Late Stage Of Sepsis Research Articles

Kinin B1 receptor: a potential therapeutic target in sepsis-induced vascular hyperpermeability

BackgroundIn sepsis, the endothelial barrier becomes incompetent, with the leaking of plasma into interstitial tissues. VE-cadherin, an adherens junction protein, is the gatekeeper of endothelial cohesion. Kinins, released during sepsis, induce vascular leakage and vasodilation. They act via two G-protein coupled receptors: B1 (B1R) and B2 (B2R). B1R is inducible in the presence of pro-inflammatory cytokines, endotoxins or after tissue injury. It acts at a later stage of sepsis and elicits a sustained inflammatory response. The aim of our study was to investigate the relationships between B1R and VE-cadherin destabilization in vivo in a later phase of sepsis.MethodsExperimental, prospective study in a university research laboratory. We used a polymicrobial model of septic shock by cecal ligation and puncture in C57BL6 male mice or C57BL6 male mice that received a specific B1R antagonist (R-954). We studied the influence of B1R on sepsis-induced vascular permeability 30 h after surgery for several organs, and VE-cadherin expression in the lung and kidneys by injecting R-954 just before surgery. The 96-h survival was determined in mice without treatment or in animals receiving R-954 as a “prophylactic” regimen (a subcutaneous injection of 200 µg/kg, prior to CLP and 24 h after CLP), or as a “curative” regimen (injection of 100 µg/kg at H6, H24 and H48 post-surgery).ResultsB1R inactivation helps to maintain MAP above 65 mmHg but induces different permeability profiles depending on whether or not organ perfusion is autoregulated. In our model, VE-cadherin was destabilized in vivo during septic shock. At a late stage of sepsis, the B1R blockade reduced the VE-cadherin disruption by limiting eNOS activation. The survival rate for mice that received R-954 after sepsis induction was higher than in animals that received an antagonist as a prophylactic treatment.ConclusionsB1R antagonizing reduced mortality in our model of murine septic shock by limiting the vascular permeability induced by VE-cadherin destabilization through maintenance of the macrohemodynamics, consequently limiting organ dysfunctions.

Subcutaneous administration of \u03b2-hydroxybutyrate improves learning and memory of sepsis surviving mice

Post-sepsis cognitive impairment is one of the major sequelae in sepsis survivors. Its prevention remains clinically challenging. Here we tested the effects and underlying mechanisms of exogenous β-hydroxybutyrate (BHB) on post-sepsis cognitive impairment. We found that subcutaneous administration of BHB increased survival and body weight recovery of sepsis mice and improved learning and memory of sepsis surviving mice in a cecal ligation and perforation-induced sepsis model. Additionally, the improvement of learning and memory of sepsis surviving mice was still detected even if BHB was administrated at the late stage of sepsis. In contrast, glucose solution did not show similar effects. Mechanistically, subcutaneous administration of BHB increased the BHB level of hippocampus, and limited neuroinflammation and neuroplasticity damage in sepsis mice. Intracerebroventricular administration of BHB also alleviated neuroinflammation and cognitive impairment of sepsis surviving mice. In the coculture of neurons, astrocytes, and BV2 cells (a microglial cell line), knocking down the expression of microglial HCA2 (BHB receptor) via a specific shRNA reduced the protection of BHB to lipopolysaccharide-induced inflammatory response and neuron damage more significantly than knocking down neuronal MCT2 (BHB transporter). These data showed that (1) BHB was a potential pharmacological adjunct treatment for prevention of post-sepsis cognitive impairment and (2) inhibiting neuroinflammation via HCA2 was an important mechanism.

Serum Levels of Mitochondrial and Microbial Metabolites Reflect Mitochondrial Dysfunction in Different Stages of Sepsis

