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Matrix metalloproteinase (MMP)‐12 has been reported to have diverse functions, including regulation of immune reactions and anti‐inflammatory effects, but the potential roles of MMP‐12 in kidney injury have not been fully elucidated. This study aimed to determine whether MMP‐12 contributes to tubulointerstitial injury in a unilateral ureteric obstruction (UUO) model. MMP‐12‐deficient (MMP‐12−/−) mice and C57BL/6J mice as controls (MMP‐12+/+) were subjected to UUO and analysed 7 days after UUO. To analyse the functions of MMP‐12 on monocytes/macrophages, we generated MMP‐12‐deficient, irradiated, chimeric mice (BM‐MMP‐12−/−) and performed UUO. Bone marrow‐derived macrophages (BMDMs) were isolated from both groups of mice and used for investigations. MMP‐12−/− mice showed exacerbation of macrophage accumulation and interstitial fibrosis in the UUO‐kidney compared with control mice. BM‐MMP‐12−/− mice also showed exacerbation of kidney injury. UUO induced accumulation of Ly6C+ macrophages in MMP‐12−/− mice compared with control mice. Increases in inflammatory cytokine (tumour necrosis factor α, interleukin [IL]‐1β, IL‐6) levels from BMDMs after lipopolysaccharide stimulation were higher in MMP‐12−/− mice than in MMP‐12+/+ mice. MMP‐12 may play protective roles against kidney injury by UUO in mice, decreasing inflammatory cytokines from BMDMs and macrophage accumulation. [ABSTRACT FROM AUTHOR]

Background & aimsThe stimulator of interferon genes (STING)/TANK-binding kinase 1 (TBK1) pathway is vital in mediating innate immune and inflammatory responses during oxidative/endoplasmic reticulum (ER) stress. However, it remains unknown whether macrophage thioredoxin-interacting protein (TXNIP) may regulate TBK1 function and cell death pathways during oxidative/ER stress.MethodsA mouse model of hepatic ischaemia/reperfusion injury (IRI), the primary hepatocytes, and bone marrow-derived macrophages were used in the myeloid-specific TXNIP knockout (TXNIPM-KO) and TXNIP-proficient (TXNIPFL/FL) mice.ResultsThe TXNIPM-KO mice were resistant to ischaemia/reperfusion (IR) stress-induced liver damage with reduced serum alanine aminotransferase (ALT)/aspartate aminotransferase (AST) levels, macrophage/neutrophil infiltration, and pro-inflammatory mediators compared with the TXNIPFL/FL controls. IR stress increased TXNIP, p-STING, and p-TBK1 expression in ischaemic livers. However, TXNIPM-KO inhibited STING, TBK1, interferon regulatory factor 3 (IRF3), and NF-κB activation with interferon-β (IFN-β) expression. Interestingly, TXNIPM-KO augmented nuclear factor (erythroid-derived 2)-like 2 (NRF2) activity, increased antioxidant gene expression, and reduced macrophage reactive oxygen species (ROS) production and hepatic apoptosis/necroptosis in IR-stressed livers. Mechanistically, macrophage TXNIP deficiency promoted cylindromatosis (CYLD), which colocalised and interacted with NADPH oxidase 4 (NOX4) to enhance NRF2 activity by deubiquitinating NOX4. Disruption of macrophage NRF2 or its target gene 2',5' oligoadenylate synthetase-like 1 (OASL1) enhanced Ras GTPase-activating protein-binding protein 1 (G3BP1) and TBK1-mediated inflammatory response. Notably, macrophage OASL1 deficiency induced hepatocyte apoptotic peptidase activating factor 1 (APAF1), cytochrome c, and caspase-9 activation, leading to increased caspase-3-initiated apoptosis and receptor-interacting serine/threonine-protein kinase 3 (RIPK3)-mediated necroptosis.ConclusionsMacrophage TXNIP deficiency enhances CYLD activity and activates the NRF2-OASL1 signalling, controlling IR stress-induced liver injury. The target gene OASL1 regulated by NRF2 is crucial for modulating STING-mediated TBK1 activation and Apaf1/cytochrome c/caspase-9-triggered apoptotic/necroptotic cell death pathway. Our findings underscore a novel role of macrophage TXNIP-mediated CYLD-NRF2-OASL1 axis in stress-induced liver inflammation and cell death, implying the potential therapeutic targets in liver inflammatory diseases.Lay summaryLiver inflammation and injury induced by ischaemia and reperfusion (the absence of blood flow to the liver tissue followed by the resupply of blood) is a significant cause of hepatic dysfunction and failure following liver transplantation, resection, and haemorrhagic shock. Herein, we uncover an underlying mechanism that contributes to liver inflammation and cell death in this setting and could be a therapeutic target in stress-induced liver inflammatory injury.

