Organ Damage In Sepsis Research Articles

MPO-DNA Complexes and cf-DNA in Patients with Sepsis and Their Clinical Value.

Background/Objectives: Neutrophils, as the first line of defense in the immune response, produce neutrophil extracellular traps (NETs) upon activation, which are significant in the pathogenesis and organ damage in sepsis. This study aims to explore the clinical value of myeloperoxidase-DNA (MPO-DNA) and cell-free DNA (cf-DNA) in sepsis patients. Methods: Clinical data were collected from 106 sepsis patients, 25 non-sepsis patients, and 51 healthy controls. Sequential Organ Failure Assessment (SOFA) scores were calculated, and levels of MPO-DNA) complexes and cf-DNA were measured using specific kits. Correlation analyses assessed relationships between indicators, while logistic regression identified independent risk factors. Receiver operating characteristic (ROC) curves calculated the area under the curve (AUC) to evaluate the diagnostic value of the biomarkers. Results: Sepsis patients exhibited significantly elevated levels of MPO-DNA and cf-DNA compared to non-sepsis patients and healthy controls. In sepsis patients, MPO-DNA and cf-DNA levels correlated with inflammation, coagulation, and organ damage indicators, as well as procalcitonin (PCT) levels and SOFA scores. Both C-reactive protein (CRP) and cf-DNA were identified as independent risk factors for sepsis, demonstrating moderate diagnostic value. ROC analysis showed that the combination of MPO-DNA and CRP (AUC: 0.837) enhances the AUC value of CRP (0.777). Conclusions: In summary, elevated serum levels of MPO-DNA and cf-DNA in sepsis patients correlate with SOFA scores and PCT levels, providing reference value for sepsis diagnosis in clinical settings.

Impact of sympathetic hyperactivity induced by brain microglial activation on organ damage in sepsis with chronic kidney disease

BackgroundSympathetic nerve activity (SNA) plays a central role in the pathogenesis of several diseases such as sepsis and chronic kidney disease (CKD). Activation of microglia in the paraventricular nucleus of the hypothalamus (PVN) has been implicated in SNA. The mechanisms responsible for the adverse prognosis observed in sepsis associated with CKD remain to be determined. Therefore, we aimed to clarify the impact of increased SNA resulting from microglial activation on hemodynamics and organ damage in sepsis associated with CKD.Methods and resultsIn protocol 1, male Sprague–Dawley rats underwent either nephrectomy (Nx) or sham surgery followed by cecal ligation and puncture (CLP) or sham surgery. After CLP, Nx-CLP rats exhibited decreased blood pressure, increased heart rate, elevated serum creatinine and bilirubin levels, and decreased platelet count compared to Nx-Sham rats. Heart rate variability analysis revealed an increased low to high frequency (LF/HF) ratio in Nx-CLP rats, indicating increased SNA. Nx-CLP rats also had higher creatinine and bilirubin levels and lower platelet counts than sham-CLP rats after CLP. In protocol 2, Nx-CLP rats were divided into two subgroups: one received minocycline, an inhibitor of microglial activation, while the other received artificial cerebrospinal fluid (CSF) intracerebroventricularly via an osmotic minipump. The minocycline-treated group (Nx-mino-CLP) showed attenuated hypotensive and increased heart rate responses compared to the CSF-treated group (Nx-CSF-CLP), and the LF/HF ratio was also decreased. Echocardiography showed larger left ventricular dimensions and inferior vena cava in the Nx-mino-CLP group. In addition, creatinine and bilirubin levels were lower and platelet counts were higher in the Nx-mino-CLP group compared to the Nx-CSF-CLP group.ConclusionsIn septic rats with concomitant CKD, SNA was significantly enhanced and organ dysfunction was increased. It has been suggested that the mechanism of exacerbated organ dysfunction in these models may involve abnormal systemic hemodynamics, possibly triggered by activation of the central sympathetic nervous system through activation of microglia in the PVN.

Targeting purinergic signaling in platelets ameliorates the bacterial burden and reduces the cytokine storm in the peritoneal cavity during intra-abdominal sepsis

