[Research progress of effect of neutrophil elastase and its inhibitors in sepsis].

Sepsis is a clinical syndrome of life-threatening organ dysfunction caused by infection. When an infection occurs, as the first line of defense of the body's immune system, neutrophils are first recruited to the site of infection to capture and kill pathogens by releasing neutrophil elastase (NE). However, a large amount of NE release will injury the surrounding normal tissues and induce organ dysfunction or failure. NE inhibitors can inhibit NE activity and reduce inflammatory response, which may be a promising drug for the treatment of sepsis. Currently, a variety of NE inhibitors have been developed and reported, but there is no systematic overview of their characteristics, and the role and underlying mechanisms of NE and related inhibitors in sepsis have not been thoroughly discussed. This article will make a review in this regard, in order to elucidate the effect of NE and its inhibitors in sepsis.

Evaluation of the rs35996865 polymorphism of the ROCK1 gene in sepsis.

OBJECTIVE: Sepsis is a complex and serious medical condition resulting from the activation of an innate host response to infections. The etiology of sepsis is complex and can be influenced by genetic susceptibility. The purpose of the present study was to investigate a possible association of Rho-kinase 1 (ROCK1) gene polymorphism with sepsis in a Turkish population. METHODS: The study group consisted of 100 unrelated patients with sepsis and 100 healthy controls. Genomic DNA was isolated from peripheral leukocytes from EDTA-containing blood using the QIAamp DNA Blood Mini Kit. ROCK1 gene rs35996865 and rs112130712 (Lys1054Arg) polymorphisms were analyzed in genomic DNA using the LightCycler 480 II real-time polymerase chain reaction. RESULTS: There were no significant differences in allele and genotype frequencies for ROCK1 gene rs35996865 polymorphism between the patients with sepsis and control group (p>0.05). Additionally, no association was detected between the rs35996865 polymorphism and mortality in the patient group. No polymorphism was detected with ROCK1 gene rs112130712 (Lys1054Arg) in our study groups. CONCLUSIONS: Our data showed that there is no marked association between the rs35996865 polymorphism and sepsis. Therefore, these results suggest that ROCK1 gene rs35996865 polymorphism is not risk factor for the development of sepsis in the Turkish population.

The role of neutrophil gelatinase-associated lipocalin and iron homeostasis in object recognition impairment in aged sepsis-survivor rats.

Older adult patients with sepsis frequently experience cognitive impairment. The roles of brain neutrophil gelatinase-associated lipocalin (NGAL) and iron in older sepsis patients remain unknown. We investigated the effects of lipopolysaccharide-induced sepsis on novel object recognition test, NGAL levels, an inflammatory mediator tumor necrosis factor-α (TNFα) levels, and iron ion levels in the hippocampus and cortex of young and aged rats. The effect of an iron chelator deferoxamine pretreatment on aged sepsis rats was also examined. Young sepsis-survivor rats did not show impaired novel object recognition, TNFα responses, or a Fe2+/Fe3+ imbalance. They showed hippocampal and cortical NGAL level elevations. Aged sepsis-survivor rats displayed a decreased object discrimination index, elevation of NGAL levels and Fe2+/Fe3+ ratio, and no TNFα responses. Pretreatment with deferoxamine prevented the reduction in the object recognition of aged sepsis-survivor rats. The elevation in hippocampal and cortical NGAL levels caused by lipopolysaccharide was not influenced by deferoxamine pretreatment. The lipopolysaccharide-induced Fe2+/Fe3+ ratio elevation was blocked by deferoxamine pretreatment. In conclusion, our findings suggest that iron homeostasis in the cortex and hippocampus contributes to the maintenance of object recognition ability in older sepsis survivors.

Geranylgeranyl diphosphate synthase 1 knockdown suppresses NLRP3 inflammasome activity via promoting autophagy in sepsis-induced acute lung injury.