BACKGROUND: Excessive activation of osteoclasts is one of the key links in the occurrence and development of osteoporosis. Exploring the function of osteoclasts and developing drugs that can inhibit osteoclast differentiation and reduce bone resorption is an important means to explore the clinical treatment of osteoporosis. OBJECTIVE: To analyze the mechanism of paroxetine on osteoclast differentiation based on the mitogen-activated protein kinase/nuclear factor-κB signal pathway. METHODS: (1) In vitro experiment: Different concentrations of paroxetine were used to interfere with bone marrow-derived macrophages in mice, and then the effect of paroxetine on cell activity was determined by cell counting kit-8. Under the induction of macrophage colony stimulating factor and receptor activator of receptor activator of nuclear factor-κB ligand, different concentrations of paroxetine were used to interfere with osteoclast differentiation. The number of osteoclasts was determined by tartrate-resistant acid phosphatase staining. The effect of paroxetine on the mRNA expression of osteoclast differentiation related cytokines was detected by real-time fluorescence quantitative real-time PCR. The effect of paroxetine on nuclear factor κB and mitogenactivated protein kinase signal pathways in the cells was detected by western blot. (2) In vivo experiment: Forty male C57BL/6 mice were randomly divided into four groups (n=10 per group): blank control group, lipopolysaccharide group, paroxetine 2 mg/kg treatment group, and paroxetine 5 mg/kg treatment group. Lipopolysaccharide was injected subcutaneously into the sagittal suture of the skull every 2 days to construct the mouse skull osteolysis model in the lipopolysaccharide group, paroxetine 2 mg/kg and 5 mg/kg groups. The corresponding dose of paroxetine was injected 1 day after each lipopolysaccharide injection in the two paroxetine groups. After 14 days of treatment, mouse skulls were isolated for Micro-CT scanning. RESULTS AND CONCLUSION: (1) In vitro experiment: there was no significant effect on the proliferation of bone marrow-derived macrophages when paroxetine concentration was ≤ 5 μmol/L, while paroxetine concentration > 10 μmol/L could inhibit cell proliferation. When paroxetine concentration was ≥ 0.5 μmol/L, there was a significant reduction in the number and morphology of osteoclast differentiation as well as the mRNA expression of c-Fos, Nfatc1, Ctsk, Mmp9, Acp5, and Atp6v0d2. Paroxetine at a dose of 0.5 μmol/L could inhibit the protein expression of c-Fos, Nfatc1, and Ctsk through activating nuclear factor-κB/ mitogen-activated protein kinase signal pathway. (2) In vivo experiment: Micro-CT scanning results showed that compared with lipopolysaccharide, 5 mg/kg paroxetine could significantly increase the number of trabeculae and bone volume fraction and reduce bone surface bone volume ratio and trabecular separation (P < 0.05). (3) To conclude, paroxetine can inhibit bone resorption by reducing osteoclast differentiation and has a potential effect in the treatment of osteoporosis. [ABSTRACT FROM AUTHOR]

BACKGROUND: Abnormal activation of osteoclast-mediated bone resorption leads to the loss of bone mass, damage and deterioration of bone microarchitecture, which is an important cause of osteoporosis. Wnt/β-catenin signaling pathway may be a potential target and tool for the prevention and treatment of osteoporosis. Overactivation of Wnt/β-catenin signaling pathway inhibits osteoclast differentiation. However, the physiological role of Wnt/ β-catenin signaling pathway in osteoclast formation remains to be clarified. OBJECTIVE: To investigate the effect of inhibitor of Wnt production 2 (IWP-2) on proliferation and differentiation of bone marrow-derived macrophages through inhibiting the Wnt/β-catenin signaling pathway. METHODS: (1) Bone marrow-derived macrophages were extracted. Cell counting kit-8 assay was performed to assess the viability of bone marrow-derived macrophages after being treated with gradient concentrations from 0 to 40 μmol/L IWP-2 for 24, 72, or 120 hours. (2) Bone marrow-derived macrophages induced by 100 μg/L receptor activator of nuclear factor kappa B ligand (RANKL) were treated with 5 or 10 μmol/L IWP-2 and equivalent solvent, and precipitated proteins were collected at 0, 1, 3 and 5 days. The activity of Wnt/β-catenin signaling pathway and the inhibitory effect of IWP-2 on it during osteoclast differentiation were detected by western blot assay. (3) Bone marrow-derived macrophages induced by 100 μg/L RANKL were treated with nontoxic IWP-2 (0, 5, or 10 μmol/L) for 6 days. The effects of IWP-2 on osteoclast differentiation were evaluated by tartrate-resistant acid phosphatase staining. The mRNA expression of osteoclast-related genes including NFATc1, Oscar, CTSK, Acp5, Mmp9, and Dcstamp were detected by real-time fluorescence quantitative PCR. RESULTS AND CONCLUSION: (1) After treatment with different concentrations of IWP-2 for 24 hours, 10 μmol/L IWP-2 promoted the proliferation of bone marrow-derived macrophages. After treatment with different concentrations of IWP-2 for 72 and 120 hours, the viability of bone marrow-derived macrophages was not significantly affected by IWP-2 at a concentration of 10 μmol/L or less, while high concentrations of IWP-2 (20, 30, and 40 μmol/L) inhibited the proliferation of bone marrow-derived macrophages. (2) After 1, 3, and 5 days of RANKL stimulation, the protein expression of active β-catenin in the control group increased gradually, while that in the 5 and 10 μmol/L IWP-2 treatment group was significantly inhibited. (3) Many large and irregular TRAP-positive multinucleated mature osteoclasts were observed in the control group, while the number and size of the cells decreased in a dose-dependent manner in the IWP-2 treatment groups, suggesting that IWP-2 could inhibit osteoclast differentiation. (4) The expression levels of osteoclast-related genes including NFATc1, Oscar, CTSK, Acp5, Mmp9, and Dcstamp were all down-regulated in a dose-dependent manner after IWP-2 treatment. (5) To conclude, IWP-2 can inhibit osteoclast differentiation in vitro and may play a role in the prevention and treatment of osteoporosis by directly or indirectly inhibiting the expression of osteoclast-elated genes [ABSTRACT FROM AUTHOR]

