Two independent modes of kidney stone suppression achieved by AIM/CD5L and KIM-1.

The prevalence of kidney stones is increasing and its recurrence rate within the first 5 years is over 50%. No treatments that prevent the occurrence/recurrence of stones have reached the clinic. Here, we show that AIM (also called CD5L) suppresses stone development and improves stone-associated physical damages. The N-terminal domain of AIM associates with calcium oxalate crystals via charge-based interaction to impede the development of stones, whereas the 2nd and C-terminal domains capture the inflammatory DAMPs to promote their phagocytic removal. Accordingly, when stones were induced by glyoxylate in mice, recombinant AIM (rAIM) injection dramatically reduced stone development. Expression of injury molecules and inflammatory cytokines in the kidney and overall renal dysfunction were abrogated by rAIM. Among various negatively charged substances, rAIM was most effective in stone prevention due to its high binding affinity to crystals. Furthermore, only AIM was effective in improving the physical complaints including bodyweight-loss through its DAMPs removal effect. We also found that tubular KIM-1 may remove developed stones. Our results could be the basis for the development of a comprehensive therapy against kidney stone disease.

Novel Complement C5 Small-interfering RNA Lipid Nanoparticle Prolongs Graft Survival in a Hypersensitized Rat Kidney Transplant Model.

BACKGROUND: Prophylaxis of antibody-mediated rejection (AMR) caused by donor-specific antibodies remains challenging. Given the critical roles of complement activity in antibody-mediated graft injury, we developed a lipid nanoparticle (LNP) formulation of small-interfering RNA against complement C5 (C5 siRNA-LNP) and investigated whether C5 siRNA-LNP could downregulate the complement activity and act as an effective treatment for AMR. METHODS: Lewis recipient rats were sensitized by skin grafting from Brown Norway donor rats. Kidney transplantation was performed at 4 wk post-skin grafting.C5 siRNA- or control siRNA-LNP was administered intravenously, and the weekly injections were continued until the study's conclusion. Cyclosporin (CsA) and/or deoxyspergualin (DSG) were used as adjunctive immunosuppressants. Complement activity was evaluated using hemolysis assays. The deposition of C5b9 in the grafts was evaluated using immunohistochemical analysis on day 7 posttransplantation. RESULTS: C5 siRNA-LNP completely suppressed C5 expression and complement activity (hemolytic activity ≤ 20%) 7 d postadministration. C5 siRNA-LNP in combination with CsA and DSG (median survival time: 56.0 d) prolonged graft survival compared with control siRNA-LNP in combination with CsA and DSG (median survival time: 21.0 d; P = 0.0012; log-rank test). Immunohistochemical analysis of the grafts revealed that downregulation of C5 expression was associated with a reduction in C5b9-positive area ( P = 0.0141, Steel-Dwass test). CONCLUSIONS: C5 siRNA-LNP combined with immunosuppressants CsA and DSG downregulated C5 activity and significantly prolonged graft survival compared with control siRNA-LNP with CsA and DSG. Downregulation of C5 expression using C5 siRNA-LNP may be an effective therapeutic approach for AMR.

Role of Fractalkine-CX3CR1 Axis in Acute Rejection of Mouse Heart Allografts Subjected to Ischemia Reperfusion Injury.

Transplantation outcomes are affected by the increase in rejection associated with ischemia reperfusion injury (IRI). Fractalkine (FKN), a chemokine for recruitment of CX3CR1+ leukocytes, contributes to the pathogenesis of various inflammatory diseases. Herein, we evaluated the importance of the FKN-CX3CR1 axis during IRI-related rejections using a mouse heterotopic heart transplantation model. FKN expression and graft survival was compared between wild-type C57BL/6 recipients transplanted with BALB/c hearts preserved for 8 (WT-IRI) and 0.5 h (WT-control) at 4°C. Graft survival of WT-IRI was shorter than that of WT-control. FKN was expressed on the vascular endothelium in WT-IRI allografts, but minimally in WT-control. The role of the FKN-CX3CR1 axis in IRI-related rejection was directly investigated using the transplant model with CX3CR1-deficient recipients (CX3CR1 KO-IRI) or treatment with anti-mouse FKN monoclonal antibodies. Graft survival of CX3CR1 KO-IRI was longer than that of WT-IRI; antibody treatment prolonged graft survival. The contribution of CX3CR1+ monocytes to IRI-related rejection was evaluated by adoptive transfer to CX3CR1 KO-IRI. Adoptive transfer of CX3CR1+ monocytes attenuated the effect of prolonged graft survival in CX3CR1 KO-IRI. Overall, the FKN-CX3CR1 axis plays a major role during IRI-related rejection; its blockade has the potential to improve the outcomes of deceased donor transplantation.

