Model Of Septic Shock Research Articles

Clinical-grade mesenchymal stem cells derived from umbilical cord improve septic shock in pigs

BackgroundSeptic shock is the leading cause of death in intensive care units. The pathophysiological complexity of this syndrome contributes to an absence of specific treatment. Several preclinical studies in murine models of septic shock have shown improvements to organ injury and survival after administration of mesenchymal stem cells (MSCs). To better mimic a clinical approach in humans, we investigated the impact of randomized controlled double-blind administration of clinical-grade umbilical cord-derived MSCs to a relevant pig model of septic shock.MethodsSeptic shock was induced by fecal peritonitis in 12 male domestic pigs. Animals were resuscitated by an experienced intensivist including fluid administration and vasopressors. Four hours after the induction of peritonitis, pigs were randomized to receive intravenous injection of thawed umbilical cord-derived MSCs (UCMSC) (1 × 106 UCMSCs/kg diluted in 75 mL hydroxyethyl starch (HES), (n = 6) or placebo (HES alone, n = 6). Researchers were double-blinded to the treatment administered. Hemodynamic parameters were continuously recorded. Gas exchange, acid-base status, organ function, and plasma cytokine concentrations were assessed at regular intervals until 24 h after the onset of peritonitis when animals were sacrificed under anesthesia.ResultsPeritonitis induced profound hypotension, hyperlactatemia, and multiple organ failure. These disorders were significantly attenuated when animals were treated with UCMSCs. In particular, cardiovascular failure was attenuated, as attested by a better mean arterial pressure and reduced lactatemia, despite lower norepinephrine requirements. As such, UCMSCs improved survival in this very severe model (60% survival vs. 0% at 24 h).ConclusionUCMSCs administration is beneficial in this pig model of polymicrobial septic shock.

Xiphoid Surface Temperature Predicts Mortality in a Murine Model of Septic Shock.

Sepsis continues to be a major challenge for modern medicine. Several preclinical models were developed to study sepsis and each has strengths and weaknesses. The cecal slurry (CS) method is a practical alternative because it does not require surgery, and the infection can be dosed. However, one disadvantage is that the dosage must be determined for each CS preparation using survival studies. Our aim was to refine a survival protocol for the CS model by determining a premonitory humane endpoint that would reduce animal suffering. Mice become hypothermic in sepsis; therefore, we tested whether reductions in surface temperature (Ts), measured by noninvasive infrared thermometry, could predict eventual death. We injected 154 C57BL/6J mice with CS (0.9-1.8 mg/g) and periodically monitored Ts at the xiphoid process over 5 days. We used, as predictors, combinations of temperature thresholds (29°C -31°C) and times, postinjection (18-36 h). A receiver-operator curve, sensitivity, and specificity were determined. A Distress Index value was calculated for the threshold conditions. The optimum detection threshold (highest Youden index) was found at Ts ≤ 30.5°C at 24 h (90% specific, 84% sensitive). This threshold condition reduced animal suffering by 41% while providing an accurate survival rate estimate. Using this threshold, only 13 of 154 mice would have died from sepsis; 67 would have been euthanized at 24 h, and only 7 of 154 would have been euthanized unnecessarily. In conclusion, using a humane endpoint of Ts ≤ 30.5°C at 24 h accurately predicts mortality and can effectively reduce animal suffering during CS survival protocols.

Dose and timing of normal saline resuscitation on endothelial glycocalyx in early septic shock

