were markedly attenuated by pharmacological or genetic suppression of A-FABP in macrophages. Furthermore, the LPS-induced elevation in A-FABP expression could also be prevented by the selective A-FABP inhibitor BMS309403. These findings support the notion that pharmacological inhibition of A-FABP represents a valid strategy for treating inflammation-related disorders such as atherosclerosis.

Regulating Immune Response Using Polyvalent Nucleic Acid−Gold Nanoparticle Conjugates

The immune response of macrophage cells to internalized polyvalent nucleic acid-functionalized gold nanoparticles has been studied. This study finds that the innate immune response (as measured by interferon-beta levels) to densely functionalized, oligonucleotide-modified nanoparticles is significantly less (up to a 25-fold decrease) when compared to a lipoplex carrying the same DNA sequence. The magnitude of this effect is inversely proportional to oligonucleotide density. It is proposed that the enzymes involved in recognizing foreign nucleic acids and triggering the immune response are impeded due to the local surface environment of the particle, in particular high charge density. The net effect is an intracelluar gene regulation agent that elicits a significantly lower cellular immune response than conventional DNA transfection materials.

Role of inflammation in right ventricular damage and repair following experimental pulmonary embolism in rats

Right ventricular (RV) dysfunction is associated with poor clinical outcome following pulmonary embolism (PE). Previous studies in our laboratory show that influx of neutrophils contributes to acute RV damage seen in an 18 h rat model of PE. The present study describes the further progression of inflammation over 6 weeks and compares the neutrophil and monocyte responses. The RV outflow tract became white in colour by day 1 with influx of neutrophils (tissue myeloperoxidase activity increased 17-fold) and mononuclear cells with characteristics of M1 phenotype (high in Ccl20, Cxcl10, CcR2, MHCII, DNA microarray analysis). Matrix metalloproteinase activities were increased and tissue was thinned to produce a translucent appearance in weeks 1 through 6 in 40% of hearts. RV contractile function was significantly reduced at 6 weeks of PE. In this later phase, there was accumulation of myofibroblasts, the presence of mononuclear cells with M2 characteristics (high in scavenger mannose receptors, macrophage galactose lectin 1, PDGFR1, PDGFRbeta), enrichment of the subendocardial region of the RV outflow tract with neovesels (alpha-smooth muscle immunohistochemistry) and deposition of collagen fibres (picrosirius red staining) beginning scar formation. Thus, while neutrophil response is associated with the early, acute inflammatory events, macrophage cells continue to be present during the proliferative phase and initial deposition of collagen in this model, changing from the M1 to the M2 phenotype. This suggests that the macrophage cell response is biphasic.

Cellular Response to Magnetic Nanoparticles “PEGylated” via Surface-Initiated Atom Transfer Radical Polymerization

A new method to PEGylate magnetic nanoparticles with a dense layer of poly(poly(ethylene glycol) monomethacrylate) (P(PEGMA)) by surface-initiated atom transfer radical polymerization (ATRP) is reported. In this approach, an initiator for ATRP was first immobilized onto the magnetic nanoparticle surface, and then P(PEGMA) was grafted onto the surface of magnetic nanoparticle via copper-mediated ATRP. The modified nanoparticles were subjected to detailed characterization using FTIR, XPS, and TGA. The P(PEGMA)-immobilized nanoparticles dispersed well in aqueous media. The saturation magnetization values of the P(PEGMA)-immobilized nanoparticles were 19 emu/g and 11 emu/g after 2 and 4 h polymerization respectively, compared to 52 emu/g for the pristine magnetic nanoparticles. The response of macrophage cells to pristine and P(PEGMA)-immobilized nanoparticles was compared. The results showed that the macrophage cells are very effective in cleaning up the pristine magnetic nanoparticles. With the P(PEGMA)-immobilized nanoparticles, the amount of nanoparticles internalized into the cells is greatly reduced to <2 pg/cell over a 5 day period. With this amount of nanoparticles uptake, no significant cytotoxicity effects were observed.

Controlled release of heparin from poly( ε-caprolactone) electrospun fibers

Sustained delivery of heparin to the localized adventitial surface of grafted blood vessels has been shown to prevent the vascular smooth muscle cell (VSMC) proliferation that can lead to graft occlusion and failure. In this study heparin was incorporated into electrospun poly( ε-caprolactone) (PCL) fiber mats for assessment as a controlled delivery device. Fibers with smooth surfaces and no bead defects could be spun from polymer solutions with 8% w/v PCL in 7:3 dichloromethane:methanol. A significant decrease in fiber diameter was observed with increasing heparin concentration. Assessment of drug loading, and imaging of fluorescently labeled heparin showed homogenous distribution of heparin throughout the fiber mats. A total of approximately half of the encapsulated heparin was released by diffusional control from the heparin/PCL fibers after 14 days. The fibers did not induce an inflammatory response in macrophage cells in vitro and the released heparin was effective in preventing the proliferation of VSMCs in culture. These results suggest that electrospun PCL fibers are a promising candidate for delivery of heparin to the site of vascular injury.