Neutrophil extracellular trap fragments stimulate innate immune responses that prevent lung transplant tolerance

Neutrophil extracellular traps (NETs) have been shown to worsen acute pulmonary injury including after lung transplantation. The breakdown of NETs by DNAse‐1 can help restore lung function, but whether there is an impact on allograft tolerance remains less clear. Using intravital 2‐photon microscopy, we analyzed the effects of DNAse‐1 on NETs in mouse orthotopic lung allografts damaged by ischemia‐reperfusion injury. Although DNAse‐1 treatment rapidly degrades intragraft NETs, the consequential release of NET fragments induces prolonged interactions between infiltrating CD4⁺ T cells and donor‐derived antigen presenting cells. DNAse‐1 generated NET fragments also promote human alveolar macrophage inflammatory cytokine production and prime dendritic cells for alloantigen‐specific CD4⁺ T cell proliferation through activating toll‐like receptor (TLR) — Myeloid Differentiation Primary Response 88 (MyD88) signaling pathways. Furthermore, and in contrast to allograft recipients with a deficiency in NET generation due to a neutrophil‐specific ablation of Protein Arginine Deiminase 4 (PAD4), DNAse‐1 administration to wild‐type recipients promotes the recognition of allo‐ and self‐antigens and prevents immunosuppression‐mediated lung allograft acceptance through a MyD88‐dependent pathway. Taken together, these data show that the rapid catalytic release of NET fragments promotes innate immune responses that prevent lung transplant tolerance.     

Induction of Alloimmune Tolerance in Heart Transplantation Through Gene Silencing of TLR Adaptors

Toll‐like receptors (TLRs) activate biochemical pathways that evoke activation of innate immunity, which leads to dendritic cell (DC) maturation and initiation of adaptive immune responses that provoke allograft rejection. We aimed to prolong allograft survival by selectively inhibiting expression of the common adaptors of TLR signaling, namely MyD88 and TRIF, using siRNA. In vitro we demonstrated that blocking expression of MyD88 and TRIF led to reduced DC maturation. In vivo treatment of recipients with MyD88 and TRIF siRNA significantly prolonged allograft survival in the BALB/c > C57BL6 cardiac transplant model. Moreover, the combination of MyD88 and TRIF siRNA along with a low dose of rapamycin further extended the allograft survival (88.8 ± 7.1 days). Tissue histopathology demonstrated an overall reduction in lymphocyte interstitium infiltration, vascular obstruction and hemorrhage in mice treated with MyD88 and TRIF siRNA vector plus rapamycin. Furthermore, treatment was associated with an increase in the numbers of CD4⁺CD25⁺FoxP3⁺ regulatory T cells and Th2 deviation. To our knowledge, this study is the first demonstration of prolonging the survival of allogeneic heart grafts through gene silencing of TLR signaling adaptors, highlighting the therapeutic potential of siRNA in clinical transplantation.     

Short‐term MyD88 inhibition ameliorates cardiac graft rejection and promotes donor‐specific hyporesponsiveness of skin grafts in mice

Recognition of evolutionarily conserved ligands by Toll‐like receptors (TLRs) triggers signaling cascades in innate immune cells to amplify adaptive immune responses. Nearly all TLRs require MyD88 to transduce downstream signaling. MyD88 deficiency has been shown to promote the allograft acceptance in mice. However, direct evidence for therapeutic potential of MyD88 inhibitors remains lacking. Herein, we used a MyD88 inhibitor, namely ST2825, to explore its therapeutic potential and mechanisms in fully allogeneic skin and heart transplant models. Phenotypic maturation of dendritic cells stimulated by TLR ligands was alleviated by ST2825 in parallel with reduced T‐cell proliferation in vitro. A short‐course treatment with ST2825 significantly prolonged cardiac graft survival (mean survival time = 18.5 ± 0.92 days vs. 7.25 ± 0.46 days). ST2825‐treated group had significantly reduced proinflammatory cytokines in allografts compared with control group. ST2825 combined with anti‐CD154 induced long‐term skin allograft acceptance in about one‐third of recipients (>100 days). ‘Skin‐tolerant’ recipients showed attenuated donor‐specific IFN‐γ responses, intact IL‐4 responses, and compromised alloantibody responses. We conclude that MyD88 inhibitor ST2825 attenuates acute cardiac rejection and promotes donor‐specific hyporesponsiveness in stringent skin transplant models. The direct evidence suggests that pharmacological inhibition of MyD88 hold promising potential for transplant rejection.     

