Mouse Macrophages In Response Research Articles

Borrelia burgdorferi Engages Mammalian Type I IFN Responses via the cGAS-STING Pathway.

Borrelia burgdorferi, the etiologic agent of Lyme disease, is a spirochete that modulates numerous host pathways to cause a chronic, multisystem inflammatory disease in humans. B. burgdorferi infection can lead to Lyme carditis, neurologic complications, and arthritis because of the ability of specific borrelial strains to disseminate, invade, and drive inflammation. B. burgdorferi elicits type I IFN (IFN-I) responses in mammalian cells and tissues that are associated with the development of severe arthritis or other Lyme-related complications. However, the innate immune sensors and signaling pathways controlling IFN-I induction remain unclear. In this study, we examined whether intracellular nucleic acid sensing is required for the induction of IFN-I to B. burgdorferi. Using fluorescence microscopy, we show that B. burgdorferi associates with mouse and human cells in culture, and we document that internalized spirochetes colocalize with the pattern recognition receptor cyclic GMP-AMP synthase (cGAS). Moreover, we report that IFN-I responses in mouse macrophages and murine embryonic fibroblasts are significantly attenuated in the absence of cGAS or its adaptor stimulator of IFN genes (STING), which function to sense and respond to intracellular DNA. Longitudinal invivo tracking of bioluminescent B. burgdorferi revealed similar dissemination kinetics and borrelial load in C57BL/6J wild-type, cGAS-deficient, or STING-deficient mice. However, infection-associated tibiotarsal joint pathology and inflammation were modestly reduced in cGAS-deficient compared with wild-type mice. Collectively, these results indicate that the cGAS-STING pathway is a critical mediator of mammalian IFN-I signaling and innate immune responses to B. burgdorferi.

Toxoplasma gondii microneme protein MIC3 induces macrophage TNF-α production and Ly6C expression via TLR11/MyD88 pathway

Toxoplasma gondii is the most successful parasite worldwide. It is of great interest to understand how T. gondii induce different immune responses in different hosts. In this study, we found that a peptide of T. gondii microneme protein MIC3 induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression in mouse macrophage RAW264.7 cells. MyD88 inhibition, small interfering RNA against Tlr11 and CRISPR/Cas9-mediated knock-out of Tlr11 all reduced MIC3-induced TNF-α production, NF-κB phosphorylation, iNOS transcription and Ly6C expression. Additionally, we determined the location of MIC3 peptide in mouse macrophages using immunofluorescence. MIC3 could both adhere to the cell membrane of mouse macrophages and enter the cells. These results suggest that MIC3 triggered the immune responses in mouse macrophages via TLR11/MyD88/NF-κB pathway. It is known that human macrophages lacking TLR11. We predicted that the immune responses induced by MIC3 in human macrophages were significantly different from those in mouse macrophages. As expected, MIC3 peptide failed to induce TNF-α expression, iNOS expression and NF-κB phosphorylation in human THP-1 derived macrophages. MIC3 induced macrophage immune responses via TLR11. Intriguingly, the amino acid sequence of MIC3 is completely different from the well-known TLR11 ligand profilin, which generates a potent IL-12p40, TNF-α and IL-6 response. In marked contrast to profilin, MIC3 could not induce IL-12p40 expression in both mouse RAW264.7 cells and human THP-1 derived macrophages. Furthermore, the simulated tertiary structure of MIC3 peptide shows poor similarity with the crystal structure of profilin, suggesting that MIC3 might be a different ligand from profilin. These findings about MIC3 and TLR11 will provide us with important insights into the pathogenesis of toxoplasmosis and coevolution during host-parasite interaction.

Degradation of HDAC10 by autophagy promotes IRF3-mediated antiviral innate immune responses.

