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Innate immune responses of primary murine macrophage-lineage cells and RAW 264.7 cells to ligands of Toll-like receptors 2, 3, and 4
Abstract
Although studies have been performed to characterize responses of macrophages from individual anatomical sites (e.g., alveolar macrophages) or of murine-derived macrophage cell lines to microbial ligands, few studies compare these cell types in terms of phenotype and function. We directly compared the expression of cell surface markers and functional responses of primary cultures of three commonly used cells of monocyte-macrophage lineage (splenic macrophages, bone marrow-derived macrophages, and bone marrow-derived dendritic cells) with those of the murine-leukemic monocyte-macrophage cell line, RAW 264.7. We hypothesized that RAW 264.7 cells and primary bone marrow-derived macrophages would be similar in phenotype and would respond similarly to microbial ligands that bind to either Toll-like receptors 2, 3, and 4. Results indicate that RAW 264.7 cells most closely mimic bone marrow-derived macrophages in terms of cell surface receptors and response to microbial ligands that initiate cellular activation via Toll-like receptors 3 and 4. However, caution must be applied when extrapolating findings obtained with RAW 264.7 cells to those of other primary macrophage-lineage cells, primarily because phenotype and function of the former cells may change with continuous culture.
... RAW 264.7 cells are widely used to study oxidative stress and inflammatory and antibacterial activities because they mimic murine bone-marrow-derived macrophage properties with similar surface receptors, the toll-like receptors (TLR). However, as with many of the cell lines, the phenotype may change with consecutive passages [129,130]. Caco-2/THP1: Over the last 15 years, several in vitro models were constructed to analyze the interaction between intestinal epithelial and macrophage-like cells. The choice to use THP-1 cells in these models was prevalent because most macrophages found in the healthy intestine are characterized by the absence of several response receptors, such as the LPS-receptor CD14 [135], as well as the absence of an inflammatory response towards pathogens [136]. ...
... In all the tri-culture setups that have been described, the culture system was established by growing Caco-2 and HT29-MTX cells onto inserts with semipermeable membranes, and RAW 264.7 or THP-1 cells in the basolateral compartment where the macrophage stimulus was added. Both these two macrophage cell types require the same culture medium, RPMI 1640 [129,141], although THP-1 cells grow in suspension and RAW 264.7 differentiate as an adherent cell line [142]. However, Raw 264.7 should not be used after passage number 30, otherwise it might have an impact on their stability [142]. ...
The use of cell models is common to mimic cellular and molecular events in interaction with their environment. In the case of the gut, the existing models are of particular interest to evaluate food, toxicants, or drug effects on the mucosa. To have the most accurate model, cell diversity and the complexity of the interactions must be considered. Existing models range from single-cell cultures of absorptive cells to more complex combinations of two or more cell types. This work describes the existing solutions and the challenges that remain to be solved.
... RAW 264.7 cells are monocyte/macrophage-like cells generated from an Abelson leukemia virus-transformed BALB/c mouse cell line. These cells have been considered a suitable macrophage type as evidence found that the phenotype and functional characteristics of RAW 264.7 remained stable through passages [17], and these cells mimic bone-marrow derived macrophages, which initiate cellular activation and responses to microorganisms and their products [18,19]. ROS plays a protective as well as destructive role; the overproduction of ROS can lead to the dysregulation of nitric oxide (NO) and intracellular calcium (Ca 2+ ), resulting in deleterious effects and damage to cellular structures in the body [20]. ...
... Therefore, they are commonly used in in vitro and in vivo assessments of pathophysiological roles in human diseases [26][27][28]. As mentioned, RAW 264.7 cell line is considered a suitable macrophage type due to its stable phenotype and functional characteristics and is ideal for studying cellular responses [17][18][19]. Our study first evaluated the effects of weak ARW (pH 8.5 and pH 9.5) on cell viability after induction of OS by H 2 O 2 or LPS stimulation. ...
Excessive oxidative stress (OS) is a common cause of various diseases such as cancer, diabetes, and obesity; thus, an anti-oxidative solution is essential for the improvement of human health. Increasing evidence suggests that alkaline reduced water (ARW), especially between pH 9.5–10.0, has antioxidant capacity; however, relatively few studies have reported the effect of weak ARW at pH 8.5 on OS, especially in vitro. This study was conducted to evaluate the anti-oxidative efficacy of weak ARW with negative oxidation-reduction potential (ORP) and relatively high hydrogen (H2) concentration, as compared to tap water (TW) and ARW at pH 9.5. RAW 264.7 murine macrophage cells, stimulated by hydrogen peroxide (H2O2) and lipopolysaccharide (LPS) to induce OS, were used as a control (Con) and then treated with TW and ARW at pH 8.5 (ARW_8.5) and pH 9.5 (ARW_9.5) at different concentrations (0.1%, 1%, and 10% v/v). Results showed that cell viability was significantly restored after treatment with both ARW_8.5 and ARW_9.5 compared to Con/H2O2 and Con/LPS, while TW treatment did not induce significant changes. Levels of reactive oxygen species (ROS), nitric oxide (NO), Ca2+, catalase, and glutathione peroxide (GPx) showed significant differences in a concentration-dependent manner in ARW_8.5 and ARW_9.5 groups compared to Con/H2O2 and Con/LPS groups. Likewise, the expression of p-p38, p-JNK, and p-ERK was also significantly reduced in the ARW-treated groups, but not in the TW group. In conclusion, ARW_8.5 exhibited anti-oxidative effects through the regulation of the MAPK signaling pathway in RAW 264.7 murine macrophage cells, indicating the health-promoting potential of weak ARW through daily intake.
... ( Figure 3B). RAW 264.7 cells are of monocyte-derived origin that has been shown to closely resemble primary BMMs (Berghaus et al., 2010). Furthermore, Zajd et al. reported that compared to PMs, primary BMMs (C57BL/6) are skewed more toward a M1 phenotype when stimulated, which may explain both increased MHCII and CD86 marker expression in RAW 264.7 cells (Zajd et al., 2020). ...
Macrophages are phagocytic innate immune cells capable of phenotypical switching in response to the local microenvironment. Studies often use either primary macrophages or immortalized cell lines for hypothesis testing, therapeutic assessment, and biomaterial evaluation without carefully considering the potential effects of cell source and tissue of origin, which strongly influence macrophage response. Surprisingly, limited information is available about how, under similar stimuli, immortalized cell lines and primary cells respond in both phenotypical and functional changes. To address this need, in this work, we cultured immortalized macrophage cell lines derived from different origins ( i.e., blood, lung, peritoneal) to understand and compare macrophage phenotypical responses, including polarization and plasticity, morphological changes, and phagocytic functionalities, as well as compared primary macrophages extracted from peritoneal and bone marrow to their immortalized cell line counterparts. We found significant differences in baseline expression of different markers (e.g., CD86, MHCII, CD206, and EGR2) amongst different cell lines, which further influence both polarization and repolarization of the cells, in addition to their phagocytic functionality. Additionally, we observed that, while RAW 264.7 cells behave similarly to the primary bone marrow-derived macrophages, there are noticeable phenotypical and functional differences in cell line (IC-21) and primary peritoneal macrophages, highlighting tissue-specific differences in macrophage response amongst cell lines and primary cells. Moving to three-dimensional (3D) culture in well-defined biomaterials, blood-derived primary and cell line macrophages were encapsulated within hydrogel-based synthetic extracellular matrices and their polarization profiles and cell morphologies were compared. Macrophages exhibited less pronounced polarization during 3D culture in these compliant, soft materials compared to two-dimensional (2D) culture on rigid, tissue culture plastic plates. Overall, our findings highlight origin-specific differences in macrophage response, and therefore, careful considerations must be made to identify the appropriate cell source for the application of interest.
... Although RAW 246.7 cells are commonly used in various studies, it is worth noting that RAW cells, which are not cloned, may undergo changes in their phenotype (surface marker and expression of macrophage characteristic gene) and function (NO production) over time in continuous culture (passage no. > 30) [254,255]. However, following the standard of 100 ng/mL of LPS, cell densities ranging from 1 to 9.9 × 10 5 cells/well and incubation periods of 12 to 24 h in an atmosphere of 5% CO2 at 37 °C are recommended as the optimum condition [255]. ...
Natural products (NPs) have played a vital role in human survival for millennia, particularly for their medicinal properties. Many traditional medicine practices continue to utilise crude plants and animal products for treating various diseases, including inflammation. In contrast, contemporary medicine focuses more on isolating drug-lead compounds from NPs to develop new and better treatment drugs for treating inflammatory disorders such as inflammatory bowel diseases. There is an ongoing search for new drug leads as there is still no cure for many inflammatory conditions. Various approaches and technologies are used in drug discoveries from NPs. This review comprehensively focuses on anti-inflammatory small molecules and describes the key strategies in identifying, extracting, fractionating and isolating small-molecule drug leads. This review also discusses the (i) most used approaches and recently available techniques, including artificial intelligence (AI), (ii) machine learning, and computational approaches in drug discovery; (iii) provides various animal models and cell lines used in in-vitro and in-vivo assessment of the anti-inflammatory potential of NPs.
... In applying this FluidFM technology to study single immune cell responses, we chose TLR-induced NF-kB activation of RAW 264.7 monocyte/macrophage-like cells as a model system. TLRinduced NF-kB translocation is commonly used as a readout of innate immune activation via TLR pathways and RAW 264.7 cells are commonly used as an in vitro model of macrophage responses (9,(22)(23)(24)(25). Macrophages play a key role in the early immune response to infection or vaccination by releasing chemical signals to initiate an inflammatory immune response in response to TLR stimulation. ...
The innate immune system initiates early response to infection by sensing molecular patterns of infection through pattern-recognition receptors (PRRs). Previous work on PRR stimulation of macrophages revealed significant heterogeneity in single cell responses, suggesting the importance of individual macrophage stimulation. Current methods either isolate individual macrophages or stimulate a whole culture and measure individual readouts. We probed single cell NF-κB responses to localized stimuli within a naïve culture with Fluidic Force Microscopy (FluidFM). Individual cells stimulated in naïve culture were more sensitive compared to individual cells in uniformly stimulated cultures. In cluster stimulation, NF-κB activation decreased with increased cell density or decreased stimulation time. Our results support the growing body of evidence for cell-to-cell communication in macrophage activation, and limit potential mechanisms. Such a mechanism might be manipulated to tune macrophage sensitivity, and the density-dependent modulation of sensitivity to PRR signals could have relevance to biological situations where macrophage density increases.
... Raw264.7 cells can elicit a robust and well-known inflammatory response upon challenges with increasing dosages of stimulants, such as lipopolysaccharide (LPS), which is often employed to screen biomaterials and to predict their potential in regulating immune responses [31]. This may ultimately activate a series of events, including the production of inflammatory and anti-inflammatory cytokines, which can be used as biomarkers to screen for possible anti-inflammatory and immunomodulatory compounds or biomaterials [32]. Both mono-and co-culture experiments of Raw264.7 cells and mBMSCs were conducted to deepen the understanding of the crosstalk between macrophages and mBMSCs mediated by Mn-implanted surfaces. ...
Manganese (Mn) is an essential micronutrient in various physiological processes, but its functions in bone metabolism remain undefined. This is partly due to the interplay between immune and bone cells because Mn plays a central role in the immune system. In this study, we utilized the plasma immersion ion implantation and deposition (PIII&D) technique to introduce Mn onto the titanium surface. The results demonstrated that Mn-implanted surfaces stimulated the shift of macrophages toward the M1 phenotype and had minimal effects on the osteogenic differentiation of mouse bone marrow mesenchymal stem cells (mBMSCs) under mono-culture conditions. However, they promoted the M2 polarization of macrophages and improved the osteogenic activities of mBMSCs under co-culture conditions, indicating the importance of the crosstalk between mBMSCs and macrophages mediated by Mn in osteogenic activities. This study provides a positive incentive for the application of Mn in the field of osteoimmunology.
... The RAW 264.7 murine macrophage cell line is extensively utilized due to its ability to faithfully replicate inflammatory reactions, thereby providing valuable insights into the immunomodulatory properties of tested substances [21]. Although more recent insights have brought into question the inherent differences between the immune responses of mouse and human cells [22], researchers have successfully employed RAW 264.7 cells as a convenient model to study macrophage biology and certain immune responses [23][24][25][26][27]. ...
