Macrophage Cell Line RAW Research Articles

In vitro anti-tuberculosis effect of probiotic Lacticaseibacillus rhamnosus PMC203 isolated from vaginal microbiota

Mycobacterium tuberculosis (M. tb), the etiological agent of tuberculosis (TB), poses a severe challenge for public health and remains the number one cause of death as a single infectious agent. There are 10 million active cases of TB per year with 1.5 million deaths, and 2–3 billion people are estimated to harbor latent M. tb infection. Moreover, the emergence of multi-drug-resistant (MDR), extremely-drug-resistant (XDR), and the recent totally drug-resistant (TDR) M. tb is becoming a global issue that has fueled the need to find new drugs different from existing regimens. In these circumstances, probiotics can be a potential choice, so we focused on developing them as an anti-tuberculosis drug candidate. Here, we report the anti-tubercular activities of Lacticaseibacillus rhamnosus PMC203 isolated from the vaginal microbiota of healthy women. PMC203 exhibited a promising intracellular killing effect against both drug-sensitive and resistant M. tb infected murine macrophage cell line RAW 264.7 without showing any cytotoxicity. Additionally, it also inhibited the growth of M. tb under broth culture medium. PMC203 did not cause weight change or specific clinical symptoms in a 2-week repeated oral administration toxicity test in a guinea pig model. Here, we also found that PMC203 induces autophagy in a dose dependent manner by increasing the signal of well-known autophagy gene markers, suggesting a possible intracellular killing mechanism.

Murine RAW Macrophages Are a Suitable Model to Study the CD3 Signaling in Myeloid Cells.

In recent years, a growing body of evidence has shown the presence of a subpopulation of macrophages that express CD3, especially in the context of mycobacterial infections. Despite these findings, the function of these cells has been poorly understood. Furthermore, the low frequency of CD3+ macrophages in humans limits the study of this subpopulation. This work aimed to evaluate the expression of CD3 in a murine macrophage cell line and its potential for the study of CD3 signaling. The murine macrophage cell line RAW was used to evaluate CD3 expression at the transcriptional and protein levels and the effect of in vitro infection with the Mycobacterium bovis Bacillus Calmette-Guérin (BCG) on these. Our data showed that RAW macrophages express CD3, both the ε and ζ chains, and it is further increased at the transcriptional level after BCG infection. Furthermore, our data suggest that CD3 can be found on the cell surface and intracellularly. However, this molecule is internalized constantly, mainly after activation with anti-CD3 stimulus, but interestingly, it is stably maintained at the transcriptional level. Finally, signaling proteins such as NFAT1, c-Jun, and IKK-α are highly expressed in RAW macrophages. They may play a role in the CD3-controlled signaling pathway to deliver inflammatory cytokines such as TNF and IL-6. Our study provides evidence to support that RAW cells are a suitable model to study the function and signaling of the CD3 complex in myeloid cells.

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.

