Macrophage-derived exosomal HMGB3 regulates silica-induced pulmonary inflammation by promoting M1 macrophage polarization and recruitment

doi:10.21203/rs.3.rs-2882038/v1

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Research Article

Macrophage-derived exosomal HMGB3 regulates silica-induced pulmonary inflammation by promoting M1 macrophage polarization and recruitment

https://doi.org/10.21203/rs.3.rs-2882038/v1

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Journal Publication

published 07 Mar, 2024

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Background:

Chronic inflammation and fibrosis are characteristics of silicosis, and the inflammatory mediators involved in silicosis have not been fully elucidated. Recently, macrophage-derived exosomes have been reported as inflammatory modulators, but their role in silicosis remains largely unexplored. The purpose of the present study was to investigate the role of macrophage-derived exosomal HMGB3 in silicosis.

Methods:

First, HMGB3 expression in macrophages (with or without silica (SiO₂) exposure) and exosomes derived from these cells was measured by western blot analysis. Second, the role of exosomal HMGB3 in the inflammatory activation and migration of macrophages was evaluated by immunofluorescence, flow cytometry and transwell assays. The expression of IL-1β, IL-6 and TNF-α was detected by RT-PCR and ELISA, and the involved signal transduction pathways were studied by western blot analysis.

Results:

HMGB3 expression in SiO₂-exposed macrophages and exosomes derived from these cells was significantly upregulated. In silicosis mouse model, upregulated HMGB3 was mainly colocalized with infiltrating macrophages. In vitro experiments demonstrated that exosomes derived from SiO₂-exposed macrophages (SiO₂-Exos) significantly upregulated the expression of TNF-α, IL-6, IL-1β, iNOS and CCR2 in monocytes or M0 macrophages, promoting M1 polarization and migration of these cells. An in vivo study demonstrated that SiO₂-Exos promoted the infiltration of pulmonary macrophages and increased the proportion of iNOS⁺/F4/80⁺ macrophages. Knockdown of exosomal HMGB3 partially reversed this phenotype, while overexpression of exosomal HMGB3 promoted this phenotype. The proinflammatory effect of exosomal HMGB3 may be mediated through the activation of the STAT3/MAPK (ERK1/2 and P38)/NF-κB pathways.

Conclusions:

Exosomal HMGB3 is a potential inflammatory modulator in silicosis that induces inflammatory activation and recruitment of monocytes/macrophages by regulating the activation of the STAT3/MAPK/NF-κB/CCR2 pathways.

HMGB3

Silicosis

Macrophage polarization

Inflammation

Silicosis is an irreversible and fatal lung disease that is caused by long-term inhalation of silica dust (1). Silicosis used to be an occupational disease among miners. With the development of science and technology, protection and measurement standards have improved, and the morbidity of silicosis has been decreasing. With the lack of attention to dust exposure during processing in some emerging industries, such as denim sandblasting processing, building stone processing and jewelry polishing, silicosis has resurfaced globally (1–3). The National Health Commission of China reported that, in 2021, there are 15,407 new cases of occupational diseases nationwide, including 111,809 cases of occupational pneumoconiosis(4). There are limited clinically available treatments for silicosis, including whole lung lavage, pirfenidone and nidanib (1, 2, 5). These measures may improve clinical symptoms but do not reverse pulmonary fibrosis and prolong survival. Therefore, further exploration of the complex mechanism of silicosis is needed to elucidate a potential therapeutic strategy to mitigate its progression and reduce mortality.

Silicosis is characterized by chronic inflammation and fibrosis (5). Macrophages are the main effector cells of chronic inflammation in silicosis (2, 6, 7). The first critical step of host immune defense is the recognition and internalization of inhaled SiO₂ dust by alveolar macrophages, which activates the autophagic lysosome system and inflammasome within macrophages, triggering pulmonary inflammation and resulting in the release of multiple inflammatory mediators, including interleukin-1 beta (IL-1β), IL-6, reactive oxygen species (ROS), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor-α (TNF-α) (8–13). These inflammatory factors further activate the inflammatory response, causing activation, proliferation and recruitment of various cells, including neutrophils, monocytes and macrophages (7, 11).

Exosomes are membranous vesicles with a diameter of 30–150 nm, which are important mediators of local and distant cell-to-cell communication (14, 15). Exosomes are involved in various diseases by carrying specific molecules or genetic information (15–18). Increasing proofs have revealed the essential role of exosomes in inflammation (14, 19–22). IL-1β and TNF-α are not only released by cells but are also secreted by exosomes (23, 24). The bronchoalveolar lavage fluid (BALF) of asthmatic patients contains exosomes, in which the expression of enzymes for leukotriene (LT) biosynthesis is higher than that of healthy individuals. LTs are potent proinflammatory factors involved in asthma (25). The extracellular cold-inducible RNA-binding protein levels increase in exosomes from sepsis mouse serum and LPS-treated macrophages, which induce TNF-α and IL-6 production and neutrophil migration (26). Therefore, exosomes may be involved in the dysregulation of inflammation in silicosis, while the potential role of macrophage-derived exosomes in silicosis inflammation remains largely unexplored.

High mobility group box 3 (HMGB3) is a nonhistone nucleoprotein belonging to the high mobility group box (HMGB) family. HMGBs usually bind to nucleosomes and nucleosome complexes in a sequence-independent manner, and they are involved in DNA repair, replication, transcription and recombination (27, 28). To date, studies related to HMGB3 have mainly focused on tumor proliferation, metastasis and drug resistance (29–31), while there are several studies related to inflammation and immunity. For example, HMGB3 acts as a plant damage-associated molecular pattern (DAMP) to activate inflammatory responses in Arabidopsis (32). HMGB3 acts as an activator of NF-κB to promote ROS production in sea cucumber (33). HMGB3 expression is upregulated in inflammatory damaged intestinal epithelial cells, and exosomal miR-200b derived from bone marrow mesenchymal stem cells attenuates inflammatory damage by targeting HMGB3 (34). HMGB3 expression is upregulated in sepsis-induced acute lung injury rats, and inhibiting HMGB3 expression alleviates sepsis-mediated apoptosis of lung epithelial cells and inflammation (35). A previous report has revealed that HMGB3 expression is increased in RAW264.7 macrophage-derived exosomes after silica exposure (36). Hence, we hypothesize that exosomal HMGB3 derived from macrophage may play an essential role in silicosis inflammation.

The present study revealed that HMGB3 is secreted in the form of exosome, which is contributed to silicosis inflammation by promoting M1 polarization and recruitment of monocytes/macrophages. Therefore, identification of exosomal HMGB3 as an important inflammatory mediator may provide a new entry point to attenuate inflammation in the early stage of silicosis.

