Renal ischemia-reperfusion injury and adenosine 2A receptor-mediated tissue protection: the role of CD4+ T cells and IFN-gamma

A(2A) adenosine receptor (A(2A)R)-expressing bone marrow (BM)-derived cells contribute to the renal protective effect of A(2A) agonists in renal ischemia-reperfusion injury (IRI). We performed IRI in mice lacking T and B cells to determine whether A(2A)R expressed in CD4+ cells mediate protection from IRI. Rag-1 knockout (KO) mice were protected in comparison to wild-type (WT) mice when subjected to IRI. ATL146e, a selective A(2A) agonist, did not confer additional protection. IFN-gamma is an important early signal in IRI and is thought to contribute to reperfusion injury. Because IFN-gamma is produced by kidney cells and T cells we performed IRI in BM chimeras in which the BM of WT mice was reconstituted with BM from IFN-gamma KO mice (IFN-gamma KO-->WT chimera). We observed marked reduction in IRI in comparison to WT-->WT chimeras providing additional indirect support for the role of T cells. To confirm the role of CD4+ A(2A)R in mediating protection from IRI, Rag-1 KO mice were subjected to ischemia-reperfusion. The protection observed in Rag-1 KO mice was reversed in Rag-1 KO mice that were adoptively transferred WT CD4+ cells (WT CD4+-->Rag-1 KO) or A(2A) KO CD4+ cells (A(2A) KO CD4+-->Rag-1 KO). ATL146e reduced injury in WT CD4+-->Rag-1 KO mice but not in A(2A) KO CD4+-->Rag-1 KO mice. Rag-1 KO mice reconstituted with CD4+ cells derived from IFN-gamma KO mice (IFN-gamma CD4+-->Rag-1 KO) were protected from IRI; ATL146e conferred no additional protection. These studies demonstrate that CD4+ IFN-gamma contributes to IRI and that A(2A) agonists mediate protection from IRI through action on CD4+ cells.     

CXCR3+CD4+ T cells mediate innate immune function in the pathophysiology of liver ischemia/reperfusion injury

Ischemia-reperfusion injury (IRI), an innate immune-dominated inflammatory response, develops in the absence of exogenous Ags. The recently highlighted role of T cells in IRI raises a question as to how T lymphocytes interact with the innate immune system and function with no Ag stimulation. This study dissected the mechanism of innate immune-induced T cell recruitment and activation in rat syngeneic orthotopic liver transplantation (OLT) model. Liver IRI was induced after cold storage (24-36 h) at 4 degrees C in University of Wisconsin solution. Gene products contributing to IRI were identified by cDNA microarray at 4-h posttransplant. IRI triggered increased intrahepatic expression of CXCL10, along with CXCL9 and 11. The significance of CXCR3 ligand induction was documented by the ability of neutralizing anti-CXCR3 Ab treatment to ameliorate hepatocellular damage and improve 14-day survival of 30-h cold-stored OLTs (95 vs 40% in controls; p < 0.01). Immunohistology analysis confirmed reduced CXCR3+ and CD4+ T cell infiltration in OLTs after treatment. Interestingly, anti-CXCR3 Ab did not suppress innate immune activation in the liver, as evidenced by increased levels of IL-1beta, IL-6, inducible NO synthase, and multiple neutrophil/monokine-targeted chemokine programs. In conclusion, this study demonstrates a novel mechanism of T cell recruitment and function in the absence of exogenous Ag stimulation. By documenting that the execution of innate immune function requires CXCR3+CD4+ T cells, it highlights the critical role of CXCR3 chemokine biology for the continuum of innate to adaptive immunity in the pathophysiology of liver IRI.     

B cell deficiency confers protection from renal ischemia reperfusion injury

Recent data have demonstrated a role for CD4(+) cells in the pathogenesis of renal ischemia reperfusion injury (IRI). Identifying engagement of adaptive immune cells in IRI suggests that the other major cell of the adaptive immune response, B cells, may also mediate renal IRI. An established model of renal IRI was used: 30 min of renal pedicle clamping was followed by reperfusion in B cell-deficient ( mu MT) and wild-type mice. Renal function was significantly improved in mu MT mice compared with wild-type mice at 24, 48, and 72 h postischemia. mu MT mice also had significantly reduced tubular injury. Both groups of mice had similar renal phagocyte infiltration postischemia assessed by myeloperoxidase levels and similar levels of CD4(+) T cell infiltration postischemia. Peritubular complement C3d staining was also similar in both groups. To identify the contribution of cellular vs soluble mechanism of action, serum transfer into mu MT mice partially restored ischemic phenotype, but B cell transfers did not. These data are the first demonstration of a pathogenic role for B cells in ischemic acute renal failure, with a serum factor as a potential underlying mechanism of action.     