Quantum dots (QDs) with photostable fluorescence are recommended for imaging applications; however, their effect on living cells is incompletely understood. We aimed to elucidate the RAW 264.7 murine macrophage cell line’s response to the Si/SiO2 QDs challenge. Cells were exposed to 5 and 15 μg/mL Si/SiO2 QDs for 6 h, 12 h, and 24 h. Cell metabolic activity and viability were assessed by MTT, live/dead, and dye-exclusion assays. Oxidative stress and membrane integrity were assessed by anion superoxide, malondialdehyde, and lactate dehydrogenase activity evaluations. Antioxidative enzyme activities were analyzed by kinetic spectrophotometric methods. Cytokines were analyzed with an antibody-based magnetic bead assay, PGE2 was assessed by ELISA, and Nrf-2, Bcl-2, Beclin 1, and the HSPs were analyzed by western blot. Autophagy levels were highlighted by fluorescence microscopy. The average IC50 dose for 6, 12, and 24 h was 16.1 ± 0.7 μg/mL. Although glutathione S-transferase and catalase were still upregulated after 24 h, superoxide dismutase was inhibited, which together allowed the gradual increase of malondialdehyde, anion superoxide, nitric oxide, and the loss of membrane integrity. G-CSF, IL-6, TNF-α, MIP-1β, MCP-1, Nrf-2, PGE2, and RANTES levels, as well as autophagy processes, were increased at all time intervals, as opposed to caspase 1 activity, COX-2, HSP60, and HSP70, which were only upregulated at the 6-h exposure interval. These results underscore that Si/SiO2 QDs possess significant immunotoxic effects on the RAW 264.7 macrophage cell line and stress the importance of developing effective strategies to mitigate their adverse impact.
... Moreover, the activity of D-CQNP more or less similar to the positive control (LPS). This result was agreed in previous studies, where CD11b and CD40 expression were found to be higher when macrophages were activated by micro bers [44]. Furthermore, the expression of CD40 was higher than CD11b, which demonstrates the stimulation of RAW 264.7 cells by non-starch polysaccharide of C. quinoa through the NF-κB and MAPK signaling pathways by the production of pro-in ammatory cytokines and NO production via CD40 expression. ...
Non-starch polysaccharides derived from natural resources play a significant role in the field of food science and human health due to their extensive distribution in nature and less toxicity. In this order, the immunostimulatory activity of a non-starch polysaccharide (CQNP) from Chenopodium quinoa was examined before and after and deproteination in murine macrophage RAW264.7 cells. The chemical composition of CQNP and deproteinated- CQNP (D-CQNP) were spectrometrically analysed that revealed the presence of carbohydrate (22.7 ± 0.8% and 39.5± 0.8%), protein (41.4± 0.8% and 20.8± 0.5%) and uronic acid (8.7±0.8% and 6.7± 0.2%). The monosaccharide composition results exposed that CQNP possesses a high amount of arabinose (34.5±0.3) followed by galactose (26.5±0.2), glucose (21.9±0.3), rhamnose (7.0±0.1), mannose (6.0±0.1) and xylose (4.2±0.2). However, after deproteination, a difference was found in the order of the monosaccharide components, with galactose (41.1±0.5) as a major unit followed by arabinose (34.7±0.5), rhamnose (10.9±0.2), glucose (6.6±0.2), mannose (3.4±0.2) and xylose (3.2±0.2). Further, D-CQNP potentially stimulate the RAW 264.7 cells through the production of nitric oxide (NO), upregulating inducible nitric oxide synthase (iNOS) and various pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α). Moreover, stimulation of RAW 264.7 cells by D-CQNP takes place along the NF-κB and the MAPKs signaling pathways through the expression of CD40.
... Recent impedance cytometry work has correlated leukocyte activation to an increase in electrical diameter (Petchakup et al., 2021), but for macrophages that possess a highly migratory phenotype, intercellular interactions can limit the size increase and stress cells to create heterogeneity in their activation response (Nomura et al., 2000). Hence, using impedance phase metrics to detect the stressed subpopulation from intercellular interaction, we optimize macrophage cell density to enable facile formation of the polarized cell morphology under activation (Berghaus et al., 2010), (Taciak et al., 2018). Using multiple frequencies to aggregate the multivariate phenotypic alterations, based on electrical diameter, capacitance of the plasma membrane, and conductivity and permittivity of the cell interior, impedance metrics are identified to quantify macrophage activation by degenerated discs and its reversal by curcumin, with dose and duration dependence. ...
... Wnt5A signaling promotes uptake and survival of common gut commensal bacteria by phagocytes. In order to evaluate the influence of Wnt5A signaling on the cellular uptake and survival of gut commensal bacteria, we initially focused on two common gut commensals, Enterococcus faecalis and Lactobacillus rhamnosus, using the macrophage line RAW264.7 and peritoneal macrophages, which comprise different types of cells, as representative phagocytes (27)(28)(29)(30). The Enterococcus faecalis strain was identified through screening of the mouse cecum-resident bacteria by Wnt5Amediated augmented internalization into macrophages and later validated by 16S sequencing (see Fig. S1A and B in the supplemental material). ...
In light of the demonstrated antagonism of Wnt5A signaling toward the growth of several bacterial pathogens, it was important to study the influence of Wnt5A on gut-resident bacteria and its outcome. Here, we demonstrate that in contrast to inhibiting the survival of the established gut pathogen Salmonella enterica, Wnt5A clearly promotes the survival of the common gut commensals Enterococcus faecalis and Lactobacillus rhamnosus within macrophages through a self-perpetuating Wnt5A-actin axis. A Wnt5A-actin axis furthermore regulates the subsistence of the natural bacterial population of the Peyer's patches, as is evident from the diminution in the countable bacterial CFU therein through the application of Wnt5A signaling and actin assembly inhibitors. Wnt5A dependency of the gut-resident bacterial population is also manifested in the notable difference between the bacterial diversities associated with the feces and Peyer's patches of Wnt5A heterozygous mice, which lack a functional copy of the Wnt5A gene, and their wild-type counterparts. Alterations in the gut commensal bacterial population resulting from either the lack of a copy of the Wnt5A gene or inhibitor-mediated attenuation of Wnt5A signaling are linked with significant differences in cell surface major histocompatibility complex (MHC) II levels and regulatory versus activated CD4 T cells associated with the Peyer's patches. Taken together, our findings reveal the significance of steady state Wnt5A signaling in shaping the gut commensal bacterial population and the T cell repertoire linked to it, thus unveiling a crucial control device for the maintenance of gut bacterial diversity and T cell homeostasis. IMPORTANCE Gut commensal bacterial diversity and T cell homeostasis are crucial entities of the host innate immune network, yet the molecular details of host-directed signaling pathways that sustain the steady state of gut bacterial colonization and T cell activation remain unclear. Here, we describe the protective role of a Wnt5A-actin axis in the survival of several gut bacterial commensals and its necessity in shaping gut bacterial colonization and the associated T cell repertoire. This study opens up new avenues of investigation into the role of the Wnt5A-actin axis in protection of the gut from dysbiosis-related inflammatory disorders.
... It should be noted that RAW 264.7 demonstrated 20 times greater incorporation of EdU than BMDM cells ( Figure 5C), reflecting the difference of the immortalized cell line from the primary BMDM culture. 51 3.5. Effect of Microcapsules on Macrophage Movement. ...
A new promising trend in personalized medicine is the use of autologous cells (macrophages or stem cells) for cell-based therapy and also as a "Trojan horse" for targeted delivery of a drug carrier. The natural ability of macrophages for chemotaxis allows them to deliver cargo to the damaged area, significantly reducing side effects on healthy organ tissues. Therefore, it is important to develop tools to track their behavior in the organism. While labeled containers can serve as anchored tags for imaging macrophages in vivo, they can affect the properties and functions of macrophages. This work demonstrates that 3 μm sized capsules based on biocompatible polyelectrolytes and fluorescently labeled with both Cy7 and RITC dyes do not affect cell functionalization in vitro, such as viability, proliferation, and movement of transformed monocyte/macrophage-like cells (RAW 264.7) and primary bone marrow derived macrophages (BMDM) at maximal loading of five capsules per cell. In addition, capsules allowed fluorescent detection of ex vivo loaded cells 24 h after the tail vein injection in vivo and visualization of microcapsule-laden macrophages ex vivo using confocal microscopy. We have delivered about 62.5% of injected BMDM containing 12.5 million capsules with 3.75 μg of high-molecular-weight cargo (0.3 pg/capsule) to the liver. Our results demonstrate that 3 μm polyelectrolyte fluorescently labeled microcapsules can be used for safe macrophage loading, allowing cell tracking and drug delivery, which will facilitate development of macrophage-based cell therapy protocols.
... The anti-inflammatory action of flavonoids in LPS-stimulated RAW 264.7 cells has been extensively investigated [47]. Previous studies have shown that quercetin inhibits the activation of the phosphorylated tyrosine motif of myeloid differentiation primaryresponse protein 88 (MyD88), thereby inhibiting the activation of molecules acting downstream of MyD88 and limiting LPS-induced inflammatory mediators [48]. ...
In sepsis, the persistence of uncontrolled inflammatory response of infected host cells eventually leads to severe lung and organ failure and, ultimately, death. Carbapenem-resistant Acinetobacter baumannii (CRAB), causative bacteria of sepsis and lung failure in acute cases, belongs to a group of critical pathogens that cannot be eradicated using the currently available antibiotics. This underlines the necessity of developing new modes of therapeutics that can control sepsis at the initial stages. In this study, we investigated the anti-inflammatory activities in vitro and in vivo and the antiseptic effects of rhamnetin, a naturally occurring flavonoid. We found that among its isoforms, the potency of rhamnetin was less explored but rhamnetin possessed superior anti-inflammatory activity with least cytotoxicity. Rhamnetin showed significant anti-inflammatory effects in lipopolysaccharide-, CRAB-, and Escherichia coli (E. coli)-stimulated mouse macrophages by inhibiting the release of interleukin-6 and nitric oxide. In a mouse model of sepsis infected with clinically isolated CRAB or E. coli, rhamnetin significantly reduced the bacterial burden in the organs. In addition, normalized pro-inflammatory cytokine levels in lung lysates and histological analysis of lung tissue indicated alleviation of lung damage. This study implies that a potent natural product such as rhamnetin could be a future therapeutic for treating carbapenem-resistant gram-negative sepsis.
... There are quite differences between RAW264.7 cell lines and primary cells. With continuous culture and passage, phenotypes and functions of the cell lines may change [55]. Therefore, responses observed in a cell line may not reflect normal physiology. ...
Heat shock protein 90β (Hsp90β, encoded by Hsp90ab1 gene) is the most abundant proteins in the cells and contributes to variety of biological processes including metabolism, cell growth and neural functions. However, genetic evidences showing Hsp90β in vivo functions using tissue specific knockout mice are still lacking. Here, we showed that Hsp90β exerted paralogue-specific role in osteoclastogenesis. Using myeloid-specific Hsp90ab1 knockout mice, we provided the first genetic evidence showing the in vivo function of Hsp90β. Hsp90β binds to Ikkβ and reduces its ubiquitylation and proteasomal degradation, thus leading to activated NF-κB signaling. Meanwhile, Hsp90β increases cholesterol biosynthesis by activating Srebp2. Both pathways promote osteoclastogenic genes expression. Genetic deletion of Hsp90ab1 in osteoclast or pharmacological inhibition of Hsp90β alleviates bone loss in ovariectomy-induced mice. Therefore, Hsp90β is a promising druggable target for the treatment of osteoporosis.
... TLR4 is considered a vital sensor of LPS simulation and is essential for the activation of downstream NF-κB and MAPK signaling pathways. Activated TLR4 can initiate downstream cell signaling through MyD88-dependent and MyD88-independent (TRIF-dependent) pathways [28], and the TLR4/MyD88/NF-κB pathway has been reported to be one of the most essential signaling pathways in the regulation of inflammation [29]. Moreover, NF-κB plays a vital role in regulating the transcription of numerous pro-inflammatory mediators and cytokines during the inflammatory process [29,30]. ...
Background:
Benzoylmesaconine (BMA), the most abundant monoester alkaloid in Aconitum plants, has some biological activities and is a potential therapeutic agent for inflammation-related diseases. However, the potential anti-inflammatory mechanisms of BMA have not been clarified.
Purpose:
This study aimed to investigate the underlying molecular mechanisms of the anti-inflammatory action of this compound using lipopolysaccharide (LPS)-activated RAW264.7 macrophages.
Methods:
The release of pro-inflammatory cytokines and mediators were detected by nitric oxide (NO) assays, reactive oxygen species (ROS) assays, and enzyme-linked immunosorbent assays (ELISA) in LPS-activated RAW264.7 macrophage cells. Quantitative real-time PCR was used to measure the gene expression of interleukin (IL)-1β, tumor necrosis factor (TNF)-α, IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2). Cell viability was determined using a cell counting kit-8 (CCK-8) assay. The expression of iNOS, COX-2, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB)-related proteins were detected by western blot, and nuclear translocation of p65 was observed by immunofluorescence.
Results:
BMA significantly decreased the production of IL-1β, IL-6, TNF-α, PGE2, NO, and ROS and inhibited the protein and mRNA levels of COX-2 and iNOS in LPS-activated RAW264.7 macrophages. Moreover, LPS-induced phosphorylation of IκBα, JNK, p38, and ERK; degradation of IκBα; and nuclear translocation of p65 were significantly suppressed by BMA treatment.