Polarized Macrophages As Potential Anti‐Endometrioid agent

Endometriosis is an estrogen‐dependent chronic inflammatory disease characterized by the presence of endometrial stroma and glands outside the endometrium. The gold standard in the treatment of endometriosis is laparoscopy but after that, the relapse rate is about 50%. Thus, the search for new effective non‐invasive methods of endometriosis therapy is an actual problem. Endometriosis has many properties similar to tumors including the fact that macrophages with an anti‐inflammatory phenotype (M2) prevail in the foci of endometriosis. It is already known that activated macrophages with a pro‐inflammatory phenotype (M1) have an antitumor effect. In this regard, we hypothesized that increasing the proportion of M1 macrophages could help in the treatment of endometriosis. The objectives of our work were (1) to assess the expression profile of macrophages in the foci of endometriosis in mice model of endometriosis; (2) to test the efficacy of administration of reprogrammed M1 macrophages as a possible anti‐endometrioid agent in the mouse.We studied the phenotypic profile of macrophages in the foci of endometriosis and the anti‐endometrioid activity of M1 macrophages on in vivoallogeneic model of mouse endometriosis induced in ovariectomized mice after intraperitoneal transplantation of the mouse uterus accompanied by 17β‐estradiol therapy. Analysis of the phenotypic profile of macrophages in the foci of endometriosis was carried out using immunohistochemical staining, western blot analysis, and qPCR. We obtained a reproducible model with lesions that had characteristic morphology. It was shown that macrophages with an anti‐inflammatory arginase1+ phenotype prevail in the foci of endometriosis compared with the intact endometrium. After verifying endometriosis development in mice, the experimental group was injected with M1‐polarized macrophages, while the control group was injected with unpolarized macrophages, and then the animals were monitored for two weeks. Macrophage cell line RAW264 was incubated with lipopolysaccharide (100 ng/ml) for 24 h to obtain M1‐polarized macrophages. M1‐polarization was verified by significant up‐regulation of MARCO, iNOS markers using western blot and qPCR analysis. After treatment of animals with M1 macrophages, we found that the number of foci and their sizes significantly decreased (p<0.05) in comparison with control animals.In our work, we proved the predominance of anti‐inflammatory macrophages in the foci of endometriosis and obtained the first results of a positive effect of M1 macrophages on the regression of foci of endometriosis.

Regulatory relationship between macrophage autophagy and PVL-positive methicillin-resistant Staphylococcus aureus

The present study intends to clarify the hypothesis that PVL-positive Methicillin-resistant S. aureus strain (PVL+-MRSA)-infected macrophages regulate autophagy and thus in turn inhibit phagocytosis through the in vitro and in vivo experiments. The autophagy of mouse macrophage cell line RAW264.7 was observed by fluorescence microscopy, and counted based on the number of each cell dot-like structure GFP-LC3. The protein levels of the phagocytic factors associated with autophagy were determined by western blotting. The phagocytosis of RAW264.7 on MRSA was determined by counting the colony. The clinically isolated and identified PVL+-MRSA strain was used to infect BALB/c mice (left nasal drip) to establish a mouse pneumonia model. PVL+-MRSA mice were then treated with 3-MA or linezolid. Bronchoalveolar lavage fluid (BALF) from mice was collected for macrophage counting by Flow cytometry assay. The right lung was aseptically isolated for counting the amount of bacteria. The results showed that PVL+-MRSA could induced the autophagy of macrophages, which in turn reduced the damage from macrophages, which were respectively alleviated by 3-MA and aggravated by rapamycin. Exogenous rPVL administrated into PVL--MRSA-infected macrophages caused the autophagy of macrophage. Exogenous rPVL, particularly A-Luk S-PV, administrated into macrophages also caused the autophagy of macrophage, which was reversed by PMX53, a C5aR antagonist. In a mouse pneumonia model, PVL+-MRSA could induced the autophagy of macrophages, which in turn reduced the damage from macrophages, which were respectively alleviated by 3-MA or linezolid. In conclusion, this study indicated PVL+-MRSA regulated macrophage autophagy, which in turns inhibit the phagocytosis of S. aureus by macrophage. This study may provide a potential target against S. aureus infection.

Exploration of potential mechanism of interleukin-33 up-regulation caused by 1,4-naphthoquinone black carbon in RAW264.7 cells