BACKGROUND: NOD-like receptor protein 3 (NLRP3) inflammasome activation has emerged as a crucial contributor to sepsis-induced lung injury. Geranylgeranyl diphosphate synthase 1 (GGPPS1) reportedly exerts the pro-inflammatory capability via activation of NLRP3 inflammasome. However, little is known about the role and mechanism of GGPPS1 in sepsis-induced lung injury. METHODS: Mice underwent cecal ligation and puncture (CLP) surgery to establish the in vivo model of sepsis. The lung injury of mice was assessed by analyzing the histological changes, the lung wet/dry ratio, PaO2/FiO2 ratio, myeloperoxidase (MPO) activity, total protein content, total cell, and polymorphonuclear leukocyte counts. Mouse alveolar macrophages MH-S were exposed to LPS for developing in vitro model of sepsis. The mRNA and protein expression levels of GGPPS1, beclin-1, and autophagy and inflammasome-related genes were detected using quantitative reverse transcription-polymerase chain reaction and western blot assays. Enzyme-linked immunosorbent assay was conducted to determine the levels of interleukin (IL)-1ß and IL-18. RESULTS: We successfully established sepsis-induced acute lung injury in vivo by CLP surgery. GGPPS1 was upregulated in the lung tissues of CLP-induced septic mice. The activation of autophagy and NLRP3 inflammasome were found in the lung tissues of CLP-induced septic mice. The addition of exogenous GGPP (synthesis products catalyzed by GGPPS1) and autophagic inhibitor 3-MA aggravated sepsis-induced hypoxemia, alveolar inflammatory response, intrapulmonary hemorrhage, and pulmonary edema, as evidenced by increased lung injury score, lung wet/dry weight ratio, MPO activity, total protein content, total cell, and PMNs counts, and decreased PaO2/FiO2 ratio. While NLRP3 inhibitor MCC950 exerted the opposite effects. Additionally, administration of exogenous GGPP could inhibit the activation of autophagy, enhance the activity of NLRP3 inflammasome, and the production of IL-1ß and IL-18. Inhibition of autophagy by 3-MA treatment also promoted the activity of NLRP3 inflammasome and the production of IL-1ß and IL-18. While MCC950 restrained the activity of NLRP3 inflammasome, but did not affect the activation of autophagy. Notably, the expression of GGPPS1 was unaltered in CLP-induced mice following GGPP, 3-MA, or MCC950 treatment. Moreover, GGPPS1 was upregulated in MH-S cells stimulated with LPS, and GGPPS1 knockdown enhanced the activation of autophagy and inhibited the activity of NLRP3 inflammasome in vitro. Importantly, depletion of GGPPS1 could alleviate LPS-induced inflammatory response by inducing autophagy-dependent NLRP3 inflammasome inhibition. CONCLUSION: GGPPS1 knockdown suppressed NLRP3 inflammasome activity via promoting autophagy and then attenuated sepsis-induced acute lung injury, revealing a novel target for treating sepsis-induced lung injury.

Global Lysine Acetylome Analysis of LPS-Stimulated HepG2 Cells Identified Hyperacetylation of PKM2 as a Metabolic Regulator in Sepsis.

Sepsis-induced liver dysfunction (SILD) is a common event and is strongly associated with mortality. Establishing a causative link between protein post-translational modification and diseases is challenging. We studied the relationship among lysine acetylation (Kac), sirtuin (SIRTs), and the factors involved in SILD, which was induced in LPS-stimulated HepG2 cells. Protein hyperacetylation was observed according to SIRTs reduction after LPS treatment for 24 h. We identified 1449 Kac sites based on comparative acetylome analysis and quantified 1086 Kac sites on 410 proteins for acetylation. Interestingly, the upregulated Kac proteins are enriched in glycolysis/gluconeogenesis pathways in the Kyoto Encyclopedia of Genes and Genomes (KEGG) category. Among the proteins in the glycolysis pathway, hyperacetylation, a key regulator of lactate level in sepsis, was observed at three pyruvate kinase M2 (PKM2) sites. Hyperacetylation of PKM2 induced an increase in its activity, consequently increasing the lactate concentration. In conclusion, this study is the first to conduct global profiling of Kac, suggesting that the Kac mechanism of PKM2 in glycolysis is associated with sepsis. Moreover, it helps to further understand the systematic information regarding hyperacetylation during the sepsis process.

Rho-Proteins and Downstream Pathways as Potential Targets in Sepsis and Septic Shock: What Have We Learned from Basic Research.

