Mouse Model Of Sepsis Research Articles

An Autophagy-Associated MITF-GAS5-miR-23 Loop Attenuates Vascular Oxidative and Inflammatory Damage in Sepsis.

Sepsis induces GAS5 expression in the vascular endothelium, but the molecular mechanism is unclear, as is the role of GAS5 in sepsis. We observed that GAS5 expression in the endothelium was significantly upregulated in a sepsis mouse model. ChIP-PCR and EMSA confirmed that the oxidative stress (OS)-activated MiT-TFE transcription factor (MITF, TFE3, and TFEB)-mediated GAS5 transcription. In vitro, GAS5 overexpression attenuated OS and inflammation in endothelial cells (ECs) while maintaining the structural and functional integrity of mitochondria. In vivo, GAS5 reduced tissue ROS levels, maintained vascular barrier function to reduce leakage, and ultimately attenuated sepsis-induced lung injury. Luciferase reporter assays revealed that GAS5 protected MITF from degradation by sponging miR-23, thereby forming a positive feedback loop consisting of MITF, GAS5, and miR-23. Despite the fact that the OS-activated MITF-GAS5-miR-23 loop boosted MITF-mediated p62 transcription, ECs do not need to increase mitophagy to exert mitochondrial quality control since MITF-mediated Nrf2 transcription exists. Compared to mitophagy, MITF-transcribed p62 prefers to facilitate the autophagic degradation of Keap1 through a direct interaction, thereby relieving the inhibition of Nrf2 by Keap1, indicating that MITF can upregulate Nrf2 at both the transcriptional and posttranscriptional levels. Following this, ChIP-PCR demonstrated that Nrf2 can also transcribe MITF, revealing that there is a reciprocal positive regulatory association between MITF and Nrf2. In sepsis, the ROS-activated MITF-GAS5-miR-23 loop integrated the antioxidant and autophagy systems through MITF-mediated transcription of Nrf2 and p62, which dynamically regulate the level and type of autophagy, as well as exert antioxidant and anti-inflammatory effects.

Epistatic interactions between the high pathogenicity island and other iron uptake systems shape Escherichia coli extra-intestinal virulence

The intrinsic virulence of extra-intestinal pathogenic Escherichia coli is associated with numerous chromosomal and/or plasmid-borne genes, encoding diverse functions such as adhesins, toxins, and iron capture systems. However, the respective contribution to virulence of those genes seems to depend on the genetic background and is poorly understood. Here, we analyze genomes of 232 strains of sequence type complex STc58 and show that virulence (quantified in a mouse model of sepsis) emerged in a sub-group of STc58 due to the presence of the siderophore-encoding high-pathogenicity island (HPI). When extending our genome-wide association study to 370 Escherichia strains, we show that full virulence is associated with the presence of the aer or sit operons, in addition to the HPI. The prevalence of these operons, their co-occurrence and their genomic location depend on strain phylogeny. Thus, selection of lineage-dependent specific associations of virulence-associated genes argues for strong epistatic interactions shaping the emergence of virulence in E. coli.

#3581 ACSS2-INDUCED FATTY ACID SYNTHESIS PROMOTES NLRP3 INFLAMMASOME AND PYROPTOSIS OF RENAL TUBULAR CELLS IN SEPSIS-INDUCED ACUTE KIDNEY INJURY

Abstract Background and Aims Cellular fatty acid metabolism was supposed to be tightly associated with immune responses. Pyroptosis is a form of programmed cell death dependent on the NLRP3 inflammasome activation. Previous studies have revealed that acyl-CoA synthetase 2 (ACSS2) promoted fatty acid synthesis under metabolic stress. However, whether ACSS2-mediated fatty acid metabolism can regulate pyroptosis and inflammatory responses of renal tubular cells in AKI remain unclear. Method lipopolysaccharide (LPS) was intraperitoneally injected to establish the sepsis mouse model, and in vitro HK-2 cell culture model was achieved by LPS stimulation. HE and PAS staining were carried out to evaluate pathological injury of kidneys in mice. The renal tissue immunostaining was conducted to detect IL-1β expression and macrophage distribution in kidney tissues of mice. Gene expression of inflammatory factors was evaluated by real-time PCR. Western Blot was conducted to the activation of the NLRP3 pathway and pyroptosis in vivo and in vitro. Results Here, we demonstrated that the expression of ACSS2 was significantly increased in the renal epithelial cells of mice with Lipopolysaccharide (LPS)-induced AKI when compared to wild-type mice. ACSS2 regulates NLRP3-mediated caspase-1 activation and IL-1β production through the stimulation of fatty acid synthase (FASN) in renal epithelial cells. The deletion of ACSS2 attenuated renal tubular pathological injury and inflammatory cell infiltration in an LPS-induced mouse model. Consistently, ACSS2-deficient mice displayed impaired FASN-mediated lipid synthesis and decreased IL-1β production in response to the LPS challenge. In HK-2 cells, ACSS2 deficiency suppressed NLRP3-mediated caspase-1 activation and decreased fatty acid synthesis through the downregulation of FASN. The treatment with the chemical inhibitor C75 suppressed NLRP3-mediated caspase-1 activation and pyroptosis of HK-2 cells under LPS treatment in renal tubular cells. Conclusion Our results suggested that ACSS2 regulated the NLRP3 inflammasome activation and pyroptosis by inducing the FASN-mediated fatty acid synthesis pathway in renal epithelial cells. These results identified ACSS2 as a potential therapeutic target in AKI.