TH1 Immune Responses to Fully MHC Mismatched Allografts are Diminished in the Absence of MyD88, a Toll-Like Receptor Signal Adaptor Protein

Toll‐like receptors (TLRs) are innate immune receptors that are critical for recognizing conserved microbial motifs by inducing TH1 immunity. The majority of TLRs utilize the adaptor protein MyD88 for signal transduction, although other adaptors have been recently described. As the role of innate immunity in transplantation is unclear, we examined the importance of the MyD88 pathway in acute rejection of fully MHC‐mismatched murine allografts and specifically investigated whether MyD88 signaling is important for DC (dendritic cell) function and TH1 alloimmune responses. Our results demonstrate that acute rejection of both fully allogeneic skin and cardiac allografts occurs in the absence of MyD88. However, priming of naïve recipient T cells by allogeneic DCs and TH1 immune responses were diminished in the absence of MyD88, although TH2 immunity remained intact. Thus, these results demonstrate that MyD88 signaling is important for DC function and TH1 responses during fully MHC‐mismatched solid‐organ transplantation, although graft rejection occurs independently of MyD88.     

Impact of TLR4-siRNA transfection on the expression of TLR4/MyD88 Signaling in rat tubular epithelial cells under high glucose condition

Objective To observe the expression of toll like receptor 4(TLR4) Signaling and the release of inflammation factors in rat tubular epithelial cell(NRK-52E) under high glucose condition after TLR4-siRNA transfection. Methods Three TLR4-siRNA sequences were designed and synthesized. The transfection efficiency was observed by fluorescence microscope after transfection, and the expression of TLR4 mRNA was detected by real time PCR. The most effective siRNA was selected to be used for forward experiments. After transfection for 24 h, cells were stimulated with 25 mmol/L glucose and/or 10-7 mmol/L Angiotension Ⅱ(AngⅡ) for 12 h, 24 h; cells without stimulation were as normal control. Real-time PCR was used to analyze TLR4 and myeloid differentiation factor 88 (MyD88) mRNA expression; Western blot was used to observe TLR4/MyD88 and NF-κB protein expression. ELISA assay was used to detect the concentration of monocyte chemoattractant protein-1(MCP-1), interleukin-6(IL-6) in cell supernatant after cells were stimulated for 24 h. Results TLR4/MyD88 mRNA and TLR4/MyD88/NF-κB protein were highly expressed under high glucose or AngⅡ co-incubated NRK-52E(P＜0.01), the MCP-1 and IL-6 levels were also increased markedly compared with normal control group (P＜0.01). TLR4/MyD88 mRNA and TLR4/MyD88/NF-kB protein expressions were obviously inhibited in cells that were transfected with TLR4-siRNA compared with high glucose group(P＜0.01), MCP-1 and IL-6 production decreased remarkably compared with high glucose or AngⅡ co-stimulated group(P＜0.01). Conclusions High glucose can lead to the activation of TLR4/MyD88/NF-kB signaling and the secretion of inflammation factors in NRK-52E, AngⅡ further augments these effects. The effect can be blocked efficiently by specific siRNA gene silence. TLR4 signaling plays a pivotal role in the innate-immune inflammatory reaction in NRK-52E.     

Angiotensin II up-regulates the release of inflammation factors via Toll-like receptor 4 signal in rat mesangial cells under high glucose

