Shuang Wang

NK Cells Induce Apoptosis in Tubular Epithelial Cells and Contribute to Renal Ischemia-Reperfusion Injury

Renal ischemia-reperfusion injury (IRI) can result in acute renal failure with mortality rates of 50% in severe cases. NK cells are important participants in early-stage innate immune responses. However, their role in renal tubular epithelial cell (TEC) injury in IRI is currently unknown. Our data indicate that NK cells can kill syngeneic TEC in vitro. Apoptotic death of TEC in vitro is associated with TEC expression of the NK cell ligand Rae-1, as well as NKG2D on NK cells. In vivo following IRI, there was increased expression of Rae-1 on TEC. FACS analyses of kidney cell preparations indicated a quantitative increase in NKG2D-bearing NK cells within the kidney following IRI. NK cell depletion in wild-type C57BL/6 mice was protective, while adoptive transfer of NK cells worsened injury in NK, T, and B cell-null Rag2−/−γc−/− mice with IRI. NK cell-mediated kidney injury was perforin (PFN)-dependent as PFN−/− NK cells had minimal capacity to kill TEC in vitro compared with NK cells from wild-type, FasL-deficient (gld), or IFN-γ−/− mice. Taken together, these results demonstrate for the first time that NK cells can directly kill TEC and that NK cells contribute substantially to kidney IRI. NK cell killing may represent an important underrecognized mechanism of kidney injury in diverse forms of inflammation, including transplantation.

Osteopontin Expressed in Tubular Epithelial Cells Regulates NK Cell-Mediated Kidney Ischemia Reperfusion Injury

Renal ischemia reperfusion injury (IRI) occurs after reduced renal blood flow and is a major cause of acute injury in both native and transplanted kidneys. Studies have shown diverse cell types in both the innate and the adaptive immune systems participate in kidney IRI as dendritic cells, macrophages, neutrophils, B cells, CD4+ NK+ cells, and CD4+ T cells all contribute to this form of injury. Recently, we have found that NK cells induce apoptosis in tubular epithelial cells (TECs) and also contribute to renal IRI. However, the mechanism of NK cell migration and activation during kidney IRI remains unknown. In this study, we have identified that kidney TECs express a high level of osteopontin (OPN) in vitro and in vivo. C57BL/6 OPN-deficient mice have reduced NK cell infiltration with less tissue damage compared with wild-type C57BL/6 mice after ischemia. OPN can directly activate NK cells to mediate TEC apoptotic death and can also regulate chemotaxis of NK cells to TECs. Taken together, our study’s results indicate that OPN expression by TECs is an important factor in initial inflammatory responses that involves NK cells activity in kidney IRI. Inhibiting OPN expression at an early stage of IRI may be protective and preserve kidney function after transplantation.

Glycyrrhizic Acid Ameliorates HMGB1-Mediated Cell Death and Inflammation after Renal Ischemia Reperfusion Injury

Background: Renal ischemia reperfusion injury (IRI) leads to acute kidney injury (AKI) and the death of tubular epithelial cells (TEC). The release of high-mobility group box-1 (HMGB1) and other damage-associated molecular pattern moieties from dying cells may promote organ dysfunction and inflammation by effects on TEC. Glycyrrhizic acid (GZA) is a functional inhibitor of HMGB1, but its ability to attenuate the HMGB1-mediated injury of TEC has not been tested. Methods/Results: In vitro, hypoxia and cytokine treatment killed TEC and resulted in the progressive release of HMGB1 into the supernatant. GZA reduced the hypoxia-induced TEC death as measured by annexin-V and propidium iodide. Hypoxia increased the expression of MCP-1 and CXCL1 in TEC, which was reduced by GZA in a dose-dependent manner. Similarly, the HMGB1 activation of effector NK cells was inhibited by GZA. To test the effect of HMGB1 neutralization by GZA in vivo, mice were subjected to renal IRI. HMGB1 protein expression increased progressively in kidneys from 4 to 24 h after ischemia and was detected in tubular cells by 4 h using immunohistochemistry. GZA preserved renal function after IRI and reduced tubular necrosis and neutrophil infiltration by histological analyses and ethidium homodimer staining. Conclusions: Importantly, these data demonstrate for the first time that AKI following hypoxia and renal IRI may be promoted by HMGB1 release, which can reduce the survival of TEC and augment inflammation. Inhibition of the interaction of HMGB1 with TEC through GZA may represent a therapeutic strategy for the attenuation of renal injury following IRI and transplantation.

Adoptive transfer of DNT cells induces long-term cardiac allograft survival and augments recipient CD4(+)Foxp3(+) Treg cell accumulation.

Regulatory T (Treg) cells play an important role in the regulation of immune responses but whether Treg will induce tolerance in transplant recipients in the clinic remains unknown. Our previous studies have shown that TCRαβ(+)CD3(+)CD4⁻CD8⁻NK1.1⁻ (double negative, DN) T cells suppress T cell responses and prolong allograft survival in a single locus MHC-mismatched mouse model. In this study, we investigated the role of DNT cells in a more robust, fully MHC-mismatched BALB/c to C57BL/6 transplantation model, which may be more clinically relevant. Adoptive transfer of DNT cells in combination with short-term rapamycin treatment (days 1-9) induced long-term heart allograft survival (101±31 vs. 39±13 days rapamycin alone, p<0.01). Furthermore adoptive transfer DNT cells augmented CD4+Foxp3+ Treg cells accumulation in transplant recipients while depletion of CD4(+) Treg cells by anti-CD25 inhibited the effect of DNT cells on long-term graft survival (48±12 days vs. 101±31 days, p<0.001). In conclusion, DNT cells combined with short-term immunosuppression can prolong allograft survival, which may be through the accumulation of CD4(+)Foxp3(+) Treg cells in the recipient. Our result suggests that allograft tolerance may require the co-existence of different type Treg cell phenotypes which are affected by current immunosuppression.

