Diacerein mitigates renal ischemia/reperfusion injury via inhibition of renal inflammation, dendritic cells maturation and apoptosis: the role of TLR4/NF-kB/NLRP3/IL-1beta signaling pathway / La diacereína mitiga la lesión por isquemia/reperfusión renal a través de la inhibición de la inflamación renal, la maduración de las células dendríticas y la apoptosis: el papel de la vía de señalización TLR4/NF-kB/NLRP3/IL-1beta

SUMMARY: One of the reasons for acute kidney damage is renal ischemia. Nevertheless, there are limited protective and therapeutic approaches for this problem. Diacerein is an anti-inflammatory drug characterized by numerous biological activities. We aimed to determine the ameliorative impact of diacerein on renal ischemia/reperfusion injury (I/R) condition, exploring the underlying mechanisms. Twenty-four male rats were allotted into four groups (n= 6): sham group; Diacerein (DIA) group; I/R group, in which a non-crushing clamp occluded the left renal pedicle for 45 min, and the right kidney was nephrectomized for 5 min before the reperfusion process; I/R + diacerein group, injected intraperitoneally with 50 mg diacerein/kg i.m 30 minutes prior to I/R operation. Ischemia/ reperfusion was found to affect renal function and induce histopathological alterations. The flow cytometry analysis demonstrated an elevated expression of innate and mature dendritic cells in I/R renal tissues. Moreover, upregulation in the expression of the inflammatory genes (TLR4, Myd88, and NLRP3), and overexpression of the pro-inflammatory cytokines (IL-1β), apoptotic (caspase-3) and pyroptotic (caspase-1) markers were observed in I/R-experienced animals. The aforementioned deteriorations were mitigated by pre-I/R diacerein treatment. Diacerein alleviated I/R-induced inflammation and apoptosis. Thus, it could be a promising protective agent against I/R.

La isquemia renal es una de los motivos del daño renal agudo. Sin embargo, los enfoques protectores y terapéuticos para este problema son limitados. La diacereína es un fármaco antiinflamatorio caracterizado por numerosas actividades biológicas. Nuestro objetivo fue determinar el impacto de mejora de la diacereína en la condición de lesión por isquemia/ reperfusión renal (I/R), explorando los mecanismos subyacentes. Veinticuatro ratas macho se distribuyeron en cuatro grupos (n= 6): grupo simulado; grupo de diacereína (DIA); grupo I/R, en el que una pinza no aplastante ocluyó el pedículo renal izquierdo durante 45 min, y el riñón derecho fue nefrectomizado durante 5 min antes del proceso de reperfusión; Grupo I/R + diacereína, inyectado por vía intraperitoneal con 50 mg de diacereína/kg i.m. 30 min antes de la operación I/R. Se encontró que la isquemia/ reperfusión afecta la función renal e induce alteraciones histopatológicas. El análisis de citometría de flujo demostró una expresión elevada de células dendríticas innatas y maduras en tejidos renales I/R. Además, se observó una regulación positiva en la expresión de los genes inflamatorios (TLR4, Myd88 y NLRP3) y una sobreexpresión de las citoquinas proinflamatorias (IL-1β), marcadores apoptóticos (caspasa-3) y piroptóticos (caspasa-1) en animales con experiencia en I/R. Los deterioros antes mencionados fueron mitigados por el tratamiento previo a la diacereína I/R. La diacereína alivió la inflamación y la apoptosis inducidas por I/R. Por lo tanto, podría ser un agente protector prometedor contra I/R.

ILC2s expanded by exogenous IL-33 regulate CD45+CD11b+F4/80high macrophage polarization to alleviate hepatic ischemia-reperfusion injury.