Histone deacetylases (HDACs) play important roles in immunity and inflammation. Through functional screening, we identified HDAC10 as an inhibitor of the type I interferon (IFN) response mediated by interferon regulatory factor 3 (IRF3). HDAC10 abundance was decreased in mouse macrophages in response to innate immune stimuli and was reduced in peripheral blood mononuclear cells (PBMCs) from patients with systemic lupus erythematosus (SLE) compared with that in PBMCs from healthy donors. Deficiency in HDAC10 in mouse embryonic fibroblasts and in mice promoted the expression of genes encoding type I IFNs and of IFN-stimulated genes (ISGs), leading to enhanced antiviral responses in vitro and in vivo. HDAC10 bound in a deacetylase-independent manner to IRF3 in uninfected cells to inhibit the phosphorylation of IRF3 at Ser396 by TANK-binding kinase 1 (TBK1). Upon viral infection, HDAC10 was targeted for autophagy-mediated degradation through its interaction with LC3-II. Consequently, IRF3 phosphorylation was increased, which resulted in enhanced type I IFN production and antiviral responses. Our findings identify a potential target for improving host defense responses against pathogen infection and for treating autoimmune disease.

Anti-inflammatory and anti-oxidant properties of Melianodiol on DSS-induced ulcerative colitis in mice.

BackgroundUlcerative colitis is a unique inflammatory bowel disease with ulcerative lesions of the colonic mucosa. Melianodiol (MN), a triterpenoid, isolated from the fruits of the Chinese medicinal plant Melia azedarach, possesses significant anti-inflammatory properties.ObjectiveThe present study investigated the protective effects of MN on lipopolysaccharide (LPS)-induced macrophages and DSS-mediated ulcerative colitis in mice.MethodsIn the study, mice were given MN (50, 100, and 200 mg/kg) and 5-ASA (500 mg/kg) daily for 9 days after induction by DSS for 1 week. The progress of the disease was monitored daily by observation of changes in clinical signs and body weight.ResultsThe results showed that MN effectively improved the overproduction of inflammatory factors (IL-6, NO, and TNF-α) and suppressed the activation of the NF-κB signalling cascade in LPS-mediated RAW264.7 cells. For DSS-mediated colitis in mice, MN can reduce weight loss and the disease activity index (DAI) score in UC mice, suppress colon shortening, and alleviate pathological colon injury. Moreover, MN treatment notably up regulated the levels of IL-10 and down regulated those of IL-1β and TNF-α, and inhibited the protein expression of p-JAK2, p-STAT3, iNOS, NF-κB P65, p-P65, p-IKKα/β, and p-IκBα in the colon. After MN treatment, the levels of MDA and NO in colonic tissue were remarkably decreased, whereas the levels of GSH, SOD, Nrf-2, Keap-1, HO-1, IκBα, and eNOS protein expression levels were significantly increased.ConclusionThese results indicate that MN can activate the Nrf-2 signalling pathway and inhibit the JAK/STAT, iNOS/eNOS, and NF-κB signalling cascades, enhance intestinal barrier function, and effectively reduce the LPS-mediated inflammatory response in mouse macrophages and DSS-induced intestinal injury in UC.

An alternative downstream translation start site in the non-TIR adaptor Scimp enables selective amplification of CpG DNA responses in mouse macrophages.

Toll‐like receptor (TLR) signaling relies on Toll/interleukin‐1 receptor homology (TIR) domain‐containing adaptor proteins that recruit downstream signaling molecules to generate tailored immune responses. In addition, the palmitoylated transmembrane adaptor protein family member Scimp acts as a non‐TIR‐containing adaptor protein in macrophages, scaffolding the Src family kinase Lyn to enable TLR phosphorylation and proinflammatory signaling responses. Here we report the existence of a smaller, naturally occurring translational variant of Scimp (Scimp TV1), which is generated through leaky scanning and translation at a downstream methionine. Scimp TV1 also scaffolds Lyn, but in contrast to full‐length Scimp, it is basally rather than lipopolysaccharide (LPS)‐inducibly phosphorylated. Macrophages from mice that selectively express Scimp TV1, but not full‐length Scimp, have impaired sustained LPS‐inducible cytokine responses. Furthermore, in granulocyte macrophage colony‐stimulating factor‐derived myeloid cells that express high levels of Scimp, selective overexpression of Scimp TV1 enhances CpG DNA‐inducible cytokine production. Unlike full‐length Scimp that localizes to the cell surface and filopodia, Scimp TV1 accumulates in intracellular compartments, particularly the Golgi. Moreover, this variant of Scimp is not inducibly phosphorylated in response to CpG DNA, suggesting that it may act via an indirect mechanism to enhance TLR9 responses. Our findings thus reveal the use of alternative translation start sites as a previously unrecognized mechanism for diversifying TLR responses in the innate immune system.

