The Role Of Macrophage Infiltration And Polarization In Intervertebral Disc Degeneration And Correlated Mechanisms

PART Ⅰ: The Effects and Mechanisms of Macrophages in Nucleus Pulposus Inflammation and CatabolismObjective: To investigate the role of macrophage infiltration and polarization in the pathogenesis of intervertebral disc(IVD)degeneration(IVDD),and the role of the Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)pathway in nucleus pulposus(NP)inflammation and catabolism induced by M1-polarized macrophages.Methods: Macrophage infiltration into human IVD tissues was detected by immunohistochemical(IHC)staining.The interactions between NP cells(NPCs)and macrophages were evaluated by the treatment of the conditioned medium(CM)of each other,followed by quantitative real-time PCR(qRT-PCR),western blot(WB)and immunofluorescence staining to detect the levels of inflammatory factors or matrix proteases.Furthermore,STAT3 inhibitor Stattic was used to evaluate the role of STAT3 activation in the inflammation and catabolism of NPCs.In vitro cultured murine lumbar IVD tissues were treated with macrophage CM and histological analysis was conducted to analyze the levels of inflammatory factors and matrix proteases.The murine coccygeal disc puncture model was constructed to evaluate the infiltration and subtype of macrophages as well as the levels of IL-1β,TNF-α and MMP13 in degenerated IVD tissues.Results: Severely degenerated human IVD samples showed massive infiltration of macrophages,especially M1 phenotype,as well as elevated levels of IL-1β,TNF-α and MMP13.The CM of IL-1β-treated NPCs enhanced the M1 polarization of macrophages.The CM of M1 macrophages but not M2 macrophages promoted the expression of inflammatory factors and matrix proteases in NPCs via activating the JAK2/STAT3 pathway.STAT3 inhibitor Stattic could represent anti-inflammatory and anti-catabolic effects.Additionally,M1-CM induced the expression of IL-1β,TNF-α and MMP13 in in vitro cultured murine IVD tissues.Murine coccygeal disc puncture models showed macrophage infiltration and elevated levels of IL-1β,TNF-α and MMP13 in degenerated IVD tissues.Conclusions: M1 macrophages induce NP inflammation and catabolism via activating the JAK2/STAT3 pathway.PART Ⅱ: The Effects and Mechanisms of Macrophages in Nucleus Pulposus Fibrosis and Pathological AngiogenesisObjective: To investigate the effects of macrophage infiltration on NP fibrosis and pathological angiogenesis,and the regulatory role of cell migration inducing protein(CEMIP).Methods: A copy of single-cell RNA sequencing data was analyzed to distinguish healthy NPC clusters and fibrotic NPC clusters in human NP tissues,and to reveal correlated biological processes.Immunostaining was conducted to detect the association between macrophage infiltration and blood vessel ingrowth,as well as the levels of vascular endothelial growth factor A(VEGFA)and CEMIP in human IVD tissues.The pro-fibrotic effects of macrophages and the anti-fibrotic effects of downregulating Cemip were examined in NPCs by qRT-PCR and WB.The migratory and angiogenic ability of endothelial cells(ECs)in different treatment groups were evaluated by wound healing assays,transwell migration assays,and tube formation assays.NP fibrosis and pathological angiogenesis were analyzed in murine puncture-induced IVDD models.Results: Single-cell RNA sequencing analysis revealed the classification of fibrotic NPC clusters and healthy NPC clusters in human NP tissues,and the fibrotic NPC clusters were possibly associated with angiogenesis-related biological processes.Immunostaining showed the spatial association between the ingrowth of blood vessels and macrophage infiltration,as well as elevated levels of CEMIP and VEGFA in severely degenerated human IVD tissues.Downregulating Cemip ameliorated macrophage-induced fibrotic phenotype of NPCs.M2 but not M1 macrophages promoted the angiogenic ability of ECs via activating the Rho A/Rho-associated coiled-coil forming kinase(ROCK)pathway and the Akt pathway.Reversing the fibrotic phenotype of NPCs by Cemip siRNA also mitigated the pro-angiogenesis effects of M2-CM-treated NPCs.Moreover,NP fibrosis and EC infiltration were detected in degenerated murine IVD tissues.Conclusion: NP fibrosis and pathological angiogenesis potentially interact with each other under macrophage stimulation.CEMIP participates in the regulation of both NP fibrosis and pathological angiogenesis.PART Ⅲ: The Effects and Mechanisms of Macrophage-derived Small Extracellular Vesicles in Nucleus Pulposus Cell SenescenceObjective: To investigate the role of macrophage-derived small extracellular vesicles(sEVs)in NPC senescence,and to screen the micro-RNA(miRNA)with pro-senescence effects in BMDM-sEV and potential targets by sequencing analysis.Methods: BMDM-sEV was isolated by the differential centrifugation method,and the phenotype of sEV was identified by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and WB.NPCs were treated with M1-sEV or M2-sEV and the cell senescence levels were examined.NPCs were detected by senescence‐associated β‐galactosidase(SA?β?Gal)staining,CCK-8,qRT-PCR and WB.Cytometric bead array(CBA)was performed to detect the inflammatory factor levels in the cell culture supernatant of NPCs.M1-sEV or M2-sEV was injected into rat coccygeal IVD tissues to evaluate the in vivo effects of BMDM-sEV on IVDD.The expression levels of miRNA in BMDM-sEV were detected by PANDORA sequencing.NPCs were transfected with miRNA mimics to overexpress the corresponding miRNA,and the cell senescence levels were evaluated to screen the miRNA with pro-senescence effects.The targets of miRNA were predicted in three databases(miRDB,Target Scan,miRWalk),and validated by qRT-PCR and WB.In various NPC viability impairment models,the cytoprotective effects of M2-CM on NPCs were evaluated by CCK-8 assays.Results: BMDM-sEV displayed the cup-shaped morphology with diameters mainly ranging from 30 nm to 150 nm.Both M1-sEV and M2-sEV impaired the viability of NPCs,and increased the rate of SA?β?Gal-positive cells.PCR and WB showed that both M1-sEV and M2-sEV stimulated the expression of multiple inflammatory factors and matrix proteases,and inhibited the anabolic process.In addition,the expression of senescence-related proteins,including p16,p21 and p53,was elevated by M1-sEV or M2-sEV treatment.Transfection of miRNA mimic indicated that let-7i-5p promoted NPC senescence.Target prediction in databases indicated that let-7i-5p might exert pro-senescence effects by inhibiting LIN28 A.M2-CM could protect the viability of NPCs.Removal or inhibition of sEV improved the cytoprotective effects of M2-CM.Conclusion: Both M1-sEV and M2-sEV could induce NPC senescence by delivering miRNA let-7i-5p.sEV-free M2-CM could protect NPC viability.