Expression of NKG2D and Its Ligand in Mouse Heart Allografts May Have a Role in Acute Rejection

Background

Ligands for the natural killer cell–activating receptor NKG2D, such as retinoic acid early inducible (Rae-1), minor histocompatibility antigen H60 (mouse), and major histocompatibility complex class I chain-related (human) may be expressed by tissues in response to stress. Because NKG2D-ligand engagement may induce natural killer cell activation and provide T-cell costimulation, we examined whether this interaction between innate and adaptive immunity occurred during heart transplant rejection.

Methods

Hearts from BALB/c mice were heterotopically transplanted into C57BL/6 mice without immunosupression. Grafts were harvested at 1, 3, and 5 days after transplantation. Rae-1, H60, and NKG2D mRNA were analyzed by RT-PCR, and the proteins were detected by immunohistochemistry.

Results

Compared with no expression in naïve BALB/c mice hearts, Rae-1 mRNA levels in heart allografts were detected from days three to five postoperative, H60 on day five, and NKG2D on day three but prominently on day five postoperative. Immunohistochemical assay showed that compared with rare expression in syngeneic cardiac grafts, there were significant protein expressions of Rae-1 and NKG2D in heart allografts from days three to five postoperative and of H60 on day 5 postoperative.

Conclusion

This study reported significant mRNA and protein expression of Rae-1, H60, and NKG2D during acute cardiac allograft rejection. The simultaneous and significant expression of NKG2D and its ligands indicated that interactions with innate immunity may promote acute rejection. The results also suggested that Rae-1 and H60 may be new targets to amelioate this immune response.     

Yisheng injection decreases the expression of H60 and RAE-1 genes in ischemic mice liver

OBJECTIVE: Major histocompatability complex class I chain-related antigen A, B (MICA, B) functions as ligands for human NKG2D receptors, which may play a role in graft rejection and cellular stress. In this study we explored the effect of ischemia/reperfusion injury (IRI) on the expression of H60 and RAE-1 (MICA, B homologues) in mice to study the protective effect of Yisheng injection (YS), an herbal preparation developed from traditional Chinese medicine. METHODS: Male BALB/c mice were divided into sham, ischemic, and YS-treated groups using 90 minutes of left liver lobe ischemia. Sham control mice underwent the same operation, but without vascular occlusion. In the treated group, YS (20 mg/kg) was given before ischemia and after reperfusion for 7 days. Liver samples collected at 7 days postoperation were used for real-time quantitative polymerase chain reaction analysis, Western blotting, and immunohistochemical assays. RESULTS: Compared with the sham group, H60 and RAE-1 mRNA levels were increased by sevenfold and 4.5-fold in the ischemic group, respectively. After YS treatment, they were reduced by 76% and 70%, respectively. Western blotting and immunohistochemical assays showed that there was absent or faint H60 and RAE-1 expression in sham liver, but they were apparently increased in ischemic liver; however, the expressions were significantly decreased in the presence of YS. CONCLUSIONS: Hepatic IRI significantly increased H60 and RAE-1 expression in mouse liver. YS treatment effectively reduced this increase, seeming to attenuate NKG2D-ligand-mediated immune responses caused by IRI. This may suggest a new concept to prevent IRI and graft rejection.     