Here, we describe the production and characterization of a novel p65fl/fl/LysMCre mouse model, which lacks canonical nuclear factor-kappaB member RelA/p65 (indicated as p65 hereafter) in bone marrow-derived macrophages. Cultured bone marrow-derived macrophages that lack p65 protein reveal NF-κB signaling deficiencies, a reduction in phagocytic ability, and reduced ability to produce nitrites. Despite abnormal bone marrow-derived macrophage function, p65fl/fl/LysMCre mice do not exhibit differences in naïve systemic immune profiles or colony forming units and time to death following Salmonella infection as compared to controls. Additionally, p65fl/fl/LysMCre mice, especially females, display splenomegaly, but no other obvious physical or behavioral differences as compared to control animals. As bone marrow-derived macrophages from this transgenic model are almost completely devoid of canonical nuclear factor-kappaB pathway member p65, this model has the potential for being very useful in investigating bone marrow-derived macrophage NF-kappaB signaling in diverse biological and biomedical studies. [ABSTRACT FROM AUTHOR]

BACKGROUND: Macrophage polarization-mediated inflammation plays an important role in chronic inflammatory diseases such as osteoarthritis and rheumatoid arthritis. Phosphocreatine-modified chitosan hydrogels show good potential application prospects in tissue repair, but the effect of this new hydrogel on polarization and inflammatory factor expression in macrophages is still unclear. OBJECTIVE: To explore the effect of phosphocreatine modified methacrylic anhydride chitosan hydrogel on macrophage polarization and inflammatory factor expression. METHODS: One-step freeze-drying method was used to prepare phosphocreatine modified methacrylic anhydride chitosan with good water solubility. Hydrogels were further prepared under low toxicity initiators and ultraviolet irradiation. Bone marrow-derived macrophages were extracted from SD rats and cultured for 7 days, and then divided into three groups. Complete cell culture medium was added in the control group. Complete cell culture medium containing lipopolysaccharide was added in the lipopolysaccharide group. Freeze-dried hydrogel samples and cell complete medium containing lipopolysaccharide were added in the chitosan hydrogel group. Cell viability was detected using CCK-8 assay. RT-PCR, ELISA, western blot assay, and immunofluorescence staining were used to detect macrophage polarization and the expression of inflammatory factors. RESULTS AND CONCLUSION: (1) CCK-8 assay showed that after 1, 3, and 5 days of culture, there was no significant difference in the cell viability among the three groups (P > 0.05). (2) Western blot assay and immunofluorescence staining showed that compared with the control group, the M2 macrophage surface marker CD206 protein expression was down-regulated (P < 0.05) and the ARG1 protein expression was up-regulated (P < 0.05) in the lipopolysaccharide group after 1 and 3 days of culture. Compared with the lipopolysaccharide group, the protein expression levels of CD206 and ARG1 in the chitosan hydrogel group were up-regulated after 3 days of culture (P < 0.05). (3) ELISA and RT-PCR results showed that compared with the control group, the protein and mRNA expression levels of pro-inflammatory factors interleukin-1β and interleukin-6 in the lipopolysaccharide group increased after 1 and 3 days of culture (P < 0.05). Compared with the lipopolysaccharide group, the protein and mRNA expression levels of interleukin-1β and interleukin-6 in the chitosan hydrogel group were decreased after 1 and 3 days of culture (P < 0.05). (4) These results have shown that phosphocreatine modified methacrylic anhydride chitosan hydrogel promotes M2-type polarization of macrophages and inhibits the expression of pro-inflammatory factors, which may provide a potential treatment for immune-related diseases in the future. [ABSTRACT FROM AUTHOR]

Here, we investigate the contribution of long-term hematopoietic stem cells (HSCs LT ) to trained immunity (TI) in the setting of chronic autoimmune disease. Using a mouse model of systemic lupus erythematosus (SLE), we show that bone marrow-derived macrophages (BMDMs) from autoimmune mice exhibit hallmark features of TI, including increased Mycobacterium avium killing and inflammatory cytokine production, which are mechanistically linked to increased glycolytic metabolism. We show that HSCs from autoimmune mice constitute a transplantable, long-term reservoir for macrophages that exhibit the functional properties of TI. However, these BMDMs exhibit reduced glycolytic activity and chromatin accessibility at metabolic genes while retaining elevated expression of TI-associated transcriptional regulators. Hence, HSC exposed to autoimmune inflammation can give rise to macrophages in which the functional and metabolic properties of TI are decoupled. Our data support a model in which TI is characterized by a spectrum of molecular and metabolic states driving augmented immune function., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

