Murine Sepsis Model Research Articles

Resveratrol attenuates TLR-4 mediated inflammation and elicits therapeutic potential in models of sepsis

Sepsis is a potentially fatal condition triggered by systemic inflammatory response to infection. Due to the heightened immune reactivity and multi-organ pathology, treatment options are limited and several clinical trials have not produced the desired outcome, hence the interest in the discovery of novel therapeutic strategies. The polyphenol resveratrol (RSV) has shown promise against several pathological states, including acute and chronic inflammation. In this study, we evaluated its therapeutic potential in a murine model of sepsis and in patients undergoing transrectal ultrasound biopsy. RSV was able to inhibit lipopolysaccharide (LPS) stimulated inflammatory responses through blocking Phospholipase D (PLD) and its downstream signaling molecules SphK1, ERK1/2 and NF-κB. In addition, RSV treatment resulted in the downregulation of MyD88, an adaptor molecule in the TLR4 signaling pathway, and this effect at least in part, involved RSV-induced autophagy. Notably, RSV protected mice against polymicrobial septic shock induced upon cecal ligation and puncture, and inhibited pro-inflammatory cytokine production by human monocytes from transrectal ultrasound (TRUS) biopsy patients. Together, these findings demonstrate the immune regulatory activity of RSV and highlight its therapeutic potential in the management of sepsis.

CD14 release induced by P2X7 receptor restricts inflammation and increases survival during sepsis.

P2X7 receptor activation induces the release of different cellular proteins, such as CD14, a glycosylphosphatidylinositol (GPI)-anchored protein to the plasma membrane important for LPS signaling via TLR4. Circulating CD14 has been found at elevated levels in sepsis, but the exact mechanism of CD14 release in sepsis has not been established. Here, we show for first time that P2X7 receptor induces the release of CD14 in extracellular vesicles, resulting in a net reduction in macrophage plasma membrane CD14 that functionally affects LPS, but not monophosphoryl lipid A, pro-inflammatory cytokine production. Also, we found that during a murine model of sepsis, P2X7 receptor activity is important for maintaining elevated levels of CD14 in biological fluids and a decrease in its activity results in higher bacterial load and exacerbated organ damage, ultimately leading to premature deaths. Our data reveal that P2X7 is a key receptor for helping to clear sepsis because it maintains elevated concentrations of circulating CD14 during infection.

A Peptide-Based Checkpoint Immunomodulator Alleviates Immune Dysfunction in Murine Polymicrobial Sepsis.

Sepsis-induced immunosuppression involves both innate and adaptive immunity and is associated with the increased expression of checkpoint inhibitors, such as programmed cell-death protein 1 (PD-1). The expression of PD-1 is associated with poor outcomes in septic patients, and in models of sepsis, blocking PD-1 or its ligands with antibodies increased survival and alleviated immune suppression. While inhibitory antibodies are effective, they can lead to immune-related adverse events (irAEs), in part due to continual blockade of the PD-1 pathway, resulting in hyperactivation of the immune response. Peptide-based therapeutics are an alternative drug modality that provide a rapid pharmacokinetic profile, reducing the incidence of precipitating irAEs. We recently reported that the potent, peptide-based PD-1 checkpoint antagonist, LD01, improves T-cell responses. The goal of the current study was to determine whether LD01 treatment improved survival, bacterial clearance, and host immunity in the cecal-ligation and puncture (CLP)-induced murine polymicrobial sepsis model. LD01 treatment of CLP-induced sepsis significantly enhanced survival and decreased bacterial burden. Altered survival was associated with improved macrophage phagocytic activity and T-cell production of interferon-γ. Further, myeloperoxidase levels and esterase-positive cells were significantly reduced in LD01-treated mice. Taken together, these data establish that LD01 modulates host immunity and is a viable therapeutic candidate for alleviating immunosuppression that characterizes sepsis and other infectious diseases.