Hepatic ischemia-reperfusion injury (IRI) is an adverse consequence of hepatectomy or liver transplantation. Recently, immune mechanisms involved in hepatic IRI have attracted increased attention of investigators working in this area. In specific, group 2 innate lymphoid cells (ILC2s), have been strongly implicated in mediating type 2 inflammation. However, their immune mechanisms as involved with hepatic IRI remain unclear. Here, we reported that the population of ILC2s is increased with the development of hepatic IRI as shown in a mouse model in initial stage. Moreover, M2 type CD45+CD11b+F4/80high macrophages increased and reached maximal levels at 24 h followed by a significant elevation in IL-4 levels. We injected exogenous IL-33 into the tail vein of mice as a mean to stimulate ILC2s production. This stimulation of ILC2s resulted in a protective effect upon hepatic IRI along with an increase in M2 type CD45+CD11b+F4/80high macrophages. In contrast, depletion of ILC2s as achieved with use of an anti-CD90.2 antibody substantially abolished this protective effect of exogenous IL-33 and M2 type CD45+CD11b+F4/80high macrophage polarization in hepatic IRI. Therefore, this exogenous IL-33 induced potentiation of ILC2s appears to regulate the polarization of CD45+CD11b+F4/80high macrophages to alleviate IRI. Such findings provide the foundation for the development of new targets and strategies in the treatment of hepatic IRI.

The Role of NLRP3 Inflammasome Activation Pathway of Hepatic Macrophages in Liver Ischemia-Reperfusion Injury.

Ischemia-reperfusion injury (IRI) is considered an inherent component involved in liver transplantation, which induce early organ dysfunction and failure. And the accumulating evidences indicate that the activation of host innate immune system, especially hepatic macrophages, play a pivotal role in the progression of LIRI. Inflammasomes is a kind of intracellular multimolecular complexes that actively participate in the innate immune responses and proinflammatory signaling pathways. Among them, NLRP3 inflammasome is the best characterized and correspond to regulate caspase-1 activation and the secretion of proinflammatory cytokines in response to various pathogen-derived as well as danger-associated signals. Additionally, NLRP3 is highly expressed in hepatic macrophages, and the assembly of NLRP3 inflammasome could lead to LIRI, which makes it a promising therapeutic target. However, detailed mechanisms about NLRP3 inflammasome involving in the hepatic macrophages-related LIRI is rarely summarized. Here, we review the potential role of the NLRP3 inflammasome pathway of hepatic macrophages in LIRI, with highlights on currently available therapeutic options.

Human Endothelial Progenitor Cells Protect the Kidney against Ischemia-Reperfusion Injury via the NLRP3 Inflammasome in Mice.

Ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI) and progression to chronic kidney disease (CKD). However, no effective therapeutic intervention has been established for ischemic AKI. Endothelial progenitor cells (EPCs) have major roles in the maintenance of vascular integrity and the repair of endothelial damage; they also serve as therapeutic agents in various kidney diseases. Thus, we examined whether EPCs have a renoprotective effect in an IRI mouse model. Mice were assigned to sham, EPC, IRI-only, and EPC-treated IRI groups. EPCs originating from human peripheral blood were cultured. The EPCs were administered 5 min before reperfusion, and all mice were killed 72 h after IRI. Blood urea nitrogen, serum creatinine, and tissue injury were significantly increased in IRI mice; EPCs significantly improved the manifestations of IRI. Apoptotic cell death and oxidative stress were significantly reduced in EPC-treated IRI mice. Administration of EPCs decreased the expression levels of NLRP3, cleaved caspase-1, p-NF-κB, and p-p38. Furthermore, the expression levels of F4/80, ICAM-1, RORγt, and IL-17RA were significantly reduced in EPC-treated IRI mice. Finally, the levels of EMT-associated factors (TGF-ß, α-SMA, Snail, and Twist) were significantly reduced in EPC-treated IRI mice. This study shows that inflammasome-mediated inflammation accompanied by immune modulation and fibrosis is a potential target of EPCs as a treatment for IRI-induced AKI and the prevention of progression to CKD.

Intravenous immunoglobulin is effective in alleviating hepatic ischemia-reperfusion injury: a rat model study.

