• Experimental and Molecular Medicine
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• 제50권11호
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• pp.1.1-1.10
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• 2018

Bone marrow-derived mesenchymal stem cells (BMMSCs) are used extensively for cardiac repair and interact with immune cells in the damaged heart. Macrophages are known to be modulated by stem cells, and we hypothesized that priming macrophages with BMMSCs would enhance their therapeutic efficacy. Rat bone marrow-derived macrophages (BMDMs) were stimulated by lipopolysaccharide (LPS) with or without coculture with rat BMCs. In the LPS-stimulated BMDMs, induction of the inflammatory marker iNOS was attenuated, and the anti-inflammatory marker Arg1 was markedly upregulated by coculture with BMMSCs. Myocardial infarction (MI) was induced in rats. One group was injected with BMMSCs, and a second group was injected with MIX (a mixture of BMMSCs and BMDMs after coculture). The reduction in cardiac fibrosis was greater in the MIX group than in the BMC group. Cardiac function was improved in the BMMSC group and was substantially improved in the MIX group. Angiogenesis was better in the MIX group, and anti-inflammatory macrophages were more abundant in the MIX group than in the BMMSC group. In the BMMSCs, interferon regulatory factor 5 (IRF5) was exclusively induced by coculture with macrophages. IRF5 knockdown in BMMSCs failed to suppress inflammatory marker induction in the macrophages. In this study, we demonstrated the successful application of BMDMs primed with BMMSCs as an adjuvant to cell therapy for cardiac repair.

• Asian-Australasian Journal of Animal Sciences
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• 제32권11호
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• pp.1789-1800
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• 2019

Objective: Although alveolar macrophages play a key role in the respiratory immunity of livestock, studies on the mechanism of differentiation and survival of alveolar macrophages are lacking. Therefore, we undertook to investigate changes in the lipid metabolism and survival rate, using 3D4/31 macrophages and Dudleya brittonii which has been used as a traditional asthma treatment. Methods: 3D4/31 macrophages were used as the in vitro porcine alveolar macrophages model. The cells were activated by exposure to phorbol 12-myristate 13-acetate (PMA). Dudleya brittonii extraction was performed with distilled water. For evaluating the cell survival rate, we performed the water-soluble tetrazolium salt cell viability assay and growth curve analysis. To confirm cell death, cell cycle and intracellular reactive oxygen species (ROS) levels were measured using flow cytometric analysis by applying fluorescence dye dichlorofluorescein diacetate and propidium iodide. Furthermore, we also evaluated cellular lipid accumulation with oil red O staining, and fatty acid synthesis related genes expression levels using quantitative polymerase chain reaction (qPCR) with SYBR green dye. Glycolysis, fatty acid oxidation, and tricarboxylic acid (TCA) cycle related gene expression levels were measured using qPCR after exposure to Dudleya brittonii extract (DB) for 12 h. Results: The ROS production and cell death were induced by PMA treatment, and exposure to DB reduced the PMA induced downregulation of cell survival. The PMA and DB treatments upregulated the lipid accumulation, with corresponding increase in the acetyl-CoA carboxylase alpha, fatty acid synthase mRNA expressions. DB-PMA co-treatment reduced the glycolysis genes expression, but increased the expressions of fatty acid oxidation and TCA cycle genes. Conclusion: This study provides new insights and directions for further research relating to the immunity of porcine respiratory system, by employing a model based on alveolar macrophages and natural materials.

• 대한본초학회지
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• 제32권2호
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• pp.17-24
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• 2017

Objective : This study was designed to evaluate candidate materials as anti-inflammation agent from extracts of various Korean Compositae herbs in Hwaak mountain. Among Korea medicinal herbs, Ainsliaea acerifolia (AA) belongs to the Compositae family, has been used for the treatment of rheumatic arthritis. However, AA has not been previously reported to have an anti-inflammatory effect. Therefore, we investigated the anti-inflammatory effects of AA and its underlying molecular mechanisms in lipopolysaccharide (LPS)-induced RAW 264.7 macrophages. Methods : Cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in RAW 264.7 macrophages. Nitric oxide (NO) was measured with Griess reagent and pro-inflammatory cytokines were detected by enzyme immunoassay (EIA) kits in LPS-stimulated RAW 264.7 macrophages. Protein expressions of inducible nitric oxide synthase, and cyclooxygenase-2 (COX-2) and p65 subunit of nuclear $factor-{\kappa}B$ ($NF-{\kappa}B$) were determined by Western blot analysis. Results : Among 8 extracts of Korean Compositae herbs tested, AA showed the inhibition of NO production without cytotoxicity. Consistent with the observation, AA reduced the expression levels of iNOS and COX-2 proteins in LPS-simulated RAW 264.7 macrophages in dose-dependent manner. In addition, AA inhibited the productions of $TNF-{\alpha}$ and IL-6 in LPS-simulated RAW 264.7 macrophages. However, AA did not inhibit activation of p65 $NF-{\kappa}B$ in LPS-simulated RAW 264.7 macrophages. Conclusion : These results suggest that down-regulation of iNOS, COX-2 protein expression and $TNF-{\alpha}$ and IL-6 production by AA are responsible for its anti-inflammatory effects.

