Mouse Macrophage Cell Line RAW264 Research Articles

Oroxyloside protects against dextran sulfate sodium-induced colitis by inhibiting ER stress via PPARγ activation

Ulcerative colitis (UC), a prevalent form of inflammatory bowel disease (IBD), may result from immune system dysfunction, leading to the sustained overproduction of reactive oxygen species (ROS) and subsequent cellular oxidative stress damage. Recent studies have identified both peroxisome proliferator-activated receptor-γ (PPARγ) and endoplasmic reticulum (ER) stress as critical targets for the treatment of IBD. Oroxyloside (C22H20O11), derived from the root of Scutellariabaicalensis Georgi, has traditionally been used in treating inflammatory diseases. In this study, we investigated the molecular mechanisms by which oroxyloside mitigates dextran sulfate sodium (DSS)-induced colitis. We examined the effects of oroxyloside on ROS-mediated ER stress in colitis, including the protein expressions of GRP78, p-PERK, p-eIF2α, ATF4, and CHOP, which are associated with ER stress. The beneficial impact of oroxyloside was reversed by the PPARγ antagonist GW9662 (1 mg·kg-1, i.v.) in vivo. Furthermore, oroxyloside decreased pro-inflammatory cytokines and ROS production in both bone marrow-derived macrophages (BMDM) and the mouse macrophage cell line RAW 264.7. However, PPARγ siRNA transfection blocked the anti-inflammatory effect of oroxyloside and even abolished ROS generation and ER stress activation inhibited by oroxyloside in vitro. In conclusion, our study demonstrates that oroxyloside ameliorates DSS-induced colitis by inhibiting ER stress via PPARγ activation, suggesting that oroxyloside might be a promising effective agent for IBD.

Abstract 486: Paclitaxel nanoformulation attenuates pancreatic tumor desmoplasia and alter tumor immune responses

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal types of cancer with a 5-year survival rate of less than 13% due to the lack of effective diagnostic/therapeutic modalities. Paclitaxel (PTX) has been tested in pancreatic cancer (PanCa) therapy with marginally better clinical outcomes, but remains limited by its poor hemocompatibility, biodistribution, and intracellular accumulation in tumor cells. Gemcitabine (GEM) is the most effective therapy for PanCa. However, it shows only a marginal survival benefit of 6 months. The poor therapy response is attributed to high desmoplasia, unfavorable tumor microenvironment (TME), and excessive recruitment of immune suppressive cells. We have recently engineered a multi-layered Pluronic F127 and polyvinyl alcohol stabilized and poly-L-lysine coated paclitaxel loaded poly(lactic-co-glycolic acid) nanoparticle formulation (PPNPs), which effectively inhibits pancreatic tumor. In this study, we demonstrate that PPNPs effectively sensitize tumor cells to GEM via attenuation of tumor desmoplasia. Our findings also show that PPNPs synergize GEM therapy response in cell lines and in vivo mouse models. Mechanistically, PPNPs target the TME via inhibition of the sonic hedgehog (SHH) pathway and other oncogenic signaling axis that inhibits bidirectional tumor-stromal cell interaction as determined by qPCR and Western blot analyses. In addition, PPNPs effectively target tumor-associated macrophages (TAM) by repolarizing M2 into M1 phenotype via inhibiting expression of M2 markers and an increase in M1 markers in mouse macrophage cell line RAW264.7. M2 polarization of RAW264.7 cells were induced by culture with IL-4 (20 ng/mL) in the presence of PPNPs or vehicle control. Furthermore, PPNPs effectively increase phagocytic capacity in murine macrophages as determined by phagocytosis assay (Vybrant Phagocytosis Assay Kit). In conclusion, we observed that our novel PPNP nano formulation effectively targets TME, and facilitates GEM uptake by inhibiting the activation of SHH signaling and reprograming the tumor immune surveillance mechanisms. This study suggests that PPNPs have great potential for future clinical use in management of pancreatic cancer. Citation Format: Vivek K. Kashyap, Godwin P. Darkwah, Neeraj Chauhan, Mohammed Sikander, Eswara N. Ghali, Meghana Kolli, Bilal B. Hafeez, Murali M. Yallapu, Subhash C. Chauhan. Paclitaxel nanoformulation attenuates pancreatic tumor desmoplasia and alter tumor immune responses [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 486.

Function of Burkholderia pseudomallei RpoS and RpoN2 in bacterial invasion, intracellular survival, and multinucleated giant cell formation in mouse macrophage cell line.