BACKGROUND: Hepatic ischemia-reperfusion injury (I/R) is an important factor affecting the prognosis of patients undergoing liver surgery. This study aimed to explore the value of intravenous immunoglobulin (IVIG) in hepatic I/R and its mechanism in a rat model. MATERIALS AND METHODS: Forty eight adult male Sprague-Dawley (SD) rats were divided into six groups randomly: (1-2) treated with normal saline (NS) without ischemia or reperfusion; (3-4) treated with NS + 30 min ischemia; (5-6) treated with IVIG + 30 min ischemia. Rats of group 1/3/5 were euthanized at 12 h after operation (sham + NS + 12 h, I/R + NS + 12 h, I/R + IVIG + 12 h group) while group 2/4/6 were euthanized at 24 h (sham + NS + 24 h, I/R + NS + 24 h, I/R + IVIG + 24 h group). Interleukin 10 (IL-10), interleukin 6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) were quantified as well as serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT). Hepatic pathological changes were observed while nuclear factor kappa B p65 (NF-κB p65), Inhibitory Subunit of NF Kappa B Alpha (IKB-alpha) and cleaved caspase-3 were detected. CONCLUSION: ALT, AST, IL-6, TNF-alpha, NF-κB p65 and cleaved caspase-3 were increased by I/R whereas IL-10 and IKB-alpha were decreased. However, IVIG pretreatment reduced ALT, AST, IL-6, TNF-alpha, NF-κB p65 and cleaved caspase-3, but increased IL-10 and IKB-alpha. IVIG treatment attenuates the infiltration of inflammatory cell and cell apoptosis which were observed in I/R groups. IVIG may alleviate hepatic I/R in rats by inhibiting the classical NF-κB signaling pathway, reducing IL-6, TNF-alpha, promoting IL-10, and inhibiting cell apoptosis.

NETosis in ischemic/reperfusion injuries: An organ-based review.

Neutrophil extracellular trap (NETosis), the web-like structures induced by neutrophil death, is an important inflammatory mechanism of the immune system leading to reactive oxygen species production/coagulopathy, endothelial dysfunction, atherosclerosis, and ischemia. NETosis exerts its role through different mechanisms such as triggering Toll-like receptors, inflammatory cytokines, platelet aggregation, neutrophil activation/infiltration, and vascular impairment. NETosis plays a key role in the prognosis of coronary artery disease, ischemic injury of kidney, lung, gastrointestinal tract and skeletal muscles. In this review, we explored the molecular mechanisms involved in NETosis, and ischemic/reperfusion injuries in body organs.

Interferon-γ-Induced Myeloid-Derived Suppressor Cells Aggravate Kidney Ischemia-Reperfusion Injury by Regulating Innate Immune Cells.

OBJECTIVE: Myeloid-derived suppressor cells (MDSCs) are heterogeneous cells which can suppress T-cell functionality. Herein, we evaluated the functional importance of MDSCs in the context of kidney ischemia-reperfusion injury (IRI) and explored their ability to regulate innate and adaptive immune cell function in this context. METHODS: The differentiation of MDSCs was induced in vitro by treating cells with GM-CSF and interferon (IFN)-γ. In a murine model of renal IRI, serum creatinine and blood urea nitrogen values were measured to monitor kidney function, while histopathological and immunohistochemical approaches were used to assess kidney injury severity. In addition, flow cytometry was employed to assess the phenotypes and apoptosis of kidney cells in these mice. RESULTS: MDSCs induced by treatment with GM-CSF + IFN-γ could suppress T-cell functionality in vitro. The adoptive transfer of these MDSCs into an IRI mouse model system enhanced kidney damage and impaired renal function following the recruitment of these cells to renal tissues in these mice. Following such adoptive transfer, the relative frequency of MDSCs with a CD11b+Ly6G-Ly6Chigh monocytic-MDSC phenotype decreased, whereas cells with a CD11b+Ly6G+Ly6Clow polymorphonuclear-MDSC phenotype become more prevalent within kidney tissues following IRI. Adoptive transfer also coincided with increased frequencies of macrophages and dendritic cells (DCs) in the kidney tissues. This suggested that M-MDSCs contributed to early-stage renal IRI damage by differentiating into these deleterious cell types. However, MDSC-induced suppression of CD4+ and CD8+ T-cell infiltration was not sufficient to prevent the deterioration of renal function in these mice. CONCLUSIONS: Herein, we successfully developed a protocol wherein MDSCs were differentiated in vitro through combination GM-CSF/IFN-γ treatment. When these MDSCs were subsequently adoptively transferred into a murine model of renal IRI, they aggravated kidney damage, likely owing to the differentiation of M-MDSCs into deleterious macrophages and DCs.

Macrophage nuclear factor erythroid 2-related factor 2 deficiency promotes innate immune activation by tissue inhibitor of metalloproteinase 3-mediated RhoA/ROCK pathway in the ischemic liver.