Sepsis and septic shock are associated with acute and sustained impairment in the function of the cardiovascular system, kidneys, lungs, liver, and brain, among others. Despite the significant advances in prevention and treatment, sepsis and septic shock sepsis remain global health problems with elevated mortality rates. Rho proteins can interact with a considerable number of targets, directly affecting cellular contractility, actin filament assembly and growing, cell motility and migration, cytoskeleton rearrangement, and actin polymerization, physiological functions that are intensively impaired during inflammatory conditions, such as the one that occurs in sepsis. In the last few decades, Rho proteins and their downstream pathways have been investigated in sepsis-associated experimental models. The most frequently used experimental design included the exposure to bacterial lipopolysaccharide (LPS), in both in vitro and in vivo approaches, but experiments using the cecal ligation and puncture (CLP) model of sepsis have also been performed. The findings described in this review indicate that Rho proteins, mainly RhoA and Rac1, are associated with the development of crucial sepsis-associated dysfunction in different systems and cells, including the endothelium, vessels, and heart. Notably, the data found in the literature suggest that either the inhibition or activation of Rho proteins and associated pathways might be desirable in sepsis and septic shock, accordingly with the cellular system evaluated. This review included the main findings, relevance, and limitations of the current knowledge connecting Rho proteins and sepsis-associated experimental models.

Proteinase 3 contributes to endothelial dysfunction in an experimental model of sepsis.

In sepsis-induced inflammation, polymorphonuclear neutrophils (PMNs) contribute to vascular dysfunction. The serine proteases proteinase 3 (PR3) and human leukocyte elastase (HLE) are abundant in PMNs and are released upon degranulation. While HLE's role in inflammation-induced endothelial dysfunction is well studied, PR3's role is largely uninvestigated. We hypothesized that PR3, similarly to HLE, contributes to vascular barrier dysfunction in sepsis. Plasma PR3 and HLE concentrations and their leukocyte mRNA levels were measured by ELISA and qPCR, respectively, in sepsis patients and controls. Exogenous PR3 or HLE was applied to human umbilical vein endothelial cells (HUVECs) and HUVEC dysfunction was assessed by FITC-dextran permeability and electrical resistance. Both PR3 and HLE protein and mRNA levels were significantly increased in sepsis patients (P < 0.0001 and P < 0.05, respectively). Additionally, each enzyme independently increased HUVEC monolayer FITC-dextran permeability (P < 0.01), and decreased electrical resistance in a time- and dose-dependent manner (P < 0.001), an effect that could be ameliorated by novel treatment with carbon monoxide-releasing molecule 3 (CORM-3). The serine protease PR3, in addition to HLE, lead to vascular dysfunction and increased endothelial permeability, a hallmark pathological consequence of sepsis-induced inflammation. CORMs may offer a new strategy to reduce serine protease-induced vascular dysfunction.

Blocking SphK1/S1P/S1PR1 Signaling Pathway Alleviates Lung Injury Caused by Sepsis in Acute Ethanol Intoxication Mice.

Acute ethanol intoxication increases the risk of sepsis and aggravates the symptoms of sepsis and lung injury. Therefore, this study aimed to explore whether sphingosine kinase 1 (SphK1)/sphingosine-1-phosphate (S1P)/S1P receptor 1 (S1PR1) signaling pathway functions in lung injury caused by acute ethanol intoxication-enhanced sepsis, as well as its underlying mechanism. The acute ethanol intoxication model was simulated by intraperitoneally administering mice with 32% ethanol solution, and cecal ligation and puncture (CLP) was used to construct the sepsis model. The lung tissue damage was observed by hematoxylin-eosin (H&E) staining, and the wet-to-dry (W/D) ratio was used to evaluate the degree of pulmonary edema. Inflammatory cell counting and protein concentration in bronchoalveolar lavage fluid (BALF) were, respectively, detected by hemocytometer and bicinchoninic acid (BCA) method. The levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6, IL-1ß, and IL-18 in BALF were detected by their commercial enzyme-linked immunosorbent assay (ELISA) kits. The myeloperoxidase (MPO) activity and expression of apoptosis-related proteins and SphK1/S1P/S1PR1 pathway-related proteins were, respectively, analyzed by MPO ELISA kit and Western blot analysis. The cell apoptosis in lung tissues was observed by TUNEL assay. Acute ethanol intoxication (EtOH) decreased the survival rate of mice and exacerbated the lung injury caused by sepsis through increasing pulmonary vascular permeability, neutrophil infiltration, release of inflammatory factors, and cell apoptosis. In addition, EtOH could activate the SphK1/S1P/S1PR1 pathway in CLP mice. However, PF-543, as a specific inhibitor of SphK1, could partially reverse the deleterious effects on lung injury of CLP mice. PF-543 alleviated lung injury caused by sepsis in acute ethanol intoxication rats by suppressing the SphK1/S1P/S1PR1 signaling pathway.

