Mouse Bone Marrow-derived Cells Research Articles

Liposomes derived from Mycobacterium smegmatis promote immune activation of mice bone marrow-derived dendritic cells.

Tuberculosis (TB) is the leading cause of mortality due to infectious diseases. The development of new generation vaccines against TB is of paramount importance for the control of the disease. In previous studies, liposomes obtained from lipids of Mycobacterium smegmatis (LMs) demonstrated their immunogenicity and protective capacity against Mycobacterium tuberculosis in mice. To characterize the immunomodulatory capacity of this experimental vaccine candidate, in the current study, the stimulatory capacity of LMs was determined on bone marrow-derived dendritic cells (BMDCs) from mice. LMs were obtained and incubated with mature BMDCs. The internalization of LMs by BMDCs was studied by confocal microscopy, and the LMs immune-stimulatory capacity was determined by the expression of surface molecules (CD86 and MHCII) and the cytokine production (interleukin [IL]-12, interferon-Υ, tumor necrosis factor-α, and IL-10) 24 h after exposure to LMs. The interaction of LMs with BMDCs and its internalization was demonstrated as well as the immune activation of BMDCs, characterized by the increased expression of CD86 and the production of IL-12. The LMs internalization and immune activation of BMDCs were blocked in the presence of cytochalasin, filipin III and chlorpromazine, which demonstrated that internalization of LMs by BMDCs is a key process for the LMs induced immune activation of BMDCs. The results obtained support the further evaluation of LMs as a mycobacterial vaccine, adjuvant, and in immunotherapy.

Gold clusters prevent breast cancer bone metastasis by suppressing tumor-induced osteoclastogenesis.

Rationale: Bone is the most frequent site for breast cancer metastasis, which accounts for the leading cause of death in advanced breast cancer patients. Serious skeletal-related events (SREs) caused by bone metastasis have a decisive impact on the life expectancy of breast cancer patients, making breast cancer almost incurable. Metastatic breast cancer cell induced pathological osteoclastogenesis is a key driver of bone metastasis and osteolytic bone lesions. We previously reported that gold clusters can prevent inflammation induced osteoclastogenesis and osteolysis in vivo. In this study, we investigated the effects of a BSA-coated gold cluster on metastatic breast cancer-induced osteoclastogenesis in vitro and tumor-induced osteolysis in vivo, and elucidated its possible mechanism.Methods: Breast cancer cell line MDA-MB-231 was used to evaluate the regulatory effects of gold clusters on breast cancer metastasis and tumor induced osteoclastogenesis in vitro. Cell counting kit-8, transwell, wound-healing and colony formation assays were performed to evaluate the effect of gold clusters on proliferation and metastasis of MDA-MB-231 cells. Tartrate-resistant acid phosphatase (TRAP) staining and filamentous-actin rings analysis were used to detect the regulatory effects of gold clusters on MDA-MB-231 cell-conditioned medium (MDA-MB-231 CM) triggered and receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastogenesis in mouse bone marrow-derived mononuclear cells (BMMs). A mouse model of breast cancer bone metastasis was used to evaluate the in vivo activity of the gold cluster on the tumor induced osteolysis.Results: The gold clusters suppressed the migration, invasion and colony formation of MDA-MB-231 cells in a dose-dependent manner in vitro. The gold clusters strongly inhibited both MDA-MB-231 CM triggered and RANKL-induced osteoclast formation from BMMs in vitro. Cell studies indicated that the gold clusters suppressed the expression of osteolysis-related factors in MDA-MB-231 cells and inhibited the subsequent activation of NF-κB pathway in BMMs. Treatment with the clusters at a dose of 10 mg Au/kg.bw significantly reduces the breast cancer cell induced osteolysis in vivo.Conclusion: Therefore, the gold clusters may offer new therapeutic agents for preventing breast cancer bone metastasis and secondary osteolysis to improve patient outcomes.

The role of bone marrow-derived cells in the origin of liver cancer revealed by single-cell sequencing.