BackgroundAs air pollution has been paid more attention to by public in recent years, effects and mechanism in particulate matter-triggered health problems become a focus of research. Lysosomes and mitochondria play an important role in regulation of inflammation. Interleukin-33 (IL-33) has been proved to promote inflammation in our previous studies. In this research, macrophage cell line RAW264.7 was used to explore the potential mechanism of upregulation of IL-33 induced by 1,4-naphthoquinone black carbon (1,4-NQ-BC), and to explore changes of lysosomes and mitochondria during the process. Results50 μg/mL 1,4-NQ-BC exposure for 24 h dramatically increased expression of IL-33 in RAW264.7 cells. Lysosomal membrane permeability was damaged by 1,4-NQ-BC treatment, and higher mitochondrial membrane potential and ROS level were induced by 1,4-NQ-BC. The results of proteomics suggested that expression of ferritin light chain was increased after cells were challenged with 1,4-NQ-BC, and it was verified by Western blot. Meanwhile, expressions of p62 and LC3B-II were increased by 50 μg/mL 1,4-NQ-BC in RAW264.7 cells. Ultimately, expression of IL-33 could return to same level as control in cells treated with 50 μg/mL 1,4-NQ-BC and 50 μM deferoxamine combined. Conclusions1,4-NQ-BC induces IL-33 upregulation in RAW264.7 cells, and it is responsible for higher lysosomal membrane permeability and ROS level, lower mitochondrial membrane potential, and inhibition of autophagy. Ferritin light chain possibly plays an important role in the upregulation of IL-33 evoked by 1,4-NQ-BC.

Fermentation of Vaccinium floribundum Berries with Lactiplantibacillus plantarum Reduces Oxidative Stress in Endothelial Cells and Modulates Macrophages Function.

Accumulating evidence suggests that high consumption of natural antioxidants promotes health by reducing oxidative stress and, thus, the risk of developing cardiovascular diseases. Similarly, fermentation of natural compounds with lactic acid bacteria (LAB), such as Lactiplantibacillus plantarum, enhances their beneficial properties as regulators of the immune, digestive, and cardiovascular system. We investigated the effects of fermentation with Lactiplantibacillus plantarum on the antioxidant and immunomodulatory effects of Pushgay berries (Vaccinium floribundum, Ericaceae family) in human umbilical vein endothelial cells (HUVECs) and macrophage cell line RAW264.7. Polyphenol content was assayed by Folin–Ciocalteu and HPLC-MS/MS analysis. The effects of berries solutions on cell viability or proliferation were assessed by WST8 (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt and Lactate dehydrogenase (LDH) release, Trypan blue exclusion test, and Alamar blue assay. Antioxidant activity was evaluated by a cell-based chemiluminescent probe for the detection of intracellular H2O2 production in HUVECs. Heme oxygenase-1 (HO-1) expression levels were investigated by RT-qPCR. Glutathione reductase (GR), glutathione peroxidase (Gpx), superoxide dismutase (SOD), and catalase (CAT) activities, as markers of intracellular antioxidant defense, were evaluated by spectrophotometric analysis. The immunomodulatory activity was examined in RAW 264.7 by quantification of inducible nitric oxide synthase (iNOS) and Tumor Necrosis Factor—alpha (TNFα) by RT-qPCR. Data showed that fermentation of Pushgay berries (i) enhances the content of quercetin aglycone, and (ii) increases their intracellular antioxidant activity, as indicated by the reduction in H2O2-induced cell death and the decrease in H2O2-induced HO-1 gene expression in HUVECs treated for 24 h with fermented berries solution (10 µg/mL). Moreover, treatment with Pushgay berries for 72 h (10 µg/mL) promotes cells growth in RAW 264.7, and only fermented Pushgay berries increase the expression of iNOS in the same cell line. Taken together, our results show that LAB fermentation of Pushgay berries enhances their antioxidant and immunomodulatory properties.

Synthesis, and anti-inflammatory activities of gentiopicroside derivatives.

