Role of gingival mesenchymal stem cell exosomes in macrophage polarization under inflammatory conditions

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Role of gingival mesenchymal stem cell exosomes in macrophage polarization under inflammatory conditions ⁎

Ru Wanga,b, Qiuxia Jia, Chenda Mengb, Hanyun Liuc, Chun Fana, Sofya Lipkindd, Zhiguo Wange, , ⁎ Quanchen Xua, a

Department of Stomatology, Affiliated Hospital of Qingdao University, 16 Jiangsu Road, Qingdao 266003, Shandong, China School of Stomatology of Qingdao University, Qingdao 266003, China c Department of Infectious Diseases, Affiliated Hospital of Qingdao University, Qingdao, Shandong, China d Department of Molecular and Cellular Biology, University of California, Davis, CA, United States e Department of Burn and Plastic Surgery, Affiliated Hospital of Qingdao University, Qingdao, Shandong, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Gingival mesenchymal stem cells Exosomes Macrophage Periodontitis Polarization

Objective: Exosomes have been shown to play a strong role in intercellular communication. While GMSCs have been extensively studied, less research exists on exosomes derived from GMSCs, especially on how exosomes affect macrophages. This study aimed to investigate the impact of GMSC-derived exosomes on macrophage polarization and phenotype under inflammatory conditions. Methods: Exosomes were isolated from GMSCs-conditioned media by ultracentrifugation (UC) and characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot (WB). In vitro, GMSC-derived exosomes were co-incubated with macrophages for 24 h in the absence or presence of M1 polarizing conditions in the six-well plate. The protein and mRNA expression levels of M1 and M2 macrophage markers were detected and the supernatants were collected for an enzyme-linked immunosorbent assay (ELISA). Results: Exosomes were successfully isolated from GMSCs. Macrophages co-cultured with exosomes showed significantly decreased levels of the M1 markers Tumor Necrosis Factor-α (TNF-α), Interleukin-12 (IL-12), CD86 and Interleukin-1β (IL-1β). By contrast, M2 marker Interleukin-10 (IL-10) levels moderately increased. Meanwhile, similar results were acquired in the cell culture supernatants. Conclusion: GMSC-derived exosomes may promote M1 macrophage transformation into M2 macrophages, reducing the pro-inflammatory factors produced by M1 macrophages.

1. Introduction Periodontitis is a common form of periodontal disease characterized by periodontal pocket formation, attachment loss and alveolar bone resorption [1]. Periodontal pathogens may stimulate a strong innate immune response [2]. Macrophages, blood cells, and lymphocytes mobilize into the periodontal tissue, causing periodontal soft tissue inflammation and alveolar bone resorption [3]. Macrophages play a critical defensive role against periodontal pathogens [4,6]. In response to different local microenvironments, naive macrophages (M0) can be polarized into two different phenotypes, pro-inflammatory (M1) or anti-inflammatory (M2), and perform different roles in different physiological or pathological conditions [5,6]. M1 macrophages show high expression of TNF-α, IL-12, IL-6, IL-1β, CD86; M2 macrophages have high expression of IL-10, CD206, CD163 [5]. These pro-inflammatory

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cytokines appear to contribute to periodontal tissue destruction [7–10]. The ideal periodontitis treatment should be to dampen the inflammatory immune response and restore periodontal structure and function. The routine treatments of periodontal disease mainly including basic treatment, guided tissue regeneration (GTR) and guided bone regeneration (GBR). However, the effects of these methods are limited and have poor clinical predictability [11]. Cell‐based therapies have become a new treatment to promote regeneration in a predictable way and expand the scope of regeneration therapy to a wide range of defects [12,13]. Accordingly, mesenchymal stem cell (MSC) has a broad application prospect in the regeneration of periodontal defects [13]. In recent years, GMSCs have been isolated and identified in the gingiva lamina propria, possessing good regeneration and immunomodulatory functions [14]. Compared with other MSCs, GMSCs are rich in source and are easily obtained by minimally invasive cell

Corresponding authors. E-mail addresses: [email protected] (Z. Wang), [email protected] (Q. Xu).

https://doi.org/10.1016/j.intimp.2019.106030 Received 8 October 2019; Received in revised form 4 November 2019; Accepted 4 November 2019 1567-5769/ © 2019 Elsevier B.V. All rights reserved.

