bMSCs and bMSCs derived exosomes induced transformation of microglial polarization towards the anti-inflammatory phenotype
Exosomes were isolated from bMSCs cells by ultracentrifugation. The exosomes were then identified using TEM, nanoparticle tracking analysis (NTA) and western blot analysis. A typical cup-shaped membrane vesicle morphology was observed (Figure 1). The size distribution profiles from the nanoparticle tracking analysis revealed that most of the vesicles had a diameter of ~130 nm. The original concentration of the exosomes was 6.3×1010 particles/ml. Western blotting analysis further revealed that exosome markers TSG101 and HSP70 were expressed in the exosomes (Figure 1).
Figure 1:Identification and NTA analysis of bMSCs derived exosomes. TEM results revealed the membrane vesicle had a typical cup-shaped morphology. Scale bar =100 nm. NTA revealed that most vesicles had a diameter of ~130 nm. The original concentration of the exosomes was 6.3×1010 particles/ml. Western Blot further revealed that exosome markers TSG101 and HSP70 were expressed in the exosomes.
BV2 microglia cells were activated using LPS (1000ng/ml) and then incubated at 37℃ for 48 hours prior to coculture with bMSCs and exosomes. They were then cocultured with bMSCs and exosomes for 48 hours to facilitate the polarization of activated BV2 cells toward the anti-inflammation type (Figure 2A). This was done to investigate the effects of bMSCs and bMSCs derived exosomes on the polarization phenotype of activated BV2 cells in a co-culture system. The expression of anti-inflammation marker Arg1 increased significantly in the MG+bMSCs and MG+Exo groups, while the expression of pro-inflammation marker iNOS decreased compared to that of the MG group. Flow cytometry analysis was then performed to determine the proportion of pro-inflammatory and anti-inflammatory phenotype cells. The BV2 cells were labelled using FITC-conjugated anti-IBA1 antibodies, APC-conjugated anti-CD86 antibodies, and PE-conjugated anti-CD206 antibodies. The Q1 area indicated Iba-1/CD86 double-positive BV2 cells while the Q3 area indicated IBA-1/CD206 double-positive BV2 cells (Figure 2B). In the same line, activated BV2 cells were transformed to the anti-inflammatory phenotype after 48 hours of co-culturing with bMSCs and exosomes (Figure 2C). Moreover, the exosomes were associated with a stronger anti-inflammatory effect.
Figure 2: Effects of bMSCs and exosomes on the phenotype of activated BV2 microglia.A. Immunofluorescence results of DAPI (blue), Arg1+ (red) and iNOS+ (green) cells. B, C. Flow cytometry analysis of the proportion of IBA1/CD86 double-positive and IBA1/CD206 double-positive cells.
bMSCs and exosomes promoted the expression of anti-inflammatory factors
Phenotype transformation is accompanied with functional change. As such, RT-qPCR was used to detect the expression of BV2 cells-related inflammatory factors after 48 hours of co-culture with bMSCs and exosomes. Both bMSCs and exosomes inhibited the expression of pro-inflammatory factors (IL1β, IL6, TNFα) but promoted the expression of anti-inflammatory factors (IL10, TGFβ). The exosomes played a stronger role in regulating the expression of related inflammatory factors (Figure 3).
Figure 3: The expression of inflammatory factors of BV2 cells in the co-culture system.
** Differences between the three groups were all statistically significant (P<0.05).
* The difference between the MG and the MG+Exo group was statistically significant (P<0.05).
Exosomes decreased neuronal apoptosis after induction of TBI in mice.
The lesion area and the neuron apoptosis in each group was determined after 7 days of TBI to detect the effects of exosomes on the nervous system after TBI in vivo (Figure 4). The damaged area of the mice in the TBI group and TBI + Saline group were larger compared with those of mice in the TBI + Exo group (Figure 4A & B). Brain sections were then subjected to TUNEL staining followed by quantification of the TUNEL-positive cells. Red staining represented the TUNEL positive cells (indicated by white arrows). The quantities of TUNEL positive cells in the sham group, TBI group, TBI + Saline group, and TBI + Exo group were 0.4 ± 0.55, 42.1 ± 4.30, 40.8 ± 5.26, and 22.2 ± 3.03, respectively. The TBI + Exo group had significantly fewer apoptotic neurons than the TBI group and the TBI+ Saline group (Figure 4C & D).
Figure 4: Exosomes induced reduction of the injured area and neuronal apoptosis. A. Nissl staining of the Ⅰ) sham group, Ⅱ) TBI group, Ⅲ) TBI + Saline group, Ⅳ) TBI + Exo group. B. The measured lesion areas of A. C. Quantification of D shows the difference of TUNEL+ cells. D. TUNEL staining of the four groups. Scale bar = 0.2 mm. *, P<0.05.
