Search

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]

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]

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]

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.

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.

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]

Abstract: Numerous studies have shown various relationships between foods with a high nutritional value and a robust immune response, particularly studies that have focused on host protection and cytokine networks. This study aimed to clarify the role played by the procyanidin trimer C1 in innate and adaptive immunity. Procyanidin C1 did not exert cytotoxicity at concentrations ranging from 7.8 to 62.5μg/ml in macrophage cells; therefore, concentration of 62.5μg/ml was used as the maximum dose of procyanidin C1 throughout subsequent experiments. Procyanidin C1 enhanced inducible nitric oxide synthase-mediated nitric oxide production in a concentration-dependent manner. In addition, procyanidin C1 functionally induced macrophage activation by augmenting the expression of cell surface molecules (CD80, CD86, and MHC II) and proinflammatory cytokine production (tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6) via activation of mitogen-activated protein kinase (MAPK), e.g., p38, ERK, and JNK and nuclear factor (NF)-κB signaling pathways. Interestingly, procyanidin C1 effectively polarized T helper type 1 (Th1) by secreting Th1-mediated cytokines (interferon-γ, IL-12p70, and IL-2) and inducing splenocyte proliferation, indicating that procyanidin C1 contributes to Th1 polarization of the immune response. Accordingly, these findings confirms that the procyanidin C1 induces macrophage activation via NF-κB and MAPK pathways, leading to Th1 polarization in murine splenocytes, which suggests that procyanidin C1 regulates innate and adaptive immunity by macrophage activation and Th1 polarization. [Copyright &y& Elsevier]

We report here that mouse macrophages undergo receptor-interacting kinase-3 (RIP3)-dependent but TNF-α-independent necrosis when Toll-like receptors (TLR) 3 and 4 are activated by poly(I:C) and LPS, respectively. An adaptor protein, Toll/IL-1 receptor domain-containing adapter inducing IFN-β (TRIF/TICAM-1), which is dispensable for TNF-α-induced necrosis, forms a complex with RIP3 upon TLR3/TLR4 activation and is essential for TLR3/TLR4-induced necrosis. Mice without RIP3 or functional TRIF did not show macrophage loss and elevation of inflammatory cytokines when they were exposed to LPS. Necrosis in mouse macrophages induced by either TNFR or TLR3/TLR4 is executed by reactive oxygen species. Taken together, these data indicate that there are multiple upstream necrosis-initiating signaling pathways converging on the RIP3 during an innate immune response to viral and bacterial infections in mammals. [ABSTRACT FROM AUTHOR]

Abstract: Colony stimulating factor-1 (CSF-1)-dependent macrophages play crucial roles in the development and progression of several pathological conditions including atherosclerosis and breast cancer metastasis. Macrophages in both of these pathologies take up increased amounts of glucose. Since we had previously shown that CSF-1 stimulates glucose uptake by macrophages, we have now investigated whether glucose metabolism is required for the survival of CSF-1-dependent macrophages as well as examined the mechanism by which CSF-1 stimulates glucose uptake. Importantly, we found that CSF-1-induced macrophage survival required metabolism of the glucose taken up in response to CSF-1 stimulation. Kinetic studies showed that CSF-1 stimulated an increase in the number of glucose transporters at the plasma membrane, including Glut1. The uptake of glucose induced by CSF-1 required intact PI3K and PLC signalling pathways, as well as the downstream effectors Akt and PKC, together with a dynamic actin cytoskeleton. Expression of constitutively active Akt partially restored glucose uptake and macrophage survival in the absence of CSF-1, suggesting that Akt is necessary but not sufficient for optimal glucose uptake and macrophage survival. Taken together, these results suggest that CSF-1 regulates macrophage survival, in part, by stimulating glucose uptake via Glut1, and PI3K and PLC signalling pathways. [Copyright &y& Elsevier]