Melatonin Attenuates Sepsis-Induced Small-Intestine Injury by Upregulating SIRT3-Mediated Oxidative-Stress Inhibition, Mitochondrial Protection, and Autophagy Induction.

Melatonin reportedly alleviates sepsis-induced multi-organ injury by inducing autophagy and activating class III deacetylase Sirtuin family members (SIRT1-7). However, whether melatonin attenuates small-intestine injury along with the precise underlying mechanism remain to be elucidated. To investigate this, we employed cecal ligation and puncture (CLP)- or endotoxemia-induced sepsis mouse models and confirmed that melatonin treatment significantly prolonged the survival time of mice and ameliorated multiple-organ injury (lung/liver/kidney/small intestine) following sepsis. Melatonin partially protected the intestinal barrier function and restored SIRT1 and SIRT3 activity/protein expression in the small intestine. Mechanistically, melatonin treatment enhanced NF-κB deacetylation and subsequently reduced the inflammatory response and decreased the TNF-α, IL-6, and IL-10 serum levels; these effects were abolished by SIRT1 inhibition with the selective blocker, Ex527. Correspondingly, melatonin treatment triggered SOD2 deacetylation and increased SOD2 activity and subsequently reduced oxidative stress; this amelioration of oxidative stress by melatonin was blocked by the SIRT3-selective inhibitor, 3-TYP, and was independent of SIRT1. We confirmed this mechanistic effect in a CLP-induced sepsis model of intestinal SIRT3 conditional-knockout mice, and found that melatonin preserved mitochondrial function and induced autophagy of small-intestine epithelial cells; these effects were dependent on SIRT3 activation. This study has shown, to the best of our knowledge, for the first time that melatonin alleviates sepsis-induced small-intestine injury, at least partially, by upregulating SIRT3-mediated oxidative-stress inhibition, mitochondrial-function protection, and autophagy induction.

Chrysin Ameliorates Sepsis-Induced Cardiac Dysfunction Through Upregulating Nfr2/Heme Oxygenase 1 Pathway.

ABSTRACT: The incidence of myocardial dysfunction caused by sepsis is high, and the mortality of patients with sepsis can be significantly increased. During sepsis, oxidative stress and inflammation can lead to severe organ dysfunction. Flavone chrysin is one of the indispensable biological active ingredients for different fruits and vegetables and has antioxidant and anti-inflammatory properties. However, it is not clear whether chrysin is an effective treatment for heart dysfunction caused by sepsis. We found that it had protective effects against the harmful effects caused by LPS, manifested in improved survival, normalized cardiac function, improved partial pathological scores of myocardial tissue, and remission of apoptosis, as well as reduced oxidative stress and inflammation. Mechanism studies have found that chrysin is an important antioxidant protein, a key regulator of heme oxygenase 1 (HO-1). We found that HO-1 levels were increased after LPS intervention, and chrysin further increased HO-1 levels, along with the addition of Nrf2, a regulator of antioxidant proteins. Pretreatment with PD98059, an extracellular signal-regulated kinase-specific inhibitor, blocked chrysin-mediated phosphorylation of Nrf2 and the nuclear translocation of Nrf2. The protective effect of chrysin on sepsis-induced cardiac dysfunction was blocked by ZnPP, which is a HO-1 blocker. Chrysin increased antioxidant activity and reduced markers of oxidative stress (SOD and MDA) and inflammation (MPO and IL-1ß), all of which were blocked by ZnPP. This indicates that HO-1 is the upstream molecule regulating the protective effect of chrysin. Thus, by upregulation of HO-1, chrysin protects against LPS-induced cardiac dysfunction and inflammation by inhibiting oxidative stress.

