Macrophage RAW264 Research Articles

The transcriptome of MHV-infected RAW264.7 cells offers an alternative model for macrophage innate immunity research.

Macrophages are the primary innate immune cells encountered by the invading coronaviruses, and their abilities to initiate inflammatory reactions, to maintain the immunity homeostasis by differential polarization, to train the innate immune system by epigenic modification have been reported in laboratory animal research. In the current in vitro research, murine macrophage RAW 264.7 cell were infected by mouse hepatitis virus, a coronavirus existed in mouse. At 3-, 6-, 12-, 24-, and 48-h post infection (hpi.), the attached cells were washed with PBS and harvested in Trizol reagent. Then The harvest is subjected to transcriptome sequencing. The transcriptome analysis showed the immediate (3 hpi.) up regulation of DEGs related to inflammation, like Il1b and Il6. DEGs related to M2 differential polarization, like Irf4 showed up regulation at 24 hpi., the late term after viral infection. In addition, DEGs related to metabolism and histone modification, like Ezh2 were detected, which might correlate with the trained immunity of macrophages. The current in vitro viral infection study showed the key innated immunity character of macrophages, which suggested the replacement value of viral infection cells model, to reduce the animal usage in preclinical research.

Seagrass as a potential nutraceutical to decrease pro-inflammatory markers

BackgroundThe Pro-inflammatory mediators such as prostaglandin E2, nitric oxide and TNF-α are the key players in the stimulation of the inflammatory responses. Thus, the pro-inflammatory mediators are considered to be potential targets for screening nutraceutical with anti-inflammatory activity.MethodsIn this context, we explored the anti-inflammatory potency of seagrass extract with western blot (Bio-Rad) analysis by using LPS induced RAW macrophages as in-vitro models, western blot analysis, In-silico methods using Mastero 13.0 software.ResultsThe anti-inflammatory activity of Seagrass was demonstrated through down regulation of Pro-inflammatory markers such as Cyclooxygenase-2, induced Nitric oxide synthase and prostaglandin E synthase-1. The results were validated by docking the phytochemical constituents of seagrass namely Isocoumarin, Hexadecanoic acid, and Cis-9 Octadecenoic acid, 1,2 Benzene dicarboxylic acid and beta-sitosterol with TNF-alpha, COX-2, iNOS and PGES-1.ConclusionThe methanolic extract of seagrass Halophila beccarii is a potential nutraceutical agent for combating against inflammation with a significant anti-inflammatory activity.

Two new pinophol derivatives from endophytic fungus Penicillium herquei JX4 hosted in Ceriops tagal

An OSMAC strategy was used to study secondary metabolites and anti-inflammatory activities of the endophytic fungus Penicillium herquei JX4 hosted in Ceriops tagal. The PDB ferment of fungus P. herquei JX4 was isolated, purified, and identified by using silica gel column chromatography, gel column chromatography, octadecylsilyl(ODS) column chromatography, and semi-preparative high-performance liquid chromatography. Two new pinophol derivatives, pinophol H(1) and pinophol I(2) were isolated and identified, and they were evaluated in terms of the inhibitory activities against the nitric oxide(NO) production induced by lipopolysaccharide(LPS) in mouse macrophage RAW264.7 cells. The results showed that compound 1 had significant inhibitory activity on NO production, with an IC_(50) value of 8.12 μmol·L~(-1).

Sophoricoside ameliorates methicillin-resistant Staphylococcus aureus-induced acute lung injury by inhibiting Bach1/Akt pathway