To observe the effect of different doses and timing of normal saline (NS) resuscitation combined with norepinephrine (NE) on endothelial glycocalyx in rabbits with early septic shock. Thirty New Zealand male rabbits were randomly divided into sham group, model group, 30 mL and 60 mL timely resuscitation groups (30 mL and 60 mL timely group), and 30 mL delayed resuscitation group (30 mL delayed group) with 6 rabbits in each group. The rabbit model of septic shock was reproduced by cecal ligation and puncture (CLP). The rabbits in sham group were only received abdominal cavity open without cecal and ligation. The rabbits in 30 mL and 60 mL timely groups and 30 mL delayed group were intravenous infused with 30 mL/kg or 60 mL/kg NS immediately or 1 hour after model reproduction for 1 hour, and the mean arterial pressure (MAP) was maintained over 75 mmHg (1 mmHg = 0.133 kPa) compared with intravenous pumping of 0.02-0.05 μg×kg-1×min-1 NE followed by 5 mL/h NS infusion till the end of the experiment. The rabbits in sham and model groups were only given 5 mL/h NS. The changes in arterial blood gas before and immediately after resuscitation were observed in three fluid resuscitation groups. The internal jugular vein blood was collected at 0, 3, 6 hours after model reproduction. The levels of syndecan-1 (polysaccharide envelope marker) in plasma were determined by enzyme linked immunosorbent assay (ELISA). The rabbits were sacrificed at 6 hours after model reproduction, and the lung tissue was harvested. Western Blot was used to determine the protein expressions of intercellular adhesion molecule-1 (ICAM-1), matrix metalloproteinase 2 (MMP-2) and syndecan-1. The positive expression of syndecan-1 in lung tissue was observed by immunohistochemical method. (1) Blood gas analysis: compared with the results before resuscitation, the levels of lactic acid (Lac) after resuscitation in three fluid resuscitation groups were significantly decreased, especially in 30 mL timely group; the central venous blood oxygen saturation (ScvO2) was significantly increased, especially in 30 mL delayed group. Oxygenation index (PaO2/FiO2) was improved in 30 mL timely and 30 mL delayed resuscitation groups, which was decreased in 60 mL delayed group. (2) Plasma marker: compared with sham group, plasma syndecan-1 level in model group was significantly increased with a time-dependent manner. Plasma syndecan-1 levels at 3 hours in 30 mL timely and 30 mL delayed groups were significantly decreased as compared with those of model group (ng/L: 138.0±2.4, 139.7±15.7 vs. 161.5±4.1, both P < 0.05), but it was significantly increased at 6 hours in 30 mL delayed group (ng/L: 213.1±19.4 vs. 206.4±15.5, P < 0.05). The plasma syndecan-1 levels at 3 hours and 6 hours in 60 mL timely group were significantly higher than those in model group (ng/L: 233.0±28.9 vs. 161.5±4.1, 252.3±27.2 vs. 206.4±15.5, both P < 0.05). (3) Protein expression in lung tissue: compared with sham group, the protein expressions of ICAM-1 and MMP-2 in lung tissue of model group were significantly increased, and syndecan-1 protein expression was significantly decreased. After 30 mL timely or 30 mL delayed resuscitation, the protein expressions of ICAM-1 and MMP-2 in lung tissue were significantly decreased, and syndecan-1 protein expression was significantly increased, especially in 30 mL timely group, which showed statistical differences as compared with those of model group (ICAM-1 protein: 0.56±0.09 vs. 1.04±0.05, MMP-2 protein: 0.83±0.15 vs. 1.06±0.06, syndecan-1 protein: 2.09±0.08 vs. 0.99±0.03, all P < 0.05). The change tendency of protein expressions in 60 mL timely group was opposite to the other two resuscitation groups. (4) Immunohistochemistry: the positive expression of syndecan-1 in lung tissues was significant in the sham group, and it was lowered in model group. The positive expression of syndecan-1 was increased after 30 mL timely or 30 mL delayed resuscitation, but further weakened in 60 mL timely group. The dose and timing of resuscitation with NS in septic shock can affect pulmonary vascular endothelial glycocalyx function. The timely resuscitation with 30 mL NS in combination with NE plays a protective effect on endothelial cell and glycocalyx. However, NS resuscitation which was not timely or excessive infusion can make the glycocalyx degradation more obvious, resulting in increased endothelial permeability, microcirculation damaged, thus aggravate lung injury.

Group 2 Innate Lymphoid Cells (ILC2s) Are Key Mediators of the Inflammatory Response in Polymicrobial Sepsis

Sepsis remains a major public health concern, characterized by marked immune dysfunction. Innate lymphoid cells develop from a common lymphoid precursor but have a role in orchestrating inflammation during innate response to infection. Here, we investigate the pathologic contribution of the group 2 innate lymphoid cells (ILC2s) in a murine model of acute septic shock (cecal ligation and puncture). Flow cytometric data revealed that ILC2s increase in number and percentage in the small intestine and in the peritoneal cells and inversely decline in the liver at 24 hours after septic insult. Sepsis also resulted in changes in ILC2 effector cytokine (IL-13) and activating cytokine (IL-33) in the plasma of mice and human patients in septic shock. Of interest, the sepsis-induced changes in cytokines were abrogated in mice deficient in functionally invariant natural killer T cells. Mice deficient in IL-13-producing cells, including ILC2s, had a survival advantage after sepsis along with decreased morphologic evidence of tissue injury and reduced IL-10 levels in the peritoneal fluid. Administration of a suppressor of tumorigenicity 2 (IL-33R) receptor-blocking antibody led to a transient survival advantage. Taken together, these findings suggest that ILC2s may play an unappreciated role in mediating the inflammatory response in both mice and humans; further, modulating ILC2 response invivo may allow development of immunomodulatory strategies directed against sepsis.

Cellular Mechanisms of Myocardial Depression in Porcine Septic Shock.