Sepsis is a complex clinical syndrome of the host response to an infection, involving a pathogenic cytokine storm that drives inflammatory injury in multiple organ systems. The abdomen is a major site of sepsis, contributing to high mortality. Platelets express the purinergic receptors P2Y1 and P2Y12; once activated, these receptors mediate the secretion of pro-inflammatory mediators that contribute to organ damage in sepsis. Systemic blockade of P2Y1 or P2Y12 decreased organ damage and inflammatory cytokine levels in a sex-related manner during sepsis. Here, we hypothesize that blocking P2Y1 or P2Y12 activity enhances pathogen clearance in the peritoneal cavity during intra-abdominal sepsis in a sex-specific manner. Male and female C57BL6/6J mice underwent sham surgery or cecal ligation and puncture (CLP) to induce sepsis. P2Y1 or P2Y12 antagonists, MRS2279 (10 mg/kg) and Ticagrelor (30 mg/kg) respectively, were administered intraperitoneally after surgery. The peritoneal cavity fluid (PCF) was collected 24 hours post-surgery by peritoneal lavage. To assess cell recruitment in the cavity, we measured leukocyte and platelet counts using Hemavet® Multispecies Hematology System. To assess inflammation, we measured PCF levels of the cytokines MIP-1α and Rantes as well as bacterial clearance. The ileum of the small intestinal tissue samples were collected 24 hours post-surgery and platelet infiltration was measured by platelet factor 4 (PF4) content in the tissue using an ELISA kit. In response to the P2Y1 or P2Y12 blocker, we noted a significant reduction in the bacteria content in the PCF of CLP mice. Blocking P2Y12 but not P2Y1 reduced leukocyte recruitment, while platelet counts were reduced only with P2Y1 blockade in septic males. The sepsis-induced increase in MIP-1α levels was not noted upon P2Y1 but not P2Y12 blockage in the PCF of septic males. However, Rantes levels in the PCF were reduced for either treatment in males compared to the untreated CLP. The platelet marker PF4 in the ileum was increased during sepsis but reduced after either treatment in sepsis males as compared to the untreated CLP. In female CLP mice, a significant reduction in bacterial burden in the PCF in response to the P2Y1 but not the P2Y12 blocker was shown. Blocking P2Y1 or P2Y12 reduced leukocyte recruitment and platelet counts in female CLP mice. As observed in males, PF4 was increased in the ileum during sepsis but reduced following exposure to either treatment in females. No change was noted for MIP-1α or Rantes levels in the females among all the septic groups. Our data indicate that blocking purinergic signaling and in particular P2Y12 in the peritoneal cavity in males prevents bacterial spreading and diminishes cytokine levels, indicating that it could be an effective treatment for intra-abdominal sepsis. However, in female mice blocking the receptor P2Y1, but not P2Y12 has shown to be optimal. Targeting purinergic signaling is a promising sex-specific therapeutic strategy for sepsis. This work was supported by the Research grant AI156627-01 (EL) from the National Institute of Health. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The role and therapeutic potential of SIRTs in sepsis.

Sepsis is a life-threatening organ dysfunction caused by the host's dysfunctional response to infection. Abnormal activation of the immune system and disturbance of energy metabolism play a key role in the development of sepsis. In recent years, the Sirtuins (SIRTs) family has been found to play an important role in the pathogenesis of sepsis. SIRTs, as a class of histone deacetylases (HDACs), are widely involved in cellular inflammation regulation, energy metabolism and oxidative stress. The effects of SIRTs on immune cells are mainly reflected in the regulation of inflammatory pathways. This regulation helps balance the inflammatory response and may lessen cell damage and organ dysfunction in sepsis. In terms of energy metabolism, SIRTs can play a role in immunophenotypic transformation by regulating cell metabolism, improve mitochondrial function, increase energy production, and maintain cell energy balance. SIRTs also regulate the production of reactive oxygen species (ROS), protecting cells from oxidative stress damage by activating antioxidant defense pathways and maintaining a balance between oxidants and reducing agents. Current studies have shown that several potential drugs, such as Resveratrol and melatonin, can enhance the activity of SIRT. It can help to reduce inflammatory response, improve energy metabolism and reduce oxidative stress, showing potential clinical application prospects for the treatment of sepsis. This review focuses on the regulation of SIRT on inflammatory response, energy metabolism and oxidative stress of immune cells, as well as its important influence on multiple organ dysfunction in sepsis, and discusses and summarizes the effects of related drugs and compounds on reducing multiple organ damage in sepsis through the pathway involving SIRTs. SIRTs may become a new target for the treatment of sepsis and its resulting organ dysfunction, providing new ideas and possibilities for the treatment of this life-threatening disease.

Platelet-derived extracellular vesicles are associated with kidney injury in patients with urosepsis

BackgroundThere is increasing evidence that platelet-derived extracellular vesicles (PEVs) may be involved in the mechanisms of inflammatory storm and organ damage in sepsis. However, there are no available studies on PEVs and renal injury in patients with urosepsis. MethodsWe analyzed the concentration and ratio of PEVs in plasma by flow cytometry and measured plasma IL-1β/IL-6/TNF-α/NGAL levels by ELISA. Correlation analysis was also used to examine the concentration of PEVs in relation to levels of inflammatory factors and indicators of kidney damage, as well as the severity of the disease. Finally, the receiver operating characteristic curves were produced for PEVs concentrations as a diagnosis of S-AKI/AKI. ResultsWe found significantly higher levels of IL-1β/IL-6/TNF-α/NGAL in patients with urogenital sepsis. Furthermore, the concentrations of PEVs in plasma were significantly elevated in patients with urosepsis, especially in patients with Gram-negative bacterial infections, which were significantly and positively correlated with IL-1β/IL-6/TNF-α/NGAL levels. The area under the curve for PEVs diagnosing S-AKI and AKI was 0.746 [0.484, 1.000] and 0.943 [0.874, 1.000] respectively. ConclusionOverall, the present study suggested that PEVs may mediate the release of inflammatory mediators in patients with urosepsis and participate in the mechanism of acute kidney injury, as well as having potential as diagnostic indicators of S-AKI and AKI and as early warning indicators of the severity of patients with urosepsis.