BackgroundThe lack of effective treatments for methicillin-resistant Staphylococcus aureus (MRSA) infection, which often leads to severe acute lung injury (ALI), poses a grave threat to human life. Sophoricoside (SOP), an isoflavone glycoside abundant in the fruit of traditional Chinese herbal Sophora japonica l., showed anti-inflammatory effects against atopic dermatitis, allergic inflammation, and lipopolysaccharide-induced ALI. However, its effect and underlying mechanism on MRSA-induced ALI remain unclear. PurposeThe aim of this study is to assess the protective effect of SOP in MRSA-induced ALI and elucidate its underlying molecular mechanisms. MethodsIn vivo experiments were conducted using wild-type mice to establish MRSA-induced ALI mouse model, and the effects of SOP on ALI were evaluated by hematoxylin-eosin staining, flow cytometry, quantitative real-time polymerase chain reaction, and several biochemical indicators. Adoptive transfer experiments and BTB and CNC homology 1 knockout (Bach1-/-) mice were also utilized in this study. In vitro studies employed murine macrophages RAW264.7 cells, primary bone marrow-derived macrophages (BMDMs), and primary lung macrophages to explore the underlying molecular mechanisms. ResultsThe administration of SOP ameliorated MRSA-induced ALI by improving pulmonary histological damages, reducing neutrophil infiltration, suppressing oxidative stress levels, and decreasing the expression of inflammatory cytokines. In isolation experiments with ALI mouse lung macrophages and macrophage adoptive transfer experiments, SOP prevented macrophage activation, thereby reducing the production of proinflammatory cytokines. In vitro experiments demonstrated that SOP decreased the expression of inflammatory mediators in lipoteichoic acid (LTA)-stimulated RAW264.7 cells, BMDMs, and primary lung macrophages. Additionally, SOP inhibited protein kinase B (Akt) phosphorylation and treatment with MK2206-a specific inhibitor of Akt-eliminated SOP's ability to suppress LTA-stimulated macrophage inflammation. Furthermore, stimulation with LTA or MRSA up-regulated Bach1 expression; however, deletion of Bach1 abolished the inhibitory effect of SOP on p-Akt activation as well as inflammation and ALI development. ConclusionThis study provides the first evidence that SOP effectively mitigates MRSA-induced ALI via suppressing macrophage activation through the inhibition of Bach1/Akt pathway. These findings highlight the potential of SOP as a novel therapeutic agent for treating MRSA-induced ALI.

IN VITRO AND IN SILICO INHIBITORY ACTIVITY OF ANGIOTENSIN-CONVERTING ENZYME II AND ANTI-INFLAMMATORY EFFECTS OF NATURAL COMPOUNDS FROM RHEUM OFFICINALE ROOTS

Rhubarb roots (Rheum officinale), a common medicinal plant in folk remedies, and their chemical compositions were investigated for their inhibitory activity of angiotensin-converting enzyme II and anti-inflammatory effects by inhibiting NO production and IL-6 expression. Four natural compounds, including two anthraquinones, chrysophanol (2) and emodin (3), and two stilbenes, rhapontigenin (1) and trans-piceatannol (4), were isolated from the roots of Rhubarb. The results showed that rhapontigenin (1) exhibited an inhibitory effect on the ACE-2 enzyme with an IC50 value of 132 mM. In an in silico study, rhapotigenin (1) had a binding energy value of -9.32 kcal/mol, lower than MLN-4760, on ACE-2. Both rhapontigenin (1) and emodin (3) exhibited inhibitory activity on NO production in primary cultures of macrophages RAW264.7, with IC50 values ranging from 5.65 to 32.3 µg/mL. This is the first study of the anti-ACE-2 and anti-inflammatory activity of Rhubarb root extract and its constituents.

The study on in vitro antioxidant properties, cellular immune activities and mice anti-fatigue effects of Sanghuangporus vanninii mycelium polysaccharides

Sanghuang mycelial polysaccharides (SVMPs) were studied using poplar sanghuang as raw material (sanghuangporus vanninii, SV). The bioactivity of SVMPs was evaluated through in vitro antioxidant and cellular immunity activity assays, and the acute oral toxicity and anti-fatigue ability of SVMPs in mice were determined using mice as an experimental animal model. The results showed that SVMPs have a good scavenging effect on 1,1-diphenyl-2-picrylhydrazyl (DPPH), hydroxyl, and 2,2′-Azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS+) radicals, with a slightly weaker reducing power. SVMPs promote, to some extent, the secretion of cytokines Tumor Necrosis Factor-α (TNF-α), Interleukin-1β (IL-1β), and Interleukin-6 (IL-6), and the release of NO from mouse macrophage RAW264.7. In mouse experiments, the mouse tolerance of SVMPs was more greater than 18.2 g/kg BW, which was 2724 times the daily dosage of humans, and it was a non-toxic substance; the high dose group of SVMPs could significantly prolong the swimming time of weight-bearing mice. Thus, the results of this study clearly demonstrate the biological activity of SVMPs and provide a valuable basis for the potential application of SVMPs as antioxidants, immunomodulators, and functional foods.

