Neuroplastin 65 regulates lipopolysaccharide-induced macrophage polarization
10.3760/cma.j.cn112309-20240116-00027
- VernacularTitle:细胞黏附分子neuroplastin 65调控脂多糖诱导巨噬细胞极化
- Author:
Xiaoxue XIA
1
;
Huan REN
;
Yalei DAI
Author Information
1. 同济大学附属上海市肺科医院结核病重点实验室，上海 200433
- Keywords:
Macrophage;
Neuroplastin 65;
Polarization;
Signal transduction
- From:
Chinese Journal of Microbiology and Immunology
2024;44(11):917-925
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the molecular mechanism by which neuroplastin 65 (Np65) regulates lipopolysaccharide (LPS)-induced macrophage polarization, and elucidate the effect of Np65 on macrophage polarization during inflammation.Methods:Bone marrow-derived macrophages from C57BL/6 wild-type mice and Np65 knockout mice with the same background were prepared. Quantitative real-time PCR was used to detect the expression of related genes including Nptn, CD80, CD86, TNF-α, IL-1β, IL-6, CD206, CD163, Ym1, Arg1, IL-10, CD14 and TLR4 in macrophages. The expression of CD86 and CD206 molecules on the surface of macrophages was measured by flow cytometry. Western blot was used to detect the expression of related proteins and their phosphorylation levels in macrophages including p38, extracellular regulated protein kinases (ERK), c-Jun N-terminal kinase (JNK), p65, signal transducers and activators of transcription 3 (STAT3), Janus kinase 1 (JAK1), JAK2 and protein kinase B (AKT). Co-immunoprecipitation was used to detect the binding of Np65 to CD14 or TLR4. Immunofluorescence confocal analysis was used to identify the colocalization of Np65 with CD14 or TLR4 in the cells. One-way analysis of variance and paired Student′s t test were used for statistical analysis. Results:LPS or IL-4 could activate macrophages and upregulate the Np65 gene expression ( P<0.05). Np65 deficiency resulted in a significant downregulation of the expression of macrophage M1 polarization-related markers CD80, and CD86 as well as the cytokines TNF-α, IL-1β and IL-6 at mRNA level ( P<0.01). However, the expression of M2 polarization markers CD206, CD163, Ym1, Arg1 and IL-10 at mRNA level were increased significantly ( P<0.05). Co-immunoprecipitation and immunofluorescence confocal analysis revealed the significant co-localization of Np65 with CD14. Furthermore, Np65 deficiency resulted in significantly down-regulated phosphorylation of p38, ERK, and p65 in M1 macrophages, and significantly increased STAT3 phosphorylation in M2 macrophages. Conclusions:Np65 participates in the polarization of macrophages towards the M1 type and inhibits M2 type polarization through affecting the phosphorylation of multiple key proteins in macrophage polarization-related signaling pathways, thereby regulating the activation of macrophages during inflammation.