Molecular hydrogen alleviates brain injury and cognitive impairment in a chronic sequelae model of murine polymicrobial sepsis.

Sepsis-related encephalopathy (SAE), which causes a series of brain injuries and long-term, potentially irreversible cognitive dysfunction, is closely associated with increased morbidity and mortality. Hydrogen (H2) is a new type of medical gas molecule that has been widely used in the treatment of various diseases in recent years. The aim of the present study was to explore the protective effects of H2 inhalation on brain injury and long-term cognitive impairment in an improved chronic septic mouse model. Male C57BL/6J mice were randomized into four groups: Control, Control + H2, SAE and SAE + H2. The SAE and Control models were established by intraperitoneal injection of human stool suspension or saline in mice. H2 (2%) was inhaled for 60min at 1h and 6h after SAE or Control treatment. The survival rates were recorded for 14days (days 1-14) and the Morris Water Maze was performed for 7 days (days 8-14). To assess the severity of the brain injury, hematoxylin and eosin staining, Nissl staining, Evans blue (EB) extravasation and the wet/dry weight ratio of brain tissue were detected 24h after SAE or Control treatment. In addition, inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin 6 (IL-6), high-mobility group box 1 (HMGB1), as well as the protein levels of nuclear factor-erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), zonula occludens-1 (ZO-1) and Occludin, were measured 6, 12 and 24h after SAE or Control treatment. The results showed that H2 treatment increased survival rates, mitigated cognitive impairment, reduced hippocampal histological damage, decreased EB and water content, and decreased the levels of TNF-α, IL-6, HMGB1, Nrf2, HO-1, ZO-1 and Occludin, as compared with the SAE group. These data revealed that 2% H2 could suppress brain damage and improve cognitive function in septic mice by inhibiting oxidative stress, inflammatory response and the sepsis-induced blood-brain barrier (BBB) disruption.

Role of Fractalkine in promoting inflammation in sepsis-induced multiple organ dysfunction.

Fractalkine, CX3CL1, is involved in the directional movement of chemokine cells, immune response, inflammatory response, tissue repair, and other processes. However, its role in sepsis is not well known. We measured circulating Fractalkine in adult patients with sepsis. Effects of Fractalkine on the survival, inflammation, tissue injury, and bacterial clearance were assessed using the WT or CX3CL-/- murine model of cecal ligation and puncture (CLP)-induced sepsis. Serum Fractalkine concentrations were significantly elevated in adult patients with sepsis compared to healthy adults. Increased Fractalkine correlated positively with the number of blood leukocytes and the level of inflammatory cytokines, including IL-6, IL-1β, IL-17A, IFN-γ, and TNF-α, and correlated negatively with IL-10 in clinical sepsis. Recombinant Fractalkine impaired survival whereas Fractalkine gene knockout or anti-Fractalkine antibody improved survival in the murine model of CLP-induced sepsis. Fractalkine administration increased inflammatory response, evident by higher levels of cytokines (TNF-α, IL-1β, IL-17A, IFN-γ, and IL-6 but not IL-10), and tissue damage (lung, liver, and kidney) in CLP-induced sepsis. Fractalkine reduced bacterial clearance in CLP-induced polymicrobial sepsis by reducing macrophage or neutrophil phagocytosis and intracellular elimination of E. coli. Fractalkine aggravates sepsis by increasing inflammation and decreasing bacterial clearance, and is a potential tool for anti-sepsis therapy.

The emergence of hypervirulent blaNDM-1-positive Klebsiella pneumoniae sequence type 395 in an oncology hospital.