Coumarins and flavones from Ficus erecta and their anti-inflammatory activity

Ethnopharmacological relevanceFicus erecta, a traditional Chinese She Ethnomedicine, has been historically utilized to treat various inflammatory conditions such as arthritis, nephritis, and osteoporosis. However, the underlying mechanisms accounting for its anti-inflammatory activity, as well as its active components, largely remain elusive. Aim of the studyThe purpose of this research was to investigate the chemical constituents of F. erecta that contribute to its anti-inflammatory effects. Materials and methodsCoumarins and flavones were obtained from the 95% EtOH extract of F. erecta using virous column chromatography and reversed-phase semipreparative HPLC. The structures of the new compounds were elucidated by extensive analysis of spectroscopic methods, including HRESIMS, 1D and 2D NMR spectra, and CD experiments. Cultured macrophage RAW264.7 cells were utilized for the anti-inflammatory experiments. MTT cell viability assay, Griess reagent method, ELISA, and Western blot experiments were employed to evaluate the anti-inflammatory activity and investigate the related mechanism. ResultsFour new (1–4) and eleven previously identified (5–16) coumarins, together with one new (17) and six known flavones (18–23) were isolated from the whole plant of F. erecta. Compounds 7 and 17 significantly reduced nitric oxide (NO) and prostaglandin E2 (PGE2) production without cytotoxic effects. Furthermore, compounds 7 and 17 reduced the production of proinflammatory cytokines including tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in a concentration-dependent manner. Western blot analysis indicated that compounds 7 and 17 suppressed the expression of iNOS, COX-2, and p-IκBα in LPS-stimulated RAW264.7 macrophage cells. ConclusionThe current phytochemical investigations revealed that coumarins and flavones represent the primary chemical constituents of F. erecta. Compounds 7 and 17 exhibit potent anti-inflammatory properties, linked with the inhibition of NF-κB activation by preventing the degradation of IκBα phosphorylation. These compounds may serve as promising candidates for treating or preventing certain inflammatory diseases.

Anti‑obesity and immunostimulatory activity of Chrysosplenium flagelliferum in mouse preadipocytes 3T3‑L1 cells and mouse macrophage RAW264.7 cells.

Chrysosplenium flagelliferum (CF) is known for its anti-inflammatory, antioxidant and antibacterial activities. However, there is a lack of research on its other pharmacological properties. In the present study, the bifunctional roles of CF in 3T3-L1 and RAW264.7 cells were investigated, focusing on its anti-obesity and immunostimulatory effects. In 3T3-L1 cells, CF effectively mitigated the accumulation of lipid droplets and triacylglycerol. Additionally, CF downregulated the peroxisome proliferator-activated receptor (PPAR)-γ and CCAAT/enhancer-binding protein α protein levels; however, this effect was impeded by the knockdown of β-catenin using β-catenin-specific small interfering RNA. Consequently, CF-mediated inhibition of lipid accumulation was also decreased. CF increased the protein levels of adipose triglyceride lipase and phosphorylated hormone-sensitive lipase, while decreasing those of perilipin-1. Moreover, CF elevated the protein levels of phosphorylated AMP-activated protein kinase and PPARγ coactivator 1-α. In RAW264.7 cells, CF enhanced the production of pro-inflammatory mediators, such as nitric oxide (NO), inducible NO synthase, interleukin (IL)-1β, IL-6 and tumor necrosis factor-α, and increased their phagocytic capacities. Inhibition of Toll-like receptor (TLR)-4 significantly reduced the effects of CF on the production of pro-inflammatory mediators and phagocytosis, indicating its crucial role in facilitating these effects. CF-induced increase in the production of pro-inflammatory mediators was controlled by the activation of c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-κB pathways, and TLR4 inhibition attenuated the phosphorylation of these kinases. The results of the pesent study suggested that CF inhibits lipid accumulation by suppressing adipogenesis and inducing lipolysis and thermogenesis in 3T3-L1 cells, while stimulating macrophage activation via the activation of JNK and NF-κB signaling pathways mediated by TLR4 in RAW264.7 cells. Therefore, CF simultaneously exerts both anti-obesity and immunostimulatory effects.