Granzyme A inhibition reduces inflammation and increases survival during abdominal sepsis.

Aims: Peritonitis is one of the most common causes of sepsis, a serious syndrome characterized by a dysregulated systemic inflammatory response. Recent evidence suggests that Granzyme A (GzmA), a serine protease mainly expressed by NK and T cells, could act as a proinflammatory mediator and could play an important role in the pathogenesis of sepsis. This work aims to analyze the role and the therapeutic potential of GzmA in the pathogenesis of peritoneal sepsis. Methods: The level of extracellular GzmA as well as GzmA activity were analyzed in serum from healthy volunteers and patients with confirmed peritonitis and were correlated with the Sequential Organ Failure Assessment (SOFA) score. Peritonitis was induced in C57Bl/6 (WT) and GzmA-/- mice by cecal ligation and puncture (CLP). Mice were treated intraperitoneally with antibiotics alone or in combination serpinb6b, a specific GzmA inhibitor, for 5 days. Mouse survival was monitored during 14 days, levels of some proinflammatory cytokines were measured in serum and bacterial load and diversity was analyzed in blood and spleen at different times. Results: Clinically, elevated GzmA was observed in serum from patients with abdominal sepsis suggesting that GzmA plays an important role in this pathology. In the CLP model GzmA deficient mice, or WT mice treated with an extracellular GzmA inhibitor, showed increased survival, which correlated with a reduction in proinflammatory markers in both serum and peritoneal lavage fluid. GzmA deficiency did not influence bacterial load in blood and spleen and GzmA did not affect bacterial replication in macrophages in vitro, indicating that GzmA has no role in bacterial control. Analysis of GzmA in lymphoid cells following CLP showed that it was mainly expressed by NK cells. Mechanistically, we found that extracellular active GzmA acts as a proinflammatory mediator in macrophages by inducing the TLR4-dependent expression of IL-6 and TNFα. Conclusions: Our findings implicate GzmA as a key regulator of the inflammatory response during abdominal sepsis and provide solid evidences about its therapeutic potential for the treatment of this severe pathology.

Transglutaminase 2 as a Marker for Inflammation and Therapeutic Target in Sepsis.

Sepsis results in lethal organ malfunction due to dysregulated host response to infection, which is a condition with increasing prevalence worldwide. Transglutaminase 2 (TG2) is a crosslinking enzyme that forms a covalent bond between lysine and glutamine. TG2 plays important roles in diverse cellular processes, including extracellular matrix stabilization, cytoskeletal function, cell motility, adhesion, signal transduction, apoptosis, and cell survival. We have shown that the co-culture of Candida albicans and hepatocytes activates and induces the translocation of TG2 into the nucleus. In addition, the expression and activation of TG2 in liver macrophages was dramatically induced in the lipopolysaccharide-injected and cecal ligation puncture-operated mouse models of sepsis. Based on these findings and recently published research, we have reviewed the current understanding of the relationship between TG2 and sepsis. Following the genetic and pharmacological inhibition of TG2, we also assessed the evidence regarding the use of TG2 as a potential marker and therapeutic target in inflammation and sepsis.

Melatonin Alleviates Cardiac Dysfunction Via Increasing Sirt1-Mediated Beclin-1 Deacetylation and Autophagy During Sepsis.

Cardiac dysfunction is a major cause leading to multiple organ failure in sepsis. Beclin-1-dependent autophagy has been evidenced to exert protective effects on hearts in sepsis. However, the mechanisms on how Beclin-1 and autophagy are regulated remains enigmatic. To explore the detailed mechanisms controlling Beclin-1-dependent autophagy in septic heart and whether melatonin could protect against sepsis via regulating cardiac autophagy, adult Sprague-Dawley (SD) rats were subjected to cecal ligation and puncture (CLP) to induce sepsis. Rats were intraperitoneally administrated with 30 mg/kg melatonin within 5-min post-CLP surgery. Our data showed that sepsis induced Becline-1 acetylation and inhibited autophagy in hearts, resulting in impaired cardiac function. However, melatonin treatment facilitated Beclin-1 deacetylation and increased autophagy in septic hearts, thus improved cardiac function. Moreover, melatonin increased the expression and activity of Sirtuin 1 (Sirt1), and inhibition of Sirt1 abolished the protective effects of melatonin on Beclin-1 deacetylation and cardiac function. In conclusion, increased Beclin-1 acetylation was involved in impaired autophagy in septic hearts, while melatonin contributed to Beclin-1 deacetylation via Sirt1, leading to improved autophagy and cardiac function in sepsis. Our study sheds light on the important role of Beclin-1 acetylation in regulating autophagy in sepsis and suggests that melatonin is a potential candidate drug for the treatment of sepsis.