Lung T Lymphocyte Trafficking and Activation during Ischemic Acute Kidney Injury

Despite advances in renal replacement therapy, the mortality rate for acute kidney injury (AKI) remains unacceptably high, likely owing to extrarenal organ dysfunction. Kidney ischemia-reperfusion injury (IRI) activates cellular and soluble mediators that facilitate organ crosstalk and induce caspase-dependent lung apoptosis and injury through a TNFR1-dependent pathway. Given that T lymphocytes mediate local IRI in the kidney and are known to drive TNFR1-mediated apoptosis, we hypothesized that T lymphocytes activated during kidney IRI would traffic to the lung and mediate pulmonary apoptosis during AKI. In an established murine model of kidney IRI, we identified trafficking of CD3(+) T lymphocytes to the lung during kidney IRI by flow cytometry and immunohistochemistry. T lymphocytes were primarily of the CD3(+)CD8(+) phenotype; however, both CD3(+)CD4(+) and CD3(+)CD8(+) T lymphocytes expressed CD69 and CD25 activation markers during ischemic AKI. The activated lung T lymphocytes did not demonstrate an increased expression of intracellular TNF-α or surface TNFR1. Kidney IRI induced pulmonary apoptosis measured by caspase-3 activation in wild-type controls, but not in T cell-deficient (T(nu/nu)) mice. Adoptive transfer of murine wild-type T lymphocytes into T(nu/nu) mice restored the injury phenotype with increased cellular apoptosis and lung microvascular barrier dysfunction, suggesting that ischemic AKI-induced pulmonary apoptosis is T cell dependent. Kidney-lung crosstalk during AKI represents a complex biological process, and although T lymphocytes appear to serve a prominent role in the interorgan effects of AKI, further experiments are necessary to elucidate the specific role of activated T cells in modulating pulmonary apoptosis.     

Ischemic Preconditioning in the Liver Is Independent of Regulatory T Cell Activity

Ischemic preconditioning (IPC) protects organs from ischemia reperfusion injury (IRI) through unknown mechanisms. Effector T cell populations have been implicated in the pathogenesis of IRI, and T regulatory cells (Treg) have become a putative therapeutic target, with suggested involvement in IPC. We explored the role of Treg in hepatic IRI and IPC in detail. IPC significantly reduced injury following ischemia reperfusion insults. Treg were mobilized rapidly to the circulation and liver after IRI, but IPC did not further increase Treg numbers, nor was it associated with modulation of circulating pro-inflammatory chemokine or cytokine profiles. We used two techniques to deplete Treg from mice prior to IRI. Neither Treg depleted FoxP3.LuciDTR mice, nor wildtyoe mice depleted of Tregs with PC61, were more susceptible to IRI compared with controls. Despite successful enrichment of Treg in the liver, by adoptive transfer of both iTreg and nTreg or by in vivo expansion of Treg with IL-2/anti-IL-2 complexes, no protection against IRI was observed.We have explored the role of Treg in IRI and IPC using a variety of techniques to deplete and enrich them within both the liver and systemically. This work represents an important negative finding that Treg are not implicated in IPC and are unlikely to have translational potential in hepatic IRI.     