Mechanisms of mitochondrial dysfunction in sepsis are being extensively studied in recent years. During our study, concentrations of microbial phenolic acids and mitochondrial metabolites (succinic, α-ketoglutaric, fumaric, itaconic acids) as indicators of sepsis and mitochondrial dysfunction, respectively, are measured by gas chromatography–mass spectrometry (GC–MS) in the blood of critically ill patients at the early and late stages of documented sepsis. The increase in levels of some phenylcarboxylic (phenyllactic (PhLA), p-hydroxyphenylacetic (p-HPhAA), p-hydroxyphenyllactic (p-HPhAA)) acids (PhCAs), simultaneously with a rise in levels of mitochondrial dicarboxylic acids, are mainly detected during the late stage of sepsis, especially succinic acid (up to 100–1000 µM). Itaconic acid is found in low concentrations (0.5–2.3 µM) only at early-stage sepsis. PhCAs in vitro inhibits succinate dehydrogenase (SDH) in isolated mitochondria but, unlike itaconic acid which acts as a competitive inhibitor of SDH, microbial metabolites most likely act on the ubiquinone binding site of the respiratory chain. A close correlation of the level of succinic acid in serum and sepsis-induced organ dysfunction is revealed, moreover the most significant correlation is observed at high concentrations of phenolic microbial metabolites (PhCAs) in late-stage sepsis. These data indicate the promise of such an approach for early detection, monitoring the progression of organ dysfunction and predicting the risk of non-survival in sepsis.

Basic Principles of Antibiotics Dosing in Patients with Sepsis and Acute Kidney Damage Treated with Continuous Venovenous Hemodiafiltration

Abstract Sepsis is the leading cause of acute kidney damage in patients in intensive care units. Pathophysiological mechanisms of the development of acute kidney damage in patients with sepsis may be hemodynamic and non-hemodynamic. Patients with severe sepsis, septic shock and acute kidney damage are treated with continuous venovenous hemodiafiltration. Sepsis, acute kidney damage, and continuous venovenous hemodiafiltration have a significant effect on the pharmacokinetics and pharmacodynamics of antibiotics. The impact dose of antibiotics is increased due to the increased volume of distribution (increased administration of crystalloids, hypoalbuminemia, increased capillary permeability syndrome to-proteins). The dose of antibiotic maintenance depends on renal, non-renal and extracorporeal clearance. In the early stage of sepsis, there is an increased renal clearance of antibiotics, caused by glomerular hyperfiltration, while in the late stage of sepsis, as the consequence of the development of acute renal damage, renal clearance of antibiotics is reduced. The extracorporeal clearance of antibiotics depends on the hydrosolubility and pharmacokinetic characteristics of the antibiotic, but also on the type of continuous dialysis modality, dialysis dose, membrane type, blood flow rate, dialysis flow rate, net filtration rate, and effluent flow rate. Early detection of sepsis and acute kidney damage, early target therapy, early administration of antibiotics at an appropriate dose, and early extracorporeal therapy for kidney replacement and removal of the inflammatory mediators can improve the outcome of patients with sepsis in intensive care units.

Park 7: A Novel Therapeutic Target for Macrophages in Sepsis-Induced Immunosuppression.

Sepsis remains a serious and life-threatening condition with high morbidity and mortality due to uncontrolled inflammation together with immunosuppression with few therapeutic options. Macrophages are recognized to play essential roles throughout all phases of sepsis and affect both immune homeostasis and inflammatory processes, and macrophage dysfunction is considered to be one of the major causes for sepsis-induced immunosuppression. Currently, Parkinson disease protein 7 (Park 7) is known to play an important role in regulating the production of reactive oxygen species (ROS) through interaction with p47phox, a subunit of NADPH oxidase. ROS are key mediators in initiating toll-like receptor (TLR) signaling pathways to activate macrophages. Emerging evidence has strongly implicated Park 7 as an antagonist for sepsis-induced immunosuppression, which suggests that Park 7 may be a novel therapeutic target for reversing immunosuppression compromised by sepsis. Here, we review the main characteristics of sepsis-induced immunosuppression caused by macrophages and provide a detailed mechanism for how Park 7 antagonizes sepsis-induced immunosuppression initiated by the macrophage inflammatory response. Finally, we further discuss the most promising approach to develop innovative drugs that target Park 7 in patients whose initial presentation is at the late stage of sepsis.

Glucose homeostasis in two degrees of sepsis lethality induced by caecum ligation and puncture in mice.