BACKGROUND: Interleukin-1 is an important pro-inflammatory cytokine that has been documented in the regulation of bone inflammation and bone remodeling. A previous study has demonstrated that interleukin-1α can induce apoptosis while inhibiting osteoblast differentiation in MC3T3-E1 cells. OBJECTIVE: To investigate the role and mechanism of interleukin-1α on osteoclast activation and bone loss in mice. METHODS: (1) Cell test: RAW264.7 cells were either treated with interleukin-1α alone or with receptor activator of nuclear factor-κB ligand (RANKL) for 1 and 4 days. Cell viability was tested by cell counting kit-8 assay. The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay. The mRNA and protein levels of osteoclast-specific genes and genes related to nuclear factor-κB pathway and Wnt/β-catenin pathway were tested by real-time fluorescence quantitative PCR, immunofluorescence staining or western blot. Bone marrow-derived macrophages were either treated with interleukin-1α alone or with RANKL and macrophage colony-stimulating factor for 7 days. The number of multinuclear osteoclasts was detected by tartrate resistant acid phosphatase assay. The protein levels of osteoclast-specific genes were tested by western blot. (2) Animal test: Twenty-four male C57BL/6J mice (6-8 weeks old) were assigned into two groups at random: control group and test group. Mice were subsequently treated with interleukin-1α solution or PBS by intraperitoneal injection twice a week for 5 weeks. Bone tissues from the femurs were performed with micro-computed tomography analysis and hematoxylin-eosin staining, tartrate resistant acid phosphatase, and immunofluorescence analysis. RESULTS AND CONCLUSION: Cell test: Interleukin-1α alone significantly increased RAW264.7 cell proliferation, but stimulated cell differentiation into osteoclasts in combination with RANKL (P < 0.05). Interleukin-1α significantly increased the expression of osteoclast-related markers and the number of tartrate resistant acid phosphatasepositive multinuclear cells in RAW264.7 cells and bone marrow-derived macrophages in the existence of RANKL or RANKL+macrophage colony-stimulating factor (both P < 0.05). Interleukin-1α was found to significantly enhance the nuclear factor-κB and Wnt/beta-catenin signaling in RAW264.7 cells (P < 0.05). Blocking of nuclear factor-κB or Wnt3 signaling not only reversed the activation of nuclear factor-κB and Wnt3 signaling but also weakened the enhanced expression of osteoclast-specific genes induced by interleukin-1α in RAW264.7 cells (P < 0.05). Animal test: interleukin1α induced bone loss in mice while also upregulating the expression of osteoclast-specific markers, RANK, TRAF6 and p65, and Wnt3 in vivo (P < 0.05). The findings indicate that interleukin-1α can induce osteoclast activation and bone loss by promoting the nuclear factor-κB and Wnt signaling pathways. [ABSTRACT FROM AUTHOR]

Postmenopausal osteoporosis (PMOP) is a kind of primary osteoporosis that is characterized by decreased bone density and strength. Berbamine is a nonbasic quaternary benzylisoquinoline plant alkaloid that has been widely used in the clinic to treat leukopenia in China. We found that berbamine inhibited RANKL-induced osteoclastogenesis of bone marrow-derived macrophages (BMMs) in vitro , which mainly occurred in the middle phase and late phase. The gene and protein expression levels of osteoclast-related molecules, including CTSK, MMP-9, NFATc1, CD44 and DC-STAMP, were also downregulated by berbamine. In vivo , we treated PMOP mice with berbamine for 8 weeks and found that the extent of osteoporosis was alleviated significantly according to micro-CT scanning, hematoxylin-eosin staining, DC-STAMP immunohistochemical staining and TRAP immunohistochemical staining in the distal femurs of the mice. Our findings demonstrate that berbamine has an inhibitory effect on the osteoclastogenesis of BMMs and can prevent bone loss after ovariectomy in vivo. This study provides evidence that berbamine is a potential drug for the prevention and treatment of PMOP. [ABSTRACT FROM AUTHOR]

