SHED-exos increase saliva secretion and ameliorate lymphocytic infiltration in the SMGs of NOD mice
SHED-exos extracted from SHEDs culture supernatant were identified and showed a standard microstructure (Figure 1A), and they expressed the exosome-associated proteins CD9, CD63, C81, and HSP70 (Figure 1B). The mean diameter of SHED-exos was 126.5±5.7 nm (Figure 1C). The stimulated saliva flow rate was significantly decreased in 14-week-old NOD mice and further dropped in 21-week-old NOD mice compared with 7-week-old NOD mice and age-matched BALB/c mice (Figure 1D). To explore the therapeutic effect of SHED-exos on sialadenitis, we injected 50 µg SHED-exos into the SMGs of 14-week-old NOD mice and sacrificed the mice at 21 weeks. SHED-exos significantly increased the stimulated saliva flow rate compared with those in age-matched untreated and PBS-treated groups. Moreover, quantitative analysis showed that the focus score and the ratio index were increased in 21-week-old NOD mice, but both of them were significantly decreased in the SHED-exo-treated group compared with the age-matched untreated and PBS-treated groups (Figures 1F and G). These results suggest that SHED-exos injected into SMGs increase saliva secretion and reduce lymphocytic infiltration in the SMGs of NOD mice.
SHED-exos are taken up by glandular epithelial cells
To investigate the distribution of SHED-exos, PKH-26-exos or DiR-exos were injected into the SMGs. We found that PKH-26-exos-positive signals were expressed intensively on the 1st day and appeared to be more uniformly distributed throughout the glandular tissues on the 3rd and 7th days (Figure 2A). In addition, DiR-exos were infused into the glands. As shown in Figures 2B-D, DiR-exos were distributed around the neck and precisely in the SMG. The heart, lung, liver, spleen, kidney, pancreas, and intestine tissues showed negative intensity (Figure 2E). Further observation showed that the positive signals in SMGs lasted for more than 40 days in 14-week-old mice (Figures 2F-J).
Next, we determined whether the exosomes were taken up by glandular epithelial cells. In vitro, PKH-26-exos were cultured with SMG-C6 cells, primary cultured acinar and duct cells of human SMG for 24 h. As shown in Figure 2K, there were positive signals in the cytoplasm of SMG-C6 cells, human acinar cells and duct cells, which suggest that SHED-exos can be ingested by salivary glandular epithelial cells to perform further functions.
SHED-exos upregulate the expression of the tight junction proteins ZO-1 and occludin
Fluid secretion can be accomplished through either the aquaporin 5 (AQP5)-mediated transcellular or tight junction-mediated paracellular route [25, 26]. We collected labial gland tissues from SS patients and found that the expression levels of AQP5, ZO-1, occludin, and claudin-4 were significantly decreased compared with those in controls. SHED-exos incubation with labial gland tissues from SS patients for 24 h remarkably increased the expression of ZO-1 and partially recovered the level of occludin but did not affect the contents of AQP5 and claudin-4 (Figures 3A-D). When SMG-C6 cells were cultured with SHED-exos for 24 h, the expression of ZO-1 and occludin was increased (Figures 3E-G). In addition, considering that the redistribution of tight junction proteins also affects their function, we further examined the location of ZO-1 and occludin. As shown in Figure 3H, SHED-exos did not change their distribution in SMG-C6 cells.
ZO-1 is required for the SHED-exo-induced increase in paracellular permeability
TER is an important indicator used to evaluate the function of tight junctions, and decreased TER is associated with increased paracellular permeability. In this study, the basal TER value of untreated SMG-C6 monolayers was 589 ± 23.31 Ω cm2, which was consistent with our previous studies . SHED-exos induced a visible drop in TER values at 24 h and 48 h (Figures 3I). These results suggest that the therapeutic effect of SHED-exos in sialadenitis may involve enhancing ZO-1 and occludin expression and improving paracellular permeability in SMGs.
ZO-1 plays crucial roles in both basal salivary epithelial barrier function and paracellular transport . To confirm that the increased paracellular permeability of SHED-exos was related to ZO-1, we conducted ZO-1 depletion and rescue experiments. Compared with control cells, ZO-1 protein expression markedly reduced in ZO-1 knockout cells and recovered in ZO-1 rescue cells (Figure 3J). Compared with the control cells, ZO-1 knockout did not affect the basic TER values, which was consistent with our previous study . Furthermore, the decreased TER values induced by SHED-exos were abolished in ZO-1 knockout cells and reappeared in ZO-1 rescue cells (Figure 3K), which suggests that ZO-1 is required for the SHED-exo-induced increase in paracellular permeability.