Sepsis is associated with high mortality. Both critically ill humans and animal models of sepsis exhibit changes in their glucose homeostasis, that is, hypoglycaemia, with the progression of infection. However, the relationship between basal glycaemia, glucose tolerance and insulin sensitivity is not well understood. Thus, we aimed to evaluate this glucose homeostasis triad at the late stage of sepsis (24h after surgery) in male Swiss mice subjected to lethal and sublethal sepsis by the caecal ligation and puncture (CLP) model. The percentage of survival 24h after CLP procedure in the Lethal and Sublethal groups was around 66% and 100% respectively. Both Lethal and Sublethal groups became hypoglycaemic in fasting and fed states 24h after surgery. The pronounced fed hypoglycaemia in the Lethal group was not due to worsening anorexic behaviour or hepatic inability to deliver glucose in relation to the Sublethal group. Reduction in insulin sensitivity in CLP mice occurred in a lethality-dependent manner and was not associated with glucose intolerance. Analysis of oral and intraperitoneal glucose tolerance tests, as well as the gastrointestinal motility data, indicated that CLP mice had reduced intestinal glucose absorption. Altogether, we suggest cessation of appetite and intestinal glucose malabsorption are key contributors to the hypoglycaemic state observed during experimental severe sepsis.

Progress in understanding of adrenal insufficiency in septic patients

Background Sepsis is one of the critical complications following serious injuries or major surgeries. It is a leading cause of death. The mortality of septic shock exceeds 40%. Accumulating studies have demonstrated that the severity of adrenal cortex dysfunction has a vital impact on the phenotype and prognosis of sepsis, while glucocorticoid(GC) may be an effective intervention. However, the mechanisms underlying adrenocortical dysfunction remain unclear. Objective To review strategies for assessment of adrenocortical function, and timely supplement of GC, aiming to appropriately compensate the impaired adrenal function, finally, to improve prognosis and to reduce mortality of sepsis. Content At the early stage of sepsis, hypothalamus-pituitary-adrenal axis(H-P-A) is activated, and is followed by release of GC and mobilization of inflammation cascades, especially mitogen-activated protein kinase(MAPK) and nuclear regulatory factor-κB pathways. GC counteracts the inflammation cascades. At the late stage of sepsis or during sepsis shock, damages of adrenocortical cells and vascular epithelia cells occur in adrenal gland, resulting in adrenocortical dysfunction, including low levels of GC and low affinity of GC receptors. The measurement of serum GC and adrenocorticotrophic hormone are combined to assess adrenocortical function: adrenal insufficiency(AI) or relative adrenal insufficiency(RAI). Low doses of GC help to reverse septic shock, improve adrenal function, and maintain normal hemodynamics in septic patients. Trend Further investigation in understanding the mechanisms underlying AI and RAI are highly demanded to optimize therapeutic application of GC in sepsis. Key words: Sepsis; Septic shock; Adrenal insufficiency; Relative adrenal insufficiency

Role of microRNAs in sepsis.

MicroRNAs have been found to be of high significance in the regulation of various genes and processes in the body. Sepsis is a serious clinical problem which arises due to the excessive host inflammatory response to infection. The non-specific clinical features and delayed diagnosis of sepsis has been a matter of concern for long time. MicroRNAs could enable better diagnosis of sepsis and help in the identification of the various stages of sepsis. Improved diagnosis may enable quicker and more effective treatment measures. The initial acute and transient phase of sepsis involves excessive secretion of pro-inflammatory cytokines which causes severe damage. MicroRNAs negatively regulate the toll-like receptor signaling pathway and regulate the production of inflammatory cytokines during sepsis. Likewise, microRNAs have shown to regulate the vascular barrier and endothelial function in sepsis. They are also involved in the regulation of the apoptosis, immunosuppression, and organ dysfunction in later stages of sepsis. Their importance at various levels of the pathophysiology of sepsis has been discussed along with the challenges and future perspectives. MicroRNAs could be key players in the diagnosis and staging of sepsis. Their regulation at various stages of sepsis suggests that they may have an important role in altering the outcome associated with sepsis.

Expression and Function of Granzymes A and B in Escherichia coli Peritonitis and Sepsis.

Escherichia (E.) coli is the most common causative pathogen in peritonitis, the second most common cause of sepsis. Granzymes (gzms) are serine proteases traditionally implicated in cytotoxicity and, more recently, in the inflammatory response. We here sought to investigate the role of gzms in the host response to E. coli-induced peritonitis and sepsis in vivo. For this purpose, we used a murine model of E. coli intraperitoneal infection, resembling the clinical condition commonly associated with septic peritonitis by this bacterium, in wild-type and gzmA-deficient (gzmA−/−), gzmB−/−, and gzmAxB−/−mice. GzmA and gzmB were predominantly expressed by natural killer cells, and during abdominal sepsis, the percentage of these cells expressing gzms in peritoneal lavage fluid decreased, while the amount of expression in the gzm+ cells increased. Deficiency of gzmA and/or gzmB was associated with increased bacterial loads, especially in the case of gzmB at the primary site of infection at late stage sepsis. While gzm deficiency did not impact neutrophil recruitment into the abdominal cavity, it was accompanied by enhanced nucleosome release at the primary site of infection, earlier hepatic necrosis, and more renal dysfunction. These results suggest that gzms influence bacterial growth and the host inflammatory response during abdominal sepsis caused by E. coli.