• Archives of Plastic Surgery
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• 제50권4호
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• pp.432-442
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• 2023

Background Macrophages play a major role in wound healing and prevent infection from the outside. Polarization conversion of macrophages regulates aspects of inflammation, and two macrophages, M1 (classically activated) and M2 (alternatively activated), exist at both ends of broad-spectrum macrophage polarization. Thus, we aimed to investigate whether macrophage polarization can be artificially regulated. To this end, MgSO4 and small-interfering RNA (siRNA) targeting magnesium transport 1 (MAGT1) were used to investigate the effects of intracellular magnesium (Mg2+) concentrations on the differentiation of macrophages in vitro. Methods THP-1 derived macrophages maintained in a culture medium containing 5 mM MgSO4 and siRNA to inhibit the expression of MAGT1. As comparative groups, THP-1 derived macrophages polarized into M1 and M2 macrophages by treatment with M1, M2 inducer cytokine. The polarization status of each group of cells was confirmed by cell surface antigen expression and cytokine secretion. Results We found that MgSO4 treatment increased CD163 and CD206, similar to the effect noted in the M2 group. The expression of CD80 and HLA-DR was increased in the group treated with MAGT1 siRNA, similar to the effect noted in the M1 group. Functional assays demonstrated that the group treated with MgSO4 secreted higher levels of IL-10, whereas the MAGT1 siRNA-treated group secreted higher levels of IL-6 cytokines. Additionally, the conditional medium of the Mg2+ treated group showed enhanced migration of keratinocytes and fibroblasts. Conclusion Mg2+ can help to end the delay in wound healing caused by persistent inflammation in the early stages.

• IMMUNE NETWORK
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• 제23권3호
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• pp.23.1-23.17
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• 2023

Inflammation is a series of host defense processes in response to microbial infection and tissue injury. Inflammatory processes frequently cause extracellular acidification in the inflamed region through increased glycolysis and lactate secretion. Therefore, the immune cells infiltrating the inflamed region encounter an acidic microenvironment. Extracellular acidosis can modulate the innate immune response of macrophages; however, its role for inflammasome signaling still remains elusive. In the present study, we demonstrated that macrophages exposed to an acidic microenvironment exhibited enhanced caspase-1 processing and IL-1β secretion compared with those under physiological pH. Moreover, exposure to an acidic pH increased the ability of macrophages to assemble the NLR family pyrin domain containing 3 (NLRP3) inflammasome in response to an NLRP3 agonist. This acidosis-mediated augmentation of NLRP3 inflammasome activation occurred in bone marrow-derived macrophages but not in bone marrow-derived neutrophils. Notably, exposure to an acidic environment caused a reduction in the intracellular pH of macrophages but not neutrophils. Concordantly, macrophages, but not neutrophils, exhibited NLRP3 agonist-mediated translocation of chloride intracellular channel protein 1 (CLIC1) into their plasma membranes under an acidic microenvironment. Collectively, our results demonstrate that extracellular acidosis during inflammation can increase the sensitivity of NLRP3 inflammasome formation and activation in a CLIC1-dependent manner. Thus, CLIC1 may be a potential therapeutic target for NLRP3 inflammasome-mediated pathological conditions.

• Biomolecules & Therapeutics
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• 제32권3호
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• pp.341-348
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• 2024

Endoplasmic reticulum (ER) stress plays a crucial role in liver diseases, affecting various types of hepatic cells. While studies have focused on the link between ER stress and hepatocytes as well as hepatic stellate cells (HSCs), the precise involvement of hepatic macrophages in ER stress-induced liver injury remains poorly understood. Here, we examined the effects of ER stress on hepatic macrophages and their role in liver injury. Acute ER stress led to the accumulation and activation of hepatic macrophages, which preceded hepatocyte apoptosis. Notably, macrophage depletion mitigated liver injury induced by ER stress, underscoring their detrimental role. Mechanistic studies revealed that ER stress stimulates macrophages predominantly via the PERK signaling pathway, regardless of its canonical substrate ATF4. hnRNPA1 has been identified as a crucial mediator of PERK-driven macrophage activation, as the overexpression of hnRNPA1 effectively reduced ER stress and suppressed pro-inflammatory activation. We observed that hnRNPA1 interacts with mRNAs that encode UPR-related proteins, indicating its role in the regulation of ER stress response in macrophages. These findings illuminate the cell type-specific responses to ER stress and the significance of hepatic macrophages in ER stress-induced liver injury. Collectively, the PERK-hnRNPA1 axis has been discovered as a molecular mechanism for macrophage activation, presenting prospective therapeutic targets for inflammatory hepatic diseases such as acute liver injury.