Please cite this article as: Ru Wang, et al., International Immunopharmacology, https://doi.org/10.1016/j.intimp.2019.106030

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2.2.2. Osteogenic differentiation GMSCs were cultured by adding MSC growth medium in six-well plates. When the cells reached 80% confluence, the osteogenic induction mediums (α-MEM containing 5% FBS, 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate and 10 mM β-glycerophosphate; SigmaAldrich) were replaced by fresh mediums [36]. The mediums were changed every three days. After 4 weeks, alizarin red S staining was used for cell mineralization in vitro.

isolation technique. Increasing evidence suggests that MSCs help to enhance immune responses during early-stage inflammation and subsequent tissue regeneration by producing cytokines or growth factors through the paracrine and autocrine pathways [15,16]. Exosomes, also called endosome-derived nanoscale vesicles, are a type of membrane vesicles released into extracellular fluid by a wide variety of cells [17]. This vesicle, ranging in size from 30 to 150 nm, carries biomolecules that serve crucial roles in local and distal cellular communication [18–21]. As a kind of membrane vesicle of paracrine or endocrine of MSCs, exosome may directly or indirectly participate in biological energy conversion, cell proliferation and immune regulation [22,23]. Exosomes derived from MSCs reduced the inflammatory response after seizures [24], traumatic spinal cord injury [25] and promoted the repair of cartilage defects [26], skin defects [27,28] and accelerate the outward growth of nerve axons after trauma [29] and strengthened the repairs of fractures and brain injury [30–32]. GMSCderived exosomes trigger the formation of blood vessels and regeneration of rat sciatica and induce the proliferation and differentiation of taste bud cells [33,34]. Based on the above findings, we hypothesize GMSC-derived exosomes may play a significant role in macrophage-dominated periodontitis. This study investigates how exosomes secreted by GMSCs affect macrophage polarization under inflammatory conditions.

2.2.3. Adipogenicity differentiation When the cells reached 80% confluence, the culture mediums were replaced by MEM fat induction culture mediums (α-MEM containing 10% FBS, 10 µM human insulin, 10 µM dexamethasone, 200 µM indomethacin, and 0.5 mM IBMX; Sigma-Aldrich) [36]. The mediums were changed every three to thirteen days. 2.2.4. Flow cytometry The fourth-generation GMSCs (1x106) were collected and washed twice with PBS. After incubated with CD45, CD73, CD90 and CD105 at 4 °C for 40 min in the dark, the cells were centrifuged and washed three times, and the suspension was analyzed by flow cytometry (Beckman Coulter, Inc., Brea, CA, USA). 2.3. Isolation and identification of exosomes

2. Material and methods

When the density of the fourth-generation cells reached approximately 70–80%, the serum-supplied mediums were replaced by fresh αMEM containing 10% exosome-depleted FBS (American SBI) mediums. The cell cultures were collected in some 15 ml of centrifuge tubes (Beckman tube, Inc., Brea, CA, USA) after 48 h and centrifuged at 300g for 10 min. The resulting precipitates were removed. The supernatants were collected, transferred to new test tubes, and centrifuged at 3000g for 20 min. This precipitates also were removed. The supernatants from the second centrifugation were collected, transferred to new test tubes, and centrifuged at 10,000g for 30 min. The supernatants from the third centrifugation were filtered through 0.22 μm filters. Finally, the supernatants from filtration were centrifuged twice in the ultracentrifuge machine (Beckman Coulter Optima L-90K ultracentrifuge) at 100,000g for 70 min. The resulting supernatant were removed from the centrifuge tubes and PBS was added to dissolve the precipitate and obtain the exosomes. The exosomes were stored at −80 °C in a cryogenic refrigerator. Exosome concentration was detected using a BCA protein kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). Exosome morphology was observed under TEM. WB was used to analyze the exosome-characteristic markers CD9, CD81, and CD63. The particle size distribution of the exosomes was seen under the NTA.