2.1 Cell lines and cell culture

Mouse leukemic monocyte/macrophage cell line (RAW264.7) and human monocytic cell line (THP-1) were obtained from the National Collection of Authenticated Cell Cultures (Shanghai, China). RAW264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, Grand Island, NY, USA), with the supply of 10% fetal bovine serum (FBS, Gibco). THP-1 cells were cultured in RPMI 1640 medium (Gibco), with the supply of 10% FBS. THP-1 monocytes were first stimulated with 100 ng/ml phorbol-12-myristate-13-acetate (PMA, Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) for 24 hours to differentiate into macrophages, and these cells were subsequently exposed to silica (SiO₂, 12.5 µg/mm², S5631, 1–5 µm, Sigma-Aldrich) for 36h, and the cell culture supernatant (SN) was harvested for exosome isolation. Similarly, the SN of RAW264.7 cells was collected for exosome isolation after 36 hours stimulation of SiO₂ (25 µg/mm²). The untreated THP-1 cells and RAW264.7 cells using as M0 phenotype.

2.2 Exosome isolation, identification and treatment

Differential centrifugation (Optimal^™ L-80 XP, Beckman Coulter, USA) was used to isolate exosomes from the BALF or the SN of the cultured cells. Exosome isolation was performed as before (37). The total protein content of exosomes was used to evaluate the quantity of exosomes, which is measured by micro-BCA assay (Sigma-Aldrich). To validate the exosomes, the expression of extracellular vesicle-related protein markers in the purified exosomes were detected by western blot analysis, including TSG101 (tumor susceptibility gene 101, 14497-1-AP, 1:1 000, Proteintech, Wuhan, China), HSP70 (heat shock protein 70, ab2787, 1:1 000, Abcam, Cambridge, UK) and CD63 (ab134045, 1:1000, Abcam). A transmission electron microscopy (TEM, FEI, Massachusetts, USA) was used to observe the morphology of exosomes, and nanoparticle tracking analysis (NTA, Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK) was used to analyze the size distribution range of exosomes. For cell treatment, 50 µg of exosomes was used to treat 1 × 10⁵ Raw264.7 cells or 1 × 10⁶ THP-1 cells. To evaluate the activation of signaling pathways, a STAT3 inhibitor (Stattic, 5 µM, Abcam, USA) and an AKT inhibitor (MK2206, 10 nM, Beyotime, Shanghai, China) were used to inhibit protein phosphorylation. Proteinase K (Sigma-Aldrich) was used to verify the location of exosomal HMGB3. The purified exosomes were diluted in 1 ml of PBS and divided evenly into 2 parts, one of which was incubated with 100 µg/ml protease K at 37°C for 10 min to digest exosome surface proteins without penetrating the membranous lipid bilayer. After termination of digestion, exosomes were extracted by centrifugation at 4°C at 110,000 × g for 2 hours.

2.3 Trafficking analysis of exosomes in vitro and vivo study

To dynamically trace exosomes, a PKH26 fluorescent kit (Sigma-Aldrich) was used to label the exosomes as described previously(37). In the in vivo study, PKH26-labeled exosomes were resuspended in 100 µl of sterile PBS and administered to C57BL/6 mice by tail intravenous injection. After 20 hours, the distribution of exosomes was detected by in vivo Xtreme II (BRUKER; Munich, Germany).

2.4 Exosome secretion inhibition assay

The neutral sphingomyelinase inhibitor (GW4869, 10 µM, Cayman Chemical, Michigan, USA) was used to block exosome secretion. Before SiO₂ exposure, THP-1 cells and RAW264.7 cells were pre-treated with GW4869 for 24 hours. These cells were then treated with SiO₂ + GW4869 (10 µM) for 36 hours and the SN was collected.

2.5 Western blot analysis

The primary antibodies used for western blot analysis are list below: anti-CD68 (28058-1-AP, 1:1000, Proteintech, Wuhan, China), anti-CD31 (28083-1-AP, 1:1000, Proteintech), anti-SP-B (sc-133143, 1:1000, Santa Cruz Biotechnology, Texas, USA), anti-podoplanin (PDPN, sc-53533, 1:1000, Santa Cruz Biotechnology), anti-caveolin-1 (sc-53564, 1:1000, Santa Cruz Biotechnology), anti-p-STAT1 (Tyr701) (340797, 1:1000, Zenbio, Chengdu, China), anti-p-STAT3 (AP0247, 1:1000, Bioworld, Nanjing, China), anti-p-AKT (ab81283, 1:500, Abcam, Cambridge, UK), anti-p-NF-κB p65 (310013, 1:1000, Zenbio), anti-p-ERK1/2 (AF1015, 1:1000, Affinity Biosciences, Jiangsu, China), anti-p-P38 MAPK (ab4822, 1:1000, Abcam), anti-STAT1 (ab4822, 1:1000, Proteintech), anti-STAT3 (10253-2-AP, 1:1000, Proteintech), anti-AKT (10176-2-AP, 1:1000, Proteintech), anti-NF-κB p65 (R25149, 1:1000, Zenbio), anti-ERK1/2 (BF8004, 1:1000, Affinity Biosciences), anti-P38 MAPK (R25239, 1:1000, Zenbio), anti-IL-1β (ab283818, 1:1000, Abcam), anti-HMGB1 (R22773, 1:1000, Zenbio), anti-HMGB2 (R26860, 1:1000, Zenbio), anti-HMGB3 (D160490, 1:1000, Sangon Biotech, Shanghai, China), anti-beta-actin (β-actin, 20536-1-AP, 1:1000, Proteintech), anti-Histone-H3 (17168-1-AP, 1:1000, Proteintech) and anti-glyceraldehyde3-phosphate dehydrogenase (GAPDH, 60004-1-AP, 1:1000, Proteintech). A chemiluminescent system (ChemiDocTM XRS+, BIO-RAD, USA) was used for detection.

2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis of mRNA

The total RNA extraction, reverse transcription and RT-PCR were performed as before (37). Using β-actin or GAPDH as reference. The primer sequences are listed in table S1.

2.7 Animal model

A 28-day silicosis mouse model was constructed by one-dose intratracheal injection of silica suspension (100 mg/kg bodyweight) as described previously(37), and the BALF and lungs were harvested for further study. For administration of exosomes, exosomes (7.5 µg/g of bodyweight) were first suspended in 50 µl of PBS and administered to each animal by intratracheal injection. Then exosomes (10 µg/g of bodyweight per two days) were subsequently administered to the mice by tail intravenous injection until they were sacrificed at Day 9, and the lungs were collected for flow cytometry analysis. The same volume of PBS was administered to the control group. Exosomes for animal treatment were derived from RAW264.7 macrophages. We transfected RAW264.7 macrophages with siRNA and isolated exosomes from the cell culture supernatant, resulting in exosomes derived from siNC-transfected SiO₂-exposed RAW264.7 macrophages (SiO₂ + siNC-Exos) and exosomes derived from siHMGB3-transfected SiO₂-exposed RAW264.7 macrophages (SiO₂ + siHMGB3-Exos). The animal protocols were in accordance with the requirements of related regulations and procedures of the National Institutes of Health Guide for the Care and Use of Laboratory Animals as well as ethical principles.

