Macrophage-Derived Exosomal HMGB3 Regulates Pulmonary Inflammation/Fibrosis By Promoting M1 Macrophage Polarization And Recruitment In Silicosis

Background:Silicosis is an irreversible respiratory occupational disease caused by long-term exposure to silica（SiO₂）dust.Chronic inflammation and fibrosis are the core pathological processes of silicosis.The key modulators of inflammation in silicosis have not been fully elucidated.In recent years,exosome has been reported as mediator of inflammation and widely involved in the progression of many diseases,such as asthma,sepsis and inflammatory bone disease.Macrophages are the key effector cells in the progression of silicosis.Our previous studies had shown that macrophage-derived exosomes promoted the progression of fibrosis by regulating phenotypic transformation,proliferation,and migration of myofibroblasts in silicosis.However,the role of macrophage-derived exosomes in the inflammatory response of silicosis remains unclear.The purpose of this study was to investigate the regulatory role of exosomal HMGB3（high mobility group box 3）derived from macrophages in the inflammatory/fibrotic response of silicosis.Methods:1.To investigate the secretion level of macrophages-derived exosomes after SiO₂ exposure in vitro and in vivo.（1）We first constructed a 28-day silicosis mouse model,and detected the expression of CD68（a macrophage-related marker）by immunohistochemistry and western blotting to analyze the number and proportion of macrophages in lung tissue and bronchoalveolar lavage fluid（BALF）.The morphology and proportion of cells derived from BALF were observed by Giemsa staining.The expression of IL-1β,IL-6,TNF-αand IL-10 in alveolar macrophages was detected by RT-PCR.（2）Western blotting detected the expression of CD63,HSP70 and TSG101 in exosomes from BALF,the morphology and structure of exosomes was observed by transmission electron microscopy（TEM）,and the size distribution range of exosomes was detected by nanoparticle tracking analysis（NTA）.（3）The total protein content of BALF-derived exosomes was detected by BCA assay,to evaluate the secretion level of exosomes.（4）The expressions of CD68,CD31（a vascular endothelial related marker）and alveolar epithelial related markers（SP-B,PDPN and Caveolin-1）in BALF-derived exosomes were detected by western blotting,to analyze the main source cells of exosomes from BALF.（5）Similarly,exosomes from the cell culture supernatant of macrophages were identified by western blotting,TEM and NTA,and exosome secretion levels of macrophages（with or without SiO₂stimulation）were analyzed by BCA assay.2.To explore whether macrophage-derived exosomes regulated pulmonary inflammation/fibrosis in silicosis.（1）PKH26 dye was used to label exosomes to observe the uptake of exosomes by macrophages.（2）Exosomes were co-cultured with M0 macrophages or monocytes.The expression of inflammatory factors（IL-1β,IL-6,TNF-αand i NOS）in monocytes or macrophages were detected by RT-PCR,enzyme linked immunosorbent assay（Elisa）,immunofluorescence and flow cytometry.（3）We used an inhibitor（GW4869）of exosome formation to block the secretion of exosome in vivo,the infiltration of inflammatory cells and fibrosis in the lung tissue were evaluated by H&E and Masson staining,and the secretion of inflammatory factors in BALF was detected by Elisa.3.To explore whether macrophage-derived exosomes regulate monocyte/macrophage migration and the mechanism.（1）Transwell assays analyzed the migration ability of monocytes/macrophages treated with exosomes.（2）RT-PCR and western blotting detected the expression of CC chemokine receptor 2（CCR2）in monocytes/macrophages treated with exosomes.4.To explore the mechanism of macrophage activation induced by macrophage-derived exosomes.（1）The activation of inflammatory related signaling pathways（JAK/STAT,NF-κB,MAPK and PI3K-AKT）in monocytes/macrophages treated with exosomes were detected by western blotting.（2）Western blotting and Elisa were used to detect the effects of STAT3 inhibitor（Stattic）and AKT inhibitor（MK2206）on exosome-induced signaling pathway activation and inflammatory cytokines（IL-1β,IL-6,and TNF-α）expression.5.To explore whether macrophage-derived exosomes mediate macrophage activation and recruitment through HMGB3.（1）Western blotting detected the expression of HMGB3 in SiO₂-stimulated macrophages and their secreted exosomes.（2）Western blotting detected HMGB3 expression in the nucleus and cytoplasm of macrophages under SiO₂ stress.（3）The expressions of CD68,α-SMA（a myofibroblast-related marker）and HMGB3 in lung tissues were detected by immunohistochemistry,and the expression and localization of HMGB3 in lung tissues were analyzed.（4）a neutral sphingomyelinase inhibitor was used to explore the regulatory pathway of HMGB3 packaging into exosomes.（5）Protease K intervention experiment was used to investigate the localization of HMGB3 in exosomes.