Puerarin suppresses macrophage M1 polarization to alleviate renal inflammatory injury through antagonizing TLR4/MyD88-mediated NF-κB p65 and JNK/FoxO1 activation

BackgroundAcute kidney injury (AKI) is a clinically common and serious renal dysfunction, characterized by inflammation and damage to tubular epithelial cells. Puerarin, an isoflavone derivative isolated from Pueraria lobata, has been proven to possess exceptional effectiveness in reducing inflammation. However, the effects and underlying mechanisms of puerarin on AKI remain uncertain. PurposeThis study investigated the possible therapeutic effects of puerarin on AKI and explored its underlying mechanism. Study design and methodsThe effects of puerarin on AKI and macrophage polarization were investigated in lipopolysaccharide (LPS)-induced or unilateral ureteral obstruction (UUO)-induced mouse models in vivo and LPS-treated macrophages (Raw264.7) in vitro. Additionally, the effects of puerarin on inflammation-related signaling pathways were analyzed. ResultsAdministration of puerarin effectively alleviated kidney dysfunction and reduced inflammatory response in LPS-induced and UUO-induced AKI. In vitro, puerarin treatment inhibited the polarization of M1 macrophages and the release of inflammatory factors in Raw264.7 cells stimulated by LPS. Mechanistically, puerarin downregulated the activities of NF-κB p65 and JNK/FoxO1 signaling pathways. The application of SRT1460 to activate FoxO1 or anisomycin to activate JNK eliminated puerarin-mediated inhibition of JNK/FoxO1 signaling, leading to suppression of macrophage M1 polarization and reduction of inflammatory factors. Further studies showed that puerarin bound to Toll/interleukin-1 receptor (TIR) domain of MyD88 protein, hindering its binding with TLR4, ultimately resulting in downstream NF-κB p65 and JNK/FoxO1 signaling inactivation. ConclusionsPuerarin antagonizes NF-κB p65 and JNK/FoxO1 activation via TLR4/MyD88 pathway, thereby suppressing macrophage polarization towards M1 phenotype and alleviating renal inflammatory damage.

Characterization of a glycosyltransferase from Paris polyphylla for application in biosynthesis of rare ophiopogonins and ginsenosides

Saponins are bioactive components of many medicinal plants, possessing complicated chemical structures and extensive pharmacological activities, but the production of high-value saponins remains challenging. In this study, a 6′-O-glucosyltransferase PpUGT7 (PpUGT91AH7) was functionally characterized from Paris polyphylla Smith var. yunnanensis (Franch.) Hand. -Mazz., which can transfer a glucosyl group to the C-6′ position of diosgenin-3-O-rhamnosyl-(1 → 2)-glucoside (1), pennogenin-3-O-rhamnosyl-(1 → 2)-glucoside (2), and diosgenin-3-O-glucoside (5). The KM and Kcat values of PpUGT7 towards the substrate 2 were 8.4 μM and 2 × 10−3 s−1, respectively. Through molecular docking and site-directed mutagenesis, eight residues were identified to interact with the sugar acceptor 2 and be crucial for enzyme activity. Moreover, four rare ophiopogonins and ginsenosides were obtained by combinatorial biosynthesis, including an undescribed compound ruscogenin-3-O-glucosyl-(1 → 6)-glucoside (10). Firstly, two monoglycosides 9 and 11 were generated using a known sterol 3-O-β-glucosyltransferase PpUGT80A40 with ruscogenin (7) and 20(S)-protopanaxadiol (8) as substrates, which were further glycosylated to the corresponding diglycosides 10 and 12 under the catalysis of PpUGT7. In addition, compounds 7–11 were found to show inhibitory effects on the secretion of TNF-α and IL-6 in macrophages RAW264.7. The findings provide valuable insights into the enzymatic glycosylation processes in the biosynthesis of bioactive saponins in P. polyphylla var. yunnanensis, and also serve as a reference for utilizing UDP-glycosyltransferases to construct high-value or rare saponins for development of new therapeutic agents.

Therapeutic Modulation of Arginase with nor-NOHA Alters Immune Responses in Experimental Mouse Models of Pulmonary Tuberculosis including in the Setting of Human Immunodeficiency Virus (HIV) Co-Infection.