BACKGROUND AND AIMS: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of reactive oxygen species (ROS) and inflammation and has been implicated in both human and murine inflammatory disease models. We aimed to characterize the roles of macrophage-specific Nrf2 in liver ischemia/reperfusion injury (IRI). APPROACH AND RESULTS: First, macrophage Nrf2 expression and liver injury in patients undergoing OLT or ischemia-related hepatectomy were analyzed. Subsequently, we created a myeloid-specific Nrf2-knockout (Nrf2M-KO ) strain to study the function and mechanism of macrophage Nrf2 in a murine liver IRI model. In human specimens, macrophage Nrf2 expression was significantly increased in liver tissues after transplantation or hepatectomy. Interestingly, lower Nrf2 expressions correlated with more severe liver injury postoperatively. In a mouse model, we found Nrf2M-KO mice showed worse hepatocellular damage than Nrf2-proficient controls based on serum biochemistry, pathology, ROS, and inflammation. In vitro, Nrf2 deficiency promoted innate immune activation and migration in macrophages on toll-like receptor (TLR) 4 stimulation. Microarray profiling showed Nrf2 deletion caused markedly lower transcriptional levels of tissue inhibitor of metalloproteinase 3 (Timp3). ChIP-seq, PCR, and luciferase reporter assay further demonstrated Nrf2 bound to the promoter region of Timp3. Moreover, a disintegrin and metalloproteinase (ADAM) 10/ROCK1 was specifically increased in Nrf2-deficient macrophages. Increasing Timp3 expression effectively inhibited ADAM10/ROCK1 expression and rescued the Nrf2M-KO -mediated inflammatory response on TLR4 stimulation in vitro. Importantly, Timp3 overexpression, recombinant Timp3 protein, or ROCK1 knockdown rescued Nrf2M-KO -related liver IRI by inhibiting macrophage activation. CONCLUSIONS: In conclusion, macrophage Nrf2 mediates innate proinflammatory responses, attenuates liver IRI by binding to Timp3, and inhibits the RhoA/ROCK pathway, which provides a therapeutic target for clinical organ IRI.

Deficiency of IKKα in Macrophages Mitigates Fibrosis Progression in the Kidney after Renal Ischemia-Reperfusion Injury.

Aims. Acute kidney injury (AKI) can lead to chronic kidney disease (CKD), and macrophages play a key role in this process. The aim of this study was to discover the role of IκB kinase α (IKKα) in macrophages in the process of AKI-to-CKD transition. Main Methods. We crossed lyz2-Cre mice with IKKα-floxed mice to generate mice with IKKα ablation in macrophages (Mac IKKα-/-). A mouse renal ischemia/reperfusion injury (IRI) model was induced by clamping the renal artery for 45 minutes. Treated mice were evaluated for blood biochemistry, tissue histopathology, and fibrosis markers. Macrophages were isolated from the peritoneal cavity for coculturing with tubular epithelial cells (TECs) and flow cytometry analysis. Key Findings. We found that fibrosis and kidney function loss after IRI were significantly alleviated in Mac IKKα-/- mice compared with wild-type (WT) mice. The expression of fibrosis markers and the infiltration of M2 macrophages were decreased in the kidneys of Mac IKKα-/- mice after IRI. The in vitro experiment showed that the IRI TECs cocultured with IKKα-/- macrophages (KO MΦs) downregulated the fibrosis markers accompanied by a downregulation of Wnt/ß-catenin signaling. Significance. These data support the hypothesis that IKKα is involved in mediating macrophage polarization and increasing the expression of fibrosis-promoting inflammatory factors in macrophages. Therefore, knockdown of IKKα in macrophages may be a potential method that can be used to alleviate the AKI-to-CKD transition after IRI.

Characterization of Novel P-Selectin Targeted Complement Inhibitors in Murine Models of Hindlimb Injury and Transplantation.