Objective: Epithelial cancers often originate from progenitor cells, while the origin of hepatocellular carcinoma (HCC) is still controversial. HCC, one of the deadliest cancers, is closely linked with liver injuries and chronic inflammation, which trigger massive infiltration of bone marrow-derived cells (BMDCs) during liver repair.Methods: To address the possible roles of BMDCs in HCC origination, we established a diethylnitrosamine (DEN)-induced HCC model in bone marrow transplanted mice. Immunohistochemistry and frozen tissue immunofluorescence were used to verify DEN-induced HCC in the pathology of the disease. The cellular origin of DEN-induced HCC was further studied by single cell sequencing, single-cell nested PCR, and immunofluorescence-fluorescence in situ hybridization.Results: Studies by using single cell sequencing and biochemical analysis revealed that HCC cells in these mice were coming from donor mice BMDCs, and not from recipient mice. Furthermore, the copy numbers of mouse orthologs of several HCC-related genes previously reported in human HCC were also altered in our mouse model. DEN-induced HCCs exhibited a similar histological phenotype and genomic profile as human HCCs.Conclusions: These results suggested that BMDCs are an important origin of HCC, which provide important clues to HCC prevention, detection, and treatments.

Selenomethionine supplementation reduces lesion burden, improves vessel function and modulates the inflammatory response within the setting of atherosclerosis

Atherosclerosis is a chronic inflammatory disease of the vasculature characterised by the infiltration of activated neutrophils and macrophages at sites of damage within the vessel wall, which contributes to lesion formation and plaque progression. Selenomethionine (SeMet) is an organic form of selenium (Se), an essential trace element that functions in the regulation of the immune response by both bolstering the endogenous thioredoxin and glutathione antioxidant defence systems and by directly scavenging damaging oxidant species. This study evaluated the effect of dietary SeMet supplementation within a high fat diet fed apolipoprotein E deficient (ApoE−/-) mouse model of atherosclerosis. Dietary supplementation with SeMet (2 mg/kg) increased the tissue concentration of Se, and the expression and activity of glutathione peroxidase, compared to non-supplemented controls. Supplementation with SeMet significantly reduced atherosclerotic plaque formation in mouse aortae, resulted in a more stable lesion phenotype and improved vessel function. Concurrent with these results, SeMet supplementation decreased lesion accumulation of M1 inflammatory type macrophages, and decreased the extent of extracellular trap release from phorbol myristate acetate (PMA)-stimulated mouse bone marrow-derived cells. Importantly, these latter results were replicated within ex-vivo experiments on cultured neutrophils isolated from acute coronary syndrome patients, indicating the ability of SeMet to alter the acute inflammatory response within a clinically-relevant setting. Together, these data highlight the potential beneficial effect of SeMet supplementation as a therapeutic strategy for atherosclerosis.

Expression and Functional Role of TRPV4 in Bone Marrow-Derived CD11c+ Cells.

The increase in cytosolic Ca2+ is essential in key effector functions of dendritic cells (DCs), including differentiation, maturation, cytokine expression, and phagocytosis. Although several Ca2+-permeable ion channels have been described in DCs, the contribution of transient receptor potential (TRP) channels remains poorly understood. Here, we investigated whether TRPV4 plays a role in the differentiation, maturation, and phagocytosis of granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced mouse bone marrow-derived cells (BMDCs). Using intracellular Ca2+ imaging experiments, we found that TRPV4 was functionally expressed in the plasma membrane of immature CD11c+ BMDCs and that its activity and expression were downregulated in CD11c+ BMDCs matured with lipopolysaccharide (LPS). Comparative analysis of the GM-CSF-stimulated cells showed that Trpv4 knockout and wild-type bone marrow cultures had a similar distribution of differentiated cells, generating a heterogenous culture population rich in CD11c+, CD11b+ cells, and low levels of F4/80+ cells. The lack of TRPV4 did not prevent the LPS-induced nuclear translocation of NF-κB, the upregulation of the proinflammatory cytokines IL-6 and IL-12, or the upregulation of the maturation markers CD40, CD80, and CD86. In contrast, TRPV4-deficient CD11c+ BMDCs exhibited a significantly reduced endocytic capacity of IgG-coated beads, but the internalization of uncoated beads in the absence of TRPV4 was not affected. Taken together, our results demonstrate that TRPV4 was dispensable in the differentiation and maturation of mouse CD11c+ BMDCs but contributed to the mechanism underlying Fc receptor-mediated phagocytosis. Overall, our results further strengthen the role of TRPV4 in immune-related processes.