Curcumin attenuates poly(I:C)-induced immune and inflammatory responses in mouse macrophages by inhibiting TLR3/TBK1/IFNB cascade

This study suggested that the involvement of TLR3/TBK1/IFN-β in the underlying mechanism of curcumin alleviation of virus-like infections. • Poly(I:C) mimicked viral infection is specifically recognized by TLR3 to induce innate immune and inflammatory response. • Curcumin alleviates virus-like infection through the mitigation of TLR3/TBK1/INF-β. • Potential anti-viral effects of curcumin was investigated by transcriptomics and multiple molecular biological approaches. Curcumin is widely used in dietary supplements due to its beneficial effects on inflammatory and immune pathways. To better understand the action of curcumin during viral infection, its effect on stimulation of RAW264.7 macrophages with polyinosinic-polycytidylic acid (poly(I:C)) was evaluated by deep sequencing transcriptome, followed by RT-qPCR, western blotting and ELISA validations. The transcriptomics results showed that 157 differentially expressed genes are in association with the activity of curcumin on the cellular response to poly(I:C) stimulation. Gene Ontology and KEGG enrichment analysis uncovered the involvement of TLRs, in particular TLR3, in curcumin’s effect on poly(I:C)-induced immune and inflammatory responses. RT-qPCR results showed that curcumin (2.5, 5, 10 μM) significantly down-regulated the mRNA expression of Tlr3, Irf-7, Ifnb1, Il-6, Tnfα, and Il1bf in a dose-dependent manner. Significant down-regulation of curcumin (5 M) on protein or phosphorylated protein expressions of TLR3 (p < 0.05), TBK1 (P < 0.005), IRF3 (P < 0.0005), IRF7 (p < 0.0005), IFN-β (P < 0.0005),TNF-α (P < 0.0001) and IL6 (P < 0.0001) were also observed in western blotting or ELISA results. Taken together, these results indicated that down-regulation on TLR3/TBK1/IFN-β cascade may involve in the underlying mechanism of curcumin alleviating virus-like infections. The findings from our study contribute to a better understanding of the action of curcumin during viral infection, suggesting the potential of curcumin preparations to support a healthy immune system.

Aeromonas sobria regulates proinflammatory immune response in mouse macrophages via activating the MAPK, AKT, and NF-κB pathways.

Aeromonas sobria, a Gram-negative bacterium that can colonize both humans and animals, is found in a variety of environments, including water, seafood, meat, and vegetables (Cahill, 1990; Galindo et al., 2004; Song et al., 2019). Aeromonas spp. are conditionally pathogenic bacteria in aquaculture, which can rapidly proliferate, causing disease and even death in fish, especially when the environment is degraded (Neamat-Allah et al., 2020, 2021a, 2021b). In developing countries, Aeromonas spp. have been associated with a wide spectrum of infections in humans, including gastroenteritis, wound infections, septicemia, and lung infections (San Joaquin and Pickett, 1988; Wang et al., 2009; Su et al., 2013). Infections caused by Aeromonas spp. are usually more severe in immunocompromised individuals (Miyamoto et al., 2017). The presence of a plasmid encoding a β‍-lactamase in A. sobria that confers resistance to β-lactam antibiotics poses a huge challenge to the treatment of diseases caused by this microorganism (Lim and Hong, 2020). Consequently, an in-depth understanding of the interaction between A. sobria and its hosts is urgently required to enable the development of effective strategies for the treatment of A. sobria infections.

Giardia duodenalis extracellular vesicles regulate the proinflammatory immune response in mouse macrophages in vitro via the MAPK, AKT and NF-\u03baB pathways