ACT001 improved cardiovascular function in septic mice by inhibiting the production of proinflammatory cytokines and the expression of JAK-STAT signaling pathway

Sepsis is a life-threatening multiple organ dysfunction syndrome (MODS) caused by a microbial infection that leads to high morbidity and mortality worldwide. Sepsis-induced cardiomyopathy (SIC) and coagulopathy promote the progression of adverse outcomes in sepsis. Here, we reported that ACT001, a modified compound of parthenolide, improved the survival of sepsis mice. In this work, we used cecal ligation and puncture (CLP) model to induce SIC. Transthoracic echocardiography and HE staining assays were adopted to evaluate the influence of ACT001 on sepsis-induced cardiac dysfunction. Our results showed that ACT001 significantly improved heart function and reduced SIC. Coagulation accelerates organ damage in sepsis. We found that ACT001 decreased blood clotting in the FeCl3-induced carotid artery thrombosis experiment. ACT001 also reduced the production of neutrophil extracellular traps (NETs). RNA-sequencing of heart tissues revealed that ACT001 significantly downregulated the expression of pro-inflammatory cytokines and the JAK-STAT signaling pathway. These results were confirmed with real-time PCR and ELISA. In summary, we found ACT001 rescued mice from septic shock by protecting the cardiovascular system. This was partially mediated by inhibiting pro-inflammatory cytokine production and down-regulating the JAK-STAT signaling.

Research progress on the activation mechanism of NLRP3 inflammasome in septic cardiomyopathy.

Sepsis is an uncontrolled host response to infection, resulting in a clinical syndrome involving multiple organ dysfunctions. Cardiac damage is the most common organ damage in sepsis. Uncontrolled inflammatory response is an important mechanism in the pathogenesis of septic cardiomyopathy (SCM). NLRP3 inflammasome promotes inflammatory response by controlling the activation of caspase-1 and the release of pro-inflammatory cytokines interleukin IL-1β and IL-18. The role of NLRP3 inflammasome has received increasing attention, but its activation mechanism and regulation of inflammation in SCM remain to be investigated.

Revealing the biological mechanism of acupuncture in alleviating excessive inflammatory responses and organ damage in sepsis: a systematic review.

Sepsis is a systemic inflammation caused by a maladjusted host response to infection. In severe cases, it can cause multiple organ dysfunction syndrome (MODS) and even endanger life. Acupuncture is widely accepted and applied in the treatment of sepsis, and breakthroughs have been made regarding its mechanism of action in recent years. In this review, we systematically discuss the current clinical applications of acupuncture in the treatment of sepsis and focus on the mechanisms of acupuncture in animal models of systemic inflammation. In clinical research, acupuncture can not only effectively inhibit excessive inflammatory reactions but also improve the immunosuppressive state of patients with sepsis, thus maintaining immune homeostasis. Mechanistically, a change in the acupoint microenvironment is the initial response link for acupuncture to take effect, whereas PROKR2 neurons, high-threshold thin nerve fibres, cannabinoid CB2 receptor (CB2R) activation, and Ca2+ influx are the key material bases. The cholinergic anti-inflammatory pathway of the vagus nervous system, the adrenal dopamine anti-inflammatory pathway, and the sympathetic nervous system are key to the transmission of acupuncture information and the inhibition of systemic inflammation. In MODS, acupuncture protects against septic organ damage by inhibiting excessive inflammatory reactions, resisting oxidative stress, protecting mitochondrial function, and reducing apoptosis and tissue or organ damage.