ABSTRACT Leptospirosis, an emerging infectious disease caused by pathogenic Leptospira spp., occurs in ecoregions with heavy rainfall and has public health implications. Macrophages are the major anti-Leptospira phagocytes that infiltrate the kidneys during renal leptospirosis, which is caused by leptospires residing in the renal tubules. The pathogenicity of Leptospira spp. in immune effector cells such as macrophages is not well understood. To evaluate this pathogenesis, we characterized and compared the transcriptome-wide alterations in macrophages infected with pathogenic and nonpathogenic Leptospira spp. Using transcriptome data and quantitative reverse transcription PCR analysis, at 2 h postinfection, the hypoxia-inducible factor-1α-dependent glycolysis pathway was implicated in pathogenic Leptospira-infected macrophages but not in nonpathogenic leptospiral infections. Immune-related biological processes were mostly activated in pathogenic Leptospira-infected macrophages, and flow cytometry investigations revealed that classically activated macrophages represent the predominant polarization status. At 24 h after infection, biological pathways associated with interleukin-10, IL-10, signaling the induction of macrophage tolerance, as well as higher levels of IL-10 mRNA and protein expression, were observed in nonpathogenic Leptospira-infected macrophages compared to in pathogenic leptospiral infection. Following leptospiral infection of macrophages, strong IL-10-expressing transcriptome signatures were observed following nonpathogenic leptospiral infection. The transcriptional programs generated in Leptospira-infected macrophages revealed an inflammatory milieu following the production of a critical anti-inflammatory cytokine, IL-10, which is implicated in controlling the pathogenicity of activated macrophages. These findings imply that IL-10-mediated anti-inflammatory responses and tolerance in activated macrophages induced by nonpathogenic Leptospira spp. infection reduce inflammation and tissue damage, thus providing a potential therapeutic target for leptospirosis. IMPORTANCE Activation of macrophages by Leptospira spp. infection is thought to be involved in the pathogenesis of leptospirosis. To evaluate the innate macrophage responses to Leptospira spp., specifically pathogenic versus nonpathogenic Leptospira spp., we characterized the entire transcriptome-wide alterations in infected macrophages. We showed that hypoxia-inducible factor-1α and immune-related pathways are activated in pathogenic leptospiral-infected macrophages. We confirmed the significantly high levels of IL-10-expressing signatures and tolerance in activated macrophages caused by nonpathogenic Leptospira infection. Furthermore, nonpathogenic leptospiral infections attenuated macrophage activation responses. These findings suggest a potential therapeutic strategy for the immune microenvironment caused by macrophage activation driven by IL-10 overexpression, which may contribute to regulating inflammation in leptospirosis.

Postmenopausal osteoporosis (PMOP) is a kind of primary osteoporosis that is characterized by decreased bone density and strength. Berbamine is a nonbasic quaternary benzylisoquinoline plant alkaloid that has been widely used in the clinic to treat leukopenia in China. We found that berbamine inhibited RANKL-induced osteoclastogenesis of bone marrow-derived macrophages (BMMs) in vitro, which mainly occurred in the middle phase and late phase. The gene and protein expression levels of osteoclast-related molecules, including CTSK, MMP-9, NFATc1, CD44 and DC-STAMP, were also downregulated by berbamine. In vivo, we treated PMOP mice with berbamine for 8 weeks and found that the extent of osteoporosis was alleviated significantly according to micro-CT scanning, hematoxylin-eosin staining, DC-STAMP immunohistochemical staining and TRAP immunohistochemical staining in the distal femurs of the mice. Our findings demonstrate that berbamine has an inhibitory effect on the osteoclastogenesis of BMMs and can prevent bone loss after ovariectomy in vivo. This study provides evidence that berbamine is a potential drug for the prevention and treatment of PMOP.

Study Design: In vivo study using immunostaining and immunoblot analysis.Objective: To determine the distribution of bone marrow-derived macrophages (BMDMs), macrophage polarization and cytokine expression in the process of intervertebral disc (IVD) degeneration.Summary Of Background Data: Knowledge of the detailed distribution of exogeneous macrophages in the disc degeneration process is important for understanding the pathomechanisms and establishing novel therapeutic targets.Methods: To distinguish BMDMs, GFP-labeled bone marrow chimeric rats (n = 12) were generated. The degenerative process of the intervertebral disc was reproduced in a rat caudal disc puncture model (n = 49). Immunofluorescence staining was performed to observe the distribution of BMDMs, Iba-1 and GFP double-positive cells, and Iba-1 and iNOS (M1 macrophage) or arginase-1 (M2 macrophage) double-positive cells. Immunoblot analysis was used to evaluate differences in cytokines (TNF-α, IL-1β, IL-6, TGF-β, IL-4, and IL-10) depending on the distribution of BMDMs.Results: BMDMs infiltrated into the outer annulus fibrosus and endplate, while increasing tissue-resident macrophage was observed inside the annulus fibrosus/nucleus pulposus. The ratio of BMDMs and the polarity change differed among the regions. Especially in the endplate, BMDMs increased gradually and the macrophage phenotype was M2 dominant. Expression of IL-1β decreased gradually at endplate, and that of IL-4 increased early after disc puncture at inside of the annulus fibrosus.Conclusion: During the disc degeneration process, BMDMs were observed mainly around the endplate and outside area of the annulus fibrosus, with few in the inside area of annulus fibrosus and nucleus pulposus. Compared to other IVD area, macrophage polarity and cytokine expression is concomitantly M2-dominant in endplate. Increased hematogenous M2-phenotype macrophages in endplate with progression of IVD degeneration could enhance our understanding of the underlying mechanisms of disc degeneration.Level of Evidence: N/A. [ABSTRACT FROM AUTHOR]