Melioidosis, a human infectious disease with a high mortality rate in many tropical countries, is caused by the pathogen Burkholderia pseudomallei (B. pseudomallei). The function of the B. pseudomallei sigma S (RpoS) transcription factor in survival during the stationary growth phase and conditions of oxidative stress is well documented. Besides the rpoS, bioinformatics analysis of B. pseudomallei genome showed the existence of two rpoN genes, named rpoN1 and rpoN2. In this study, by using the mouse macrophage cell line RAW264.7 as a model of infection, the involvement of B. pseudomallei RpoS and RpoN2 in the invasion, intracellular survival leading to the reduction in multinucleated giant cell (MNGC) formation of RAW264.7 cell line were illustrated. We have demonstrated that the MNGC formation of RAW264.7 cell was dependent on a certain number of intracellular bacteria (at least 5 × 104). In addition, the same MNGC formation (15%) observed in RAW264.7 cells infected with either B. pseudomallei wild type with multiplicity of infection (MOI) 2 or RpoN2 mutant (∆rpoN2) with MOI 10 or RpoS mutant (∆rpoS) with MOI 100. The role of B. pseudomallei RpoS and RpoN2 in the regulation of type III secretion system on bipB-bipC gene expression was also illustrated in this study.

Immunotoxicity Evaluation of Trihalophenolic Disinfection By-Products in Mouse and Human Mononuclear Macrophage Systems: The Role of RNA Epitranscriptomic Modification in Mammalian Immunity.

2,4,6-Trichlorophenol (TCP), 2,4,6-tribromophenol (TBP) and 2,4,6-triiodophenol (TIP) are three widely detected trihalophenolic disinfection by-products (DBPs). Previous studies have mainly focused on the carcinogenic risk and developmental toxicity of 2,4,6-trihalophenols. Very little is known about their immunotoxicity in mammals. We investigated the effects of 2,4,6-trihalophenols on mammalian immunity using a mouse macrophage model infected with bacteria or intracellular parasites and aimed to elucidate the underlying mechanisms from an epitranscriptomic perspective. The identified mechanisms were further validated in human peripheral blood mononuclear cells (PBMCs). The mouse macrophage cell line RAW264.7 and primary mouse peritoneal macrophages were exposed to different concentrations of TCP, TBP, and TIP. The pro-inflammatory marker Ly6C, the survival of the bacterium Escherichia coli (E. coli), and the parasite burden of Toxoplasma gondii (T. gondii) were assessed. Furthermore, the global gene expression profiling of macrophages following exposure to 2,4,6-trihalophenols was obtained through RNA-sequencing (RNA-seq). The effects of 2,4,6-trihalophenols on RNA () methyltransferases and total RNA levels were evaluated using Western blotting and dot blot, respectively. Transcriptome-wide methylome was analyzed by . In addition, expression of regulators and total RNA levels in human PBMCs exposed to 2,4,6-trihalophenols were detected using quantitative reverse transcriptase polymerase chain reaction and dot blot, respectively. Mouse macrophages exposed to TCP, TBP, or TIP had lower expression of the pro-inflammatory marker Ly6C, with a greater difference from control observed for TIP-exposed cells. Consistently, macrophages exposed to such DBPs, especially TIP, were susceptible to infection with the bacterium E. coli and the intracellular parasite T. gondii, indicating a compromised ability of macrophages to defend against pathogens. Intriguingly, macrophages exposed to TIP had significantly greater levels, which correlated with the greater expression levels of methyltransferases. Macrophages exposed to each of the three 2,4,6-trihalophenols exhibited transcriptome-wide redistribution of . In particular, the peaks in genes associated with immune-related pathways were altered after exposure. In addition, differences in were also observed in human PBMCs after exposure to 2,4,6-trihalophenols. These findings suggest that 2,4,6-trihalophenol exposure impaired the ability of macrophages to defend against pathogens. This response might be associated with notable differences in after exposure. To the best of our knowledge, this study presents the first landscape across the transcriptome of immune cells exposed to pollutants. However, significant challenges remain in elucidating the mechanisms by which mediates immune dysregulation in infected macrophages after 2,4,6-trihalophenol exposure. https://doi.org/10.1289/EHP11329.

Diosgenin alters LPS-induced macrophage polarization by activating PPARγ/NF-κB signaling pathway

Diosgenin (DG) is a steroidal saponin derived from plants, and it exhibits anti-inflammatory properties. In this study, we employed an in vitro model of P.g.-LPS-stimulated mouse macrophage cell line RAW264.7 to investigate the anti-inflammatory effects and mechanism of DG under the condition of altered polarization of macrophages. The RAW264.7 cells were subjected to pre-treatment with DG with or without P.g.-LPS. In cultured macrophages, DG inhibited P.g.-LPS-induced pro-inflammatory M1 macrophages, and increased anti-inflammatory M2 macrophages. Notably, DG reduced the expression of phosphorylation levels of NF-κB p65 and IκB while increasing the expression of PPARγ. Further studies revealed that PPARγ inhibitor GW9662 or PPARγ siRNA reversed the inhibitory effect of DG on M1 phenotype. Collectively, the anti-inflammatory mechanism of DG is related to altering macrophage polarization by activating PPARγ and inhibiting NF-κB signaling pathways.