Objective To observe the regulation of Toll-like receptor 4 (TLR4) signal and the release of inflammation factors after angiotensin II (AngⅡ) stimulation in rat mesangial cells under high glucose condition, revealing the innate immune-related mechanism of injury by AngⅡ on mesangial cells under high glucose. Methods After synchronization, cells incubated with AngⅡ(10-7 mmo/L) and/or high glucose (25 mmol/L) were used as the stimulation group, cells without stimulation were as normal control (5.6 mmol/L glucose). To determine the role of TLR4 and the adaptor myeloid differentiation factor 88 (MyD88), equal number of HBZY-1 cells were added with 10-5 mmol/L irbesartan and/or TLR4 blocker (10 mg/L) for 1 h and then incubated with AngⅡ (10-7 mmo/L) and/or high glucose (25 mmol/L) for 12 h or 24 h respectively. Real-time PCR was used to analyze TLR4 mRNA and MyD88 mRNA expression after 12 h. Immunofluorescence was used to observe TLR4 protein expression after 24 h; Western blotting was used to observe TLR4, MyD88 and nuclear factor κB (NF-κB) protein; ELISA was used to detect the concentration of MCP-1, IL-6 in cell supernatant respectively. Results Compared with normal control group, TLR4 mRNA and MyD88 mRNA were highly expressed in high glucose or AngⅡ-induced HBZY-1 cells (P＜0.01), TLR4, MyD88 and NF-κB protein as well as MCP-1, IL-6 were also up-regulated significantly (P＜0.01). Compared with high glucose or AngⅡ group, MyD88 and NF-κB protein as well as MCP-1, IL-6 were further up-regulated markedly in AngⅡ and high glucose costimulated group (P＜0.01)．In HBZY-1 cells that were preincubated with irbesartan and/or TLR4 blocker, TLR4 and MyD88 protein expression were obviously inhibited, IL-6 and MCP-1 production were also decreased remarkably compared with high glucose and/or AngⅡ group (P＜0.01). Conclusions High glucose and AngⅡ stimulate the release of proinflammatory factors in rat glomerular mesangial cells via TLR4-MyD88 pathway. This process is inhibited by irbesartan or TLR4 blocker via modulation of the signal. AngⅡ has the positive-regulation potential on the release of inflammation factors via TLR4 signal in rat mesangial cells under high glucose condition.     

B cell–derived IL‐1β and IL‐6 drive T cell reconstitution following lymphoablation

Understanding the mechanisms of T cell homeostatic expansion is crucial for clinical applications of lymphoablative therapies. We previously established that T cell recovery in mouse heart allograft recipients treated with anti‐thymocyte globulin (mATG) critically depends on B cells and is mediated by B cell–derived soluble factors. B cell production of interleukin (IL)‐1β and IL‐6 is markedly upregulated after heart allotransplantation and lymphoablation. Neutralizing IL‐1β or IL‐6 with mAb or the use of recipients lacking mature IL‐1β, IL‐6, IL‐1R, MyD88, or IL‐6R impair CD4⁺ and CD8⁺ T cell recovery and significantly enhance the graft‐prolonging efficacy of lymphoablation. Adoptive co‐transfer experiments demonstrate a direct effect of IL‐6 but not IL‐1β on T lymphocytes. Furthermore, B cells incapable of IL‐1β or IL‐6 production have diminished capacity to mediate T cell reconstitution and initiate heart allograft rejection upon adoptive transfer into mATG treated B cell deficient recipients. These findings reveal the essential role of B cell–derived IL‐1β and IL‐6 during homeostatic T cell expansion in a clinically relevant model of lymphoablation.     

Comprehensive analysis of the immunomodulatory effects of rapamycin on human T cells in graft-versus-host disease prophylaxis

Graft-versus-host disease (GVHD) is a major cause of toxicity after allogeneic hematopoietic cell transplantation (allo-HCT). While rapamycin (RAPA) is commonly used in GVHD prophylaxis in combination with a calcineurin inhibitor (CNI), the understanding of its mechanism of action on human T cells is still incomplete. Here, we performed an extensive analysis of RAPA effects on human T cells in a humanized mouse model of GVHD, in ex-vivo T cell cultures and in patients given RAPA plus tacrolimus as GVHD prophylaxis after nonmyeloablative allo-HCT. We demonstrate that RAPA mitigates GVHD by decreasing T cell engraftment and differentiation, inhibiting CD8⁺ T cell activation and increasing the long-term IL-2 secretion, thereby supporting regulatory T cell (Treg) proliferation. In contrast, graft-versus-leukemia effects were not abrogated, as RAPA-treated T cells had increased resistance to apoptosis and retained their effector function and proliferative capacity upon re-stimulation. Importantly, we found that RAPA impact on Treg and CD8⁺ T cells was closely dependent upon IL-2 signaling and that therapeutic options interfering with IL-2, such as calcineurin inhibitors, antagonize the IL-2-dependent promotion of Treg mediated by RAPA. Our results suggest that RAPA immunological efficacy could be improved in combination with drugs having possible synergistic effects such as the hypomethylating agent 5-azacytidine.     