Abstract: Previously, we designed and synthesized a new NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), and found that racemic DHMEQ inhibited cytokine secretion and phagocytosis by cells of the macrophage cell line RAW264.7. In the present research, we looked into the effect of optically active (−)-DHMEQ on the NO production, inflammatory cytokine secretion, and prostaglandin secretion in mouse bone marrow-derived macrophages (BMMs). We also studied the effect of (−)-DHMEQ on the differentiation of macrophages. DHMEQ inhibited lipopolysaccharide (LPS)-induced NF-κB activation. It also inhibited the expression of inducible NO synthase (iNOS) and NO production induced by LPS. Using enzyme-linked immunosorbent assays, we showed DHMEQ to inhibit LPS-induced secretion of IL-6 and TNF-α. It also inhibited COX-2 expression and prostaglandin E2 production and secretion. It did not inhibit the phagocytosis of fluorescently labeled Escherichia coli by BMMs treated with LPS, unlike in the case of RAW264.7 cells. Next we examined the effect of the inhibitor on M-CSF-induced differentiation of bone marrow cells to macrophages. DHMEQ showed no effect on the differentiation in terms of reactive oxygen species production and F4/80 expression. However, although BMM incorporated oxidized LDL to give rise to foam cells, the (−)-DHMEQ-treated bone marrow cells did not take up oxidized LDL. Taken together, our data show that (−)-DHMEQ inhibited LPS-induced activation of BMM in terms of NO and cytokine secretion, but its effect on phagocytosis differed between BMMs and RAW264.7 cells. We also found that the functional differentiation into macrophages was inhibited by (−)-DHMEQ. [Copyright &y& Elsevier]

The article studies whether 4-1BB and 4-1BB ligand interaction plays a role in osteoclastogenesis, using 4-1BB-deficient mice. 4-1BB is an inducible T-cell constimulatory molecule and a member of the tumor necrosis factor (TNF) receptor family. Enhanced osteoclastogenesis was observed in bone marrow-derived macrophage cells from 4-1BB-deficient mice than in those from wildtype mice.

Abstract: The innate immune system is composed of neutrophils and monocyte/macrophages. As a cell type, bone marrow-derived macrophage (BMM) are easier to study than neutrophils since they are still capable of cell division and have a longer life span. However, in comparison with neutrophils, few methodological studies on the production of reactive oxygen species (ROS) by such macrophages have been reported. Here we present studies on ROS production of this cell type under various conditions including the use of different priming and stimulating agents. In addition, we report that the de novo adhesion of BMM to tissue culture plates induces superoxide anion production and this can be further enhanced by stimulation with PMA. BMM are able to adhere to endothelial cells that have been activated by TNF-α exposure, and under these circumstances also generate ROS. We explored different methods to introduce gene products into BMM without activating them to avoid complicating subsequent studies of ROS production. Infection with lentiviral vectors was very efficient, allowed long-term expression and did not activate the BMM. We conclude that BMM are very suitable for the biochemical study of the oxidative burst. [Copyright &y& Elsevier]

Abstract: We performed adoptive transfer of bone marrow-derived (BM) macrophages following pharmacological depletion of leukocytes in a mouse model of unilateral ureteral obstruction (UUO). Treatment with cyclophosphamide (CPM) caused marked decrease in the numbers of F4/80-positive interstitial macrophages as well as in peripheral blood leukocyte counts, and adoptive transfer of BM macrophages to CPM-treated mice resulted in significant increase in the numbers of interstitial macrophages both at day 5 and at day 14 after UUO. At day 5 after UUO, no significant change was observed in the degree of renal interstitial fibrosis either by treatment with CPM or with CPM+macrophage. However, at day 14 after UUO, treatment with CPM caused significant increase in the degree of interstitial fibrosis, and adoptive macrophage transfer to these mice attenuated this enhancement in renal fibrosis. Our result suggests the role of infiltrating macrophages on facilitating tissue repair at late stage of UUO. [Copyright &y& Elsevier]

The aim of this study was to investigate the influence of Voluven on the NLRP3 inflammasome-mediated pyroptosis in bone marrow-derived macrophage (BMDM). Separated BMDMs were cultured and treated with different concentration of Voluven (0, 0.1, and 0.5 µg) dissolved in 10 µL 0.9% NaCl solution for 24 h. Both wild-type and nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) -/- C57BL/6J mice ( n  = 18) were intravenously injected with 0.2 mL of 0%, 5%, and 10% Voluven through femoral vein, respectively. Pyroptosis was inspected with flow cytometry. The mRNA levels of NLRP3 and caspase-1 were detected with quantitative real-time polymerase chain reaction (qRT-PCR). The levels of NLRP3, pro-caspase-1, and cleaved caspase-1 (p10) in serum were determined with Western blot. The expression of IL-17A in peripheral blood CD4 + T cells was analyzed with flow cytometry. The expression of cleaved caspase-1 (p10) in mice spleens was inspected with immunofluorescence. Compared with control group, the ratio of pyroptosis in all Voluven-treated groups rose significantly. The levels of NLRP3 and caspase-1 were increased after Voluven treatment. The expression of interleukin (IL)-17A in Voluven-treated CD4 + T cells was also increased, exhibiting a dose-dependent pattern. In wild-type mice, Voluven-treated mice had higher levels of IL-17A, NLRP3, and cleaved caspase-1 (p10) in a dose-dependent manner. The effects of Voluven were diminished in NLRP3 -/- mice.