非转移性黑色素瘤糖蛋白B在急性肾缺血再灌注损伤中的表达及与巨噬细胞表型的关系

目的 观察非转移性黑色素瘤糖蛋白B(glycoprotein non-metastatic melanoma protein B,Gpnmb)在急性肾缺血再灌注损伤(IRI)肾脏和尿液中的表达,分析其与M1、M2表型巨噬细胞的相关性,探讨其在IRI免疫炎性反应损伤中的作用.方法 C57BL/6J小鼠被随机分为3组:正常对照组(n=4)、假手术组(n=4)、IRI组(n=12).以双侧肾蒂血管阻断30min建立IRI模型.PAS染色观察肾脏病理改变;免疫荧光双染色及流式细胞术检测Gpnmb和巨噬细胞标志物F4/80蛋白的表达及分布;实时荧光定量PCR检测Gpnmb及M1型巨噬细胞表型(CD40、CCR7)及M2型巨噬细胞表型(CD163、MMR)在肾脏中的mRNA表达;Western印迹和ELISA法检测小鼠尿液中Gpnmb的表达.结果 IRI小鼠肾脏肾小管上皮细胞脱落,肾间质内可见大量炎性细胞浸润.实时荧光定量PCR结果显示,与正常对照组、假手术组比较,IRI组小鼠肾脏Gpnmb mRNA于造模后第1天和第2天升高,差异有统计学意义(P＜ 0.01),第3天下降至接近正常水平.免疫荧光双染色及流式细胞术检测结果均显示Gpnmb蛋白主要表达在F4/80阳性的巨噬细胞内.Gpnmb mRNA表达与M1型巨噬细胞表型(CD40、CCR7)的mRNA表达无相关性,与M2型巨噬细胞表型(CD163、MMR)的mRNA表达呈正相关.Western印迹和ELISA结果示IRI组小鼠在造模后第1天尿液中的Gpnmb表达显著升高,与正常对照组和假手术组比较差异有统计学意义(P＜0.01),第3天降至接近正常水平.结论 Gpnmb在IRI肾脏的巨噬细胞和尿液中呈高表达,并与M2型巨噬细胞相关,提示Gpnmb可能参与了巨噬细胞的分型和急性肾损伤的炎性反应过程,临床上也可用作AKI早期诊断的生物学标志物.     

The natural killer cell activating receptor,NKG2D,is critical to antibody-dependent chronic rejection in heart transplantation

Chronic rejection is among the most pressing clinical challenges in solid organ transplantation. Interestingly, in a mouse model of heterotopic heart transplantation, antibody-dependent, natural killer (NK) cell-mediated chronic cardiac allograft vasculopathy occurs in some donor–recipient strain combinations, but not others. In this study, we sought to identify the mechanism underlying this unexplained phenomenon. Cardiac allografts from major histocompatibility complex (MHC) mismatched donors were transplanted into immune-deficient C57Bl/6.rag^−/− recipients, followed by administration of a monoclonal antibody against the donor MHC class I antigen. We found marked allograft vasculopathy in hearts from C3H donors, but near-complete protection of BALB/c allografts from injury. We found no difference in recipient NK cell phenotype or intrinsic responsiveness to activating signals between recipients of C3H versus BALB/c allografts. However, cardiac endothelial cells from C3H allografts showed an approximately twofold higher expression of Rae-1, an activating ligand of the NK cell receptor natural killer group 2D (NKG2D). Importantly, the administration of a neutralizing antibody against NKG2D abrogated the development of allograft vasculopathy in recipients of C3H allografts, even in the presence of donor-specific antibodies. Therefore, the activating NK cell receptor NKG2D is necessary in this model of chronic cardiac allograft vasculopathy, and strain-dependent expression of NK activating ligands correlates with the development of this disease.     

Expression of H60 on mice heart graft and influence of cyclosporine

OBJECTIVES: It has been reported that the MHC class I chain-related antigen (MIC), a ligand of NKG2D, an activating receptor of natural killer cells, is expressed on rejected renal allografts. This study investigated whether heart transplantation induced expression of a homologue of MIC (H60) in outbred Kun Ming (KM) mice, widely used in China, and whether CsA had an influence on the process. METHODS: Forty-eight KM female mice were divided into untreated and CsA-treated groups, after cervical heart allotransplantation. Grafts were harvested 1, 3, 5, and 7 days postoperatively. H60 mRNA was analyzed by RT-PCR, and the protein detected by immunohistochemistry. RESULTS: Compared with no mRNA expression in the normal heart, H60 mRNA was observed at day 5 and upregulated on day 7 in the untreated group. It was detected on day 3, peaked on day 5, but was lower than untreated group, and decreased on day 7 in the CsA-treated group. H60 protein was detected in cardiocytes only on day 7 in the untreated group. CONCLUSION: Our study suggested that expression of the NKG2D ligand, H60, may activate natural killer cells playing a significant role in innate immunity associated with transplantation. The early expression of H60 mRNA on day 3 in the CsA-treated group might relate to the toxicity of CsA. The expression peaked on day 5 and decreased on day 7, possibly induced by CsA. The results suggested that H60 might be a new target for prevention of rejection.     