Conclusion:
These findings demonstrate that the anti-inflammatory effect of BMA was through the suppression of the NF-κB and MAPK signaling pathways and that it may be a therapeutic agent targeting specific signal transduction events required for inflammation-related diseases.
... The aporphine alkaloid magnoflorine exhibited anti-inflammatory activity in LPS-activated human THP-1 macrophages by inactivating the MyD88/NF-κB pathway (Zhao et al. 2021). In terms of the response patterns to microbial ligands, surface markers, and functional characteristics, RAW264.7 and THP-1 macrophages closely mimic primary human macrophages and have been used to explore the immunomodulatory effects of various compounds or drugs (Berghaus et al. 2010;Chanput et al. 2014). Previously, we reported that (-)-stephanine and dehydrostephanine isolated from S. venosa tubers can reduce LPS-activated inflammatory cytokine production in murine macrophages (Chulrik et al. 2020). ...
Plant-derived medicinal compounds are increasingly being used to treat acute and chronic inflammatory diseases, which are generally caused by aberrant inflammatory responses. Stephania pierrei Diels, also known as Sabu-lueat in Thai, is a traditional medicinal plant that is used as a remedy for several inflammatory disorders. Since aporphine alkaloids isolated from S. pierrei tubers exhibit diverse pharmacological characteristics, we aimed to determine the anti-inflammatory effects of crude extracts and alkaloids isolated from S. pierrei tubers against lipopolysaccharide (LPS)-activated RAW264.7 macrophages. Notably, the n-hexane extract strongly suppressed nitric oxide (NO) while exhibiting reduced cytotoxicity. Among the five alkaloids isolated from the n-hexane extract, the aporphine alkaloid oxocrebanine exerted considerable anti-inflammatory effects by inhibiting NO secretion. Oxocrebanine also significantly suppressed prostaglandin E2, tumour necrosis factor-α, interleukin (IL)-1β, IL-6, inducible nitric oxide synthase, and cyclooxygenase (COX)-2 protein expression by inactivating the nuclear factor κB, c-Jun NH2-terminal kinase, extracellular signal-regulated kinase 1/2, and phosphatidylinositol 3-kinase/Akt inflammatory signalling pathways. Molecular docking analysis further revealed that oxocrebanine has a higher affinity for toll-like receptor 4/myeloid differentiation primary response 88 signalling targets and the COX-2 protein than native ligands. Thus, our findings highlight the potential anti-inflammatory effects of oxocrebanine and suggest that certain alkaloids of S. pierrei could be used to treat inflammatory diseases.
... Recent impedance cytometry work has correlated leukocyte activation to an increase in electrical diameter (Petchakup et al., 2021), but for macrophages that possess a highly migratory phenotype, intercellular interactions can limit the size increase and stress cells to create heterogeneity in their activation response (Nomura et al., 2000). Hence, using impedance phase metrics to detect the stressed subpopulation from intercellular interaction, we optimize macrophage cell density to enable facile formation of the polarized cell morphology under activation (Berghaus et al., 2010), (Taciak et al., 2018). Using multiple frequencies to aggregate the multivariate phenotypic alterations, based on electrical diameter, capacitance of the plasma membrane, and conductivity and permittivity of the cell interior, impedance metrics are identified to quantify macrophage activation by degenerated discs and its reversal by curcumin, with dose and duration dependence. ...
Measurement of macrophage activation and its modulation for immune regulation is of great interest to arrest inflammatory responses associated with degeneration of intervertebral discs that cause chronic back pain, and with transplants that face immune rejection. Due to the phenotypic plasticity of macrophages that serve multiple immune functions, the net disease outcome is determined by a balance of subpopulations with competing functions, highlighting the need for single-cell methods to quantify heterogeneity in their activation phenotypes. However, since macrophage activation can follow several signaling pathways, cytometry after fluorescent staining of markers with antibodies does not often provide dose-dependent information on activation dynamics. We present high throughput single-cell impedance cytometry for multiparametric measurement of biophysical changes to individual macrophages for quantifying activation in a dose and duration dependent manner, without relying on a particular signaling pathway. Impedance phase metrics measured at two frequencies and the electrical diameter from impedance magnitude at lower frequencies are used in tandem to benchmark macrophage activation by degenerated discs against that from lipopolysaccharide stimulation at varying dose and duration levels, so that reversal of the activation state by curcumin can be ascertained. This label-free single-cell measurement method can form the basis for platforms to screen therapies for inflammation, thereby addressing the chronic problem of back pain.
... Despite the widespread use of these cells in numerous fields of study, the authors concluded that when utilizing RAW 246.7 cells, caution should be exercised when interpreting the data due to alterations during continuous culture. Various cross-reactions should also be considered when merging human and mouse cells (e.g., co-culture of Caco-2/RAW 246.7) [143]. ...
Inflammatory bowel disease (IBD) is a chronic, relapsing gastrointestinal condition. Ulcerative colitis and Crohn’s disease are types of inflammatory bowel disease. Over many decades, the disease has been a topic of study, with experts still trying to figure out its cause and pathology. Researchers have established many in vivo animal models, in vitro cell lines, and ex vivo systems to understand its cause ultimately and adequately identify a therapy. However, in vivo animal models cannot be regarded as good models for studying IBD since they cannot completely simulate the disease. Furthermore, because species differences are a crucial subject of concern, in vitro cell lines and ex vivo systems can be employed to recreate the condition properly. In vitro models serve as the starting point for biological and medical research. Ex vivo and in vitro models for replicating gut physiology have been developed. This review aims to present a clear understanding of several in vitro and ex vivo models of IBD and provide insights into their benefits and limits and their value in understanding intestinal physiology.
... To investigate how the S100a9/Cxcl12 signaling affect myeloid cells, we cultured cells of RAW264.7 cell line, a murine leukemic monocyte/macrophage cell line 43 , with various types of conditioned medium (CM) for 48 h and monitored the proliferation, migration, and molecular actions of the macrophages. While CM from WT mammary epithelial cells (B477) had very little effect, CM from MT mammary epithelial cells (G600) significantly increased the colony size and migration of RAW264.7 cells. ...
Immune checkpoint blockade (ICB) is a powerful approach for cancer therapy although good responses are only observed in a fraction of cancer patients. Breast cancers caused by deficiency of breast cancer-associated gene 1 (BRCA1) do not have an improved response to the treatment. To investigate this, here we analyze BRCA1 mutant mammary tissues and tumors derived from both BRCA1 mutant mouse models and human xenograft models to identify intrinsic determinants governing tumor progression and ICB responses. We show that BRCA1 deficiency activates S100A9-CXCL12 signaling for cancer progression and triggers the expansion and accumulation of myeloid-derived suppressor cells (MDSCs), creating a tumor-permissive microenvironment and rendering cancers insensitive to ICB. These oncogenic actions can be effectively suppressed by the combinatory treatment of inhibitors for S100A9-CXCL12 signaling with αPD-1 antibody. This study provides a selective strategy for effective immunotherapy in patients with elevated S100A9 and/or CXCL12 protein levels.
... While these findings suggest a role for uc.48+ in promoting macrophage-mediated inflammation under diabetic conditions, the underlying molecular mechanism remains to be elucidated [149]. Similarly, the studies for the other two lncRNAs, Dnm3os and Lethe, characterized their inflammatory effects in RAW264.7 cells, which are not necessarily representative of the primary macrophages contributing to insulitis and β-cell destruction [150]. Further investigation of these three lncRNAs, ideally using in vivo T1D models or human cadaveric pancreatic samples, are required to validate their functional effects on macrophagemediated inflammation during T1D pathogenesis. ...
Type 1 diabetes (T1D) is an autoimmune disease in which immune cells mediate the specific destruction of the insulin-producing β cells in the pancreatic islets. Genetic and transcriptome studies for T1D indicate that a relatively large number of long noncoding RNAs (lncRNAs), detected in both immune cells and β cells, contribute to the underlying inflammation and autoimmune pathology. Although lncRNAs do not encode proteins, their biochemical versatility as RNA molecules enables them to interact with proteins, DNA or RNA to exert regulatory effects on various cellular processes. Recent studies have begun to determine these effects for a small number of lncRNAs in modulating specific immune cell and β-cell responses to elevated glucose levels and pro-inflammatory cytokines that are present within the islets during T1D pathogenesis. These findings are reviewed here and highlight the potential for different lncRNAs to act in concert to inhibit or exacerbate inflammatory and autoimmune responses. Despite this progress to date, additional investigations are required for a more in-depth understanding of their individual functional roles in this interplay, as well as identifying which lncRNAs are likely diagnostic biomarkers or therapeutic targets for autoimmune diseases such as T1D.
... Our nanofibers functionalized with the natural cell membranes possess cell surface antigens, which may initiate immune response to different extent. The RAW 264.7 cells more precisely resemble BMDMs and P-macrophages related to the existence of surface proteins and reaction to microbial ligands that instigate cellular trigger via Toll-like receptors 3 and 4 [59] . In order to evaluate the macrophage polarization, the release profiles of cytokines and gene expression of macrophages on the nanofibers functionalized with macrophage cell membranes were evaluated. ...
Immunomodulation is an important phenomenon in the normal mammalian host response toward an injury, and plays a critical role in tissue regeneration and regenerative medicine. Different phenotypes of macrophages show an array of activation states compassing pro-inflammatory to pro-alleviating cells, which are the critical players to modulate immune response and tissue regeneration. In this study, macrophage membranes of different phenotypes (macrophages (M0), classically activated macrophages (M1) and alternatively activated macrophages (M2)) were coated onto poly-ε-caprolactone (PCL) nanofibers to acquire exterior surface proteins and similar functions of the natural membranes. In vitro results unveiled that these nanofibers, especially the M2-PCL nanofibers, can suppress the activities of inflammatory markers such as TNF-α and IL-1β, and stimulate anti-inflammatory markers such as Arg-1, IL-10 and TGF-β. In a C57BL/6 mouse model, the macrophage membrane-coated nanofibers, especially the M2-PCL nanofibers, displayed minimal cellular infiltration and low collagen deposition, increased anti-inflammatory CD206 and decreased inflammatory CD86 levels. The M2-PCL nanofibers most effectively neutralized inflammatory chemokines, regulated the expression of inflammation-associated genes as well as anti-inflammatory genes, and showed strong immunomodulatory effects than the PCL, M0-PCL and M1-PCL nanofibers.
Statement of Significance
Different types of macrophage membrane-functionalized PCL nanofibers were successfully prepared and well characterized. They inherited the surface proteins imitating the source macrophages, and played an important role in limiting cellular infiltration and collagen deposition. These different macrophages and their membrane-coated nanofibers (M0-PCL, M1-PCL and M2-PCL) behaved like their respective source cells. The M2 mimicking M2-PCL nanofibers effectively polarized macrophages to M2 phenotype and decreased the expression of inflammation-associated chemokines and promoted the anti-inflammation in vitro and in vivo, which is critical for tissue regeneration. The mice implanted with the bio-mimicking M2-PCL nanofibers effectively inhibited toll like receptors signaling induced NF-kB and IRF-5 and their target genes such as Edn-1, IL-6, iNOS, TNF-α, etc. compared to the PCL, and M0-PCL and M1-PCL macrophage membrane-coated nanofibers.
... Moreover, the activity of D-CQNP more or less similar to the positive control (LPS). This result was agreed in previous studies, where CD11b and CD40 expression were found to be higher when macrophages were activated by microfibers [44]. Furthermore, the expression of CD40 was higher than CD11b, which demonstrates the stimulation of RAW 264.7 Fig. 5 The protein expression of p-p38, p-JNK, p-ERK, and p-p65 in CQNP and D-CQNP treated RAW 264.7 cells. ...
Non-starch polysaccharides derived from natural resources play a significant role in the field of food science and human health due to their extensive distribution in nature and less toxicity. In this order, the immunostimulatory activity of a non-starch polysaccharide (CQNP) from Chenopodium quinoa was examined before and after deproteination in murine macrophage RAW 264.7 cells. The chemical composition of CQNP and deproteinated-CQNP (D-CQNP) were spectrometrically analysed that revealed the presence of carbohydrate (22.7 ± 0.8% and 39.5 ± 0.8%), protein (41.4 ± 0.5% and 20.8 ± 0.5%) and uronic acid (8.7 ± 0.3% and 6.7 ± 0.2%). The monosaccharide composition results exposed that CQNP possesses a high amount of arabinose (34.5 ± 0.3) followed by galactose (26.5 ± 0.2), glucose (21.9 ± 0.3), rhamnose (7.0 ± 0.1), mannose (6.0 ± 0.1) and xylose (4.2 ± 0.2). However, after deproteination, a difference was found in the order of the monosaccharide components, with galactose (41.1 ± 0.5) as a major unit followed by arabinose (34.7 ± 0.5), rhamnose (10.9 ± 0.2), glucose (6.6 ± 0.2), mannose (3.4 ± 0.2) and xylose (3.2 ± 0.2). Further, D-CQNP potentially stimulate the RAW 264.7 cells through the production of nitric oxide (NO), upregulating inducible nitric oxide synthase (iNOS) and various pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α). Moreover, stimulation of RAW 264.7 cells by D-CQNP takes place along the NF-κB and the MAPKs signaling pathways through the expression of cluster of differentiation 40 (CD40). This results demonstrate that RAW 264.7 cells are effectively stimulated after removal of the protein content in C. quinoa non-starch polysaccharides, which could be useful for develop a new immunostimulant agent.