Investigation on the role of temperature in macrophage responses

Abstract This study investigated temperature-associated changes in macrophage activation and metabolism across species. Macrophages were isolated from C57BL/6J mice, broiler chicken spleens, and rainbow trout head kidneys, treated with lipopolysaccharide (LPS) and nitric oxide (NO) responses measured. Mouse macrophage NO production was similar at 37°C (normal body temp.) and 35°C. However at 33°C and 28°C responses dropped notably. At 39°C, Mouse bone marrow-derived macrophages (BMDM) and mouse cell line, RAW264.7, showed higher NO production versus 37°C. In chicken splenic macrophages (CSM) LPS-induced NO responses were similar at 41°C (normal for avian) 37°C, and 39°C. A mild fever of 42°C had a large stimulatory effect on NO production compared to 41°C. During infection, trout induce fever by swimming to warmer water. We found NO responses in LPS-treated head kidney macrophages to be higher at 15, 28, and 37°C than at 4°C. Total protein synthesis was evaluated via fluorescence. Without LPS stimulation, peritoneal resident mouse macrophages (PRM) from young mice showed similar protein synthesis at 37°C and 39°C. In both young and aged LPS-stimulated PRM, significantly higher protein synthesis was observed at 39°C compared to 37°C, though the increase was smaller in aged PRM. However, this effect was not uniform across all tissues. LPS stimulated young (BMDM) showed an increased protein synthesis at 37°C but cells from aged did not. A further increase in LPS stimulation was observed at 39°C. Consistent with fever increasing protein synthesis, LPS-stimulated CSM and chicken cell line HTC, showed higher protein synthesis at 42°C compared with 41°C.

Intra-hippocampal injection with mouse bone marrow-derived microglia-like cells contribute to amyloid-β clearance and improvement of memorial dysfunction in a mouse model of Alzheimer's disease.

Intra-hippocampal injection with mouse bone marrow-derived microglia-like cells contribute to amyloid-β clearance and improvement of memorial dysfunction in a mouse model of Alzheimer's disease.

The comparative study of the effects of Fe2 O3 and TiO2 micro- and nanoparticles on oxidative states of lung and bone marrow tissues and colony stimulating factor secretion.

Nowadays, increased use of nanomaterials in industry and biomedicine poses potential risks to human health and the environment. Studying their possible toxicological effects is therefore of great significance. The present investigation was designed to examine the status of oxidative stress induced by nanoparticles (NPs) of ferric oxide (Fe2 O 3 ) and titanium oxide (TiO 2 ) with their micro-sized counterpart on mouse lung and bone marrow-derived normal tissue cells. We assessed the induction of oxidative stress by measuring its indicators such as antioxidant scavenging activity of superoxide dismutase and catalase as well as malondialdehyde concentration. Moreover, colony formation of bone marrow cells was assayed following induction with colony stimulating factor (CSF) from lung cells. NPs had a more potent stimulatory effect on the oxidative stress status than their micron-sized counterparts. In addition, the highest level of oxidative stress derived from TiO 2 NPs was observed in both tissue types. Cotreatment with NPs and the antioxidant α-tocopherol reduced antioxidant activities and membrane lipid peroxidation (LPO) in the lung cells, but increased CSF-induced colony formation activity of bone marrow cells, suggesting that oxidative stress may be the cause of the cytotoxic effects of NPs. It is concluded that free radicals generated following exposure to NPs resulted in significant oxidative stress in mouse cells, indicated by increased LPO and antioxidant enzyme activity and decreased colony formation.