Revealing the role of gut microbiota in immune regulation and organ damage in sepsis using 16s rRNA and untargeted metabolomics

To analyze the composition and metabolites of gut microbiota in septic rats by fecal 16s rRNA sequencing and untargeted metabolomics, and to preliminarily explore the effect and potential mechanism of gut microbiota and its metabolites on inflammatory response and multiple organ damage in sepsis. Ten males healthy male Wistar rats were randomly divided into a sham operated group (Sham group) and sepsis model group (CLP group) using a random number table method, with 5 rats in each group. A rat sepsis model was established by cecal ligation and perforation (CLP) method. The animals were sacrificed 24 hours after modeling, the levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in peripheral blood were detected by enzyme-linked immunosorbent assay (ELISA). Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung and kidney tissues, and the pathological scores were evaluated. Fecal samples were collected, and 16s rRNA high-throughput sequencing and non-targeted metabolomics were used to screen microbiota, metabolites and potential signal pathways that may play an important role in disease outcomes. Spearman correlation analysis was conducted to jointly analyze the gut microbiota and non-targeted metabolism. Compared with the Sham group, the degree of pathological damage to lung and kidney tissues in the CLP group was significantly increased (lung tissue score: 3.60±0.80 vs. 0.00±0.00, kidney tissue score: 2.40±0.80 vs. 0.00±0.00, both P < 0.01), the level of IL-6 and TNF-α in peripheral blood significantly increased [TNF-α (ng/L): 248.12±55.98 vs. 143.28±36.57, IL-6 (ng/L): 260.26±39.47 vs. 116.01±26.43, both P < 0.05], the species diversity of intestinal flora of rats in the CLP group was significantly reduced, the relative abundance of Morganella, Bacteroides and Escherichia-Shigella were significantly increased, and the relative abundance of Lachnospiraceae NK4A136, Ruminococcus, Romboutsia and Roseburia were significantly reduced. In addition, the biosynthesis and bile secretion of phenylalanine, tyrosine, and tryptophan in the gut microbiota of the CLP group were significantly increased, while the biosynthesis of secondary bile acids was significantly reduced. There was a significant correlation between differential metabolites and differential microbiota. Sepsis can cause significant changes in the characteristics of gut microbiota and fecal metabolites in rats, which provides a basis for translational research to seek new targets for the treatment of sepsis.

Dual Role of Extracellular Vesicles in Sepsis-Associated Kidney and Lung Injury

Extracellular vesicles form a complex intercellular communication network, shuttling a variety of proteins, lipids, and nucleic acids, including regulatory RNAs, such as microRNAs. Transfer of these molecules to target cells allows for the modulation of sets of genes and mediates multiple paracrine and endocrine actions. EVs exert broad pro-inflammatory, pro-oxidant, and pro-apoptotic effects in sepsis, mediating microvascular dysfunction and multiple organ damage. This deleterious role is well documented in sepsis-associated acute kidney injury and acute respiratory distress syndrome. On the other hand, protective effects of stem cell-derived extracellular vesicles have been reported in experimental models of sepsis. Stem cell-derived extracellular vesicles recapitulate beneficial cytoprotective, regenerative, and immunomodulatory properties of parental cells and have shown therapeutic effects in experimental models of sepsis with kidney and lung involvement. Extracellular vesicles are also likely to play a role in deranged kidney-lung crosstalk, a hallmark of sepsis, and may be key to a better understanding of shared mechanisms underlying multiple organ dysfunction. In this review, we analyze the state-of-the-art knowledge on the dual role of EVs in sepsis-associated kidney/lung injury and repair. PubMed library was searched from inception to July 2022, using a combination of medical subject headings (MeSH) and keywords related to EVs, sepsis, acute kidney injury (AKI), acute lung injury (ALI), and acute respiratory distress syndrome (ARDS). Key findings are summarized into two sections on detrimental and beneficial mechanisms of actions of EVs in kidney and lung injury, respectively. The role of EVs in kidney-lung crosstalk is then outlined. Efforts to expand knowledge on EVs may pave the way to employ them as prognostic biomarkers or therapeutic targets to prevent or reduce organ damage in sepsis.

Protective effect of Astragalus membranaceus and Astragaloside IV in sepsis-induced acute kidney injury.

Background: Acute kidney injury (AKI) is the most common target organ damage in sepsis. Sepsis-associated AKI (SA-AKI) may be characterized by damage to the renal tubular epithelium. In this study, the pharmacological mechanisms of Astragalus membranaceus and its active monomer Astragaloside IV (AS-IV) were predicted based on a network pharmacology approach and validated both in vitro and in vivo using the SA-AKI model.Method: We constructed an in vivo sepsis model using a mouse cecum ligation puncture (CLP) and HK-2 cells were treated with lipopolysaccharide (LPS) to mimic Gram (–) induced sepsis to assess the renal-protective efficacy of Astragalus membranaceus and AS-IV.Results: The findings demonstrated that Astragalus membranaceus and AS-IV attenuate renal tubular injury in mice with polymicrobial sepsis, including vacuolization, loss of brush border, mitochondrial ultrastructural changes, and increased staining of kidney injury molecule-1 (KIM-1). AS-IV protected human proximal tubular epithelial (HK-2) cells against LPS induced cell viability loss. Both Astragalus membranaceus and AS-IV activated the PI3K/AKT pathway both in vitro and in vivo, as shown by Western blot and immunohistochemistry analysis.Conclusion: The findings demonstrate that Astragalus membranaceus and AS-IV protect against sepsis-induced kidney tubular injury by activating the PI3K/AKT pathway.

