Differential immune-stimulatory effects of LTAs from different lactic acid bacteria via MAPK signaling pathway in RAW 264.7 cells

Lipopolysaccharide (LPS) treatment of monocytic cells has been shown to activate the Raf-1/mitogen-activated protein kinase (MAPK) signaling pathway and to increase secretory interleukin-1 receptor antagonist (sIL-1Ra) gene expression. The significance of the activation of the Raf-1/MAPK signaling pathway to LPS regulation of sIL-1Ra gene expression, however, has not been determined. This study addresses the role of the Raf-1/MAPK signaling pathway in regulation of sIL-1Ra gene expression by LPS. Cotransfection of the murine macrophage cell line RAW 264.7 with a 294-bp sIL-1Ra promoter/luciferase construct (pRA-294-luc) and a constitutively active Raf-1 kinase expression vector (pRSV-Raf-BXB) resulted in induction of sIL-1Ra promoter activity, indicating that Raf-1, like LPS, can regulate sIL-1Ra promoter activity. An in vitro MAPK analysis indicated that both LPS treatment and pRSV-Raf-BXB transfection of RAW 264.7 cells increases p42 MAPK activity. An in vitro Raf-1 kinase assay, however, failed to detect LPS-induced Raf-1 kinase activity in RAW 264.7 cells, suggesting that in RAW 264.7 cells, Raf-1 kinase is not an activating component of the LPS signaling pathway regulating MAPK activity or sIL-1Ra promoter activity. This observation was supported by results from transfection studies which demonstrated that expression of a dominant-inhibitory Raf-1 mutant in RAW 264.7 cells does not inhibit LPS-induced MAPK activity or sIL-1Ra promoter activity, indicating that LPS-induced sIL-1Ra promoter activation occurs independent of the Raf-1/MAPK signaling pathway. In additional studies, cotransfection of RAW 264.7 cells with pRA-294-luc and increasing amounts of pRSV-Raf-BXB caused a dose-dependent inhibition of LPS-induced sIL-1Ra promoter activity, indicating that the role of the Raf-1 pathway in the regulation of sIL-1Ra promoter activity by LPS is as an antagonizer. Interestingly, LPS treatment of RAW 264.7 cells, cotransfected with pRA-294-luc and pRSV-Raf-BXB, also inhibited pRSV-Raf-BXB-induced sIL-1Ra promoter activity, suggesting that inductions of sIL-1Ra promoter activity by LPS and Raf-1 actually occur by mutually antagonistic mechanisms. In support of this conclusion, sIL-1Ra promoter mapping studies indicated that LPS and Raf-1 responses localized to different regions of the sIL-1Ra promoter. Further studies demonstrated that mutual antagonism between the LPS and Raf-1 kinase pathways is not promoter specific, as the same phenomenon is observed in assays using a c-fos enhancer/thymidine kinase promoter/luciferase construct (pc-fos-TK81-luc). Additionally, mutual antagonism with regard to sIL-1Ra promoter activity also was observed between the LPS and MEK kinase pathways, indicating that mutual antagonism can occur in more than one MAPK activation pathway.

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Corrigendum to “Isosteroid alkaloids with different chemical structures from Fritillariae cirrhosae bulbus alleviate LPS-induced inflammatory response in RAW 264.7 cells by MAPK signaling pathway” [Int. Immunopharmacol. 78 (2020) 106047]

International Immunopharmacology ◽

10.1016/j.intimp.2020.106482 ◽

2020 ◽

Vol 84 ◽

pp. 106482

Author(s):

Simei Liu ◽

Tiechui Yang ◽

Tse Wai Ming ◽

Tse Kathy Wai Gaun ◽

Ting Zhou ◽

...

Keyword(s):

Inflammatory Response ◽

Signaling Pathway ◽

Mapk Signaling ◽

Mapk Signaling Pathway ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Chemical Structures

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Flavonoids Identified from KoreanScutellaria baicalensisGeorgi Inhibit Inflammatory Signaling by Suppressing Activation of NF-κB and MAPK in RAW 264.7 Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2013/912031 ◽

2013 ◽

Vol 2013 ◽

pp. 1-11 ◽

Cited By ~ 17

Author(s):

Gyeong-Eun Hong ◽

Jin-A. Kim ◽

Arulkumar Nagappan ◽

Silvia Yumnam ◽

Ho-Jeong Lee ◽

...