CD4^＋T细胞在大鼠心肌缺血再灌注损伤中的作用

目的通过大鼠心肌缺血再灌注损伤的动物模型,分析CD4^＋T细胞在心肌组织损伤中的作用。方法结扎大鼠冠状动脉左前降支45min,随后恢复再灌的方法,制作缺血再灌损伤的动物模型,随机分为再灌注0、2、6、9、12h组及相应的对照组。II导联心电图及TTC确定模型,组织病理学观察心肌细胞的损伤情况,免疫荧光染色计数浸润的炎性细胞,半定量PCR进一步验证各型T细胞的表达。结果心肌的梗死面积与心肌缺血再灌时间成正相关,至观察结束未出现峰值;组织中浸润的中型粒细胞和T细胞分别在2h和12h有峰值出现,但CD4^＋T/CD3^＋T的比率几乎保持不变;观察所见CD4^＋T细胞是组织中存在最多的T细胞。结论大鼠缺血再灌注损伤中,心肌组织中浸润的CD4^＋T细胞作为主要的效应细胞,参与了持续稳定的心肌损伤过程。     

NKT cell activation mediates neutrophil IFN-gamma production and renal ischemia-reperfusion injury

Previous work has shown that ischemia-reperfusion (IR) injury (IRI) is dependent on CD4(+) T cells from naive mice acting within 24 h. We hypothesize that NKT cells are key participants in the early innate response in IRI. Kidneys from C57BL/6 mice were subjected to IRI (0.5, 1, 3, and 24 h of reperfusion). After 30 min of reperfusion, we observed a significant increase in CD4(+) cells (145% of control) from single-cell kidney suspensions as measured by flow cytometry. A significant fraction of CD4(+) T cells expressed the activation marker, CD69(+), and adhesion molecule, LFA-1(high). Three hours after reperfusion, kidney IFN-gamma-producing cells were comprised largely of GR-1(+)CD11b(+) neutrophils, but also contained CD1d-restricted NKT cells. Kidney IRI in mice administered Abs to block CD1d, or deplete NKT cells or in mice deficient of NKT cells (Jalpha18(-/-)), was markedly attenuated. These effects were associated with a significant decrease in renal infiltration and, in activation of NKT cells, and a decrease in IFN-gamma-producing neutrophils. The results support the essential role of NKT cells and neutrophils in the innate immune response of renal IRI by mediating neutrophil infiltration and production of IFN-gamma.     

Phenotypic and functional characterization of kidney-infiltrating lymphocytes in renal ischemia reperfusion injury

T and B lymphocytes have been implicated in the pathogenesis of renal ischemia reperfusion injury (IRI). The trafficking of lymphocytes into kidneys during IRI has been postulated to underlie this effect, but has not been rigorously studied. We therefore characterized the lymphocyte populations infiltrating into mouse kidneys 3 and 24 h after renal IRI. Immunohistochemistry and flow cytometry staining of kidney lymphocytes showed increased trafficking of CD3+ T cells and CD19+ B cells in both sham-operated and IRI mice 3 h after renal IRI. In the IRI mice, increased infiltration of NK1.1+ and CD4+ NK1.1+ cells compared with normal and sham-operated mice was observed 3 and 24 h after renal IRI, respectively. After 24 h of renal IRI, the decreased percentages of CD3+, CD19+, and NK1.1+ populations in the IRI mice compared with control groups were observed. Increased TNF-alpha and IFN-gamma production of kidney infiltration CD3+ T cells in IRI mice but not sham-operated mice was found. Unexpectedly, isolation and transfer of kidney-infiltrating lymphocytes 24 h after renal IRI into T cell-deficient mice reduced their functional and histological injury after renal IRI, suggesting that kidney-infiltrating lymphocytes could have a protective function. These quantitative, qualitative, and functional changes in kidney lymphocytes provide mechanistic insight into how lymphocytes modulate IRI, as well as demonstrating that abdominal surgery alone leads to lymphocyte changes in kidney.     

Extracellular micro/nanovesicles rescue kidney from ischemia-reperfusion injury

Acute renal failure (ARF) is a clinical challenge that is highly resistant to treatment, and its high rate of mortality is alarming. Ischemia–reperfusion injury (IRI) is the most common cause of ARF. Especially IRI is implicated in kidney transplantation and can determine graft survival. Although the exact pathophysiology of renal IRI is unknown, the role of inflammatory responses has been elucidated. Because mesenchymal stromal cells (MSCs) have strong immunomodulatory properties, they are under extensive investigation as a therapeutic modality for renal IRI. Extracellular vesicles (EVs) play an integral role in cell-to-cell communication. Because the regenerative potential of the MSCs can be recapitulated by their EVs, the therapeutic appeal of MSC-derived EVs has dramatically increased in the past decade. Higher safety profile and ease of preservation without losing function are other advantages of EVs compared with their producing cells. In the current review, the preliminary results and potential of MSC-derived EVs to alleviate kidney IRI are summarized. We might be heading toward a cell-free approach to treat renal IRI.     