L-arginine metabolism is strongly linked with immunity to mycobacteria, primarily through the antimicrobial activity of nitric oxide (NO). The potential to modulate tuberculosis (TB) outcomes through interventions that target L-arginine pathways are limited by an incomplete understanding of mechanisms and inadequate in vivo modeling. These gaps in knowledge are compounded for HIV and Mtb co-infections, where activation of arginase-1 due to HIV infection may promote survival and replication of both Mtb and HIV. We utilized in vitro and in vivo systems to determine how arginase inhibition using Nω-hydroxy-nor-L-arginine (nor-NOHA) alters L-arginine pathway metabolism relative to immune responses and disease outcomes following Mtb infection. Treatment with nor-NOHA polarized murine macrophages (RAW 264.7) towards M1 phenotype, increased NO, and reduced Mtb in RAW macrophages. In Balb/c mice, nor-NOHA reduced pulmonary arginase and increased the antimicrobial metabolite spermine in association with a trend towards reduced Mtb CFU in lung. In humanized immune system (HIS) mice, HIV infection increased plasma arginase and heightened the pulmonary arginase response to Mtb. Treatment with nor-NOHA increased cytokine responses to Mtb and Mtb/HIV in lung tissue but did not significantly alter bacterial burden or viral load. Our results suggest that L-arginine pathway modulators may have potential as host-directed therapies to augment antibiotics in TB chemotherapy.

Musa basjoo Sieb polysaccharide improves inflammation in RAW264.7 cells and zebrafish colitis

Musa basjoo Sieb polysaccharide (MBSP) is the active ingredient of Musa basjoo Sieb. The purpose of this study was to preliminarily characterize the structural properties of MBSP, and to explore its ameliorating effect LPS-induced macrophage RAW264.7 inflammation and its therapeutic effect on DSS-induced zebrafish colitis. The data indicated that the average molecular weight of MBSP was 393.4 kDa, which was an acidic heteropolysaccharide composed of seven monosaccharides. In the LPS-induced macrophage RAW264.7 inflammation model, we found that MBSP significantly reduced the level of inflammatory mediators (p < 0.001). The release of NO, IL-6, and TNF-α was reduced by 48.21%, 65.45%, and 86.49%, respectively. And MBSP modulated intracellular ROS levels. This might be related to the activation of NF-κB/MAPK signaling pathway by MBSP. Additionally, MBSP significantly improved the oxidative stress status of UC zebrafish. SOD, CAT, GSH-Px were increased by 50.58%, 39.07% and 65.86%, and MDA, MPO were decreased by 26.00% and 50.98%, respectively (p < 0.001). Meanwhile, MBSP was also found to be effective in improving UC by activating the TLR4/NF-κB/MAPK signaling pathway in vivo. In conclusion, the present study characterized the structural properties of MBSP from various aspects, confirmed the anti-inflammatory effect of MBSP in vitro, and further verified the in vivo anti-inflammatory activity of MBSP using UC zebrafish as an animal model. The results of this study provide a reference for the development and application of MBSP in the field of treating inflammatory diseases.

The Marine Factor 3,5-dihydroxy-4-methoxybenzyl Alcohol Suppresses Cell Growth, Inflammatory Cytokine Production, and NF-κB Signaling-enhanced Osteoclastogenesis in In vitro Mouse Macrophages RAW264.7 Cells.

The novel marine factor 3,5-dihydroxy-4- methoxybenzyl alcohol (DHMBA) was originally identified in the Pacific oyster Crassostrea Gigas. DHMBA has been shown to prevent oxidative stress by scavenging radicals and enhance the production of antioxidant proteins. However, the pharmacologic role of DHMBA has been poorly understood. Inflammation is implicated in the pathogenesis of many diseases. Inflammatory cytokines are produced in macrophages with stimulation of lipopolysaccharide (LPS) and are used as biomarkers that cause diverse disease conditions. Therefore, this study has been undertaken to elucidate whether DHMBA expresses anti-inflammatory effects in in vitro mouse macrophage RAW264.7 cells. Mouse macrophage RAW264.7 cells were cultured in a medium containing 10% fetal bovine serum (FBS) with or without DHMBA (1-1000 μM). Culturing with DHMBA (1-1000 μM) suppressed the growth and stimulated the death of RAW264.7 cells in vitro, leading to a decrease in cell number. Treatment with DHMBA reduced the levels of Ras, PI3K, Akt, MAPK, phospho-MAPK, and mTOR, which are signalling factors to promote cell proliferation, and it raised the levels of p53, p21, Rb, and regucalcin, which are cell growth suppressors. DHMBA treatment elevated caspase-3 and cleaved caspase-3 levels. Interestingly, DHMBA treatment repressed the production of inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, interleukin-1β, or prostaglandin E2, which were enhanced by LPS stimulation. Notably, the levels of NF-κB p65 were increased by LPS treatment, and this augmentation was repres-sed by DHMBA treatment. Moreover, LPS treatment stimulated osteoclastogenesis of RAW264.7 cells. This stimulation was blocked by DHMBA treatment, and this effect was not caused by the presence of an NF-κB signalling inhibitor. DHMBA was found to potentially suppress the activity of inflammatory macrophages in vitro, suggesting its therapeutic usefulness in inflammatory conditions.