Deferoxamine preconditioning activated hypoxia-inducible factor-1α and MyD88-dependent Toll-like receptor 4 signaling in intestinal stem cells

Background/Purpose

Toll-like receptors (TLRs) are important regulators of innate immunity, and TLR4 pathway can regulate the survival, migration, and differentiation of stem cells, including intestinal stem cells (ISCs). Deferoxamine (DFO), a hypoxia-mimic compound, can activate the proliferation of ISCs. In this study, we investigated the response of TLR4 signaling to DFO-induced hypoxia in cultured ISCs in vitro.

IL-6 and TNF-α Synergistically Inhibit Allograft Acceptance

Previous studies suggested that activation of the innate immune system impairs the induction of transplantation tolerance, but the responsible inflammatory mediators have not been identified. In this study, we examined whether IL-6 and TNF-α promote resistance to transplantation tolerance. Using a highly immunogenic murine skin allograft model, we found that the absence of both IL-6 and TNF-α in the graft recipient synergized with co-stimulatory blockade to induce tolerance. Furthermore, IL-6 and TNF-α acted together to promote T cell alloimmune responses both in vitro and in vivo and to impair the ability of regulatory T cells to suppress effector T cell alloimmunity. In addition, deficiency of recipient IRAK-M, a negative regulator of certain innate immune pathways, augmented cellular IL-6 and TNF-α responses and impaired the ability of co-stimulatory blockade to extend allograft survival. In summary, IL-6 and TNF-α synergistically impair the efficacy of therapies that promote allograft acceptance.Studies have demonstrated that innate immunity and inflammation are involved in acute allograft rejection and transplantation tolerance.^1–4 Specifically, activation of Toll-like receptors (TLRs), innate immune receptors, on dendritic cells (DCs) is critical for acute allograft rejection in certain experimental models.² Furthermore, the administration of TLR ligands prevents the induction of transplantation tolerance,^3,4 whereas the genetic deletion of MyD88, a key TLR signal adaptor, leads to allograft acceptance induced by co-stimulatory blockade.¹ Importantly, humans who exhibit operational tolerance of kidney transplants exhibit reduced peripheral expression of MyD88 compared with transplant recipients with evidence of chronic rejection.⁵ Thus, high expression of MyD88 predisposes patients to a phenotype of resistance to transplant tolerance, whereas low expression indicates a greater chance that transplant tolerance will occur; however, to test these findings in clinical transplantation trials, it is necessary to elucidate the inflammatory mediators downstream of TLR/MyD88 activation that leads to the resistance of transplant tolerance.Previous work demonstrated that inflammatory cytokines, in particular IL-6, can impair the function of regulatory T cells (Tregs) by promoting the proliferation of effector T cells.⁶ This is supported by a study demonstrating that IL-6 is necessary for the induction of experimental allergic encephalitis⁷; however, other inflammatory cytokines play important roles on immune regulation.⁸ Indeed, TNF-α, like IL-6, promotes T cell proliferation⁹ and also impairs peripheral tolerance of islets.¹⁰ It is likely that there are redundant pathways between inflammatory cytokines in immune regulation,¹¹ but it is not clear whether IL-6 and TNF-α cooperate in preventing transplant tolerance.Our previous work demonstrated that DCs produce both IL-6 and TNF-α during acute allograft rejection in a MyD88-dependent manner.¹ In particular, the administration of co-stimulatory blockade and the absence of MyD88 led to an abrogated ability of DCs to produce these cytokines during acute allograft rejection.¹ We further demonstrated that inflammatory cytokines produced by TLR-activated DCs augment T cell alloimmune responses and subsequently impair the function of Tregs.¹ Given these findings and previous work indicating that IL-6 and TNF-α impair immune regulation, we examined whether the absence of IL-6, TNF-α, or both is necessary for allograft acceptance induced by co-stimulatory blockade antibody treatment. We provide in vivo evidence that the absence of both IL-6 and TNF-α but neither alone is critical for the ability of co-stimulatory blockade treatment to induce indefinite allograft survival. These data demonstrate that the inflammatory cytokines IL-6 and TNF-α cooperate to prevent immune tolerance of allografts.     

