Mesenchymal stem cell-derived exosomes inhibit Aβ1-42 induced microglia polarization by TLR2/MYD88/NF-κB pathway

In recent years, more and more research evidence indicates that the causes of various neurodegeneration diseases are related to neuroinammation in the brain, Aβ1–42 may be a key factor in the development of neuroinammation in neurodegenerative diseases. Therefore, it is urgent to nd an effective and targeted alleviation of neuroinammation caused by Aβ1–42. Our study found that exogenous administration of Aβ1–42 has a very obvious polarizing effect on microglia in the brain compared with the PBS-treated group. After the intervention of Aβ1–42, we obtained the mesenchymal stem cells derived exosome by ultracentrifugation. Microglia were treated with MSC-Exo in vivo and in vitro.


Background
In recent years, more and more research evidence indicates that the causes of various neurodegeneration diseases are related to neuroin ammation in the brain, Aβ1-42 may be a key factor in the development of neuroin ammation in neurodegenerative diseases. Therefore, it is urgent to nd an effective and targeted alleviation of neuroin ammation caused by Aβ1-42.

Methods
Our study found that exogenous administration of Aβ1-42 has a very obvious polarizing effect on microglia in the brain compared with the PBS-treated group. After the intervention of Aβ1-42, we obtained the mesenchymal stem cells derived exosome by ultracentrifugation. Microglia were treated with MSC-Exo in vivo and in vitro.

Results
The MSC-Exo were found have the effect of inhibiting microglial polarization in vivo and in vitro experiments. It was further found that its target gene was TLR2 on the surface of microglial cells to inhibit the expression of the downstream protein MYD88/NF-κB and inhibit the microglia polarization and the development of neuroin ammation.

Background
Neuroin ammation is an important pathogenic factor affecting neurotrauma and neurodegenerative diseases [1]. In recent years, more and more research evidences have shown that the causes of death in a variety of neurological diseases are related to neuroin ammation in the brain, even if the main cause is not in ammation. After an acute neurological event (whether it is a traumatic disease or a non-traumatic disease), in ammatory markers increase in the brain and the whole body within a few minutes [2]. This process can be harmful, but it also promotes repairment of brain. Similarly, chronic neurological diseases, such as Alzheimer's disease and chronic traumatic encephalopathy, will have long-term and continuous neuroin ammation [3]. The chronic damage of neurons is accumulating and increasing. The regulatory cells of neuroin ammation are derived from the glial cells in brain. Microglia are the most important neuroimmune cells [4]. 22]. The substances and effects of exosomes are mainly determined by their source. Therefore, exosomes derived from mesenchymal stem cells have become safer and more targeted drugs that can replace stem cell transplantation [23].
Our research focus on the effect of Aβ1-42 on the polarization of microglia and the use of mesenchymal stem cell-derived exosomes for interventional treatment. We found that injection of Aβ1-42 into the lateral ventricle can signi cantly cause the polarization of microglial cells and the administration of exosomes derived from mesenchymal stem cells can effectively alleviate the activation of microglia and inhibit the occurrence and development of neuroin ammation. After biosynthesis analysis and in vitro and in vivo experiments, we have con rmed that MSC-Exo can inhibit the polarization of microglia and the release of neuroin ammatory factors maybe through the TLR2/MYD88/NF-KB signaling pathway.

Materials And Methods
All experimental procedures were performed in accordance with the Guide for the Care and Use of immuno uorescence staining, the mouse was sacri ced by trans cardiac perfusion with PBS followed by 4% paraformaldehyde. The samples were xed, dehydrated and embedded in the optimum cutting temperature medium (Sakura, Torrance, CA, USA). After that, mouse coronal sections were cut using a cryostat, and the tissue sections were stored at -20 ℃. For qRT-PCR and Western Blot, the cerebral cortex and hippocampus were acquired from the injected side brain. The brain tissue was stored at -80℃ for subsequent experiments.

Exosome preparation and identi cation
The collected cell culture supernatants were used to isolate exosomes by ultracentrifugation as described previously [25]. Brie y, the medium was removed free cells by centrifugations at 300g for 10 min. Subsequently, the supernatants were spun at 2000g for 10 minutes at 4 ° C to remove cell debris, and at 10,000 g for 30 minutes at 4 ° C to remove cell pellets. Next, the supernatant was ltered through a 0.22 mm lter (Millipore-Sigma, USA) to remove dead cells and particles larger than 200 nm. After that, the exosomes were harvested by ultracentrifugation at 100,000g for 70 min in a swing rotor (SW32Ti, XPN-100, Beckman, California, USA). All centrifugation steps were performed at 4℃. After the supernatant was discarded, the precipitation was resuspended with PBS pellets and stored at 4°C temporarily (<24 h) for further experiments. The total protein content of exosome was quanti ed by bicinchoninic acid (BCA) assay (Solarbio, Beijing, China). For the identi cation of exosomes, transmission electron microscope (TEM, HT8700; Hitachi, Tokyo, Japan) was used to observe the morphology of isolated particles. Brie y, twenty microliters of the sample were applied to a carbon coated formvar lm that was attached to a metal sample grid. The grid was incubated with 50 ul of 2% phosphotungstic acid for 2 minutes at room temperature. After drying with lter paper, the sample was examined by TEM. Further, western blot analysis the biomarkers of exosomes including CD63, HSP70 and TSG101(1:1000, Abcam, USA). The size distribution of the particles in the particles was measured and analyzed using Nano Particle Tracking (NTA).