2.1. Reagents Lipopolysaccharides (LPS) (Escherichia coli O111: B4) were obtained from Sigma-Aldrich (USA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Mce (USA). Recombinant human interferon-γ (rhIFN-γ) was acquired from PeproTech (USA). Antibodies were purchased from suppliers as follows: CD90, CD105, CD73, CD45 and CD163 were supplied by Biolegend (USA); CD86, CD81, CD9 and anti-GAPDH were acquired from Elabscience Biotechnology Co., Ltd (Wuhan, China); CD63 was from Wan lei Biotechnology Co., Ltd (Shenyang, China). 1′dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (Dil) and 4′,6-diamidino-2-phenylindole (DAPI) were from Solarbio Science & Technology Co., Ltd. (Beijing, China).

2.2. Cell culture and GMSC multipotent differentiation 2.2.1. Cell culture This study was approved of the ethics committee of Qingdao University, and the participants' informed consent was obtained. Healthy human subjects (19–26 years old) followed routine dental procedures to generate gingival samples from discarded tissue. The tissues were sterilized and immersed in Dispase Ⅱ (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) (2 mg/ml) and Collagenase Ⅰ (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) (2 mg/ml) and in a 37 °C water bath for 40 min. The cell suspensions were separated at 1000 rpm for 5 min, and the supernatants were removed. The solutions were added with 3 ml of 15% fetal bovine serum (American Hyclone) of Alpha MEM (American Hyclone) and incubated at 37 °C and 5% CO2. These cell mediums were changed every three days. When the cells fusion reached 80%, the cells were digested with trypsin/EDTA solution (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) and subcultured. The cells from the third to fifth generation were used in the experiment. As previously described, the cells were subjected to colony forming unit-fibroblast assay (CFU-F) and pluripotent differentiation of GMSCs from a single community source [35]. Human acute monocytic leukemia cell line (THP-1) (ATCC, USA) and PMA were co-cultured in six-well plates for 48 h to differentiate into naive macrophages (M0).

2.4. Exosomes transfer to macrophages After being stained by Dil, exosomes were again centrifuged at 100,000g for 70 min and co-incubated with macrophages. After 24 h, the culture solutions were abandoned and 4% paraformaldehyde was added to the macrophages for 20 min. After washing with PBS, macrophages then were stained by DAPI for 3–5 min. Fluorescence signals were observed under a fluorescence microscope and a laser confocal microscope. 2.5. The incubation of macrophages with GMSC-derived exosomes After monocytes were induced into M0 macrophages by PMA, they were placed in cell culture mediums containing 10% exosome-depleted FBS. The addition of LPS plus IFN-γ induced M0 macrophages to polarize to M1 macrophages. Exosomes were added to the M1 macrophages to form the experimental groups. The control groups of M1 macrophages did not receive exosomes. The blank groups comprised 2

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3.2. Identification of exosomes

Table 1 Primer sequences of qRT-PCR analysis.

The morphologies of the exosomes by TEM (Fig. 2A) were elliptic and displayed a typical cup-shaped morphology. WB analysis showed that GMSC-derived exosomes expressed the exosome markers CD9, CD81, and CD63 (Fig. 2B). The size distributions of the purified exosomes were measured by NTA (Fig. 2C).