（6）The role of HMGB3 in inflammatory activation and migration of monocytes or macrophages was evaluated by knockdown or overexpression of exosomal HMGB3.Elisa was used to determine the expression levels of IL-1β,IL-6 and TNF-α.The migration ability of monocytes or macrophages was detected by transwell assay,and the signal transduction involved was studied by western blotting.Results:1.Silica dust induced exosome secretion of macrophages.（1）Compared with the saline control group,the number of macrophages in lung tissue and BALF of silicosis mice was significantly increased.Alveolar macrophages in the early stage of silicosis were mainly inflammatory macrophages,and the expression of IL-1β,IL-6 and TNF-αwas up-regulated,while the expression of IL-10 shown no significantly difference.（2）The expression of CD63,HSP70 and TSG101 in BALF-derived exosomes of silicosis mice was abundant.Under TEM,exosomes were found to be round membranous vesicles with a"cup-shape"structure,with a diameter of 60-100 nm.The results of NTA showed that the size of exosomes was mainly distributed in the range of 30-150 nm.（3）Compared with the saline control group,the number of exosomes in BALF of silicosis mice was increased,and CD68 was highly expressed in these exosomes,while CD31,SP-B,PDPN and Caveolin-1 were low expressed,suggesting that exosomes in BALF of silicosis mice were mainly derived from alveolar macrophages.（4）Exosomes isolated from the cell culture supernatant of macrophages shown high expression of CD63,HSP70 and TSH101.Under TEM,exosomes were round membranous vesicles with a concave side,with a diameter of 60-100 nm.The results of NTA showed that the size of exosomes was mainly distributed in the range of 80-200 nm.（5）SiO₂-stimulated macrophages secreted more exosomes.2.Macrophage-derived exosomes regulated pulmonary inflammation/fibrosis in silicosis.（1）PKH26-labeled exosomes are taken up by macrophages.（2）SiO₂-stimulated macrophage-derived exosomes（SiO₂-Exos）up-regulated the expression of inflammatory factors（IL-1β,IL-6,TNF-αand i NOS）in M0 macrophages or monocytes.（3）The exosome secretion inhibitor GW4869 effectively alleviated the degree of inflammatory cell infiltration and fibrosis in the lung tissues of silicosis mice,and decreased the expression levels of IL-1β,IL-6 and TNF-αin BALF.3.SiO₂-Exos might promoted monocyte/macrophage migration by up-regulating CCR2 expression.（1）SiO₂-Exos significantly promoted monocyte/macrophage migration.（2）SiO₂-Exos up-regulated CCR2expression in monocytes/macrophages;After inhibition of exosome secretion,CCR2 expression was down-regulated.4.SiO₂-Exos-induced macrophage activation might be related to the activation of STAT3/MAPK/NF-κB signaling pathway.（1）SiO₂-Exos up-regulated the phosphorylation of STAT3,MAPK（ERK1/2 and P38）and NF-κB p65 in M0 macrophages and monocytes.（2）Stattic effectively inhibited the phosphorylation of STAT3 induced by SiO₂-Exos in monocytes/macrophages,accompany with the decrease of pro IL-1β.MK2206 effectively inhibited the level of p-AKT in monocytes/macrophages,accompany with the decrease of pro IL-1β.Stattic significantly inhibited the expression of IL-1β,IL-6 and TNF-αin the supernatant of RAW264.7 cells treated with exosomes.MK2206 also decreased the levels of IL-1βand IL-6 in the supernatant of RAW264.7cells treated with exosomes,while there was no significant difference in TNF-αexpression.5.Macrophage-derived exosomes mediate macrophage activation and recruitment through HMGB3.（1）The expression of HMGB3 was increased in SiO₂-stimulated macrophages and their secreted exosomes.（2）HMGB3 transferred from nucleus to cytoplasm under SiO₂ stress.（3）Compared with the saline control group,the expression of HMGB3 in the lung tissues of silicosis mice was increased.The up-regulated HMGB3 was mainly localized in macrophages.（4）The neutral sphingomyelinase inhibitor could partially decrease HMGB3 expression in exosomes.（5）Protease K could effectively digest exosomal HMGB3 protein.（6）In vitro,knockdown of exosomal HMGB3 decreased the expressions of IL-1β,IL-6 and TNF-αinduced by exosomes,reduced the migration of monocytes and macrophages,and down-regulated the levels of p-STAT3,p-MAPK（p-ERK1/2 and p-P38）,p-NF-κB p65 and CCR2.（7）In vivo,knockdown of exosomal HMGB3 could reduce macrophages infiltration in lung tissue and down-regulate the proportion of inflammatory macrophages.（8）Overexpression of exosomal HMGB3 could promote inflammatory activation and migration of monocytes or macrophages treated with exosomes,increase the transcription and secretion of IL-1β,IL-6 and TNF-α,and up-regulate the levels of p-STAT3,p-MAPK（p-ERK1/2 and p-P38）,p-NF-κB p65 and CCR2.Conclusion:1.SiO₂ stimulation enhanced exosome secretion of macrophages.2.The expression of HMGB3 was increased in SiO₂-stimulated macrophages and their secreted exosomes.3.Macrophage-derived exosomes mediated the activation and recruitment of monocytes/macrophages through HMGB3,which played pro-inflammatory and pro-fibrotic roles mainly by regulating the activation of STAT3/MAPK/NF-κB/CCR2 signaling pathway in silicosis.