EXTENDED CYTOPROTECTIVE EFFECT OF AUTOPHAGY IN THE LATE STAGES OF SEPSIS AND FLUCTUATIONS IN SIGNAL TRANSDUCTION PATHWAYS IN A RAT EXPERIMENTAL MODEL OF KIDNEY INJURY.

The impact of a potential autophagy (LC3a/b) deregulation in hyper and in hypo stages during sepsis-induced kidney injury and the temporal profile of phosphorylated extracellular signal-related kinase, P38 (pP38), Akt (pAKT), and 13-3-3β protein were investigated in the current study, using a rat cecal ligation and puncture (CLP) model, by means of flow cytometry and immunohistochemistry. Cell viability was assessed by protein C zymogen concentrate (PC), 7-aminoactinomycin D (7-AAD) staining and inflammation by S100 protein immunostaining. The impact of reduced kidney inflammation in autophagy was assessed by PC administration, an anti-inflammatory and cytoprotective substance. Sepsis induction increased LC3a/b expression, which presented two peaks at 6 and 36 h after CLP, both in the percentage of positive cells (P = 0.024, P = 0.025, respectively) and in fluorescence intensity. At 6 h when inflammation was already apparent, LC3a/b increase was escorted by phosphorylated extracellular signal-related kinase stimulation and high cell viability (65%), designating autophagy as a cytoprotective mechanism against microbial infection. The phosphorylation of P38 was delayed to 12 h after CLP, when autophagy was reduced. pAkt and 14-3-3β expression was stimulated between 6 and 36 h after CLP, although a slight inhibition of pAkt within each cell was detected (lower MnIX value). During the second peak, inflammation was intensified, necrosis was significantly increased with LC3a/b+/7-AAD + cells to present a 1.5-fold increase. Protein C zymogen concentrate administration declined autophagy at 6 and 36 h after CLP and reduced necrosis, whereas double positive LC3a/b and 7-AAD cells were increased by 1.68 and 2.78-fold, respectively. These data open new prospectives in sepsis treatment, since they further support that autophagy represents a cytoprotective mechanism triggered by stress conditions, rather than an alternative cell death pathway.

The NET Effect: Platelet Factor 4 and DNA-Histone Interactions in Sepsis

In response to infection and inflammation, neutrophils release neutrophil extracellular traps (NETs), web like structures composed of nuclear DNA associated with histones that may have both beneficial and deleterious effects. The formation of NETs alters the course of late-stage sepsis and the associated release of histones has been shown to contribute to many of the observed pathologic complications of sepsis. Histones are octamers comprised of two copies of H2A, H2B, H3 and H4, each of which is highly positively charged. NET formation is dependent on chromatin decondensation mediated by the enzyme peptidylarginine deiminase 4 (PAD4). PAD4 potentiates chromatin decondensation by decreasing the overall positive charge of histones through citrullinating many of their lysines residues, forming Cit-histones, which have a decreased affinity for negatively charged DNA. Platelets also contribute to events in late sepsis by undergoing significant activation and degranulation. We propose that one way platelets may affect outcomes in late sepsis is through the release of large amounts of the highly positively charged chemokine platelet factor 4 (PF4, CXCL4). After its release, we believe that PF4 can displace histones and cit-histones from cell free DNA, altering the composition of NETs. We chose to investigate whether PF4 might liberate cit-histones from NET fibers more effectively than non-citrullinated histones. We initially sought to examine the effect of PF4 on histone attachment to DNA. In a competitive binding assay, we found that PF4 binds to DNA with greater affinity than histones. Of note, cit-histones were approximately 5 times more easily displaced from DNA than non-cit-histones consistent with a model of decreased DNA affinity of cit-histones. Furthermore, using immunofluorescence studies and confocal microscopy, we showed that when NETs are generated in the presence of platelets, endogenous PF4 adheres readily to NET DNA. We have also demonstrated that exogenous PF4 avidly binds to NETs generated from neutrophils isolated with minimal platelet contamination. Based on the results of these experiments, we decided to investigate the interaction between PF4, NETs and histones in a novel microfluidic system that is designed to mimic intravascular flow conditions. We isolated neutrophils from fresh whole blood samples obtained from healthy human donors and stimulated them with TNFα to promote adherence to fibronectin coated microfluidic channels. After the neutrophils had firmly bound to the channels, we exposed them to NET stimuli, including lipopolysaccharide (LPS) and calcium ionophore and visualized NET formation. Extracellular DNA was detected using the cell membrane impermeable dye, SYTOX Green. After NET formation occurred, PF4 was flowed through the channels at 25-100 μg/mL, concentrations similar to those observed in terminal sepsis. Exposure to PF4 at these concentrations lead to the dissolution of NET fibers. Interestingly, although the residual NET fibers continued to stain positive for non-cit-histones, they no longer stained positive for cit-histones. In conclusion, cit-histones are present in NETs and may contribute to the pathobiology of late sepsis. We propose that cit-histones are competitively displaced from NETs by PF4. This may be due to their decreased relative affinity for DNA binding. These studies provide new insights into how histones are released from NET fibers into the circulation during sepsis. This information sheds new light on the interaction of chemokines and NETs and may lead to the identification of new therapeutic strategies in the treatment of sepsis. DisclosuresNo relevant conflicts of interest to declare.