BackgroundGiardia duodenalis is an extracellular protozoan parasite that causes giardiasis in mammals. The presentation of giardiasis ranges from asymptomatic to severe diarrhea, and the World Health Organization lists it in the Neglected Diseases Initiative. Extracellular vesicles (EVs) are a key mediator of intracellular communication. Although previous studies have shown that G. intestinalis can regulate a host’s innate immune response, the role of G. intestinalis EVs (GEVs) in triggering a G. intestinalis-induced innate immune response remains to be further explored.MethodsIn this study, GEVs, G. intestinalis and GEVs + G. intestinalis were inoculated into macrophages, respectively. The transcription and secretion levels of proinflammatory cytokines, including interleukin (IL)-1β, IL-6 and tumor necrosis factor alpha (TNF-α), were measured using real-time quantitative PCR (qPCR) and enzyme-linked immunosorbent assays (ELISAs). The phosphorylation levels of the MAPK, AKT and NF-κB signaling pathways in GEV-stimulated mouse macrophages were examined using western blotting and immunofluorescence methods. The roles of activated pathways in the GEV-triggered inflammatory response were determined using inhibition assays, western blotting and ELISAs.ResultsThe results showed that pretreatment with GEVs enhanced with G. intestinalis (GEVs + G. intestinalis) induced IL-1β, IL-6 and TNF-α transcription and secretion from mouse macrophages compared to stimulation with either GEVs or G. intestinalis alone. Inoculation of mouse macrophages with GEVs upregulated the phosphorylation levels of the p38 MAPK, p44/42 MAPK (Erk1/2), AKT and NF-κB signaling pathways and led to the nuclear translocation of NF-κB p65. Blocking the activated p38, Erk and NF-κB signaling pathways significantly downregulated the secretion of proinflammatory cytokines, and blocking the activated AKT signaling pathway demonstrated reverse effects.ConclusionsThe results of this study reveal that GEVs can enhance G. intestinalis-induced inflammatory response levels in mouse macrophages through activation of the p38, ERK and NF-κB signaling pathways. The role of GEVs in regulating host cell immune responses may provide insights into exploring the underlying mechanisms in G. intestinalis–host interactions.Graphical abstract

Glycolipid-Anchored Proteins on Bioengineered Extracellular Vesicles for Lipopolysaccharide Neutralization.

Extracellular vesicles (EVs) with native membrane proteins possess a variety of functions. EVs have become increasingly important platforms for incorporating a new peptide/protein with additional functions on their membranes using genetic manipulation of producer cells. Although directly harnessing native membrane proteins on EVs for functional studies is promising, limited studies have been conducted to confirm its potential. This study reports bioengineered EVs with CD14, a natural glycosylphosphatidylinositol (GPI)-anchored protein and a selectively enriched native membrane protein on EVs. We demonstrated that producer cells transfected with genes encoding for GPI-anchored and transmembrane glycoproteins selectively display the former over the latter on bioengineered EVs. Furthermore, using specific enzyme cleavage studies, we characterized and validated that CD14 is indeed GPI-anchored on bioengineered EV membranes. Natural GPI-anchored proteins are conserved receptors for bacterial toxins; for example, CD14 is an innate immune receptor for lipopolysaccharide (LPS), a gram-negative bacterial endotoxin. We reported that unlike soluble CD14, bioengineered EVs harboring CD14 reduce (50-90%) LPS-induced cytokine responses in mouse macrophages, including primary cells, possibly by reduced cell surface binding of LPS. These findings highlight the importance of harnessing the native EV membrane proteins, like GPI-anchored proteins, for functional studies such as toxin neutralization. The GPI-anchoring platform can display various natural GPI-anchored proteins and other full-length proteins as GPI-anchored proteins on EV membranes.

RUVBL1/2 Complex Regulates Pro-Inflammatory Responses in Macrophages via Regulating Histone H3K4 Trimethylation.

Macrophages play an important role in the host defense mechanism. In response to infection, macrophages activate a genetic program of pro-inflammatory response to kill any invading pathogen, and initiate an adaptive immune response. We have identified RUVBL2 - an ATP-binding protein belonging to the AAA+ (ATPase associated with diverse cellular activities) superfamily of ATPases - as a novel regulator in pro-inflammatory response of macrophages. Gene knockdown of Ruvbl2, or pharmacological inhibition of RUVBL1/2 activity, compromises type-2 nitric oxide synthase (Nos2) gene expression, nitric oxide production and anti-bacterial activity of mouse macrophages in response to lipopolysaccharides (LPS). RUVBL1/2 inhibitor similarly inhibits pro-inflammatory response in human monocytes, suggesting functional conservation of RUVBL1/2 in humans. Transcriptome analysis further revealed that major LPS-induced pro-inflammatory pathways in macrophages are regulated in a RUVBL1/2-dependent manner. Furthermore, RUVBL1/2 inhibition significantly reduced the level of histone H3K4me3 at the promoter region of Nos2 and Il6, two prototypical pro-inflammatory genes, and diminished the recruitment of NF-kappaB to the corresponding enhancers. Our study reveals RUVBL1/2 as an integral component of macrophage pro-inflammatory responses through epigenetic regulations, and the therapeutic potentials of RUVBL1/2 inhibitors in the treatment of diseases caused by aberrant activation of pro-inflammatory pathways.