The complex pathogenesis of sepsis and septic shock involves myocardial depression, the pathophysiology of which, however, remains unclear. In this study, cellular mechanisms of myocardial depression were addressed in a clinically relevant, large animal (porcine) model of sepsis and septic shock. Sepsis was induced by fecal peritonitis in eight anesthetized, mechanically ventilated, and instrumented pigs of both sexes and continued for 24 h. In eight control pigs, an identical experiment but without sepsis induction was performed. In vitro analysis of cardiac function included measurements of action potentials and contractions in the right ventricle trabeculae, measurements of sarcomeric contractions, calcium transients and calcium current in isolated cardiac myocytes, and analysis of mitochondrial respiration by ultrasensitive oxygraphy. Increased values of modified sequential organ failure assessment score and serum lactate levels documented the development of sepsis/septic shock, accompanied by hyperdynamic circulation with high heart rate, increased cardiac output, peripheral vasodilation, and decreased stroke volume. In septic trabeculae, action potential duration was shortened and contraction force reduced. In septic cardiac myocytes, sarcomeric contractions, calcium transients, and L-type calcium current were all suppressed. Similar relaxation trajectory of the intracellular calcium-cell length phase-plane diagram indicated unchanged calcium responsiveness of myofilaments. Mitochondrial respiration was diminished through inhibition of Complex II and Complex IV. Defective calcium handling with reduced calcium current and transients, together with inhibition of mitochondrial respiration, appears to represent the dominant cellular mechanisms of myocardial depression in porcine septic shock.

Clinical and bacteriological features and prognosis of ascitic fluid infection in Chinese patients with cirrhosis

BackgroundSpontaneous bacterial peritonitis (SBP) and bacterascites (BA) represent frequent and serious complications in cirrhosis patients with ascites. However, few detailed data are available regarding the clinical and bacteriological feature of SBP or BA patients in China.MethodsWe retrospectively analyzed bacteriological and clinical characteristics of patients with SBP and BA at Beijing 302 Hospital in China from January 2012 to December 2015.ResultsA total of 600 patients with SBP (n = 408) or BA (n = 192) were enrolled. Patients with BA appeared to have a less severe clinical manifestation and lower mortality rate than patients with SBP. Gram-negative bacteria formed the majority of pathogens in SBP (73.9%) and BA (55.8%) cases. Higher ascitic fluid polymorphonuclear leucocytes (PMN) count and hepatocellular carcinoma were independent risk factors for BA episode progressing to SBP. The concentration of blood urea nitrogen (BUN) was independent risk factor for 30-day mortality of BA patients. For patients with SBP, the independent risk factors for 30-day mortality were age, Model for End-Stage Liver Disease (MELD) score, septic shock and hepatocellular carcinoma. Patients with third-generation cephalosporin or carbapenems resistant infection had a significantly lower survival probability. There were significant differences in clinical characteristics and outcome among the major bacteria. Multivariate analysis showed that patients infected with Klebsiella spp. had higher hazard ratio of 30-day mortality.ConclusionOur study reported the bacteriological and clinical characteristics of patients with SBP and BA. Higher ascitic fluid PMN count and hepatocellular carcinoma were found to be independent risk factors for BA episode progressed to SBP. Outcome of ascitic fluid infection in patients with cirrhosis was influenced by the type of bacteria and antimicrobial susceptibility.

Effects of resuscitation with human albumin 5%, hydroxyethyl starch 130/0.4 6%, or crystalloid on kidney damage in an ovine model of septic shock

BackgroundColloid solutions have been associated with kidney dysfunction in septic animals and humans. The present study investigated the influence of resuscitation with human albumin (HA) 5%, hydroxyethyl starch (HES) 130/0.4 6%, and balanced crystalloids on ultrastructural kidney damage, kidney function, and survival in a model of ovine septic shock. MethodsAfter induction of peritoneal septic shock, animals were randomised to one of the following groups: (1) HA 5%, (2) HES 130/0.4 6%, (3) balanced crystalloid, and (4) control (each n=10). Causal therapy included re-laparotomy, peritoneal lavage, and antimicrobial therapy. Sequential kidney biopsies were obtained for the assessment of the electron microscopic tubular injury (EMTI) score. ResultsSerum creatinine and urea were highest in the control group, and there were no differences between the intervention groups. Cumulative diuresis was significantly higher in the HA group [1.0 ml kg−1 h−1 (0.6; 1.2)] compared with control [0.7 ml kg−1 h−1 (0.6; 0.9), P<0.05]. Creatinine clearance was highest in the HA and crystalloid groups. Ultrastructural kidney damage was highest in the control group [EMTI score 7.8 (6.7; 9.0)] without differences between intervention groups. Survival was 100% in the colloid groups vs 90% (crystalloid) and 60% (control, all P<0.05). ConclusionIn an ovine model of septic shock, kidney function and cumulative diuresis were preserved in the 5% albumin and crystalloid resuscitation groups, whereas HES 130/0.4 6% resulted in diminished creatinine clearance. Differences in kidney function between resuscitation fluids could not be explained by differences in ultrastructural kidney damage. Clinical trial registration84-02.04.2011.A300.