Toll‐like receptors and their adaptors are regulated in macrophages after phagocytosis of lipopolysaccharide‐coated titanium particles

Macrophages phagocytose metallic wear particles and produce mediators, which can induce cellular host response and aseptic implant loosening. Lipopolysaccharide (LPS) on the wear debris can stimulate macrophages via Toll‐like receptor 4 (TLR4) and enhance the response. However, the precise functional role and interaction of TLRs and their adaptor molecules is still unclear. Rat bone marrow macrophages were stimulated with titanium particle (Ti) coated by LPS (Ti/LPS+) and LPS‐free Ti (Ti/LPS?). mRNA levels of cytokines, TLRs and their adaptor molecules were measured using real time PCR. mRNA levels of TNF‐α, IL‐1β, and IL‐6 increased in Ti/LPS+ than Ti/LPS?. In contrast, mRNA levels of TLR4, TLR5, and TLR9 decreased in Ti/LPS+ compared to Ti/LPS?. mRNA levels of MyD88, IRAK1, IRAK4 decreased gradually, and TRAF6 underwent an initial transient increase, followed by suppression in Ti/LPS+. However, mRNA levels of TLR2 and IRAK2 increased after phagocytosis of Ti/LPS+ than Ti/LPS?. The increased expressions of proinflammatory cytokines found in Ti/LPS+ indicated that their productions cytokines could be enhanced by phagocytosis of LPS‐coated particles. Subsequent down‐regulation of TLR4, TLR5, TLR9, MyD88, IRAK1, and IRAK4 suggests that self‐protective mechanisms to regulate excessive host responses are activated in macrophages. Increase of TLR2 and IRAK2 and a transient increase of TRAF6 in Ti/LPS+ suggest that another possible pathway to modulate TLR‐mediated cellular response to prolong inflammatory response in foreign body reaction of aseptic loosening. This down‐ and/or up‐regulation of the potential TLR‐mediated responses to LPS‐coated particles reflects the proactive behavior of effector cells. © 2011 Orthopaedic Research Society Published by Wiley Periodicals, Inc. J Orthop Res 29: 984–992, 2011     

Toll-like receptor 3 is an endogenous sensor of cell death and a potential target for induction of long-term cardiac transplant survival

Inflammation posttransplant is directly linked to cell death programs including apoptosis and necrosis. Cell death leads to the release of cellular contents which can promote inflammation. Targeting of these pathways should be an effective strategy to prevent transplant rejection. Toll-like receptor 3 (TLR3) is emerging as a major endogenous sensor of inflammation. In this study, we assessed the role of TLR3 on cell death and transplant rejection. We showed that TLR3 is highly expressed on mouse microvascular endothelial cell (ECs) and the endothelium of cardiac grafts. We demonstrated that TLR3 interacting with dsRNA or self-RNA triggered apoptosis and necroptosis in ECs. Interestingly, TLR3-induced necroptosis led mitochondrial damage. Inhibition of the mitochondrial membrane permeability molecule Cyclophilin D prevented necroptosis in ECs. In vivo, endothelium damage and activities of caspase-3 and mixed lineage kinase domain-like protein were inhibited in TLR3^−/− cardiac grafts compared with C57BL/6 grafts posttransplant (n = 5, p < .001). Importantly, TLR3^−/− cardiac grafts had prolonged survival in allogeneic BALB/c mice (mean survival = 121 ± 67 vs. 31 ± 6 days of C57BL/6 grafts, n = 7, p = .002). In summary, our study suggests that TLR3 is an important cell death inducer in ECs and cardiac grafts and thus a potential therapeutic target in preventing cardiac transplant rejection.     

High glucose induces toll-like receptor expression in human monocytes: mechanism of activation