• Asian Pacific Journal of Cancer Prevention
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• 제15권14호
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• pp.5767-5772
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• 2014

Background and Aim: B7-H1, a co-inhibitory molecule of the B7 family, is found aberrantly expressed in ovarian cancer cells and infiltrating macrophage/dendritic-like cells, and plays a critical role in immune evasion by ovarian cancer. IL-12, an inducer of Th1 cell development, exerts immunomodulatory effects on ovarian cancer. However, whether IL-12 regulates B7-H1 expression in human ovarian cancer associated-macrophages has not been clarified. Therefore, we investigated the effects of IL-12 on the expression of B7-H1 in ovarian cancer-associated macrophages and possible mechanisms. Methods: PMA induced THP-1-derived macrophages or human monocyte-derived macrophages were treated with recombinant IL-12 (rIL-12) or infected with adenovirus carrying human IL-12 gene (Ad-IL-12-GFP) for 24 h, then cocultured with the SKOV3 ovarian cancer cell line for another 24 h. Macrophages were collected for real-time PCR and Western blot to detect the expression of B7-H1, and activation of the NF-${\kappa}B$ signaling pathway. Moreover, supernatants were collected to assay for IL-12, IFN-${\gamma}$ and IL-10 by ELISA. In addition, monocyte-derived macrophages treated with IFN-${\gamma}$ were cocultured with SKOV3 and determined for the expression of B7-H1. Furthermore, the expression of B7-H1 in monocyte-derived macrophages was also evaluated after blocking NF-${\kappa}B$ signaling. Results: The expression of B7-H1 was significantly upregulated in monocyte-derived macrophages treated with rIL-12 or Ad-IL-12-GFP compared with the control groups (p<0.05), accompanied by a remarkable upregulation of IFN-${\gamma}$ (p<0.05), a marked downregulation of IL-10 (p<0.05) and activation of NF-${\kappa}B$ signaling. However, the upregulation of B7-H1 was inhibited by blocking the NF-${\kappa}B$ signaling pathway (p<0.05). Expression of B7-H1 was also increased (p<0.05) in monocyte-derived macrophages treated with IFN-${\gamma}$ and cocultured with SKOV3. By contrast, the expression of B7-H1 in THP-1-derived macrophages was significantly decreased when treated in the same way as monocyte-derived macrophages (p<0.05), and IL-10 was also significantly decreased but IFN-${\gamma}$ was almost absent. Conclusions: IL-12 upregulates the expression of B7-H1 in monocyte-derived macrophages, which is possible though inducing the secretion of IFN-${\gamma}$ and further activating the NF-${\kappa}B$ signal pathway. However, IL-12 downregulates the expression of B7-H1 in THP-1-derived macrophages, associated with a lack of IFN-${\gamma}$ and inhibition of expression of IL-10.

• Tuberculosis and Respiratory Diseases
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• 제39권6호
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• pp.526-535
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• 1992

연구배경 : 산소반응성 대사물에 의한 결핵균 살해능은 대식세포의 활성화 상태에 의해 좌우되는데 체외에서의 이러한 효과를 priming 이라 한다. priming 효과를 유도하는 물질에는 IFN-$\gamma$와 LPS가 대표적인 물질인데 이들 자체가 superoxide와 같은 산소반응성 물질의 생성을 증가시키지는 않지만 식균작용이나 PMA와 같은 화학적 물질에 반응하여 증강된 산소반응성 물질의 생성을 유도한다. 혈액단구세포와는 달리 폐포대식세포는 일상적인 외부환경에 노출되어 있기 때운에 priming 효과에 대한 논란이 있고 결핵균 세포벽의 각 성분이 대식세포의 활성화에 상반되는 결과를 보인다는 보고들이 있어 본 연구에서는 사람의 폐포대식세포와 혈액단구세포, 그리고 백서의 폐포대식세포에서 IFN-$\gamma$에 의한 priming 효과를 비교 관찰하였고 결핵균증 H37Ra 균증이 폐포대식세포에 노출되었을 때 나타나는 superoxide의 생성능의 변화를 비교하여 보았다. 방법 : 사람과 백서에서 얻은 기관지폐포세척액을 Petri dish에 부착시키고 cold shock 방법으로 부착된 대식세포를 분리하여 24시간 IFN-$\gamma$로 전처치한 후의 priming 효과와 H37Ra 결핵균종을 노출시켰을 시의 superoxide의 생성능을 ferricytochrome reduction 방법으로 측정하여 비교하였다. 결과 : 1) 사람 폐포대식세포에서는 고농도의 IFN-$\gamma$ 전처치로도 priming 효과가 관찰되지 않은 반면 혈액단구세포와 백서 폐포대식세포에서는 priming 효과를 관찰할 수 있었다. 2) 폐포대식세포를 비독력 결핵균종인 H37Ra 생균에 노출시킨 결과 사람과 백서 공히 triggering 효과를 나타내었고 그 분해물에 의한 노출 역시 유사한 결과를 나타내었다. 결론 : 사람의 폐포대식세포는 다른 대식세포와는 달리 일상적인 외부환경에 노출되어 있으으로 priming 효과가 관찰되지 않았으며 비독력 결핵균종인 H37Ra에 의해서는 폐포대식세포의 활성화를 억제하는 효과를 관찰할 수 없었다.