NFAT5 Amplifies Antipathogen Responses by Enhancing Chromatin Accessibility, H3K27 Demethylation, and Transcription Factor Recruitment.

The ability of innate immune cells to respond to pathogen-associated molecular patterns across a wide range of intensities is fundamental to limit the spreading of infections. Studies on transcription responses to pathogen-activated TLRs have often used relatively high TLR ligand concentrations, and less is known about their regulation under mild stimulatory conditions. We had shown that the transcription factor NFAT5 facilitates expression of antipathogen genes under TLR stimulation conditions corresponding to low pathogen loads. In this study, we analyze how NFAT5 optimizes TLR-activated responses in mouse macrophages. We show that NFAT5 was required for effective recruitment of central effectors p65/NF-κB and c-Fos to specific proinflammatory target genes, such as Nos2, Il6, and Tnf in primary macrophages responding to low doses of the TLR4 ligand LPS. By contrast, NFAT5 was not required for p65/NF-κB recruitment in response to high LPS doses. Using the transposase-accessible chromatin with high-throughput sequencing assay, we show that NFAT5 facilitated chromatin accessibility mainly at promoter regions of multiple TLR4-responsive genes. Analysis of various histone marks that regulate gene expression in response to pathogens identified H3K27me3 demethylation as an early NFAT5-dependent mechanism that facilitates p65 recruitment to promoters of various TLR4-induced genes. Altogether, these results advance our understanding about specific mechanisms that optimize antipathogen responses to limit infections.

Human monoclonal anti‑TLR4 antibody negatively regulates lipopolysaccharide‑induced inflammatory responses in mouse macrophages.

Previous studies have revealed that activation of the Toll-like receptor 4 (TLR4)-mediated proinflammatory signaling pathway plays an important role in acute inflammation, sepsis and chronic inflammatory disorders. Moreover, TLR4 significantly contributes to lipopolysaccharide (LPS)-induced immune response. Thus, modulation of the TLR4 pathway is an important strategy to specifically target these pathologies. The aim of the present study was to develop a complete human anti-TLR4 IgG2 antibody by screening human TLR4 Fab from a phage-display library and integrating it with constant regions of the heavy chain of human IgG2 via antibody engineering. ELISA, a BLItz system and fluorescence-activated cell sorting were used to assess its affinity. Furthermore, mouse-derived peritoneal macrophages were treated with human anti-TLR4 IgG2 and induced with LPS in vitro. Reverse transcription-quantitative PCR and western blotting were used to determine mRNA expression levels of cytokines and phosphorylation levels of signaling pathways, respectively. It was found that human anti-TLR4 IgG2 bound to TLR4 with a high affinity of 8.713×10−10 M, and that preincubation with anti-TLR4 IgG2 inhibited the LPS-induced production of tumor necrosis factor-α, interferon-β and interleukin-6 mRNA expression levels in mouse peritoneal macrophages. It was also demonstrated that human anti-TLR4 IgG2 inhibited LPS-induced TLR4 signaling by reducing the phosphorylation of the NF-κB, mitogen-activated protein kinase and interferon regulatory factor 3 signaling pathways. In addition, human anti-TLR4 IgG2 protected mice from LPS challenge with a survival rate of 40% and also significantly increased the survival time in the cecal ligation and puncture model. Therefore, it was speculated that human anti-TLR4 IgG2 plays a protective role against sepsis-associated injury and is potentially applicable for the treatment of infection-associated immune dysfunction.

Wound Matrix Stiffness Imposes on Macrophage Activation.

The immune system depends on two major paths-the innate and the adaptive immunity. Macrophage, with its unique features as the first line of immune defense to engulf and digest invaders, serves as the key effector cells integrating those two paths. The dynamic plasticity of macrophage activation during wound repair, inflammation resolution, and tissue remodeling are emerging biomedical and bioengineering hot topics in immune function studies such as the various secretions of cytokines and chemokines and the signaling pathways with ligands and their cognate receptors. Better knowledge on how physical/mechanical and multicellular microenvironment on the modulation of macrophage functions will create innovative therapies to boost host defense mechanism and assist wound healing. In this, we describe an easy method to measure functions (gene expressions) of human and mouse macrophages in response to mechanical microenvironment changes by embedding isolated macrophages in polymerized hyaluronan gel with different wound matrix stiffness.