Group B Streptococcus Cas9 variants provide insight into programmable gene repression and CRISPR-Cas transcriptional effects

Group B Streptococcus (GBS; S. agalactiae) causes chorioamnionitis, neonatal sepsis, and can also cause disease in healthy or immunocompromised adults. GBS possesses a type II-A CRISPR-Cas9 system, which defends against foreign DNA within the bacterial cell. Several recent publications have shown that GBS Cas9 influences genome-wide transcription through a mechanism uncoupled from its function as a specific, RNA-programmable endonuclease. We examine GBS Cas9 effects on genome-wide transcription through generation of several isogenic variants with specific functional defects. We compare whole-genome RNA-seq from Δcas9 GBS with a full-length Cas9 gene deletion; dcas9 defective in its ability to cleave DNA but still able to bind to frequently occurring protospacer adjacent motifs; and scas9 that retains its catalytic domains but is unable to bind protospacer adjacent motifs. Comparing scas9 GBS to the other variants, we identify nonspecific protospacer adjacent motif binding as a driver of genome-wide, Cas9 transcriptional effects in GBS. We also show that Cas9 transcriptional effects from nonspecific scanning tend to influence genes involved in bacterial defense and nucleotide or carbohydrate transport and metabolism. While genome-wide transcription effects are detectable by analysis of next-generation sequencing, they do not result in virulence changes in a mouse model of sepsis. We also demonstrate that catalytically inactive dCas9 expressed from the GBS chromosome can be used with a straightforward, plasmid-based, single guide RNA expression system to suppress transcription of specific GBS genes without potentially confounding off-target effects. We anticipate that this system will be useful for study of nonessential and essential gene roles in GBS physiology and pathogenesis.

Inhibition of interleukin-6 trans-signaling improves survival and prevents cognitive impairment in a mouse model of sepsis.

Sepsis-associated encephalopathy (SAE) manifests clinically as acute and chronic cognitive impairments, which is associated with increased morbidity and mortality. Interleukin-6 (IL-6), a pro-inflammatory cytokine, is consistently up-regulated in sepsis. IL-6 initiates proinflammatory effects after binding to soluble IL-6 receptor (IL-6R) through trans-signalling, which requires the transducer gp130. In this study, we investigated whether inhibition of IL-6 trans-signalling is a putative therapeutic target for sepsis and SAE. Twenty-five patients (12 septic and 13 non-septic patients) were recruited for the study. A significant increase of IL-6, IL-1β, IL-10, and IL-8 was observed in the septic patients 24h after ICU admission. In animal study, cecal ligation and puncture (CLP) was used to induce sepsis in male C57BL/6J mice. One hour before or after inducing sepsis, mice were treated with sgp130, a selective IL-6 trans-signaling inhibitor, respectively. Survival rate, cognition, levels of inflammatory cytokines, integrity of blood-brain barrier (BBB), and oxidative stress were assessed. In addition, immune cells activation and transmigration were evaluated in peripheral blood and brains. Sgp130 improved survival rate and cognitive functions, reduced levels of inflammatory cytokines, including IL-6, TNF-α, IL-10, and MCP-1, in plasma and hippocampus (hipp), mitigated BBB disruption, and ameliorated sepsis-induced oxidative stress. Sgp130 also affected monocytes/macrophages and lymphocytes transmigration and activation in septic mice. Our results indicate that selective inhibition of IL-6 trans-signaling by sgp130 exerts protective effects against SAE in a mouse model of sepsis, suggesting a potential therapeutic strategy.

Extracellular protein homeostasis: The dawning of a new age for human disease therapies?