Fifteen hypermucoviscous isolates (13 blaNDM-1-positive) obtained from 11 oncology patients were analyzed by whole genome sequencing, and selected isolates were assessed in a murine model of sepsis. ST395/K2 isolates harboring rmpA, rmpA2, peg-344, aerobactin, enterobactin, yersiniabactin, type I fimbriae, etc. displayed maximal virulence in the mouse lethality assay (LD50=102CFU). ST147/K20 isolates lacking yersiniabactins were relatively less virulent (LD50=104CFU), ST395/K2 isolates lacking rmpA, rmpA2, peg-344, and aerobactin, but harboring yersiniabactin demonstrated minimal virulence (LD50=105CFU). Isolates represent various paths and stages of evolution directed towards convergence of multidrugresistant classical Klebsiella pneumoniae and hypervirulent K. pneumoniae.

Targeting Complement C5a Receptor 1 for the Treatment of Immunosuppression in Sepsis

Complement factor C5a was originally identified as a powerful promoter of inflammation through activation of the C5a receptor 1 (C5ar1). Recent evidence suggests involvement of C5a not only in pro- but also in anti-inflammatory signaling. The present study aims to unveil the role of C5ar1 as potential therapeutic target in a murine sepsis model. Our study discloses a significantly increased survival in models of mild to moderate but not severe sepsis of C5ar1-deficient mice. The decreased mortality of C5ar1-deficient mice is accompanied by improved pathogen clearance and largely preserved liver function. C5ar1-deficient mice exhibited a significantly increased production of the pro-inflammatory mediator interferon-γ (IFN-γ) and a decreased production of the anti-inflammatory cytokine interleukin-10 (IL-10). Together, these data uncover C5a signaling as a mediator of immunosuppressive processes during sepsis and describe the C5ar1 and related changes of the IFN-γ to IL-10 ratio as markers for the immunological (dys)function accompanying sepsis.

3153 – IMPACT OF SEPSIS ON EPIGENETIC REGULATION OF HEMATOPOIETIC STEM CELLS (HSC)

Sepsis is a systemic inflammatory over-response to microbial infection associated with high mortality rates. In addition, survivors of sepsis exhibit long-lasting effects of the disease, remaining immunosuppressed and at high risk for infections for the rest of their life. Hence, new therapies for sepsis are an unmet clinical need and novel ideas and approaches are needed. Using a murine model of severe sepsis induced by P. aeruginosa (P. aer.) bacteria or LPS, our group has previously reported that bone marrow (BM) responds to infections with a significant expansion of HSC associated with a blockage in their differentiation and inability to continuously generate mature neutrophils. As a result, the most critical defense against bacterial pathogens is suppressed, leading to profound neutropenia. Furthermore, when transplanted into healthy mice, HSC that have been exposed to a septic environment showed reduced ability to contribute to the myeloid lineage and to engraft at long-term, suggesting that they retain memory of the septic injury and maintain long-lasting changes. We hypothesize that the latters are due to sepsis-induced rapid and permanent epigenetic modifications in HSC, reshaping their transcriptional programs and affecting cell fate decisions and differentiation. To address our hypothesis, we challenged wild-type mice with P.aer. LPS and performed ATAC-seq on sorted HSC at 24 hrs or 108 hrs after challenge. Immunophenotypic analysis showed that HSC levels renormalized after 108 hr following their rapid expansion and differentiation arrest in response to LPS. However, alterations in the abundance and cell-cycle of myeloid progenitors and mature cells still persevered at long-term. Interestingly, ATAC-seq analysis revealed the persistence of euchromatic regions at 108 hrs from challenge in genomic regions involved in cell fate decisions and cell-cycle, suggesting that inflammation has long-lasting effects on epigenetic regulation, despite apparent HSC phenotypic normalization. Differently from previous works, our studies focus on epigenetic changes at HSC level, during the initial stages of the disease, when opportunity for effective therapeutic intervention may still be available. Our ultimate goal is to identify strategies to re-direct "septic" HSC to a normal differentiation program, thus allowing an efficient innate host response.

Proprotein Convertase Subtilisin/Kexin Type 9 Loss-of-Function Is Detrimental to the Juvenile Host With Septic Shock.