2.8 Immunohistochemical staining

The anti-CD68 (28058-1-AP, 1:200, Proteintech), anti-HMGB3 (D160490, 1:100, Sangon Biotech) and anti-α-SMA (14395-1-AP, 1:200, Proteintech) antibodies were used to perform immunohistochemical staining.

2.9 Immunofluorescence

After fixation and permeabilization with 4% paraformaldehyde and 0.2% Triton X-100, the adherent cells and smears were blocked with 3% BSA-PBS, and subsequently labeled with anti-CD68 (1:100, 12-0689-42, Invitrogen, Thermo Fisher Scientific, Massachusetts, USA) and anti-iNOS (1:100, 53-5920-82, Invitrogen) antibodies at 4°C overnight. DAPI was used to stain nuclei. The fluorescence was observed with an inverted fluorescence microscope (Olympus, Tokyo, Japan).

2.10 Flow cytometry

To perform flow cytometry analysis, THP-1 cells were first stimulated with 100 ng/ml PMA for 24 hours and then cocultured with exosomes for 36 hours. RAW264.7 cells were surface stained with anti-F4/80 (0.2 µg, 12-4801-82, Invitrogen) antibodies at 4°C for 1 hour and then fixed with ice-cold methyl alcohol for 30 min. these cells were subsequently labeled with an anti-iNOS (0.125 µg, 53-5920-82, Invitrogen) antibody at 4°C for 2 hours. THP-1 cells were stained with anti-iNOS (0.25 µg, 53-5920-82, Invitrogen) and anti-CD68 (0.5 µg, 12-0689-42, Invitrogen) antibodies at 4°C for 2 hours after cell fixation and permeabilization. The cells were resuspended in 500 µl of PBS and detected by BD FACSAria™ Fusion (Becton, Dickinson and Company, USA). For lung tissue, cardiopulmonary lavage was performed with PBS containing 0.5 M EDTA to remove residual blood from the lung tissue, and the tissue was then digested with 2 mg/ml collagenase at 37°C for 30 min with mixing (125 rpm/min). After adding serum to terminate digestion, cells were filtered using a nylon strainer with an aperture of 70 µm and then centrifuged at 500 x g for 5 min. Using 40% and 80% Percoll to isolate monocytes, then cells were surface stained with CD11b (0.2 µg, 56-0112-82, Invitrogen) and F4/80 (0.2 µg, 12-4801-82, Invitrogen) at 4°C for 1 hour. After washing, an intracellular fixation and permeabilization buffer set (88-8824-00, Invitrogen) was used to fix and permeabilize cells according to the instructions. Cells were then incubated with iNOS (0.25 µg, 53-5920-82, Invitrogen) and CD206 (0.25 µg, 17-2061-80, Invitrogen) antibodies at 4°C for 2 h. After washing, 500 µl of PBS was used to resuspend the cells, and flow cytometry analysis was performed.

2.11 Enzyme-linked immunosorbent assay (ELISA)

ELISA was used to analyze the expression levels of murine IL-1β, IL-6 and TNF-α in the SN or BALF according to the instruction. These ELISA kits were obtained from proteintech.

2.12 Cell migration

THP-1 cells and RAW264.7 cells were centrifuged and resuspended in FBS-free conditioned medium. A Transwell chamber (3422, Corning Costar, USA) with an aperture size of 8 µm was utilized. In brief, 150 µl of cell suspension (1–2 x 10⁵ cells) was added to the upper chamber, and 700 µl of conditioned medium with 10% FBS (with or without 150 µg of exosomes) was added to the lower chamber. Cystal violet staining (C0121, Beyotime, Shanghai, China) was used for observation ater 24 hours, and ImageJ software was used to analyze the results.

2.13 Plasmid construction and transfection

Murine HMGB3 cDNA were cloned into the pcDNA3.1(+) vector at the Pme I and Not I sites. A total of 1.5 µg plasmid with 3 µl of Lipofectamine 2000 (Invitrogen, Thermo Fisher Scientific, Massachusetts, USA) was mixed in 100 µl of OPTI-MEM (Gibco), and the mixture was used to transfect cells (1.5 µg of plasmid per 10⁵ cells) for 24–36 hours.

2.14 RNA interference

Three siRNA against the mouse HMGB3 and their negative controls were constructed and generated by RiboBio (Guangzhou, China). The siRNA sequences were listed below. si-HMGB3#1, CATGCAGGGAAGAACATAA; si-HMGB3#2, GGCAGATAAAGTCCGATAT; and si-HMGB3#3, AGCAGCCTTATGTCACCAA. siRNA (100 nM) was mixed with the transfection reagent (RiboBio; Guangzhou, China), and the mixture was used to transfect cells for 24–36 hours according to the instructions. The short hairpin RNAs (shRNAs) against the human HMGB3 and their negative controls were generated by RiboBio (Guangzhou, China). The following shRNA sequences were used: shHMGB3#1: gatcccAAGGAAAGTTTGATGGTGCAActcgagTTGCACCATCAAACTTTCCTTtttttggat, shHMGB3#2: gatcccGGCTCCATCATGATCTTCGACGATActcgagCCGAGGTAGTACTAGAAGCTGCTATtttttggat, and shHMGB3#3: gatcccGCAGATAAAGTGCGCTATGATctcgagCGTCTATTTCACGCGATACTAtttttggat.

2.15 Statistics

Using GraphPad Prism software (La Jolla, CA, USA) to analyze data. All the results are expressed as the mean ± SEM. For numerical data, Student's t test (unpaired, two-tailed) was used for comparison between two groups, and two-way ANOVA followed by Tukey's multiple comparisons test was used for multiple comparisons. Statistical significance was set as P < 0.05.