MFG-E8 facilitates heart repair through M1/M2 polarization after myocardial infarction by inhibiting CaMKII

BackgroundM1/M2 macrophage polarization affects patient outcomes after myocardial infarction (MI). The relationship between milk fat globule-epidermal growth factor 8 (MFG-E8) and Ca2+/calmodulin-dependent protein kinase II (CaMKII) on macrophage polarization after MI is unknown. To investigate the functional role of MFG-E8 in modulating cardiac M1/M2 macrophage polarization after MI, especially its influence on CaMKII signaling. MethodsHuman ventricular tissue and blood were obtained from patients with MI and controls. MFG-E8-KO mice were constructed (C57BL/6). The mice were randomized to WT-sham, sham-MFG-E8-KO, WT-PBS, rmMFG-E8 (WT injected with rmMFG-E8 10 min after MI), and MFG-E8-KO. The mouse macrophage cell line RAW264.7 was obtained. CaMKII, p-CaMKII, Akt, and NF-κB p65 were determined by qRT-PCR, western blot, and immunofluorescence. ResultsThe MFG-E8 levels were significantly enhanced after MI in the hearts and plasma of patients with MI compared with controls. The MFG-E8 levels were significantly increased in the hearts and plasma of mice after MI. MFG-E8 was derived from cardiac fibroblasts. The administration of rmMFG-E8 improved ventricular remodeling and cardiac function after MI. rmMFG-E8 did not suppress infiltrating monocyte/macrophages into the peri-infarct area. rmMFG-E8 suppressed the polarization of macrophages to the M1 phenotype and promoted the polarization of macrophages to the M2 phenotype. rmMFG-E8 suppressed CaMKII-dependent signaling in macrophages. ConclusionsMFG-E8 and CaMKII appear to collaboratively regulate myocardial remodeling and M1/M2 macrophage polarization after MI. These observations suggest new roles for MFG-E8 in inhibiting M1 but promoting M2 macrophage polarization.

Producing genetically engineered macrophages with enhanced immunity via microinjection.

Many virus-mediated and chemical-based methods for delivering foreign genes into target cells, such as recombinant lentivirus transfection and cationic lipid transfection, are remarkably challenging to use on immune cells because of low efficiency and high toxicity. Microinjection is a promising method to deliver foreign gene expression plasmids into single macrophages directly. This paper reports a new method that can be used to produce a genetically engineered macrophage cell line with enhanced immunity through a home-made high-throughput microinjection system. Microinjection of the expression plasmid carrying a mouse-derived toll-like receptor 4 (Tlr4) gene into a mouse macrophage cell line (Raw264.7) can construct a new stable cell line overexpressing the target gene. The expression efficiency of the target gene in the injected Raw264.7 cells reached 90%, which was measured by injecting a particular plasmid carrying a fused enhanced green fluorescent protein (eGFP) gene fragment with the Tlr4 gene and counting the proportion of cells that emitted green fluorescence. Further assessment of the messenger RNA (mRNA) and protein produced by the Tlr4 gene indicated that its expression was up-regulated remarkably in successfully injected cells. The expression of downstream genes of Tlr4 in injected cells was higher than in untouched cells. Microinjection can avoid polarization effects, which are common when traditional transfection methods are used. A case study was conducted to verify that the injected macrophages overexpressing Tlr4 could activate downstream signaling pathways and showed enhanced inhibition effect on tumor cell migration and invasion. The success of this research will verify that microinjection can be an efficient and safe method in cell transfection applications.

Exosomes from Cyclic Stretched Periodontal Ligament Cells Induced Periodontal Inflammation through miR-9-5p/SIRT1/NF-κB Signaling Pathway.

Abundant evidence demonstrates that mechanical stress could induce an inflammatory response in periodontal tissue, but the precise mechanism remains unclear. In the past few years, periodontal ligament cells (PDLCs), as the most force-sensitive cells, have been investigated in depth as local immune cells, associated with activation of inflammasomes and secretion of inflammatory cytokines in response to mechanical stimuli. However, this study innovatively inspected the effect of PDLCs on the other immune cells after stretch loading to reveal the detailed mechanism by which mechanical stimuli initiate immunoreaction in periodontium. In the present study, we found that cyclic stretch could stimulate human PDLCs to secret exosomes and that these exosomes could further induce the increase of phagocytic cells in the periodontium in Sprague-Dawley rats and the M1 polarization of the cultured macrophages (including the mouse macrophage cell line RAW264.7 and the bone marrow-derived macrophages from C57BL/6 mice). Furthermore, the exosomal miR-9-5p was detected to be overexpressed after mechanical stimuli in both invivo and invitro experiments and could trigger M1 polarization via the SIRT1/NF-κB signaling pathway in the cultured macrophages. In summary, this study revealed that PDLCs could transmit the mechanobiological signals to immune cells by releasing exosomes and simultaneously enhance periodontal inflammation through the miR-9-5p/SIRT1/NF-κB pathway. We hope that our research can improve understanding of force-related periodontal inflammatory diseases and lead to new targets for treatment.