Graphical Abstract
... Incubation with free cholesterol caused structural alterations in macrophages. Immortalized mouse macrophage cells RAW246.7 exhibited similar phenotype and responses to microbial ligands that bind to either Toll-like receptors 2, 3, and 4 to that of primary bone marrow-derived macrophages [22], thus utilized in this study to examine the macrophage properties and functions. Previous studies reported that cell viability and inflammatory phenotypes triggered by the cholesterol enriched environment were concentration-dependent and time-dependent in macrophages [18]. ...
Hypercholesterolemia induces intracellular accumulation of cholesterol in macrophages and other immune cells, causing immunological dysfunctions. On cellular levels, cholesterol enrichment might lead to mitochondrial metabolic reprogramming and change macrophage functions. Additionally, as cholesterol is permeable to the plasma membrane and might integrate into the membranous organelles, such as endoplasmic reticulum or mitochondria, cholesterol enrichment might change the functions or properties of these organelles, and ultimately alters the cellular functions. In this study, we investigate the mitochondrial alterations and intracellular oxidative stress induced by accumulation of cholesterol in the macrophages, and the possible immunological impacts caused by these alterations. Macrophage cells RAW264.7 were treated with cholesterol to induce intracellular accumulation of cholesterol, which further triggered the reduced production of reactive oxygen/nitrogen species, as well as decrease of oxidative phosphorylation. Basal respiration rate, ATP production and non-mitochondrial oxygen consumption are all suppressed. In contrast, glycolysis remained unaltered in this cholesterol-enriched condition. Previous studies demonstrated that metabolic profiles are associated with macrophage polarization. We further verified whether this metabolic reprogramming influences the macrophage responses to pro-inflammatory or anti-inflammatory stimuli. Our results showed the changes of transcriptional regulations in both pro-inflammatory and anti-inflammatory genes, but not specific toward M1 or M2 polarization. Collectively, the accumulation of cholesterol induced mitochondrial metabolic reprogramming and suppressed the production of oxidative stress, and induced the alterations of macrophage functions.
... Whereas, CD40 acts as a co-stimulatory molecule that stimulates the production of IL-2 and TNF-α via T-cell activation [51,52]. According to previous studies, macrophages are activated by microbial ligands, which possibly increases the expression of CD11b and CD40 [53]. In this study, the expression of CD11b and CD40 was determined as 37.55% and 89.87%, respectively, when RAW 264.7 cells were treated with LPS (Fig. 6). ...
The structural elucidation and investigation of the immunostimulatory effects of crude polysaccharides (ECPs), non-starch polysaccharides (ECPs-I), and deproteinated non-starch polysaccharides (ECPs-II) derived from finger millet (Eleusine coracana) were performed. The extracted ECPs, ECPs-I, and ECPs-II primarily comprised different levels of carbohydrate (82.9, 41.9 and 62.0 %), protein (5.3, 38.4 and 22.0 %), and uronic acid (0.5, 2.4 and 0.7 %). Monosaccharide composition analysis showed that glucose (88.2 and 40.6) was the main sugar unit present in ECPs and ECPs-I, followed by galactose, arabinose, xylose, rhamnose, and mannose; however, ECPs-II contained galactose (42.2 %) as a major unit. The average molecular weights of ECPs, ECPs-I, and ECPs-II were found to be 120, 78 and 43 kDa, respectively. The polysaccharides as well as non-starch polysaccharides did not show any toxic effects on RAW 264.7 cells. ECPs-II treatment significantly enhanced the activation of RAW 264.7 cells by inducing nitric oxide (NO) production, upregulating inducible nitric oxide synthase (iNOS) and various pro-inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor alpha (TNF-α). Furthermore, the RAW 264.7 cells were activated after ECPs-II treatment via the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and mitogen-activated protein kinase signaling (MAPK) pathways induced by a cluster of differentiation 40 (CD40) expression. The structure of ECPs-II mainly consists of (1→3,4)-linked galactopyranosyl and (1→3,5) arabinopyranosyl.
... Until now, five adaptor proteins have been identified, namely, TIRAP, MyD88, TRAM, TRIF, and SARM [4]. Cell signaling is initiated by activated TLR4 via MyD88-dependent or MyD88-independent pathways to synthesize several downstream products [29]. The NF-κB family, which consists of NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel proteins, was previously shown to be closely related to various human diseases and plays a crucial role in inflammatory processes [30]. ...
Previous studies have shown that benzoylaconine (BAC), a representative monoester alkaloid, has a potential anti-inflammatory effect. This study investigated the underlying molecular mechanisms using the mode of LPS-activated RAW264.7 macrophage cells. Our findings showed that BAC significantly suppressed the release of pro-inflammatory cytokines and mediators, including IL-6, TNF-α, IL-1β, ROS, NO, and PGE2. BAC treatment also effectively downregulated the elevated protein levels of iNOS and COX-2 induced by LPS in a dose-dependent manner. In this study, we found that BAC inhibited LPS-induced NF-κB activation by reducing the phosphorylation and degradation of IκBα by western blotting and blocking the nuclear translocation of p65 using an immunofluorescence assay. The elevated protein levels of JNK, p38, and ERK phosphorylation after LPS stimulation were restored effectively by BAC treatment. The protein expression of Toll-like receptor 4 (TLR4) and LPS-induced phosphorylation of TAK1, which is a crucial upstream regulatory factor of TLR-induced MAPK and NF-κB signaling, were inhibited by BAC in activated RAW264.7 macrophages. Moreover, BAC decreased the levels of TAK1 phosphorylation and pro-inflammatory cytokines and mediators associated with MAPK and NF-κB activation, similar to TLR4 inhibitor TAK-242. These findings demonstrated that BAC exhibited an anti-inflammatory effect by the inhibition of TLR-induced MAPK and NF-κB pathways, indicating that it could potentially be used for treating inflammatory diseases.
... Cellular uptake of antigens by innate immune cells provides antigen-processing and subsequent costimulatory signals that are crucial to trigger acquired immune responses, especially for low immunogenic peptide antigens. Macrophage-like RAW 264.7 cells (47) are often used to study cellular responses to microbes and their products (48). We selected this cell model to assess in vitro cellular internalization of our nanoformulations, using 1 mg DMSNs-57 and 1 mg DMSNs-156 loaded with 200 μg B 2 T labeled with a dye (i.e., fluoro-B 2 T) (see section §SI-2 and Figure SI-10). ...
Mesoporous silica nanoparticles have drawn increasing attention as promising candidates in vaccine delivery. Previous studies evaluating silica-based vaccine delivery systems concentrated largely on macromolecular antigens, such as inactivated whole viruses. In this study, we synthesized dendritic mesoporous silica nanoparticles (DMSNs), and we evaluated their effectiveness as delivery platforms for peptide-based subunit vaccines. We encapsulated and tested in vivo an earlier reported foot-and-mouth disease virus (FMDV) peptide vaccine (B2T). The B2T@DMSNs formulation contained the peptide vaccine and the DMSNs without further need of other compounds neither adjuvants nor emulsions. We measured in vitro a sustained release up to 930 h. B2T@DMSNs-57 and B2T@DMSNs-156 released 23.7% (135 µg) and 22.8% (132 µg) of the total B2T. The formation of a corona of serum proteins around the DMSNs increased the B2T release up to 61% (348 µg/mg) and 80% (464 µg/mg) for B2T@DMSNs-57 and B2T@DMSNs-156. In vitro results point out to a longer sustained release, assisted by the formation of a protein corona around DMSNs, compared to the reference formulation (i.e., B2T emulsified in Montanide). We further confirmed in vivo immunogenicity of B2T@DMSNs in a particle size-dependent manner. Since B2T@DMSNs elicited specific immune responses in mice with high IgG production like the reference B2T@Montanide™, self-adjuvant properties of the DMSNs could be ascribed. Our results display DMSNs as efficacious nanocarriers for peptide-based vaccine administration.
... HaCaT and HDF were chosen because they are the representative cells used in many applications, including wound healing [34][35][36][37][38]. RAW264.7 is the most commonly used cell line as in vitro model system evaluating the anti-inflammatory activity and safety of natural compound [39][40][41]. This macrophage generates robust inflammatory responses when challenged by an inflammatory stimulant, including lipopolysaccharide (LPS) [42,43]. To assess the cytotoxicity of Cabex and Rabex, HaCaT, HDF, and RAW264.7 cells were treated with 1 × 10 11 and 2 × 10 11 particles mL − 1 of Cabex and Rabex, respectively, and cell viability was measured 72 h after treatment. ...
There are extensive studies on the applications of extracellular vesicles (EVs) produced in cell culture for therapeutic drug development. However, large quantities of EVs are needed for in vivo applications, which requires high production costs and time. Thus, the development of new EV sources is essential to facilitate their use. Accordingly, plant-derived exosome-like nanovesicles are an emerging alternative for culture-derived EVs. Until now, however, few studies have explored their biological functions and uses. Therefore, it is necessary to elucidate biological activities of plant-derived exosome-like nanovesicles and harness vesicles for biomedical applications. Herein, cabbage and red cabbage were used as nanovesicle sources owing to their easy cultivation. First, an efficient method for nanovesicle isolation from cabbage (Cabex) and red cabbage (Rabex) was developed. Furthermore, isolated nanovesicles were characterized, and their biological functions were assessed. Both Cabex and Rabex promoted mammalian cell proliferation and, interestingly, suppressed inflammation in immune cells and apoptosis in human keratinocytes and fibroblasts. Finally, therapeutic drugs were encapsulated in Cabex or Rabex and successfully delivered to human cells, demonstrating the potential of these vesicles as alternative drug delivery vehicles. Overall, the current results provide strong evidence for the wide application of Cabex and Rabex as novel therapeutic biomaterials.
... It appeared that the expression levels of IL-6 and IFN-γ were below detection limit in resting cells, whereas IL-1β and more evidently TNF-α appeared to be expressed at basal levels (Table S1). Given that stimulation with lipopolysaccharide (LPS) is known to produce a strong inflammatory response in RAW 264.7 cells [60], we first measured the expression levels of immune-related targets (IL-1β, IL-6, TNF-α, TGF-β1, CXCL2) in cells exposed to LPS (100 ng/mL) and we detected a strong upregulation following a 6 h exposure to LPS that, albeit reduced, was still present after a 24 h exposure to the endotoxin, with the sole exception of IFN-γ that remained undetected ( Figure S1 and Supplementary Results 1). ...
Carnosine (β-alanyl-L-histidine) is a naturally occurring endogenous peptide widely distributed in excitable tissues such as the brain. This dipeptide has well-known antioxidant, anti-inflammatory, and anti-aggregation activities, and it may be useful for treatment of neurodegenerative disorders such as Alzheimer’s disease (AD). In this disease, peripheral infiltrating macrophages play a substantial role in the clearance of amyloid beta (Aβ) peptides from the brain. Correspondingly, in patients suffering from AD, defects in the capacity of peripheral macrophages to engulf Aβ have been reported. The effects of carnosine on macrophages and oxidative stress associated with AD are consequently of substantial interest for drug discovery in this field. In the present work, a model of stress induced by Aβ1-42 oligomers was investigated using a combination of methods including trypan blue exclusion, microchip electrophoresis with laser-induced fluorescence, flow cytometry, fluorescence microscopy, and high-throughput quantitative real-time PCR. These assays were used to assess the ability of carnosine to protect macrophage cells, modulate oxidative stress, and profile the expression of genes related to inflammation and pro- and antioxidant systems. We found that pre-treatment of RAW 264.7 macrophages with carnosine counteracted cell death and apoptosis induced by Aβ1-42 oligomers by decreasing oxidative stress as measured by levels of intracellular nitric oxide (NO)/reactive oxygen species (ROS) and production of peroxynitrite. This protective activity of carnosine was not mediated by modulation of the canonical inflammatory pathway but instead can be explained by the well-known antioxidant and free-radical scavenging activities of carnosine, enhanced macrophage phagocytic activity, and the rescue of fractalkine receptor CX3CR1. These new findings obtained with macrophages challenged with Aβ1-42 oligomers, along with the well-known multimodal mechanism of action of carnosine in vitro and in vivo, substantiate the therapeutic potential of this dipeptide in the context of AD pathology.