LPS protects macrophages from AIF-independent parthanatos by downregulation of PARP1 expression, induction of SOD2 expression, and a metabolic shift to aerobic glycolysis

In inflamed tissues or during ischemia-reperfusion episodes, activated macrophages produce large amounts of reactive species and are, thus, exposed to the damaging effects of reactive species. Here, our goal was to investigate the mechanism whereby activated macrophages protect themselves from oxidant stress-induced cell death. Hydrogen peroxide-treated mouse bone marrow-derived macrophages (BMDM) and THP-1 human monocyte-derived cells were chosen as models. We found a gradual development of resistance: first in monocyte-to-macrophage differentiation, and subsequently after lipopolysaccharide (LPS) exposure. Investigating the mechanism of the latter, we found that exposure to intense hydrogen peroxide stress causes poly(ADP-ribose) polymerase-1 (PARP-1) dependent programmed necrotic cell death, also known as parthanatos, as indicated by the protected status of PARP-1 knockout BMDMs and the protective effect of the PARP inhibitor PJ-34. In hydrogen peroxide-treated macrophages, however, apoptosis inducing factor (AIF) proved dispensable for parthanatos; nuclear translocation of AIF was not observed. A key event in LPS-mediated protection against the hydrogen peroxide-induced AIF independent parthanatos was downregulation of PARP1 mRNA and protein. The importance of this event was confirmed by overexpression of PARP1 in THP1 cells using a viral promoter, which lead to stable PARP1 levels even after LPS treatment and unresponsiveness to LPS-induced cytoprotection. In BMDMs, LPS-induced PARP1 suppression lead to prevention of NAD+ depletion. Moreover, LPS also induced expression of antioxidant proteins (superoxide dismutase-2, thioredoxin reductase 1 and peroxiredoxin) and triggered a metabolic shift to aerobic glycolysis, also known as the Warburg effect. In summary, we provide evidence that in macrophages intense hydrogen peroxide stress causes AIF-independent parthanatos from which LPS provides protection. The mechanism of LPS-mediated cytoprotection involves downregulation of PARP1, spared NAD+ and ATP pools, upregulation of antioxidant proteins, and a metabolic shift from mitochondrial respiration to aerobic glycolysis.

Cultivation and identification of mouse peritoneal and bone marrow-derived mast cells

Objective To explore the in vitro culture methods for oriented differentiation of peritoneal cells and bone marrow cells into high-purity mast cells, and to identify the function of these mast cells. Methods Peritoneal cells and bone marrow cells were isolated from the peritoneal cavity lavages and femur of C57BL/6 mice, and cultured with both interleukin-3 (IL-3) and stem cell factor for 2 and 4 weeks respectively. Light microscopy was performed to observe the morphology of these cells, toluidine blue staining to identify the degree of maturity of these mast cells, and flow cytometry to measure the expression of cell surface markers CD117 and FceRⅠα. After the stimulation with compound 48/80 at different concentrations, the degranulation rate of mast cells was counted under the microscope, and β-hexosaminidase release rate was measured by spectrophotometry. Results After 2- or 4-week culture, the mouse peritoneal and bone marrow cells all manifested as refractive suspension cells of uniform size. Toluidine blue staining showed violaceous metachromatic granules in the cytoplasm of the two kinds of cells. The proportions of CD117 or FceR Ⅰ α single-positive peritoneal and bone marrow-derived mast cells were all more than 95%, and the proportions of CD117/FceRⅠα double-positive peritoneal and bone marrow-derived mast cells were 97.68% ± 0.80% and 96.12% ± 0.76% respectively. The degranulation rates of mast cells in the 100- and 1 000-mg/L compound 48/80 groups significantly differed from those in the blank control group (all P < 0.01) . Compared with the blank control group, the β-hexosaminidase release rates significantly increased in bone marrow-derived mast cells in the 100-mg/L compound 48/80 group and peritoneal mast cells in the 10- and 100-mg/L compound 48/80 groups (P < 0.01 or 0.05) . Conclusion IL-3 and stem cell factor can co-induce the directed differentiation and proliferation of mouse bone marrow stem cells and peritoneal cells, so as to harvest high-purity mature degranulated mast cells, and lay a foundation for subsequent cell biology research. Key words: Mast cells; Myeloid progenitor cells; Induced pluripotent stem cells; Mice; Cells, cultured; Interleukin-3; Stem cell factor