Abstract 823: Piperlongumine nanoformulation enhances gemcitabine therapeutic response in pancreatic cancer

Abstract Pancreatic cancer (PanCa) is characterized by lack of early diagnosis, poor response to available therapeutic modalities and chemoresistance. Gemcitabine (GEM) is currently considered most effective therapy for PanCa; however, it shows only a marginal survival benefit of 6 months. This poor drug response has been attributed to desmoplasia, causes suboptimal drug delivery, alters tumor microenvironment (TME), which includes tumor surrounding blood vessels, fibroblasts, immune cells, extracellular matrix, and other signaling molecules and induces chemo-resistance in tumors. To overcome these existing issues associated with chemotherapy, identification and development of novel therapeutic modalities are a pressing need. Piperlongumine (PL) is a natural alkaloid isolated from the long pepper, Piper longum L., and has shown substantial cancer-preventive and therapeutic efficacy against a variety of cancers. However, delivering its effective concentration in pancreatic tumors has been challenging. We have recently engineered a multi-layered Pluronic F127 and polyvinyl alcohol stabilized and poly-L-lysine coated piperlongumine loaded poly(lactic-co-glycolic acid) nanoparticle formulation (PLGA-PL), which effectively inhibits the growth of PanCa cells. In this study, we demonstrate that PLGA-PL effectively sensitize tumor cells to GEM via decreased desmoplasia, altered TME, SHH/CXCL12/CXCR4 and immune surveillance. Our finding show that PLGA-PL synergizes with GEM in inhibiting PanCa cell (HPAF-II and Panc-1) growth, migration, and invasion compared to free PL. Mechanistically, PLGA-PL targets the TME via inhibition of sonic hedgehog (SHH) pathway and oncogenic CXCR4/CXCL12 signaling axis that inhibits bidirectional tumor-stromal cells interaction. We have also found that PLGA-PL alone and in combination with GEM targets cancer stem cells by inhibiting pluripotency maintaining stemness factors (Nanog, Sox2, c-Myc, CD133, and Oct-4) as determined by qRT-PCR, Western blotting, and immunofluorescence analysis, and further confirmed by restricting tumor sphere formation. Furthermore, PLGA-PL also effectively targets tumor-associated macrophages (TAM) by repolarizing M2 into M1 phenotype via inhibiting expression of M2 markers and an increase in M1 markers in mouse macrophage cell line RAW264.7. M2 polarization of RAW264.7 cells were induced by culture with IL-4 (20 ng/mL) in presence of PLGA-PL or vehicle control. In additions, PLGA-PL effectively increases phagocytic capacity in murine macrophages as determined by phagocytosis assay (Vybrant Phagocytosis Assay Kit). In conclusion, we observed that PLGA-PL effectively targets TME, facilitates GEM uptake by inhibiting the activation of CXCR4/CXCL12/SHH signaling, and reprograming the tumor immune surveillance. This study suggests that PLGA-PL has great potential for future clinical use in management of PanCa. Citation Format: Vivek Kumar Kashyap, Neeraj Chauhan, Mohammed Sikander, FNU Pranav, Rahul Tiwari, Bilal B. Hafeez, Murali M. Yallapu, Meena Jaggi, Subhash C. Chauhan. Piperlongumine nanoformulation enhances gemcitabine therapeutic response in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 823.

Synthesis and Biological Evaluation of Gentiopicroside Derivatives as Novel Cyclooxygenase-2 Inhibitors with Anti-Inflammatory Activity.

Nonsteroidal anti-inflammatory drugs cause a series of adverse reactions. Thus, the search for new cyclooxygenase-2 selective inhibitors have become the main direction of research on anti-inflammatory drugs. Gentiopicroside is a novel selective inhibitor of cyclooxygenase-2 from Chinese herbal medicine. However, it is highly hydrophilic owing to the presence of the sugar fragment in its structure that reduces its oral bioavailability and limits efficacy. This study aimed to design and synthesize novel cyclooxygenase-2 inhibitors by modifying gentiopicroside structure and reducing its polarity. We introduced hydrophobic acyl chloride into the gentiopicroside structure to reduce its hydrophilicity and obtained some new derivatives. Their in vitro anti-inflammatory activities were evaluated against NO, TNF-α, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by lipopolysaccharide. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Molecular docking predicted that whether new compounds could effectively bind to target protein cyclooxygenase-2. The inhibitory activity of new compounds to cyclooxygenase-2 enzyme were verified by the in vitro experiment. A total of 21 novel derivatives were synthesized, and exhibit lower polarities than the gentiopicroside. Most compounds have good in vitro anti-inflammatory activity. The in vivo activity results demonstrated that 8 compounds were more active than gentiopicroside. The inhibition rate of some compounds was higher than celecoxib. Molecular docking predicted that 6 compounds could bind to cyclooxygenase-2 and had high docking scores in accordance with their potency of the anti-inflammatory activity. The confirmatory experiment proved that these 6 compounds had significant inhibitory effect against cyclooxygenase-2 enzyme. Structure-activity relationship analysis presumed that the para-substitution with the electron-withdrawing groups may benefit the anti-inflammatory activity. These gentiopicroside derivatives especially PL-2, PL-7 and PL-8 may represent a novel class of cyclooxygenase-2 inhibitors and could thus be developed as new anti-inflammatory agents.