Proprotein convertase subtilisin/kexin type 9 is a central regulator of lipid metabolism and has been implicated in regulating the host response to sepsis. Proprotein convertase subtilisin/kexin type 9 loss-of-function is associated with improved sepsis outcomes in the adult host through increased hepatic bacterial clearance. Thus, there is interest in leveraging proprotein convertase subtilisin/kexin type 9 inhibitors as a therapeutic strategy in adults with sepsis. We sought to validate this association in children with septic shock and in a juvenile murine model of sepsis. Prospectively enrolled cohort of children with septic shock; experimental mice. Seventeen participating institutions; research laboratory. Five-hundred twenty-two children with septic shock; juvenile (14 d old) and adult (10-14 wk) mice with constitutive proprotein convertase subtilisin/kexin type 9 null and wildtype control mice (C57BL/6). Proprotein convertase subtilisin/kexin type 9 single-nucleotide polymorphisms, serum proprotein convertase subtilisin/kexin type 9, and lipid profiles in patients. Cecal slurry murine model of sepsis; survival studies in juvenile and adult mice, assessment of lipoprotein fractions, bacterial burden, and inflammation in juvenile mice. PCSK9 loss-of-function genetic variants were independently associated with increased odds of complicated course and mortality in children with septic shock. PCSK9, low-density lipoprotein, and high-density lipoprotein concentrations were lower among patients with complicated course relative to those without. PCSK9 concentrations negatively correlated with proinflammatory cytokine interleukin-8. Proprotein convertase subtilisin/kexin type 9 loss-of-function decreased survival in juvenile mice, but increased survival in adult mice with sepsis. PCSK9 loss-of-function resulted in low lipoproteins and decreased hepatic bacterial burden in juvenile mice. In contrast to the adult host, proprotein convertase subtilisin/kexin type 9 loss-of-function is detrimental to the juvenile host with septic shock. PCSK9 loss-of-function, in the context of low lipoproteins, may result in reduced hepatic bacterial clearance in the juvenile host with septic shock. Our data indicate that children should be excluded in sepsis clinical trials involving proprotein convertase subtilisin/kexin type 9 inhibitors.

Impact of the immune response modification by lysophosphatidylcholine in the efficacy of antibiotic therapy of experimental models of peritoneal sepsis and pneumonia by Pseudomonas aeruginosa: LPC therapeutic effect in combined therapy

IntroductionImmune response stimulation may be an adjuvant to antimicrobial treatment. Here, we evaluated the impact of immune response modification by lysophosphatidylcholine (LPC), combined with imipenem or ceftazidime, in murine models of peritoneal sepsis (PS) and pneumonia induced by Pseudomonas aeruginosa. MethodsThe imipenem and ceftazidime-susceptible strain (Pa39) and imipenem and ceftazidime-resistant strain (Pa238) were used. Ceftazidime pharmacokinetic and pharmacodynamic parameters were determined. The therapeutic efficacy and TNF-α and IL-10 levels were determined in murine models of PS and pneumonia induced by Pa39 and Pa238 and treated with LPC, imipenem or ceftazidime, alone or in combination. ResultsIn the PS model, LPC+ceftazidime reduced spleen and lung Pa238 concentrations (−3.45 and −3.56log10CFU/g; P<0.05) to a greater extent than ceftazidime monotherapy, while LPC+imipenem maintained the imipenem efficacy (−1.66 and −1.45log10CFU/g; P>0.05). In the pneumonia model, LPC+ceftazidime or LPC+imipenem reduced the lung Pa238 concentrations (−2.37log10CFU/g, P=0.1, or −1.35log10CFU/g, P=0.75). For Pa39, no statistically significant difference was observed in the PS and pneumonia models between combined therapy and monotherapy. Moreover, LPC+imipenem and LPC+ceftazidime significantly decreased and increased the TNF-α and IL-10 levels, respectively, in comparison with the untreated controls and monotherapies. ConclusionsThese results demonstrate the impact of immune response modification by LPC plus antibiotics on the prognosis of infections induced by ceftazidime-resistant P. aeruginosa.

