Production of proinflammatory mediators in activated microglia is synergistically regulated by Notch-1, glycogen synthase kinase (GSK-3β) and NF-κB/p65 signalling

Abstract

Microglia activation and associated inflammatory response are involved in the pathogenesis of different neurodegenerative diseases. We have reported that Notch-1 and NF-κB/p65 signalling pathways operate in synergy in regulating the production of proinflammatory mediators in activated microglia. In the latter, there is also evidence by others that glycogen synthase kinase 3β (GSK-3β) mediates the release of proinflammatory cytokines but the interrelationships between the three signalling pathways have not been fully clarified. This is an important issue as activated microglia are potential therapeutic target for amelioration of microglia mediated neuroinflammation. Here we show that blocking of Notch-1 with N-[(3,5-Difluorophenyl) acetyl]-L-alanyl-2-phenylglycine-1,1-dimethylethyl ester (DAPT) in LPS activated BV-2 microglia not only suppressed Notch intracellular domain (NICD) and Hes-1 protein expression, but also that of GSK-3β. Conversely, blocking of the latter with lithium chloride (LiCl) decreased NICD expression in a dose-dependent manner; moreover, Hes-1 immunofluorescence was attenuated. Along with this, the protein expression level of p-GSK-3β and p-AKT protein expression was significantly increased. Furthermore, DAPT and LiCl decreased production of IL-1β, TNF-α, IL-6, iNOS, Cox2 and MCP-1; however, IL-10 expression was increased notably in LiCl treated cells. The effects of DAPT and LiCl on changes of the above-mentioned biomarkers were confirmed by immunofluorescence in both BV-2 and primary microglia. Additionally, NF-κB/p65 immunofluorescence was attenuated by DAPT and LiCl; as opposed to this, IκBα protein expression was increased. Taken together, it is suggested that Notch-1, NF-κB/p65 and GSK-3β operate in synergy to inhibit microglia activation. This may be effected via increased expression of phospho-GSK-3β (p-GSK-3β), phospho-protein kinase B (PKB) (p-AKT) and IκBα. It is concluded that the three signalling pathways are functionally interlinked in regulating microglia activation.

Lipid transporter Spns2 promotes microglia pro‐inflammatory activation in response to amyloid‐beta peptide

“…Pro‐inflammatory microglia are generally identified by expression of pro‐inflammatory cytokines and chemokines, such as tumor necrosis factor α (TNFα), interleukin‐6 (IL6), IL‐1β, and nitric oxide, while anti‐inflammatory‐polarized microglia identified by high expression of Arginase‐1 (Arg1), Interleukin‐4 (IL4), and CD206 (Hirbec et al, ; Parisi et al, ). Treatment with endotoxin LPS, amyloid beta peptide (Aβ), and other agents lead to pro‐inflammatory polarization, whereas treatment with anti‐inflammatory agents, such as IL4 and glatiramer acetate, result in anti‐inflammatory polarization (Cao, Karthikeyan, Dheen, Kaur, & Ling, ; Lee et al, ; McGeer & McGeer, ; Minett et al, ). However, many aspects of the microglia biology, especially the intrinsic molecular mechanisms leading to different activation states and their corresponding effect on cellular and neuronal function in different pathophysiological context, remain elusive and demand further investigation (Cherry, Olschowka, & O'Banion, ; McGeer & McGeer, ; Minett et al, ; Moller, Contos, Musante, Chun, & Ransom, ; Ramesh, MacLean, & Philipp, ; Wang, Tan, Yu, & Tan, ).…”

“…polarization (Cao, Karthikeyan, Dheen, Kaur, & Ling, 2017;Lee et al, 2008;McGeer & McGeer, 2002;Minett et al, 2016). However, many aspects of the microglia biology, especially the intrinsic molecular mechanisms leading to different activation states and their corresponding effect on cellular and neuronal function in different pathophysiological context, remain elusive and demand further investigation (Cherry, Olschowka, & O'Banion, 2014;McGeer & McGeer, 2002;Minett et al, 2016;Moller, Contos, Musante, Chun, & Ransom, 2001;Ramesh, MacLean, & Philipp, 2013;.…”

Triticum vulgare Extract Modulates Protein-Kinase B and Matrix Metalloproteinases 9 Protein Expression in BV-2 Cells: Bioactivity on Inflammatory Pathway Associated with Molecular Mechanism Wound Healing

“…In 7 Mediators of Inflammation particular, we highlighted the induction of proinflammatory messengers mentioned above (TNF-α, IL-1β, NO, and NF-κB) [5]. Nevertheless, addition of Triticum vulgare extract (TVE), in combination with LPS showed an antiinflammatory action on specific markers of inflammation, restoring cytoplasmic levels of p65 and reducing its nuclear expression [5]; this type of mechanism has been reported by other works, in which the p65 restoration (reduction of nuclear staining of p65) is well considered an antiinflammatory signal [11,[27][28][29][30][31][32]. However, no other markers have been associated with "early inflammatory molecule" concept, except the four markers mentioned above.…”

“…Looking at the literature regarding the in vitro model simulating the molecular inflammatory mechanism, BV-2 cells [5,9,11,18]. Previously, we demonstrated that this cell culture system modulated inflammatory mediators, such as tumor necrosis factoralpha (TNF-α), interleukin 1beta (IL-1β), nitric oxide (NO), and nuclear factor kappa beta (NF-κB), after LPS stimuli [5].…”

“…So far, to investigate in vitro this process, many scientists have used the BV-2 cell cultures, derived from mouse microglia. In fact, stimulation by lipopolysaccharide (LPS) in BV-2 cells affects protein modulation of other messengers of mitogen activation such as glycogen synthase kinase (GSK-3β) protein and phosphoprotein-kinase B (PKB or pAKT) [11]. Additionally, the molecular scenario of inflammation includes important proteins/enzymes.…”