Cloning, DNA sequence, and expression of flagellins from high and low virulence strains of Edwardsiella tarda and their macrophage-stimulating activities.

Edwardsiella tarda is a causative pathogen of edwardsiellosis in fish. Our previous studies on high (NUF251) and low (NUF194) virulent strains of E. tarda demonstrated that NUF251 strain induced significantly higher levels of NO and TNF-α from fish and mouse macrophages than NUF194 strain. Subsequent studies suggested that a flagellin-like protein secreted from E. tarda might be a responsible factor for the macrophage-stimulating activities. To evaluate the activities of flagellins of E. tarda, in this study, the flagellin genes of NUF251 and NUF194 strains were isolated by PCR and cloned into pQE-30 and pCold I expression vectors, and then the recombinant flagellins of two strains were overexpressed in E. coli JM109 and pG-Tf/BL21, respectively. The molecular weight of the purified recombinant flagellins of NUF251 and NUF194 strains were estimated to be 45kDa and 37kDa, respectively on SDS-PAGE analysis. Referring the three-dimensional structure of Salmonella flagellin, which has been reported to have 4 domains (D0, D1, D2, and D3), high sequence homology between two flagellins of E. tarda was observed at conservative domain (D0 and D1) regions, whereas the sequences equivalent to D2 and D3 domains were different, and even equivalent to 57 amino acids were deleted in NUF194. Both recombinant flagellins induced NO production, mRNA expression level of inducible NO synthase (iNOS), and intercellular ROS production in mouse macrophage cell line RAW264.7cells. Also, the secretion of TNF-α and its mRNA expression level were increased by treatment of both recombinant flagellins. These results indicate that the recombinant flagellins from different virulent E. tarda strains can stimulate macrophages with nearly equal levels as judged by the parameters tested, even though they are differences in the structure and molecular weight, suggesting that conservative D0 and D1 domains are sufficient structural elements for the recombinant flagellins to induce a certain level of macrophage-stimulation in vitro. Further studies are necessary focusing on the role of D2 and D3 domain regions of the recombinant flagellins as macrophage-stimulating agent as well as their influence on host immune system in vivo.

Adenosine receptor activation promotes macrophage class switching from LPS-induced acute inflammatory M1 to anti-inflammatory M2 phenotype

Lipopolysaccharide induced monocytes/macrophages exhibit a pro-inflammatory M1 phenotype. Elevated levels of the purine nucleoside adenosine play a major role in this response. The role of adenosine receptor modulation in directing the macrophage phenotype switch from proinflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype is investigated in this study. The mouse macrophage cell line RAW 264.7 was used as the experimental model and stimulated with Lipopolysaccharide (LPS) at a dose of 1 μg/ml. Adenosine receptors were activated by treating cells with the receptor agonist NECA (1 μM). Adenosine receptor stimulation in macrophages is found to suppress LPS-induced production of proinflammatory mediators (pro-inflammatory cytokines, Reactive Oxygen Species and nitrite levels). M1 marker CD38 (Cluster of Differentiation 38) and CD83 (Cluster of Differentiation 83) were significantly decreased while M2 markers Th2 cytokines, Arginase, TIMP (Tissue Inhibitor of Metalloproteinases) and CD206 (Cluster of Differentiation 206) exhibited an increase. Hence from our study we observed that activation of adenosine receptors can program the macrophages from a pro-inflammatory classically activated M1 phenotype to an anti-inflammatory alternatively activated M2 phenotype. We report the significance and a time course profile of phenotype switching by receptor activation. Adenosine receptor targeting may be explored as a therapeutic intervention strategy in addressing acute inflammation.

Curcumin Ameliorates Particulate Matter-Induced Pulmonary Injury through Bimodal Regulation of Macrophage Inflammation via NF-κB and Nrf2.