Octreotide Ameliorates Renal Ischemia/Reperfusion Injury via Antioxidation and Anti-inflammation

Oxidative stress, calcium overload, inflammation, cellular necrosis, and apoptosis are implicated in renal ischemic/reperfusion injury (RIRI). Because octreotide (OCT) is protective in retinal IRI, the effect of OCT on mouse RIRI and the mechanisms involved were investigated. The RIRI model was induced in male C57BL/6 mice, and the mice were then treated with saline or OCT. Serum and kidneys were subjected to periodic acid–Schiff staining, terminal deoxynucleotidyl transferase dUTP nick end labeling assay, enzyme-linked immunosorbent assay, western blotting, and immunohistochemistry. Treatment with OCT restored the renal functions and histologic changes induced by RIRI. The administration of OCT reduced tumor necrosis factor–α and interleukin-6 levels in kidney tissues, protected the kidney from apoptosis, and significantly downregulated the expression of nuclear factor–κB p65. In addition, OCT treatment upregulated the expression of nuclear factor erythroid 2–related factor 2, heme oxygenase–1, and NAD(P)H quinone oxidoreductase 1 and enhanced the renal antioxidant capacity. These results cumulatively indicate that OCT may protect the kidneys against IRI in a mouse model through the regulation of antioxidation and anti-inflammation.     

AMPK attenuates inflammation to reduce fibrosis induced by acute ischemia reperfusion injury in mice

Objective To observe the effect of adenosine monophosphate activated protein kinase (AMPK) on attenuating inflammation in fibrosis induced by acute ischemia reperfusion injury (IRI) in mice. Methods Forty eight male C57BL/6 mice were randomly divided into four groups: sham operation group (sham group), IRI group, AMPK inhibitor+IRI group (AMPK/IRI group) and normal saline+IRI group (NS/IRI group), 12 mice each group. The mice with renal IRI were occluded for 30 min through clipping bilateral renal pedicle, then released renal perfusion. Mice in sham group were performed the separation of renal pedicle without clipping. Mice in AMPK/IRI group and NS/IRI group were respectively intraperitoneal injected AMPK inhibitor and normal saline before IRI. At the 2 d after operation, 6 randomly-selected mice from each group were blooded by extraction eyeball to detect BUN and Scr. The renal histopathological changes were observed through HE staining. The mRNA expression of IL-1β, IL-6 and TNF-α was detected by real time PCR, and the level of AMPK phosphorylation was detected by Western blotting. At the 14 d after operation, Collagen 1 (COL1), α-SMA and fibronectin (FN) were detected by immunofluorescence and Western blotting in 6 remained mice from each group. The degree of kidney fibrosis was observed through sirus red staining. Results Compared with those in sham group, tubular interstitial damage was aggravated (P＜0.05), BUN and Scr were increased (P＜0.05), the mRNA expression of IL-1β, IL-6 and TNF-α was increased at the 2 d after operation (all P＜0.05), and the level of AMPK phosphorylation was activated in IRI group and NS/IRI group (all P＜0.05); the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d (all P＜0.05). Compared with those in IRI group, in AMPK/IRI group tubular interstitial damage was aggravated (P＜0.05), BUN and Scr were increased (all P＜0.05), the mRNA expression of IL-1β, IL-6 and TNF-α was increased at the 2 d (all P＜0.05), and the level of AMPK phosphorylation was decreased (P＜0.05). Moreover, the degree of kidney fibrosis and the expression of COL1, α-SMA and FN were increased obviously at the 14 d in AMPK/IRI group (all P＜0.05). Conclusions AMPK can ameliorate the acute renal ischemia reperfusion injury induce fibrosis in mice, and the mechanism may be related to the decrease of inflammatory reaction.     