... RAW 264.7 cells are a murine-leukemic macrophage-like cell line that has been widely used to study macrophage-biomaterial interactions [134][135][136][137] . While RAW 264.7 cells phenotypically resemble bone-marrowderived primary macrophages in some markers like CD11b, CD11c, CD14, F4/80 and in their response to microbial molecules through TLR3 and TLR4, they do not mimic the phenotypic markers and microbial response of splenic macrophages [138] . There are also significant differences in cell surface receptors between bone marrow derived macrophages and common macrophage-like immortalized cell lines (IC-21, J774A.1, and RAW 264.7) in response to polystyrene, poly-l-lactic acid (PLLA), and Teflon-AF [139] . ...
As the focus of implantable biomaterials has shifted from bioinert implants to bioactive designs, recent research has highlighted the complex interactions between cell physiologic systems and material properties, particularly physical cues. From the cells known to interact with implanted biomaterials, the response of the immune system has been a critical target of study recently. Here, we review studies characterizing the response of innate immune cells to various material cues, particularly of those at the surface of implanted materials.
The innate immune system consists of cell types with various roles in inflammation. Neutrophils and macrophages serve both phagocytic and signaling roles, especially early in the inflammatory phase of biomaterial implantation. These cell types ultimately dictate the outcome of implants as chronic inflammation, fibrosis, or integration. Other cell types like dendritic cells, mast cells, natural killer cells, and innate lymphoid cells, may also serve an immunomodulatory role in the biomaterial context. This review highlights recent advances in our understanding of the role of innate immunity in the response to implantable biomaterials as well as key mechanobiological findings in innate immune cells underpinning these advances.
... Here, we utilized a murine-leukemic monocyte-macrophage cell line, RAW264.7, which most closely mimic BMDMs (Berghaus et al., 2010). RAW264.7 cells pretreated with FPR2-neutralizing antibody (Fig. 1A) showed significantly attenuated internalization of bacteria 0 and 30 min postinfection compared with its uptake by the control cells. ...
Here, we explore the potential role of formyl peptide receptor 2 (FPR2) during Brucella abortus infection. FPR2 manipulation affected B. abortus internalization but not its growth within macrophages. During the activation of FPR2 induced by its agonist AGP-8694, a high level of Brucella uptake was accompanied by an increase in ERK phosphorylation, while intracellular survival at 24 h postincubation was observed to be associated with slightly reduced nitrite accumulation but augmented superoxide anion production. Attenuated secretion of IL-6 and IL-10 were observed 48 h postincubation in the bone marrow-derived macrophages (BMDMs) treated with the FPR2 antagonist WRW4. An opposite pattern of bacterial uptake was observed upon treatment with the FPR2 antagonist, but no significant changes in the activation of MAPKs or the production of nitrite or superoxide anion were observed. Interestingly, AGP-8694 treatment of mice did not lead to differences in spleen or liver weight but slightly enhanced bacterial proliferation was observed in the spleen. Although the weights of the spleen or liver did not differ, WRW4 treatment led to reduced bacterial proliferation in the spleen. Furthermore, FPR2 antagonist treatment was associated with high serum levels of the proinflammatory cytokines IL-12, TNF-α, IFN-γ and MCP-1, while the production of TNF-α was inhibited in AGP-8694-treated mice. IL-6 and IL-10 levels were slightly increased in AGP-8694-treated mice at 24 h postinfection. Our findings demonstrated the contribution of FPR2 via manipulating this receptor using its reported agonist AGP-8694 and antagonist WRW4 in both in vitro and in vivo systems. Although activation of the receptor did not consistently induced Brucella infection, FPR2 inhibition may be a promising strategy to treat brucellosis in animals which encourages further investigation.
... Until now, ve adaptor proteins have been identi ed, namely TIRAP, MyD88, TRAM, TRIF, and SARM [4]. Cell-signaling is initiated by activated TLR4 via MyD88-dependent or independent pathways to synthesize several downstream products [29]. The NF-κB family, which consists of NF-κB1 (p50), NF-κB2 (p52), RelA (p65), RelB, and c-Rel proteins, was previously shown to be closely related to various human diseases and plays a crucial role in in ammatory processes [30]. ...
Previous studies have shown that benzoylaconine (BAC), a representative monoester alkaloid, has a potential anti-inflammatory effect. This study investigated the underlying molecular mechanisms using the mode of LPS-activated RAW264.7 macrophage cells. Our findings showed that BAC significantly suppressed the release of pro-inflammatory cytokines and mediators, including IL-6, TNF-α, IL-1β, ROS, NO, and PGE 2 . BAC treatment also effectively downregulated the elevated protein levels of iNOS and COX-2 induced by LPS in a dose-dependent manner. In this study, we found that BAC inhibited LPS-induced NF-κB activation by reducing the phosphorylation and degradation of IκBα by Western blotting and blocking the nuclear translocation of p65 using an immunofluorescence assay. The elevated protein levels of JNK, p38, and ERK phosphorylation after LPS stimulation were restored effectively by BAC treatment. Moreover, LPS-induced phosphorylation of TAK1, which is a crucial upstream regulatory factor of Toll-like receptor-induced MAPK and NF-κB signaling, was inhibited by BAC in activated RAW264.7 macrophages. These findings demonstrated that BAC exhibited an anti-inflammatory effect by inhibition of Toll-like receptor-induced MAPK and NF-κB pathways, indicating that it could potentially be used for treating inflammatory diseases.
The hallmark characteristic of macrophages lies in their inherent plasticity, allowing them to adapt to dynamic microenvironments. Leishmania strategically modulates the phenotypic plasticity of macrophages, creating a favorable environment for intracellular survival and persistent infection through regulatory cytokine such as interleukin (IL)-10. Nevertheless, these effector cells can counteract infection by modulating crucial cytokines like IL-12 and key components involved in its production. Using sophisticated tool of single-cell assay for transposase accessible chromatin (ATAC) sequencing, we systematically examined the regulatory axis of IL-10 and IL-12 in a time-dependent manner during Leishmania major infection in macrophages Our analysis revealed the cellular heterogeneity post-infection with the regulators of IL-10 and IL-12, unveiling a reciprocal relationship between these cytokines. Notably, our significant findings highlighted the presence of sleepy macrophages and their pivotal role in mediating reciprocity between IL-10 and IL-12. To summarize, the roles of cytokine expression, transcription factors, cell cycle, and epigenetics of host cell machinery were vital in identification of sleepy macrophages, which is a transient state where transcription factors controlled the epigenetic remodeling and expression of genes involved in pro-inflammatory cytokine expression and recruitment of immune cells.
IMPORTANCE
Leishmaniasis is an endemic affecting 99 countries and territories globally, as outlined in the 2022 World Health Organization report. The disease’s severity is compounded by compromised host immune systems, emphasizing the pivotal role of the interplay between parasite and host immune factors in disease regulation. In instances of cutaneous leishmaniasis induced by L. major , macrophages function as sentinel cells. Our findings indicate that the plasticity and phenotype of macrophages can be modulated to express a cytokine profile involving IL-10 and IL-12, mediated by the regulation of transcription factors and their target genes post -L. major infection in macrophages. Employing sophisticated methodologies such as single-cell ATAC sequencing and computational genomics, we have identified a distinctive subset of macrophages termed “sleepy macrophages.” These macrophages exhibit downregulated housekeeping genes while expressing a unique set of variable features. This data set constitutes a valuable resource for comprehending the intricate host-parasite interplay during L. major infection.
Bone regeneration is complex and involves multiple cells and systems, with macrophage-mediated immune regulation being critical for the development and regulation of inflammation, angiogenesis, and osteogenesis. Biomaterials with modified physical and chemical properties (e.g., modified wettability and morphology) effectively regulate macrophage polarization. This study proposes a novel approach to macrophage-polarization induction and -metabolism regulation through selenium (Se) doping. We synthesized Se-doped mesoporous bioactive glass (Se-MBG) and demonstrated its macrophage-polarization regulation toward M2 and its enhancement of the macrophage oxidative phosphorylation metabolism. The underlying mechanism is the effective scavenging of excessive intracellular reactive oxygen species (ROS) by the Se-MBG extracts through the promotion of peroxide-scavenging enzyme glutathione peroxidase 4 expression in the macrophages; this, in turn, improves the mitochondrial function. Printed Se-MBG scaffolds were implanted into rats with critical-sized skull defects to evaluate their immunomodulatory and bone regeneration capacity in vivo. The Se-MBG scaffolds demonstrated excellent immunomodulatory function and robust bone regeneration capacity. Macrophage depletion with clodronate liposomes impaired the Se-MBG-scaffold bone regeneration effect. Se-mediated immunomodulation, which targets ROS scavenging to regulate macrophage metabolic profiles and mitochondrial function, is a promising concept for future effective biomaterials for bone regeneration and immunomodulation.
Background:
Imperatorin (IMP) is a secondary metabolite of plants and is the most abundant in Angelica dahurica. Previously studies showed that IMP exhibited anti-inflammatory activity in RAW264.7 cell line. Here, we aim to investigate the roles and mechanisms of IMP in bone marrow derived macrophages (BMDMs), in view of the difference between primary macrophages and cell line.
Methods:
BMDMs were stimulated with LPS for the inflammation model. Flow cytometry was performed with BMDMs treated with different doses of IMP (0-20mg/L) within staining Annexin V-APC for 5 minutes. The cytokines and inflammatory mediators were detected by RT-PCR or ELISA. RNA-seq was performed in IMP-treated BMDMs or control, stimulated with LPS for 6h. Western blotting is carried out to determine the phosphorylation of p65, ERK1/2, JNK1, p38 and Akt.
Results:
Our results showed that IMP inhibited IL-12p40, IL-6, TNFα and IL-1β in LPS-stimulated BMDMs. RNA-seq analysis suggested that IMP inhibits Toll-like receptor signaling pathway (KEGG), TNF signaling pathway (KEGG), NF-κB signaling pathway (KEGG), Inflammatory Response (GO). In addition, IMP inhibited myd88, tpl2, cxcl1, ptgs2(COX-2) expression in mRNA level. Finally, we found decreased phosphorylation of NF-κB p65 in IMP-treated BMDMs, after stimulated with LPS.
Conclusion:
IMP inhibits IL-12p40, IL-6, TNFα and IL-1β expression in LPS-stimulated BMDMs. IMP inhibits macrophages activation, which maybe resulted in decreased phosphorylation of NF-κB p65. Furthermore, IMP may protect against the progress of inflammatory-related diseases.
Drug delivery by innovative nanoparticles into osteogenesis-associated cells has excellent potential for periodontitis therapy. However, a deep understanding of how the nanoparticles boost the bioactivity of delivered drugs is lacking. In this study, we evaluated the potential of echinacoside (ECH), a phenolic chemical, for periodontitis therapy by investigating how it regulates macrophages, human periodontal ligament cells (hPDLCs), and osteoclasts. Furthermore, we created ECH-based poly(-caprolactone) (PCL-ECH) nanoparticles to compare their efficacy to that of free ECH. In vitro data showed that the ECH significantly decreased the inflammatory responses in lipopolysaccharide-stimulated RAW264.7 macrophages but upregulated osteogenic factor BMP-2 expression. By modulating their crosstalk with macrophages and inhibiting osteoclast activities, ECH promoted inflammation-induced osteogenic differentiation of hPDLCs. Compared with the free ECH, the PCL-ECH nanoparticles enhanced the osteogenic differentiation of the hPDLCs by inducing ECH-modulated macrophage-hPDLC crosstalk. However, the bioactivities of the nanoparticles were comparable to the free ECH only in the macrophages or osteoclasts. The PCL-ECH nanoparticles induced more advanced alveolar bone remodeling (bone mineral density) than the free ECH In a ligature-induced periodontitis mouse model. This study provides deep insights into the nano-bio effect of ECH-based nanoparticles to investigate ways of promoting robust periodontitis therapy.
Propolis is a resinous substance extracted from the plants around the honeycomb, which can be used to construct, adjust and protect the bee colony. Propolis has been known to have various benefits as traditional medicine. It contains flavonoid, caffeic acid phenethyl ester, cinnamic acid, and artepillin C, which may play a role in the immune system and promote lymphocyte proliferation. Thus, it is referred to as an immunomodulator. However, the use of liquid propolis as an active substance faces solubility problems. Propolis powder is present to overcome the limitations of the formulation of pharmaceutical preparations and can be developed as a traditional medicine with immunomodulatory activity. The purpose of this investigation was to see if propolis powder has any immunomodulatory effects using the phagocytosis macrophages method. The RAW 264.7 macrophage cell lines which had been cultured, were used in this study. Propolis samples at dosages of 100 g/ml, 50 g/ml, 25 g/ml, 12.5 g/ml, and 6.25 g/ml were used to determine the percentage of activity in RAW 264.7 macrophage cells. According to the results of this study, giving propolis powder at a dosage of 12.5 g/ml resulted in the highest macrophage capacity for phagocytosis (31±3%). In other words, propolis powder at that concentration had the greatest stimulating impact or increased phagocytosis activity the most.