• Tuberculosis and Respiratory Diseases
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• 제70권2호
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• pp.113-124
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• 2011

Background: Macrophages are one of the most important inflammatory cells in innate immunity. PU.1 is a macrophage-specific transcription factor. Ubiquitins are the ultimate regulator of eukaryotic transcription. The ubiquitination process for PU.1 is unknown. This study investigated the lipopolysaccharide (LPS)-induced activation of PU.1 and its relation to ubiquitins in the macrophages. Methods: Raw264.7 cells, the primary cultured alveolar, pulmonary, and bone marrow derived macrophages were used. The Raw264.7 cells were treated with MG-132, $NH_4Cl$, lactacytin and LPS. Nitric oxide and prostaglandin D2 and E2 were measured. Immunoprecipitation and Western blots were used to check ubiquitination of PU.1. Results: The PU.1 ubiquitination increased after LPS ($1{\mu}g$/mL) treatment for 4 hours on Raw264.7 cells. The ubiquitination of PU.1 by LPS was increased by MG-132 or $NH_4Cl$ pretreatment. Two hours of LPS treatment on macrophages, PU.1 activation was not induced nor increased with the inhibition of proteasomes and/or lysosomes. The ubiquitination of PU.1 was increased in LPS-treated Raw264.7 cells at 12- and at 24 hours. LPS-treated cells increased nitric oxide production, which was diminished by MG-132 or $NH_4Cl$. LPS increased the production of $PGE_2$ in the alveolar and peritoneal macrophages of wild type mice; however, $PGE_2$ was blocked or diminished in Rac2 null mice. Pretreatment of lactacystin increased $PGE_2$, however it decreased the $PGD_2$ level in the macrophages derived from the bone marrow of B57/BL6 mice. Conclusion: LPS treatment in the macrophages ubiquitinates PU.1. Ubiquitination of PU.1 may be involved in synthesis of nitric oxide and prostaglandins.

• Korean Journal of Acupuncture
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• 제21권2호
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• pp.125-137
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• 2004

Objectives : Recently, Herbal-acupuncture therapeutics has been used for the treatment of inflammatory diseases such as rheumatoid arthritis. Especially, we have been interested in chemical mediators concerned with inflammation such as prostaglandin, cytokine, nitrous oxide. The purpose of this study is investigated that the effect of Chelidonii Herbal-acupuncture solution in lipopolysaccharide-stimulated RAW 264.7 macrophages, performed several expeimental items : those are prostaglandin $E_2$, nitric oxide and cyclooxygenase-2. Methods : The cytotoxicity of Chelidonii Herbal-acupuncture solution in RAW 264.7 macrophages were measured by MTT-based cytotoxicity assay. In order to observe cyclooxygenase-2 mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages, RT-PCR was used. Prostaglandin $E_2$ formation and nitric oxide production was measured by competitive enzyme immunoassay and Griess assay. Results : 1.The MTT assay demonstrated that cytotoxic effect of Chelidonii Herbal-acupuncture solution in RAW 264.7 macrophages were not appeared before concentration of 1mg/ml. 2.Chelidonii Herbal-acupuncture solution inhibited cyclooxygenase-2 mRNA expression in lipopolysaccharide-stimulated RAW 264.7 macrophages. 3. Chelidonii Herbal-acupuncture solution inhibited nitric oxide production in lipopolysaccharide-stimulated RAW 264.7 macrophages. 4. Chelidonii Herbal-acupuncture solution inhibited prostaglandin $E_2$ formation in lipopolysaccharide-stimulated RAW 264.7 macrophages. Conclusions : On the basis of these results, It was shown that Chelidonii Herbal-acupuncture solution is significantly able to inhibit the production of $PGE_2$ and NO, as well as COX-2 mRNA expression. Our results may provide new mechanism by which Chelidonii Herbal-acupuncture solution accounts for its beneficial effect on accelerating wound healing and anti-inflammation.