Effects of Delayed Rapamycin Treatment on Renal Fibrosis and Inflammation in Experimental Ischemia Reperfusion Injury

Ischemia reperfusion injury (IRI) has long-term sequelae on kidney allograft function. Early initiation of rapamycin can retard surgical wound healing and recovery from IRI. In contrast, rapamycin may paradoxically retard long-term fibrotic effects of kidney IRI. We, therefore, hypothesized that delayed initiation of rapamycin after kidney ischemia, started after the initial week of wound healing, would decrease the long-term inflammation and fibrosis caused by IRI. C57BL/6 male mice were subjected to either 45 or 60 minutes of unilateral kidney ischemia or a sham operation. Mice were given rapamycin (subcutaneous, 1.5 mg/kg/d) or vehicle starting at 1 week after IRI surgery for 3 weeks. Urine albumin excretion, kidney histology, and kidney cytokine proteins were examined at 4 weeks after surgery. The 3-week treatment course of rapamycin significantly reduced body weight gain in all 3 groups and reduced postischemic kidney weight in both the 45- and 60-minute ischemia groups, but unexpectedly increased urine albumin excretion in all rapamycin-treated sham or IRI mice compared with vehicle-treated mice. Rapamycin treatment showed minimal effects on postischemic kidney fibrosis with variable effects on various cytokine/chemokine protein expressions, namely, decreasing interleukin (IL)-1α, IL-6, tumor necrosis factor (TNF)-α, and regulated on activation normal T cell expressed and secreted (RANTES) while increasing IL-4, keratinocyte-derived chemokine (KC), macrophage inflammatory protein (MIP-1α), and IL-10 in the ischemic kidney. These data demonstrated that rapamycin reduced mouse body weight and ischemic kidney weight, while increasing urinary albumin excretion. Delayed initiation of rapamycin after IRI had a minimal effect on renal fibrosis and mixed effects on proinflammatory mediator production. These data do not support delayed initiation of rapamycin after IRI to attenuate IRI-induced progressive fibrosis and inflammation, and They raise further caution regarding rapamycin and albuminuria.     

Persistent renal and extrarenal immune changes after severe ischemic injury

BACKGROUND: Renal ischemia/reperfusion (I/R) injury is associated with delayed graft function and decreased long-term allograft function. However, most experimental studies evaluating renal I/R injury have focused on acute events after ischemia. T cells are potential candidates to link preservation injury, alloimmunity, and fibrosis. We hypothesized that severe renal I/R injury would generate long-term kidney damage and immune changes. METHODS: C57BL/6 mice underwent 60 minutes of warm unilateral I/R injury or sham surgery and were studied for 6 weeks. Serum creatinine, renal histology, and albumin excretion were measured. Phagocyte infiltration, CD4+ infiltration, renal cytokine expression, and splenic lymphocyte intracellular cytokine production were also measured in mice at 6 weeks. RESULTS: Serum creatinine levels rose following 60 minutes of unilateral I/R injury compared to sham mice. Histologic analysis of ischemic kidneys at 6 weeks revealed a pronounced loss of tubular architecture and infiltration of inflammatory cells. Phagocyte and CD4+ T-cell infiltration were significantly increased in ischemic kidneys. This was accompanied by a significant increase in interleukin (IL)-1beta and regulated upon activation, normal T-cell expressed and secreted (RANTES) expression. Despite similar splenic CD4 and CD8 numbers, intracellular cytokine staining of T cells revealed a significant increase in interferon-gamma (IFN-gamma) in I/R injury mice compared to sham mice. CONCLUSION: Persistent renal and extrarenal immune responses occur after a single episode of severe I/R injury. These immune processes resulting from injury could in turn have long-term consequences on progression of renal disease in transplanted and native kidneys.     

地塞米松预处理减轻肾缺血再灌注损伤

目的探讨地塞米松（dexamethasone,DEX）对小鼠肾缺血再灌注损伤的作用及其机制。方法建立小鼠肾缺血再灌注损伤模型。18只雄性C57BL／6小鼠随机分为3个组（n=6）,分别为假手术组（Sham）、肾缺血再灌注损伤模型组（IRI）和DEX预处理组。Sham和IRI组缺血前60min予生理盐水（腹腔注射）,DEX组缺血前60min给予DEX（4mg／kg）,IRI组和DEX组血管夹夹闭左侧肾蒂,置于32℃温箱后1h松开血管夹,去除右肾。Sham组操作同上,但不夹闭左侧肾蒂,再灌注24h后处死小鼠,收集血清和肾脏标本。PAS染色后观察肾脏病理形态学变化,PCR检测白细胞介素6（interleukin6,IL-6）、干扰素γ（interferonγ,IFN-γ）和肿瘤坏死因子（tumornecrosisfactorOt,TNF-a）Westernblotting检测pAkt和总的Akt。结果与Sham组相比,IRI组血清肌酐和血尿素氮明显升高。病理检查可见肾脏内肾小管上皮细胞明显肿胀坏死、蛋白管型形成明显,还可观察到炎症细胞浸润明显增加。PCR显示IL-6、IFN-γ和TNF-a mRNA水平明显上调,Westernblotting显示p-Akt蛋白表达量明显增加,但Akt蛋白表达量无明显差异。与IRI组相比,DEX治疗组血清肌酐、血尿素氮明显下降,肾小管上皮细胞肿胀坏死减轻、炎症细胞浸润减少,IL-6、IFN-γ和TNF-a表达降低,p-Akt表达减少,但Akt蛋白表达量无明显差异。结论DEX预处理可通过抑制Akt信号通路激活,从而抑制炎症反应,从而减轻肾缺血再灌注损伤。     