Effect of dexmedetomidine on inflammatory mediators in different periods of sepsis in rats

Objective To investigate the effect of dexmedetomidine (DEX) on inflammatory mediators in different periods of sepsis and its impact on survival rate of rats with sepsis. Methods Ninety male Wistar rats were randomly divided into three groups: control group (group C) , sepsis model group (group S) , and DEX treatment group (group D). Lipopolysaccharide (LPS) was injected to the vein to reproduce a classic sepsis model, while in control group, equivalent volume of NS was used instead. Different treatments were given to the corresponding groups, and then venous blood was taken at given intervals. Behavioral changes and mortality rate of rats within 48 hours after experiment were recorded. The levels of the early inflammatory mediators such as tumor necrosis factor-α (TNF-α) , interleukin-6 (IL-6) and the late inflammatory mediators such as high-mobility group boxl protein (HMGB1) in blood serum were detected by ELISA. Results Among three groups, there were significant differences in mortality rate of rats, the serum level of TNF-α, IL-6 and HMGB1 (all P<0.05). Conclusion DEX has a certain anti-inflammatory effects on early and late stages of sepsis in rats with sepsis, and can also reduce the mortality rate. Key words: Dexmedetomidine; Sepsis; Inflammatory mediators; High-mobility group boxl protein

Platelet and endothelial cell P-selectin are required for host defense against Klebsiella pneumoniae-induced pneumosepsis

Sepsis is associated with activation of platelets and endothelial cells accompanied by enhanced P-selectin surface expression. Both platelet- and endothelial P-selectin have been associated with leukocyte recruitment and induction of inflammatory alterations. Klebsiella (K.) pneumoniae is a common human sepsis pathogen, particularly in the context of pneumonia. Wild-type (WT) and P-selectin-deficient (Selp(-/-) ) mice or bone marrow chimeric mice were infected with K. pneumoniae via the airways to induce pneumosepsis. Mice were sacrificed during early (12 h after infection) or late-stage (44 h) sepsis for analyses, or followed in a survival study. Selp(-/-) mice displayed 10-1000-fold higher bacterial burdens in the lungs, blood and distant organs during late-stage sepsis. P-selectin deficiency did not influence leukocyte recruitment to the lungs, but was associated with decreased platelet-monocyte complexes and increased cytokine release. Bone marrow transfer studies revealed a role for both platelet and endothelial cell P-selectin as mice deficient in platelet or endothelial cell P-selectin displayed an intermediate phenotype in bacterial loads and survival compared with full wild-type or full knockout control mice. Both platelet and endothelial cell P-selectin contribute to host defense during Klebsiella pneumosepsis.

Redox signaling and splicing dependent change in myosin phosphatase underlie early versus late changes in NO vasodilator reserve in a mouse LPS model of sepsis.