Examination of intestinal ultrastructure, bowel wall apoptosis and tight junctions in the early phase of sepsis

Gut hyperpermeability can be caused by either apoptosis of the intestinal epithelium or altered status, permeability or porosity of tight junctions. This project aims to elucidate these mechanisms in the early phase of sepsis. Eighteen male wild type mice were randomized to two groups. All mice received one single gavage of fluorescein isothiocyanate (FITC) dextran 30 min before intervention. One group (n = 10) underwent cecal ligation and puncture to induce sepsis. The other group (n = 8) was sham operated. Septic animals exhibited significantly increased permeability for FITC 8 h post-operatively. Significantly increased serum interleukin-6, tumor-necrosis-factor-alpha and interleukin-1-beta confirmed sepsis. Septic animals showed significant bowel wall inflammation of ileum and colon samples. PCR revealed significantly increased expression of claudin-2 and decreased expressions of claudin-4, tight-junction-protein-1 and occludin-1 resembling increased permeability of tight junctions. However, these alterations could not be confirmed at the protein level. Light microscopy revealed significant dilatation of intercellular spaces at the basal sections of intestinal epithelial cells (IEC) in septic animals confirmed by increased intercellular spaces at the level of tight junctions and adherens junctions in electron microscopy (TEM). In small angle X-ray scattering no increase in number or size of nanopores could be shown in the bowel wall. HOECHST staining and PCR of ileum samples for apoptosis markers proofed no relevant differences in intestinal epithelial cell apoptosis between the groups. Intestinal hyperpermeability in septic animals was most likely caused by alterations of the intercellular contacts and not by apoptosis or increased size/number of nanopores of intestinal epithelial cells in this murine model of early sepsis.

Antimicrobial peptide LL-37 ameliorates a murine sepsis model via the induction of microvesicle release from neutrophils.

Sepsis is a life-threatening disease caused by systemic dys-regulated inflammatory response to infection. We previously revealed that LL-37, a human cathelicidin antimicrobial peptide, improves the survival of cecal ligation and puncture septic mice. Ectosomes, microvesicles released from neutrophils, are reported to be elevated in sepsis survivors; however, the functions of ectosomes in sepsis remain largely unknown. Therefore, we herein elucidated the protective action of LL-37 on sepsis, by focusing on LL-37-induced ectosome release in a cecal ligation and puncture model. The results demonstrated the enhancement of ectosome levels by LL-37 administration, accompanied by a reduction of bacterial load. Importantly, ectosomes isolated from LL-37-injected cecal ligation and puncture mice contained higher amounts of antimicrobial proteins/peptides and exhibited higher antibacterial activity, compared with those from PBS-injected cecal ligation and puncture mice, suggesting that LL-37 induces the release of ectosomes with antibacterial potential in vivo. Actually, LL-37 stimulated mouse bone-marrow neutrophils to release ectosomes ex vivo, and the LL-37-induced ectosomes possessed antibacterial potential. Furthermore, administration of LL-37-induced ectosomes reduced the bacterial load and improved the survival of cecal ligation and puncture mice. Together these observations suggest LL-37 induces the release of antimicrobial ectosomes in cecal ligation and puncture mice, thereby reducing the bacterial load and protecting mice from lethal septic conditions.

