In the present experiments, we first evaluated delivery of DFAT cells intravenously infused in SCG mice. Almost all of the DFAT cells were trapped in the lung and did not reach the kidney. Despite the non-delivery of DFAT cells into the kidney, the implantation of DFAT cells improved renal function (decrease in BUN), suppressed the expression of inflammatory cytokine MCP-1, and decreased urinary excretion of protein in SCG mice. We also previously showed that DFAT cells intravenously infused were trapped mainly in the lungs without reaching the kidneys, and implantation of DFAT cells reduced proteinuria and improved glomerulosclerosis and interstitial fibrosis in mAb 1-22-3-induced glomerulonephritis in rats. Moreover, the systematic implantation of DFAT cells through the vein was more effective in improving mAb 1-22-3-induced glomerulonephritis than direct implantation of DFAT cells through the renal artery . Thus, it is surmised that the DFAT cells trapped in lungs released substrates that may have then reached the kidney to improve ANCA glomerulonephritis in the SCG mice. Mechanisms underlying the immunosuppressive effects of the MSCs trapped in the lungs after intravenous implantation have been reported to be associated with exosomes including cytokines, miRNAs and peptides that improve acute graft-versus-host disease and immune-induced acute kidney injury [14, 15]. In terms of the mechanisms underlying the induction of immunosuppressive and anti-inflammatory effects by lung-trapped MSCs and DFAT cells, these effects of the implantation of MSCs are associated with the secretion of soluble factors with paracrine actions that are mediated by exosomes. Exosomes are predominantly released from the endosomal compartment and contain miRNA, cytokines, and proteins from MSCs. Recent studies in animal models suggest that exosomes have significant potential as a novel alternative to whole-cell therapies . Bruno et al.  showed that exosomes derived from MSCs improve acute tubular injury. Chaubey et al.  reported that implantation of MSCs improved experimental bronchopulmonary dysplasia in part via exosome-associated factor TSG-6. Thus, DFAT cell-derived exosomes may improve ANCA glomerulonephritis by increasing TSG-6 in kidney. It is thought that the direct infusion of exosomes from ex vivo-cultured DFAT cells will be effective immunosuppressive therapy for ANCA glomerulonephritis.
In the present experiments, the single implantation of DFAT cells was rather more effective in improving renal function, decreasing MPO-ANCA titer, and increasing the expression of TSG-6 in the kidney of SCG mice than the triple implantations of DFAT cells. Recently, single implantation has been also reported to show rather effective immunosuppressive effects on autoimmune-induced colitis in mice  and osteoarthritis in a clinical study . Thus, the single implantation of DFAT cells effectively induced the immunosuppression of ANCA glomerulonephritis, which may be associated with exosome release from the lungs.
We confirmed the ANCA glomerulonephritis as cellular crescent formation in kidney from the 12-week-old SCG mice. Neumann et al.  showed that the number of glomerular crescent formations increased along with aging. In addition, plasma levels of MPO-ANCA concentrations in SCG mice were higher than those in ICR mice. Implantation of DFAT cells decreased the GIS but did not affect the TIS of kidney from ICR mice. These results indicate that implantation of DFAT cells mainly improved the glomerular injury whereas their implantation did not appreciably affect nephrotubular degeneration.
Concerning the immunosuppressive effects of MSCs, two mechanisms have been considered to regulate immunoactivity . One mechanism has been reported in which MCSs release TSG-6 that suppresses adhesion molecule CD44 on T cells to inhibit T-cell activity and cell infiltrations, by which the regulatory T cells are increased to obtain immune tolerance . In the other reported mechanism, MSCs release PGE2 that induces the transition of M1 to M2 macrophages .
In our previous study, we observed that the systematic implantation of DFAT cells effectively ameliorated mAb 1-22-3-induced glomerulonephritis through immunosuppressive effects accompanied by the suppression of macrophage infiltration and expression of IL-6, IL-10 and IL-12β, and increased the production of serum and renal TSG-6, which improved the mAb 1-22-3-induced renal degeneration through the immunosuppressive effects of TSG-6 .
In the present experiments, the implantation of DFAT cells markedly increased the expression of TSG-6 mRNA and proteins in kidney from SCG mice, even though no DFAT cells were delivered into the kidney. The implantation of DFAT cells did not affect the number of regulatory T cells in spleen from SCG mice. However, implantation of DFAT cells decreased the expression of CD44 mRNA in kidney from these mice, thus suggesting that DFAT cells may regulate immunoactivity by suppression of the activity and invasion of T cells.
Moreover, expression of PGE2 and IL-10 mRNAs in the present experiments was increased in kidney from SCG mice after the implantation of DFAT cells. After the implantation of DFAT cells, the expression of MCP-1 as M1 macrophage chemokines was decreased in kidney of SCG mice, and that of CCL17 as a chemokine for M2 macrophages was increased in kidney of SCG mice. These results indicate that mechanisms underlying the immunoregulatory effects of DFAT cells appear to be associated with induction of the transition of M1 to M2 macrophages with production of inflammatory cytokine IL-10.
Because the implantation of DFAT cells may improve ANCA glomerulonephritis through increases in TSG-6 in kidney, which may be mediated by exosomes, we analyzed the expression of miRNAs in plasma, kidney and lungs in SCG mice after the implantation of DFAT cells. Expression of miR23b-3p was obviously higher in plasma, kidney and lung from these mice with implantation of DFAT cells compared to those in plasma, kidney and lung from SCG mice without implantation of DFAT cells. These results indicate that the increases in TSG-6 in kidney from SCG mice may be mediated by miR23b-3p delivered by the exosomes.
In the present experiments, the survival rate was higher in SCG mice with rather than without the implantation of DFAT cells. Longer-term investigations of the survival rate and side effects such as tumor genesis are needed for application of the implantation of DFAT cells for ANCA glomerulonephritis as the average survival period of SCG mice is only 120 days.