Exosomes inhibited inflammation of the brain tissue after TBI
An in-vivo experiment was designed in which exosomes were injected through the tail vein followed by induction of the TBI model to examine whether exosomes could inhibit inflammation in the CNS after induction of TBI.The expression levels of Arg1, iNOS, and inflammatory factors were then detected by immunofluorescence, western blotting and real time qPCR (Figure 5). The proportion of Arg1+ cells in the TBI+Exo group was significantly higher than that of the other groups (Figures 5A & B). In the same line, the expression level of Arg1 in the TBI+Exo group was significantly higher than that of the other groups. However, the expression of iNOS in the TBI+Exo group was significantly lower than that of the TBI and TBI+Saline group (Figure 5C, P<0.05). Figure 5D-(Ⅰ, Ⅱ, Ⅲ) shows the expression levels of the related inflammatory factors after 1 day, 3 days, and 7 days, respectively from the onset of TBI.Evidently, the expression levels of IL-10 and TGF-β in the TBI +Exo group were higher than that of the other two groups on day1, 3, and 7 post TBI. Moreover, the expression levels of IL-1β and TNF-α in the TBI+Exo group were lower than those of the TBI and TBI+Saline groups on day 3 and 7 post TBI. In the same line, injection of exosomes upregulated the expression of STAT3 but inhibited the expression of NFκB (Figure 5D-Ⅳ).
Figure 5: Effects of exosomes on Neuroinflammation. A. Immunofluorescence assay of Arg1 on brain slices 7 days post TBI. Scale bar = 200 um B. The proportion of Arg1+ cells in A. (*. P<0.05) C. The relative expression of Arg1 and iNOS tested by Western Blotting. (*. P<0.05) D. Ⅰ, Ⅱ, and Ⅲ respectively shows the expression levels of inflammatory factors on day 1, 3, and 7 post TBI (*. P<0.05 between TBI+Exo and TBI group; **. P<0.05 between TBI+Exo and the other two groups.). Ⅳ) The different expression levels of NFκB and STAT3. (**.P<0.05 between TBI+Exo and the other two groups).
miRNA-181b was highly expressed in both exosomes and brain tissues
miRNA sequencing of bMSCs cells and exosomes derived from bMSCs cells was done to explore the effective components of exosomes. Sequencing results revealed that there were more than 500 miRNAs highly expressed and more than 300 microRNAs lowly expressed in the exosomes compared to the bMSCs cells. Parts of the differentially expressed microRNAs are shown in the heat map and volcano plot (Figures 6A & B). Based on microRNAs reported in other relevant studies, let-7c, miR-124, miR-21a, and miR-181b were chosen to be intensively studied. The expression levels of these microRNAs in brain tissues of mice in the TBI + Exo group at day 7 post TBI were thus determined. miR-181b was found to be highly expressed in the TBI + Exo group (Figure 6C). This strongly suggested that miR-181b could inhibit neuroinflammation and regulate the phenotype of the microglia by up-regulating the STAT3 related pathway.
Figure 6: Differential expression of microRNAs. A. heat map of differentially expressed microRNAs between bMSCs and exosomes. B. volcano plot of the differentially expressed microRNAs (Blue represents low expression while red denotes high expression in exosomes. Gray denotes lack of differences between them.) C. The differentially expressed microRNAs in vivo. *, P<0.05
Up-regulation of microRNA-181b inhibited neuronal apoptosis, reduced neuroinflammation and regulated the phenotype of microglia
Lentiviral vectors were used to obtain different expression levels of miR-181b to verify the role of miR-181b in the neuroinflammation process post TBI. The expression level of miR-181b was detected 7 days after transfection of the mice with the lentivirus. The expression level of miR-181b in the up-regulation and down-regulation group was 2.7 times higher and 0.4 times lower than that of the normal C57BL/6 mice, respectively (Figure 7B). Neuron apoptosis detected in each group at day 7 post TBI revealed that the numbers of TUNEL-positive cells in the three groups were 38.2±3.76, 35.6±4.50, and 24.8±1.94, respectively (Figures 7A & C). Notably, the number of TUNEL-positive cells in the miR-181b up-regulation group was significantly low than the other two groups. In the same line, the microglia markers Arg1 and iNOS, and the transcriptional regulator STAT3 were detected by Western Blotting (Figure 7D). The TBI-up group had a higher expression level of Arg1 and STAT3, but a lower expression level of iNOS. Inflammatory factors were also detected at day 1, 3, and 7 post TBI (Figure 7 E- I, II & III). The expression level of IL-10 and TGF-β in the TBI-up group was significantly higher than that of the other two groups on the 1st, 3rd, and 7th day post TBI (P<0.05). However, the expression level of IL-1β in the TBI-up group was significantly lower than that of the other two groups on the 1st, 3rd and 7th day post TBI (P<0.05). Similarly, the expression level of TNF-α in the TBI-up group was significantly lower than that of the other two groups on the 3rd and 7th day post TBI (P<0.05). Nonetheless, the expression level of IL-6 was not significantly different among the groups on the 1st, 3rd, and 7th day post TBI. Further analysis of the TBI-up group revealed that the expression levels of IL-10 and TGF-β gradually increased after TBI (Figure 7E-Ⅳ).
Figure 7: The effect of different expression levels of miR-181b on neuronal apoptosis and neuroinflammation. A. TUNEL staining showing the levels of neuronal apoptosis in the three groups. Scale bar = 0.2 mm. B. The expression level of miR-181b in the three groups 7 days after the transfection of lentivirus. C. Quantification of TUNEL-positive cells of A (*. P<0.05). D. The expression of microglia markers Arg1 and iNOS as well as the transcriptional regulator STAT3. E. The expression of inflammatory factors on the 1st, 3rd, and 7th day post TBI in the three group (Ⅰ, Ⅱ, Ⅲ) (**. P<0.05 between TBI-up and the other two groups.) The dynamic changes of inflammatory factors in TBI-up group (Ⅳ). Dpi: days post injury.