Type I pili, CsuA/B and FimA induce a protective immune response against Acinetobacter baumannii

BackgroundAcinetobacter baumannii, a nosocomial pathogen, is considered as a common cause of hospital and community-acquired infections. Emerging multidrug-resistance in this pathogen followed by subsequent problems in treatment has been increasing to alarming levels that warrant investigation of new therapeutic approaches. One strategy to reduce antibiotic resistance is to use of vaccines. Although there is no vaccine currently in development for this pathogen, different attempts have been made to develop one. MethodsIn this study, we used two different recombinant pilus proteins (CsuA/B and FimA) either singly or in combination to evaluate protective efficacy against A. baumannii in lethal and sub-lethal murine sepsis models. FindingsActive immunization with recombinant proteins in combination elicited high levels of IgG antibody after the first immunization and provided 62% (five of eight mice; p < 0·001) protection against a lethal dose (1·2 × 106 CFU) of A. baumannii along with efficient clearance of bacteria in internal organs viz. spleen, liver, and lungs at sub-lethal challenge. Immunization with CsuA/B alone conferred partial protection as demonstrated by low survival rate (three [37%] of eight mice; p < 0·05) after lethal challenge and reduction of bacteria in internal organs of the mice after 24 h post-sub-lethal infection. Immunization with FimA, in comparison to CsuA/B, showed better protection (four [50%] of eight mice; p < 0·01) and reduction in CFU after 14 h. InterpretationOur results showed that pilus proteins in combination as a single immunogen could potentially impart protection against A. baumannii. FundingShahed University.

Mesenchymal stromal/stem cells modulate response to experimental sepsis-induced lung injury via regulation of miR-27a-5p in recipient mice

IntroductionMesenchymal stromal cell (MSC) therapy mitigates lung injury and improves survival in murine models of sepsis. Precise mechanisms of therapeutic benefit remain poorly understood.ObjectivesTo identify host-derived regulatory elements that may...

Soluble dietary fiber protects intestinal mucosal barrier by improving intestinal flora in a murine model of sepsis

The study investigated the effect of soluble dietary fiber supplementation on gut microbiota, mucosal barrier function, inflammatory markers, and survival in a murine model of sepsis established by cecal ligation and puncture (CLP). The expression of muc2 was down-regulated in septic mice. However, muc2 expression was significantly increased in the septic mice that received soluble dietary fiber. Survival was also improved in the septic mice treated with dietary fiber. The survival benefit was associated with decreased serum concentrations of pro-inflammatory cytokines and reduced neutrophil infiltration in the gut. Furthermore, the mucus thickness, inflammatory response, and degree of gut injury were remarkably alleviated in septic mice treated with soluble dietary fiber, while it was aggravated in septic mice treated with antibiotics in addition to the soluble fiber. Bacterial 16S ribosomal RNA gene sequencing also showed that fiber supplementation caused an increase in the relative abundance of probiotics, commonly associated with metabolic health. Administration of antibiotics to mice fed fiber diet negated the enrichment of probiotics as well as the survival benefit, following cecal ligation and puncture. Dietary supplementation with cellulose offers a microbe-mediated survival advantage in a murine model of sepsis. Improved understanding of the connection between diet, microbiota, and systemic illness may yield new therapeutic strategies for patients with sepsis.

Sulfatide Inhibits HMGB1 Secretion by Hindering Toll-Like Receptor 4 Localization Within Lipid Rafts.

The high mobility group box 1 (HMGB1) is a well-known late mediator of sepsis, secreted by multiple stimuli, involving pathways, such as the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, and reactive oxygen species (ROS) under inflammation. Sulfatide, in contrast, is a sphingolipid commonly found in myelin sheets with a disputed immunological role. We sought to determine the immunological characteristics of sulfatide in the periphery by analyzing the secretion of HMGB1 triggered by lipopolysaccharide (LPS) stimulation in Raw 264.7 cells. Suppression of HMGB1 secretion by inhibiting its cytosolic translocation was observed after pre-treatment with sulfatide before LPS stimulation. Further analysis of the downstream molecules of toll-like receptor (TLR) signaling revealed suppression of c-Jun N-terminal kinase (JNK) phosphorylation and p65 translocation. LPS-mediated ROS production was also decreased when sulfatide pre-treatment was provided, caused by the down-regulation of the phosphorylation of activators, such as IRAK4 and TBK1. Investigation of the upstream mechanism that encompasses all the aforementioned inhibitory characteristics unveiled the involvement of lipid rafts. In addition to the co-localization of biotinylated sulfatide and monosialotetrahexosylganglioside, a decrease in LPS-induced co-localization of TLR4 and lipid raft markers was observed when sulfatide treatment was given before LPS stimulation. Overall, sulfatide was found to exert its anti-inflammatory properties by hindering the co-localization of TLR4 and lipid rafts, nullifying the effect of LPS on TLR4 signaling. Similar effects of sulfatide were also confirmed in the LPS-mediated murine experimental sepsis model, showing decreased levels of serum HMGB1, increased survivability, and reduced pathological severity.