OBJECTIVE—Hyperglycemia-induced inflammation is central in diabetes complications, and monocytes are important in orchestrating these effects. Toll-like receptors (TLRs) play a key role in innate immune responses and inflammation. However, there is a paucity of data examining the expression and activity of TLRs in hyperglycemic conditions. Thus, in the present study, we examined TLR2 and TLR4 mRNA and protein expression and mechanism of their induction in monocytic cells under high-glucose conditions.RESEARCH DESIGN AND METHODS—High glucose (15 mmol/l) significantly induced TLR2 and TLR4 expression in THP-1 cells in a time- and dose-dependent manner (P < 0.05). High glucose increased TLR expression, myeloid differentiation factor 88, interleukin-1 receptor–associated kinase-1, and nuclear factor-κB (NF-κB) p65-dependent activation in THP-1 cells. THP-1 cell data were further confirmed using freshly isolated monocytes from healthy human volunteers (n = 10).RESULTS—Pharmacological inhibition of protein kinase C (PKC) activity and NADPH oxidase significantly decreased TLR2 and TLR4 mRNA and protein (P < 0.05). Knocking down both TLR2 and TLR4 in the cells resulted in a 76% (P < 0.05) decrease in high-glucose–induced NF-κB activity, suggesting an additive effect. Furthermore, PKC-α knockdown decreased TLR2 by 61% (P < 0.05), whereas inhibition of PKC-δ decreased TLR4 under high glucose by 63% (P < 0.05). Small inhibitory RNA to p47Phox in THP-1 cells abrogated high-glucose–induced TLR2 and TLR4 expression. Additional studies revealed that PKC-α, PKC-δ, and p47Phox knockdown significantly abrogated high-glucose–induced NF-κB activation and inflammatory cytokine secretion.CONCLUSIONS—Collectively, these data suggest that high glucose induces TLR2 and -4 expression via PKC-α and PKC-δ, respectively, by stimulating NADPH oxidase in human monocytes.The major cause of death in type 1 and type 2 diabetic patients is atherosclerosis (1–3). The pathogenesis of the accelerated atherosclerosis is multifactorial. Inflammation is pivotal in the development of atherosclerosis. Recent studies have shown that diabetes is a proinflammatory state (4–6). We and others have shown that the proinflammatory phenotype in diabetes is characterized by elevated plasma C-reactive protein (CRP), cytokines, chemokines, adhesion molecules, monocytic activity, etc. (4–6). Hyperglycemia contributes to vascular complications of diabetes. High glucose has been shown to induce inflammatory cytokines, chemokines, p38 mitogen-activated protein kinase, reactive oxygen species (ROS), protein kinase C (PKC), and nuclear factor-κB (NF-κB) activity in both clinical and experimental systems (7–12). Several lines of evidence support a role for oxidative stress in the development of diabetes complications (13,14). Diabetic patients have increased O₂⁻ production in monocytes and neutrophils (8,13,15); however, the mechanism of the interactions among these mediators remain unclear.Toll-like receptors (TLRs) recognize conserved pathogen-associated molecular patterns and induce innate immune responses that are essential for host defenses (16). TLRs are activated by both endogenous and exogenous agonists of microbial and nonmicrobial origin. TLR activation by their agonists triggers a signaling cascade, leading to cytokine production and initiation of an adaptive immune response (17). TLR expression is increased in a plethora of inflammatory disorders, including atherosclerosis and diabetes (18–20). Some of the endogenous ligands for TLR2 and TLR4 include high-mobility group B1, biglycan, hyaluronic acid fragments, necrotic cells, serum amyloid A, advanced glycation end products, and extracellular matrix components (18). Among the TLRs, TLR2 and TLR4 play an important role in atherosclerosis. TLR2 and TLR4 bind to components of the Gram-positive and -negative bacteria, respectively (17). They are expressed in multiple cells and tissues, primarily in monocytes. TLR2 and TLR4 expression is increased in atherosclerotic plaque macrophages and in animal models of atherosclerosis (21–25). Plaques of TLR4 knockout mice on a high-fat diet show reduced lesion size, lipid content, and macrophage infiltration (22). TLR2/LDLR^−/− and TLR2/ApoE^−/− double knockout mice are protected from the development of atherosclerosis (24). In addition, total loss of myeloid differentiation factor 88 (MyD88), a common adapter molecule of TLR2 and TLR4 in the cell, results in reduced plaque size, lipid content, inflammation, and plasma interleukin (IL)-1 and tumor necrosis factor-α (TNF-α) (25).The interactions among inflammation, hyperglycemia, and diabetes have clear implications for the immune system. Mohammad et al. (26) reported increased TLR2 and TLR4 expression in type 1 diabetic NOD mice, correlating with increased NF-κB activation in response to endotoxin, and increased proinflammatory cytokines. Kim et al. (27) using TLR2^−/−, TLR4^−/− knockouts, and NOD mice have demonstrated that TLR2 senses β-cell death and contributes to the instigation of autoimmune diabetes. Devaraj et al. (20) showed increased TLR2 and TLR4 expression, intracellular signaling, and TLR-mediated inflammation in monocytes with significant correlation to A1C levels in type 1 diabetic patients. Also, Song et al. (28) reported increased TLR4 mRNA expression in differentiating adipose tissue of db/db mice. Creely et al. (29) showed increased TLR2 expression in the adipose tissue of type 2 diabetic patients with strong correlates to endotoxin levels. These observations taken together suggest a potential role for TLR2 and TLR4 in the pathology of diabetes with limited mechanistic details.However, data examining the mechanism of increased TLR2 and TLR4 expression in diabetes are unknown. Therefore, this study aimed to test the ability of high glucose, one of the key abnormalities of the diabetic condition, to induce TLR expression in human monocytes.     