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.

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. [ABSTRACT FROM AUTHOR]

Abstract Background Tuberculosis (TB), a bacterial infection caused by Mycobacterium tuberculosis (Mtb) remains a significant health problem worldwide with a third of the world population infected and nearly nine million new cases claiming 1.1 million deaths every year. The outcome following infection by Mtb is determined by a complex and dynamic host-pathogen interaction in which the phenotype of the pathogen and the immune status of the host play a role. However, the molecular mechanism by which Mtb strains induce different responses during intracellular infection of the host macrophage is not fully understood. To explore the early molecular events triggered upon Mtb infection of macrophages, we studied the transcriptional responses of murine bone marrow-derived macrophages (BMM) to infection with two clinical Mtb strains, CDC1551 and HN878. These strains have previously been shown to differ in their virulence/immunogenicity in the mouse and rabbit models of pulmonary TB. Results In spite of similar intracellular growth rates, we observed that compared to HN878, infection by CDC1551 of BMM was associated with an increased global transcriptome, up-regulation of a specific early (6 hours) immune response network and significantly elevated nitric oxide production. In contrast, at 24 hours post-infection of BMM by HN878, more host genes involved in lipid metabolism, including cholesterol metabolism and prostaglandin synthesis were up-regulated, compared to infection with CDC1551. In association with the differences in the macrophage responses to infection with the 2 Mtb strains, intracellular CDC1551 expressed higher levels of stress response genes than did HN878. Conclusions In association with the early and more robust macrophage activation, intracellular CDC1551 cells were exposed to a higher level of stress leading to increased up-regulation of the bacterial stress response genes. In contrast, sub-optimal activation of macrophages and induction of a dysregulated host cell lipid metabolism favored a less stressful intracellular environment for HN878. Our findings suggest that the ability of CDC1551 and HN878 to differentially activate macrophages during infection probably determines their ability to either resist host cell immunity and progress to active disease or to succumb to the host protective responses and be driven into a non-replicating latent state in rabbit lungs.

Gaucher disease (GD) is caused by a deficiency in glucocerebrosidase (GC). Enzyme replacement for GD disease is effective but expensive and requires life-long treatment. Development of alternative therapeutic strategies is therefore important. One approach is an enzyme delivery system which could supply GC into the circulation continuously. We have previously reported that human GC cDNA in a retroviral vector (MFG-GC) efficiently transduced a murine myoblast line (C2C12) and expressed GC intracellularly and extracellularly. Now we have demonstrated that primary murine and human myoblasts are transduced at very high efficiency by MFG-GC (five to ten copies of human GC gene per cell at a multiplicity of infection of 5–10), 100% of MFG-GC transduced cells expressed human GC. The transduced primary murine and human myoblasts had an intracellular GC activities about five to ten times above nontransduced controls. Furthermore, transduced primary myoblasts secreted human GC extracellularly for up to 35 weeks in vitro. The secreted human GC is specifically taken up by bone marrow derived macrophages, the cell type most important to the pathogenesis of GD. These data suggest that transduced primary myoblasts may be useful in supplying GC as an alternative approach to the treatment of GD. [ABSTRACT FROM AUTHOR]