Background: Despite extensive work on macrophage heterogeneity, the mechanisms driving activation induced heterogeneity (AIH) in macrophages remain poorly understood. Here, we aimed to develop mathematical models to explore theoretical cellular states underpinning the empirically observed responses of macrophages following lipopolysaccharide (LPS) challenge.
Methods: We obtained empirical data following primary and secondary responses to LPS in two in vitro cellular models (bone marrow-derived macrophages or BMDMs, and RAW 264.7 cells) and single-cell protein measurements for four key inflammatory mediators: TNF, IL-6, pro-IL-1β, and NOS2, and used mathematical modelling to understand heterogeneity.
Results: For these four factors, we showed that macrophage community AIH is dependent on LPS dose and that altered AIH kinetics in macrophages responding to a second LPS challenge underpin hypo-responsiveness to LPS. These empirical data can be explained by a mathematical three-state model including negative, positive, and non-responsive states (NRS), but they are also compatible with a four-state model that includes distinct reversibly NRS and non-responsive permanently states (NRPS). Our mathematical model, termed NoRM (Non-Responsive Macrophage) model identifies similarities and differences between BMDM and RAW 264.7 cell responses. In both cell types, transition rates between states in the NoRM model are distinct for each of the tested proteins and, crucially, macrophage hypo-responsiveness is underpinned by changes in transition rates to and from NRS.
Conclusions: Overall, we provide a mathematical model for studying macrophage ecology and community dynamics that can be used to elucidate the role of phenotypically negative macrophage populations in AIH and, primary and secondary responses to LPS.
Background: Despite extensive work on macrophage heterogeneity, the mechanisms driving activation induced heterogeneity (AIH) in macrophages remain poorly understood. Here, we aimed to develop mathematical models to explore theoretical cellular states underpinning the empirically observed responses of macrophages following lipopolysaccharide (LPS) challenge.
Methods: We obtained empirical data following primary and secondary responses to LPS in two in vitro cellular models (bone marrow-derived macrophages or BMDMs, and RAW 264.7 cells) and single-cell protein measurements for four key inflammatory mediators: TNF, IL-6, pro-IL-1β, and NOS2, and used mathematical modelling to understand heterogeneity.
Results: For these four factors, we showed that macrophage community AIH is dependent on LPS dose and that altered AIH kinetics in macrophages responding to a second LPS challenge underpin hypo-responsiveness to LPS. These empirical data can be explained by a mathematical three-state model including negative, positive, and non-responsive states (NRS), but they are also compatible with a four-state model that includes distinct reversibly NRS and non-responsive permanently states (NRPS). Our mathematical model, termed NoRM (Non-Responsive Macrophage) model identifies similarities and differences between BMDM and RAW 264.7 cell responses. In both cell types, transition rates between states in the NoRM model are distinct for each of the tested proteins and, crucially, macrophage hypo-responsiveness is underpinned by changes in transition rates to and from NRS.
Conclusions: Overall, we provide a mathematical model for studying macrophage ecology and community dynamics that can be used to elucidate the role of phenotypically negative macrophage populations in AIH and, primary and secondary responses to LPS.
Monocytes/macrophages play a prominent role in cutaneous wound healing. Persistent inflammation in diabetic wounds is associated with the inability of monocytic cells to switch from a phagocytic M1 (classically activated) to an anti-inflammatory, pro-regenerative M2 (alternatively activated) phenotype and as consequence, the proliferative phase of healing does not commence. A targeted cell therapy approach could potentially restore the pathological wound microenvironment through paracrine signalling to enable healing. This study investigated whether in vitro pre-treatment of monocytic (J774.1 A) cells - using a combination of endotoxin-induced immune tolerance (Pam3CSK4) and M2 polarization (IL-4) - could make these cells impervious to the pathological wound microenvironment and enhance the release of anti-inflammatory cytokines/growth factors. The effect of Pam3CSK4-induced tolerance and IL-4-associated polarization was assessed independently and in combination, on the expression of intracellular (flow cytometry) and secreted (ELISA) cytokines (TNF-ɑ, IL-6, IL-10, TGF-β) with and without re-stimulation to define the optimal pre-treatment conditions. Successive pre-treatment approach consisting of endotoxin tolerance followed by IL-4 priming, dampened TNF-ɑ release and induced intracellular TGF-β production upon re-stimulation. To mimic a chronic wound microenvironment, the J774A.1 monocytes were differentiated into macrophages using GM-CSF prior to pre-treatment (optimal condition) and subsequently exposed to diabetic wound fluid. The data demonstrated that in the presence of wound fluid, the successive pre-treatment, promoted M2 polarization (CD206) of monocytic cells and significantly dampened the intracellular production of both pro-inflammatory (TNF-ɑ, IL-6) and anti-inflammatory (IL-10, TGF-β) cytokines.
Blueberries have been extensively studied for the health benefits associated with their high phenolic content. The positive impact of blueberry consumption on human health is associated in part with modulation of pro-inflammatory molecular pathways and oxidative stress. Here, we review in vitro studies examining the anti-inflammatory and antioxidant effects of blueberry phytochemicals, discuss the results in terms of relevance to disease and health, and consider how different blueberry components modulate cellular mechanisms. The dampening effects of blueberry-derived molecules on inflammation and oxidative stress in cell models have been demonstrated through down-regulation of the NF-κB pathway and reduction of ROS and lipid peroxidation. The modulatory effects of blueberry phytochemicals on the MAPK pathway and antioxidant system are not as well described, with inconsistent observations reported on immune cells and between models of endothelial, dermal, and ocular inflammation. Although anthocyanins are often reported as the being the main bioactive compound in blueberries, no individual phytochemical has emerged as the primary compound when different fractions are compared; rather, an effect of whole blueberry extracts or synergy between different phenolic and non-phenolic extracts seems apparent. The major molecular mechanisms of blueberry phytochemicals are increasingly defined in cell models, but their relevance in more complex human systems needs further investigation using well-controlled clinical trials, in which systemic exposures to blueberry-associated molecules are measured concurrently with physiologic indices of inflammation and oxidative stress.
Gold nanodendrite (AuND)-based nanotheranostic agents with versatile capabilities were fabricated by optimizing the geometrical configurations (dendrite length and density) of AuND to achieve localized surface plasmon resonance (LSPR) in near-infrared biowindow II (NIR-II), and then subsequently functionalizing with a mitochondria-targeting compound (triphenylphosphonium, TPP), loading with an NIR-photosensitizer (indocyanine green, ICG) and coating with the macrophage cell membrane (MCM) to trap ICG within AuND and selectively interact with MDA-MB-231 cells. The novel AuND-TPP-ICG@MCM system enabled the integration of multimodal fluorescence/photoacoustic/surface-enhanced Raman imaging with synergistic therapies of NIR-II photothermal therapy and NIR-I photodynamic therapy for cancer treatment. Enhanced hyperthermia and elevated production of reactive oxygen species within the tumors via MCM coating and mitochondria targeting afforded a synergistic efficacy for tumor eradication with limited side effects. The demonstrated biocompatibility, multi-imaging capability, and high therapeutic efficiency under NIR laser irradiation indicate the potentials of this multifunctional nanotheranostic platform for clinical utility in cancer therapy.
We have shown that endogenous neurosteroids, including pregnenolone and 3α,5α-THP inhibit toll-like receptor 4 (TLR4) signal activation in mouse macrophages and the brain of alcohol-preferring (P) rat, which exhibits innate TLR4 signal activation. The current studies were designed to examine whether other activated TLR signals are similarly inhibited by 3α,5α-THP. We report that 3α,5α-THP inhibits selective agonist-mediated activation of TLR2 and TLR7, but not TLR3 signaling in the RAW246.7 macrophage cell line. The TLR4 and TLR7 signals are innately activated in the amygdala and NAc from P rat brains and inhibited by 3α,5α-THP. The TLR2 and TLR3 signals are not activated in P rat brain and they are not affected by 3α,5α-THP. Co-immunoprecipitation studies indicate that 3α,5α-THP inhibits the binding of MyD88 with TLR4 or TLR7 in P rat brain, but the levels of TLR4 co-precipitating with TRIF are not altered by 3α,5α-THP treatment. Collectively, the data indicate that 3α,5α-THP inhibits MyD88- but not TRIF-dependent TLR signal activation and the production of pro-inflammatory mediators through its ability to block TLR-MyD88 binding. These results have applicability to many conditions involving pro-inflammatory TLR activation of cytokines, chemokines, and interferons and support the use of 3α,5α-THP as a therapeutic for inflammatory disease.
Dendritic cells (DCs) are antigen-presenting cells with a unique ability to induce primary immune responses. DCs capture and transfer information from the outside world to the cells of the adaptive immune system. DCs are not only critical for the induction of primary immune responses, but may also be important for the induction of immunological tolerance, as well as for the regulation of the type of T cell–mediated immune response. Although our understanding of DC biology is still in its infancy, we are now beginning to use DC-based immunotherapy protocols to elicit immunity against cancer and infectious diseases.
The molecular phagocytic lineage comprises, in order of increasing maturity, the committed macrophage precursor cell, the
monoblast, promonocyte, monocyte, and the macrophage. Methods for the preparation and culture of bone marrow-derived macrophages,
developed by Stanley and colleagues (1,2; Tushinski, R. J., personal communication), provide large numbers of mononuclear phagocytes that are capable of extensive
cell proliferation. Since their proliferation can be stimulated by colony-stimulating factor-1 (CSF-1), granulocyte macrophage
colony stimulating factor (GM-CSF), or interleukin-3 (IL-3), they represent an important primary cell source for studies of
the actions and interactions of these three growth factors. The principles underlying the method are:
1.
To generate and expand primitive mononuclear phagocyte precursor cells by culturing bone marrow cells in a combination of
partially purified CSF-1 and IL-3 for a period of 3 d;
2.
To remove contaminating red cells, fibroblasts, and mature macrophages and disrupt aggregates of proliferating cells by proteolytic
digestion of the nonadherent cells at d 1 and 3 of culture; and
3.
To obtain a population of mononuclear phagocytes that is relatively homogeneous with respect to their state of differentiation
by recovering only those cells (i.e., monoblasts, promonocytes) that acquire the capacity to adhere to tissue-culture plastic
during d 4–5 of culture.
The molecular phagocytic lineage comprises, in order of increasing maturity, the committed macrophage precursor cell, the monoblast, promonocyte, monocyte and the macrophage. Methods for the preparation and culture of bone marrow-derived macrophages, developed by Stanley and colleagues (1-3), provide large numbers of mononuclear phagocytes that are capable of extensive cell proliferation. Since their proliferation can be stimulated by colony stimulating factor-1 (CSF-1), granulocyte macrophage colony stimulating factor (GM-CSF), or interleukin-3 (IL-3), they represent an important primary cell source for studies of the actions and interactions of these three growth factors. The principles underlying the method are: (1) to generate and expand primitive mononuclear phagocyte precursor cells by culturing bone marrow cells in a combination of partially purified CSF-1 and IL-3 for a period of 3 d, (2) to remove contaminating red cells, fibroblasts and mature macrophages and disrupt aggregates of proliferating cells, by proteolytic digestion of the nonadherent cells at d 1 and 3 of culture and, (3) to obtain a population of mononuclear phagocytes that is relatively homogeneous with respect to their state of differentiation by recovering only those cells (i.e., mono-blasts, promonocytes) that acquire the capacity to adhere to tissue culture plastic during d 4-5 of culture.
Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.
Stimulation of Toll-like receptors (TLRs) triggers activation of a common MyD88-dependent signaling pathway as well as a MyD88-independent
pathway that is unique to TLR3 and TLR4 signaling pathways leading to interferon (IFN)-β production. Here we disrupted the
gene encoding a Toll/IL-1 receptor (TIR) domain-containing adaptor, TRIF. TRIF-deficient mice were defective in both TLR3-
and TLR4-mediated expression of IFN-β and activation of IRF-3. Furthermore, inflammatory cytokine production in response to
the TLR4 ligand, but not to other TLR ligands, was severely impaired in TRIF-deficient macrophages. Mice deficient in both
MyD88 and TRIF showed complete loss of nuclear factor kappa B activation in response to TLR4 stimulation. These findings demonstrate
that TRIF is essential for TLR3- and TLR4-mediated signaling pathways facilitating mammalian antiviral host defense.