Microcirculatory dysfunction may cause tissue malperfusion and progression to organ failure in the later stages of sepsis, but the role of smooth muscle contractile dysfunction is uncertain. Mice were given intraperitoneal LPS, and mesenteric arteries were harvested at 6-h intervals for analyses of gene expression and contractile function by wire myography. Contractile (myosin and actin) and regulatory [myosin light chain kinase and phosphatase subunits (Mypt1, CPI-17)] mRNAs and proteins were decreased in mesenteric arteries at 24 h concordant with reduced force generation to depolarization, Ca(2+), and phenylephrine. Vasodilator sensitivity to DEA/nitric oxide (NO) and cGMP under Ca(2+) clamp were increased at 24 h after LPS concordant with a switch to Mypt1 exon 24- splice variant coding for a leucine zipper (LZ) motif required for PKG-1α activation of myosin phosphatase. This was reproduced by smooth muscle-specific deletion of Mypt1 exon 24, causing a shift to the Mypt1 LZ+ isoform. These mice had significantly lower resting blood pressure than control mice but similar hypotensive responses to LPS. The vasodilator sensitivity of wild-type mice to DEA/NO, but not cGMP, was increased at 6 h after LPS. This was abrogated in mice with a redox dead version of PKG-1α (Cys42Ser). Enhanced vasorelaxation in early endotoxemia is mediated by redox signaling through PKG-1α but in later endotoxemia by myosin phosphatase isoform shifts enhancing sensitivity to NO/cGMP as well as smooth muscle atrophy. Muscle atrophy and modulation may be a novel target to suppress microcirculatory dysfunction; however, inactivation of inducible NO synthase, treatment with the IL-1 antagonist IL-1ra, or early activation of α-adrenergic signaling did not suppressed this response.

An Endotoxin Tolerance Signature Predicts Sepsis and Organ Dysfunction at Initial Clinical Presentation

BackgroundSepsis involves aberrant immune responses to infection, but the exact nature of this immune dysfunction remains poorly defined. Bacterial endotoxins like lipopolysaccharide (LPS) are potent inducers of inflammation, which has been associated with the pathophysiology of sepsis, but repeated exposure can also induce a suppressive effect known as endotoxin tolerance or cellular reprogramming. It has been proposed that endotoxin tolerance might be associated with the immunosuppressive state that was primarily observed during late-stage sepsis. However, this relationship remains poorly characterised. Here we clarify the underlying mechanisms and timing of immune dysfunction in sepsis. MethodsWe defined a gene expression signature characteristic of endotoxin tolerance. Gene-set test approaches were used to correlate this signature with early sepsis, both newly and retrospectively analysing microarrays from 593 patients in 11 cohorts. Then we recruited a unique cohort of possible sepsis patients at first clinical presentation in an independent blinded controlled observational study to determine whether this signature was associated with the development of confirmed sepsis and organ dysfunction. FindingsAll sepsis patients presented an expression profile strongly associated with the endotoxin tolerance signature (p<0.01; AUC 96.1%). Importantly, this signature further differentiated between suspected sepsis patients who did, or did not, go on to develop confirmed sepsis, and predicted the development of organ dysfunction. InterpretationOur data support an updated model of sepsis pathogenesis in which endotoxin tolerance-mediated immune dysfunction (cellular reprogramming) is present throughout the clinical course of disease and related to disease severity. Thus endotoxin tolerance might offer new insights guiding the development of new therapies and diagnostics for early sepsis.

Hematopoietic but not endothelial cell MyD88 contributes to host defense during gram-negative pneumonia derived sepsis.

Klebsiella pneumoniae is an important cause of sepsis. The common Toll-like receptor adapter myeloid differentiation primary response gene (MyD)88 is crucial for host defense against Klebsiella. Here we investigated the role of MyD88 in myeloid and endothelial cells during Klebsiella pneumosepsis. Mice deficient for MyD88 in myeloid (LysM-Myd88−/−) and myeloid plus endothelial (Tie2-Myd88−/−) cells showed enhanced lethality and bacterial growth. Tie2-Myd88−/− mice reconstituted with control bone marrow, representing mice with a selective MyD88 deficiency in endothelial cells, showed an unremarkable antibacterial defense. Myeloid or endothelial cell MyD88 deficiency did not impact on lung pathology or distant organ injury during late stage sepsis, while LysM-Myd88−/− mice demonstrated a strongly attenuated inflammatory response in the airways early after infection. These data suggest that myeloid but not endothelial MyD88 is important for host defense during gram-negative pneumonia derived sepsis.