Leucodin attenuates inflammatory response in macrophages and lipid accumulation in steatotic hepatocytes via P2x7 receptor pathway: A potential role in alcoholic liver disease

The current study was aimed to reveal that leucodin, a sesquiterpene lactone from Artemisia capillaris could inhibit the inflammatory response in macrophages and the lipid accumulation in hepatocytes via P2x7R-NLRP3 inflammasome activation. Several types of macrophages including mouse peritoneal macrophages, mouse bone marrow-derived macrophages and human macrophages THP-1 cells were pretreated with different concentrations of leucodin for 1 h and then stimulated with LPS and ATP. LPS plus ATP initiated IL-1β cleavage and release in mouse peritoneal macrophages and peaked at 4 h. Leucodin did not show significant toxicity within 200 μM and effectively inhibited pro-IL-1β cleavage and release of mature-IL-1β in macrophages. Also, P2x7R antagonist and caspase-1 inhibitor also decreased IL-1β release and cleavage. Additionally, leucodin suppressed P2x7R, TLR4 and NLRP3 expression in LPS/ATP-stimulated macrophages. HepG2 cells were pretreated with different concentrations of leucodin for 1 h and then exposed to ethanol for 24 h. Leucodin suppressed lipid accumulation and enhanced phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC) in HepG2 cells exposed to ethanol. In addition, leucodin inhibited the expression of sterol regulatory element binding protein-1 (SREBP1) and ACC in ethanol-treated HepG2 cells. Leucodin possessed the capacity for inhibiting inflammatory response in macrophages and suppressing lipid accumulation in hepatocytes, suggesting a promising therapeutic potential targeting inflammation and lipid metabolism in alcoholic liver disease.

Ibrutinib has a novel immunomodulatory effect by enhancing DC maturation both in vivo and in a model of inflammation

Abstract Myeloid derived suppressor cells (MDSCs) can inhibit T cell activation by altering the characteristics and activation of dendritic cells (DCs). We had previously reported that Ibrutinib enhances the expression of co-stimulatory molecules on DCs and promotes DC-mediated T cell activation. In the present study we further determined the mechanisms of DC immunomodulation by Ibrutinib. We evaluated the effect of Ibrutinib on (i) DC maturation and migration from the injection site to draining lymph nodes, (ii) T cell proliferation in vivo, and (iii) monocyte maturation to DCs using a mouse inflammation model. DCs were generated from mouse bone marrow-derived cells in the presence of vehicle or 10μM lbrutinib. CFES-labelled DCs were injected subcutaneously into the footpads of naive mice, and their migration to the regional lymph nodes and maturation profile was analyzed by flow cytometry. To evaluate T cell priming, CFSE-labelled OVA-specific T cells isolated from OT-II mice were injected into the tail vein of syngeneic mice that were prior injected with mature DCs loaded with OVA peptide. After four days, T cell proliferation was evaluated by CFSE dilution. In the inflammation model, C57BL6 mice were treated with vehicle or 6mg/kg/day Ibrutinib orally for a week. A sub-lethal dose of LPS was injected in the dorsal skin and the frequency of inflammatory monocytes and mature DCs was determined. We found that Ibrutinib significantly improves DC maturation and migration, inducing T cell activation and proliferation in vivo. In addition, Ibrutinib enhances monocyte maturation to DCs in the skin. The results suggest a novel effect of Ibrutinib, which may have therapeutic use for cancers characterized by a high MDSC infiltration.