MSC-Exo Labeling and administration
For tracking studies, exosomes were labelled with PKH67 (green, Sigma-Aldrich, USA) and PKH26 (red, Sigma-Aldrich, USA) according to the manufacturer's protocol. Brie y, 4μl PKH67 or PKH26 dye was mixed with exosome suspension in diluent C and incubated for 10 min at 37 °C. The labelling reaction was stopped by adding 20 ml chilled PBS. Labelled exosomes were ultracentrifuged at 100,000×g for 70 min, washed with PBS, ultracentrifuged again at 100,000×g and the pellet were resuspended in PBS. For in vitro experiments, BV2 cells were treated with 10μg/ml exosomes for 12 h. For in vivo experiments, mouse was intranasally administered with exosome at a concentration of 4ug/ul per nostril alternately [27][28][29]. Each mouse was treated three days with 10μL exosome once a day.

Preparation of brain extracts
Mouse were i.c.v. injected with 3µL Aβ1-42 peptide (3μg/3μl) or an equivalent volume of the PBS using a 10μl Hamilton Syringe (Bregma: -1.0 mm, Midline: ±1.5 mm, Depth: 2.5 mm) [30]. After 3 days, each mouse was treated with an equivalent volume(10ul*4ug/ul) PBS or MSC-Exo intranasally. For immuno uorescence staining, the mouses were sacri ced by trans cardiac perfusion with PBS followed by 4% paraformaldehyde. The samples were xed, dehydrated and embedded in the optimum cutting temperature medium (Sakura, Torrance, CA, USA). After that, mouse coronal sections were cut using a cryostat, and the tissue sections were stored at -20 ℃. For qRT-PCR and Western Blot, the cerebral cortex and hippocampus were acquired from the injected side brain. The brain tissue was stored at -80℃ for subsequent experiments.

Western blot
The total protein extracted from cell and brain tissue was for western blotting analysis as we previously described [25,31]. The protein concentration was measured using the BCA Protein Assay Kit (Solarbio, China). Brie y, normalized protein samples were subjected to SDS acrylamide gel treatment, and then transferred to nitrocellulose membranes (Millipore, MA, USA). 5% skim milk in Tris Buffered saline Tween (TBST) were used to block nonspeci c staining at room temperature for 2 h and incubated with primary antibodies (Table 1)

Real-time quantitative PCR
Total RNA from BV 2 cells and mouse brain tissue were performed using the Trizol reagent (Invitrogen, Carlsbad, CA). Both concentration and purity were estimated using a NanoDrop One Spectrophotometer (ThermoFisher Scienti c). cDNA was synthesized using a cDNA reverse transcription kit (Tian Gen, China) on a CFX Connect Real-Time PCR Detection System (Bio-Rad, California, USA). GAPDH was used as an internal control. The primer sequences are listed in Table 2. The data were analyzed with the2 -△△ Ct formula.

Immuno uorescence
The cell samples were xed in 4% PFA for 20 min at room temperature, and animal samples were prepared as described previously [32]. For blocking nonspeci c staining, the post-xed samples were treated with 3% BSA for 30 min at 37°C. After that, the samples were incubated with primary antibodies overnight. The primary antibodies (Table 1)

Statistical analysis
Statistical analysis was performed with Graph-Pad Prism 9(GraphPad Software). All data are presented as means ± S.E.M. and were analyzed using Student's t test (two groups), one-way ANOVA followed by Bonferroni's multiple comparison test (more than two groups). Differences between means were considered statistically signi cant when P < 0.05. Animal weight was used for randomization and group allocation. No animals were excluded from analysis.

2.The bioinformatics analysis of Aβ1-42 treatment on microglia
In order to explore the target gene of Aβ1-42 in promoting the polarization of microglia, we download chip data GSE55627 on the Geo platform, and compared the changes in the genome of the primary microglia after the treatment of Aβ1-42 and the normal group. First, the distribution of differential genes was analyzed by using the OmicStudio tools at https://www.omicstudio.cn/tool (Figure 2A), GO and KEGG enrichment analysis was performed on the DAVID data platform ( Figure 2B-C). The enrichment analysis of KEGG showed that the differential genes were in the TNF signal pathway and Toll-like receptor pathway which the enrichment is the most obvious ( Figure 2D). The GO analysis results showed that in the biological process part, the differential genes were concentrated in the in ammatory response, and in the molecular biological function part, the differential genes were concentrated in factor activation ( Figure 2E). Finally, by comprehensive analysis, we focus the signal pathway on the TLR2/MYD88/NF-κB( Figure 2F).