The quantity of exosomes secreted by macrophages is significantly increased in silicosis mice

Many researches have shown that macrophages are important effective cells in the progression of silicosis, and the infiltration and aggregation of macrophages in lung tissue are the main characteristics of the early stage of silicosis (5, 9, 38). To verify the distribution of macrophages, a 28-day silicosis mouse model was constructed, and immunohistochemical staining was used to detect CD68 expression (a macrophage marker) in lung tissue. Compared with control mice, CD68 expression was markedly upregulated in the lung tissue of silicosis mice (Fig. 1A). At the same time, cells in the BALF were collected for counting and Giemsa staining, and the number and proportion of alveolar macrophages (AMs) were calculated, which suggested that the number and proportion of AMs in the BALF of silicosis mice were significantly increased (Fig. 1B-C). RT-PCR analysis demonstrated that inflammatory cytokines (IL-1β, IL-6 and TNF-α) in AMs were upregulated, but IL-10 expression was not significantly different (Fig. 1D), which indicated that AMs in the early stage of silicosis were mainly inflammatory macrophages. Exosomes are important mediators of intercellular communication. Previous studies have reported that exosomes are involved in inflammation by transporting inflammatory factors, such as TNF-α and HMGB1 (19–21). Our previous results indicate that inhibition of exosome secretion with the neutral sphingomyelinase inhibitor, GW4869, reduces inflammatory cells infiltration in the lung tissue and the levels of IL-1β, IL-6 and TNF-α in the BALF of silicosis mice (37). Therefore, we next explore the potential role of macrophage-derived exosomes in silicosis inflammation. We collected the BALF and extracted exosomes from the fluid by differential centrifugation. Western blot analysis indicated that exosome markers (HSP70, TSG101 and CD63) were highly expressed in the extracted exosomes (Fig. 1E). We use a transmission electron microscopy (TEM) to observe the morphology of exosomes, and found that the exosomes were membrane-like structures showing a "cup shape" (Fig. 1F). Through nanoparticle tracking analysis (NTA), the purified exosomes were mainly distributed between 30 and 150 nm in size, with a peak value of 83.9 nm (Fig. 1G). The total protein content of exosomes was detected by BCA assay, which was used to evaluate the quantity of exosomes. Compared with control mice, exosome secretion in the BALF of silicosis mice was significantly increased (Fig. 1H). The expression of the macrophage marker (CD68), the vascular endothelial cell marker (CD31) and the alveolar epithelial cell markers (PDPN, SP-B and caveolin-1) in exosomes was detected. The results indicated that the secreted exosomes in the BALF of silicosis mice were mainly derived from macrophages (Fig. 1I). However, in the saline-treated control group, exosomes in the BALF were mainly derived from alveolar epithelial cells (Fig. 1I). These results showed that the infiltration of macrophages and exosome secretion of macrophages were significantly increased in silicosis.

The quantity of exosomes secreted by SiO₂-exposed macrophages increased significantly in vitro

Next, we investigated the secretion of exosome from SiO₂-exposed macrophages in vitro. Macrophages were exposed to SiO₂ for 36h, and the SN was harvested. Then exosomes in the SN were extracted by differential centrifugation. Under TEM, exosomes showed a membrane-like structure, with a diameter of 60-100nm (Fig. 2A-B). Western blotting results showed that the extracted exosomes highly expressed TSG101, HSP70 and CD63 (Fig. 1C). NTA assay showed that the particle sizes of purified exosomes were mainly distributed in the range of 80-200nm, with peaks of 152.5nm and 149.4nm (Fig. 2D). The total protein concentration of exosomes was determined by BCA assay, which indicated that the quantity of exosomes secreted by SiO₂-exposed macrophages was significantly increased (Fig. 2E).

SiO₂-Exos promote the polarization of M0 macrophage to M1 macrophage in vitro

We next investigated the potential role of SiO₂-exposed macrophage-derived exosomes (SiO₂-Exos) in inflammation. First, we explored whether exosomes could be taken up by macrophages. PKH26 dye was used to label the exosomes derived from SiO₂-exposed macrophages, and these exosomes were cocultured with M0 macrophages for 12 hours. We used a fluorescence microscope to observe the uptake of exosomes, and found that red fluorescence was visible in the membrane and cytoplasm of macrophages, which indicated that exosomes were taken up by macrophages (Fig. 3A). To explore whether SiO₂-Exos regulate inflammatory activation of macrophages, we treated macrophages with different treatments and isolated exosomes from the SN, resulting in exosomes derived from macrophages without SiO₂ exposure (NC-Exos), exosomes derived from SiO₂-exposed macrophages (SiO₂-Exos) and exosomes derived from SiO₂-exposed macrophages treated with GW4869 (10 nM) (SiO2 + GW4869-Exos). Because exosome formation is regulated by the neutral sphingomyelinase pathway (39), we utilized the neutral sphingomyelinase inhibitor, GW4869, to block exosome secretion. GW4869 effectively inhibited the secretion of exosomes in SiO₂-exposed macrophages (Figure S1). These exosomes were cocultured with M0 RAW264.7 macrophages or THP-1 monocytes for 36 hours, and changes in cell morphology were observed under a microscope. In the SiO₂-Exos-treated group, THP-1 cells changed from a suspension state to an adherent state, and a large number of THP-1 cells and RAW264.7 cells increased in volume and had polygonal morphology accompanied by filamentous pseudopods (Fig. 3B), which was consistent with the M1 macrophage activation state. However, in the NC-Exos-treated group and SiO₂ + GW4869-Exos-treated group, the number and proportion of macrophages with polygonal morphology and filamentous pseudopodia were significantly decreased (Fig. 3B). RT-PCR analysis indicated that SiO₂-Exos markedly upregulated the expression of IL-1β, IL-6 and TNF-α, whereas IL-10 expression was slightly upregulated. In contrast, when the secretion of exosomes is blocked by GW4869, the expression of IL-1β, IL-6 and TNF-α was significantly downregulated, while IL-10 expression showed no significant differences (Fig. 3C-D). ELISA analysis showed consistent results (Fig. 3E). Immunofluorescence analysis suggested that SiO₂-Exos promoted the differentiation of monocytes into macrophages in THP-1 cells as well as up regulated CD68 expression, but CD68 expression was downregulated in the SiO2 + GW4869-Exos-treated group (Fig. 4A). SiO₂-Exos also up regulated iNOS expression in THP-1 cells and RAW264.7 cells while iNOS expression was decreased in the SiO2 + GW4869-Exos-treated group (Fig. 4A-B). Flow cytometry analysis showed that the iNOS⁺/CD68⁺ cells in THP-1 macrophages had the highest proportion (53.8%) (Fig. 4C) and that the iNOS⁺/F4/80⁺ cells in RAW264.7 macrophages showed the highest proportion (93.1%) in the SiO₂-Exos treated group (Fig. 4D). In contrast, the proportion of iNOS⁺/CD68⁺ cells and iNOS⁺/F4/80⁺ cells decreased in macrophages treated with SiO₂ + GW4869-Exos with proportions of 43.2% in THP-1 cells and 76.8% in RAW264.7 cells, respectively (Fig. 4C-D). These results suggested that SiO₂-Exos play a proinflammatory role in silicosis.

