Dataset Information

Macrophage GIT1 Contributes to Bone Regeneration by Regulating Inflammatory Responses in an ERK/NRF2-Dependent Way.

ABSTRACT: Despite the best treatment, approximately 10% of fractures still face undesirable repair. Recently, many studies have focused on the importance of macrophages in bone repair; however, the cellular mechanisms by which they work are not yet fully understood. In this study, we explored the functions of macrophage G-protein-coupled receptor interacting protein 1 (GIT1) in healing a tibial monocortical defect model. Using GIT1^flox/flox Lyz2-Cre (GIT1 CKO) mice, we observed that a GIT1 deficiency in the macrophages led to an exacerbation of interleukin 1? (IL1?) production, more M1-like macrophage infiltration, and impaired intramembranous ossification in vivo. The results of in vitro assays further indicated that the macrophage GIT1 plays a critical role in several cellular processes in response to lipopolysaccharide (LPS), such as anti-oxidation, IL1? production alleviation, and glycolysis control. Although GIT1 has been recognized as a scaffold protein, our data clarified that GIT1-mediated extracellular-signal-regulated kinase (ERK) phosphorylation could activate nuclear factor (erythroid-derived 2)-like 2 (NRF2) in macrophages after LPS treatment. Moreover, we demonstrated that macrophage GIT1-activated ERK/NRF2 negatively regulates the 6-phosphofructo-2-kinase/fructose-2, 6-biphosphatase 3 (PFKFB3), facilitating the decrease of glycolysis. Our findings uncovered a previously unrecognized role of GIT1 in regulating ERK/NRF2 in macrophages to control the inflammatory response, suggesting that macrophage GIT1 could be a potential target to improve bone regeneration. © 2020 The Authors. Journal of Bone and Mineral Research published by American Society for Bone and Mineral Research..

SUBMITTER: Zhao SJ

PROVIDER: S-EPMC7689802 | biostudies-literature | 2020 Oct

REPOSITORIES: biostudies-literature

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Macrophage GIT1 Contributes to Bone Regeneration by Regulating Inflammatory Responses in an ERK/NRF2-Dependent Way.

Zhao Shu-Jie SJ Liu Hao H Chen Jian J Qian Ding-Fei DF Kong Fan-Qi FQ Jie Jian J Yin Guo-Yong GY Li Qing-Qing QQ Fan Jin J

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20200709 10

Despite the best treatment, approximately 10% of fractures still face undesirable repair. Recently, many studies have focused on the importance of macrophages in bone repair; however, the cellular mechanisms by which they work are not yet fully understood. In this study, we explored the functions of macrophage G-protein-coupled receptor interacting protein 1 (GIT1) in healing a tibial monocortical defect model. Using GIT1<sup>flox/flox</sup> Lyz2-Cre (GIT1 CKO) mice, we observed that a GIT1 defi ...[more]

PMID: 32460388

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Project description:BackgroundIt has recently become evident that activating/inhibitory cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). The immunoreceptor CD300f expressed on monocytes, neutrophils, and mast cells modulates inflammation, phagocytosis, and outcome in models of autoimmune demyelination, allergy, and systemic lupus erythematosus. On the other hand, a finely regulated inflammatory response is essential to induce regeneration after injury to peripheral nerves since hematogenous macrophages, together with resident macrophages and de-differentiated Schwann cells, phagocyte distal axonal and myelin debris in a well-orchestrated inflammatory response. The possible roles and expression of CD300f and its ligands have not been reported under these conditions.MethodsBy using quantitative PCR (QPCR) and CD300f-IgG2a fusion protein, we show the expression of CD300f and its ligands in the normal and crush injured sciatic nerve. The putative role of CD300f in peripheral nerve regeneration was analyzed by blocking receptor-ligand interaction with the same CD300f-IgG2a soluble receptor fusion protein in sciatic nerves of Thy1-YFP-H mice injected at the time of injury. Macrophage M1/M2 polarization phenotype was also analyzed by CD206 and iNOS expression.ResultsWe found an upregulation of CD300f mRNA and protein expression after injury. Moreover, the ligands are present in restricted membrane patches of Schwann cells, which remain stable after the lesion. The lesioned sciatic nerves of Thy1-YFP-H mice injected with a single dose of CD300f-IgG2a show long lasting effects on nerve regeneration characterized by a lower number of YFP-positive fibres growing into the tibial nerve after 10 days post lesion (dpl) and a delayed functional recovery when compared to PBS- or IgG2a-administered control groups. Animals treated with CD300f-IgG2a show at 10 dpl higher numbers of macrophages and CD206-positive cells and lower levels of iNOS expression than both control groups. At later time points (28 dpl), increased numbers of macrophages and iNOS expression occur.ConclusionsTaken together, these results show that the pair CD300f ligand is implicated in Wallerian degeneration and nerve regeneration by modulating both the influx and phenotype of macrophages.

Project description:BackgroundBone regeneration is a complex procedure that involves an interaction between osteogenesis and inflammation. Macrophages in the microenvironment are instrumental in bone metabolism. Amount evidence have revealed that exosomes transmitting lncRNA is crucial nanocarriers for cellular interactions in various biotic procedures, especially, osteogenesis. However, the underlying mechanisms of the regulatory relationship between the exosomes and macrophages are awaiting clarification. In the present time study, we aimed to explore the roles of human umbilical vein endothelial cells (HUVECs)-derived exosomes carrying nuclear enrichment enriched transcript 1 (NEAT1) in the osteogenesis mediated by M2 polarized macrophages and elucidate the underlying mechanisms.ResultsWe demonstrated HUVECs-derived exosomes expressing NEAT1 significantly enhanced M2 polarization and attenuated LPS-induced inflammation in vitro. Besides, the conditioned medium from macrophages induced by the exosomes indirectly facilitated the migration and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Mechanically, Exos carrying NEAT1 decreased remarkably both expression of dead-box helicase 3X-linked (DDX3X) and nod-like receptor protein 3 (NLRP3). The level of NLRP3 protein increased significantly after RAW264.7 cells transfected with DDX3X overexpression plasmid. Additionally, the knockdown of NEAT1 in exosomes partially counteracted the aforementioned effect of Exos. The results of air pouch rat model demonstrated that HUVECs-derived exosomes increased anti-inflammatory cytokines (IL-10) and decreased pro-inflammatory cytokines (IL-1β and IL-6) significantly in vivo, contributing to amelioration of LPS-induced inflammation. Afterwards, we further confirmed that the HUVECs-derived exosomes encapsulated in alginate/gelatin methacrylate (GelMA) interpenetrating polymer network (IPN) hydrogels could promote the bone regeneration, facilitate the angiogenesis, increase the infiltration of M2 polarized macrophages as well as decrease NLRP3 expression in the rat calvarial defect model.ConclusionsHUVECs-derived exosomes enable transmitting NEAT1 to alleviate inflammation by inducing M2 polarization of macrophages through DDX3X/NLRP3 regulatory axis, which finally contributes to osteogenesis with the aid of alginate/GelMA IPN hydrogels in vivo. Thus, our study provides insights in bone healing with the aid of HUVECs-derived exosomes-encapsulated composite hydrogels, which exhibited potential towards the use of bone tissue engineering in the foreseeable future.

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Macrophage GIT1 Contributes to Bone Regeneration by Regulating Inflammatory Responses in an ERK/NRF2-Dependent Way.

Publications

Macrophage GIT1 Contributes to Bone Regeneration by Regulating Inflammatory Responses in an ERK/NRF2-Dependent Way.

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