In Vitro Cell Culture Model for Osteoclast Activation during Estrogen Withdrawal.

Estrogen (17β-estradiol) deficiency post-menopause alters bone homeostasis whereby bone resorption by osteoclasts exceeds bone formation by osteoblasts, leading to osteoporosis in females. We established an in vitro model to examine the consequences of estrogen withdrawal (E2-WD) on osteoclasts derived from the mouse macrophage RAW 264.7 cell line and utilized it to investigate the mechanism behind the enhanced osteoclast activity post-menopause. We found that a greater population of osteoclasts that underwent E2-WD contained a podosome belt necessary for osteoclasts to adhere and resorb bone and possessed elevated resorptive activity compared to osteoclasts exposed to estrogen (E2) continuously. Our results show that compared to osteoclasts that received E2 continuously, those that underwent E2-WD had a faster rate of microtubule (MT) growth, reduced RhoA activation, and shorter podosome lifespan. Thus, altered podosome and MT dynamics induced by the withdrawal of estrogen supports podosome belt assembly/stability in osteoclasts, which may explain their enhanced bone resorption activity.

PTHrP participates in the bone destruction of middle ear cholesteatoma via promoting macrophage differentiation into osteoclasts induced by RANKL.

Progressive bone resorption and destruction is one of the most critical clinical features of middle ear cholesteatoma, potentially leading to various intracranial and extracranial complications. However, the mechanisms underlying bone destruction in middle ear cholesteatoma remain unclear. This study aims to explore the role of parathyroid hormone-related protein (PTHrP) in bone destruction associated with middle ear cholesteatoma. A total of 25 cholesteatoma specimens and 13 normal external auditory canal skin specimens were collected from patients with acquired middle ear cholesteatoma. Immunohistochemical staining was used to detect the expressions of PTHrP, receptor activator for nuclear factor-kappa B ligand (RANKL), and osteoprotegerin (OPG) in cholesteatoma and normal tissues. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the presence of TRAP positive multi-nucleated macrophages in cholesteatoma and normal tissues. Mono-nuclear macrophage RAW264.7 cells were subjected to interventions, divided into a RANKL intervention group and a PTHrP+ RANKL co-intervention group. TRAP staining was used to detect osteoclast formation in the 2 groups. The mRNA expression levels of osteoclast-related genes, including TRAP, cathepsin K (CTSK), and nuclear factor of activated T cell cytoplasmic 1 (NFATc1), were measured using real-time polymerase chain reaction (real-time PCR) after the interventions. Bone resorption function of osteoclasts was assessed using a bone resorption pit analysis. Immunohistochemical staining showed significantly increased expression of PTHrP and RANKL and decreased expression of OPG in cholesteatoma tissues (all P<0.05). PTHrP expression was significantly positively correlated with RANKL, the RANKL/OPG ratio, and negatively correlated with OPG expression (r=0.385, r=0.417, r=-0.316, all P<0.05). Additionally, the expression levels of PTHrP and RANKL were significantly positively correlated with the degree of bone destruction in cholesteatoma (r=0.413, r=0.505, both P<0.05). TRAP staining revealed a large number of TRAP-positive cells, including multi-nucleated osteoclasts with three or more nuclei, in the stroma surrounding the cholesteatoma epithelium. After 5 days of RANKL or PTHrP+RANKL co-intervention, the number of osteoclasts was significantly greater in the PTHrP+RANKL co-intervention group than that in the RANKL group (P<0.05), with increased mRNA expression levels of TRAP, CTSK, and NFATc1 (all P<0.05). Scanning electron microscopy of bone resorption pits showed that the number (P<0.05) and size of bone resorption pits on bone slices were significantly greater in the PTHrP+RANKL co-intervention group compared with the RANKL group. PTHrP may promote the differentiation of macrophages in the surrounding stroma of cholesteatoma into osteoclasts through RANKL induction, contributing to bone destruction in middle ear cholesteatoma.