SiO₂-Exos promote macrophage migration by upregulating CCR2 expression in vitro

The formation of fibrotic silicotic nodules in lung tissue is the main characteristic of silicosis. Immature silicotic nodules consist of a mass of dust-laden macrophages, fibroblasts and collagen fibers (40). Figure 1A shows that a lot of macrophages infiltrated in alveolar spaces and immature silicotic nodules in the lung tissue of the early stage of silicosis (Fig. 1A). Many studies have revealed that after SiO₂ exposure, a large number of circulating monocytes migrate into lung tissue to participate in the inflammatory response (7, 41). Therefore, we continued to explore whether SiO₂-Exos regulate monocyte or macrophage migration and recruitment. Transwell assays showed that SiO₂-Exos significantly promoted the migration of THP-1 cells and RAW264.7 cells, while the number of migrating cells in the SiO₂ + GW4869-Exos-treated group was decreased (Fig. 4E-F). Recruitment of monocytes into inflammatory tissue is regulated by chemokines. Monocyte chemokines are the most critical chemokines that attract cells by activating their homologous receptors, including CC chemokine receptor 2 (CCR2) (42, 43). CCR2-deficient mice have significantly reduced recruitment of monocytes in peritonitis, autoimmune encephalitis and atherosclerosis (44). It is reported that CCR2 is required for the migration of Ly6C^hi monocytes from the bone marrow to the infectious site. Circulating Ly6C^hi monocytes are also known as inflammatory monocytes (43, 45–48). Therefore, we next evaluated the expression of CCR2 in SiO₂-Exos-treated monocytes/macrophages. RT-PCR analysis showed that SiO₂-Exos up regulated CCR2 expression in THP-1 cells and RAW264.7 cells, while CCR2 expression was decreased in the SiO2 + GW4869-Exos-treated group (Fig. 4G-H). Western blot analysis showed that CCR2 in THP-1 cells and RAW264.7 cells had the highest expression in the SiO₂-Exos-treated group, while GW4869 reversed this effect (Fig. 4I-J). These results indicated that SiO₂-Exos recruit monocytes or macrophages via the upregulation of CCR2.

SiO ₂ -Exos promote inflammatory activation of monocytes/macrophages mainly by regulating the activation of the STAT3/MAPK/NF-κB signaling pathways

To further clarify the mechanism by which exosomes regulate the inflammatory response in monocytes/macrophages, we examined the activation of signaling pathways related to the inflammatory response, such as nuclear factor kappa-B (NF-κB), signal transducers and activators of transcription (STAT), mitogen-activated protein kinase (MAPK, including extracellular signal-regulated kinase 1/2 (ERK 1/2) and p38 MAPK), and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT) (PI3K/AKT) signaling pathways (49–58). The results suggested that SiO₂-Exos markedly up regulated the phosphorylation levels of p65 (NF-κB), STAT1, STAT3, AKT, ERK1/2 and P38 in RAW264.7 cells; these changes were accompanied by an increase in pro IL-1β. On the contrary, when the exosome secretion was blocked by GW4869, the phosphorylation levels of p65, STAT1, STAT3, ERK1/2 and P38 was decreased, while p-AKT expression showed no significant differences; these changes were accompanied by a decrease in pro IL-1β (Fig. 5A-B). Different from RAW264.7 cells, SiO₂-Exos selectively up regulated the expression level of CD68, and the phosphorylation levels of p65, STAT3, AKT and P38 in THP-1 cells, while the phosphorylation levels of STAT1 and ERK1/2 showed no significant differences; these changes were also accompanied by an increase in pro IL-1β. And the phosphorylation levels of p65, STAT3, ERK1/2 and P38 was downregulated in the SiO₂ + GW4869-Exos-treated group, while the expression of p-STAT1 and p-AKT showed no significant differences; these changes were accompanied by a decrease in pro IL-1β (Fig. 5C-D). These results indicated that SiO₂-Exos promote inflammatory activation of monocytes/macrophages mainly by inducing the activation of the STAT3/MAPK (ERK1/2 and P38)/NF-κB signaling pathways. The signaling cascade mediated by SiO₂-Exos in mouse leukemic monocyte/macrophages (RAW264.7 cells) and human monocytes (THP-1 cells) showed partial differences. STAT3 and Akt play a dual role in inflammation. Accumulating evidence suggests that the IL-6/STAT3 signaling pathway is activated under LPS stimulation, which is critical for the transcription of inflammatory cytokines (53, 59). Akt is the upstream signaling molecule that activates the NF-κB signaling pathway (57, 58). Moreover, the activation of STAT3 and PI3K/AKT also promotes M2 macrophage polarization, which decreases the inflammatory response (60–63). Therefore, to further verify the contribution of these two signaling pathways to the process of macrophage activation induced by SiO₂-Exos, we treated THP-1 cells and RAW264.7 cells with SiO₂-Exos in the presence of a STAT3 inhibitor (Stattic) or AKT inhibitor (MK2206), and analyzed the activation of the related signaling pathways. The addition of Stattic significantly inhibited the phosphorylation of STAT3 and downregulated pro-IL-1β expression. When MK2206 was added, the expression of p-AKT and pro IL-1β was decreased (Fig. 5E-H). ELISA analysis showed that Stattic significantly attenuated SiO₂-Exos-induced release of TNF-α, IL-6 and IL-1β. MK2206 also decreased the secretion levels of IL-6 and IL-1β, but TNF-α secretion showed no significant differences (Fig. 5I). Of note, the STAT3 inhibitor had a stronger inhibitory effect. These results suggested that p-STAT3 and p-AKT may promote the transcription of inflammatory cytokines during SiO₂-Exos-induced inflammatory activation of monocytes/macrophages.

HMGB3 expression is upregulated in SiO₂-Exos, which may be localized on the exosome membrane

