Pyroptosis occures in microglia and Treg cell is increased after spinal cord injury
To investigate the spatiotemporal characteristics of pyroptosis after spinal cord injury, we performed a serious of experiments. Bioinformatics analysis of GSE5296 revealed the expression of pyroptosis-related genes increased in mice with spinal cord injury than in sham-operated mice on day 1 and was highest on day 7, suggesting that pyroptosis peaked on day 7 after SCI(Fig. 1A). GSDMD protein levels were also markly increased on day 7(Fig. 1B). Since the spinal cord contains a variety of cell types, including astrocytes (GFAP+), microglia (IBA1+), and neurons (NeuN+), we investigated whether pyroptosis occurs in one or more kinds of cells after spinal cord injury. Immunofluorescence analysis revealed that pyroptosis mainly occured in microglia in the injured spinal cord and was lessly occured in other cell types(Fig. 1C, D). Furthermore,We ascertained a increas population of the Foxp3 + Treg cell in the injured spinal cord using flow cytometry at days 7 after spinal cord injury(Fig. 1E). These results suggest that Treg cell may be important in the regulation of microglia pyroptosis following spinal cord injury.
Specific Treg-cell ablation in Foxp3DTR mice promotes microglia pyroptosis in vivo
To assess the effect of Treg cells on microglial pyroptosis after spinal cord injury, we selectively depleted Treg cells by diphtheria toxin (DT) injections in Foxp3DTR transgenic mice that express the DT receptor under control of the Foxp3 promoter. Immunofluorescence of injury regions revealed that Foxp3DTR + DT mice had more IBA1+/GSDMD+ microglia than Foxp3DTR + PBS mice(Fig. 2A, B). Moreover, The severity of pyroptosis was also determined by using westernblot to detect NLRP3, GSDMD, GSDMD-N, Pro-CASP-1,caspase1(p20) and IL-1β levels in the injury center 7 days after spinal cord injury(Fig. 2C, D). Foxp3DTR + DT mice have significantly more extensive microglia pyroptosis than Foxp3DTR + PBS mice one week after SCI, implying that Treg cell knockout promote microglial pyroptosis in vivo. BMS score demonstrated that Foxp3DTR + DT mice recovered significantly less than Foxp3DTR + PBS mice during the 4-week post-SCI recovery process (Fig. 2E). Foxp3DTR + DT mice had less movementr coordination and less effective gait recovery, as shown by footprint assessment, supporting the findings of the BMS (Fig. 2F, G). Foxp3DTR + DT mice also had poorer functional recovery in Swimming test (Fig. 2H, I).Furthermore, electrophysiological analysis revealed that Foxp3DTR + DT mice had smaller amplitudes and longer latencies of motor evoked potentials (MEPs) after SCI than Foxp3DTR + PBS mice(Fig. 2J, K). These behavioral tests indicate that deletion of Treg cell in mice following SCI results in poor functional recovery. These findings suggest that Treg cell deficiency causes extensive microglial pyroptosis and impairs functional recovery following spinal cord injury.
Increased Treg-cell infiltration in spinal cord attenuates microglia pyroptosis in vivo
To further assess the effect of Treg cells on microglial pyroptosis after spinal cord injury, We injected Treg cells in the tail vein of mices immediately after spinal cord injury, and the infiltration of Foxp3 + cells in the spinal cord was significantly increased on day 7 after spinal cord injury (Figure S1A).BMS score demonstrated that SCI + PBS mice recovered significantly less than SCI + Treg mice during the 4-week post-SCI recovery process (Fig. 3A). SCI + PBS mice had less movementr coordination and less effective gait recovery, as shown by footprint assessment, supporting the findings of the BMS ((Fig. 3B, C). Rotarod testing for posterior limb and trunk equilibrium exhibited superior motor recovery in SCI + Treg mice (Fig. 3D). Electrophysiologic test also revealed that SCI + PBS mice had smaller amplitudes and longer latencies of motor evoked potentials (MEPs) after SCI than SCI + Treg mice (Fig. 3E, F). These behavioral tests indicate that increased infiltration of Treg cell in mice following SCI results in better functional recovery. SCI + Treg mice have significantly less microglia pyroptosis than SCI + PBS mice one week after SCI (Fig. 3G). A quantitative analysis of IBA1+/GSDMD+ regions at the injury site revealed that SCI + Treg mice had less IBA1+/GSDMD+ microglia than SCI + PBS mice (Additional file 1: Figure S1B). Moreover, The severity of pyroptosis was also determined by using westernblot to detect NLRP3, GSDMD, GSDMD-N, Pro-CASP-1,p20 and IL-1β levels in the injury center 7 days after spinal cord injury(Fig. 3H). SCI + Treg mice had significantly lower levels of NLRP3, GSDMD, GSDMD-N, caspase1(p20) and IL-1β expression than SCI + PBS mice (Additional file 1: Figure S1C). These findings suggest that increased Treg cell promotes functional recovery following spinal cord injury and attenuates microglial pyroptosis.