Combination therapy of an iNKT cell ligand and CD40-CD154 blockade establishes islet allograft acceptance in nonmyeloablative bone marrow transplant recipients.

AIMS: Islet transplantation is an effective therapeutic option for type 1 diabetes. Although maintenance immunosuppression therapy is required to prevent allogeneic rejection and recurrence of autoimmunity, long-term allograft survival has not yet been achieved partly because of its adverse effects. The induction of donor-specific immunotolerance is a promising approach for long-term allograft survival without maintenance immunosuppression therapy. We previously reported that combination therapy using a liposomal ligand for invariant natural killer T cells, RGI-2001, and anti-CD154 antibody established mixed hematopoietic chimerism for the induction of donor-specific immunotolerance. This study investigated whether the protocol could promote islet allograft acceptance in experimental diabetes. METHODS: Streptozotocin-induced diabetic BALB/c mice were transplanted with bone marrow cells from C57BL/6 donors and received combination therapy of RGI-2001 and anti-CD154 antibody after 3-Gy total body irradiation. 3 Weeks after bone marrow transplantation, islets isolated from C57BL/6 donors were transplanted under the kidney capsule. RESULTS: Mixed chimerism was established in diabetic mice receiving the tolerance induction protocol. After islet transplantation, blood glucose levels improved and normoglycemia persisted for over 100 days. Hyperglycemia recurred after islet grafts were removed. Histopathological examinations showed insulin-positive staining and absence of cellular infiltration in the islet grafts. T cells of recipients showed donor-specific hyporesponsiveness, and anti-donor antibodies were not detected. CONCLUSIONS: The tolerance induction protocol with combination therapy of RGI-2001 and anti-CD154 antibody promoted islet allograft acceptance in a mouse diabetic model. This protocol may be clinically applied to islet transplantation for type 1 diabetes mellitus.

Impact of activated invariant natural killer T cells on the expansion of regulatory T cell precursors in murine thymocytes in vitro.

Tolerance induction is a goal of clinical transplantation to prevent graft rejection without the lifelong use of immunosuppressive drugs. In a series of mouse studies, we previously reported that the establishment of mixed chimerism by treatment with a ligand for invariant natural killer T (iNKT) cells with CD40 signal blockade makes it possible to prevent allograft rejection without immunosuppressants, and this approach fails in thymectomized recipient mice. In this study, we showed that iNKT cells in murine thymocyte cultures are indispensable for the expansion of CD4+CD25+Foxp3+ regulatory T (Treg) cells as well as CD4+CD25+Foxp3- cells, which contained precursor Tregs (preTregs). After the culture of BALB/c mouse-derived thymocytes in the presence of α-galactosylceramide (α-GalCer), a representative ligand for iNKT cells, the ratio of CD4+CD25+Foxp3- preTregs to total CD4+CD8- T cells was much higher than that of CD4+CD25+Foxp3+ Treg cells, regardless of anti-CD40 L mAb treatment. The proliferation of CD4+CD25+Foxp3- cells, but not Treg cells, was significantly augmented, and the stability of Treg cells was not affected by α-GalCer. The expansion of thymocyte-derived Tregs was not inhibited by cytokine neutralization. However, in vitro thymus-derived CD4+CD25+Foxp3- cells expressed Foxp3 after IL-2 stimulation in a dose-dependent manner. These results collectively suggest that in vitro thymus-derived Treg cell expansion by α-GalCer treatment was caused by the proliferation of CD4+CD25+Foxp3- preTregs but not existing Treg cells.