Prolactin-releasing Peptide (PrRP) is a neuropeptide whose receptor is GPR10. Recently, the regulatory role of PrRP in the neuroendocrine field has attracted increasing attention. However, the influence of PrRP on macrophages, the critical housekeeper in the neuroendocrine field, has not yet been fully elucidated. Here, we investigated the effect of PrRP on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) with RNA sequencing, bioinformatics, and molecular simulation. BMDMs were exposed to PrRP (18 h) and were subjected to RNA sequencing. Differentially expressed genes (DEGs) were acquired, followed by GO, KEGG, and PPI analysis. Eight qPCR-validated DEGs were chosen as hub genes. Next, the three-dimensional structures of the proteins encoded by these hub genes were modeled by Rosetta and Modeller, followed by molecular dynamics simulation by the Gromacs program. Finally, the binding modes between PrRP and hub proteins were investigated with the Rosetta program. PrRP showed no noticeable effect on the morphology of macrophages. A total of 410 DEGs were acquired, and PrRP regulated multiple BMDM-mediated functional pathways. Besides, the possible docking modes between PrRP and hub proteins were investigated. Moreover, GPR10 was expressed on the cell membrane of BMDMs, which increased after PrRP exposure. Collectively, PrRP significantly changed the transcriptome profile of BMDMs, implying that PrRP may be involved in various physiological activities mastered by macrophages. [ABSTRACT FROM AUTHOR]

Neuropeptide FF (NPFF) is a neuropeptide that regulates various biological activities. Currently, the regulation of NPFF on the immune system is an emerging field. However, the influence of NPFF on the transcriptome of primary macrophages has not been fully elucidated. In this study, the effect of NPFF on the transcriptome of mouse bone marrow-derived macrophages (BMDMs) was explored by RNA sequencing, bioinformatics, and molecular simulation. BMDMs were treated with 1 nM NPFF for 18 h, followed by RNA sequencing. Differentially expressed genes (DEGs) were obtained, followed by GO, KEGG, and PPI analysis. A total of eight qPCR-validated DEGs were selected as hub genes. Subsequently, the three-dimensional (3-D) structures of the eight hub proteins were constructed by Modeller and Rosetta. Next, the molecular dynamics (MD)-optimized 3-D structure of hub protein was acquired with Gromacs. Finally, the binding modes between NPFF and hub proteins were studied by Rosetta. A total of 2655 DEGs were obtained (up-regulated 1442 vs. down-regulated 1213), and enrichment analysis showed that NPFF extensively regulates multiple functional pathways mediated by BMDMs. Moreover, the 3-D structure of the hub protein was obtained after MD-optimization. Finally, the docking modes of NPFF-hub proteins were predicted. Besides, NPFFR2 was expressed on the cell membrane of BMDMs, and NPFF 1 nM significantly activated NPFFR2 protein expression. In summary, instead of significantly inhibiting the expression of the immune-related gene transcriptome of RAW 264.7 cells, NPFF simultaneously up-regulated and down-regulated the gene expression profile of a large number of BMDMs, hinting that NPFF may profoundly affect a variety of cellular processes dominated by BMDMs. Our work provides transcriptomics clues for exploring the influence of NPFF on the physiological functions of BMDMs. [ABSTRACT FROM AUTHOR]

Lysophosphatidic acid receptor 1 (LPA1) contributes to brain injury following transient focal cerebral ischemia. However, the mechanism remains unclear. Here, we investigated whether nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3 (NLRP3) inflammasome activation might be an underlying mechanism involved in the pathogenesis of brain injury associated with LPA1 following ischemic challenge with transient middle cerebral artery occlusion (tMCAO). Suppressing LPA1 activity by its antagonist attenuated NLRP3 upregulation in the penumbra and ischemic core regions, particularly in ionized calcium-binding adapter molecule 1 (Iba1)-expressing cells like macrophages of mouse after tMCAO challenge. It also suppressed NLRP3 inflammasome activation, such as caspase-1 activation, interleukin 1β (IL-1β) maturation, and apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) speck formation, in a post-ischemic brain. The role of LPA1 in NLRP3 inflammasome activation was confirmed in vitro using lipopolysaccharide-primed bone marrow-derived macrophages, followed by LPA exposure. Suppressing LPA1 activity by either pharmacological antagonism or genetic knockdown attenuated NLRP3 upregulation, caspase-1 activation, IL-1β maturation, and IL-1β secretion in these cells. Furthermore, nuclear factor-κB (NF-κB), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 were found to be LPA1-dependent effector pathways in these cells. Collectively, results of the current study first demonstrate that LPA1 could contribute to ischemic brain injury by activating NLRP3 inflammasome with underlying effector mechanisms. [ABSTRACT FROM AUTHOR]