The complement system has long been recognized as a potential druggable target for a variety of inflammatory conditions. Very few complement inhibitors have been approved for clinical use, but a great number are in clinical development, nearly all of which systemically inhibit complement. There are benefits of targeting complement inhibition to sites of activation/disease in terms of efficacy and safety, and here we describe P-selectin targeted complement inhibitors, with and without a dual function of directly blocking P-selectin-mediated cell-adhesion. The constructs are characterized in vitro and in murine models of hindlimb ischemia/reperfusion injury and hindlimb transplantation. Both constructs specifically targeted to reperfused hindlimb and provided protection in the hindlimb ischemia/reperfusion injury model. The P-selectin blocking construct was the more efficacious, which correlated with less myeloid cell infiltration, but with similarly reduced levels of complement deposition. The blocking construct also improved tissue perfusion and, unlike the nonblocking construct, inhibited coagulation, raising the possibility of differential application of each construct, such as in thrombotic vs. hemorrhagic conditions. Similar outcomes were obtained with the blocking construct following vascularized composite graft transplantation, and treatment also significantly increased graft survival. This is outcome may be particularly pertinent in the context of vascularized composite allograft transplantation, since reduced ischemia reperfusion injury is linked to a less rigorous alloimmune response that may translate to the requirement of a less aggressive immunosuppressive regime for this normally nonlife-threatening procedure. In summary, we describe a new generation of targeted complement inhibitor with multi-functionality that includes targeting to vascular injury, P-selectin blockade, complement inhibition and anti-thrombotic activity. The constructs described also bound to both mouse and human P-selectin which may facilitate potential translation.

A potent truncated form of human soluble CR1 is protective in a mouse model of renal ischemia-reperfusion injury.

The complement system is a potent mediator of ischemia-reperfusion injury (IRI), which detrimentally affects the function and survival of transplanted kidneys. Human complement receptor 1 (HuCR1) is an integral membrane protein that inhibits complement activation by blocking the convertases that activate C3 and C5. We have previously reported that CSL040, a truncated form of recombinant soluble HuCR1 (sHuCR1), has enhanced complement inhibitory activity and improved pharmacokinetic properties compared to the parent molecule. Here, we compared the capacity of CSL040 and full-length sHuCR1 to suppress complement-mediated organ damage in a mouse model of warm renal IRI. Mice were treated with two doses of CSL040 or sHuCR1, given 1 h prior to 22 min unilateral renal ischemia and again 3 h later. 24 h after reperfusion, mice treated with CSL040 were protected against warm renal IRI in a dose-dependent manner, with the highest dose of 60 mg/kg significantly reducing renal dysfunction, tubular injury, complement activation, endothelial damage, and leukocyte infiltration. In contrast, treatment with sHuCR1 at a molar equivalent dose to 60 mg/kg CSL040 did not confer significant protection. Our results identify CSL040 as a promising therapeutic candidate to attenuate renal IRI and demonstrate its superior efficacy over full-length sHuCR1 in vivo.

Expanded renal lymphatics improve recovery following kidney injury.

Acute kidney injury (AKI) is a major cause of patient mortality and a major risk multiplier for the progression to chronic kidney disease (CKD). The mechanism of the AKI to CKD transition is complex but is likely mediated by the extent and length of the inflammatory response following the initial injury. Lymphatic vessels help to maintain tissue homeostasis through fluid, macromolecule, and immune modulation. Increased lymphatic growth, or lymphangiogenesis, often occurs during inflammation and plays a role in acute and chronic disease processes. What roles renal lymphatics and lymphangiogenesis play in AKI recovery and CKD progression remains largely unknown. To determine if the increased lymphatic density is protective in the response to kidney injury, we utilized a transgenic mouse model with inducible, kidney-specific overexpression of the lymphangiogenic protein vascular endothelial growth factor-D to expand renal lymphatics. "KidVD" mouse kidneys were injured using inducible podocyte apoptosis and proteinuria (POD-ATTAC) or bilateral ischemia reperfusion. In the acute injury phase of both models, KidVD mice demonstrated a similar loss of function measured by serum creatinine and glomerular filtration rate compared to their littermates. While the initial inflammatory response was similar, KidVD mice demonstrated a shift toward more CD4+ and fewer CD8+ T cells in the kidney. Reduced collagen deposition and improved functional recovery over time was also identified in KidVD mice. In KidVD-POD-ATTAC mice, an increased number of podocytes were counted at 28 days post-injury. These data demonstrate that increased lymphatic density prior to injury alters the injury recovery response and affords protection from CKD progression.

Endogenous Interleukin-33 Acts as an Alarmin in Liver Ischemia-Reperfusion and Is Associated With Injury After Human Liver Transplantation.