Effects of the Non-Neutralizing Humanized Monoclonal Anti-Adrenomedullin Antibody Adrecizumab on Hemodynamic and Renal Injury in a Porcine Two-Hit Model.

Adrenomedullin is a vasoactive peptide that improves endothelial barrier function in sepsis, but may also cause hypotension and organ failure. Treatment with a non-neutralizing monoclonal anti-adrenomedullin antibody showed improvement in murine sepsis models. We tested the effects of the humanized monoclonal anti-adrenomedullin antibody Adrecizumab in a porcine two-hit model of hemorrhagic and septic shock.In this randomized, blinded study 12 German Landrace pigs were bled to half of baseline mean arterial pressure for 45 min. Sepsis was induced using an Escherichia coli clot placed into the abdominal cavity 6 h after hemorrhagic shock. Animals received either 2 mg/kg BW anti-adrenomedullin antibody or vehicle solution immediately after sepsis induction. After 4 h, resuscitation was initiated using balanced crystalloids and noradrenalin to maintain a central venous pressure of 8 to 12 mm Hg, a mean arterial pressure ≥ 65 mm Hg, and a ScvO2 ≥70% for another 8 h. Hemodynamic parameters, laboratory parameters, and kidney histology were assessed.The amount of volume resuscitation was significantly lower and significantly less animals developed a septic shock in the antibody-treated group, compared with the vehicle group. Kidney histology showed significantly lower granulocytes in both cortex and medulla in antibody-treated animals, while the remaining four kidney measures (serum creatinine and urine output and cortical and medullary injury in histopathology) did not reach the significance levels. After induction of sepsis, plasma adrenomedullin increased immediately in both the groups, but increased quicker and more pronounced in the antibody group.In this two-hit shock model, treatment with an anti-adrenomedullin antibody significantly increased plasma adrenomedullin levels, while significantly less animals developed septic shock and renal granulocyte extravasation was significantly reduced. Thus, therapy with Adrecizumab may provide benefit in sepsis, and clinical investigation of this drug candidate is warranted.

Myricetin ameliorates sepsis-associated acute lung injury in a murine sepsis model.

Myricetin belongs to the flavonoid family which is derived from plant source. It is well-known for the anti-inflammatory and anti-oxidative properties yet the clinical use of myricetin awaits further discovery. Acute lung injury (ALI) is commonly caused by sepsis which leads to enormous burden with high morbidity and mortality. In this study, a murine sepsis model was constructed by cecal ligation and puncture (CLP). The indicated dose of myricetin (100mg/kg) was further administrated intragastrically. The survival rate test indicated that myricetin significantly improved the vitality of CLP-operated mice. The pathological changes in morphology, biomarkers of inflammatory response, oxidative stress response, and mitochondrial damage were further detected. Myricetin showed significant inhibitory effects on these changes in CLP-induced mice. Furthermore, expression levels of transcription factor Nrf2 and heme oxygenase-1 (HO-1) along with DNA binding activity of Nrf2 were analyzed by western blot and EMSA, indicating that myricetin positively regulates the Nrf2/HO-1 pathway in CLP-induced sepsis mice. Murine sepsis models with knockdown of Nrf2 and control were further established and suggested that myricetin might exert protective effects on sepsis lung injury in dependent with Nrf2. Our study provides novel mechanisms for the protective effect of myricetin in sepsis-associated acute lung injury.