Long-distance relationships - regulation of systemic host defense against infections by the gut microbiota.

Despite compartmentalization within the lumen of the gastrointestinal tract, the gut microbiota has a far-reaching influence on immune cell development and function throughout the body. This long-distance relationship is crucial for immune homeostasis, including effective host defense against invading pathogens that cause systemic infections. Herein, we review new insights into how commensal microbes that are spatially restricted to the gut lumen can engage in long-distance relationships with innate and adaptive immune cells at systemic sites to fortify host defenses against infections. In addition, we explore the consequences of intestinal dysbiosis on impaired host defense and immune-mediated pathology during infections, including emerging evidence linking dysbiosis with aberrant systemic inflammation and immune-mediated organ damage in sepsis. As such, therapeutic modification of the gut microbiota is an emerging target for interventions to prevent and/or treat systemic infections and sepsis by harnessing the long-distance relationships between gut microbes and systemic immunity.

Correlation between thrombopoietin and inflammatory factors, platelet indices, and thrombosis in patients with sepsis: A retrospective study.

BACKGROUNDThrombopoietin (TPO) is a primary regulator of thrombopoiesis in physiological conditions. TPO, in combination with its specific cytokine receptor c-Mpl, drives platelet production by inducing the proliferation and differentiation of megakaryocytes. However, the role of TPO in sepsis is not well determined. The elevated levels of TPO are often accompanied by a decrease of platelet count (PLT) in systemic infected conditions, which is contrary to the view that TPO promotes platelet production under physiological conditions. In addition, whether TPO mediates organ damage in sepsis remains controversial.AIMTo explore the relationships between TPO and inflammatory factors, platelet indices, and thrombotic indicators in sepsis.METHODSA total of 90 patients with sepsis diagnosed and treated at the emergency medicine department of The First People’s Hospital of Foshan between January 2020 and March 2021 were enrolled in this study. In addition, 110 patients without sepsis who came to the emergency medicine department were included as controls. Clinical and laboratory parameters including age, gender, TPO, blood cell count in peripheral blood, platelet indices, inflammatory factors such as high-sensitivity C-reactive protein (hs-CRP), interleukin (IL)-21, and IL-6, organ damage indicators, and thrombotic indicators were collected and analyzed by using various statistical approaches.RESULTSThe results showed that the TPO levels were higher in the sepsis group than in controls [86.45 (30.55, 193.1) vs 12.45 (0.64, 46.09) pg/mL, P < 0.001], but PLT was lower (P < 0.001). Multivariable analysis showed that white blood cell count (WBC) [odds ratio (OR) = 1.32; 95% confidence interval (CI): 1.01-1.722; P = 0.044], TPO (OR = 1.02; 95%CI: 1.01-1.04; P = 0.009), IL-21 (OR = 1.02; 95%CI: 1.00-1.03; P = 0.019), troponin I (OR = 55.20; 95%CI: 5.69-535.90; P = 0.001), and prothrombin time (PT) (OR = 2.24; 95%CI: 1.10-4.55; P = 0.027) were independent risk factors associated with sepsis. TPO levels were positively correlated with IL-21, IL-6, hs-CRP, creatinine, D-dimer, PT, activated prothrombin time, international normalized ratio, fibrinogen, WBC count, and neutrophil count, and negatively correlated with PLT, thrombin time, red blood cell count, and hemoglobin concentration (P < 0.05). Receiver operating characteristic analysis showed that TPO had fair predictive value in distinguishing septic patients and non-septic patients (the area under the curve: 0.788; 95%CI: 0.723-0.852; P < 0.001). With an optimized cutoff value (28.51 pg/mL), TPO had the highest sensitivity (79%) and specificity (65%).CONCLUSIONTPO levels are independently associated with sepsis. High TPO levels and low PLT suggest that TPO might be an acute-phase response protein in patients with infection.

Effects of Thrombomodulin in Reducing Lethality and Suppressing Neutrophil Extracellular Trap Formation in the Lungs and Liver in a Lipopolysaccharide-Induced Murine Septic Shock Model.

Neutrophil extracellular trap (NET) formation, an innate immune system response, is associated with thrombogenesis and vascular endothelial injury. Circulatory disorders due to microvascular thrombogenesis are one of the principal causes of organ damage. NET formation in organs contributes to the exacerbation of sepsis, which is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection. We have previously reported that recombinant human soluble thrombomodulin (rTM) reduces lipopolysaccharide (LPS)-induced NET formation in vitro. Here, we aimed to show that thrombomodulin (TM)-mediated suppression of NET formation protects against organ damage in sepsis. Mice were injected intraperitoneally (i.p.) with 10 mg/kg LPS. rTM (6 mg/kg/day) or saline was administered i.p. 1 h after LPS injection. In the LPS-induced murine septic shock model, extracellular histones, which are components of NETs, were observed in the liver and lungs. In addition, the serum cytokine (interleukin-1β (IL-1β), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), macrophage chemotactic protein-1 (MCP-1), and interleukin-10 (IL-10)) levels were increased. The administration of rTM in this model prevented NET formation in the organs and suppressed the increase in the levels of all cytokines except IL-1β. Furthermore, the survival rate improved. We provide a novel role of TM in treating inflammation and NETs in organs during sepsis.