Berberine inhibits free fatty acid and LPS-induced inflammation via modulating ER stress response in macrophages and hepatocytes.

Inflammation plays an essential role in the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Berberine (BBR), an isoquinoline alkaloid isolated from Chinese medicinal herbs, has been widely used to treat various diseases, including liver diseases for hundreds of years. The previous studies have shown that BBR inhibits high fat-diet-induced steatosis and inflammation in rodent models of NAFLD. However, the underlying molecular mechanisms remain unclear. This study is aimed to identify the potential mechanisms by which BBR inhibits free fatty acid (FFA) and LPS-induced inflammatory response in mouse macrophages and hepatocytes. Mouse RAW264.7 macrophages and primary mouse hepatocytes were treated with palmitic acid (PA) or LPS or both with or without BBR (0-10 μM) for different periods (0-24 h). The mRNA and protein levels of proinflammatory cytokines (TNF-α, IL-6, IL-1β, MCP-1) and ER stress genes (CHOP, ATF4, XBP-1) were detected by real-time RT-PCR, Western blot and ELISA, respectively. The results indicated that BBR significantly inhibited PA and LPS-induced activation of ER stress and expression of proinflammatory cytokines in macrophages and hepatocytes. PA/LPS-mediated activation of ERK1/2 was inhibited by BBR in a dose-dependent manner. In summary, BBR inhibits PA/LPS-induced inflammatory responses through modulating ER stress-mediated ERK1/2 activation in macrophages and hepatocytes.

Cryptotanshinone from Salvia miltiorrhiza Bunge (Danshen) inhibited inflammatory responses via TLR4/MyD88 signaling pathway

BackgroundCryptotanshinone (CPT), as a major component of Salvia miltiorrhiza Bunge (Danshen), displays many pharmacological activities including anti-inflammatory effects. However, the exact cellular and molecular mechanisms of the anti-inflammatory activities of CPT remain to be elucidated. The present study was aimed to clarify its mechanisms on lipopolysaccharide (LPS)-induced inflammatory responses in mouse macrophages, RAW264.7 cells.MethodsIn the current study, the anti-inflammatory properties of CPT were evaluated using LPS-stimulated RAW264.7 cell model. MTT assay was used to determine the viability of RAW264.7 cells. The anti-inflammatory effects of CPT were measured based on the detection of nitric oxide (NO) production (Griess and flow cytometry assay), and tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) release (ELISA). Cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) enzyme expressions were also determined by western blotting. Besides, by using flow cytometry, we also evaluated the effect of CPT on LPS-induced calcium influx. Finally, the underlying anti-inflammatory mechanisms of CPT were investigated using western blotting to assess the protein levels of toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88), phosphatidylinositol 3-kinase (PI3K)/AKT, nuclear factor erythroid 2 related factor 2 (Nrf2), mitogen-activated protein kinase (MAPK), and nuclear factor-kappa B (NF-κB) pathways.ResultsOur data showed that CPT inhibited LPS-induced pro-inflammatory cytokine release like IL-6, and TNF-α, as well as NO production. It displayed a significant inhibitory effect on the protein expressions such as iNOS, COX-2, NF-κB pathway like inhibitor of kappa B kinase (IKK)α/β, inhibitor of kappa B (IκB)-α and NF-κB/p65, PI3K/AKT pathway like PI3K and AKT, and MAPK pathway like c-Jun N-terminal kinase (JNK)1/2, extracellular signal-regulated kinase (ERK)1/2, and p38, in LPS-stimulated RAW264.7 macrophages. Moreover, the immunofluorescence results indicated that CPT suppressed NF-κB/p65 translocation from the cytoplasm into the nucleus. Further investigations showed that CPT treatment increased NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygenase-1 (HO-1) expressions together with its upstream mediator, Nrf2. In addition, CPT inhibited LPS-induced toll-like receptor 4 (TLR4) and MyD88 expressions in RAW264.7 macrophages.ConclusionsCollectively, we suggested that CPT exerted significant anti-inflammatory effects via modulating TLR4-MyD88/PI3K/Nrf2 and TLR4-MyD88/NF-κB/MAPK pathways.