Evaluation of the impact of conventional immunosuppressant on the establishment of murine transplantation tolerance - an experimental study.

Regulatory T cells (Tregs) play a significant role in immune tolerance. Since Treg function deeply depends on Interleukin-2 signaling, calcineurin inhibitors could affect their suppressive potentials, whereas mammalian target of rapamycin (mTOR) inhibitors may have less impact, as mTOR signaling is not fundamental to Treg proliferation. We previously reported a novel mixed hematopoietic chimerism induction regimen that promotes Treg proliferation by stimulating invariant natural killer T cells under CD40 blockade. Here, we use a mouse model to show the impact of tacrolimus (TAC) or everolimus (EVL) on the establishment of chimerism and Treg proliferation in the regimen. In the immunosuppressive drug-dosing phase, peripheral blood chimerism was comparably enhanced by both TAC and EVL. After dosing was discontinued, TAC-treated mice showed gradual graft rejection, whereas EVL-treated mice sustained long-term robust chimerism. Tregs of TAC-treated mice showed lower expression of both Ki67 and cytotoxic T lymphocyte antigen-4 (CTLA-4), and lower suppressive activity in vitro than those of EVL-treated mice, indicating that TAC negatively impacted the regimen by interfering with Treg proliferation and activation. Our results suggest that the usage of calcineurin inhibitors should be avoided if utilizing the regimen to induce Tregs in vivo for the establishment of mixed hematopoietic chimerism.

iNKT cell activation plus T-cell transfer establishes complete chimerism in a murine sublethal bone marrow transplant model.

Transplant tolerance induction makes it possible to preserve functional grafts for a lifetime without immunosuppressants. One powerful method is to generate mixed hematopoietic chimeras in recipients by adoptive transfer of donor-derived bone marrow cells (BMCs). In our murine transplantation model, we established a novel method for mixed chimera generation using sublethal irradiation, CD40-CD40L blockade, and invariant natural killer T-cell activation. However, numerous BMCs that are required to achieve stable chimerism makes it difficult to apply this model for human transplantation. Here, we show that donor-derived splenic T cells could contribute to not only the reduction of BMC usage but also the establishment of complete chimerism in model mice. By cotransfer of T cells together even with one-fourth of the BMCs used in our original method, the recipient mice yielded complete chimerism and could acquire donor-specific skin-allograft tolerance. The complete chimeric mice did not show any remarks of graft versus host reaction in vivo and in vitro. Inhibition of the apoptotic signal resulted in increase in host-derived CD8+ T cells and chimerism brake. These results suggest that donor-derived splenic T cells having veto activity play a role in the depletion of host-derived CD8+ T cells and the facilitation of complete chimerism.

Transitional B Cells Predominantly Reconstituted After a Desensitization Therapy Using Rituximab Before Kidney Transplantation.

The role of B cells in graft rejection and tolerance has aroused great interest. We previously reported that rituximab (RIT) induction prior to kidney transplantation (KTx) reduced the incidence rate of chronic rejection. Here, we performed a cross sectional investigation to determine the characteristics of B cells after RIT induction for KTx. We sampled blood from 29 patients with (N = 16) and without (N = 13) RIT induction 3 to 18 months after KTx. In the RIT group, the majority of repopulating B cells was the transitional type, while memory B cells were scarce. Although transitional B cells are believed to have immune-regulatory functions by producing IL-10, transcriptional levels of IL-10 in the peripheral blood mononuclear cells were similar in both groups. In contrast, transcription levels of BAFF-receptor relatively increased in patients with RIT induction. In conclusion, BAFF-receptor expressing highly proliferating transitional B cell was the major subset after RIT induction for KTx.

Donor bone marrow cells are essential for iNKT cell-mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance.

Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti-CD40 ligand (anti-CD40L) and liposomal formulation of a ligand for invariant natural killer T cells administered to sub-lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3-positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)-γ-producing CD8+ T cells, expand host Ki67+ CD4+ CD25+ Foxp3+ Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC-recipients accepted skin grafts in an allo-specific manner. Myeloid-derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.