As HMGB3 belongs to the HMGB family (HMGBs), it has high homology with HMGB1 and HMGB2 (27, 64). Approximately 80% of the amino acid sequences of the proteins encoded by HMGB1, HMGB2 and HMGB3 are identical, and they also have a similar structure, indicating that they may have similar functions (65). HMGB1 is a DAMP widely involved in inflammatory responses (64, 66). A previous report has revealed that HMGB3 expression is upregulated in exosomes derived from SiO₂-exposed RAW264.7 cells (36). Therefore, we subsequently explored whether HMGB3 is involved in the inflammatory activation and recruitment of monocytes/macrophages regulated by SiO₂-Exos. Firstly, we detected HMGB3 expression in exosomes derived from THP-1 cells or RAW264.7 cells (with or without SiO₂ exposure) through western blot analysis, indicating that HMGB3 expression in SiO₂-Exos was higher than that in NC-Exos (Fig. 6A-B). We continued to measure the expression of HMGB1 and HMGB2 in macrophage-derived exosomes. HMGB1 was expressed at low levels in exosomes from RAW264.7 cells with or without SiO₂ exposure, and HMGB2 showed no significant difference between NC-Exos and SiO₂-Exos (Figure S2A). Both HMGB1 and HMGB2 were underexpressed in THP-1 cell-derived exosomes (Figure S2B). We next explored the expression and distribution of HMGB3 in SiO₂-exposed macrophages after treating macrophages with SiO₂ for different time points. The results suggested that HMGB3 expression increased with time and decreased after reaching a peak at 24 hours in RAW264.7 cells (Fig. 6C-D), while HMGB3 expression first increased (within 30 min) and then slowly decreased with time in THP-1 cells (Fig. 6E-F). We also measured the expression of HMGB1 and HMGB2 in SiO₂-exposed macrophages. After SiO₂ exposure, HMGB1 expression in RAW264.7 cells immediately increased and then decreased with time, and HMGB2 expression increased from 6 hours and then decreased after reaching a peak at 12 hours (Figure S2C). In THP-1 cells, HMGB1 expression showed no significant difference after SiO₂ exposure, while HMGB2 expression first slightly increased and then decreased with time (Figure S2D). HMGBs are rapidly transferred from the nucleus to the cytoplasm under stress or elevated ROS production (28, 67). Many reports have revealed that ROS are upregulated in macrophages after phagocytosis of silica particles (12, 13). Therefore, we next extracted cytoplasmic proteins and nuclear proteins from SiO₂-exposed macrophages to detect HMGB3 expression. The results suggested that HMGB3 expression was markedly increased in the cytoplasm after SiO₂ exposure, while there was no significant difference in the nucleus (Fig. 6G-H). These results indicated that HMGB3 expression is increased in macrophages and that HMGB3 is rapidly transferred from the nucleus to the cytoplasm under SiO₂ stress, and HMGB3 in the cytoplasm is then packaged into exosomes and secreted into the extracellular environment. We next investigated the expression and localization of HMGB3 in vivo. Western blot analysis showed that the expression of collagen type I and HMGB3 in the lung tissues was markedly increased in silicosis mice (Fig. 6I). Immunohistochemical staining suggested that HMGB3 was expressed at low levels in normal mice but was significantly upregulated in silicosis mice (Fig. 6J), and it was mainly located in the infiltrating area of macrophages (Fig. 6K) rather than in myofibroblasts (Fig. 6L). Moreover, HMGB3 was distributed in both the nucleus and cytoplasm of macrophages (Fig. 6J-K). Similarly, HMGB3 expression was upregulated in alveolar macrophages of silicosis mice (Fig. 6M). In a word, these findings revealed that HMGB3 is transferred from the nucleus to the cytoplasm under SiO₂ stress, which suggests that HMGB3, as a nucleoprotein, is packaged into exosomes. Because exosomes are vesicles with a lipid bilayer membrane structure, the biomolecules carried by exosomes may be localized on or encapsulated in the exosome membrane (68, 69). Previous studies have reported that HMGB1 and HMGB3 can be packaged into exosomes (36, 70, 71) and that HMGB1 can be localized on cell surface membranes involved in cell differentiation, adhesion, innate immunity and inflammation through a different mechanism (64). Therefore, protease K was used in the present study to treat SiO₂-Exos to verify the localization of HMGB3 in exosomes. Western blot analysis showed that protease K effectively digested HMGB3 protein and only partially affected the expression of HSP70 and β-actin, which were located in the exosome membrane (Fig. 6N). These results suggested that HMGB3 may be localized on the exosome membrane, but its specific localization has not yet been explored.

HMGB3 deficiency attenuates SiO₂-Exos-induced inflammatory activation and recruitment of monocytes/macrophages in vitro and in vivo

To investigate whether SiO₂-exposed macrophage-derived exosomal HMGB3 regulates inflammatory activation and recruitment of monocytes/macrophages, we constructed three siRNAs or shRNA to knock down HMGB3. The knockdown efficiency of siRNA or shRNA in THP-1 cells or RAW264.7 cells was assessed through western blot analysis and RT-PCR (Fig. 7A-D), which indicated that siHMGB3#1 and shHMGB3#1 had the most gene-silencing effect and were selected for the subsequent experiments. We then transfected macrophages with siRNA or shRNA and isolated exosomes from the SN, resulting in exosomes derived from siNC-transfected or shNC-transfected SiO₂-exposed macrophages (SiO₂ + siNC-Exos or SiO₂ + shNC-Exos) and exosomes derived from siHMGB3-transfected or shHMGB3-transfected SiO₂-exposed macrophages (SiO₂ + siHMGB3-Exos or SiO₂ + shHMGB3-Exos). HMGB3 expression in exosomes was measured through western blot analysis, which demonstrated that HMGB3 expression in SiO₂ + siHMGB3-Exos and SiO₂ + shHMGB3-Exos was lower than that in SiO₂ + siNC-Exos and SiO₂ + shNC-Exos (Fig. 7E-F). We next evaluated the role of HMGB3 in the SiO₂-Exos-induced inflammatory response by treating THP-1 cells or RAW264.7 cells with these exosomes and measuring the release of inflammatory cytokines and cell migration with ELISA and transwell assays, respectively. ELISA analysis showed a marked increase of the expression of IL-1β, IL-6 and TNF-α in the SiO₂ + siNC-Exos-treated group compare with the control group, while knockdown of exosomal HMGB3 partially reversed this effect (Fig. 7G), which suggested that exosomal HMGB3 regulated the inflammatory activation of monocytes/macrophages. Transwell assays showed that SiO₂ + NC-Exos significantly promoted the migration of THP-1 cells and RAW264.7 cells, and the migration ability of these cells decreased after knocking down exosomal HMGB3 (Fig. 7H-I), which suggested that exosomal HMGB3 regulated monocyte/macrophage migration. These exosomes were then cocultured with THP-1 cells or RAW264.7 cells, and the activation of signaling pathways was measured through western blot analysis. Coculture with SiO₂ + siNC-Exos up regulated the expression of p-p65, p-STAT3, p-ERK1/2, p-P38, CCR2 and pro IL-1β in RAW264.7 macrophages. When exosomal HMGB3 was knocked down, the expression of p-p65, p-STAT3, p-ERK1/2, p-P38, CCR2 and pro IL-1β was decreased (Fig. 7J-K). In THP-1 monocytes, the expression of p-p65, p-STAT3, p-P38, CCR2 and pro IL-1β was significantly upregulated by SiO₂ + shNC-Exos, while p-ERK1/2 expression was downregulated. When exosomal HMGB3 was knocked down, the expression of p-p65, p-STAT3, p-P38, CCR2 and pro IL-1β was downregulated (Fig. 7L-M). We next investigated the role of exosomal HMGB3 in vivo by constructing a mouse model of pulmonary inflammation induced by exosomes by intratracheal injection combined with tail intravenous injection (Fig. 7N). PKH26-labeled exosomes were administered to the mice by tail intravenous injection, and the distribution of exosomes was observed 20 hours later by an in vivo Xtreme II system, which demonstrated that PKH26-labeled exosomes or cells that ingested PKH26-labeled exosomes were distributed in lung tissue (Fig. 7O). PKH26 dye were also found in nucleated cells from peripheral blood by fluorescence microscopy (Fig. 7O). The lungs were harvested for flow cytometry analysis after the mice were sacrificed. The results demonstrated that the infiltration of pulmonary macrophages and the proportion of iNOS⁺/F4/80⁺ macrophages are increased in the mice treated with SiO₂ + siNC-Exos, while the proportion of CD206⁺/F4/80⁺ cells is decreased. However, knockdown of exosomal HMGB3 partially reduced the infiltration of pulmonary macrophages and decreased the proportion of iNOS⁺/F4/80⁺ macrophages (Fig. 7P, Figure S3A-C).