Genes

Primer sequence (5′–3′)

TNF-α

FORWARD: AGC TGG TGG TGC CAT CAG AGG REVERSE: TGG TAG GAG ACG GCG ATG CG

IL-10

FORWARD: GCC AAG CCT TGT CTG AGA TGA TCC REVERSE: GCT CCA CGG CCT TGC TCT TG

IL-1β

FORWARD: TGG CTT ATT ACA GTG GCA ATG AGG ATG REVERSE: TGT AGT GGT GGT CGG AGA TTC GTA G

3.3. Exosomes transfer to macrophages

Il-12

FORWARD: TGC CTT CAC CAC TCC CAA AAC C REVERSE: CAA TCT CTT CAG AAG TGC AAG GG

GRAPDH

FORWARD: TGC ACC ACC AAC TGC TTA GC REVERSE: GGC ATG GAC TGT GGT CAT GAG

After labeling the exosomes with Dil and incubating with the macrophages, blue fluorescence contains red fluorescent. These results suggest that macrophages (blue fluorescence) absorbed the exosomes (red fluorescent) (Fig. 3). 3.4. GMSC-derived exosomes induce polarization of M2 macrophages

M0 macrophages. After 24 h, the supernatants, RNA from all of the macrophages and all of the macrophages were collected.

To investigate the effect of GMSC-derived exosomes on inflammatory macrophages, we analyzed M1 and M2 macrophage polarization markers (Fig. 4C–F) (Fig. 5). The qRT-PCR results showed that the mRNA levels of TNF-α, IL-12, IL-1β and IL-10 in the control groups were higher than in the blank groups. Compared with the control groups, the mRNA levels of the expressions of TNF-α, IL-12, and IL1β decreased and that of IL-10 increased in the experimental groups. Flow cytometry analysis showed that CD86 expression was increased in macrophages in the control groups compared with the blank groups. Compared with the control groups, the expression of CD86 in macrophages significantly decreased in the experimental groups. No significant difference was found in CD163 expression between the experimental groups and control groups.

2.6. Enzyme-linked immunosorbent assay (ELISA) The levels of IL-10 and TNF-α in cell supernatants were detected using an ELISA kit (Dakewe, China). 2.7. Quantitative real-time PCR (qRT-PCR) TRIzol reagent (Takara, Kyoto, Japan) was used to extract total RNA from macrophages. cDNA was reverse transcribed using a PrimeScript TM RT reagent kit (Takara, Kyoto, Japan) according to the manufacturer’s instructions. The mRNA levels were detected using SYBR Premix Ex Taq (Takara, Japan) with 2−Δ ΔCT method on Light Cycler 480 real-time PCR system. The PCR primers were amplified (Table 1), with GAPDH as the internal parameter.

3.5. GMSC-derived exosomes reduce pro-inflammatory factors in supernatant and increase anti-inflammatory factors

2.8. Flow cytometric analysis

Then, we examined anti-inflammatory and pro-inflammatory factors in the supernatant of each groups (Fig. 4A and B). ELISA results revealed the levels of TNF-α and IL-10 in the control groups were higher than that in the blank groups. TNF-α level decreased and IL-10 increased in the experimental groups as compared to the control groups. The results revealed that co-incubation of GMSC-derived exosomes with macrophages may inhibit the production of pro-inflammatory cytokines and stimulate the production of anti-inflammatory cytokines.

After washing with PBS, the centrifuged cells and human CD86 and CD163 were placed in the dark at 4 °C for 30 min. Then cells were washed once, resuspended in PBS, and analyzed by FACS Calibur (Beckman Coulter, USA). Flow cytometry data analyses used FlowJo software v10.2 (TreeStar). 2.9. Statistical analysis Each experiment was repeated at least three times. GraphPad Prism 7 (GraphPad Software Inc.) was used for statistical analysis. Data were expressed as the mean ± standard error of the mean (SEM). Differences between experimental and control groups were analyzed by a two-tailed unpaired Student’s t test. Values of p < 0.05 were considered statistically significant.