Cigarette smoke sustains immunosuppressive microenvironment inducing M2 macrophage polarization and viability in lung cancer settings.

It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.

Sulfate glycosaminoglycan from swim bladder exerts immunomodulatory potential on macrophages via toll-like receptor 4 mediated NF-κB signaling pathways

This study explored the immunomodulatory impact and potential mechanisms on macrophages RAW264.7 using a purified macromolecular sulfate glycosaminoglycan (SBSG) from the swim bladder, whose structure was similar to chondroitin sulfate A. The results showed that SBSG at 0.25–1 mg/mL increased the viability and phagocytosis of RAW264.7 cells. Meanwhile, SBSG promoted the secretion of tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), and nitric oxide (NO), as well as the production of reactive oxygen species (ROS). According to the RT-PCR and Western blot data, SBSG activated TLR4-nuclear factor kappa B (NF-κB) signaling pathways, which decreased the relative mRNA and protein levels of Toll-like receptor 4 (TLR4), IκB kinase β (IKKβ), NF-κB p65, and p-NF-κB p65. The molecular docking and molecular dynamic simulation findings revealed that the main binding force between TLR4 and SBSG was conventional hydrogen bond interaction, resulting in more stable ligand receptor complexes. In summary, SBSG exhibits significant immunomodulatory potential, similar to chondroitin sulfate C. The underlying molecular mechanism involved the binding of SBSG through hydrogen bonding to TLR4 receptors, triggering the NF-κB signaling pathway to downregulate the expression of related genes and proteins. This, in turn, regulated the secretion of various cytokines that were mediated by macrophages to exert the immunity of the body.

Leishmania exploits host cAMP/EPAC/calcineurin signaling to induce an IL-33–mediated anti-inflammatory environment for the establishment of infection

Host anti-inflammatory responses are critical for the progression of visceral leishmaniasis and the pleomorphic cytokine IL-33 was found to be upregulated in infection. The underlying mechanism is not yet known. Here, we documented that IL-33 induction is a consequence of elevated cAMP-mediated EPAC/calcineurin-dependent signaling and is essential for the sustenance of infection. L. donovani-infected RAW and bone marrow-derived macrophages showed significant up-regulation of IL-33 and its neutralization by anti-IL-33 antibody resulted in decreased parasite survival and increased inflammatory responses. Infection-induced elevated cAMP was involved in IL-33 production and of its two downstream effectors PKA and EPAC, only the latter was responsible for elevated IL-33 levels. EPAC initiated Rap-dependent phospholipase C activation, which triggered the release of intracellular calcium followed by calcium/calmodulin complex formation. Screening of calmodulin-dependent enzymes affirmed the involvement of the phosphatase calcineurin in cAMP/EPAC/calcium/calmodulin signaling-induced IL-33 production and parasite survival. Activated calcineurin thereby ensured nuclear localization of the transcription factors NFATc1 and HIF-1α for IL-33 transcription and we further ascertained their role in IL-33 transcription using a ChIP assay. Administering specific inhibitors of NFATc1 and HIF-1α in a BALB/c mouse model of visceral leishmaniasis led to decreased liver and spleen parasite burden with concomitant decrease in IL-33 levels. Splenocyte supernatants of inhibitor-treated infected mice further documented an increase in TNF-α and IL-12 with simultaneous decrease of IL-10, thereby indicating an overall disease-escalating effect of IL-33. Thus, this study demonstrates that cAMP/EPAC/calcineurin signaling is crucial for the activation of IL-33 and in effect creates anti-inflammatory responses, essential for infection.