Disrupted eNOS activity and expression account for vasodilator dysfunction in different stage of sepsis.

AIMS: Sepsis is a severe endothelial dysfunction syndrome. The role of endothelial nitric oxide synthase (eNOS) in endothelial dysfunction induced by sepsis is controversial. To explore the role of eNOS in vascular dysfunction. MAIN METHODS: The effect of sepsis on vasodilation and eNOS levels was examined in septic mouse arteries and in cell models. KEY FINDINGS: In early sepsis mouse arteries, endothelium-dependent relaxation decreased and phosphorylation of the inhibitory Thr495 site in endothelial nitric oxide synthase increased. Mechanically, the phosphorylation of endothelial nitric oxide synthase at Thr497 in bovine aortic endothelial cells occurred in a protein kinase C-α dependent manner. In late sepsis, both nitric oxide-dependent relaxation responses and endothelial nitric oxide synthase levels were decreased in septic mice arteries. Endothelial nitric oxide synthase levels expression levels decreased in tumor necrosis factor-α-treated human umbilical vein endothelial cells and this could be prevented by the ubiquitin proteasome inhibitor (MG-132). MG-132 could reverse the decrease in endothelial nitric oxide synthase expression and improve nitric oxide-dependent vasodilator dysfunction in septic mice arteries. SIGNIFICANCE: These data indicate that vasodilator dysfunction is induced by the increased phosphorylation of endothelial nitric oxide synthase in early sepsis and its degradation in late sepsis.

Bruton's tyrosine kinase inhibition attenuates oxidative stress in systemic immune cells and renal compartment during sepsis-induced acute kidney injury in mice.

Sepsis is a life-threatening condition which affects multiple organs including the kidney. Sepsis-induced acute kidney injury (AKI) is a major health burden throughout the globe. Pathogenesis of sepsis-induced AKI is complex; however, it involves both innate and adaptive immune cells such as B cells, T cells, dendritic cells (DCs), macrophages, and neutrophils. Bruton's tyrosine kinase (BTK) is reportedly involved in inflammatory and oxidative signaling in different immune cells, however its contribution with respect to sepsis-induced AKI has not been delineated. This study attempted to investigate the role of BTK and its inhibition on oxidizing enzymes NADPH oxidase (NOX-2) and inducible nitric oxide synthase (iNOS) in DCs, neutrophils, and B cells during AKI. Our data reveal that BTK is activated in DCs, neutrophils, and B cells which causes an increase in AKI associated biochemical markers such as serum creatinine/blood urea nitrogen, renal myeloperoxidase activity, and histopathological disturbances in renal tubular structures. Activation of BTK causes upregulation of NOX-2/iNOS/nitrotyrosine in these immune cells and kidney. Treatment with BTK inhibitor, Ibrutinib causes attenuation in AKI associated dysfunction in biochemical parameters (serum creatinine/blood urea nitrogen, renal myeloperoxidase activity) and oxidative stress in immune cells and kidney (iNOS/NOX2/lipid peroxides/nitrotyrosine/protein carbonyls). In summary, the current investigation reveals a compelling role of BTK signaling in sepsis-induced AKI which is evident from amelioration of AKI associated renal dysfunction after its inhibition.

Lipopolysaccharide reduces USP13 stability through c-Jun N-terminal kinase activation in Kupffer cells.