Comparison of first-line and second-line terlipressin versus sole norepinephrine in fulminant ovine septic shock

The Surviving Sepsis Guidelines suggest the use of vasopressin in case of catecholamine-refractory septic shock. Terlipressin (TP) as a V1-selective AVP analogue is a potential alternative, though data regarding the first-line administration in septic shock are scarce. The present study explored and compared the effects of first-line vs. second-line infusion of TP or sole norepinephrine regarding organ function, fluid and norepinephrine requirements and survival in fulminant ovine septic shock. Peritoneal sepsis was induced in 23 ewes after laparotomy and faecal withdrawal from the caecum. After onset of shock, causal and supportive sepsis therapy (antibiotics, peritoneal lavage, fluids and open-label norepinephrine) was performed in all animals. Concurrently, animals were randomized to receive 0.9% sodium chloride (control group) or TP (2 µg∙kg−1∙h−1, first-line group) after shock onset. In the second-line TP group, TP (2 µg∙kg−1∙h−1) was started once norepinephrine requirements exceeded 0.5 µg∙kg−1∙min−1. No significant differences were found between groups regarding survival, haemodynamics as well as fluid- and catecholamine-requirements. Kidney function and electron microscopic kidney injury were comparable between groups. In the present model of fulminant ovine septic shock, first-line TP infusion had no significant effect on fluid and norepinephrine requirements or organ dysfunction as compared to second-line TP infusion or placebo.

Midazolam increases preload dependency during endotoxic shock in rabbits by affecting venous vascular tone

BackgroundSeptic patients often require sedation in intensive care unit, and midazolam is one of the most frequently used sedatives among them. But the interaction between midazolam and septic shock is not known. The aim of this study is to investigate the effects of midazolam on preload dependency in an endotoxic shock model by evaluating systemic vascular tone and cardiac function.MethodsEighteen rabbits were randomly divided into three groups: Control group, MID1 group and MID2 group. Rabbits underwent ketamine anaesthesia and mechanical ventilation, and haemodynamic assessments were recorded in three groups (T0). Endotoxic shock was induced by lipopolysaccharide intravenously, and fluid resuscitation and norepinephrine were administered to obtain the baseline mean arterial pressure (MAP) (T1). Rabbits received equivalent normal saline (Control) and two consecutive dosages of midazolam: 0.3 mg kg−1 h−1 (MID1) and 3 mg kg−1 h−1 (MID2) (T2). Rabbits received another round of fluid challenge and norepinephrine infusion to return the MAP to normal (T3).ResultsNo significant differences in haemodynamic parameters were observed in three groups at T0, T1 or T3. Midazolam infusion significantly increased pulse pressure variation (PPV) and stroke volume variation (SVV) compared to the values in Control group, and MAP, central venous pressure (CVP), mean systemic filling pressure (Pmsf) and cardiac output (CO) decreased at T2. Same effects were observed with increasing doses of midazolam, and resistance for venous return (Rvr) decreased (MID1 vs. MID2) at T2. PPV and SVV increased significantly at T2 compared to the values at T1. MAP, CVP, Pmsf and CO decreased in MID1 and MID2 groups. Rvr also decreased in MID2 group (T2 vs. T1). Midazolam did not affect cardiac function index, systemic vascular resistance or artery resistance (T2 vs. T1).ConclusionsMidazolam administration promoted preload dependency in septic shock models via decreased venous vascular tone without affecting cardiac function.

Is Heparin-Binding Protein Inhibition a Mechanism of Albumin's Efficacy in Human Septic Shock?