A series of 26 novel derivatives have been synthesized through structural modification of gentiopicroside, a lead COX-2 inhibitor. And their in vivo and in vitro anti-inflammatory activities have been investigated. The in vitro anti-inflammatory activities were evaluated against NO, PGE2, and IL-6 production in the mouse macrophage cell line RAW264.7 stimulated by LPS. Results showed that most compounds had good inhibitory activity. The in vivo inhibitory activities were further tested against xylene-induced mouse ear swelling. Results demonstrated that several compounds were more active than the parent compound gentiopicroside. The inhibition rate of the most active compound P23 (57.26%) was higher than positive control drug celecoxib (46.05%) at dose 0.28 mmol·kg-1. Molecular docking suggested that these compounds might bind to COX-2 and iNOS. Some of them, e.g P7, P14, P16, P21, P23, and P24, had high docking scores in accordance with their potency of the anti-inflammatory activitiy, that downregulation of the inflammatory factors, NO, PGE2, and IL-6, was possibly associated with the suppression of iNOS and COX-2. Therefore, these gentiopicroside derivatives may represent a novel class of COX-2 and iNOS inhibitors.

A Bioreactor for 3D In Vitro Modeling of the Mechanical Stimulation of Osteocytes.

The bone is a mechanosensitive organ that is also a common metastatic site for prostate cancer. However, the mechanism by which the tumor interacts with the bone microenvironment to further promote disease progression remains to be fully understood. This is largely due to a lack of physiological yet user-friendly models that limit our ability to perform in-depth mechanistic studies. Here, we report a tunable bioreactor which facilitates the 3D culture of the osteocyte cell line, MLO-Y4, in a hydroxyapatite/tricalcium phosphate (HA/TCP) scaffold under constant fluidic shear stress and tunable hydrostatic pressure within physiological parameters. Increasing hydrostatic pressure was sufficient to induce a change in the expression of several bone remodeling genes such as Dmp1, Rankl, and Runx2. Furthermore, increased hydrostatic pressure induced the osteocytes to promote the differentiation of the murine macrophage cell line RAW264.7 toward osteoclast-like cells. These results demonstrate that the bioreactor recapitulates the mechanotransduction response of osteocytes to pressure including the measurement of their functional ability in a 3D environment. In conclusion, the bioreactor would be useful for exploring the mechanisms of osteocytes in bone health and disease.

MicroRNA-23a-3p targeting of HMGB1 inhibits LPS-induced inflammation in murine macrophages in vitro.

Inflammatory cytokines, including high mobility group box 1 (HMGB1), play a key role in sepsis via various mechanisms, some of which remain unknown. Sepsis is a common cause of death in patients admitted to the intensive care unit. MicroRNAs (miRs) serve an important role in the inflammatory response. The present study aimed to investigate the role of miR-23a-3p in macrophage inflammation and the targeted regulation of HMGB1 expression. The murine macrophage cell line RAW264.7 was subjected to lipopolysaccharide (LPS) treatment to mimic the inflammation involved in sepsis in vitro. Reverse transcription-quantitative PCR was performed to measure miR-23a-3p expression and mRNA expression. Protein levels were determined using ELISA and western blotting. The target binding relationship between miR-23a-3p and the HMGB1 3'untranslated region was predicted and validated with a dual luciferase reporter assay. HMGB1 expression was increased and miR-23a-3p expression significantly reduced in patients with sepsis and in LPS-treated RAW264.7 cells in comparison with controls. Overexpression of miR-23a-3p reduced interleukin (IL)-6 and tumor necrosis factor (TNF)-α expression in RAW264.7 cells under LPS stimulation, while silencing of miR-23a-3p elevated the expression of IL-6 and TNF-α in comparison with controls. The inhibitory effect of miR-23a-3p on LPS-induced inflammation could be abolished by HMGB1 upregulation in RAW264.7 cells. HMGB1 was targeted by miR-23a-3p. miR-23a-3p is expressed at reduced levels during inflammation in sepsis, and overexpression of miR-23a-3p inhibits LPS-induced inflammation in murine macrophages in vitro by directly downregulating HMGB1. The results of the present study provided a novel insight into the molecular mechanism underlying HMGB1 expression at the post-transcriptional level in sepsis.