Effects of moderate intensity static magnetic fields on osteoclastic differentiation in mouse bone marrow cells.

Although we recently demonstrated that static magnetic fields (SMFs) of 3, 15, and 50 mT stimulate osteoblastic differentiation, the effects of SMFs on osteoclastogenesis are still poorly understood. This study focused on the suppressive effects of SMFs on receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis and bone resorption. Direct SMFs inhibit RANKL-induced multinucleated osteoclast formation, tartrate-resistant acid phosphatase activity, and bone resorption in mouse bone marrow-derived macrophage cells. The conditioned medium from osteoblasts treated with SMFs also resulted in the inhibition of osteoclast differentiation as well as resorption. The RANKL-induced expression of osteoclast-specific transcription factors, such as c-Fos and NFATc1, was remarkably downregulated by SMF at 15 mT. In addition, SMF inhibited RANKL-activated Akt, glycogen synthase kinase 3β (GSK3β), extracellular signal-regulated kinase, c-jun N-terminal protein kinase, mitogen-activated protein kinase (MAPK), and nuclear factor-κB (NF-κB) formation. These findings indicate that SMF-mediated attenuation of RANKL-induced Akt, GSK3β, MAPK, and NF-κB pathways could contribute to the direct and indirect inhibition of osteoclast formation and bone resorption. Therefore, SMFs could be developed as a therapeutic agent against periprosthetic or peri-implant osteolysis. Additionally, these could be used against osteolytic diseases such as osteoporosis and rheumatoid arthritis. Bioelectromagnetics. 39:394-404, 2018. © 2018 Wiley Periodicals, Inc.

Transfection of bone marrow derived cells with immunoregulatory proteins

In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%–72%) and minimal cell death. The transfection efficiencies and cell death varied depending on the culture duration of the DCs and Mφ. Moreover, the electroporation efficiency was increased when conditioning medium was used for culturing the cells. Furthermore, we demonstrated that measuring the plasmid-encoded secreted proteins is a highly sensitive method for determining the transfection efficiency. In summary, electroporation with plasmid vectors is an efficient method for producing DCs and Mφ with transient expression of immunoregulatory proteins.

Transcriptional and posttranscriptional repression of histone deacetylases by docosahexaenoic acid in macrophages

Histone deacetylation is one of the posttranslational modifications of histones by which eukaryotic cells alter gene transcription. Although fatty acids are the best known macronutrients that modulate gene expression in inflammatory pathways, it is unclear whether common fatty acids in diets can regulate the expression of histone deacetylases (HDACs) in macrophages. We determined the effects of fatty acids, including palmitic acid (PA), oleic acid (OA), linoleic acid, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), on the expression of HDAC isoforms in RAW 264.7 macrophages, mouse bone marrow-derived macrophages (BMDM) and human THP-1 cells. In RAW 264.7 macrophages, OA significantly increased mRNA levels of Hdac1, 2 and 3, and EPA induced Hdac2 expression compared with control. Marked repression of Hdac9 mRNA levels by EPA and DHA, with DHA being more potent, was observed in RAW 264.7 macrophages and BMDM. DHA also decreased HDAC3, 4 and 9 protein levels. EPA and DHA facilitated the proteasomal degradation of HDAC3 and 4 protein, while the transcriptional repression of HDAC9 by DHA may be mediated by the repression of myocyte enhancer factor 2 or by the activation of retinoid X receptor. Functionally, inhibition of HDAC activity or knockdown of Hdac9 in macrophages reduced lipopolysaccharide-induced inflammatory gene expression. Our results demonstrate that DHA represses the expression of HDAC3, 4 and 9 at the transcriptional or posttranscriptional levels in murine macrophages. This suggests that the anti-inflammatory effect of DHA may be mediated by the reduction of HDACs.