Our objectives were to determine first whether albumin prevents heparin-binding protein-induced increased endothelial cell permeability and renal cell inflammation and second, whether a plasma heparin-binding protein-to-albumin ratio predicts risk of acute kidney injury, fluid balance, and plasma cytokine levels in septic shock. In vitro human endothelial and renal cell model and observation cohort of septic shock. Research laboratory and multicenter clinical trial (Vasopressin and Septic Shock Trial). Adult septic shock (norepinephrine dose > 5 μg/min for > 6 hr). In vitro: heparin-binding protein (or thrombin) was added with or without albumin to 1) human endothelial cell monolayers to assess permeability and 2) to human renal tubular epithelial cells to assess inflammation. Transendothelial electrical resistance-a marker of permeability-of human endothelial cells was measured using a voltohmmeter. We measured plasma heparin-binding protein-to-albumin ratio and a panel of cytokines in septic shock patients (n = 330) to define an heparin-binding protein-to-albumin ratio that predicts risk of acute kidney injury. Albumin inhibited heparin-binding protein (and thrombin-induced) increased endothelial cell permeability at a threshold concentration of 20-30 g/L but increased renal tubular cell interleukin-6 release. Patients who developed or had worsened acute kidney injury had significantly higher heparin-binding protein-to-albumin ratio (1.6 vs 0.89; p < 0.001) and heparin-binding protein (38.2 vs 20.8 ng/mL; p < 0.001) than patients without acute kidney injury. The highest heparin-binding protein-to-albumin ratio (> 3.05), heparin-binding protein quartiles (> 69.8), and heparin-binding protein > 30 ng/mL were significantly associated with development or worsening of acute kidney injury (p < 0.001) in unadjusted and adjusted analyses and were robust to sensitivity analyses for death as a competing outcome. Heparin-binding protein and heparin-binding protein-to-albumin ratio were directly associated with positive fluid balance (p < 0.001) and with key inflammatory cytokines. Increasing quartiles of heparin-binding protein-to-albumin ratio and heparin-binding protein (but not albumin) were highly significantly associated with days alive and free of acute kidney injury and renal replacement therapy (p < 0.001), vasopressors (p < 0.001), ventilation (p < 0.001), and with 28-day mortality. Albumin inhibits heparin-binding protein-induced increased human endothelial cell permeability and heparin-binding protein greater than 30 ng/mL and heparin-binding protein-to-albumin ratio greater than 3.01-but not serum albumin-identified patients at increased risk for acute kidney injury in septic shock.

Pyruvate-enriched resuscitation for shock.

This commentary addresses the recent retraction of an article which reported favorable outcomes in septic patients treated with intravenous pyruvate. The retracted report was cited in the authors' recent minireview on the cellular mechanisms and clinical application of pyruvate to improve cardiac performance. Because the retracted article reports pyruvate-enhanced resuscitation of critically ill patients, the authors wish to inform the readership, especially critical care providers, that this particular clinical application of pyruvate is not now supported by robust evidence. After discussing the retraction's implications for the clinical application of pyruvate-enriched resuscitation for sepsis, this commentary summarizes the extensive preclinical evidence of the efficacy and mechanisms of pyruvate resuscitation in animal models of hemorrhagic and septic shock, which argues for renewed clinical investigation of pyruvate-enriched resuscitation. Impact statement This commentary addresses the recent retraction of a clinical report of significant benefits of intravenous pyruvate resuscitation in septic patients, including sharply lowered mortality and decreased circulating pro-inflammatory cytokines, which was cited in the authors' minireview in Experimental Biology and Medicine. The potential implications of the retraction, and the extensive preclinical evidence supporting the use of pyruvate-enriched resuscitation for shock states, are summarized and discussed.

MicroRNA-326 aggravates acute lung injury in septic shock by mediating the NF-κB signaling pathway.

Our previous studies have demonstrated that the activation of the nuclear factor-kappa B (NF-κB) signaling pathway contributes to the development of lipopolysaccharide (LPS)-induced acute lung injury (ALI) as well as an inflammatory reaction, and its inhibition may provide future therapeutic values. Thereby, this study aims to explore the effects of miR-326 on inflammatory response and ALI in mice with septic shock via the NF-κB signaling pathway. The study included normal mice and LPS-induced mouse models of septic shock with ALI. Modeled mice were transfected with the blank plasmid, miR-326 mimic, miR-326 inhibitor, si-BCL2A1 and miR-326 inhibitor + si-BCL2A1. Mean arterial pressure (MAP), airway pressure (AP), heart rate (HR) and lung wet dry (W/D) ratio were determined. Serum levels of interleukin (IL)-6, IL-10, IL-1β, and tumor necrosis factor-α (TNF-α) were detected using ELISA. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were performed to detect the miR-326 expression and expression levels of BCL2A1, related genes of inflammatory response and the NF-κB signaling pathway in lung tissues. Cell viability and apoptosis were measured using the CCK-8 assay and flow cytometry, respectively. Compared to the ALI models and those transfected with blank plasmid, the up-regulated miR-326 expression and silenced BCL2A1 lead to decreased levels of MAP, increased AP, HR and lung W/D, increased serum levels of IL-6, IL-10, IL-1β and TNF-α, increased expressions of IL-6, IL-1β, TNF-α, NF-κB p65 (p-NF-κB p65), and iNOS with decreased expressions of BCL2A1s as well as inhibition of cell viability and enhanced cell apoptosis; the down-regulated miR-326 expression reversed the aforementioned situation. MiR-326 targeting the BCL2A1 gene activated the NF-κB signaling pathway, resulting in aggravated inflammatory response and lung injury of septic shock with ALI in mice.