Protein ubiquitination regulates protein stability, cellular localization, and enzyme activity. Deubiquitinases catalyze the removal of ubiquitin from target proteins and reverse ubiquitination. USP13, a deubiquitinase, has been shown to regulate a variety of cellular responses including inflammation; however, the molecular regulation of USP13 has not been demonstrated. In this study, we revealed that USP13 is degraded in response to lipopolysaccharide (LPS) in Kupffer cells. USP13 levels are significantly decreased in inflamed organs, including liver tissues from septic mice. LPS reduces USP13 protein stability, not transcription, in Kupffer cells. Furthermore, LPS increases USP13 polyubiquitination. Inhibition of proteasome, but not lysosome or immunoproteasome, attenuates LPS-induced USP13 degradation, suggesting USP13 degradation is mediated by the ubiquitin-proteasome system. A catalytically inactive form of USP13 exhibits similar degree of degradation compared with USP13 wild-type, suggesting that USP13 degradation is not dependent on its activity. Furthermore, USP13 degradation is dependent on new protein synthesis. Inhibition of c-Jun N-terminal kinase (JNK) attenuates USP13 degradation, indicating that JNK-dependent new protein synthesis is necessary for USP13 degradation. This study reveals a molecular mechanism of regulation of USP13 degradation in Kupffer cells in response to bacterial endotoxin.

WNK1-TAK1 signaling suppresses lipopolysaccharide-induced cytokine production and classical activation in macrophages.

With-no-lysine kinase (WNK) plays important roles in regulating electrolyte homeostasis, cell signaling, survival, and proliferation. It has been recently demonstrated that WNK1, a member of the WNK family, modifies the function of immune cells. Here we report that in macrophages, WNK1 has suppressive effects on lipopolysaccharide (LPS)-induced inflammatory responses via TGFß-activated kinase 1 (TAK1)-mediated activation of nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathway. We found that WNK1 heterozygous (WNK1+/-) mice produced excessive proinflammatory cytokines in an experimental LPS-induced sepsis model, and peritoneal macrophages isolated from WNK1+/- mice produced higher levels of LPS-induced cytokines and NOS2 expression as canonical proinflammatory M1 macrophage markers. We confirmed that small hairpin RNA (shRNA)-mediated knockdown of WNK1 activated LPS-induced cytokine production and NOS2 expression in RAW 264.7 macrophages. Moreover, we demonstrated that WNK1 knockdown increased the nuclear translocation of NF-κB and activated the p38 and Jun N-terminal kinase (JNK) MAPK signaling pathway and that a TAK1 inhibitor diminished these effects of WNK1 knockdown. These results suggest that WNK1 acts as a physiologic immune modulator via interactions with TAK1. WNK1 may be a therapeutic target against the cytokine storm caused by sepsis.

Inhibition of circulating dipeptidyl-peptidase 3 restores cardiac function in a sepsis-induced model in rats: A proof of concept study.

Sepsis is a global economic and health burden. Dipeptidyl peptidase 3 (DPP3) is elevated in the plasma of septic patients. The highest levels of circulating DPP3 (cDPP3) are found in non-survivor septic shock patients. The aim of this study was to evaluate the benefits of inhibiting cDPP3 by a specific antibody, Procizumab (PCZ), on cardiac function in an experimental model of sepsis, the caecal ligature and puncture (CLP) model. Rats were monitored by invasive blood pressure and echocardiography. Results are presented as mean ± SD, with p <0.05 considered significant. PCZ rapidly restored left ventricular shortening fraction (from 39 ± 4% to 51 ± 2% before and 30 min after PCZ administration (p = 0.004)). Cardiac output and stroke volume were higher in the CLP + PCZ group when compared to the CLP + PBS group (152 ± 33 mL/min vs 97 ± 25 mL/min (p = 0.0079), and 0.5 ± 0.1 mL vs 0.3 ± 1.0 mL (p = 0.009), respectively) with a markedly reduced plasma DPP3 activity (138 ± 70 U/L in CLP + PCZ group versus 735 ± 255 U/L (p = 0.048) in the CLP + PBS group). Of note, PCZ rapidly reduced oxidative stress in the heart of the CLP + PCZ group when compared to those of the CLP + PBS group (13.3 ± 8.2 vs 6.2 ± 2.5 UI, p = 0.005, 120 min after administration, respectively). Our study demonstrates that inhibition of cDPP3 by PCZ restored altered cardiac function during sepsis in rats.

Possible contribution of the neprilysin/ACE pathway to sepsis in mice.