Myeloid differentiation protein-88 (MyD88) is a signal adaptor protein required for cytokine production following engagement of Toll-like receptors (TLRs) by their cognate ligands. Activation of both TLR-3 and TLR-4, however, can engage signaling events independent of MyD88 expression. The relative importance of these MyD88-dependent and -independent signaling pathways in the macrophage response to lipopolysaccharide (LPS) is unknown. Here we define these events using microarray expression profiling of LPS-stimulated macrophages taken from MyD88-null and wild-type mice. Of the 1,055 genes found to be LPS responsive, only 21.5% were dependent on MyD88 expression, with MyD88-independent genes constituting 74.7% of the genetic response. This MyD88-independent gene expression was predominantly transcriptionally regulated, as it was unaffected by cycloheximide blockade of new protein synthesis. A previously undescribed group of LPS-regulated genes (3.8%), whose induction or repression was significantly greater in the absence of MyD88, was also identified by these studies. The regulation of these genes suggested that MyD88 could serve as a molecular brake, constraining gene activity in a subset of LPS-responsive genes. The findings generated with LPS stimulation were recapitulated by exposure of macrophages to live Escherichia coli. These expression-profiling studies redefine the current dogma of TLR-4 signaling and establish that MyD88, although essential for some of the best-characterized macrophage responses to LPS, is not required for the regulation of the majority of genes engaged by macrophage exposure to endotoxin or live bacteria.
Macrophages express a broad range of plasma membrane receptors that mediate their interactions with natural and altered-self components of the host as well as a range of microorganisms. Recognition is followed by surface changes, uptake, signaling, and altered gene expression, contributing to homeostasis, host defense, innate effector mechanisms, and the induction of acquired immunity. This review covers recent studies of selected families of structurally defined molecules, studies that have improved understanding of ligand discrimination in the absence of opsonins and differential responses by macrophages and related myeloid cells.
The mouse macrophage-like cell line RAW264.7, the most commonly used mouse macrophage cell line in medical research, was originally reported to be free of replication-competent murine leukemia virus (MuLV) despite its origin in a tumor induced by Abelson MuLV containing Moloney MuLV as helper virus. As currently available, however, we find that it produces significant levels of ecotropic MuLV with the biologic features of the Moloney isolate and also MuLV of the polytropic or MCF class. Newborn mice developed lymphoma following inoculation with the MuLV mixture expressed by these cells. These findings should be considered in interpretation of increasingly widespread use of these cells for propagation of other viruses, studies of biological responses to virus infection and use in RNA interference and cell signalling studies.
Lentiviral genomes encode multiple structural and regulatory proteins. Expression of the full complement of viral proteins is accomplished in part by alternative splicing of the genomic RNA. Caprine arthritis encephalitis virus (CAEV) and maedi-visna virus (MVV) are two highly related small-ruminant lentiviruses (SRLVs) that infect goats and sheep. Their genome seems to be less complex than those of primate lentiviruses since SRLVs encode only three auxiliary proteins, namely, Tat, Rev, and Vif, in addition to the products of gag, pol, and env genes common to all retroviruses. Here, we investigated the central part of the SRLV genome to identify new splice elements and their relevance in viral mRNA and protein expression.
We demonstrated the existence of a new 5' splice (SD) site located within the central part of CAEV genome, 17 nucleotides downstream from the SD site used for the rev mRNA synthesis, and perfectly conserved among SRLV strains. This new SD site was found to be functional in both transfected and infected cells, leading to the production of a transcript containing an open reading frame generated by the splice junction with the 3' splice site used for the rev mRNA synthesis. This open reading frame encodes two major protein isoforms of 18- and 17-kDa, named Rtm, in which the N-terminal domain shared by the Env precursor and Rev proteins is fused to the entire cytoplasmic tail of the transmembrane glycoprotein. Immunoprecipitations using monospecific antibodies provided evidence for the expression of the Rtm isoforms in infected cells. The Rtm protein interacts specifically with the cytoplasmic domain of the transmembrane glycoprotein in vitro, and its expression impairs the fusion activity of the Env protein.
The characterization of a novel CAEV protein, named Rtm, which is produced by an additional multiply-spliced mRNA, indicated that the splicing pattern of CAEV genome is more complex than previously reported, generating greater protein diversity. The high conservation of the SD site used for the rtm mRNA synthesis among CAEV and MVV strains strongly suggests that the Rtm protein plays a role in SRLV propagation in vivo, likely by competing with Env protein functions.
Academic Writing and Publishing will show academics (mainly in the social sciences) how to write and publish research articles. Its aim is to supply examples and brief discussions of recent work in all aspects of the area in short, sharp chapters. It should serve as a handbook for postgraduates and lecturers new to publishing. The book is written in a readable and lively personal style. The advice given is direct and based on up-to-date research that goes beyond that given in current textbooks. For example, the chapter on titles lists different kinds of titles and their purposes not discussed in other texts. The chapter on abstracts instructs the reader on writing structured abstracts from the start.
Bacterial endotoxin (lipopolysaccharide [LPS]) causes fatal shock in humans and experimental animals. The shock is mediated by cytokines released by direct LPS stimulation of cells of monocytic origin (monocyte/macrophage [MO]). Recent studies have supported the concept that the plasma protein, LPS binding protein (LBP), plays an important role in controlling MO responses to LPS. Specifically, evidence has been presented to suggest that CD14, a membrane protein present in MO, serves as a receptor for complexes of LPS and the plasma protein LPS binding protein (LBP). In this function CD14 mediates attachment of LPS-bearing particles opsonized with LBP and appears to play an important role in regulating cytokine production induced by complexes of LPS and LBP. The CD14-, murine pre-B cell line 70Z/3 responds to LPS by synthesis of kappa light chains and consequent expression of surface IgM. To better understand the role of CD14 in controlling cellular responses to LPS, we investigated the effect of transfection of CD14 into 70Z/3 cells on LPS responsiveness. We report here that transfection of human or rabbit CD14 cDNA into 70Z/3 cells results in membrane expression of a glycosyl-phosphatidylinositol-anchored CD14. When LPS is complexed with LBP, CD14-bearing 70Z/3 cells bind more LPS than do the parental or 70Z/3 cells transfected with vector only. Remarkably, the expression of CD14 lowers the amount of LPS required to stimulate surface IgM expression by up to 10,000-fold when LPS dose-response curves in the CD14-, parental and CD14-bearing, transfected 70Z/3 cells are compared. In contrast, the response of CD14-bearing 70Z/3 cells and the parental 70Z/3 cell line (CD14-) to interferon gamma is indistinguishable. LPS stimulation of the parental and CD14-bearing 70Z/3 cells results in activation of NF-kB. These data provide evidence to support the concept that the LPS receptor in cells that constitutively express CD14 may be a multiprotein complex containing CD14 and membrane protein(s) common to a diverse group of LPS-responsive cells.
The primary anthropogenic factor causing amphibian declines in Europe is habitat loss and fragmentation. Here we explore the effects of aquatic and landscape habitat variables on the adult counts of the Common Toad (Bufo bufo) in 43 ponds in central Romania surveyed between 2000 and 2005. Principal components analysis (PCA) identified two main factors, with which the adult counts can be related the first related to landscape variables (percentage of forest cover, presence/absence of roads and habitat corridors) and the second, to pond variables, in particular the presence/absence of fish, together accounting for 49% of the total variance. Analysis of variance (ANOVA) showed differences between ponds where toads were present or absent in factor loadings of both principal components, but only the landscape factor was significantly correlated with toad counts. These results highlight the role of landscape composition and configuration in maintaining toad populations in this area, and suggest a negative effect of landscape fragmentation. To efficiently protect amphibians in Romania, appropriate legislation and a close collaboration between landowners, landscape planners and herpetologists are needed.
The field of chemokines represented by the β-chemokine RANTES began just 20 years ago ([1][1]). Several of these small basic proteins were discovered about the same time and defined the process of chemotaxis, which today is an important component of our understanding of immune responses to
Our understanding of the innate immune response in the horse has been limited by a lack of definitive data concerning cell signaling in response to microbial products. Toll-like receptors (TLRs) recognize conserved molecular motifs of microbes and elicit immune responses through their coupling with intracellular adaptor molecules, particularly MyD88 and TRIF. To provide a more definitive characterization of TLR signaling in the horse, the objectives of this study were to: (1) characterize the responses of equine monocytes to TLR ligands that signal through MyD88, TRIF or both in other species, and (2) determine the profiles of gene expression initiated utilizing these adaptor molecules. Monocytes were used to establish concentration response curves for Escherichia coli lipopolysaccharide (LPS; TLR4 ligand) and N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-[R]-cysteinyl-[S]-seryl-[S]-lysyl-[S]-lysyl-[S]-lysyl-[S]-lysine x 3 HCl (Pam(3)CSK(4); TLR2 ligand) based on expression of procoagulant activity (PCA) and production of tumor necrosis factor-alpha (TNF-alpha); effects of polyinosine-polycytidylic acid (Poly I:C; TLR3 ligand) were determined by quantifying expression of mRNA for interferon-beta (IFN-ss). Expression of genes associated with the MyD88- (TNF-alpha, IL-1ss, IL-6 and IL-10) and TRIF-dependent pathways (IFN-ss, IP-10, RANTES and TRAF1) were measured at intervals spanning 20 h. LPS and Pam(3)CSK(4) induced significantly higher expression of TNF-alpha, IL-1ss, and IL-10 than did Poly I:C. Poly I:C induced significantly higher expression of IFN-ss, IP-10 and RANTES than did either the TLR2 or TLR4 ligands. High concentrations of E. coli LPS did not significantly increase expression of genes associated with the TRIF-dependent pathway. The results of this study suggest that equine monocytes utilize a common intracellular pathway in response to TLR2 and TLR4 ligands, but a distinct pathway in response to TLR3 ligands.
Dendritic cells have been considered an immune cell type that is specialized for the presentation of Ag to naive T cells. Considerable effort has been applied to separate their lineage, pathways of differentiation, and effectiveness in Ag presentation from those of macrophages. This review summarizes evidence that dendritic cells are a part of the mononuclear phagocyte system and are derived from a common precursor, responsive to the same growth factors (including CSF-1), express the same surface markers (including CD11c), and have no unique adaptation for Ag presentation that is not shared by other macrophages.
In this study, we established an efficient in vitro culture method for generating mature splenic macrophages (Sp-Mphi). Splenocytes were cultured in the presence of conditioned medium containing macrophage colony-stimulating factor (M-CSF) for 7 days post post-isolation and the generated Sp-Mphi were characterized phenotypically and functionally. Through this method, 9 x 10(6)/mouse Sp-Mphi were obtained in comparison to 2 x 10(5)/mouse when Mphi were cultured in regular medium. In addition, the purity of these cells was as high as 80% by day 5 and >90% by day 7 of culturing, confirmed with Mphi-specific markers. The increased Sp-Mphi yields, in the presence of M-CSF, point towards the existence of a precursor population in the spleen that can be influenced to differentiate into Sp-Mphi. Moreover, we compared the maturation of generated Sp-Mphi to conventional bone marrow-derived Mphi (BM-Mphi) in vitro. Interestingly, Sp-Mphi exhibited lower capacity to phagocytose dead cells after 3 days of maturation, but showed similar internalizing capacity after 5 and 7 of maturation to BM-Mphi cultured for the same time period. Importantly, Sp-Mphi upregulated the expression of several surface markers such as MOMA-2 and CD68 while downregulating SIGN-R1 after 7 days, indicating that these Sp-Mphi undergo further maturation in vitro due to culturing in M-CSF. Taken together, we describe and validate a method for generating Sp-Mphi in large quantities and high purity. These data should prove valuable in future studies characterizing the functions and maturation of Sp-Mphi.
Macrophage activation was examined in resistant C57BL/6 and susceptible A/J mice during the course of blood-stage infection with Plasmodium chabaudi AS. Three parameters of macrophage activation (lipopolysaccharide [LPS]- and malaria antigen-induced tumor necrosis factor [TNF] production in vitro, phorbol myristate acetate [PMA]-induced production of oxygen metabolites in vitro, and Ia antigen expression) were assessed during infection in populations of peritoneal and splenic macrophages recovered from infected mice of the two strains. The peak level of LPS-induced TNF production in vitro by splenic macrophages from both infected C57BL/6 and infected A/J mice occurred on day 7, which was 3 days before the peak of parasitemia. Although the kinetics of TNF production in vitro in response to either LPS, soluble malaria antigen, or intact parasitized erythrocytes varied in some of the other macrophage populations during infection, there was no significant difference in the peak level of production. Peritoneal and splenic macrophages from infected C57BL/6 mice exhibited significantly increased PMA-induced production of H2O2 in vitro on day 7. Peritoneal macrophages from infected A/J mice also exhibited significant PMA-induced H2O2 production on day 7, while production by splenic macrophages from these hosts was not increased in comparison with production by cells from normal animals. Only peritoneal macrophages from infected C57BL/6 mice produced significantly increased levels of O2-, and this occurred on day 7 postinfection. Ia antigen expression by both peritoneal and splenic macrophages from resistant C57BL/6 and susceptible A/J mice was significantly increased during P. chabaudi AS infection. However, the percentage of Ia+ peritoneal macrophages on days 8 and 10 postinfection and Ia+ splenic macrophages on day 3 postinfection was significantly higher in C57BL/6 than in A/J mice. Thus, these results demonstrate that macrophages from P. chabaudi AS-infected A/J mice exhibit defects in oxygen metabolism and Ia antigen expression which may contribute to the susceptibility of these hosts to this intraerythrocytic parasite. The cause-and-effect relationship between these defects and the susceptibility of A/J mice to P. chabaudi AS is unknown.