Notch1 provides myocardial protection by improving mitochondrial quality control

Mitochondrial quality control is a new target for myocardial protection. Notch signaling plays an important role in heart development, maturation, and repair. However, the role of Notch in the myocardial mitochondrial quality control remains elusive. In this study, we isolated myocardial cells from rats and established myocardial ischemia reperfusion injury (IRI) model. We modulated Notch1 expression level in myocardial cells via infection with recombinant adenoviruses Ad-N1ICD and Ad-shN1ICD. We found that IR reduced myocardial cells viability, but Notch1 overexpression increased the viability of myocardial cells exposed to IRI. In addition, Notch1 overexpression improved ATP production, increased mitochondrial fusion and decreased mitochondrial fission, and inhibited mitophagy in myocardial cells exposed to IRI. However, N1ICD knockdown led to opposite effects. The myocardial protection role of Notch1 was related to the inhibition of Pink1 expression and Mfn2 and Parkin phosphorylation. In conclusion, Notch1 exerts myocardial protection and this is correlated with the maintenance of mitochondrial quality control and the inhibition of Pink1/Mfn2/Parkin signaling.     

CD11c+ Cells Partially Mediate the Renoprotective Effect Induced by Bone Marrow-Derived Mesenchymal Stem Cells

Previous studies have shown that induction of immune tolerance by mesenchymal stem cells (MSCs) is partially mediated via monocytes or dendritic cells (DCs). The purpose of this study was to determine the role of CD11c⁺ cells in MSC-induced effects on ischemia/reperfusion injury (IRI). IRI was induced in wildtype (WT) mice and CD11c⁺-depleted mice following pretreatment with or without MSCs. In the in-vitro experiments, the MSC-treated CD11c⁺ cells acquired regulatory phenotype with increased intracellular IL-10 production. Although splenocytes cocultured with MSCs showed reduced T cell proliferation and expansion of CD4⁺FoxP3⁺ regulatory T cells (Tregs), depletion of CD11c⁺ cells was associated with partial loss of MSCs effect on T cells. In in-vivo experiment, MSCs’ renoprotective effect was also associated with induction of more immature CD11c⁺ cells and increased FoxP3 expression in I/R kidneys. However all these effects induced by the MSCs were partially abrogated when CD11c⁺ cells were depleted in the CD11c⁺-DTR transgenic mice. In addition, the observation that adoptive transfer of WT CD11c⁺ cells partially restored the beneficial effect of the MSCs, while transferring IL-10 deficient CD11c⁺ cells did not, strongly suggest the important contribution of IL-10 producing CD11c⁺ cells in attenuating kidney injury by MSCs. Our results suggest that the CD11c⁺ cell-Tregs play critical role in mediating renoprotective effect of MSCs.     

Irisin is induced in renal ischemia-reperfusion to protect against tubular cell injury via suppressing p53

Renal ischemia-reperfusion is a major cause of acute kidney injury, a disease currently without effective treatments. Irisin was initially identified as an important factor produced by muscles to mediate the health benefits of exercise, and recent work has further suggested its protective effect against lung and liver injury. However, the role of Irisin in kidney diseases, including renal ischemia-reperfusion injury (IRI), remains unknown. In the present study, we found that the Irisin precursor, fibronectin type III domain-containing protein 5 (Fndc5), was induced in renal tubules in a mouse model of renal IRI and in cultured mouse renal proximal tubular cells subjected ATP depletion injury. Functionally, silencing Fndc5 in cultured proximal tubular cells increased the sensitivity to ATP depletion-induced apoptosis, whereas both Fndc5 overexpression and supplementation of recombinant Irisin alleviated ATP depletion-induced apoptosis. In vivo, administration of recombinant Irisin dramatically attenuated kidney dysfunction, tissue damage, tubular cell apoptosis, and inflammation during renal IRI in mice. Mechanistically, Irisin suppressed the activation of p53 in renal IRI, a critical factor in tubular cell death. Together, these results indicate that Irisin is induced in renal IRI as a protective mechanism for renal tubular cells, suggesting the therapeutic potential of recombinant Irisin in renal IRI and related kidney diseases.     