AIM: Omapatrilat is an antagonist of angiotensin-converting (ACE) and neprilysin-neuropeptidase (NEP) enzymes. The aim of our study is to show that omapatrilat may have beneficial effects as a treatment for polymicrobial sepsis. MAIN METHODS: A cecal ligation and puncture (CLP) sepsis model was used to evaluate 10 and 20 mg/kg doses of omapatrilat in mice (n = 30) fasted for 12 h. The lungs were removed 12 h after CLP, and lung levels of cytokines (tumor necrosis factor-alpha [TNF-α], interleukin-6 [IL-6], NF-κB), iNOS and eNOS mRNA expression, GSH and MDA levels, and ACE and NEP activities were determined. Histopathological examinations were also performed. KEY FINDINGS: Omapatrilat treatment provided a dose-dependent reduction in oxidative stress and inflammatory parameters in lung tissues. Omapatrilat administration decreased lung iNOS and eNOS mRNA levels at 20 mg/kg dose. Histopathological analysis revealed a decline in the thickening and edema areas in the alveolar septa in the Sepsis+OMA20 group. SIGNIFICANCE: Omapatrilat, a dual ACE and NEP inhibitor, protected lung tissue from sepsis damage by reducing ACE and NEP activities, by decreasing the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-6, and NF-κB), by suppressing leukocyte infiltration and edema, by restoring iNOS and eNOS levels, and by restoring SOD activity and GSH and MDA levels, thereby reducing oxidative stress.

Myeloperoxidase instigates proinflammatory responses in a cecal ligation and puncture rat model of sepsis.

Myeloperoxidase (MPO)-derived hypochlorous (HOCl) reacts with membrane plasmalogens to yield α-chlorofatty aldehydes such as 2-chlorofatty aldehyde (2-ClFALD) and its metabolite 2-chlorofatty acid (2-ClFA). Recent studies showed that 2-ClFALD and 2-ClFA serve as mediators of the inflammatory responses to sepsis by as yet unknown mechanisms. Since no scavenger for chlorinated lipids is available and on the basis of the well-established role of the MPO/HOCl/chlorinated lipid axis in inflammatory responses, we hypothesized that treatment with MPO inhibitors (N-acetyl lysyltyrosylcysteine amide or 4-aminobenzoic acid hydrazide) would inhibit inflammation and proinflammatory mediator expression induced by cecal ligation and puncture (CLP). We used intravital microscopy to quantify in vivo inflammatory responses in Sham and CLP rats with or without MPO inhibition. Small intestines, mesenteries, and lungs were collected to assess changes in MPO-positive staining and lung injury, respectively, as well as free 2-ClFA and proinflammatory mediators levels. CLP caused neutrophil infiltration, 2-ClFA generation, acute lung injury, leukocyte-/platelet-endothelium interactions, mast cell activation (MCA), plasminogen activator inhibitor-1 (PAI-1) production, and the expression of several cytokines, chemokines, and vascular endothelial growth factor, changes that were reduced by MPO inhibition. Pretreatment with a PAI-1 inhibitor or MC stabilizer prevented CLP-induced leukocyte-endothelium interactions and MCA, and abrogated exogenous 2-ClFALD-induced inflammatory responses. Thus, we provide evidence that MPO instigates these inflammatory changes in CLP and that chlorinated lipids may serve as a mechanistic link between the enzymatic activity of MPO and PAI-1- and mast cell-dependent adhesive interactions, providing a rationale for new therapeutic interventions in sepsis.NEW & NOTEWORTHY Using two distinct myeloperoxidase (MPO) inhibitors, we show for the first time that MPO plays an important role in producing increases in free 2-chlorofatty aldehyde (2-ClFALD)-a powerful proinflammatory chlorinated lipid in plasma and intestine-a number of cytokines and other inflammatory mediators, leukocyte and platelet rolling and adhesion in postcapillary venules, and lung injury in a cecal ligation and puncture model of sepsis. In addition, the use of a plasminogen activator inhibitor-1 (PAI-1) inhibitor or a mast cell stabilizer prevented inflammatory responses in CLP-induced sepsis. PAI-1 inhibition also prevented the proinflammatory responses to exogenous 2-ClFALD superfusion. Thus, our study provides some of the first evidence that MPO-derived free 2-ClFA plays an important role in CLP-induced sepsis by a PAI-1- and mast cell-dependent mechanism.