Neuronal Nitric Oxide Synthase and Its Interaction With Soluble Guanylate Cyclase Is a Key Factor for the Vascular Dysfunction of Experimental Sepsis*

Vascular dysfunction plays a central role in sepsis, and it is characterized by hypotension and hyporesponsiveness to vasoconstrictors. Nitric oxide is regarded as a central element of sepsis vascular dysfunction. The high amounts of nitric oxide produced during sepsis are mainly derived from the inducible isoform of nitric oxide synthase 2. We have previously shown that nitric oxide synthase 2 levels decrease in later stages of sepsis, whereas levels and activity of soluble guanylate cyclase increase. Therefore, we studied the putative role of other relevant nitric oxide sources, namely, the neuronal (nitric oxide synthase 1) isoform, in sepsis and its relationship with soluble guanylate cyclase. We also studied the consequences of nitric oxide synthase 1 blockade in the hyporesponsiveness to vasoconstrictors. Randomized controlled prospective experimental study. Academic research laboratory. Female Wistar rats submitted to cecal ligation and puncture method. 1) Six, 12, and 24 hours after cecal ligation and puncture, vascular reactivity to phenylephrine (3 and 30 nmol/kg) before and after 7-nitroindazole (45 μmol/kg, s.c.) or aminoguanidine (30 μmol/kg, s.c.) administration was evaluated. 2) Protein levels and interaction between nitric oxide synthase 1 and soluble guanylate cyclase were determined. 3) Six, 12, and 24 hours after cecal ligation and puncture, thoracic aorta segments were stimulated with phenylephrine in the presence or absence of 7-nitroindazole and cyclic guanosine monophosphate accumulation was determined. 4) After 24 hours of cecal ligation and puncture, norepinephrine was infused (10 μg/kg/min) in the presence or absence of 7-nitroindazole or S-methyl-L-thiocitrulline (1 μmol/kg, IV) and mean arterial pressure was registered. 1) Both nitric oxide synthase 1 and soluble guanylate cyclase are expressed in higher levels in vascular tissues during sepsis; 2) both proteins physically interact and nitric oxide synthase 1 blockade inhibits cyclic guanosine monophosphate production; 3) pharmacological blockade of nitric oxide synthase 1 using 7-nitroindazole or S-methyl-L-thiocitrulline reverts the hyporesponsiveness to phenylephrine and increases the vasoconstrictor effect of norepinephrine and phenylephrine. Sepsis induces increased expression and physical association of nitric oxide synthase 1/soluble guanylate cyclase and a higher production of cyclic guanosine monophosphate that together may help explain sepsis-induced vascular dysfunction. In addition, selective inhibition of nitric oxide synthase 1 restores the responsiveness to vasoconstrictors. Therefore, inhibition of nitric oxide synthase 1 (and possibly soluble guanylate cyclase) may represent a valuable alternative to restore the effectiveness of vasopressor agents during late sepsis.

Morphine exacerbates experimental sepsis by modulating IL17/IL22 response (IRC8P.488)

Abstract Sepsis ranks as the top 10 cause of mortality in the intensive care units (ICU) in USA and the standard management of pain in these patients are opioids. However, very few studies have investigated the influence of opioids on development and progression of sepsis. In the present study, we determined the effects of morphine on sepsis progression using the murine cecal ligation and puncture model, which resembles many aspects of the human pathology. Our studies demonstrate that morphine treatment increases bacterial dissemination at the early stage of sepsis (24 hours) and significantly attenuates pathogen clearance at late stage of sepsis (72 hours). In addition, IL17a and IL22 responses to pathogen stimulation are impaired following morphine treatment, resulting in delayed neutrophil recruitment to peritoneal cavity, thereby compromising pathogen clearance. This is the first study to demonstrate that IL17a and IL22 responses play important roles in host defense against polymicrobial sepsis, and that this crucial response is impaired following opioid treatment. These studies imply that IL17a and IL22 may be potential therapeutic targets to treat or prevent sepsis, especially in opioid use and abuse population.