Ischemia and reperfusion injury is an early inflammatory process during liver transplantation that impacts on graft function and clinical outcomes. Interleukin (IL)-33 is a danger-associated molecular pattern involved in kidney ischemia/reperfusion injury and several liver diseases. The aims were to assess whether IL-33 was released as an alarmin responsible for ischemia/reperfusion injury in a mouse model of warm hepatic ischemia, and whether this hypothesis could also apply in the setting of human liver transplantation. First, a model of warm hepatic ischemia/reperfusion was used in wild-type and IL-33-deficient mice. Severity of ischemia/reperfusion injury was assessed with ALT and histological analysis. Then, serum IL-33 was measured in a pilot cohort of 40 liver transplant patients. Hemodynamic postreperfusion syndrome, graft dysfunction (assessed by model for early allograft scoring >6), renal failure, and tissue lesions on time-zero biopsies were assessed. In the mouse model, IL-33 was constitutively expressed in the nucleus of endothelial cells, immediately released in response to hepatic pedicle clamping without neosynthesis, and participated in the recruitment of neutrophils and tissue injury on site. The kinetics of IL-33 in liver transplant patients strikingly matched the ones in the animal model, as attested by serum levels reaching a peak immediately after reperfusion, which correlated to clinical outcomes including postreperfusion syndrome, posttransplant renal failure, graft dysfunction, and histological lesions of ischemia/reperfusion injury. IL-33 was an independent factor of graft dysfunction with a cutoff of IL-33 at 73 pg/ml after reperfusion (73% sensitivity, area under the curve of 0.76). Taken together, these findings establish the immediate implication of IL-33 acting as an alarmin in liver I/R injury and provide evidence of its close association with cardinal features of early liver injury-associated disorders in LT patients.

Glycinergic Signaling in Macrophages and Its Application in Macrophage-Associated Diseases.

Accumulating evidences support that amino acids direct the fate decision of immune cells. Glycine is a simple structural amino acid acting as an inhibitory neurotransmitter. Besides, glycine receptors as well as glycine transporters are found in macrophages, indicating that glycine alters the functions of macrophages besides as an inhibitory neurotransmitter. Mechanistically, glycine shapes macrophage polarization via cellular signaling pathways (e.g., NF-κB, NRF2, and Akt) and microRNAs. Moreover, glycine has beneficial effects in preventing and/or treating macrophage-associated diseases such as colitis, NAFLD and ischemia-reperfusion injury. Collectively, this review highlights the conceivable role of glycinergic signaling for macrophage polarization and indicates the potential application of glycine supplementation as an adjuvant therapy in macrophage-associated diseases.

Hydralazine attenuates renal inflammation in diabetic rats with ischemia/reperfusion acute kidney injury.

Acute kidney injury (AKI) is one of the major complications with increased oxidative stress and inflammation in diabetic patients. Hyperglycemia stimulates the formation of advanced glycation end products (AGEs). However, hyperglycemia directly triggers the interaction between AGEs and transmembrane AGEs receptors (RAGE), which enhances oxidative stress and increases the production of inflammatory substances. Therefore, diabetes plays a pivotal role in kidney injury. Hydralazine, a vasodilator and antihypertensive drug, was found to have the ability to reduce ROS, oxidative stress, and inflammation. We applied Hydralazine co-culture with AGEs in rat mesangial cells (RMC) and to renal ischemia/reperfusion(I/R) injury models in streptozotocin-induced diabetic rats. Hydralazine significantly decreased AGEs-induced RAGE, iNOS, and COX-2 expressions in RMC. Compared to the diabetic with AKI group, hydralazine decreased inflammation-related protein, and JAK2, STAT3 signaling in rat kidney tissue. Our studies indicate that Hydralazine has the potential to become a beneficial drug in the treatment of diabetic acute kidney injury.

Properdin Deficiency Impairs Phagocytosis and Enhances Injury at Kidney Repair Phase Post Ischemia-Reperfusion.