Dendritic cell sphingosine 1-phosphate receptor-3 regulates Th1-th2 polarity in kidney ischemia-reperfusion injury

Dendritic cells (DCs) are central to innate and adaptive immunity of early kidney ischemia-reperfusion injury (IRI), and strategies to alter DC function may provide new therapeutic opportunities. Sphingosine 1-phosphate (S1P) modulates immunity through binding to its receptors (S1P1-5), and protection from kidney IRI occurs in S1P3-deficient mice. Through a series of experiments we determined that this protective effect was owing in part to differences between S1P3-sufficient and -deficient DCs. Mice lacking S1P3 on bone marrow cells were protected from IRI, and S1P3-deficient DCs displayed an immature phenotype. Wild-type (WT) but not S1P3-deficient DCs injected into mice depleted of DCs prior to kidney IR reconstituted injury. Adoptive transfer (i.e., i.v. injection) of glycolipid (Ag)-loaded WT but not S1P3-deficient DCs into WT mice exacerbated IRI, suggesting that WT but not S1P3-deficient DCs activated NKT cells. Whereas WT DC transfers activated the Th1/IFN-γ pathway, S1P3-deficient DCs activated the Th2/IL-4 pathway, and an IL-4-blocking Ab reversed protection from IRI, supporting the concept that IL-4 mediates the protective effect of S1P3-deficient DCs. Administration of S1P3-deficient DCs 7 d prior to or 3 h after IRI protected mice from IRI and suggests their potential use in cell-based therapy. We conclude that absence of DC S1P3 prevents DC maturation and promotes a Th2/IL-4 response. These findings highlight the importance of DC S1P3 in modulating NKT cell function and IRI and support development of selective S1P3 antagonists for tolerizing DCs for cell-based therapy or for systemic administration for the prevention and treatment of IRI and autoimmune diseases.     

The Fifth Domain of Beta 2 Glycoprotein I Protects from Natural IgM Mediated Cardiac Ischaemia Reperfusion Injury

Reperfusion after a period of ischemia results in reperfusion injury (IRI) which involves activation of the inflammatory cascade. In cardiac IRI, IgM natural antibodies (NAb) play a prominent role through binding to altered neoepitopes expressed on damaged cells. Beta 2 Glycoprotein I (β2GPI) is a plasma protein that binds to neoepitopes on damaged cells including anionic phospholipids through its highly conserved Domain V. Domain I of β2GPI binds circulating IgM NAbs and may provide a link between the innate immune system, IgM NAb binding and cardiac IRI. This study was undertaken to investigate the role of Β2GPI and its Domain V in cardiac IRI using wild-type (WT), Rag-1 ^-/- and β2GPI deficient mice. Compared with control, treatment with Domain V prior to cardiac IRI prevented binding of endogenous β2GPI to post-ischemic myocardium and resulted in smaller myocardial infarction size in both WT and β2GPI deficient mice. Domain V treatment in WT mice also resulted in less neutrophil infiltration, less apoptosis and improved ejection fraction at 24 h. Rag-1 -/- antibody deficient mice reconstituted with IgM NAbs confirmed that Domain V prevented IgM NAb induced cardiac IRI. Domain V remained equally effective when delivered at the time of reperfusion which has therapeutic clinical relevance.Based upon this study Domain V may function as a universal inhibitor of IgM NAb binding in the setting of cardiac IRI, which offers promise as a new therapeutic strategy in the treatment of cardiac IRI.     