Myosin phosphatase isoforms in the microvasculature during endotoxemia (678.2)

Sepsis is characterized by widespread inflammation and severe hypotension caused by the induction of iNOS as well as other vasodilators. We hypothesized that altered contractility of resistance artery smooth muscle could play a role in the later stages of sepsis. Sepsis was modeled via a single IP injection of lipopolysaccharides (LPS; 20mg/kg), and mouse vascular tissues were harvested 24hrs post injection. We examined the expression of the myosin phosphatase (MP) regulatory (Mypt1) and inhibitory (CPI‐17) subunits. Expression of Mypt1 Exon24/leucine zipper variants may determine sensitivity to vasodilator NO, while the level of expression of CPI‐17 may determine sensitivity to vasoconstrictor NE. Mesenteric and femoral arteries and portal veins switched from the E24+/LZ‐ isoform to the E24‐/LZ+ isoform of Mypt1 (p&lt;0.05) with decreases in total Mypt1 and CPI‐17 transcript expression (p&lt;0.05). Relaxation to cGMP via wire myography was enhanced in α‐toxin permeabilized mesenteric arteries from LPS injected mice compared to saline injected control. Constriction to phenylephrine activation was significantly depressed in LPS injected mice compared to control. We propose that endotoxemia triggers specific changes in MP subunit expression thereby enhancing vasodilatory mechanisms and suppressing vasoconstrictor mechanisms thereby contributing to the severe hypotension of the later stages of sepsis.Grant Funding Source: HL066171

Recovery Of Myeloid Derived Suppressor Cell Subsets Following Allogeneic Hematopoietic Stem/Progenitor Cell Transplantation

Myeloid derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that expand during many inflammatory conditions and malignancies. MDSCs may play an important role following allogeneic hematopoietic stem/progenitor cell transplant (HSCT). MDSCs suppress T-cell, B-cell and dendritic cell responses by a number of mechanisms, including promoting regulatory T cell expansion and producing soluble mediators such as Arginase 1 (Arg-1) and iNOS. MDSCs are divided into two subsets: monocytic (M-MDSCs) and granulocytic (G-MDSCs). MDSC morphology and function differ in various tissues under different inflammatory conditions. In a murine asthma model, M-MDSCs inhibit airway inflammation, but the other subset of MDSCs exacerbated airway inflammation. In a sepsis model, MDSCs exaggerated inflammation in the early stage, but suppressed inflammation in the later stage of sepsis. As the early post-transplant period is characterized by the rapid expansion of immature myeloid cells, we postulated this time period may also be a time when MDSCs might play a major role in modulating immune recovery post-transplant, and aid in the development of immune regulatory networks potentially important in the pathophysiology of graft-versus-host disease (GVHD). In nine patients undergoing allogeneic HSCT, peripheral blood was drawn on the day prior to the start of conditioning, days +4-5, +7-9, +14-16, +21-23, +27-29 and +80-100 post HSCT. White blood cells were quantified, red cell depleted using HetaSep (Stem Cell Technologies), then stained with fluorescence-labelled antibodies against CD45, CD15, CD14, HLA-DR, CD33 and CD66b and analyzed by flow cytometry for MDSC subsets. The soluble mediators iNOS and Arg-1were evaluated by intracellular staining for iNOS and Arg-1 and analyzed by flow cytometry. Four of the nine patients developed acute GVHD (II-IV) and/or extensive chronic GVHD. Early recovery of CD33+CD14+HLA-DR-/low M-MDSCs and CD33+CD15+CD66b+ G-MDSCs was seen post-transplant. Compared to healthy donors, the percentage of M- and G-MDSCs was increased by 3 weeks post-transplant. Interestingly, the patients who went on to develop GVHD had lower percentage and number of M-MDSCs, but inversely had higher numbers of G-MDSCs by day +27-29 and day +80-100 post-transplant (Fig. 1). When compared with healthy donors, the expression of Arg-1 in G-MDSCs, a measure of activation of MDSCs, was increased in patients pre- and post-HSCT, especially at day +80-100; while there was no difference seen iNOS expression in G-MDSCs (Fig 2). The expression of Arg-1 and iNOS in M-MDSCs was increased pre-transplant but fell by day +80-100 post HSCT (Fig 2). Taken together, our pilot data indicates that both M- and G- MDSCs recover early post HSCT and may contribute to the pathophysiology of GVHD. Patients with lower numbers of M-MDSCs and higher numbers of G-MDSCs at earlier time points post-transplant might be at greater risk for developing GVHD. Disclosures: No relevant conflicts of interest to declare.