Properdin, a positive regulator of complement alternative pathway, participates in renal ischemia-reperfusion (IR) injury and also acts as a pattern-recognition molecule affecting apoptotic T-cell clearance. However, the role of properdin in tubular epithelial cells (TECs) at the repair phase post IR injury is not well defined. This study revealed that properdin knockout (PKO) mice exhibited greater injury in renal function and histology than wild-type (WT) mice post 72-h IR, with more apoptotic cells and macrophages in tubular lumina, increased active caspase-3 and HMGB1, but better histological structure at 24 h. Raised erythropoietin receptor by IR was furthered by PKO and positively correlated with injury and repair markers. Properdin in WT kidneys was also upregulated by IR, while H2O2-increased properdin in TECs was reduced by its small-interfering RNA (siRNA), with raised HMGB1 and apoptosis. Moreover, the phagocytic ability of WT TECs, analyzed by pHrodo Escherichia coli bioparticles, was promoted by H2O2 but inhibited by PKO. These results were confirmed by counting phagocytosed H2O2-induced apoptotic TECs by in situ end labeling fragmented DNAs but not affected by additional serum with/without properdin. Taken together, PKO results in impaired phagocytosis at the repair phase post renal IR injury. Properdin locally produced by TECs plays crucial roles in optimizing damaged cells and regulating phagocytic ability of TECs to effectively clear apoptotic cells and reduce inflammation.

Role of cellular prion protein in splenic CD4+ T cell differentiation in cerebral ischaemic/reperfusion.

OBJECTIVE: Cellular prion protein (PrPC ), the primary form of prion diseases pathogen, has received increasing attention for its protective effect against ischaemic stroke. Little is known about its role in peripheral immune responses after cerebral ischaemia/reperfusion (I/R) injury. This study is to detect the variation of splenic CD4+ T lymphocytes differentiation and the concentration of inflammatory cytokines after murine cerebral I/R injury in the context of PRNP expression as well as its influence on the ischaemic neuronal apoptosis. METHODS: We established the cerebral ischaemic murine model of different PRNP genotypes. We detected the percentage of splenic CD4+ PrPC+ T cells of PRNP wild-type mice and the ratio of splenic Th1/2/17 lymphocytes of mice of different PRNP expression. The relevant inflammatory cytokines were then measured. Oxygen glucose deprivation/reperfusion (OGD/R) HT22 mouse hippocampal neurons were co-cultured with the T-cell-conditioned medium harvested from the spleen of modelled mice and then the neuronal apoptosis was detected. RESULTS: CD4+ PrPC+ T lymphocytes in wild-type mice elevated after MCAO/R. PRNP expression deficiency led to an elevation of Th1/17 phenotypes and the promotion of pro-inflammatory cytokines, while PRNP overexpression led to the elevation of Th2 phenotype and upregulation of anti-inflammatory cytokines. In addition, PrPC -overexpressed CD4+ T cells weakened the apoptosis of OGD/R HT-22 murine hippocampal neurons caused by MCAO/R CD4+ T-cell-conditioned medium, while PrPC deficiency enhanced apoptosis. INTERPRETATION: PrPC works as a neuron protector in the CNS when I/R injury occurs and affects the peripheral immune responses and defends against stroke-induced neuronal apoptosis.

Single-cell profiling reveals distinct immune phenotypes that contribute to ischaemia-reperfusion injury after steatotic liver transplantation.

OBJECTIVES: The discrepancy between supply and demand of organ has led to an increased utilization of steatotic liver for liver transplantation (LT). Hepatic steatosis, however, is a major risk factor for graft failure due to increased susceptibility to ischaemia-reperfusion (I/R) injury during transplantation. MATERIALS AND METHODS: To assess the plasticity and phenotype of immune cells within the microenvironment of steatotic liver graft at single-cell level, single-cell RNA-sequencing (scRNA-Seq) was carried out on 23 675 cells from transplanted rat livers. Bioinformatic analyses and multiplex immunohistochemistry were performed to assess the functional properties, transcriptional regulation, phenotypic switching and cell-cell interactions of different cell subtypes. RESULTS: We have identified 11 different cell types in transplanted livers and found that the highly complex ecosystem was shaped by myeloid-derived cell subsets that transit between different states and interact mutually. Notably, a pro-inflammatory phenotype of Kupffer cells (KCs) with high expression of colony-stimulating factor 3 (CSF3) that was enriched in transplanted steatotic livers was potentially participated in fatty graft injury. We have also detected a subset of dendritic cells (DCs) with highly expressing XCR1 that was correlated with CD8+ T cells, mediating the severer steatotic liver damage by I/R injury. CONCLUSIONS: The findings of our study provide new insight into the mechanisms by which steatosis exacerbates liver damage from I/R injury. Interventions based on these observations create opportunities in attenuating fatty liver graft injury and expanding the donor pool.