SUN-651 Murine Cecal Ligation and Puncture (CLP) Perturbs Phosphorylation of Insulin Receptor Substrate 2 (IRS-2)

Hyperglycemia is a characteristic finding in sepsis, and its presence worsens outcome (1). Patients with sepsis need larger doses of insulin to reduce glucose levels. This abnormality has been termed “insulin resistance” but the molecular mechanism by which sepsis attenuates the insulin signaling pathway is unknown. Previous work has shown impairment of phosphorylation in several intracellular signaling pathways following CLP, a well-validated murine model of sepsis (2). Phosphorylation of tyrosine in IRS-2 is essential for functional insulin signaling in hepatocytes (3). Therefore the aim of this study was to investigate the effects of CLP on IRS-2 phosphorylation. Hypothesis: CLP attenuates phosphorylation of IRS-2. Methods: All studies were approved by the Feinstein IACUC and conformed to ARRIVE guidelines. CLP was performed on C57Bl6 mice. Before CLP, animals were identified for sacrifice at specific post-procedure time points. To stimulate phosphorylation of IRS-2, insulin was injected in control and CLP mice at 24 and 48 hours post CLP. Following sacrifice, protein was isolated from liver tissue. Protein abundance was determined using immunoblotting. The detection of the phosphorylated form of these proteins was determined by enzyme-linked immunosorbent assay (ELISA) with a phospho-insulin receptor antibody. Statistical significance was determined using ANOVA for repeated measures with a Sidak post-hoc correction. Results: Relative to the control, tyrosine phosphorylation of IRS-2 was significantly (p<0.05) reduced at 24 and 48 hours following CLP. Conclusions: Tyrosine phosphorylation of hepatic IRS2 is attenuated at early time points following CLP. These results are consistent with other studies examining the effects of CLP on intra-cellular signal transduction pathways (1). Further, these results provide evidence that changes in the insulin signaling transduction underlie CLP-induced “insulin resistance”.

Immunomodulatory dose of clindamycin in combination with ceftriaxone improves survival and prevents organ damage in murine polymicrobial sepsis.

Sepsis is a life-threatening organ dysfunction resulting from inflammatory responses instigated by toxins secreted by bacteria. Immunomodulatory effect of clindamycin is earlier reported in a murine lipopolysaccharide (LPS)-induced sepsis model. There are no studies demonstrating the immunomodulatory effect of clindamycin in combination with ceftriaxone in a clinically relevant murine polymicrobial sepsis model induced by cecal ligation and puncture (CLP). Ceftriaxone is combined to control the bacterial growth. Following 3h of CLP challenge, Swiss albino mice were administered vehicle, ceftriaxone alone (100mg/kg, subcutaneously), and in combination with clindamycin at immunomodulatory dose (200mg/kg, intraperitoneally). Survival was assessed for 5days, and bacterial count and biochemical and physiological parameters were measured after 18h of CLP challenge. Ceftriaxone alone caused significant reduction in bacterial count in blood, peritoneal fluid, lung, liver, and kidney homogenate which was not further substantially reduced by ceftriaxone and clindamycin combination. Day 5 survival was greatly improved by combination compared with ceftriaxone alone which was also evident through marked drop in blood glucose, total white blood cell (WBC) count, and body temperature. The combination group significantly mitigated the cytokine (tumor necrosis factor (TNF)-α and interleukin (IL)-6) and myeloperoxidase (MPO) levels in plasma, lung, liver, and kidney of CLP-challenged mice, which further helped in significantly suppressing the elevated levels of liver and kidney function parameters. Clindamycin at immunomodulatory dose in combination with ceftriaxone attenuated organ damage and improved survival of septic mice by suppressing infection, inflammatory responses, and oxidative stress.