Advanced Glycation End Products (AGEs) Inhibit Macrophage Efferocytosis of Apoptotic β Cells through Binding to the Receptor for AGEs.

Pancreatic β cell apoptosis is important in the pathogenesis of type 2 diabetes mellitus (T2DM). Generally, apoptotic β cells are phagocytosed by macrophages in a process known as "efferocytosis." Efferocytosis is critical to the resolution of inflammation and is impaired in T2DM. Advanced glycation end products (AGEs), which are increased in T2DM, are known to suppress phagocytosis function in macrophages. In this study, we found that AGEs inhibited efferocytosis of apoptotic β cells by primary peritoneal macrophages in C57BL/6J mice or mouse macrophage cell line Raw264.7. Mechanistically, AGEs inhibit efferocytosis by blocking Ras-related C3 botulinum toxin substrate 1 activity and cytoskeletal rearrangement through receptor for advanced glycation end products/ras homolog family member A/Rho kinase signaling in macrophages. Furthermore, it was observed that AGEs decreased the secretion of anti-inflammatory factors and promoted the proinflammatory ones to modulate the inflammation function of efferocytosis. Taken together, our results indicate that AGEs inhibit efferocytosis through binding to receptor for advanced glycation end products and activating ras homolog family member A/Rho kinase signaling, thereby inhibiting the anti-inflammatory function of efferocytosis.

Interleukin-17 promotes osteoclastogenesis and periodontal damage via autophagy in vitro and in vivo

ObjectiveBecause of its potent pro-inflammatory properties, interleukin-17 (IL-17) contributes to the pathogenesis of various inflammatory diseases. This study explored the effects of IL-17 on osteoclastogenesis in an osteoclast monoculture and osteoblast-osteoclast co-culture system, as tools to investigate the molecular mechanisms underlying the interactions between osteoclastogenesis and autophagy. MethodsVarious ratios of calvarial osteoblasts (OB) and osteoclast precursor cells (mouse macrophage cell line RAW264.7, hereinafter referred to as OC) were tested. Tartrate-resistant acid phosphatase (TRAP) staining was used to detect the optimum osteoblasts:osteoclasts ratio. IL-17 was added to the co-culture system to test its effects on multinucleated osteoclast formation and osteoclast-related proteins. We assessed the effects of IL-17 on receptor activator of nuclear factor-kappa B ligand (RANKL) expression in osteoblasts, and determined if IL-17 alone could modulate osteoclast formation in an osteoclast monoculture. Administration of exogenous RANKL combined with IL-17 was employed to stimulate RAW264.7 cells osteoclastogenesis and to determine production of osteoclasts and autophagy-related proteins. We knocked down Beclin1 expression in RAW264.7 cells and examined the expression of autophagy-related and osteoclast-related proteins in RAW264.7 cells and the co-culture system, and the TAK1-binding protein 3 (TAB3)/ extracellular signal regulated kinase (ERK) pathway. ResultsA ratio of 20 OB : 1 OC yielded the highest rate of osteoclast formation. Low IL-17 concentrations increased osteoclastogenesis in co-cultures significantly, but high levels of IL-17 had the opposite effect. IL-17 alone could not induce formation of TRAP+ multinucleated cells in RAW264.7 cells. Low IL-17 concentrations promoted osteoclast differentiation and autophagy in RAW264.7 cells induced by exogenous RANKL, but high IL-17 concentrations inhibited this process. Knockdown of Beclin1 reversed the enhanced effects of 0.1 ng/mL IL-17 on osteoclastogenesis and autophagy in RAW264.7 cells. The TAB3/ERK pathway was also blocked after autophagy inhibition. ConclusionIn the co-culture model used in this study, a ratio of 20 OB:1 OC proved to be the optimal ratio to facilitate osteoclast formation. IL-17 regulated RANKL-induced osteoclastogenesis via autophagy. The Beclin1/TAB3/ERK pathway was involved in osteoclast autophagy.