低氧诱导因子1α高表达对小鼠急性缺血性肾损伤的影响

目的 探讨低氧预处理诱导低氧诱导因子1α（HIF-1α）高表达对小鼠肾缺血再灌注损伤（IRI）的影响及其可能机制。 方法 雄性C57BL/6N小鼠35只，随机分为健康对照组、氯化钴（CoCl2）组和8%O2组，每组10只；预处理12 h后，以上3组各取5只，分为缺血再灌注（IR）组、CoCl2＋IR组、8%O2＋IR组；另设5只作为假手术对照组。采用夹闭双侧肾蒂30 min的方法建立肾缺血动物模型，观察CoCl2和8％O2预处理对小鼠IR 24 h后肾功能、肾组织病理和相关肾损伤指标的影响及与CoCl2和8％O2诱导HIF-1α及其保护性靶基因血红素氧化酶1（HO-1）表达之间的关系。 结果 CoCl2＋IR组小鼠的肾功能[BUN (35.2±12.2) mmol/L，Scr (34.0±9.7) μmol/L]和8%O2＋IR组小鼠的肾功能[BUN (31.8±9.1) mmol/L，Scr (41.6±10.6) μmol/L]均较IR组[BUN (65.8±2.6) mmol/L，Scr (229.5±11.2) μmol/L]显著改善（P < 0.01）；与此相一致，CoCl2＋IR组和8%O2＋IR组的病理学改变、细胞凋亡程度和波形蛋白的表达均明显低于IR组。另外，CoCl2组和8%O2组中HIF-1α及其靶基因HO-1的表达明显高于健康对照组。 结论 低氧预处理可上调体内HIF-1α表达，对小鼠IRI肾脏具有良好保护效果     

Deletion of the activating NK cell receptor NKG2D accelerates rejection of cardiac allografts

It has already been shown that neutralization of the activating NK cell receptor NKG2D in combination with co‐stimulation blockade prolongs graft survival of vascularized transplants. In order to clarify the underlying cellular mechanisms, we transplanted complete MHC‐disparate BALB/c‐derived cardiac grafts into C57BL/6 wildtypes or mice deficient for NKG2D (Klrk1^?/?). Although median survival was 8 days for both recipient groups, we detected already at day 5 posttransplantation significantly greater intragraft frequencies of NKp46⁺ NK cells in Klrk1^?/? recipients than in wildtypes. This was followed by a significantly greater infiltration of CD4⁺, but a lesser infiltration of CD8⁺ T cell frequencies. Contrary to published observations, co‐stimulation blockade with CTLA4‐Ig resulted in a significant acceleration of cardiac rejection by Klrk1^?/? recipients, and this result was confirmed by applying a neutralizing antibody against NKG2D to wildtypes. In both experimental setups, grafts derived from Klrk1^?/? recipients were characterized by significantly higher levels of interferon‐γ mRNA, and both CD4⁺ and CD8⁺ T cells displayed a greater capacity for degranulation and interferon‐γ production. In summary, our results clearly illustrate that NKG2D expression in the recipient is important for cardiac allograft survival, thus supporting the hypothesis that impairment of NK cells prevents the establishment of graft acceptance.     