The direct effects of particulate matter (PM) on lung injury and its specific molecular mechanisms are unclear. However, experimental evidence has shown that oxidative stress-mediated inflammation in macrophages is the main pathological outcome of PM exposure. Curcumin has been reported to protect organs against the disturbance of homeostasis caused by various toxic agents through anti-inflammatory and antioxidative effects. However, the protective action of curcumin against PM-induced pulmonary inflammation and the underlying mechanism have not been thoroughly investigated. In this study, we established a PM-induced pulmonary inflammation mouse model using the intratracheal instillation method to investigate the protective ability of curcumin against PM-induced pulmonary inflammation. Compared to the mice treated with PM only, the curcumin-treated mice showed alleviated alveolar damage, decreased immune cell infiltration, and reduced proinflammatory cytokine production in both lung tissue and BALF. To evaluate the underlying mechanism, the mouse macrophage cell line RAW264.7 was used. Pretreatment with curcumin prevented the production of PM-induced proinflammatory cytokines by deactivating NF-κB through the suppression of MAPK signaling pathways. Furthermore, curcumin appears to attenuate PM-induced oxidative stress through the activation of Nrf2 and downstream antioxidant signaling. Our findings demonstrate that curcumin protects against PM-induced lung injury by suppressing oxidative stress and inflammatory activation in macrophages.

Amphiphilic Phosphorus Dendrons Associated with Anti-inflammatory siRNA Reduce Symptoms in Murine Collagen-Induced Arthritis.

Small interfering RNA (siRNA) holds promise for treating rheumatoid arthritis by inhibiting major cytokines such as tumor necrosis factor-α (TNF-α). We developed original cationic amphiphilic phosphorus dendrons to produce dendriplexes associated with TNF-α siRNA. The dendrons were made of 10 pyrrolidinium end groups and a C17 aliphatic chain. The dendriplexes demonstrated the ability to protect siRNA from nuclease degradation and to promote macrophage uptake. Moreover, they led to potent inhibition of TNF-α expression in the lipopolysaccharide-activated mouse macrophage cell line RAW264.7 in vitro model. A significant anti-inflammatory effect in the murine collagen-induced arthritis model was observed through arthritis scoring and histological observations. These results open up essential perspectives in using this original amphiphilic dendron to reduce the disease burden and improve outcomes in chronic inflammatory diseases.

Pyroptosis induced by TLR9 ligand, ODN1826, requires ROS production and caspase-11 activation in Raw264.7 cells.

Toll-like receptor 9 (TLR9), located in the endosomal compartment, is known to play a role in inflammation by recognizing oligonucleotides that contain CpG motive (CpG-ODN). Signaling by TLR9 leads to the production of proinflammatory cytokines and can trigger cell death. This study aims to investigate the molecular mechanism of pyroptosis induced by ODN1826 in the mouse macrophage cell line (Raw264.7). The protein expression and the amount of lactate dehydrogenase (LDH) of ODN1826-treated cells were determined by immunoblotting and LDH assay, respectively. In addition, the level of cytokine production was observed by ELISA assay and the ROS production was determined by flow cytometry. Our results showed that ODN1826 induced pyroptosis as judged by LDH releases. Furthermore, caspase-11 and gasdermin D activation, which are the key molecules in pyroptosis, were also observed in ODN1826-activated cells. Moreover, we also demonstrated that Reactive Oxygen Species (ROS) production by ODN1826 is essential for caspase-11 activation and gasdermin D release, which leads to pyroptosis. ODN1826 induces pyroptosis in Raw264.7 cells via caspase-11 and GSDMD activation. Moreover, the production of ROS by this ligand plays an essential role in the regulation of caspase-11 and GSDMD activation, which then controls pyroptosis in TLR9 activation.

TLR9 Negatively Regulates Intracellular Bacterial Killing by Pyroptosis in Burkholderia pseudomallei-Infected Mouse Macrophage Cell Line (Raw264.7).

ABSTRACTMelioidosis is a serious infectious disease caused by Burkholderia pseudomallei. This bacterium is able to survive and multiply inside the immune cells such as macrophages. It is well established that Toll-like receptors (TLRs), particularly surface TLRs such as TLR2, TLR4, and TLR5, play an essential role in defending against this bacterial infection. However, the involvement of endosomal TLRs in the infection has not been elucidated. In this study, we demonstrated that the number of intracellular bacteria is reduced in TLR9-depleted RAW264.7 cells infected with B. pseudomallei, suggesting that TLR9 is involved in intracellular bacterial killing in macrophages. As several reports have previously demonstrated that pyroptosis is essential for restricting intracellular bacterial killing, particularly in B. pseudomallei infection, we also observed an increased release of cytosolic enzyme lactate dehydrogenase (LDH) in TLR9-depleted cells infected with B. pseudomallei, suggesting TLR9 involvement in pyroptosis in this context. Consistently, the increases in caspase-11 and gasdermind D (GSDMD) activations, which are responsible for the LDH release, were also detected. Moreover, we demonstrated that the increases in pyroptosis and bacterial killing in B. pseudomallei-infected TLR9-depleted cells were due to the augmentation of the IFN-β, one of the key cytokines known to regulate caspase-11. Altogether, this finding showed that TLR9 suppresses macrophage killing of B. pseudomallei by regulating pyroptosis. This information provides a novel mechanism of TLR9 in the regulation of intracellular bacterial killing by macrophages, which could potentially be leveraged for therapeutic intervention.IMPORTANCE Surface TLRs have been well established to play an essential role in Burkholderia pseudomallei infection. However, the role of endosomal TLRs has not been elucidated. In the present study, we demonstrated that TLR9 plays a crucial role by negatively regulating cytokine production, particularly IFN-β, a vital cytokine to control pyroptosis via caspase-11 activation. By depletion of TLR9, the percentage of pyroptosis was significantly increased, leading to suppression of intracellular survival in B. pseudomallei-infected macrophages. These findings provide a new role of TLR9 in macrophages.