Selenium-Rich Yeast Peptide Fraction Ameliorates Imiquimod-Induced Psoriasis-like Dermatitis in Mice by Inhibiting Inflammation via MAPK and NF-κB Signaling Pathways.

Psoriasis, a chronic and immune-mediated inflammatory disease, adversely affects patients’ lives. We previously prepared selenium-rich yeast peptide fraction (SeP) from selenium-rich yeast protein hydrolysate and found that SeP could effectively alleviate ultraviolet radiation-induced skin damage in mice and inhibited H2O2-induced cytotoxicity in cultured human epidermal keratinocyte (HaCaT) cells. This study aimed to investigate whether SeP had a protective effect on imiquimod (IMQ)-induced psoriasis-like dermatitis in mice and the underlying mechanisms. Results showed that SeP significantly ameliorated the severity of skin lesion in IMQ-induced psoriasis-like mouse model. Moreover, SeP treatment significantly attenuated the expression of key inflammatory cytokines, including interleukin (IL)-23, IL-17A, and IL-17F, in the dorsal skin of mice. Mechanistically, SeP application not only inhibited the activation of JNK and p38 MAPK, but also the translocation of NF-κB into the nucleus in the dorsal skin. Furthermore, SeP treatment inhibited the levels of inflammatory cytokines and the activation of MAPK and NF-κB signaling induced by lipopolysaccharide in HaCaT cells and macrophage cell line RAW264.7. Overall, our findings showed that SeP alleviated psoriasis-like skin inflammation by inhibiting MAPK and NF-κB signaling pathways, which suggested that SeP would have a potential therapeutic effect against psoriasis.

POS-458 PRELIMINARY STUDY ON THE REGULATORY EFFECT OF HIGH GLUCOSE ON MACROPHAGE POLARIZATION THROUGH miR-125b

Taking the mouse macrophage cell line RAW264.7 as the research object, to explore the effect of high glucose microenvironment on the polarization of mouse macrophages and the regulatory role of miR-125b in vitro.

P104 Icariin Attenuates Intestinal Inflammation through Inhibition of the Nuclear Factor-ĸB signaling

Abstract Background Icariin is a natural flavonoid glucoside from plants in Epimedium family, known to have anti-oxidative and anti-inflammatory activities. This study investigated the effects of Icariin on nuclear factor-kappa B (NF-ĸB) signaling pathway in intestinal epithelial cells (IEC) and macrophages, and in acute and chronic murine colitis models. Methods Human IEC line COLO 205 and murine macrophage cell line RAW 264.7 were pretreated with Icariin and then stimulated with tumor necrosis factor-α (TNF-α) and lipopolysaccharide respectively for in vitro studies. The expression of pro- and anti-inflammatory cytokines were evaluated by real-time RT-PCR (TNF- α, IL-6, IL-8, IL-12, and IL-10), and the effect of Icariin on NF-ĸB signaling pathway was examined by immunoblot analysis to detect IĸBα phosphorylation and degradation. Dextran sulfate sodium (DSS)-induced acute colitis in C57BL/6 wild-type mice and chronic colitis in IL-10-/- mice were treated with or without Icariin for in vivo studies. The severity of colitis was evaluated by daily body weight change, colon length, and colonic tissue histopathology. Results Icariin significantly attenuated the production of pro-inflammatory cytokines and degeneration of anti-inflammatory cytokines, and alleviated IĸBα phosphorylation and degradation in both IEC and murine macrophages stimulated by TNF-α and LPS, respectively. Icariin also significantly reduced the severity of colitis in both DSS-induced acute colitis model and chronic colitis in IL-10-/- mice models. Conclusion Icariin inhibits NF-ĸB signaling in both IECs and macrophages, and attenuates experimental murine colitis. These results suggest that Icariin may be a potential novel therapeutic agent for inflammatory bowel disease.