Growth differentiation factor 11 relieves acute lung injury in mice by inhibiting inflammation and apoptosis.

Acute lung injury (ALI) is the most common organ damage in sepsis and sepsis-induced ALI is a clinically extremely dangerous disease. Therefore, it is essential to find an effective way to treat ALI. We hope to provide a new target for the treatment of clinical ALI by studying the effect of GDF11 on LPS-induced ALI. C57BL/6 male mice and lipopolysaccharide (LPS) were used to induce mouse ALI. Recombinant GDF11 protein was used to treat mice to detect the effect of GDF11 on mouse ALI. In addition, BEAS-2B cells were used to further validate the effects of GDF11 on inflammation and apoptosis of alveolar epithelial cells. Recombinant GDF11 protein significantly reduced the expression of inflammatory factors and apoptosis-related pathways in mouse lung tissues. Overexpression of GDF11 in BEAS-2B cells also significantly attenuated the levels of inflammation and apoptosis in the cells. In addition, GDF11 can reduce the activity of TLR2/HMGB1/NF-κB signaling pathway, which is an important mechanism for GDF11 to play a role in lung protection. GDF11 can exert lung protection effects by inhibiting the TLR2/HMGB1/NF-κB signaling pathway and reduce the level of inflammation and apoptosis of the lung.

Therapeutic effects of extracellular vesicles obtained from bone marrow-derived, adipose tissue-derived, and lung-derived mesenchymal stromal cells on the lung and distal organs in experimental sepsis

Background & Aim The beneficial effects of mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) in experimental sepsis have been described. However, to date, there is no information regarding the best source of MSC-EVs to enhance survival rate and mitigate lung and distal organ damage in sepsis. In this context, we compared the effects of MSC-EVs obtained from bone marrow (BM), adipose tissue (AD), and lung (L) on mortality rate and lung and distal organ histology and molecular biology in a murine model of experimental polymicrobial sepsis. Methods, Results & Conclusion Twenty-four C57BL/6 mice (weight 20-25g) underwent cecal ligation and puncture (CLP) to induce sepsis. A sham-operated group was used as control (SHAM). Animals received antibiotics (imipenem/cilastatin, 10 mg/kg) at 6 and 24 hours. Twenty-four hours after CLP induction, mice were treated with saline (SAL) or EVs from 3 × 106 BM-, AD- or L-derived MSCs. Total protein content, intensity, and hydrodynamic diameter of the EV fraction were measured and compared according to source. Twenty-four hours after therapy, lung, liver, and kidney samples were obtained for histology and molecular biology. Tumor-necrosis factor (TNF)-α protein level was measured in lung tissue. Interleukin (IL)-18, kidney injury molecule (KIM)-1 and heme oxygenase-1 (HO)-1 mRNA expressions were measured in kidney. Survival rate was evaluated at 48 hours. EVs from BM-, AD-, and L-derived MCs exhibited similar total protein content and hydrodynamic diameters. In septic mice, EVs from all sources were able to reduce TNF-α in lung tissue, neutrophils in alveolar septa, and diffuse alveolar damage compared to SAL (p<0.01). BM-EVs reduced liver congestion (p=0.01) and Kupffer cells in sinusoids (p=0.03). EVs from all sources decreased interstitial edema in kidney (p<0.001), but only BM-EVs were able to preserve the brush border, which is damaged in sepsis. Regarding kidney inflammation, BM-EVs and L-EVs, but not AD-EVs, reduced mRNA expression of IL-18 and KIM-1 (p<0.0125). HO-1 (p<0.0125), a marker of acute kidney injury in mice, increased only after administration of AD-EVs. In addition, AD-EVs were associated with a trend (p=0.10) toward lower survival (60%) compared to groups treated with BM- (89%) and L- (88%) derived EVs. In the model of sepsis used herein, EVs obtained from BM-derived MSCs seem to be associated with reduced lung, liver, and kidney damage compared with EVs obtained from AD- and L-MSCs. The beneficial effects of mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) in experimental sepsis have been described. However, to date, there is no information regarding the best source of MSC-EVs to enhance survival rate and mitigate lung and distal organ damage in sepsis. In this context, we compared the effects of MSC-EVs obtained from bone marrow (BM), adipose tissue (AD), and lung (L) on mortality rate and lung and distal organ histology and molecular biology in a murine model of experimental polymicrobial sepsis. Twenty-four C57BL/6 mice (weight 20-25g) underwent cecal ligation and puncture (CLP) to induce sepsis. A sham-operated group was used as control (SHAM). Animals received antibiotics (imipenem/cilastatin, 10 mg/kg) at 6 and 24 hours. Twenty-four hours after CLP induction, mice were treated with saline (SAL) or EVs from 3 × 106 BM-, AD- or L-derived MSCs. Total protein content, intensity, and hydrodynamic diameter of the EV fraction were measured and compared according to source. Twenty-four hours after therapy, lung, liver, and kidney samples were obtained for histology and molecular biology. Tumor-necrosis factor (TNF)-α protein level was measured in lung tissue. Interleukin (IL)-18, kidney injury molecule (KIM)-1 and heme oxygenase-1 (HO)-1 mRNA expressions were measured in kidney. Survival rate was evaluated at 48 hours.