Treg cell-derived exosomes attenuates pyroptosis in BV2 microglia in vitro
To further investigate the mechanism of microglia pyroptosis inhibition by Treg cells, we collected Treg-cell conditioned medium(TCM) after Treg cells were stimulated with anti-CD3 (1ug/ml) and anti-CD28 (10ug/ml). BV2 cells were co-cultured with TCM before being treated with LPS + ATP. microglia were immunofluorescently stained for IBA1 and GSDMD to assess pyroptosis. GSDMD expression was considerably lower in the LPS + ATP + TCM group in comparison to the group that received only LPS + ATP treatment. Because exosomes contribute significantly in intercellular communication by transferring hereditary material, we speculated Treg cells attenuates pyroptosis in microglia by secreting exosomes. In order to prevent Treg cells from secreting exosomes, we used GW4869. Co-cultured TCM pre-treated GW4869 restored the expression levels of GSDMD (Fig. 4A, B).
Exosomes derived from Treg cells aid in the recovering of motor performance and attenuates pyroptosis following SCI
To further explore the function of Treg cell-derived exosomes in the spinal cord injury micro-environment and the underlying effect on microglia pyroptosis, we extracted exosomes (Treg-Exos) from Treg cells culture supernatant and identified them using transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot. TEM showed classic nanoparticles with diameters between 50 and 150 nm, and NTA showed a semblable size distribution (Fig. 4C, D). Exosome surface markers like CD9, CD63, and CD81 were detected by Western blot (Fig. 4E). Exosomes that were Dil-labeled were taken up by microglia (Fig. 4F). The above investigations verified the exosome seperated from Treg supernatants.
Exosomes were injected at once following SCI, and behavioral tests were conducted at the times specified (Fig. 5A). According to BMS behavioral analysis, Treg-Exos injection improved motor function scores in the hind limbs following spinal cord injury (Fig. 5B). Footprint analysis, swimming tests and MEPs all yielded similar results, with Treg-Exos injection resulting in longer strides, higher MEP amplitude, smaller body and water surface angles, and a more upturned tail(Fig. 5C–H). When Treg-exos-treated group were compared to PBS group, immunofluorescence staining revealed a decrease in IBA-1 + /GSDMD + cells and fluorescence intensity of GSDMD (Fig. 5I, J).Moreover, Treg-exos-treated group had significantly lower levels of NLRP3, GSDMD, GSDMD-N,p20 and IL-1β expression than PBS group(Fig. 5K and Additional file 1: Figure S2A).
Moreover, The results of immunofluorescence showed that Treg-exosomes had better therapeutic efficacy in terms of treating microglia pyroptosis compare with Treg cell treatment, which may be due to the characteristics of smaller particle size and higher membrane permeability of exosomes, which allowed them to easily cross the blood-spinal barrier, thus exerting a better effect on inhibiting microglia pyroptosis (Additional file 1: Figure S2B-C).
Taken together, these findings suggest that Treg-Exos can promotes functional recovery after SCI and decrease the onset of microglial pyroptosis.
Exosomes deliver miR-709 to microglia
Treg-Exos promotes functional recovery after SCI and inhibits the onset of microglial pyroptosis, according to in vivo and in vitro studies. Exosomal miRNAs have been demonstrated in prior research to have regulating actions on target cells and may be crucial for the adjust of biological processes. miR-709 was screened by taking the intersection of the top 5 expressed miRNAs in Treg cells and Treg-Exos (Figure S3A). Moreover, miR-709 was markly increased in Treg-Exos-treated BV2 cells, indicating that exosomes can transfer miR-709 from Treg cells to BV2 cells (Additional file 1: Figure S3B).
Exosomes suppressed microglia pyroptosis and motor function recovery after SCI by delivering miR-709
Because the above study demonstrated that Treg-derived exosomal miR-709 can be transmitted to microglia, we wondered if miR-709 could act as a biological messenger between Treg cells and microglia, regulating microglia pyroptosis and motor function recovery after SCI. To explore the function of exosomal miR-709 in the Treg-Exos-regulated microglia pyroptosis after SCI, miR-709 overexpression (miR-709OE) and knockdown (miR-709KD) Treg cells using a lentiviral-based method as well as the corresponding negative control (miR-NCOE and miR-NCKD) were established.