Inducible nitric oxide synthase during the late phase of sepsis is associated with hypothermia and immune cell migration

Hypothermia is a significant sign of sepsis, which is associated with poor prognosis, but few mechanisms underlying the regulation of hypothermia are known. Inducible nitric oxide synthase (iNOS) is a key inflammatory mediator of sepsis. However, the therapeutic benefit of iNOS inhibition in sepsis is still controversial, and requires elucidation in an accurate model system. In this study, wild-type (WT) mice showed temperature drops in a biphasic manner at the early and late phase of sepsis, and all mice died within 48 h of sepsis. In contrast, iNOS-knockout (KO) mice never showed the second temperature drop and exhibited improved mortality. Plasma nitric oxide (NO) levels of WT mice increased in the late phase of sepsis and correlated to hypothermia. The results indicate that iNOS-derived NO during the late phase of sepsis caused vasodilation-induced hypothermia and a lethal hypodynamic state. The expression of the iNOS mRNA was high in the lung of WT mice with sepsis, which reflects the pathology of acute respiratory distress syndrome (ARDS). We obtained the results in a modified keyhole-type cecal ligation and puncture model of septic shock induced by minimally invasive surgery. In this accurate and reproducible model system, we transplanted the bone marrow cells of GFP transgenic mice into WT and iNOS-KO mice, and evaluated the role of increased pulmonary iNOS expression in cell migration during the late phase of sepsis. We also investigated the quantity and type of bone marrow-derived cells (BMDCs) in the lung. The number of BMDCs in the lung of iNOS-KO mice was less than that in the lung of WT mice. The major BMDCs populations were CD11b-positive, iNOS-negative cells in WT mice, and Gr-1-positive cells in iNOS-KO mice that expressed iNOS. These results suggest that sustained hypothermia may be a beneficial guide for future iNOS-targeted therapy of sepsis, and that iNOS modulated the migratory efficiency and cell type of BMDCs in septic ARDS.

Revisiting the role of IRF3 in inflammation and immunity by conditional and specifically targeted gene ablation in mice

IFN regulatory factor 3 (IRF3) is a transcription regulator of cellular responses in many cell types that is known to be essential for innate immunity. To confirm IRF3's broad role in immunity and to more fully discern its role in various cellular subsets, we engineered Irf3-floxed mice to allow for the cell type-specific ablation of Irf3 Analysis of these mice confirmed the general requirement of IRF3 for the evocation of type I IFN responses in vitro and in vivo. Furthermore, immune cell ontogeny and frequencies of immune cell types were unaffected when Irf3 was selectively inactivated in either T cells or B cells in the mice. Interestingly, in a model of lipopolysaccharide-induced septic shock, selective Irf3 deficiency in myeloid cells led to reduced levels of type I IFN in the sera and increased survival of these mice, indicating the myeloid-specific, pathogenic role of the Toll-like receptor 4-IRF3 type I IFN axis in this model of sepsis. Thus, Irf3-floxed mice can serve as useful tool for further exploring the cell type-specific functions of this transcription factor.

Recombinant Fasciola hepatica Fatty Acid Binding Protein Downregulates Inflammatory Cytokines/Chemokines in a Mouse Model of Septic Shock and Shows a Wide Spectrum of Action

Sepsis is the leading cause of death from infections. It involves, both pathogen and host factors and it is characterized by an exacerbation of inflammatory cytokines and chemokines and organ dysfunction and failure. Recently, we reported that a recombinant Fasciola hepatica fatty acid binding protein, termed Fh15, is capable of suppressing the LPS induced expression of inflammatory cytokines such as IL‐1β and TNFα in murine bone marrow derived macrophages in vitro. We also demonstrated that such anti‐inflammatory effect is achieved by targeting specifically the CD14 co‐receptor, which results in a complete suppression of TLR4 activation and makes macrophages irresponsive to subsequent LPS‐stimuli. In the current study, we evaluated the capacity of Fh15 to suppress the TLR4‐activation in vitro, as well as its capacity to therapeutically suppress the cytokine storm in a mouse model of septic shock. We also tested the ability of Fh15 to suppress the activation of multiple TLRs in response to Gram‐positive and Gram‐negative whole bacteria extracts in vitro. For in vivo studies, we used female BALB/c mice that received a lethal intraperitoneal (i.p.) injection of LPS‐E. coli and 1h later received a single i.p. injection with 50μg Fh15. Animals were sacrificed 12h after the LPS challenge. For in vitro experiments, we used THP1‐CD14 cells, which were cultured with Fh15 prior to stimulation with optimized concentrations of 1×108 cells of Klebsiella pneumoniae and Enterococcus faecalis. Results demonstrated that, although Fh15 was unable to stimulate the secretion of any inflammatory cytokine, it significantly suppressed the levels of serum IFNγ (p=0.0425), TNFα (p=0.0008), IL‐1β (p=0.0021) in all animals exposed to lethal doses of LPS. Fh15 also significantly suppressed the levels in serum of two macrophage inflammatory proteins MIP‐1α (p=0.0027) and MIP‐1β (p&lt;0.0001), two chemotactic chemokines that play relevant roles in the inflammatory process. Moreover, we also demonstrated that Fh15 inhibits the stimulation of various TLRs in response to whole bacteria extracts, suggesting that Fh15 could have a broad spectrum of action. These results support the possibility of using Fh15 as an excellent alternative for an anti‐inflammatory drug in pre‐clinical studies in the near future.Support or Funding InformationThis study was supported by MBRS‐RISE R25GM061838‐13, and NIH grants G12MD007600 and 2P40OD012217.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Skeletal Muscle Produces Acute Phase Proteins in Response to Polymicrobial Sepsis