Genomic profiling of kidney ischemia-reperfusion reveals expression of specific alloimmunity-associated genes: Linking "immune" and "nonimmune" injury events

Increased organ ischemia time leads to delayed graft function (DGF), increased acute rejection (AR), enhanced chronic allograft nephropathy (CAN), and reduced long-term allograft survival. The mechanisms by which IRI predisposes to AR and CAN are unknown. We hypothesized that gene expression profiling of ischemia-reperfusion injury (IRI)-affected kidney would identify how IRI predisposes to AR and CAN. Furthermore, we examined how current immunosuppressive drug molecular targets are altered by IRI. C57BL/6J mice were exposed to 30 (n = 3) or 60 (n = 3) minutes of bilateral kidney ischemia or sham surgery (n = 5). At 36 hour kidney tissue was collected and analyzed using Affymetrix 430MOEA (22626 genes) array and GC-RMA-SAM pipeline. Genes with the false discovery rate (q < 1%) and +/-50% fold change (FC) were considered affected by IRI. Genes coding for histocompatibility and antigen-presenting factors, calcineurin, and mammalian target of rapamycin (mTOR) pathway-associated proteins were selected using Gene Ontology (GO) analysis. GO analysis identified 10 and 17 alloimmunity-related genes affected by IRI induced by 30 and 60 minutes of ischemia, respectively, including Traf6 (FC = 2.99) and H2-D1 (FC = 2.58). We also detected significant IRI genomic responses in calcineurin and mTOR pathways represented by Fkbp5 (FC = 4.18) and Fkbp1a (FC = 2.0), and Eif4ebp1 (FC = 16.8) and Akt1 (FC = 3.64), respectively. These data demonstrated that IRI up-regulates expression of several alloimmunity-associated genes, which can in turn enhance alloimune responses. Our discovery of IRI-induced up-regulation of genes associated with calcineurin and mTOR pathways are consistent with clinical observations that FK506 and Rapamycin can alter the course of DGF. Further validation and dissection of these pathways can lead to novel approaches by which improved management of early "nonimmune" transplant events can decrease susceptibility to more classic "immune" changes and CAN.     

Expression of stromal cell-derived factor 1 in the acute ischemia-reperfusion injury and its relationship with macrophages

Objective To observe the expression of stromal cell-derived factor 1 (SDF-1) in the kidney after ischemic reperfusion injury (IRI), and explore its relationship with macrophage during the IRI kidney. Methods A total of 28 healthy C57BL/6 male mice were used to establish renal IRI model by clamping both pedicles for 35 min followed by reperfusion. Kidney tissue samples were collected at indicated time points. Renal histological changes were estimated. The expression of SDF-1 was determined by immunohistochemistry, ELISA and real-time PCR. After the liposomal clodronate was injected intraperitoneally, the location of CD68 was observed by immunofluorescence. Renal histology and protein expression of SDF-1 were also detected. Results Compared with sham-operated group, classical tubular damage was found in IRI group, accompanied by a large number of inflammatory cells. The expression of total renal SDF-1 peaked on day 1 and decreased to control levels in the following days. SDF-1 in healthy kidney was localized at cortex, but spread to the corticomedullary area of the kidney during IRI. Compared with IRI groups, elimination of macrophage by injection of liposomal clodronate alleviated renal IRI and down-regulated the expressions of CD68 while up-regulating SDF-1. Conclusions SDF-1 expression is up-regulated in IRI kidney and is associated with macrophage. SDF-1 may play a role in the early phase of acute kidney injury and it may be a new marker in diagnosis of AKI.     

Natural Killer Cell Receptor Repertoire and Their Ligands,and the Risk of CMV Infection After Kidney Transplantation

Cytomegalovirus (CMV) infection is the most common viral complication after solid organ transplantation (SOT). Whilst current immunosuppression is known to impair antiviral‐specific T‐cell immunity in SOT, a potential role for natural killer (NK) cells not affected by immunosuppressive therapy remains to be determined. To address this, we compared the genotype of the NK immunoglobulin‐like receptor (KIR) genes and their HLA cognate ligands to the rate of CMV infection in 196 kidney transplant recipients. We have shown that the absence of the HLA‐C ligand for inhibitory KIR and the presence of activating KIR genes in the recipients were both associated with a lower rate of CMV infection after transplantation. In a cohort of 17 recipients with acute CMV infection, NK cells were phenotyped over a period of time after diagnosis by their expression profile of C‐type lectin receptors and capacity to secrete IFN‐γ. The increased expression of the activating C‐type lectin receptors NKG2C and NKG2D was paralleled by the decreased IFN‐γ secretion during the early phase of CMV infection. In conclusion, our findings suggest that KIR/HLA genotype and expression of NKG2C and NKG2D might play a significant role in regulating NK cell function and anti‐CMV immunity after kidney transplantation.     