Inhibitory Effects of Myrtucommuacetalone 1 (MCA-1) from Myrtus communis on Inflammatory Response in Mouse Macrophages.

(1) Introduction: Reactive oxygen species (ROS) and nitric oxide (NO) are key signaling molecules that play important roles in the progression of inflammatory disorders. The objective of this study was to explore the use of myrtucommuacetalone-1 (MCA-1), as a novel compound of natural origin and a potential anti-inflammatory agent. (2) Methodology: The anti-inflammatory potential of MCA-1, which was isolated from Myrthus communis Linn, was determined by assaying superoxide, hydrogen peroxide, and nitric oxide production in macrophages. Furthermore, the effects of the compound were analyzed via phosphorylation and translocation of the transcription factor NF kappa B, which is a key regulator of iNOS activation. The effect of MCA-1 on the inducible nitric oxide synthase (iNOS) enzyme was also examined using in silico docking studies. The anticancer potential for MCA-1 was evaluated with an MTT cytotoxic assay. (3) Results: In stimulated macrophages, MCA-1 inhibited superoxide production by 48%, hydrogen peroxide by 53%, and nitric oxide (NO) with an IC50 of <1 µg/mL. MCA-1 also showed a very strong binding pattern within the active site of the inducible nitric oxide synthase enzyme. Furthermore, 25 µg/mL of MCA-1 inhibited inducible nitric oxide synthase expression and abolished transcription factor (NFκB) phosphorylation and translocation to the nucleus. Cytotoxicity analyses of MCA-1 on 3T3 mouse fibroblasts, CC1 liver cell line, J774.2, macrophages and MDBK bovine kidney epithelial cell, yielded IC50 values of 6.53 ± 1.2, 4.6 ± 0.7, 5 ± 0.8, and 4.6 ± 0.7, µg/mL, respectively. (4) Conclusion: Our results suggest that MCA-1, a major phloroglucinol-type compound, shows strong anti-inflammatory activity and has a potential to be a leading therapeutic agent in the future.

Control (Native) and oxidized (DeMP) mitochondrial RNA are proinflammatory regulators in human

Inflammation is implicated in a wide range of disorders, and thought to be involved in most leading causes of death today in the United States with high associated costs. New insights into better understanding its etiology, detection and prevention are thus of major importance in health care. One emerging field providing such insights has been the identification of DAMPs, or damage-associated molecular patterns. We have studied DAMPs within the context of degraded and oxidized mitochondrial DNA and RNA (“DeMP”), most recently demonstrating potent mitochondrial RNA (mtRNA) immunogenic response in mouse macrophages. Here, we extend these studies to assess the proinflammatory role of mitochondrial control (native) and oxidized RNA using human RNA and cells. THP-1 macrophage mtRNA triggered a proinflammatory response (induction of IL-6 and TNFα) when transfected into the same cells. Modestly oxidized mtRNA (DeMP RNA) but not cytoplasmic RNA induced a similar response, in contrast to attenuated immunogenicity previously observed with more oxidized DeMP RNA. This DeMP RNA may also cause a mild prooxidant stress. The proinflammatory effects of mtRNA was significantly reduced following pretreatment with RNases specific for single and double stranded RNA, implicating these forms of mtRNA in proinflammatory response. The natural nucleic acid-encapsulating peptide LL-37 also triggered a proinflammatory effect in the presence of control mtRNA and DeMP RNA. Finally, human blood plasma RNA exhibits proinflammatory activity. These results provide new insights into the immunostimulation of mitochondrial RNA including its activity in human cells; identify human plasma RNA as proinflammatory; and provide further evidence that oxidized DeMP mtRNA acts as a sensitive and broad-spectrum sensor and regulator of mitochondrial oxidative stress.