Hepatocyte-Specific Deletion of AMPKα1 Results in Worse Outcomes in Mice Subjected to Sepsis in a Sex-Specific Manner.

Alterations in the energy homeostasis contribute to sepsis-mediated multiple organ failure. The liver plays a central role in metabolism and participates to the innate immune and inflammatory responses of sepsis. Several clinical and experimental studies have suggested that females are less susceptible to the adverse outcome of sepsis. However, underlying mechanisms of organ damage in sepsis remain largely undefined. AMP-activated protein kinase (AMPK) is an important regulator of mitochondrial quality control. The AMPK catalytic α1 isoform is abundantly expressed in the liver. Here, we determined the role of hepatocyte AMPKα1 in sepsis by using hepatocyte-specific AMPKα1 knockout mice (H-AMPKα1 KO) generated with Cre-recombinase expression under the control of the albumin promoter. Using a clinically relevant model of polymicrobial sepsis by cecal ligation and puncture (CLP), we observed that male H-AMPKα1 KO mice had higher plasma levels of tumor necrosis factor-α and interleukin-6 and exhibited a more severe liver and lung injury than male H-AMPKα1 WT mice, as evaluated by histology and neutrophil infiltration at 18 h after CLP. Plasma levels of interleukin-10 and the keratinocyte-derived chemokine were similarly elevated in both KO and WT male mice. At transmission electron microscopy analysis, male H-AMPKα1 KO mice exhibited higher liver mitochondrial damage, which was associated with a significant decrease in liver ATP levels when compared to WT mice at 18 h after sepsis. Mortality rate was significantly higher in the male H-AMPKα1 KO group (91%) when compared to WT mice (60%) at 7 days after CLP. Female H-AMPKα1 WT mice exhibited a similar degree of histological liver and lung injury, but significantly milder liver mitochondrial damage and higher autophagy when compared to male WT mice after CLP. Interestingly, H-AMPKα1 KO female mice had lower organ neutrophil infiltration, lower liver mitochondrial damage and lower levels of cytokines than WT female mice. There was no significant difference in survival rate between WT and KO mice in the female group. In conclusion, our study demonstrates that AMPKα1 is a crucial hepatoprotective enzyme during sepsis. Furthermore, our results suggest that AMPK-dependent liver metabolic functions may influence the susceptibility to multiple organ injury in a sex-dependent manner.

Exercise Preconditioning-Induced Modification of Gut Microbiota Increases Survival in Cecal Ligation and Perforation (CLP)-Induced Sepsis

PURPOSE: To investigate whether exercise preconditioning-induced modification of gut microbiota increases survival in cecal ligation and puncture (CLP)-induced sepsis. METHODS: C57BL/6N male mice at age of 4 weeks were randomly assigned to either control (n=10) or exercise preconditioning (n=10). Mice in the exercise group were subjected to an 8-week-high-intensity treadmill running. Prior to sepsis, the effect of exercise preconditioning on gut microbiota was explored by 16S ribosomal RNA amplification sequencing. Survival probability, organ damage, and pro- and anti-inflammatory cytokines were assessed to investigate whether or not exercise preconditioning-induced changes in gut microbiota modulate host response to CLP-induced sepsis. RESULTS: Along with increased β diversity, exercise preconditioning induced the modification of gut microbiota at species level dominated by the phyla Bacteriodetes, Firmicutes, and Verrucomicrobia and to a less extent by the phylum Cyanobacteria. Preconditioned mice had higher survival (p<0.05) and less organ damage in sepsis compared to control mice. At 8-hr post-sepsis, the preconditioned mice had lower peritoneal interleukin (IL)-1β (p=0.020) and IL-6 (p<0.001) but higher peritoneal interferon (IFN)-γ (p=0.002), IL-10 (p<0.001), and transforming growth factor (TGF)-1β (p<0.001) compared to the control mice. In addition, the preconditioned mice has lower blood growth-regulated oncogene (GRO)-α (p=0.001), monocyte chemoattractant protein (MCP)-1 (p<0.001), macrophage inflammatory protein (MIP) 2 (p<0.001), and tumor necrosis factor (TNF)-α (p=0.003) compared to the control mice. At 20-hr post-sepsis, the preconditioned mice had lower peritoneal IL-1β (p<0.001) and Il-6 (p<0.001) but higher peritoneal TGF-1β (p<0.001) in conjunction with higher blood IL-17A (p=0.002) and TGF-1β (p=0.029) compared to the control mice. CONCLUSIONS: The current findings suggested that exercise preconditioning-induced modification of gut microbiota might contribute to increased survival in sepsis by modulating host response toward the establishment of a balance between pro- and anti-inflammation. Supported by the National Research Foundation funded by the Korean Government (NRF-2018R1D1A1B07048153 and NRF-2016R1A6A3A11932432).