Prolongation of cardiac allograft survival by inhibition of ERK1/2 signaling in a mouse model.

Background. It has been demonstrated that in vitro the presence of extracellular signal-regulated kinase 1 and 2 (ERK1/2) signaling inhibitor suppresses T cell activation and Th1 development. However, pharmacological interference of ERK1/2 signaling by administration of its small molecule inhibitor has not been tested as a therapeutic target in the prevention of allograft rejection. Methods. The immunosuppressive effect of targeting ERK1/2 signaling was tested on cardiac allograft survival in C57BL/6 (H-2b) to Balb/c (H-2d) murine model using PD98059 inhibitor. Phosphorylation/activation of ERK 1/2 and STAT6 proteins were assessed by Western blot. Results. Blockade of ERK1/2 using PD98059 had significant immunosuppressive effect and prolonged survival of mouse cardiac allografts from 8.3±0.5 days (vehicle) to 12.6±1.3 days (100 mg/kg PD98059; P<0.0001). Combination therapy of PD98059 (100 mg/kg) with cyclosporine (CsA, 15 mg/kg for 20 days) additionally enhanced graft survival (34.4±1.2 days) compared to CsA (14.9±1.1 days; P<0.0001) or PD98059 monotherapy (P<0.0001). Attenuation of graft rejection by PD98059 correlated to reduction of intragraft ERK phosphorylation and leukocyte infiltration, and to increase in interleukin (IL)-4 or decrease in interferon-&ggr; production within the grafts. In vitro inhibition of ERK1/2 by PD98059 promoted Th2 differentiation by upregulation IL-4 production but not altering IL-4 stimulating STAT6 pathway. Conclusion. Targeting ERK1/2 signaling results in suppression of alloimmune responses by an unique mechanism that involves Th1/Th2 skewing, suggesting a therapeutic potential of inhibition of ERK1/2 signaling for transplant rejection, particularly in combination with CsA.

Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T-cell clonal deletion and suppressing NK cell function.

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft‐versus‐host‐disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation‐free condition. Our data indicate that adoptive transfer of donor‐derived T‐cell receptor (TCR) αβ+CD3+CD4–CD8–NK1.1– (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable‐mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third‐party C3H (H‐2k) skin grafts. Adoptive transfer of CD4+ and CD8+ T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T‐cell alloreactive responsiveness was reduced in the DN Treg cell‐treated group and clonal deletions of TCRVβ2, 7, 8.1/2, and 8.3 were observed in both CD4+ and CD8+ T cells. Furthermore, DN Treg‐cell treatment suppressed NK cell‐mediated BM rejection in a perforin‐dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable‐mixed chimerism and donor‐specific tolerance in the irradiation‐free regimen.

Reduction of chronic allograft nephropathy by inhibition of extracellular signal-regulated kinase 1 and 2 signaling

Chronic allograft nephropathy (CAN), the most common cause of late kidney allograft failure, is not effectively prevented by immunosuppressive regimens. Activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) via MEK mediates actions of various growth factors, including transforming growth factor (TGF)-beta1, which plays a key role in CAN. Hence, we tested the therapeutic potential of MEK-ERK1/2 signaling disruption to prevent CAN. Kidneys from C57BL/6J (H-2(b)) mice were transplanted to bilaterally nephrectomized BALB/c (H-2(d)) mice. At 14 days after transplantation, the recipients were subjected to 28 days of treatment with the MEK inhibitor CI-1040. All six CI-1040-treated allografts survived, while two of seven grafts in the vehicle-treated group were lost. At the end of the experiment, the function and structure of grafts in the CI-1040-treated group were significantly preserved, as indicated by lower levels of serum creatinine or blood urea nitrogen than in the vehicle-treated group [30 +/- 6 vs. 94 +/- 39 microM creatinine (P = 0.0015) and 22 +/- 8 vs. 56 +/- 25 mM BUN (P = 0.0054)] and reduced CAN in the CI-1040-treated group compared with vehicle controls (CAN score = 4.2 vs. 10.3, P = 0.0119). The beneficial effects induced by CI-1040 were associated with reduction of ERK1/2 phosphorylation and TGFbeta1 levels in grafts. Also, CI-1040 potently suppressed not only TGFbeta biosynthesis in kidney cell cultures but also antiallograft immune responses in vitro and in vivo. Our data suggest that interference of MEK-ERK1/2 signaling with a pharmacological agent (e.g., CI-1040) has therapeutic potential to prevent CAN in kidney transplantation.

RENAL TUBULAR CELL (TEC) EXPRESSION OF SPI-6 (SERPIN PROTEASE INHIBITOR-6) IS REQUIRED FOR PROTECTION FROM GRANZYME B MEDIATED EFFECTOR CELL INJURY FOLLOWING TRANSPLANTATION: 2669

K. Shek1, Z. Zhang2, K. Khan3, S. Wang4, A. Lau5, Z. Yin4, W. Liu6, B. Garcia6, B. Singh3, H. Wang7, A.M. Jevnikar8 1Microbiology And Immunology, University Of Western Ontario, London/Ontario/CANADA, 2Medicine, Unuiversity of Western Ontario, London/Ontario/CANADA, 3, University of Western Ontario, London/CANADA, 4Department Of Medicine, Unuiversity of Western Ontario, London/CANADA, 5, Robarts Research Institute, London/ Ontario/CANADA, 6Department Of Pathology, University of Western Ontario, London/CANADA, 7Department Of Surgery, London Health Sciences Center-University Hospital, London/CANADA, 8, Multi-Organ Transplant Program-London Health Sciences Centre, London/ON/ CANADA