The effect of mesenchymal stem cells on chemotaxis of osteoclast precursor cells

Regeneration of tissue, including bone, using mesenchymal stem cells (MSCs) has been progressing rapidly. Regeneration of bone requires the presence of an appropriate environment and efficient chemotaxis of cells to the target site. Differentiation of MSCs into mesenchymal cells has received considerable attention, but the effect of MSCs on chemotaxis is not well understood. In this study, we investigated the effect of MSCs on chemotaxis of RAW264 cells via C-C motif chemokine ligand 2 (CCL2). Balb/c mouse bone marrow-derived MSCs and RAW264 cells, which are osteoclast precursor cells, were co-cultured without cell contact. The gene expression of CCL2 in MSCs and CC-chemokine receptor 2 (CCR2) in RAW264 cells was determined using quantitative real-time PCR. Analysis of RAW264 cell chemotaxis was performed using the Boyden chamber assay. mRNAs for CCL2 and CCR2 were significantly upregulated upon co-culture in comparison to culture of either cell type alone, and the number of chemotactic RAW264 cells was significantly increased by co-culture. MSCs enhanced the chemotaxis of RAW264 cells, possibly via CCL2-CCR2 interaction, suggesting the potential utility of MSCs for tissue regeneration.

Synthesis of 1,5-Anhydro-d-fructose derivatives and evaluation of their inflammasome inhibitors

Synthesis of several 1,5-Anhydro-d-fructose (1,5-AF) derivatives to evaluate inhibitory activities of the inflammasome was carried out. Recently, 1,5-AF reported to suppress the inflammasome, although with only low activity. We focused on the hydration of 2-keto form of 1,5-AF and speculated that this hydration was the cause of low activity. Therefore, we synthesized some 1,5-AF derivatives that would not be able to form the dimer conformation and can be expected to have high activity against inflammasome, and then evaluated their inhibitory activities with respect to the NLRP3 inflammasome by using mouse bone marrow-derived macrophages and human THP-1 cells. As a result, some synthesized 2-keto form compounds had much higher inhibitory activities with respect to the NLRP3 inflammasome than did 1,5-AF.

Mycobacterium tuberculosis ESAT6 induces IFN-β gene expression in Macrophages via TLRs-mediated signaling

Mycobacterium tuberculosis is a highly virulent bacterium that causes tuberculosis. It infects about one third of the world’s population. Type I interferons (IFNs) play a detrimental role in host defense against M. tuberculosis infection. Proteins secreted by M. tuberculosis through ESX-1 secretion system contribute to type I IFNs production. However, the precise mechanism by which 6-kDa early secretory antigen target (ESAT6), one of ESX-1-mediated secretory proteins, induces type I IFNs production in host cells is currently unclear. Therefore, the objective of the present study was to determine the underlying molecular mechanism regulating ESAT6-mediated gene expression of IFN-β in macrophages. Recombinant ESAT6 produced from E. coli expression system induced IFN-β gene expression in various types of macrophages such as mouse bone marrow-derived macrophages (BMDMs), peritoneal macrophages, and MH-S cells (murine alveolar macrophage cell line). Deficiency of TLR4 and TRIF absolutely abrogated ESAT6-induced IFN-β gene expression. TLR2 and MyD88 were partially involved in IFN-β gene expression in response to low dose of ESAT6. Another recombinant ESAT6 produced from baculovirus system also upregulated IFN-β gene expression via TLR4-dependent pathway. Polymyxin B (PMB) treatment impaired LPS-induced IFN-β expression. However, IFN-β expression induced by ESAT6 was not influenced by PMB. This suggests that ESAT6-mediated IFN-β expression is not due to LPS contamination. Treatment with ESAT6 resulted in activation of TBK1 and IRF3 in macrophages. Such activation was abolished in TLR4- and TRIF-deficient cells. Moreover, inhibition of IRF3 and TBK1 suppressed IFN-β gene expression in response to ESAT6. Our results suggest that ESAT6 might contribute to virulence of M. tuberculosis by regulating type I IFNs production through TLR4-TRIF signaling pathway.