Silencing YY1 Alleviates Ox-LDL-Induced Inflammation and Lipid Accumulation in Macrophages through Regulation of PCSK9/ LDLR Signaling.

The formation of macrophage foam cells stimulated by oxidized low-density lipoprotein (ox-LDL) is deemed an important cause of atherosclerosis. Transcription factor Yin Yang 1 (YY1), which is a universally expressed multifunctional protein, is closely related to cell metabolism disorders such as lipid metabolism, sugar metabolism, and bile acid metabolism. However, whether YY1 is involved in macrophage inflammation and lipid accumulation still remains unknown. After mouse macrophage cell line RAW264.7 cells were induced by ox-LDL, YY1 and proprotein convertase subtilisin/kexin type 9 (PCSK9) expressions were found to be increased while low-density lipoprotein receptor (LDLR) expression was lowly expressed. Subsequently, through reverse transcription-quantitative polymerase chain reaction (RT-qPCR), Western blot analysis, Oil Red O staining and cholesterol quantification, it turned out that silencing of YY1 attenuated the inflammatory response and lipid accumulation in RAW264.7 cells caused by ox-LDL. Moreover, results from the JASPAR database, chromatin immunoprecipitation (ChIP) assay, luciferase reporter assay and Western blot analysis suggested that YY1 activated PCSK9 by binding to PCSK9 promoter and modulated the expression of LDLR in the downstream of PCSK9. In addition, the results of functional experiments demonstrated that the inhibitory effects of YY1 interference on ox-LDL-mediated macrophage inflammation and lipid accumulation were reversed by PCSK9 overexpression. To sum up, YY1 depletion inhibited its activation of PCSK9, thereby reducing cellular inflammatory response, cholesterol homeostasis imbalance, and lipid accumulation caused by ox-LDL.

Reduning Injection protects flu-infected mice by inhibiting infiltration of inflammatory cells in lung and down-regulating cytokine storm

This study aimed to explore the protective effect of Reduning Injection(RDN) on mice infected by influenza virus A/PR/8(PR8) and its immune regulatory roles during viral infection. In in vivo experiments, female C57 BL/6 mice were randomly divided into phosphate buffered saline(PBS) group, PR8-infected group, oseltamivir treatment group(OSV) and RDN treatment group. After 2 h of PR8 infection, mice in the oseltamivir group were gavaged with oseltamivir 30 mg·kg~(-1), and those in the RDN treatment group were injected intraperitoneally with RDN 1.5 mL·kg~(-1)once per day for seven consecutive days. The body weight of mice in each group was recorded at the same time every morning for 16 consecutive days. The line chart of body weight change was created to analyze the protective effect of RDN on flu-infected mice. The relative mRNA expression of different cytokines(IL-6, TNF-α, MCP-1, IL-1β, MIP-2, IP-10 and IL-10) in lung samples of flu-infected mice was detected by PCR. Flow cytometry was utilized to analyze the composition of immune cells of mouse BALF samples on day 5 after infection. Mouse macrophage cell line RAW264.7 was planted and treated by different concentrations of RDN(150, 300, 600 μg·mL~(-1)) for 24 h or 48 h, and cell proliferation was detected by CCK-8 assay. RAW264.7 cells and mouse primary peritoneal macrophages were stimulated with synthetic single stranded RNA(R837), which elicited the inflammatory response by mimicking the infection of single-stranded RNA viruses. The expression of cytokines and chemokines in the supernatants of above culture system was detected by ELISA and qPCR. On days 4, 5, 6, 7 and 15 after infection, the body weight loss of mice in the RDN treatment group was alleviated compared with that of PR8-infected mice(P<0.05). RDN treatment obviously reduced lung index and the production of IL-6, TNF-α, MCP-1 and MIP-2 in lung tissues of flu-infected mice(P<0.05). The proportions of macrophages, neutrophils and T cells in mouse BALF samples were analyzed by flow cytometry, and compared with PR8-infected mice, RDN decreased the proportion of macrophages in BALF of flu-infected mice(P<0.05), and the proportion of T cells was recovered dramatically(P<0.001). In CCK-8 assay, the concentrations of RDN(150, 300, 600 μg·mL~(-1)) failed to cause cytotoxicity to RAW264.7 cells. In addition, RDN lowered the expression of inflammatory cytokines such as IL-6, TNF-α,MCP-1, IL-1β, RANTES, and IP-10 and even anti-inflammatory cytokine IL-10 in R837-induced macrophages. RDN reduced the infiltration of inflammatory macrophages and the production of excessive inflammatory cytokines, alleviated the body weight loss of flu-infected mice. What's more, RDN restored the depletion of T cells, which might prevent secondary infection and deteriorative progression of the disease. Taken together, RDN may inhibit cytokine production and therefore down-regulate cytokine storm during the infection of influenza virus.