3.MSC-Exo can signi cantly inhibit the polarization of microglia in vitro
In this part, we veri ed the effect of MSC-Exo on inhibiting the polarization of microglia. First,we extracted and identi ed rat bone marrow mesenchymal stem cells (Supplementary Fig2A-E) ,then we identi ed the exosomes extracted by ultracentrifugation (Fig. 3D). The results of NTA showed that the peak range of the particles was between 30-150nm (Fig. 3A). The peak value is 85nm, which is in line with the diameter distribution of exosomes. Western blot was used to detect the expression of CD63, Tsg101, and HSP70.
Compared with the supernatant group, the expression of each protein in the exosome group is signi cantly increased (Fig. 3B). The results of transmission electron microscopy show that the particles we extracted are basically in about 100nm, the shape is round-shaped, which is fully in line with the standard shape of exosomes (Fig. 3C). Besides, the PKH67-labeled exosomes were added to the BV2 cells culture medium. Immuno uorescence was used to demonstrate that MSC-Exo (green uorescence) were engulfed by BV2 cells (red uorescence) (Fig. 3E).
Then, we divide the group into: control+PBS, control+MSC-Exo, Aβ1-42, Aβ1-42+MSC-Exo. The results of western blot show that compared with the Aβ1-42 treatment group, the Aβ1-42+MSC-Exo group signi cantly inhibited the expression of iNOS (Fig. 4A, C), The results of immuno uorescence show that iNOS+ positive cells in the MSC-Exo group were signi cantly reduced (Fig. 4B, E),The results of MTT show that compared with control group, the cytoactive of the Aβ1-42 group was signi cantly reduced. After exosomal treatment, the cytoactive of Aβ1-42+Exo was improved compared with the Aβ1-42 group.Besides, the PCR results showed the expression of related pro-in ammatory factors. After MSC-Exo treatment, there is a decrease expression of the pro-in ammatory factors IL-1β, IL-6, TNF-α,and increase expression of the anti-in ammatory factor IL-10( Fig. 4G-I). Similarly, we tested the expression of Aβ1-42 ( Supplementary Fig.3A,3B )and Mature-IL-1β( Supplementary Fig.3A,3D)by westernblot ,The above results con rm that MSC-Exo inhibits M1 polarization of microglia and regulates the release of related in ammatory factors in vitro.

4.MSC-Exo can decrease the expression of TLR2/MYD88/Nf-κB in vitro.
In order to con rm the expression of related signal pathways, we detected the expression of TLR2 and RELA in each group by PCR (Fig 4L). At the same time, WB results con rmed the expression changes of related proteins. Compared with the Aβ1-42 treatment group, the expression of TLR2 and Nf-κB in MSC-Exo treatment group was signi cantly decreased (Fig.4D, 4E). The above results are basically consistent with the results of the biometric analysis.

5.Aβ1-42 can promote M1 polarization of microglia in vivo
In order to verify the results of in vitro experiments, stereotactic injection device were used to inject Aβ1-42 with green uorescent labeled into the lateral ventricle (Fig.5A). Immuno uorescence showed the distribution range of green uorescence (Fig.5B) and the expression of Aβ1-42 in cortex and hippocampus after injected 1day, 3day and 14day (Fig.5C).Besides, the results of Immuno uorescence and western blot showed that compared with control group, the Aβ1-42 group signi cantly increase the expression of iNOS (Fig.6A,6B,6C,6H) 6.MSC-Exo nasal administration can inhibit the polarization of microglia through the TLR2/MYD88/Nf-κB pathway Three days after the injection of Aβ1-42 into the lateral ventricle, we administered MSC-Exo to the mouse by na sal injection. Three days after treatment, the mRNA and protein in brain tissues were taken for PCR and WB analysis. Frozen section of brain tissue was used for Immuno uorescence. PCR results showed that the exosomes intervention group signi cantly reduced the expression of IL-1β and TNF-α, and increased the expression of IL-10( Fig.6I-L). At the same time, the expression of related pathway genes and proteins were detected. Compared with the normal group, the Aβ1-42 group signi cantly increased the expression of TLR2,MYD88 and P-p65 protein (Fig.6A,6E-G). Compared with the Aβ1-42 group, Aβ1-42+MSC -Exo group signi cantly reduced the expression of the above proteins (Fig.6A,6E-G). The above results con rmed that TLR2/MYD88/Nf-κB may be an important pathway for MSC-Exo to inhibit microglia activation and neuroin ammation.