Skeletal muscle has been implicated as an endocrine organ, as observed by the secretion of cytokines in response to multiple stimuli such as exercise, heat stress and infection. However, whether skeletal muscles can produce acute phase proteins (APPs) in response to polymicrobial sepsis is controversial. This is relevant since APPs play an important role in the transition from innate to adaptive immunity and, thus far, the liver has been considered the primary source of these proteins. Our aim was to determine whether skeletal muscles are capable of producing APPs in response to polymicrobial sepsis.METHODSWe injected (IP) 37 female C57BL/6J mice with cecal slurry (1.6 g/ml) and harvested the soleus, gastrocnemius and livers at 6, 12, and 24 h after the injection. Respective muscles and livers from non‐septic mice were used as controls. Specimens were then homogenized and analyzed for protein content. Immunoblot assays were then conducted using polyclonal antibodies for, serum amyloid a1 (SAA1), fibrinogen, lipocalin, and c‐reactive protein (CRP). For quantification, membranes were scanned for fluorescence intensity and normalized for total protein.RESULTSWe observed elevated expression of fibrinogen, lipocalin and SAA1 in the livers of septic mice across all time points in comparison to non‐septic mice. We observed an increased signal for SAA1 and fibrinogen in skeletal muscle of septic mice. Notably, the signal intensity was higher in soleus in comparison to gastrocnemius muscle, which suggests a muscle‐specific response. We did not observe any signal for lipocalin, regardless of the muscle type. We also detected the presence of CRP in the soleus muscles of septic mice. Our results suggest that the cecal slurry model of septic shock induces an acute phase response in both liver and skeletal muscle. Though skeletal muscles produce a smaller spectrum of known APPs, these may contribute to the transition from innate to adaptive immunity, and the overall response to severe bacterial infection.Support or Funding InformationSupported by NIH RO1GM118895‐01 and the BK and Betty Stevens Endowment.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Role of baroreflex during endotoxemic shock

Septic shock is a prevalent and life‐threatening condition which is often associated with major hypotension, resulting in multiple organs failure. The infusion of high doses of endotoxin lipopolysaccharide (LPS) is a well‐accepted experimental model of septic shock. Considering that baroreflex is a mandatory mechanism involved in the beat‐to‐beat hemodynamic regulation, we hypothesized herein that baroreflex plays a role buffering endotoxin‐induced hypothension in conscious freely moving rats. Thus, in the present study, we evaluated the cardiovascular outcomes of iv injection of endotoxin (1.5 mg.kg−1 or 2.5 mg.kg−1) or saline in male Wistar rats (280–350g, CEUA #2017.1.585.58.9). The baseline levels of mean arterial pressure and heart rate were not different among all groups. As expected, endotoxin induced hypotension (P&lt;0.001) and tachycardia (P &lt; 0.001). These effects were independent of dose. Interestingly, the drop of mean arterial pressure was significantly higher in rats previously submitted to baroreceptors afferents removal (4 days before recordings) than in (sham) rats with intact baroreceptor afferents (P&lt;0.001). The present data are consistent with the notion that baroreflex is key lessening endotoxin‐induced hypothension. The neural mechanisms involved in this control deserve further investigation aiming new strategic therapeutic tools to revert this critical condition.Support or Funding InformationFinancial support: FAPESP, CAPES and CNPqThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