Ischemia/Reperfusion-Induced CHOP Expression Promotes Apoptosis and Impairs Renal Function Recovery: The Role of Acidosis and GPR4

Endoplasmic reticulum (ER) stress-induced apoptosis is implicated in a wide range of diseases, including ischemia/reperfusion injury (IRI). As a common feature of ER stress, the role of CCAT/enhancer-binding protein homologous protein (CHOP) in renal IRI has not been thoroughly investigated. We found that IR led to renal CHOP expression, accompanied by apoptosis induction. Renal IRI was markedly alleviated in CHOP^−/− mice. Observations from bone marrow chimeras showed that this was based on CHOP inactivation in renal parenchymal cells rather than inflammatory cells. In vivo and in vitro studies demonstrated that IRI induced CHOP expression in both endothelial and epithelial cells, which was responsible for apoptosis induction. These results were reinforced by the observation that CHOP knockout led to improvement of the postischemic microcirculatory recovery. In vitro studies revealed hypoxia-induced acidosis to be a major inducer of CHOP in endothelial cells, and neutralizing acidosis not only diminished CHOP protein, but also reduced apoptosis. Finally, knockdown of a proton-sensing G protein-coupled receptor GPR4 markedly reduced CHOP expression and endothelial cell apoptosis after hypoxia exposure. These results highlight the importance of hypoxia-acidosis in ER stress signaling regulation in ischemic kidneys and suggest that GPR4 inhibitors or agents targeting CHOP expression may be promising in the treatment of renal IRI.     

Hypothermic oxygenated perfusion inhibits HECTD3-mediated TRAF3 polyubiquitination to alleviate DCD liver ischemia-reperfusion injury

Ischemia-reperfusion injury (IRI) is an inevitable and serious clinical problem in donations after heart death (DCD) liver transplantation. Excessive sterile inflammation plays a fateful role in liver IRI. Hypothermic oxygenated perfusion (HOPE), as an emerging organ preservation technology, has a better preservation effect than cold storage (CS) for reducing liver IRI, in which regulating inflammation is one of the main mechanisms. HECTD3, a new E3 ubiquitin ligase, and TRAF3 have an essential role in inflammation. However, little is known about HECTD3 and TRAF3 in HOPE-regulated liver IRI. Here, we aimed to investigate the effects of HOPE on liver IRI in a DCD rat model and explore the roles of HECTD3 and TRAF3 in its pathogenesis. We found that HOPE significantly improved liver damage, including hepatocyte and liver sinusoidal endothelial cell injury, and reduced DCD liver inflammation. Mechanistically, both the DOC and HECT domains of HECTD3 directly interacted with TRAF3, and the catalytic Cys (C832) in the HECT domain promoted the K63-linked polyubiquitination of TRAF3 at Lys138. Further, the ubiquitinated TRAF3 at Lys138 increased oxidative stress and activated the NF-κB inflammation pathway to induce liver IRI in BRL-3A cells under hypoxia/reoxygenation conditions. Finally, we confirmed that the expression of HECTD3 and TRAF3 was obviously increased in human DCD liver transplantation specimens. Overall, these findings demonstrated that HOPE can protect against DCD liver transplantation-induced-liver IRI by reducing inflammation via HECTD3-mediated TRAF3 K63-linked polyubiquitination. Therefore, HOPE regulating the HECTD3/TRAF3 pathway is a novel target for improving IRI in DCD liver transplantation.Subject terms: Molecular biology, Diseases     

大鼠心肌缺血再灌注损伤中诱导CD4＋T细胞募集的趋化因子的表达

目的检测大鼠心肌缺血再灌注中诱导CD4＋T细胞趋化的趋化因子的表达。方法建立大鼠心肌缺血再灌注损伤的实验动物模型,冠状动脉左前降支结扎45 min后恢复再灌,在不同的再灌注时间点0、2、6、9、12 h获取心脏组织（每组4只）,相应时间点的假手术组作为对照。RT-PCR检测趋化CD4＋T细胞的趋化因子的表达;免疫荧光检测趋化因子诱导的CD4＋T细胞的募集;组织学评价心肌细胞的损伤。结果诱导CD4＋T细胞募集的趋化因子的表达在再灌注2 h即迅速增高,随后出现下降。其中,CXCL-10（IP-10）的表达高峰出现在2 h,与其共用同一受体CXCR3的其它两个趋化因子CXCL9（Mig）和CXCL11（I-TAC）的表达,分别在再灌注12 h和9 h出现另一高峰。受趋化因子的诱导,组织中浸润的CD4＋T细胞逐渐增多;心肌的缺血再灌注损伤也随之不断加重。结论诱导CD4＋T细胞募集的趋化因子,通过CD4＋T细胞介导的免疫损伤作用,最终导致了心肌的梗死。