Discussion
Our research has found that in vivo and in vitro experiments have con rmed that exogenous administration of Aβ1-42 can promote the M1 polarization of microglia, and MSC-Exo can inhibit the effect of Aβ1-42 in promoting microglia polarization and reduce the release of in ammatory factors. Through Geo-shared chip biometric analysis and further con rmatory experiments, it is con rmed that the speci c pathway of this effect is: TLR2/MYD88/Nf-κB. Based on our research, we can have a closer understanding of the polarization of microglia caused by Aβ1-42, and have a deeper exploration of the mechanism by which MSC-EXO inhibits the polarization of glial cells and the development of neuroin ammation (Fig. 7). It has played a guiding role in the treatment of neuroin ammation and nerve damage caused by chronic degenerative diseases.
Aβ1-42 is a very important protein involved in neurodegenerative diseases, especially Alzheimer's disease [33,34]. A large number of previous studies focused on the study of Aβ1-42 on neuronal damage and cognitive dysfunction. With the deepening of research, more and more studies have found that neuroin ammation is also an important factor in the development of neurodegenerative diseases [35]. In a variety of brain injuries including traumatic brain injury and chronic traumatic encephalopathy, Aβ participates in the occurrence and development of neuroin ammation in the acute phase, especially in the chronic phase [36]. The speci c mechanism of action is still not very clear. Our research has veri ed the effect of Aβ1-42 in promoting the polarization of microglia both in vivo and in vitro. In vitro experiments found that this effect has a concentration-related effect of lipopolysaccharide, and the evidence of activation of related pathways was obtained through in vivo and in vitro experiments of bioinformatics analysis.
Mesenchymal stem cells (MSCs) have strong potential for self-renewal and multi-directional differentiation, as well as low immunogenicity and immunomodulatory properties. They are the most promising research objects [37,38]. Bone marrow mesenchymal stem cells (BMSCs) have signi cant tissue regeneration potential and can secrete signal molecules. For example, neurotrophic factors, growth factors and cytokines play a role in tissue regeneration and injury protection through paracrine action [39].
In recent years, a large number of experiments have veri ed the effect of MSC cell transplantation on Aβ metabolism and glial cell polarization in the brain, and some clinical trials have begun to progress, but the use of mesenchymal stem cells still has certain limitations [40,41]. The rst is the source of cells and the contamination of foreign bodies in the process of culturing and cryopreservation brings a certain risk of infection in the body. Some patients have symptoms such as fever and arrhythmia, and then the targeted injection of certain organs (heart and brain and other important organs). There is a certain degree of di culty, and it is di cult for cell therapy to reach a speci c target location that needs to be treated. The discovery of exosomes provides a safer and more targeted option for MSC research. As same as our research, some studies have found that MSC-Exo can alleviate the occurrence and development of in ammation in cardiac reperfusion injury, which can be regulated by the polarization of astrocytes in the brain alleviates the neuroin ammation caused by spinal cord injury [42]. In addition, it also has a more obvious therapeutic effect in the research of skin injury and bronchitis [43,44]. Compared with exosomes derived from other cells, the exosomes derived from mesenchymal stem cells have a single composition, a more obvious therapeutic effect, and are easier to obtain in vitro and massproduced [45,46]. For the use of mesenchymal stem cell exosomes, we used exosomes extracted from untreated simple cells. In the current study, some experimenters also used exosomes obtained after intervention with other substances, such as interferon gamma [47][48][49]. As with other related secretionpromoting substances, whether the use of activated exosomes is more bene cial to the treatment of diseases remains to be veri ed. The changes in the contents of activated exosomes still need to be further clari ed, so we chose to use simple exosomes derived from processed mesenchymal stem cells.
Toll-like receptors (TLR) are an important class of protein molecules involved in non-speci c immunity (natural immunity), and they are also a bridge between non-speci c immunity and speci c immunity. At present, there are different experimental results on the activation of TLR receptors of microglia by Aβ [50]. The most studied is the activation of TLR2 and TLR4 [51,52], and some studies believe that it is the coactivation of multiple TLR receptors [53][54][55]. So it needs to be closer to explore the synergy of receptor activation.
There are several de ciencies in our research. The rst is the use of exogenous Aβ1-42 to interfere with cells and brain tissues, which may deviate from the amount, time and range of Aβ1-42 produced by itself, and then Toll like receptors is affected by it.

Conclusion
In conclusion, this study not only demonstrates the effect of Aβ1-42 on microglia polarization, but also veri ed the mechanism of treatment of MSC-Exo on neuroin ammation induced by Aβ1-42. In subsequent studies, we will focus on exploring the speci c substance in MSC-Exo acts on microglia. In addition, we will also study the effect of MSC-Exo in neuronal cells and astrocytes after Aβ1-42 treatment.