PARP inhibitor PJ34 ameliorates cognitive impairments induced by transient cerebral ischemia/reperfusion through its anti-inflammatory effects in a rat model.

Cerebral ischemia is a major health threat to humankind around the world, and the reperfusion methods may provoke irreversible damages to brain tissues, causing impairment of neurological function. The goal of this study is to investigate the potential neurological protective effect of PJ34, a well-characterized poly (ADP-ribose) polymerase 1 (PARP-1) inhibitor, on cerebral ischemia-reperfusion (I/R)-induced injury of the rat model. The cerebral I/R rats were received (3, 6, or 12 mg/kg) injections of PJ34 or saline at 24 h, 6 h before middle cerebral artery occlusion (MCAO) and 1 h, 24 h, and 48 h after MCAO. All rats were subject to the neurological behavior tests by open field test and Morris water maze test. The expression of pro-inflammatory cytokines, Cyclooxygenase 2 (COX-2) and inducible nitric oxide synthase (iNOS) in cerebral tissues was also determined. Our results demonstrated that the administration of PJ34 dose-dependently ameliorated cerebral I/R-induced injury and improved neurological performance of cerebral I/R rats. We also revealed that PJ34 treatment effectively reduced COX2, iNOS, and pro-inflammatory cytokine levels in the I/R-induced injury tissues. Our finding further supports that inhibition of PARP-1 activity is beneficial for reducing post-I/R-induced brain damage via targeting inflammatory response.

6-Gingerol protects against cerebral ischemia/reperfusion injury by inhibiting NLRP3 inflammasome and apoptosis via TRPV1 / FAF1 complex dissociation-mediated autophagy.

BACKGROUND: Our previous studies demonstrated that autophagy alleviates cerebral I/R injury by inhibiting NLRP3 inflammasome-mediated inflammation. 6-Gingerol, a phenolic compound extracted from ginger, was reported to possess potent antiapoptotic and anti-inflammatory activities and is associated with autophagy. However, the effects of 6-Gingerol in cerebral I/R injury have not been elucidated, and whether they involve autophagy-induced NLRP3 inflammasome inhibition remains unclear. METHODS: Adult male Sprague-Dawley (SD) rats were subjected to middle cerebral artery occlusion (MCAO) for 1 h, followed by reperfusion for 24 h. 6-Gingerol and 3-methyladenine (3-MA) were injected intraperitoneally, and si-TRPV1 was injected via the lateral ventricle. Cerebral infarct volume, brain edema, neurological deficits, HE and Nissl were used to evaluate the morphological and functional changes of brain tissue, respectively. TRPV1, FAF1, autophagy related (LC3II/I, P62, Beclin1), inflammation related (NLRP3, cleaved-caspase-1, caspase-1, cleaved-IL-1ß, IL-1ß, cleaved-IL-18, IL-18) and apoptosis related (Bcl-2, Bax, cleaved-caspase-3) proteins were assessed by Western blot, immunofluorescence staining and coimmunoprecipitation, respectively. Enzyme linked immunosorbent assay (ELISA) was used to evaluate the changes in the expression levels of interleukin-1 (IL-1ß) and interleukin-18(IL-18), respectively. The degree of neuronal apoptosis was evaluated by TUNEL staining. Neuronal ultrastructure was examined by transmission electron microscopy. RESULT: 6-Gingerol treatment significantly reduced cerebral infarct volume, improved brain edema and neurological scores, and reversed brain histomorphological damage after I/R injury. In addition, 6-Gingerol significantly reduced NLRP3 inflammasome-derived inflammation and neuronal apoptosis and upregulated autophagy. The autophagy inhibitor 3-MA rescued the effects of 6-Gingerol on the NLRP3 inflammasome and apoptosis. Moreover, the findings illustrated that 6-Gingerol inhibited autophagy-induced NLRP3 inflammasome activation and apoptosis through the dissociation of TRPV1 from FAF1. CONCLUSION: In brief, 6-Gingerol exerts antiapoptotic and anti-inflammatory effects via TRPV1/FAF1 complex dissociation-mediated autophagy during cerebral I/R injury. Therefore, 6-Gingerol may be an effective drug for the treatment of I/R injury.