Lysosomal iron recycling in mouse macrophages is dependent upon both LcytB and Steap3 reductases

AbstractIron that is stored in macrophages as ferritin can be made bioavailable by degrading ferritin in the lysosome and releasing iron back into the cytosol. Iron stored in ferritin is found as Fe3+ and must be reduced to Fe2+ before it can be exported from the lysosome. Here we report that the lysosomal reductase Cyb561a3 (LcytB) and the endosomal reductase six-transmembrane epithelial antigen of prostate 3 (Steap3) act as lysosomal ferrireductases in the mouse macrophage cell line RAW264.7 converting Fe3+ to Fe2+ for iron recycling. We determined that when lysosomes were loaded with horse cationic ferritin, reductions or loss of LcytB or Steap3 using CRISPR/Cas9-mediated knockout technology resulted in decreased lysosomal iron export. Loss of both reductases was additive in decreasing lysosomal iron export. Decreased reductase activity resulted in increased transcripts for iron acquisition proteins DMT1 and transferrin receptor 1 (Tfrc1) suggesting that cells were iron limited. We show that transcript expression of LcytB and Steap3 is decreased in macrophages exposed to Escherichia coli pathogen UTI89, which supports a role for these reductases in regulating iron availability for pathogens. We further show that loss of LcytB and Steap3 in macrophages infected with UTI89 led to increased proliferation of intracellular UTI89 suggesting that the endolysosomal system is retaining Fe3+ that can be used for proliferation of intravesicular pathogens. Together, our findings reveal an important role for both LcytB and Steap3 in macrophage iron recycling and suggest that limiting iron recycling by decreasing expression of endolysosomal reductases is an innate immune response to protect against pathogen proliferation and sepsis.

GINS2 affects activity/differentiation, apoptosis and proliferation of osteoblast and osteoclast in steroid-induced osteonecrosis of the femoral head by regulating P53/GADD45A signaling pathway

To investigate the function and mechanism of GINS complex subunit 2 (GINS2) in osteoblast and osteoclast in steroid-induced osteonecrosis of the femoral head (SINFH). Clinical specimens were collected from 40 cases of SINFH patients and 40 cases of control patients. Then, SINFH mouse model was built. Immediately, osteoblasts and osteoclasts were induced and differented from the pre-osteoblast cell line MC3T3-E1 and the mononuclear macrophage cell line RAW264.7 of mice, respectively. The qRT-PCR assay was used for detecting the expressions of GINS2, p53 and growth arrest and DNA damage 45A (GADD45A). The cytoactive, proliferation and apoptosis of osteoblasts and osteoclasts of SINFH mice were measured by tartaric acid phosphatase staining, CCK-8 and Annexin V-FITC/PI dual staining, respectively. GINS2 was up-regulated while p53/GADD45A was down-regulated in femoral tissues in SINFH patients and mice. GINS2 was down-regulated while p53 and GADD45A were up-regulated in osteoblasts of SINFH mice, which presented opposite trends in osteoclasts formations. GINS2 decreased osteoblasts activities and increased osteoclasts activities, inhibited apoptosis and promoted proliferation in osteoblasts and osteoclasts via p53/GADD45A pathway. GINS2 affects activity/differentiation, apoptosis and proliferation of osteoblast and osteoclast in steroid-induced osteonecrosis of the femoral head by regulating p53/GADD45A signaling pathway.

Measuring the Lateral Diffusion of Plasma Membrane Receptors Using Raster Image Correlation Spectroscopy.

Raster image correlation spectroscopy (RICS) enables detecting and quantifying diffusion in live cells using standard commercial laser scanning confocal microscopes. Here, we describe a protocol based on RICS for measuring the lateral diffusion of two immunoreceptors within the plasma membrane of the macrophage cell line RAW 264.7. The sample images and measurements presented in this chapter were obtained from RICS analysis of Toll-like receptor 2 (TLR2) and cluster of differentiation 14 (CD14), which are transmembrane and membrane-anchored receptors, respectively. A step-by-step guideline is provided to acquire raster-scanned images and to extract the diffusion coefficients using RICS analysis.