Keyword(s):

Signaling Pathway ◽

Inflammatory Mediators ◽

Inflammatory Diseases ◽

Mapk Signaling ◽

Raw 264.7 Cells ◽

Raw 264.7 ◽

Two Dimensional Gel Electrophoresis ◽

Necrosis Factor Alpha ◽

Mitogen Activated Protein ◽

Anti Inflammatory

Scutellaria baicalensisGeorgi has been used as traditional medicine for treating inflammatory diseases, hepatitis, tumors, and diarrhea in Asia. Hence, we investigated the anti-inflammatory effect and determined the molecular mechanism of action of flavonoids isolated from KoreanS. baicalensisG. in lipopolysaccharide- (LPS-) stimulated RAW 264.7 macrophages. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was performed to examine cytotoxicity of the flavonoids at various concentrations of 10, 40, 70, and 100 µg/mL. No cytotoxicity was observed in RAW 264.7 cells at these concentrations. Furthermore, the flavonoids decreased production of inflammatory mediators such as inducible nitric oxide synthase, cyclooxygenase-2, interleukin-6, and tumor necrosis factor-alpha and inhibited phosphorylation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) in LPS-induced RAW 264.7 cells. Moreover, to identify the differentially expressed proteins in RAW 264.7 cells of the control, LPS-treated, and flavonoid-treated groups, two-dimensional gel electrophoresis and mass spectrometry were conducted. The identified proteins were involved in the inflammatory response and included PRKA anchor protein and heat shock protein 70 kD. These findings suggest that the flavonoids isolated fromS. baicalensisG. might have anti-inflammatory effects that regulate the expression of inflammatory mediators by inhibiting the NF-κB signaling pathway via the MAPK signaling pathway in RAW 264.7 cells.

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Anti-inflammatory effect of the six compounds isolated from Nauclea officinalis Pierrc ex Pitard, and molecular mechanism of strictosamide via suppressing the NF-κB and MAPK signaling pathway in LPS-induced RAW 264.7 macrophages

Journal of Ethnopharmacology ◽

10.1016/j.jep.2016.12.007 ◽

2017 ◽

Vol 196 ◽

pp. 66-74 ◽

Cited By ~ 23

Author(s):

DongYu Li ◽

JiaQuan Chen ◽

JiQing Ye ◽

XiaoTing Zhai ◽

Jie Song ◽

...

Keyword(s):

Signaling Pathway ◽

Molecular Mechanism ◽

Mapk Signaling ◽

Mapk Signaling Pathway ◽

Raw 264.7 ◽

Raw 264.7 Macrophages ◽

Anti Inflammatory ◽

Inflammatory Effect

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Immunostimulatory Activity of Synbiotics Using Lactococcus lactis SG-030 and Glucooligosaccharides from Weissella cibaria YRK005

Microorganisms ◽

10.3390/microorganisms9122437 ◽

2021 ◽

Vol 9 (12) ◽

pp. 2437

Author(s):

Ayeon Kwon ◽

Young-Seo Park

Keyword(s):

Nitric Oxide ◽

Lactic Acid ◽

Lactic Acid Bacteria ◽

Lactococcus Lactis ◽

Raw 264.7 Cells ◽

No Production ◽

Raw 264.7 ◽

Immunostimulatory Activity ◽

Simultaneous Treatment ◽

Weissella Cibaria

Much attention has been recently paid to the health benefits of synbiotics, a combination of probiotics and prebiotics. In this study, synbiotics were prepared by combining lactic acid bacteria with potential as probiotics and purified glucooligosaccharides, and their immunostimulatory activity was evaluated using RAW 264.7 macrophage cells. A lactic acid bacteria strain with high antioxidant activity, acid and bile salt tolerance, adhesion to Caco-2 cells, and nitric oxide (NO) production was selected as a potential probiotic strain. The selected strain, isolated from forsythia, was identified as Lactococcus lactis SG-030. The purified glucooligosaccharides produced from Weissella cibaria YRK005 were used as prebiotics. RAW 264.7 cells were treated with synbiotics in two ways. One way was a simultaneous treatment with lactic acid bacteria and glucooligosaccharides. The other way was to pre-culture the lactic acid bacteria with glucooligosaccharides followed by treatment with synbiotic culture broth or synbiotic culture supernatant. In both cases, synbiotics synergistically increased NO production in RAW 264.7 cells. In addition, synbiotics treatment increased the expression of tissue necrosis factor-α, interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase genes. Synbiotics also increased the expression of P38, extracellular signal-regulated kinases, c-Jun N-terminal kinases, phosphoinositide 3-kinase, and Akt proteins. The results confirmed that the synbiotics prepared in this study exhibited synergistic immunostimulatory activity.

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