Objective: To research the effect of glutaminase inhibitor CB-839 on the polarization of macrophages. Methods: Following treatment of bone mar row-de rived macrophages (BMDM) and RAW264.7 cells with PBS or CB-839 for 48 h, the treated cells were divided into the the control group and the CB-839 group. The proliferation of cells was detected by CCK 8 kit, the expression levels of tumor necrosis factor α(TNF-α), interleukin-1β(IL-1β), TGF-β and IL-10 were detected by ELISA, the expression levels of CD86 and CD2O6 were detected by flow cytometry, and the expression levels of iNOS and Arg1 were detected by Western blot. Results: CCK8 detection showed that CB-839 almost had no effect on the proliferation of macrophages. The expression levels of TNF-α, IL-1β, CD86 and iNOS in the CB-839 group significantly elevated as compared with those of the control group, the expression levels of TGF-α and IL-10 as well as CD2O6 and Arg1 markedly down-regulated as compared with those of the control group. Conclusion: CB-839 could enhance macrophage polarization towards M1, based on the premise that it did not inhibit the proliferation of cells. [ABSTRACT FROM AUTHOR]

Abstract Background Spinal cord injury (SCI) has a very disabling central nervous system impact but currently lacks effective treatment. Bone marrow-derived macrophages (BMDMs) are recruited to the injured area after SCI and participate in the regulation of functional recovery with microglia. Previous studies have shown that M2 microglia-derived small extracellular vesicles (SEVs) have neuroprotective effects, but the effects of M2 BMDM-derived sEVs (M2 BMDM-sEVs) have not been reported in SCI treatment. Results In this study, we investigated the role of M2 BMDM-sEVs in vivo and in vitro for SCI treatment and its mechanism. Our results indicated that M2 BMDM-sEVs promoted functional recovery after SCI and reduced neuronal apoptosis in mice. In addition, M2 BMDM-sEVs targeted mammalian target of rapamycin (mTOR) to enhance the autophagy level of neurons and reduce apoptosis. MicroRNA-421-3P (miR-421-3p) can bind to the 3′ untranslated region (3′UTR) of mTOR. MiR-421-3p mimics significantly reduced the activity of luciferase-mTOR 3′UTR constructs and increased autophagy. At the same time, tail vein injection of inhibitors of SEVs (Inh-sEVs), which were prepared by treatment with an miR-421-3p inhibitor, showed diminished protective autophagy of neuronal cells in vivo. Conclusions In conclusion, M2 BMDM-sEVs inhibited the mTOR autophagy pathway by transmitting miR-421-3p, which reduced neuronal apoptosis and promoted functional recovery after SCI, suggesting that M2 BMDM-sEVs may be a potential therapy for SCI.

HIF-1α plays a critical role in shaping macrophage phenotype and effector function. We have previously shown that tissue-resident alveolar macrophages (TR-AMs) have extremely low glycolytic capacity at steady-state, but can shift toward glycolysis under hypoxic conditions. Here, using inducible HIF-1α knockout ( Hif1a -/- ) TR-AMs and bone marrow-derived macrophages (BMDMs) and show that TR-AM HIF-1α is required for the glycolytic shift under prolyl hydroxylase inhibition, but is dispensable at steady-state for inflammatory effector function. In contrast, HIF-1α deletion in BMDMs led to diminished glycolytic capacity at steady-state and reduced inflammatory capacity, but higher mitochondrial function. Gene set enrichment analysis revealed enhanced c-Myc transcriptional activity in Hif1a -/- BMDMs, and upregulation of gene pathways related to ribosomal biogenesis and cellular proliferation. The findings highlight the heterogeneity of HIF-1α function in distinct macrophage populations and provide new insight into how HIF-1α regulates gene expression, inflammation, and metabolism in macrophages., Competing Interests: Declaration of interests The authors declare no competing interests.

Adaptive immunity, which plays an important role in the development of atherosclerosis, is mediated by major histocompatibility complex (MHC)-dependent antigen presentation. In atherosclerotic lesions, macrophages constitute an important class of antigen-presenting cells that activate adaptive immune responses to oxidized low-density lipoprotein (OxLDL). It has been reported that autophagy regulates adaptive immune responses by enhancing antigen presentation to MHC class II (MHC-II). In a previous study, we have demonstrated that SYK (spleen tyrosine kinase) regulates generation of reactive oxygen species (ROS) and activation of MAPK8/JNK1 in macrophages. Because ROS and MAPK8 are known to regulate autophagy, in this study we investigated the role of SYK in autophagy, MHC-II expression and adaptive immune response to OxLDL. We demonstrate that OxLDL induces autophagosome formation, MHC-II expression, and phosphorylation of SYK in macrophages. Gene knockout and pharmacological inhibitors of NOX2 and MAPK8 reduced OxLDL-induced autophagy. Using bone marrow-derived macrophages isolated from wild-type and myeloid-specific SYK knockout mice, we demonstrate that SYK regulates OxLDL-induced ROS generation, MAPK8 activation, BECN1-BCL2 dissociation, autophagosome formation and presentation of OxLDL-derived antigens to CD4(+) T cells. ldlr(-/-) syk(-/-) mice fed a high-fat diet produced lower levels of IgG to malondialdehyde (MDA)-LDL, malondialdehyde-acetaldehyde (MAA)-LDL, and OxLDL compared to ldlr(-/-) mice. These results provide new insights into the mechanisms by which SYK regulates MHC-II expression via autophagy in macrophages and may contribute to regulation of adaptive immune responses in atherosclerosis.