The myocardial infarct-exacerbating effect of cell-free DNA is mediated by the high-mobility group box 1–receptor for advanced glycation end products–Toll-like receptor 9 pathway

IntroductionDamage-associated molecular patterns, such as high-mobility group box 1 (HMGB1) and cell-free DNA (cfDNA), play critical roles in mediating ischemia-reperfusion injury (IRI). HMGB1 activates RAGE to exacerbate IRI, but the mechanism underlying cfDNA-induced myocardial IRI remains unknown. We hypothesized that the infarct-exacerbating effect of cfDNA is mediated by HMGB1 and receptor for advanced glycation end products (RAGE).MethodsC57BL/6 wild type mice, RAGE knockout (KO), and Toll-like receptor 9 KO mice underwent 20- or 40-minute occlusions of the left coronary artery followed by up to 60 minutes of reperfusion. Cardiac coronary perfusate was acquired from ischemic hearts without reperfusion. Exogenous mitochondrial DNA was acquired from livers of normal C57BL/6 mice. Myocardial infarct size (IS) was reported as percent risk region, as measured by 2,3,5-triphenyltetrazolium chloride and Phthalo blue (Heucotech, Fairless Hill, Pa) staining. cfDNA levels were measured by Sytox Green assay (Thermo Fisher Scientific, Waltham, Mass) and/or spectrophotometer.ResultsFree HMGB1 and cfDNA levels were increased in the ischemic myocardium during prolonged ischemia and subsequently in the plasma during reperfusion. In C57BL/6 mice undergoing 40′/60′ IRI, deoxyribonuclease I, or anti-HMGB1 monoclonal antibody reduced IS by approximately half to 29.0% ± 5.2% and 24.3% ± 3.5% (P < .05 vs control 54.3% ± 3.4%). However, combined treatment with deoxyribonuclease I + anti-HMGB1 monoclonal antibody did not further attenuate IS (29.3% ± 4.9%). In C57BL/6 mice undergoing 20′/60′ IRI, injection of 40′/5′ plasma upon reperfusion increased IS by more than 3-fold (to 19.9 ± 4.3; P < .05). This IS exacerbation was abolished by pretreating the plasma with deoxyribonuclease I or by depleting the HMGB1 by immunoprecipitation, or by splenectomy. The infarct-exacerbating effect also disappeared in RAGE KO mice and Toll-like receptor 9 KO mice. Injection of 40′/0′ coronary perfusate upon reperfusion similarly increased IS. The levels of HMGB1 and cfDNA were significantly elevated in the 40′/0′ coronary perfusate and 40′/reperfusion (min) plasma but not in those with 10′ ischemia. In C57BL/6 mice without IRI, 40′/5′ plasma significantly increased the interleukin-1β protein and messenger RNA expression in the spleen by 30 minutes after injection. Intravenous bolus injection of recombinant HMGB1 (0.1 μg/g) or mitochondrial DNA (0.5 μg/g) 5 minutes before reperfusion did not exacerbate IS (P = not significant vs control). However, combined administration of recombinant HMGB1 + mitochondrial DNA significantly increased IS (P < .05 vs individual treated groups) and this infarct-exacerbating effect disappeared in RAGE KO mice and splenectomized C57BL/6 mice. The accumulation of cfDNA in the spleen after combined recombinant HMGB1 + mitochondrial DNA treatment was significantly more elevated in C57BL/6 mice than in RAGE KO mice.Conclusions:Both HMGB1 and cfDNA are released from the heart upon reperfusion after prolonged ischemia and both contribute importantly and interdependently to post-IRI by a common RAGE-Toll-like receptor 9–dependent mechanism. Depleting either of these 2 damage-associated molecular patterns suffices to significantly reduce IS by approximately 50%.