HMGB3-enriched exosomes promote inflammatory activation and migration of monocytes/macrophages in vitro

We next constructed a pcDNA3.1 (+)-HMGB3 overexpression plasmid, and the transfection efficiency of pcDNA3.1-HMGB3 in RAW264.7 cells was evaluated through western blot analysis and RT-PCR (Fig. 8A-B). We transfected macrophages with plasmids and isolated exosomes from the SN, resulting in Vector-Exos derived from macrophages transfected with pcDNA3.1-vector and HMGB3-Exos derived from macrophages transfected with pcDNA3.1-HMGB3. HMGB3 expression in these exosomes was assessed through western blot analysis, which suggested that HMGB3 expression in HMGB3-Exos was upregulated compared with Vector-Exos (Fig. 8C). These exosomes were then cocultured with RAW264.7 macrophages, and the activation and migration of macrophages were measured. ELISA analysis showed a significant increase of the release levels of IL-1β, IL-6 and TNF-α from the HMGB3-Exos-treated macrophages (Fig. 8D). Transwell assays showed that HMGB3-Exos significantly promoted RAW264.7 macrophage migration (Fig. 8E-F). We then detected the activation of signaling pathways in macrophages by western blot analysis, which demonstrated that HMGB3-Exos up regulated the expression of p-p65, p-STAT3, p-ERK1/2, p-P38, CCR2 and pro IL-1β (Fig. 8G-H). These results revealed that exosomal HMGB3 participate in SiO₂-Exos-induced inflammatory activation and recruitment of monocytes/macrophages.

Macrophages play a key role in chronic inflammation and fibrosis in silicosis (5, 9, 38, 41, 72). The repeated activation of inflammatory macrophages and the infiltration of circulating inflammatory monocytes are the main factors causing chronic inflammation (7, 41). The resent results showed that after silica exposure, the number of macrophages in lung tissue increased (Fig. 1A-C), and the alveolar macrophages in the early stage of silicosis were mainly inflammatory macrophages (Fig. 1D). Additionally, the number of exosomes derived from alveolar macrophages increased in BALF, and the quantity of exosomes secreted by SiO₂-exposed macrophages also increased in the cultured cells (Fig. 1H, Fig. 2E). Many reports have revealed that hypersecretory exosome is a dangerous signal, resulting in the progression of many diseases, such as asthma, IPF and sepsis (20, 22, 73–75), and exosomes are relevant to the regulation of macrophage polarization and inflammatory cytokine transcription (14, 19–23). Therefore, we hypothesized that the abnormal increase in macrophage-derived exosomes may be contributed to inflammation dysregulation in the early stage of silicosis. We cocultured SiO₂-Exos with monocytes or M0 macrophages and found that SiO₂-Exos promoted the inflammatory activation and migration of monocytes or macrophages. A previous report has suggested that HMGB3 expression is upregulated in exosomes derived from SiO₂-exposed RAW264.7 macrophages (36). HMGB3 belongs to the HMGB family and has an 80% homologous amino acid sequence and a similar structure to HMGB1 and HMGB2 (65). HMGB1, as a DAMP, regulates the inflammatory response in sepsis and lung injury (64, 66, 76). Therefore, we hypothesized that SiO₂-Exos may mediate the inflammatory activation and migration of monocytes/macrophages through HMGB3. We first detected the expression of HMGB3 in exosomes derived from macrophages with or without SiO₂ exposure, which demonstrated that HMGB3 was increased in SiO₂-Exos (Fig. 6A-B), agreeing with a previous report (36). Knockdown of exosomal HMGB3 attenuated exosome-induced inflammatory activation and migration of monocytes/macrophages (Fig. 7), whereas overexpression of exosomal HMGB3 promoted this process (Fig. 8). We also found that exosomal HMGB3 promoted the inflammatory activation and migration of monocytes/macrophages mainly through the activation of the STAT3/MAPK (ERK1/2 and P38)/NF-κB/CCR2 signaling pathways (Fig. 9).

HMGB1 is rapidly transferred from the nucleus to the cytoplasm under stress and ROS upregulation (28, 66). Previous studies have reported that intracellular ROS are upregulated after macrophage phagocytosis of silica particles (10, 12). Therefore, we hypothesized that HMGB3 may be transferred from the nucleus to the cytoplasm under SiO₂ stress. The present findings verified that SiO₂ stress triggered the transfer of HMGB3 from the nucleus to the cytoplasm (Fig. 6G-H) and HMGB3 participated in inflammation via exosome. Because exosomes are vesicles with a lipid bilayer membrane structure, the bioactive molecules carried by exosomes may be located on or encapsulated in the exosome membrane. Once released, exosomes deliver their contents to the recipient cells mainly through endocytosis, membrane fusion and receptor-ligand mediated interactions (15, 17, 18). In addition, exosomal surface proteins integrate with receptors on the surface membrane of recipient cells to trigger intracellular signaling pathways (68, 69, 77). Numerous researches have demonstrated that the released HMGBs can bind to TLRs and RAGE to induce cytokine transcription (78, 79). To clarify the localization of HMGB3 in SiO₂-Exos, we treated purified SiO₂-Exos with protease K and then detected the expression of HMGB3. The results showed that protease K effectively digested HMGB3 protein (Fig. 6N), which suggested that HMGB3 may be localized on the exosome membrane. However, the specific membrane binding mode of HMGB3 and whether it mediates intracellular signal transduction by integrating with receptors on the surface membrane of recipient cells remain unclear.