Background: The balance between bone formation and bone resorption which is attributed to osteoblast and osteoclast is required to maintain skeleton homeostasis. Osteoclast differentiation is regulated by a tumor necrosis factor–receptor activator of nuclear factor NF-kB ligand (RANKL). The dysregulation of bone-resorbing osteoclast differentiation can lead to osteoporosis. The adverse effects of the long-term use of bone resorption inhibitors are of concern and so the development of new osteoporosis therapy treatment is desirable. Objective: In this study, 67 plants (70 samples) were screened for osteoclastogenesis inhibitory activities on RAW264.7 mouse macrophages and bone marrow-derived macrophages (BMMs). Materials and Methods: The RAW264.7 cells and the BMMs isolated from male ICR mice were treated with various doses of plant extracts and TRAP histochemical staining of the cells was performed. TRAP-positive multinucleated cells were photographed under microscopy to observe the effects of the extracts on osteoclast differentiation. Results: Among 70 methanol extracts, we found that nine samples exhibited significant inhibitory effects in RANKL-induced osteoclast differentiation. They included Aleurites moluccana (S16 and S17), Aporosa dioca (S19), Antidesma bunius (S21), Cinnamomum balansae (S32), Macrosolen cochinchinensis (S41), Pinus kesiya (S52), Photinia benthamiana (S54), and Mischocarpus pentapetalus (S59). Conclusion: In the present study, 70 plant extracts were screened for the osteoclastogenesis inhibitory effects in RAW264.7 murine macrophages and BMMs. Nine extracts have the potential as effective agents against osteoclastogenesis. This is the first report on the anti-osteoclastogenetic activity of these plants. [ABSTRACT FROM AUTHOR]

Obesity-induced inflammation in adipose tissue (AT) promotes the development of metabolic dysregulations by increasing macrophage recruitment in the stromal vascular fraction (SVF). The activation of nuclear factor-κB (NF-κB) signaling in macrophages serves as a pivotal mediator of AT inflammatory responses by increasing the expression of proinflammatory genes in obesity. Given the purported anti-inflammatory effects of berry consumption in humans, we evaluated if anthocyanin-rich aronia berry extract (ARN) can prevent obesity-induced AT inflammation in vivo. We also examined whether ARN suppresses lipopolysaccharide (LPS)-induced NF-κB activation in RAW 264.7 macrophages and mouse bone marrow-derived macrophages (BMDMs). Male C57BL/6J mice were fed a low-fat diet, a high-fat (HF), and high-sucrose (HS) diet or HF/HS diet supplemented with 0.2% ARN (HF/HS + ARN) for 14 weeks. Compared to HF-/HS-fed mice, ARN supplementation tended to decrease fasting serum glucose (P = .07). Furthermore, ARN supplementation significantly inhibited the phosphorylation of NF-κB p65 in epididymal AT with a concomitant decrease in the expression of Cd11b and Tnfα mRNAs in epididymal SVF isolated, compared with those from HF-/HS-fed mice. Consistent with these in vivo findings, ARN treatment significantly decreased the phosphorylation of p65 in LPS-stimulated RAW 264.7 macrophages and BMDMs. Moreover, ARN suppressed LPS-induced mRNA expression of inflammation mediators (iNos, Cox-2, Tnfα, Mcp-1, and Il-6) and glycolysis markers (Glut1, G6pdh, and Hk1) in both cell types. Taken together, our in vivo and in vitro results suggest that ARN supplementation may attenuate obesity-induced AT inflammation by inhibiting NF-κB signaling and glycolytic pathway in macrophages. [ABSTRACT FROM AUTHOR]

Background: Spinal cord injury (SCI) has a very disabling central nervous system impact but currently lacks effective treatment. Bone marrow-derived macrophages (BMDMs) are recruited to the injured area after SCI and participate in the regulation of functional recovery with microglia. Previous studies have shown that M2 microglia-derived small extracellular vesicles (SEVs) have neuroprotective effects, but the effects of M2 BMDM-derived sEVs (M2 BMDM-sEVs) have not been reported in SCI treatment. Results: In this study, we investigated the role of M2 BMDM-sEVs in vivo and in vitro for SCI treatment and its mechanism. Our results indicated that M2 BMDM-sEVs promoted functional recovery after SCI and reduced neuronal apoptosis in mice. In addition, M2 BMDM-sEVs targeted mammalian target of rapamycin (mTOR) to enhance the autophagy level of neurons and reduce apoptosis. MicroRNA-421-3P (miR-421-3p) can bind to the 3′ untranslated region (3′UTR) of mTOR. MiR-421-3p mimics significantly reduced the activity of luciferase-mTOR 3′UTR constructs and increased autophagy. At the same time, tail vein injection of inhibitors of SEVs (Inh-sEVs), which were prepared by treatment with an miR-421-3p inhibitor, showed diminished protective autophagy of neuronal cells in vivo. Conclusions: In conclusion, M2 BMDM-sEVs inhibited the mTOR autophagy pathway by transmitting miR-421-3p, which reduced neuronal apoptosis and promoted functional recovery after SCI, suggesting that M2 BMDM-sEVs may be a potential therapy for SCI. [ABSTRACT FROM AUTHOR]

