Effect Of Nell-1 On LPS Induced Polarization Of Macrophages And The Underlying Mechanism

ObjectivesMacrophages are key cellular components of the innate immunity in the dental pulp which have high plasticity.They are usually can be polarized into two phenotypes:classically activated M1 macrophages(pro-inflammatory)and alternatively activated M2 macrophages(anti-inflammatory).Macrophages are closely related to dental pulp injury and repair.Regulating the functions of macrophages is of great significance for dental pulp repair and regeneration.Neural EGFL like 1(Nell-1)is a novel osteogenic specific growth factor which has multiple known functions including promoting chondrogenesis,suppressing osteoclastic activity,promoting osteogenesis,suppressing inflammation,promoting vascularization.Our previous study found that Nell-1 plays a positive regulatory role in dentin regeneration and nerve regeneration,and Nell-1 can inhibit early pulpitis and promote restorative dentin regeneration in rat pulp injury model,but the regulatory effect of Nell-1 on macrophages has not been reported.Further exploration of the anti-inflammatory mechanism of Nell-1 will provide theoretical reference for the clinical application of Nell-1.Our aim was to explore the effects of Nell-1 on macrophage polarization and possible cellular signaling mechanism to provide theoretical references for vital pulp therapy and pulpitis control.Methods1.Culture and induction of human myeloid cell line(THP-1)derived macrophagesThe human monocyte THP-1 cell line was purchased from the Stem Cell Bank,Chinese Academy of Sciences and the THP-1 cells were treated with 100 ng/ml phorbol-12-myristate-13-acetate(PMA)for 48 h to induce differentiation into macrophages.2.Effect of Nell-1 on THP-1 derived macrophages viability and the best concentration determinationCell counting kit 8(CCK-8)assay was used to detect the effect of different concentrations(0,100,200,400 and 800 ng/ml)of Nell-1 on THP-1 derived macrophages viability.The messenger ribonucleic acid(mRNA)expression levels of cluster of differentiation 86(CD86)and tumor necrosis factor-alpha(TNF-α)were detected by quantitative real time polymerase chain reaction(qRT-PCR)after the macrophages stimulated with different concentrations(0,100,200,400 and 800 ng/ml)of Nell-1 and 100 ng/ml lipopolysaccharide(LPS)for 24h and find out the best concentrations for subsequent experiments.3.Effects of Nell-1 on M1/M2 polarization induced by LPS in THP-1 derived macrophagesTHP-1-derived macrophages were stimulated with different concentrations of Nell-1(400 and 800 ng/ml)alone or in combination with 100 ng/ml LPS for 24h.qRT-PCR was used to detect the expression of M1 polarization related genes interleukin-6(IL-6),interleukin-1 beta(IL-1β)and M2 polarization related genes cluster of differentiation 163(CD 163),vascular endothelial growth factor(VEGF),arginase-1(Arg-1),interleukin-10(IL-10),transforming growth factor β1(TGF-β1)in THP-1 derived macrophages.The effect of Nell-1 on the secretion of M1 polarization related factors(IL-1β and TNF-α)in THP-1 derived macrophages was detected by enzyme-linked immunosorbent assay(ELISA).Western blot was used to detect the effect of Nell-1 on the expression of M2 polarization related proteins VEGF and CD206 in THP-1 derived macrophages.The effect of Nell-1 on the expression of M1-polarized surface markers CD86 and M2-polarized surface markers CD 163,CD206 in macrophages was detected by flow cytometry and immunofluorescence.4.Effects of Nell-1 on the nuclear factor-kappa B(NF-κB)signaling pathway induced by LPS in macrophages.THP-1-derived macrophages were stimulated with different concentrations of Nell-1(400 and 800 ng/ml)alone or in combination with 100 ng/ml LPS for 24 h.Phosphorylation level of NF-κB signaling pathway related proteins p65,IKα,IKKβ,IKKa was detected by Western blot.The nuclear translocation of NF-κB signaling pathway p65 was detected by immunofluorescence.Results1.THP-1 monocytes showed adherent growth after being induced by 100 ng/ml PMA for 48 h.The cell size was enlarged and some cells deformed and the parapodium stretched out,the morphology was irregular.2.CCK-8 results showed that compared with the control group,the cell viability of THP-1 derived macrophages had no significant change with the increase of Nell-1 concentration.The results of qRT-PCR showed that Nell-1 inhibited the mRNA expression of CD86 and TNF-α which upregulated by LPS in a dose-dependent manner.And the 400ng/mL and 800ng/mL of Nell-1 were selected as the optimal concentrations for subsequent experimental studies.3.The results of qRT-PCR and ELISA showed that Nell-1 down-regulated the expression of the Ml macrophages related genes IL-6,IL-1β and inflammatory factors TNF-α and IL-1β under inflammatory environment.The results of qRT-PCR and Western blot showed that Nell-1 up-regulated the expression levels of related genes CD206,VEGF,Arg-1 and related proteins VEGF and CD206 in M2 macrophages under inflammatory conditions.4.Western blot and immunofluorescence showed that Nell-1 inhibited LPS-induced activation of NF-κB signaling pathway in macrophages.ConclusionsIn this study,we preliminarily confirmed that Nell-1 can inhibit LPS-induced M1 polarization in macrophages and promote the polarization of macrophages to M2 type in the inflammatory microenvironment.Its mechanism may be related to inhibiting the activation of NF-κB signaling pathway.And this study will provide theoretical references for the clinical application of Nell-1.