The Alleviation of LPS-Induced Murine Acute Lung Injury by GSH-Mediated PEGylated Artesunate Prodrugs.

Acute lung injury (ALI) or its aggravated stage acute respiratory distress syndrome (ARDS) is a common severe clinical syndrome in intensive care unit, may lead to a life-threatening form of respiratory failure, resulting in high mortality up to 30–40% in most studies. Nanotechnology-mediated anti-inflammatory therapy is an emerging novel strategy for the treatment of ALI, has been demonstrated with unique advantages in solving the dilemma of ALI drug therapy. Artesunate (ART), a derivative of artemisinin, has been reported to have anti-inflammatory effects. Therefore, in the present study, we designed and synthesized PEGylated ART prodrugs and assessed whether ART prodrugs could attenuate lipopolysaccharide (LPS) induced ALI in vitro and in vivo. All treatment groups were conditioned with ART prodrugs 1 h before challenge with LPS. Significant increased inflammatory cytokines production and decreased GSH levels were observed in the LPS stimulated mouse macrophage cell line RAW264.7. Lung histopathological changes, lung W/D ratio, MPO activity and total neutrophil counts were increased in the LPS-induced murine model of ALI via nasal administration. However, these results can be reversed to some extent by treatment of ART prodrugs. The effectiveness of mPEG2k-SS-ART in inhibition of ALI induced by LPS was confirmed. In conclusion, our results demonstrated that the ART prodrugs could attenuate LPS-induced ALI effectively, and mPEG2k-SS-ART may serve as a novel strategy for treatment of inflammation induced lung injury.

Copper oxide nanoparticles trigger macrophage cell death with misfolding of Cu/Zn superoxide dismutase 1 (SOD1)

BackgroundCopper oxide (CuO) nanoparticles (NPs) are known to trigger cytotoxicity in a variety of cell models, but the mechanism of cell death remains unknown. Here we addressed the mechanism of cytotoxicity in macrophages exposed to CuO NPs versus copper chloride (CuCl2).MethodsThe mouse macrophage cell line RAW264.7 was used as an in vitro model. Particle uptake and the cellular dose of Cu were investigated by transmission electron microscopy (TEM) and inductively coupled plasma mass spectrometry (ICP-MS), respectively. The deposition of Cu in lysosomes isolated from macrophages was also determined by ICP-MS. Cell viability (metabolic activity) was assessed using the Alamar Blue assay, and oxidative stress was monitored by a variety of methods including a luminescence-based assay for cellular glutathione (GSH), and flow cytometry-based detection of mitochondrial superoxide and mitochondrial membrane potential. Protein aggregation was determined by confocal microscopy using an aggresome-specific dye and protein misfolding was determined by circular dichroism (CD) spectroscopy. Lastly, proteasome activity was investigated using a fluorometric assay.ResultsWe observed rapid cellular uptake of CuO NPs in macrophages with deposition in lysosomes. CuO NP-elicited cell death was characterized by mitochondrial swelling with signs of oxidative stress including the production of mitochondrial superoxide and cellular depletion of GSH. We also observed a dose-dependent accumulation of polyubiquitinated proteins and loss of proteasomal function in CuO NP-exposed cells, and we could demonstrate misfolding and mitochondrial translocation of superoxide dismutase 1 (SOD1), a Cu/Zn-dependent enzyme that plays a pivotal role in the defense against oxidative stress. The chelation of copper ions using tetrathiomolybdate (TTM) prevented cell death whereas inhibition of the cellular SOD1 chaperone aggravated toxicity. Moreover, CuO NP-triggered cell death was insensitive to the pan-caspase inhibitor, zVAD-fmk, and to wortmannin, an inhibitor of autophagy, implying that this was a non-apoptotic cell death. ZnO NPs, on the other hand, triggered autophagic cell death.ConclusionsCuO NPs undergo dissolution in lysosomes leading to copper-dependent macrophage cell death characterized by protein misfolding and proteasomal insufficiency. Specifically, we present novel evidence for Cu-induced SOD1 misfolding which accords with the pronounced oxidative stress observed in CuO NP-exposed macrophages. These results are relevant for our understanding of the consequences of inadvertent human exposure to CuO NPs.