The activation of NLRP3 inflammasome is a key factor for various inflammatory diseases. Here, we provide experimental evidence supporting the regulatory role of sphingosine-1-phosphate (S1P) in NLRP3 inflammasome activation in mouse bone-marrow-derived macrophages (BMDMs), along with the S1P receptor subtype involved and underlying regulatory mechanisms. During the priming stage, S1P induced NLRP3 upregulation in BMDMs only when primed with lipopolysaccharide (LPS). In this event, S1P2, but not S1P1, was involved based on the attenuated NLRP3 upregulation with JTE013 (S1P2 antagonist) or S1P2 knockdown. During the activation stage, S1P induced NLRP3 inflammasome activation in LPS-primed BMDMs via caspase-1 activation, interleukin 1β maturation, apoptosis-associated speck-like protein containing a CARD (ASC) speck formation, and IL-1β secretion. Such NLRP3 inflammasome activation was blocked by either pharmacological inhibition or genetic knockdown of S1P2. NF-κB, PI3K/Akt, and ERK1/2 were identified as effector pathways underlying S1P/S1P2 signaling in the regulation of NLRP3 upregulation in LPS-primed BMDMs. Further, reactive oxygen species (ROS) production was dependent on the S1P/S1P2 signaling axis in these cells, and the ROS generated regulate NLRP3 inflammasome activation, but not NLRP3 priming. Collectively, our findings suggest that S1P promotes NLRP3 upregulation and NLRP3 inflammasome activation in LPS-primed BMDMs via S1P2 and subsequent effector pathways.

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Chrysanthemum zawadskii (C. zawadskii) is used in traditional East Asian medicine for the treatment of various diseases, including inflammatory disease. However, it has remained unclear whether extracts of C. zawadskii inhibit inflammasome activation in macrophages. The present study assessed the inhibitory effect of an ethanol extract of C. zawadskii (CZE) on the activation of the inflammasome in macrophages and the underlying mechanism. Bone marrow-derived macrophages were obtained from wild-type C57BL/6 mice. The release of IL-1β and lactate dehydrogenase in response to nucleotide-binding oligomerization domain-like receptor (NLR) family pyrin domain containing 3 (NLRP3) inflammasome activators, such as ATP, nigericin and monosodium urate (MSU) crystals, was significantly decreased by CZE in lipopolysaccharide (LPS)-primed BMDMs. Western blotting revealed that CZE inhibited ATP-induced caspase-1 cleavage and IL-1β maturation. To investigate whether CZE inhibits the priming step of the NLRP3 inflammasome, we confirmed the role of CZE at the gene level using RT-qPCR. CZE also downregulated the gene expression of NLRP3 and pro-IL-1β as well as NF-κB activation in BMDMs in response to LPS. Apoptosis-associated speck-like protein containing a caspase-recruitment domain (CARD) oligomerization and speck formation by NLRP3 inflammasome activators were suppressed by CZE. By contrast, CZE did not affect NLR family CARD domain-containing protein 4 or absent in melanoma 2 inflammasome activation in response to Salmonella typhimurium and poly(dA:dT) in LPS-primed BMDMs, respectively. The results revealed that three key components of CZE, namely linarin, 3,5-dicaffeoylquinic acid and chlorogenic acid, decreased IL-1β secretion in response to ATP, nigericin and MSU. These findings suggest that CZE effectively inhibited activation of the NLRP3 inflammasome. [ABSTRACT FROM AUTHOR]

Background: Portulaca oleracea (PO) has been widely used as traditional medicine because of its pharmacological activities. However, the effects of PO on osteoclasts that modulate bone homeostasis are still elusive. Methods: In this study, we examined the effects of PO ethanol extract (POEE) on receptor activator of nuclear factor-κB ligand (RANKL)-mediated Ca2+ mobilization, nuclear factor of activated T-cell d (NFATc1) amplification, tartrate-resistant acid phosphatase-positive (TRAP+) multinucleated cell (MNC) formation, and cytotoxicity. Results: Our results demonstrated that POEE suppressed RANKL-induced Ca2+ oscillations by inhibition of Ca2+ release from internal Ca2+ stores, resulting in reduction of NFATc1 amplification. Notably, POEE attenuated RANKL-mediated cytotoxicity and cleavage of polyadenosine 5'-diphosphate-ribose polymerase (PARP), resulted in enhanced formation of TRAP+ MNCs. Conclusions: These results present in vitro effects of POEE on RANKL-mediated osteoclastogenesis and suggest the possible use of PO in treating bone disorders, such as osteopetrosis. [ABSTRACT FROM AUTHOR]