4. Discussion Extensive tissue engineering studies have found that MSCs have tissue repair and anti-inflammatory abilities that may allow them to be used as a method to promote periodontal tissue regeneration [12,38–42]. Compared with MSCs and periodontal membrane stem cells, GMSCs are easy to obtain and culture, and they grow quickly [35]. The previous study has demonstrated that GMSCs may trigger the macrophage inflammatory response and promote the transformation of M1 macrophages to M2 macrophage [43]. Although there is a good deal of literatures on the efficacy of cell-based therapies for periodontitis [12,42,44–46], development of a treatment of periodontitis using exosomes generated by GMSCs represents a novel and possibly a safer therapeutic approach. Meanwhile, more and more studies have shown that exosomes may be used to be a new alternative free cell-therapy of inflammatory diseases and mediate paracrine functions of stem cells [47–49]. It has been demonstrated that the exosomes extraction methods include UC, ExoQuick and size exclusion chromatography, especially UC, which has greater purity and less protein contamination than the others [50]. We used UC to extract GMSC-derived exosomes in this study. In general, at least two different techniques should be used to

3. Results 3.1. Characteristics of GMSCs and macrophages Alizarin red and oil red O staining showed the GMSCs differentiation into osteoblasts and adipocytes (Fig. 1A and B). GMSCs were isolated from the gingivae and presented as long fusiform cells (Fig. 1C). GMSCs formed cell colonies measured by CFU-F (Fig. 1D). In addition, the surface markers expressions by analyzed flow cytometric such as CD90 (96.5%), CD105 (94%) CD73 (95.6%) and CD45 (1.22%) (Fig. 1G) were in line with the definition of GMSCs by the international society for cell therapeutics [37]. The naive macrophages (M0) derived from THP-1 (Fig. 1E) were attached to the 6-well plate, presenting the morphology of macrophages (Fig. 1F). 3

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Fig. 1. Characteristics of GMSCs and macrophages. A: Mineralization was assessed by Alizarin red staining. Magnification, ×200. B: Adipocytes were identified by oil red O staining. Magnification, ×200. C: Adherent GMSCs showed a spindle-shaped, fibroblast-like morphology. Magnification, ×40. D: Colony-forming ability, as identified by staining 1% crystal violet. Magnification, ×40. E: Monocytes. Magnification ×100. F: Naive macrophage (M0). Magnification, ×400. G: Flow cytometric analysis of surface markers in GMSCs: CD90 (96.5%), CD105 (94%), CD73 (95.6%), CD45 (1.22%). Data are representative of five independent experiments.

significantly when LPS plus IFN-γ were added [63]. The expression of macrophage inflammatory factors after stimulation is consistent with observed conditions in inflamed periodontal tissues [64,65]. Emerging researches have revealed the therapeutic effects of stem cell-derived exosomes in several IRI models based on their role in tissue repair, however, their immunomodulatory function in inflammatory diseases has rarely been reported [66–69]. In this study, qRT-PCR results from the experimental groups showed that IL-12, TNF-α and IL-1β expression were lower than in the control groups, and that IL-10 expression was higher than in the control groups. ELISA results showed that TNF-α decreased and IL-10 increased in the supernatant of experimental groups. The flow cytometry tests revealed lowered CD86 expression (M1 surface protein). Overall, GMSC-derived exosomes diminished the secretion and expression level of pro-inflammatory cytokines (TNF-α, IL-12, IL-1β, CD86), and enhanced that of the anti-inflammatory cytokine IL-10. Confusingly, CD163 macrophage expression levels did not significantly differ between the control groups and the experimental groups. This may be due to GMSC-derived exosomes possessing limited ability to change proinflammatory phenotypes. This study showed that in vitro GMSC-derived exosomes could inhibit the activation of M1 macrophages that had been stimulated by LPS plus IFN-γ, and instead induce them to convert to M2 macrophages. These findings also suggest that GMSC-derived exosomes may provide a possible treatment for macrophage-dominated periodontitis. In summary, the satisfactory immunomodulation regulated by GMSC-derived exosomes makes it possible for exosomes to replace GMSCs when treating macrophage-related inflammation. Although it has been shown that GMSC-derived exosomes help regulate macrophage polarization, more researches are needed to understand the detailed mechanisms of this process.