A Traditional Medicine-Based Combination of Bamboo Salt and Egg White-Chalcanthite Ameliorates Atopic Dermatitis

Bamboo salt and egg white-chalcanthite mixture has been prescribed for relieving the inflammation of atopic dermatitis in Hamyang, Gyeongsangnam-do, Korea. This study was conducted to demonstrate the effect of atopic dermatitis on cell and atopic animal models using a combination of bamboo salt with egg white-chalcanthite, which have been used as folk remedies for inflammatory conditions. Based on the in vitro experiment, the 1:20 mixture of egg white-chalcanthite and bamboo salt was shown to inhibit the release of beta-hexosaminidase and histamine in rat basophilic leukemia cell line RBL-2H3 by sensitizing immunoglobulin E and inducing allergic responses by dinitrophenyl as well as the expression of inflammatory cytokines and inflammatory mediators in a dose-dependent manner in lipopolysaccharide-treated mouse macrophage cell line RAW 264.7 cells. Furthermore, egg white-chalcanthite with bamboo salt and its combination at different mixing ratios showed a decrease in inflammatory mediators. Dinitrochlorbenzene was applied to NC/Nga mice for 6 w to induce the atopic in vivo model. After 4 w administration of the test substance, the dermatitis decreased visually and dermal hyperplasia/inflammation, mast cell/eosinophils counts and degree of nerve fiber infiltration were decreased according to histopathological examination. In addition, the secretion rate of interferon gamma in high-dose treated group was lower than that in atopic dermatitis control group. These results show that bamboo salt with egg white-chalcanthite effectively relieves skin inflammation in atopic dermatitis and it might be a promising mineral agent for relieving the symptoms of atopic dermatitis.

Cinacalcet Targets the Neurokinin-1 Receptor and Inhibits PKCδ/ERK/P65 Signaling to Alleviate Dextran Sulfate Sodium-Induced Colitis.

Objective: Inflammatory bowel disease is an immune-mediated chronic inflammatory disease of the gastrointestinal tract for which curative drugs are currently not available. This study was performed to assess the therapeutic effects of cinacalcet on dextran sulfate sodium (DSS)-induced colitis. Methods: Primary macrophages obtained from bone marrow and the macrophage cell line RAW264.7 were used to examine the inhibitory effect of cinacalcet on cytokine production, the PKCδ/ERK/P65 signaling pathway, and NF-κB P65 translocation. Colitis was induced using DSS to assess the treatment effect of cinacalcet. Bioinformatics approaches were adopted to predict potential targets of cinacalcet, and a drug affinity responsive target stability (DARTs) assay was performed to confirm binding between cinacalcet and potential target. Results: In vivo analysis showed that cinacalcet reduced the disease activity score, prevented shortening of the colon, diminished inflammatory cell infiltration, and protected the structural integrity of the intestinal wall. Cinacalcet also reduced production of the inflammatory cytokines TNFα, IL-1β, and IL-6 in the colon and sera of mice with DSS-induced colitis. In vitro studies revealed that cinacalcet suppressed the translocation of P65 and inhibited production of the inflammatory cytokines IL-1β and IL-6. Mechanistic studies revealed that the target of cinacalcet was neurokinin-1 receptor (NK1R) and their binding was confirmed by a DARTs assay. Furthermore, the inhibition of NK-κB P65 activation was found to occur via the suppression of PKCδ/ERK/P65 signaling mediated by cinacalcet. Conclusion: Cinacalcet inhibits the activation of NF-κB and reduces the production of inflammatory cytokines by suppressing the PKCδ/ERK/P65 signaling pathway via targeting NK1R, suggesting that it can be used to treat inflammatory diseases, particularly colitis.