We have studied the effects of highly purified rabbit lipopolysaccharide (LPS)-binding protein (LBP) on the ability of murine bone marrow-derived macrophages to respond to bacterial LPS. Macrophage responses studied include the secretion of tumor necrosis factor alpha, production of arginine-derived nitrite (NO2-), and killing of an intracellular pathogen, Leishmania enriettii. Macrophages from either CBA or LPS-hyporesponsive C3H/HeJ mice exhibited significantly greater sensitivity to LPS in the presence of LBP. Furthermore, both CBA and C3H/HeJ macrophages demonstrated an LBP-dependent enhancement of LPS binding. These results suggest that C3H/HeJ macrophages are capable of binding LPS-LBP complexes and support the hypothesis that hyporesponsiveness in this strain involves a step subsequent to LPS binding. Furthermore, these findings provide additional evidence of the important role played by the acute-phase plasma protein LBP in modifying host response to LPS.
The present paper describes cytochemical, membrane, functional, and mitotic characteristics of monoblasts, promonocytes, monocytes, and macrophages of the mouse, guinea pig, rat, and man. For all of these species the results show that after staining for nonspecific esterase, with alpha-naphthylbutyrate as substrate, and for lysozyme, mononuclear phagocytes can be distinguished from other cells, e.g., T and B lymphocytes. However, it must be kept in mind that immature and mature granulocytic cells are also lysozyme positive. The presence of Fc and C receptors is dependent on the maturity of the cells and the duration of incubation in vitro; with respect to the former, an in vivo population of immature mononuclear phagocytes may have a lower percentage of positive cells than is the case in a mature population, and with respect to the latter, the percentage of positive cells rises during incubation. Phagocytosis of opsonized bacteria and red cells is a reliable criterion for the distinction between mononuclear phagocytes and other cell types, e.g. lymphocytes and fibroblasts. In all of the species studied, the majority of both immature and mature mononuclear phagocytes ingested particles opsonized with IgG; the proportion of phagocytosis of red cells via C3 receptors is usually very small. Incorporation studies with [3H] thymidine have shown that immature mononuclear phagocytes (i.e., monoblasts and promonocytes) divided and that monocytes and macrophages do not. The small number of macrophages that incorporate [3H] thymidine are immature mononuclear phagocytes which have very recently arrived in the tissues from the bone marrow. Comparison of mononuclear phagocytes in different organs of various species has shown unequivocally that these cells belong to one cell line, called the mononuclear phagocyte system.
Normal, thioglycollate-stimulated and BCG-activated mouse peritoneal macrophages were cultivated in vitro with the conditioned medium of mouse L-929 cells. The thioglycollate- and BCG-macrophages rapidly proliferated, whereas normal macrophages grew more slowly. A clear morphological difference between the three types of macrophages in the culture was observed. Glucocorticoids inhibited the growth of the macrophages at pharmacological concentrations. Other steroids, progesterone, diethylstilbestrol and testosterone in that order, had a far lower growth-inhibiting effect. Macrophages cultured with 10(-6) M dexamethasone had a reduced antimicrobial effect on Candida parapsilosis compared with that of the untreated cells. Choleragen had the same effect on the macrophages as glucocorticoids. The toxin inhibited growth at a concentration as low as 10 pg/ml and cells treated with 1 ng of choleragen per ml had decreased antifungal activity. Similarly, Escherichia coli lipopolysaccharide at 10 ng/ml inhibited the growth of thioglycollate-macrophages. However, macrophages incubated with the lipopolysaccharide had enhanced anticandida activity. Thus, the immunosuppressors glucocorticoid and choleragen inhibited both the increase in the number of macrophages and the microbicidal activity of the phagocytes. Lipopolysaccharide, an immunostimulant, stimulated macrophage activity, but was toxic for cell growth.
The S-form lipopolysaccharide (LPS) was effectively separated from a native preparation of smooth-type Salmonella abortus equi LPS by means of the centrifugal partition chromatography (CPC). To clarify the mechanisms by which LPS activates macrophages, CPC-separated S-form LPS was assessed for its ability to induce the secretion of tumor necrosis factor-alpha (TNF-alpha) by murine macrophage-like J774.1 cells in comparison with other fractions of LPS which lacks most of O-polysaccharides. LPS dose-response and time-kinetics studies showed that serum factor(s) regulated especially the onset of TNF-alpha secretion in stimulation with S-form LPS. These results strongly suggest that the native (unfractionated) LPS activates macrophages in both O-polysaccharide/serum-dependent and -independent pathways.
As dendritic cells (DC) are rare populations in all organs, their generation from hematopoietic precursors in large quantities has proven critical to study their biology. From murine bone marrow about 5 x 10(6) cells at 70% purity are obtained per mouse after 8 days of culture with GM-CSF. We have improved this standard method and routinely achieve a 50-fold higher yield, i.e., 1-3 x 10(8) immature and mature DC per mouse at 90-95% purity. The major modifications were: (i) the avoidance of any active depletion of bone marrow cell subpopulations to circumvent loss of precursors, (ii) a lower plating density of bone marrow cells, (iii) a prolonged culture period of 10-12 days, (iv) the reduction of the GM-CSF dose from day 8 or 10 onwards to reduce granulocyte contaminations. The final non-adherent population at day 10-12 constitutes a mixture of immature and mature DC. Further maturation of DC could be induced by high doses of LPS or TNF-alpha for the last 24 h, where 50-70% of the non-adherent fraction represented mature DC with high levels of NLDC-145, CD86 and CD40. This method allows by simple means the generation of high numbers of murine DC with very low B cell or granulocyte contaminations. It will be valuable to study DC biology notably at the molecular level.
The innate immune response is the first line of defense against infectious disease. It is vital that the host detect the pathogen and rapidly mount a defense. The Toll-like family of receptors have evolved to detect conserved patterns on pathogens. When stimulated, these receptors activate the inflammatory response.
Mammalian Toll-like receptor (TLR) proteins are pattern recognition receptors for a diverse array of bacterial and viral products. Gram negative bacterial lipopolysaccharide (LPS) activates cells through TLR4, whereas the mycobacterial cell wall glycolipids, lipoarabinomannan (LAM) and mannosylated phosphatidylinositol (PIM), activate cells through TLR2. Furthermore, short term culture filtrates of M. tuberculosis bacilli contain a TLR2 agonist activity, termed soluble tuberculosis factor (STF), that appears to be PIM. It was recently shown that stimulation of RAW264.7 murine macrophages by LPS, LAM, STF, and PIM rapidly activated NF-kappaB, AP1, and MAP kinases.
This study shows that signalling by TLR2 and TLR4 also activates the protein kinase Akt, a downstream target of phosphatidylinositol-3'-kinase (PI-3-K). This finding suggests that activation of PI-3-K represents an additional signalling pathway induced by engagement of TLR2 and TLR4. Subsequently, the functional responses induced by the different TLR agonists were compared. LPS, the mycobacterial glycolipids, and the OspC lipoprotein (a TLR2 agonist) all induced macrophages to secrete tumour necrosis factor alpha (TNFalpha), whereas only LPS could induce nitric oxide (NO) secretion. Human alveolar macrophages also exhibited a distinct pattern of cellular response after stimulation with TLR2 and TLR4 agonists. Specifically, LPS induced TNFalpha, MIP-1beta, and RANTES production in these cells, whereas the TLR2 agonists induced only MIP-1beta production.
Together, these data show that different TLR proteins mediate the activation of distinct cellular responses, despite their shared ability to activate NF-kappaB, AP1, MAP kinases, and PI-3-K.
One of the mechanisms by which the innate immune system senses the invasion of pathogenic microorganisms is through the Toll-like receptors (TLRs), which recognize specific molecular patterns that are present in microbial components. Stimulation of different TLRs induces distinct patterns of gene expression, which not only leads to the activation of innate immunity but also instructs the development of antigen-specific acquired immunity. Here, we review the rapid progress that has recently improved our understanding of the molecular mechanisms that mediate TLR signalling.
Macrophages recognize lipopolysaccharide (LPS) by Toll-like receptor 4 and activate inflammatory responses by inducing expression of various genes. TLR4 activates intracellular signaling pathways via TIR domain containing adaptor molecules, MyD88, and Toll/IL-1 domain containing adaptor inducing IFN-beta (TRIF). Although macrophages lacking MyD88 or TRIF showed impaired cytokine production, activation of intracellular signaling molecules still occurred in response to LPS in these cells. In the present study, we implemented cDNA microarrays to investigate the contribution of MyD88 and TRIF in gene expression induced by LPS stimulation. Whereas wild-type macrophages induced 148 genes in response to LPS, macrophages lacking both MyD88 and TRIF did not upregulate any genes in response to LPS. Surprisingly, 80 LPS-inducible genes were redundantly regulated by either MyD88 or TRIF. In contrast, proinflammatory cytokines and chemokines were critically regulated by MyD88 or TRIF alone. Genes critically regulated by MyD88 alone tend to be induced quickly after LPS stimulation and regulated by mRNA stability as well as transcription. Genes known to be induced by type I interferons were simply dependent on TRIF for their expression. Taken together, MyD88 and TRIF play both redundant and distinct roles in LPS-induced gene expression.
It has demonstrated that the recognition of triacylated lipopeptides by Toll-like receptor (TLR) 2 requires TLR1 as a coreceptor. In the NF-kappaB reporter assay system in which human embryonic kidney 293 cells were transfected with TLR2 and TLR1 together with an NF-kappaB luciferase reporter gene, S-(2,3-bispalmitoyloxypropyl)-N-palmitoyl-Cys-Lys-Lys-Lys-Lys (Pam(3)CSK(4)) and Pam(3)CSSNA were recognized by TLR2/TLR1, but the recognition level was unexpectedly very low. However, cotransfection of CD14 drastically enhanced the recognition of triacylated lipopeptides by TLR2/TLR1. The CD14-induced enhancement did not occur without cotransfection of TLR1. Both CD14(dS39-A48), a mutant with deletion of the part of possible N-terminal ligand-binding pocket, and anti-CD14 monoclonal antibody reduced the CD14-induced enhancement. Transfection of a TIR domain-deficient mutant of TLR2 (TLR2(dE772-S784)) or TLR1 (TLR1(dQ636-K779)) completely abrogated the CD14-induced enhancement. Soluble recombinant CD14 added extracellularly enhanced the recognition of Pam(3)CSSNA by TLR2/TLR1. Immunoprecipitation analysis demonstrated that CD14 was not associated with TLR2 but that TLR1 was associated with TLR2. In addition, surface plasmon resonance-based assay demonstrated that CD14 binds to Pam(3)CSK(4) at a dissociation constant of 5.7 microM. This study suggests that CD14 directly binds to triacylated lipopeptides and facilitates recognition of the lipopeptides by the TLR2/TLR1 complex without binding to the receptor complex.
Cells of the mononuclear phagocytic system including monocytes and macrophages (e.g., pooled human monocytes, bone marrow-derived macrophages, etc.) are often employed for in vitro assessment of novel biomaterials and to assay anti-inflammatory drug activity. In this context, numerous macrophage cells are treated interchangeably in the literature despite a lack of demonstrated equivalence among immortalized cell lines and further, between cell lines and primary-derived macrophages of different species. Three murine (monocyte-) macrophage cell lines (IC-21, J774A.1, and RAW 264.7), commonly utilizedin biomaterial and pharmaceutical screening research, have been compared with primary-derived murine bone marrow macrophages. Significant differences were discovered in the expression of cell surface proteins requisite for cell adhesion and activation among cell lines and primary-derived cells as well as between the different cell lines. Results demonstrate activation but with reduced cytokine expression to chemical stimulus (lipopolysaccharide) by cell lines compared with that of primary-derived macrophages. Limited correlation between cultured primary and immortalized cells in cytokine production, phenotype and intrinsic activation states has relevance to fidelity for in vitro testing. These differences warrant justification for selection of various cell lines for specific assay purposes, and merit caution if comparisons to primary cell types (i.e., for biocompatibility) are required.
An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow
- Mb Lutz
- N Kukutsch
- Al Ogilvie
- S Rossner
- F Koch
- N Romani
- G Schuler
Lutz MB, Kukutsch N, Ogilvie AL, Rossner S, Koch F, Romani N, Schuler G. An advanced
culture method for generating large quantities of highly pure dendritic cells from mouse bone
marrow. J Immunol Methods 1999;223:77–92. [PubMed: 10037236]
An advanced culture method for generating large quantities of highly pure dendritic cells from mouse bone marrow
- Lutz