O-GlcNAc stimulation is beneficial in septic shock models

Septic shock is a worldwide burden, without efficient treatments, killing one person every 3 s. Recently, O-GlcNAc stimulation (a post-translational modification) appeared to be beneficial in acute pathologies. This study has been designed to evaluate if O-GlcNAc stimulation shows the same benefits in two relevant models of septic shock. Impact of NButGT (an OGlcNAcase inhibitor – 10 mg/kg) has been evaluated in LPS or CLP (caecal ligation and puncture) and their respective controls with or without fluidotherapy (FR). Cardiovascular function (mean arterial blood pressure (MAP), echocardiography) and biochemical markers (creatinine, lactates) were measured at 3 h and 24 h after shock induction respectively, then heart and blood were harvested to evaluate impact of the treatment. Also, O-GlcNAc impact on calcium actor was evaluated by western blot. LPS study was completed with a Kaplan–Meier mortality evaluation. NButGT treatments efficiently increased cardiac O-GlcNAc in both model (+300% vs. Ctrl or Sham). In both group, NButGT treatment induced an amelioration of the outcome and organs function specifically: cardiac (heart rate −9% vs. LPS; −10% vs. CLP), renal (creatinine −35% vs. LPS; −27% vs. CLP) and beneficial impact on blood pressure (MAP + 10% vs. CLP). Treatment had a significant impact on lactate in LPS group (−36% vs. LPS) consistent with a 3 times higher survival rate. Treatment impacted on SERCA2a protein (−150% vs. LPS, NS vs. ctrl) without modification of its regulator (phospholamban) or ryanodine receptor. Our results support the fact that acute O-GlcNAc stimulation is a potential new therapeutic target at the early phase of septic shock. Potentially through improvement of cardiomyocytes’ calcium handling. We are currently validating our approach at different dose and age to be able to transfer our results to the clinic.

The Trend of β3-Adrenergic Receptor in the Development of Septic Myocardial Depression: A Lipopolysaccharide-Induced Rat Septic Shock Model

Septic shock with low cardiac output is very common in children. However, the mechanism underlying myocardial depression is unclear. The role of β₃-AR in the development of myocardial depression in sepsis is unknown. In the present study, we generated an adolescent rat model of hypodynamic septic shock induced by lipopolysaccharide (LPS). Neonatal cardiomyocytes were also treated with LPS to mimic myocardial depression in sepsis, which was confirmed via an in vivo left ventricular hemodynamic study, and measurements of contractility and the Ca²⁺ transient in isolated adolescent and neonatal cardiomyocytes. After 16 h of LPS treatment, cultured neonatal cardiomyocytes showed a diminished Ca²⁺ transient amplitude associated with an increase in the β₃-AR level. With the addition of a β₃-AR agonist, the Ca²⁺ transient in LPS-treated neonatal rat cardiomyocytes gradually decreased over time; such a change was absent in cells treated with nitric oxide synthase (NOS) inhibitors prior to treatment with a β₃-AR agonist. In adolescent rats with septic myocardial depression, cardiac function declined as indicated by decreased MAP, dP/dt_max, and dP/dt_mix for 6 h after LPS injection; however, the β₃-AR level first increased 2 h after LPS treatment and then decreased 6 h after LPS treatment in the absence of exogenous catecholamines. The results indicate that, in vitro, at the cellular level β₃-AR may be involved in the development of myocardial depression (Ca²⁺ transient depression) in sepsis through NOS signaling pathways; however, in vivo, a complicated mechanism for modulating β₃-AR may exist.

Low-dose hydrocortisone prolongs survival in a lethal sepsis model in adrenalectomized rats

BackgroundControversial clinical findings of low-dose hydrocortisone supplementation in septic shock led us to investigate the impact of administration in lethal septic shock in adrenalectomized rats. Materials and methodsAfter preliminary experiments, to define the intravenous dose of hydrocortisone delivered in bilaterally adrenalectomized rats with serum cortisol level similar to sham rats, survival experiments were run in 75 rats after intraperitoneal challenge with Escherichia coli. Rats were treated with placebo, ertapenem, hydrocortisone, and a combination. Sacrifice experiments were run to measure gene transcripts in whole blood and in the liver and to assess cytokine stimulation of splenocytes and tissue overgrowth. ResultsThe combination of hydrocortisone and ertapenem was superior to any single treatment and mandatory to achieve survival benefit. Splenocytes from infected rats had decreased production of tumor necrosis factor-alpha (TNFα); this was reversed with hydrocortisone treatment. Hydrocortisone increased the expression of TNF, Il1r2, and Hdac4 and decreased that of Dnmt3a. Bacterial burden of E. coli in kidney was decreased after hydrocortisone treatment. ConclusionsLow dose of hydrocortisone is a mandatory adjunctive to antimicrobial therapy in a rat model of septic shock after bilateral adrenalectomy. The mechanism of action is related to reversal of sepsis-induced immunosuppression through interaction with histone deacetylases and de novo DNA methyltransferases.