The transfection efficiency was confirmed using qRT-PCR (Fig. 6A). Exosomes were isolated from miR-NCKD-Tregs, miR-709KD-Tregs, miR-NCOE-Tregs, and miR-709OE-Tregs named miR-NCKD-Exos, miR-709KD-Exos, miR-NCOE-Exos, and miR-709OE-Exos, respectively. A significant decrease in the expression of miR-709 in miR-709KD-Exos compared with the miR-NCKD-Exos,while an evident increase in the expression of miR-709 in miR-709OE-Exos when compared with the miR-NCOE-Exos was observed (Fig. 6B). Furthermore, the miR-709 expression level in the target BV2 microglia in the miR-709KD-Exos treatment group showed a dramatic decrease in expression compared with the miR-NCKD-Exos treatment group. The 709 expression levels in the target BV2 microglia in the miR-709OE-Exos treatment group showed an increase in expression compared with the miR-NCOE-Exos treatment group (Fig. 6C). To assess pyroptosis, BV2 cells were then treated with LPS + ATP. Fluorescent intensity of GSDMD was markedly decreased by addition of miR-709OE-Exos compared with miR-NCOE-Exos treatment group, while miR-709KD-Exos treatment strongly enhanced GSDMD expression compared with miR-NCKD-Exos treatment group (Fig. 6D, E). Furthermore, expression of pyroptosis proteins detected by western blot demonstrated similar results with those discussed above (Additional file 1: Figure S4A-D).
miR-709OE-Exosomes, miR-709KD-Exosomes and their negative control were respectively injected WT mice immediately after SCI, and behavioral assessments were conducted at the indicated times. According to BMS behavioral analysis, the miR-709OE-Exosomes enhanced the effect of Treg-Exos on improving hindlimb motor function following spinal cord injury, while miR-709KD-Exos treatment reduced the effect of Treg-Exosomes (Fig. 7A). Rotarod testing and MEPs all produced similar results (Fig. 7B–D). Immunofluorescence staining in the miR-709OE-Exos group compared to the miR-NCOE-Exos group revealed an decrease fluorescence intensity of GSDMD, while miR-709KD-Exos group compared miR-NCKD-Exos group exhibited an increased fluorescence intensity (Fig. 7E, F). These findings suggest that Treg-Exos, by delivering miR-709, inhibits microglia pyroptosis and promotes motor function recovery after spinal cord injury.
miR-709 negatively regulates NKAP
To investigate the underlying mechanism of action of exosomal miR-709. According to the online database of miRNA targets was used to search the predicted mRNA targets for miR-709. NKAP may be a potential target of miR-709 (Fig. 8A). Moreover, NKAP has been shown to play an active role in inflammation. To confirm that the NKAP 3'UTR is a direct target of miR-709, NKAP wild-type (WT) and mutant (MUT) 3'UTR sequences were created and cotransfected into 293T cells with miR-709 sequences.The luciferase reporter assay showed that miR-709 overexpression greatly reduced luciferase activity when co-transfected with WT-3' UTR of NKAP compared to control, but no inhibition activity of miR-709 was observed when co-transfected with MUT-3' UTR of NKAP (Fig. 8B). Further analysis using qRT-PCR and western blot assays showed that miR-709 knockdown increased NKAP mRNA and protein levels while miR-709 overexpression decreased NKAP expression(Fig. 8C, D).
The impacts of miR-709KD-Exos on microglia are restored by NKAP silencing
To further explore the connection between exosomal miR- 709 and NKAP, some in vitro rescue trails were carried out.Using shRNA, the expression of NKAP was suppressed in BV2 cell. Results revealed that inhibiting NKAP during co-treatment with miR-709KD-Exos reduced the activation of microglia pyroptosis (Fig. 8E, F). Moreover, western blot supported these findings (Fig. 8G). As a result of these rescue experiments, it was demonstrated that shNKAP in microglia can abolish the facilitating role of miR709KD-Exos in promoting the microglia pyroptosis.
The effects of miR-709OE-Exos on microglia are eliminated by overexpressing NKAP
NKAP was overexpressed by transfection with a NKAP lentivirus in microglia. Results demonstrated that overexpression of NKAP promoted the activation of microglia pyroptosis during co-treatment with miR-709OE-Exos(Fig. 8H, I). Western blot analysis also confrmed these results (Fig. 8J). Therefore, it was concluded that exosomal miR-709 suppresses the microglia pyroptosis by targeting NKAP.