Abstract PO4-28-11: Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells

Abstract Several studies in the past have demonstrated the role of cell-to-cell interaction (crosstalk) between tumor cells and normal (nontumorigenic) cells in cancer progression and metastasis. Within the tumor microenvironment (TME), these interactions have a potential to transform the phenotypes and the behaviors of normal cells. Application of the 2D in-vitro cultures has been limited due to its inability to replicate the complex in-vivo TME. Conditioned Medium (CM) obtained from cultured cancer cells contains secreted growth factors that potentially regulate the phenotype and the functionality of normal cells. In this study, a culture of normal murine fibroblast NIH3T3 and macrophage RAW 264.7 cells with conditioned medium (CM) obtained from malignant mammary epithelial 4T1 cells (4T1CM) resulted in an altered phenotype with increased cell viability. 4T1CM treated NIH3T3 and RAW 264.7, in comparison with the respective control cells, resulted in an upregulation of the genes including- α-smooth muscle actin (αSMA), IL-10, CD206, and vascular endothelial growth factor (VEGF). In addition, 4T1.CM treated NIH3T3 showed an EMT phenotype as indicated by the regulation of EMT markers such as, E-cadherin, β-catenin, N-cadherin, and Vimentin. Interestingly, an upregulation of cyclooxygenase-2 (COX-2) and programmed death-ligand 1 (PDL-1) was observed in 4T1CM treated RAW 264.7, a tendency towards exhibiting an inhibitory immune response. Intriguingly, NIH3T3 cells conditioned with 4T1CM demonstrated an upregulation of stemness markers including- sex determining region Y-box 2, and Aldehyde dehydrogenase. Collectively, our study suggested a role for 4T1CM in transforming the normal NIH3T3 and RAW 264.7 into cancer-associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). RNAseq experiments are underway to map differentially expressed genes (DEGs) that potentially regulate the 4T1CM induced transformation of NIH3T3 and RAW 264.7 cells. Furthermore, invitro drug testing in 3D model of these transformed cells to target the pathway intermediates may provide novel therapeutic intervention strategies. Citation Format: Biplov Sapkota, Naveen Chintalaramulu, Abhishek Pandit, Shilpa Thota, Rizwana Begum, Joseph Francis. Murine Breast Cancer Cell Culture Supernatant Induces CAF-like and TAM-like Traits in Normal Cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-28-11.

Salvimulticanol from Salvia multicaulis suppresses LPS-induced inflammation in RAW264.7 macrophages: in vitro and in silico studies.

Sustained inflammatory responses can badly affect several vital organs and lead to chronic inflammation-related disorders, such as atherosclerosis, pneumonia, rheumatoid arthritis, obesity, diabetes, Alzheimer's disease, and cancers. Salvia multicaulis is one of the widely distributed plants that contains several biologically active phytochemicals and diterpenoids with anti-inflammatory effects. Therefore, finding alternative and safer natural plant-extracted compounds with good curative anti-inflammatory efficiencies is an urgent need for the clinical treatment of inflammation-related diseases. In the current study, S. multicaulis Vahl was used to extract and isolate two compounds identified as salvimulticanol and candesalvone B methyl ester to examine their effects against inflammation in murine macrophage RAW264.7 cells that were induced by lipopolysaccharide (LPS). Accordingly, after culturing RAW264.7 cells and induction of inflammation by LPS (100ng/ml), cells were exposed to different concentrations (9, 18, 37.5, 75, and 150µM) of each compound. Then, Griess assay for detection of nitric oxide (NO) levels and western blotting for the determination of inducible nitric oxide synthase (iNOS) expression were performed. Molecular docking and molecular dynamics (MD) simulation studies were employed to investigate the anti-inflammatory mechanism. Our obtained results validated that the level of NO was significantly decreased in the macrophage cell suspensions as a response to salvimulticanol treatment in a dose-dependent manner (IC50: 25.1 ± 1.2µM) as compared to the methyl ester of candesalvone B which exerted a weaker inhibition (IC50: 69.2 ± 3.0µM). This decline in NO percentage was comparable with a down-regulation of iNOS expression by western blotting. Salvimulticanol strongly interacted with both the Toll-like receptor 4 (TLR4)/myeloid differentiation factor 2 (MD-2) complex and the inhibitor of nuclear factor kappa-B (NF-κB) kinase subunit beta (IKKβ) to disrupt their inflammatory activation due to the significant hydrogen bonds and effective interactions with amino acid residues present in the target proteins' active sites. S.multicaulis is a rich natural source of the aromatic abietane diterpenoid, salvimulticanol, which exerted a strong anti-inflammatory effect through targeting iNOS and diminishing NO production in LPS-induced RAW264.7 cells in a mechanism that is dependent on the inhibition of TLR4-MD-2 and IKKβ as activators of the classical NF-κB-mediated inflammatory pathway.