In RAW264.7 macrophages, exosomal HMGB3 up regulated the phosphorylation levels of p65, STAT3 and MAPK (ERK1/2 and P38). While in THP-1 cells, exosomal HMGB3 selectively up regulated the phosphorylation levels of p65, STAT3 and P38 (Fig. 7). These discrepancies probable as a result of the difference among different cell types or species. These activated signaling pathways constitute a complex cross-talk network and feedback loop. Therefore, we hypothesized that SiO₂-Exos, as inflammatory regulators, trigger a signal network, including intracellular signal transduction directly activated by HMGB3 and continuous induction of indirect autocrine and paracrine signaling events. All of these different pathways are integrated into the response to inflammatory stimuli, which comprises highly complex cross-talk. It is still unknown whether these signaling pathways participate in the further polarization of M2 macrophages in the activation process of macrophages in silicosis. To better understand the outcome of these signaling pathways, different time points should be set to elucidate the association between activation of signaling pathways and macrophage activation. In addition, future studies should focus on how exosomal HMGB3 activates the signaling cascade network in recipient cells.

In summary, the present research indicated that SiO₂-Exos promote the inflammatory activation and recruitment of macrophages or monocytes in the early stage of silicosis. These processes are mainly regulated by the activation of the STAT3/MAPK (ERK1/2 and P38)/NF-κB/CCR2 signaling pathways mediated by exosomal HMGB3. These findings may provide a potential therapeutic target for early intervention in silicosis inflammation.

In this study, silica stimulation enhanced exosome secretion from macrophages, and the released exosomes regulated silicosis inflammation by promoting M1 polarization and recruitment of macrophages. Importantly, the expression of HMGB3 was increased in the exosomes from SiO₂-exposed macrophages, and exosomal HMGB3 induced the inflammatory activation and recruitment of monocytes/macrophages by regulating the activation of STAT3/MAPK/NF-κB/CCR2 signaling pathways. Our work provides new insights into the chronic inflammation of silicosis.

SiO₂	Silica
MCP-1	Monocyte chemotactic protein-1
TNF-α	Tumor necrosis factor-α
IL-1β	Interleukin-1 beta
LT	Leukotriene
HMGB3	High mobility group box 3
DAMP	Damage-associated molecular pattern
SN	Cell culture supernatant
TSG101	Tumor susceptibility gene 101
HSP70	Heat shock protein 70
TEM	Transmission electron microscopy
NTA	Nanoparticle tracking analysis
PDPN	Podoplanin
GAPDH	Glyceraldehyde3-phosphate dehydrogenase
ELISA	Enzyme-linked immunosorbent assay
RT-PCR	Reverse transcription-polymerase chain reaction
AMs	Alveolar macrophages
BALF	Bronchoalveolar lavage fluid
Exos	Exosomes
NC-Exos	Exosomes derived from macrophages without SiO₂ exposure
SiO₂-Exos	Exosomes derived from SiO₂-exposed macrophages
SiO2+GW4869-Exos	Exosomes derived from SiO₂-exposed macrophages treated with GW4869
CCR2	CC chemokine receptor 2
STAT	Signal transducers and activators of transcription
NF-κB	Nuclear factor kappa-B
PI3K/AKT	Phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/AKT)
MAPK	Mitogen-activated protein kinase
ERK 1/2	Extracellular signal-regulated kinase 1/2
HMGBs	HMGB family
ROS	Reactive oxygen species
TLRs	Toll-like receptors
RAGE	Receptor of advanced glycation endproducts

Authors' contributions

Xiaofeng Qin, Zhiyuan Niu and Hui Chen performed experiments and data analysis. Yongbin Hu and Xiaofeng Qin designed experiments, oversaw all data analysis and drafted the manuscript. All the authors have critically revised the manuscript. All authors have read, reviewed and approved the final manuscript as submitted to take public responsibility for it.

Funding

This work was supported by grants from the National Natural Science Foundation of China, Grant/Award Number: 81673120.

Availability of data and materials

The datasets supporting the conclusions of this article are included within the article and can be retrieved from the corresponding author upon reasonable request.

Ethics approval and consent to participate

All animal experiments were in accordance with the requirements of related regulations and procedures of the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The animal protocols were performed according to the ethical rules of the Institutional Animal Care and Use Committee of Central South University (number: CSU-2022-0001-0037).

Consent for publication

All coauthors granted consent for their respective contributions to be included in publication.

Competing interests

The authors declare that there is no conflict of interest.

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No competing interests reported.

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published 07 Mar, 2024

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Macrophage-derived exosomal HMGB3 regulates silica-induced pulmonary inflammation by promoting M1 macrophage polarization and recruitment

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Materials and methods

2.1 Cell lines and cell culture

2.2 Exosome isolation, identification and treatment

2.3 Trafficking analysis of exosomes in vitro and vivo study

2.4 Exosome secretion inhibition assay

2.5 Western blot analysis

2.6 Reverse Transcription-Polymerase Chain Reaction (RT-PCR) analysis of mRNA

2.7 Animal model

2.8 Immunohistochemical staining

2.9 Immunofluorescence

2.10 Flow cytometry

2.11 Enzyme-linked immunosorbent assay (ELISA)

2.12 Cell migration

2.13 Plasmid construction and transfection

2.14 RNA interference

2.15 Statistics

Results

The quantity of exosomes secreted by macrophages is significantly increased in silicosis mice

The quantity of exosomes secreted by SiO2-exposed macrophages increased significantly in vitro

SiO2-Exos promote the polarization of M0 macrophage to M1 macrophage in vitro

SiO2-Exos promote macrophage migration by upregulating CCR2 expression in vitro

SiO 2 -Exos promote inflammatory activation of monocytes/macrophages mainly by regulating the activation of the STAT3/MAPK/NF-κB signaling pathways

HMGB3 expression is upregulated in SiO2-Exos, which may be localized on the exosome membrane

HMGB3 deficiency attenuates SiO2-Exos-induced inflammatory activation and recruitment of monocytes/macrophages in vitro and in vivo

HMGB3-enriched exosomes promote inflammatory activation and migration of monocytes/macrophages in vitro

Discussion

Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1

The quantity of exosomes secreted by SiO₂-exposed macrophages increased significantly in vitro

SiO₂-Exos promote the polarization of M0 macrophage to M1 macrophage in vitro

SiO₂-Exos promote macrophage migration by upregulating CCR2 expression in vitro

SiO ₂ -Exos promote inflammatory activation of monocytes/macrophages mainly by regulating the activation of the STAT3/MAPK/NF-κB signaling pathways

HMGB3 expression is upregulated in SiO₂-Exos, which may be localized on the exosome membrane

HMGB3 deficiency attenuates SiO₂-Exos-induced inflammatory activation and recruitment of monocytes/macrophages in vitro and in vivo