Abstract B77: Low-density neutrophils (LDN) in circulating blood of postoperative patients may participate in the development of distant recurrence through the production of neutrophil extracellular traps (NETs)

Abstract Background: Low density neutrophils (LDN) which are co-purified with mononuclear cells by Ficoll-Hypaque density gradient preparations, have been reported to be increased in circulating blood in patients with autoimmune diseases, sepsis and cancer. Recent studies have suggested the LDN contributes to lupus pathogenesis as well as the development of organ damage in sepsis. Method: In patients who underwent curative surgery for gastrointestinal malignancy, peripheral blood was obtained at the timing of laparotomy (preoperative) and just before wound closure (postoperative) and the ratios of LDN were evaluated with flowcytometry. The LDN and mononuclear cells (MNC) in postoperative samples were separated using CD66b microbeads and MACS column and the proliferation of T cells were evaluated by in vitro culture on anti-CD3 coated plate using CFSE dilution method. The LDN were cultured for 2 hours and neutrophil extracellular traps (NETs) were observed with the addition of SYTOX green, dye for extracellular double strand (ds)-DNA. In adhesion experiments, human colon cancer, DLD-1, were stained by PKH-26 and added on the cultured LDN for 5 min. After gentle washing, the remaining cells were counted under fluorescein microscope. Results: 1. Ratios of CD66b(+) LDG in CD45(+) leukocytes were markedly increased in postoperative as compared with preoperative blood samples. 2. Division of CD3(+) T cells were significantly suppressed by the addition autologous CD66b(+) LDN. 3. Short term culture of the LDN produced massive thread-like structures stained with SYTOX green. Immunostaining with specific mAbs revealed that histone and myeloperoxidase (MPO) were colocalized on the fibrous structure, indicating NETs 3. DLD-1, selectively attached to the NETs, which was totally abrogated by the pretreatment with 100u/ml DNAse. Conclusion: During abdominal surgery, immunosuppressive LDN are markedly increased in circulating blood. The LDN extensively produces NETs which support the metastasis formation by trapping the circulating tumor cells in target organ. Functional blockade of the LDN might effectively reduce the recurrence rate after curative surgery. Citation Format: Yuko Kumagai, Rihito Kanamaru, Hideyuki Ohzawa, Hisanaga Horie, Yoshinori Hosoya, Naohiro Sata, Joji Kitayama. Low-density neutrophils (LDN) in circulating blood of postoperative patients may participate in the development of distant recurrence through the production of neutrophil extracellular traps (NETs) [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B77.

MicroRNA-146a protects against LPS-induced organ damage by inhibiting Notch1 in macrophage

MicroRNA-146a is a well-studied microRNA participating in immune and inflammatory diseases, but its role in sepsis has not been investigated. Here in our study, we found increased level of microRNA-146a in macrophage stimulated by lipopolysaccharide. In addition, the mRNA level of Notch1 was also increased. Up-regulation of microRNA-146a by miR-146a-mimic alleviated inflammatory responses of macrophage, for the levels of IL-1β, IL-6 and CCL-2 were decreased, and the activation of NF-κB signaling was inhibited. The histological examination showed that microRNA-146a protected against organ damage in mice with lipopolysaccharide injection, and the level of inflammatory factors, Cr, BUN, AST and ALT in serum were all decreased, reflecting the alleviated inflammation and recovered organ function. Then predicting databases were used and Notch1 was predicted as one of the potential targets of microRNA-146a. Knockout of Notch1 in macrophage showed reduced secretion of inflammatory factors and attenuated activation of NF-κB signaling in response to lipopolysaccharide. Specifically knockout of Notch1 in macrophage protected mice from LPS induced organ damage and dysfunction. Therefore, we prove that miR-146a acts as an inhibitor of inflammation by targeting Notch1 in macrophage, therefore protects mice from organ damage in sepsis.