describe individual extracellular vesicles. Therefore, TEM, NTA and WB were used to confirm the existence of exosomes. Using TEM (Fig. 2A) and NTA (Fig. 2C), we isolated the particles within the exosomes (30–150 nm), that displayed morphological traits consistent with exosomes [51–54]. The typical cup-shaped morphology of exosomes was also observed under TEM (Fig. 2A). Tang et al found that exosomes obtained by UC showed high expression of CD9 and CD63 as identified by WB [50], which is consistent with our results. The specific composition of exosomes should be compared with that of the secreting cells as indicated by Lotvall et al. [55]. CD81 has been found on the surface of GMSCs [56]. Meanwhile, WB results also found CD81 expression in GMSC-derived exosomes in this study. In summary, we identified the extracellular vesicles extracted by UC as exosomes (Fig. 2). Exosomes may be directly fused with the receptor cytoplasmic membranes and transfer designative information to be internalized by endocytosis or phagocytosis [57–59]. Exosomes can be engulfed by macrophages, and this is required for them to act on said macrophages. In this experiment, we detected exosomes using exosome-tracers and fluorescence microscopy, which confirmed that exosomes labeled with Dil could be engulfed by macrophages (Fig. 3A), which is consistent with previous research [60]. The fluorescence hotspots from Dil observed under fluorescence microscope likely represent exosome clusters, as the size of a single exosome (mean 135 nm) is lower than the detection threshold of optical microscope. The three-dimensional confocal microscopy analysis further showed that exosomes had entered into the macrophages (Fig. 3B). M1 macrophages got the dominate role in patients with chronic periodontitis, who also had higher levels of the M1-related pro-inflammatory factor IL-1β [61,62]. In this study, we demonstrated that LPS plus IFN-γ act on macrophages to induce high levels of M1-related IL-12, TNF-α and IL-1β, promoting macrophage inflammatory responses. Consistent with previous studies, macrophages released few cytokines without stimulation, and increased cytokine secretion 4

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Fig. 2. Characteristics of GMSC-derived exosomes. A: Exosomes were observed by transmission electron microscopy (TEM) (original magnification ×120,000). B: Western blot analysis shows the proteins of CD9, CD81 and CD63 in GMSC-derived exosome. C: Nanoparticle tracking analysis (NTA) shows particle size distribution of GMSC-exosomes. Data are representative of at least three independent experiments.

Fig. 3. Exosomes transfer to macrophages. GMSC-derived exosomes were labeled with Dil (red fluorescence) and cocultured with macrophages (blue fluorescence) for 24 h. Fluorescence signals were visualized under fluorescence microscope (A, Magnification ×100) and Laser confocal (B, Magnification ×400). Data are representative of at least three independent experiments.

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Fig. 4. GMSCs exosomes induce M2 macrophages polarization. M0:Naive macrophages; M1: M0 + LPS + IFN-γ; M1 + exosome: M0 + LPS + IFN-γ + exosome (20 ug/ml). Secretory TNF-α (A) and IL-10 (B) levels were test by ELISA from different groups. The mRNA level of M2-related IL-10 (E) and M1 macrophages-related TNF-α (C), IL-12 (D) and IL-1β (F) were examined by qRT-PCR. The data in this groups represented at least three independent experiments, expressed by mean ± SEM of three replicates (A-F). *P < 0.05, VS M0; #P < 0.05 Compared with M1, n ≥ 3.

Fig. 5. The expression of surface markers in macrophages by flow cytometry. A: The expression of CD86 in macrophages exposed to M1 polarized stimulation (51.72%) was increased, and the expression of CD86 after exosome co-incubation (33.14%) was decreased significantly. B: The data represent the mean ± SEM (*P < 0.05, compared with M0; #P < 0.05 VS M1, n ≥ 3). C: There was no significant difference in the expression of CD163 between M1 and M1 exosome coincubation.

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Declaration of Competing Interest

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