Transcriptome profiling of poly(I:C)-induced RAW 264.7 mouse macrophages in response to panaxadiol

Panaxadiol is an active compound in steamed ginseng that possesses anti-cancer properties. Nonetheless, the effect of panaxadiol on inflammation remains unclear. Double-stranded RNA (dsRNA) acts as a molecular mimic associated with viral infection. It is recognized by Toll-like receptor-3 (TLR3), and it induces pro-inflammatory cytokines production of immune cells-like macrophages. Synthetic dsRNA polyriboinosinic polyribocytidylic acid [Poly(I:C)] is structurally similar to double-stranded RNA, which is used to simulate dsRNA in inducing immune response. In this study, we used an RNA-Seq based approach to analyze changes in mRNA level of poly (I:C)-induced RAW264.7 treated by panaxadiol. First, our data suggested that 20 μM and 40 μM panaxadiol may reduce poly(I:C)-induced RAW264.7 cell viability, while the concertation of 10 μM panaxadiol was enough to inhibit the cell production of TNF-α and IL-6. Next, 10 μM panaxadiol was applied to investigate the transcriptome profile change of Poly(I:C)-induced RAW264.7 cells. Transcriptome profile showed that inflammatory related genes (such as Cxcl2, Csf3, Ptgs2, Acod1, Lif and Il1b) could be downregulated by panaxadiol, which was confirmed by real-time PCR. Moreover, Gene Ontology enrichment analysis showed that downregulated genes were associated with immune responses, while many upregulated genes were related to DNA replication. In addition, KEGG enrichment showed changes of pathways like NF-kappa B signaling pathway and DNA replication after treatment with panaxadiol. To sum up, our results suggested that panaxadiol may inhibit inflammatory associated genes in macrophages induced by poly(I:C). Considering its anti-virus effects, panaxadiol could be used to treat a variety of virus related diseases.

Differential Immune Activation in Fetal Macrophage Populations

Distinct macrophage subsets populate the developing embryo and fetus in distinct waves. However little is known about the functional differences between in utero macrophage populations or how they might contribute to fetal and neonatal immunity. Here we tested the innate immune response of mouse macrophages derived from the embryonic yolk sac and from fetal liver. When isolated from liver or lung, CD11bHI fetal liver derived macrophages responded to the TLR4 agonist LPS by expressing and releasing inflammatory cytokines. However F4/80HI macrophages from the yolk sac did not respond to LPS treatment. While differences in TLR4 expression did not appear to explain these data, F4/80HI macrophages had much lower NLRP3 inflammasome expression compared to CD11bHI macrophages. Gene expression profiling also demonstrated LPS-induced expression of inflammatory genes in CD11bHI macrophages, but not in F4/80HI cells. Genes expressed in LPS-treated CD11bHI macrophages were more likely to contain predicted NF-κB binding sites in their promoter regions. Our data show that CD11bHI macrophages derived from fetal liver are the major pro-inflammatory cells in the developing fetus. These findings could have important implications in better understanding the fetal inflammatory response and the unique features of neonatal immunity.

In vitro inhibition of tumor growth by low-dose iron oxide nanoparticles activating macrophages.

Macrophages as immunocyte are attracting more and more attention in cancer therapy. Our previous study observed that dimercaptosuccinic acid (DMSA)-coated Fe3O4 magnetic nanoparticles triggered comprehensive immune responses of mouse macrophages (RAW264.7 cells) and induced production of many kinds of cytokines. This study investigated the effects of Fe3O4 magnetic nanoparticles on RAW264.7 cells proliferation, migration, and inhibition of tumor growth in vitro. Fe3O4 magnetic nanoparticles had an average size of about 11 nm with good dispersibility and uniformity. Fe3O4 magnetic nanoparticles internalized efficiently into RAW264.7 cells. Through Cell Counting Kit-8 (CCK-8) detection, the proliferation of RAW264.7 cells significantly increased by the low-dose Fe3O4 magnetic nanoparticles (50 µg/mL) treatment. The results of wound-healing and Transwell assays both displayed a significant promotion of the RAW264.7 cells migratory capability compared with control group ( P<0.01). It is interesting to find that a large number of proliferated RAW264.7 cells were activated to surround quickly and attack mouse liver cancer cell (Hepa1-6) cells by Fe3O4 magnetic nanoparticles. The growth of Hepa1-6 cells was effectively inhibited according to microscope imaging and flow cytometry analysis. The inhibition may be cooperative effects of RAW264.7 cells proliferation, migration, and immune activation. The results suggest potential clinical value of low-dose iron oxide nanomaterials in cancer therapy.