The Akt/GSK-3β pathway mediates SHED-exo-induced ZO-1 expression and increased paracellular permeability
To explore the regulatory mechanism of SHED-exos on ZO-1, we performed a miRNA microarray of SHED-exos, and 180 exosomal miRNAs were identified and profiled, as shown in Table 1. KEGG pathway classification analysis was performed (Figure 4A left panel). The majority of the target genes mediating signal transduction were further listed in the right panel of Figure 4A, which suggested that the phosphatidylinositol 3 kinase (PI3K)-protein kinase B (PKB; Akt) pathway might play an important role. Moreover, in SHED-exo-treated SMGs of NOD mice, the ratios of p-Akt/Akt and p-GSK-3β/GSK-3β were decreased, but ZO-1 was increased (Figures 5A-E). In vitro, SHED-exos incubation for 24 h decreased the expression of p-Akt/Akt and p-GSK-3β/GSK-3β and increased the ZO-1 levels in SMG-C6 cells. Pretreatment with insulin-like growth factor 1 (IGF1), an Akt upstream molecule PI3K agonist, abolished SHED-exo-induced responses. IGF1 alone increased p-Akt/Akt and p-GSK-3β/GSK-3β expression but decreased ZO-1 expression (Figures 5F-J). These results suggest that Akt/GSK-3β signaling molecules negatively regulates ZO-1 expression and that SHED-exos increase ZO-1 expression via the Akt/GSK-3β pathway.
Furthermore, SHED-exos decreased the TER level, which could be attenuated by IGF1 preincubation. IGF1 treatment alone increased TER levels (Figure 4K). To further reveal whether ZO-1 was involved in the SHED-exo-induced increase in paracellular permeability via the Akt/GSK-3β pathway, a TER assay was performed on ZO-1 knockout cells. As shown in Figure 5L, SHED-exos- or IGF1-induced changes in the TER value disappeared in ZO-1 knockout SMG-C6 cells. These results suggest that the increased paracellular permeability induced by SHED-exos is related to Akt/GSK-3β pathway targeting at ZO-1.
Slug is involved in the SHED-exo-induced decrease in ZO-1
Slug, a Snail family transcription factor and the downstream signaling molecule of GSK-3β, is reported to act as a transcriptional repressor of several tight junction proteins, such as claudin-1, occludin, and ZO-1 in MDCK cells and claudin-3 in SMG-C6 cells [29, 30]. In the present study, SHED-exos decreased the Slug level in both SMG tissues (Figures 5A and D) and SMG-C6 cells (Figures 5M and N) but did not change Snail expression (Figures 5M-O). Moreover, IGF1 preincubation abolished the SHED-exo-induced Slug decrease and ZO-1 increase responses. IGF1 alone increased Slug expression but downregulated ZO-1 levels (Figures 5F and I-J). These results suggest that Slug acts as a transcriptional repressor of ZO-1 expression. To further reveal whether Slug binds to the ZO-1 gene directly, the promoter region of the rat ZO-1 gene was isolated and fused to the luciferase reporter vector. Transient transfection assays in the presence of pCMV6-Slug revealed that Slug significantly repressed wild-type ZO-1 promoter activity (Figure 5P). These results suggest that SHED-exos suppress Slug expression by inhibiting the Akt/GSK-3β pathway, thereby decreasing the transcriptional inhibition of Slug to ZO-1 and finally enhancing ZO-1 expression.
SHED-exo intraductal infusion into the SMG restores saliva secretion in NOD mice
Stem cell-based cell therapy is commonly performed using intravenous injection or local administration. For the exocrine glands, intraductal infusion is also a good choice. To facilitate the clinical application of SHED-exos, we infused SHED-exos through the orifice of the submandibular duct in 14-week-old NOD mice and collected the SMGs at 21 weeks. As shown in Figure 6A, DiR-exo intensities were observed in the neck area on the 1st day and 14th day after infusion and even on the 49th day. As expected, the saliva flow rate was significantly increased in SHED-exo-infused mice compared with that in the PBS group (Figure 6B). After treatment, inflammatory cell infiltration in the SMG was alleviated. The focus scores and ratio index were decreased in the glands infused with SHED-exos compared with those in the PBS group (Figures 6C-E). Moreover, p-Akt/Akt, p-GSK-3β/GSK-3β, and Slug expression was decreased, and ZO-1 was increased in SHED-exo-infused glands compared with PBS controls (Figures 6F-J).