七氟醚预处理对大鼠肺缺血-再灌注损伤的保护作用及对TLR4/MyD88/NF-κB信号通路的影响

目的  探讨七氟醚预处理对肺缺血-再灌注损伤（IRI）的保护作用及对Toll样受体4（TLR4）/髓样分化因子88（MyD88）/核因子（NF）-κB信号通路的影响。 方法  将40只健康成年SD大鼠随机分为对照组（Sham组）、模型组（LIRI组）、七氟醚预处理组（Sev组）和TLR4抑制剂TAK-242联合七氟醚预处理组（TAK+Sev组）,每组各10只。采用苏木素-伊红（HE）染色观察肺组织病理学变化并进行病理损伤评分;采用脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记（TUNEL）法检测肺组织细胞凋亡并计算细胞凋亡率;测定肺组织湿重/干重（W/D）比值以确定肺组织含水量;检测肺组织中氧化应激相关指标水平以及肺组织和血清中炎症因子水平;采用蛋白质印迹法检测肺组织中TLR4/MyD88/NF-κB信号通路相关蛋白的表达水平。 结果  与Sham组比较,LIRI组和Sev组大鼠肺组织病理损伤评分、W/D比值、细胞凋亡率、丙二醛（MDA）水平、炎症因子水平以及TLR4、MyD88、NF-κB p65蛋白相对表达量均升高,超氧化物歧化酶（SOD）水平和NF-κB抑制蛋白α（IκBα）相对表达量均降低（均为P < 0.05）;与LIRI组比较,Sev组和TAK+Sev组大鼠肺组织病理损伤评分、W/D比值、细胞凋亡率、MDA水平、炎症因子水平以及TLR4、MyD88、NF-κB p65蛋白相对表达量均降低,SOD水平和IκBα相对表达量均升高（均为P < 0.05）;与Sev组比较,TAK+Sev组大鼠肺组织病理损伤评分、W/D比值、细胞凋亡率、MDA水平、炎症因子水平以及TLR4、MyD88、NF-κB p65蛋白相对表达量均降低,IκBα相对表达量升高（均为P < 0.05）。 结论  七氟醚预处理能够抑制TLR4/MyD88/NF-κB信号通路的激活,抑制炎症反应和氧化应激,从而有效减轻肺IRI。     

Innate Lymphoid Cells Groups 1 and 3 in the Epithelial Compartment of Functional Human Intestinal Allografts

We examined intraepithelial lymphocytes (IELs) in 213 ileal biopsies from 16 bowel grafts and compared them with 32 biopsies from native intestines. During the first year posttransplantation, grafts exhibited low levels of IELs (percentage of CD103⁺ cells) principally due to reduced CD3⁺CD8⁺ cells, while CD103⁺CD3^– cell numbers became significantly higher. Changes in IEL subsets did not correlate with histology results, isolated intestine, or multivisceral transplants, but CD3^– IELs were significantly higher in patients receiving corticosteroids. Compared with controls, more CD3^– IELs of the grafts expressed CD56, NKp44, interleukin (IL)‐23 receptor, retinoid‐related orphan receptor gamma t (RORγt), and CCR6. No difference was observed in granzyme B, and CD3^–CD127⁺ cells were more abundant in native intestines. Ex vivo, and after in vitro activation, CD3^– IELs in grafts produced significantly more interferon (IFN)‐γ and IL‐22, and a double IFNγ⁺IL‐22⁺ population was observed. Epithelial cell–depleted grafts IELs were cytotoxic, whereas this was not observed in controls. In conclusion, different from native intestines, a CD3^– IEL subset predominates in grafts, showing features of natural killer cells and intraepithelial ILC1 (CD56⁺, NKp44⁺, CCR6⁺, CD127^–, cytotoxicity, and IFNγ secretion), ILC3 (CD56⁺, NKp44⁺, IL‐23R⁺, CCR6⁺, RORγt⁺, and IL‐22 secretion), and intermediate ILC1–ILC3 phenotypes (IFNγ⁺IL‐22⁺). Viability of intestinal grafts may depend on the balance among proinflammatory and homeostatic roles of ILC subsets.