Bone regenerative effect of aqueous Cynanchum wilfordii extract in receptor activator of nuclear factor-κB ligand-induced osteoclast differentiation and estrogen deficiency-induced osteoporosis

&lt;p&gt;Osteoporosis increases with age, most frequently in postmenopausal women because of reduced ovarian hormone levels. Furthermore, estrogen deficiency impairs trabecular metaphyseal bone. Although efficacious, long-term hormone replacement therapy (HRT) has estrogen-like side effects including breast and endometrial cancers, and non-hormonal or herbal therapies may be safer alternatives. Therefore, the aim of this study was to investigate the effects of aqueous extracts of &lt;em&gt;Cynanchum wilfordii&lt;/em&gt; (CWW) on receptor activator of nuclear factor-κ B (NF-κ B) ligand (RANKL)-induced osteoclast differentiation &lt;em&gt;in vitro&lt;/em&gt; and ovariectomy-mediated osteoporosis &lt;em&gt;in vivo&lt;/em&gt;. CWW inhibited RANKL-induced osteoclast formation and tartrate-resistant acid phosphatase (TRAP) activity in primary mouse bone marrow-derived cells. We investigated the osteoprotective effect of CWW in an ovariectomized (OVX) Sprague-Dawley rat model treated with vehicle (OVX/vehicle), 17&lt;span style="font-family: 맑은 고딕;"&gt;β&lt;/span&gt;-estradiol (OVX/E2), or three CWW doses (100, 200, and 400 mg/kg). After a 24-week treatment, the body and uterus weights were not affected except in the OVX/E2 group. Additionally, bone mineral density (BMD) and histological analyses showed that the BMD of the femurs of CWW400-treated rats was significantly higher than that of the OVX/vehicle rats, and comparable to that of the OVX/E2 group rats. Serum levels of bone turnover markers alkaline phosphatase (ALP), osteocalcin, collagen type I C-telopeptide, and TRAP significantly decreased in the CWW400 group. Our results show that compared to the vehicle, CWW had a significant anti-osteoporotic effect in the OVX model. Taken together, CWW exhibited inhibitory effects on osteoclastogenesis &lt;em&gt;in vitro,&lt;/em&gt; and we confirmed its in vivo efficacy in the prevention of osteoporosis.&lt;/p&gt;

Native and bone marrow-derived cell mosaicism in gastric carcinoma in H. pylori-infected p27-deficient mice.

ObjectiveChronic Helicobacter pylori (H. pylori) infection promotes non-cardia gastric cancer. Some mouse models suggest that bone marrow derived cells (BMDC) contribute to Helicobacter-associated gastric carcinogenesis. We determined whether this increased susceptibility to Helicobacter-induced gastric carcinogenesis of p27-deficient mice is dependent upon their p27-null BMDC or their p27-null gastric epithelial cells.DesignFemale mice (recipients) were irradiated and transplanted with BMDC from male donors. Wild type (WT) mice in group 1 (control) received BMDC from male GFP-transgenic mice. Female WT and p27 KO mice were engrafted with male p27KO mice BMDC (Group 2) or GFP-transgenic WT BMDC (Group 3). Recipients were infected with H. pylori SS1 for one year.ResultsMice lacking p27 in either the BM pool or gastric epithelium developed significantly more advanced gastric pathology, including high-grade dysplasia. Co-staining of donor BMDC in dysplastic gastric glands was confirmed by immunofluorescence. Gastric expression of IL-1 beta protein was reduced in groups 2 and 3 (p < 0.05 vs control) whereas expression of IFN-γ and chemokines MIP-1 beta, MIG, IP-10 and RANTES in group 2 were significantly higher than group 3.ConclusionsBoth bone marrow-derived and gastric epithelial cells contribute to the increased gastric cancer susceptibility of p27-deficient H. pylori-infected mice.