Extracellular protein homeostasis: The dawning of a new age for human disease therapies?

Tectorigenin relieved sepsis-induced myocardial ferroptosis by inhibiting the expression of Smad3.

Myocardial injury is a serious consequence of sepsis that contributes to high rates of death. Currently, the pathophysiology of cardiac damage in sepsis is still unknown, and treatment approaches are limited. The sepsis mouse model was established inducing by Lipopolysaccharide (LPS) in vivo and Tectorigenin was pretreated to explore whether it contributed to alleviated myocardial injury. Hematoxylin-eosin (HE) stain was employed to evaluate the myocardial injury severity. TUNEL assay measured the number of apoptosis cells and the levels of B-cell lymphoma-2 associated X (Bax) and Cleaved Caspase-3 were assessed by western blot. The contents of iron and related ferroptosis molecules (acyl-CoA synthetase long-chain family (ACSL4), Glutathione Peroxidase 4 (GPX4)) were assessed. Then, interleukin-1β (IL-1β), IL-18, IL-6, tumor necrosis factor-α (TNF-α), and other inflammatory-related cytokines were detected by ELISA. The expression of the mother against decapentaplegic homolog 3 (Smad3) in heart tissues was evaluated by western blot and immunofluorescence. Tectorigenin alleviated myocardial dysfunction and myofibrillar disruption in LPS-related sepsis groups. Tectorigenin ameliorated cardiomyocyte apoptosis and myocardial ferroptosis in LPS-stimulated sepsis mice. Tectorigenin reduced inflammatory-relevant cytokines in the cardiac tissues of LPS stimuli mice. In addition, we further confirm that Tectorigenin relieved myocardial ferroptosis by inhibiting the expression of Smad3. Tectorigenin ameliorates myocardial damage stimulated by LPS and this effect exerts by inhibiting ferroptosis and the inflammation of the myocardium. Furthermore, the inhibitory effect of Tectorigenin on ferroptosis may deregulate Smad3 expression. Taken together, Tectorigenin may be a viable method for alleviating myocardial damage in sepsis.

The combination of mesenchymal stem cell- and hepatocyte-derived exosomes, along with imipenem, ameliorates inflammatory responses and liver damage in a sepsis mouse model

AimSepsis is a medical emergency with no definitive treatment. Animal experiments have confirmed the therapeutic characteristics of exosomes in reducing inflammation and tissue damage. The study investigates the effect of MSC and hepatocyte-derived exosomes along with imipenem in controlling systemic and local (liver) inflammation in a mouse model of sepsis. Main methodsTo induce sepsis in C57BL/6 mice, the Cecal Ligation and Puncture (CLP) model was used. The mice were given various treatments, including imipenem, MSC-derived exosomes, hepatocyte-derived exosomes, and a mixture of exosomes. Blood and liver samples were collected and analyzed for cell blood count, liver enzymes, NO levels, cytokine concentrations, and bacterial presence. The percentages of TCD3 + CD4+/CD8+ and Treg in the spleen and mesenteric lymph nodes were also assessed using flow cytometry. The pathological changes were assessed in the liver, lung, and heart tissues. In addition, the cytokine content of exosomes was measured by ELISA. Key findingsOur results demonstrated that MSC-derived exosomes+imipenem could control systemic and local inflammation and increase the TCD4+ and Treg populations. Hepatocyte-derived exosomes+imipenem reduced inflammation in the liver and increased the TCD8+ and Treg populations. The mixture of exosomes+imipenem had the best function in reducing inflammation, maintaining all T lymphocyte populations, reducing liver damage, and ultimately increasing the survival rate. SignificanceThe mixture of exosomes derived from MSCs and hepatocytes, along with imipenem, in the inflammatory phase of sepsis could be a promising therapeutic strategy in sepsis treatment.

Cx43-Delivered miR-181b Negatively Regulates Sirt1/FOXO3a Signalling Pathway-Mediated Apoptosis on Intestinal Injury in Sepsis

Introduction: Gap junctions can transmit signals between cells, including miRNAs, leading to the amplification of adjacent cell damage. No previous study has addressed gap junctions and miRNAs in sepsis because the internal mechanism of sepsis-induced intestinal injury is complex. Therefore, we studied the relationship between connexin43 (Cx43) and miR-181b and provided a research direction for further study of sepsis. Methods: A mouse caecal ligation and puncture method was used to construct a mouse sepsis model. Firstly, damage to intestinal tissues at different time points was analysed. The levels of Cx43, miR-181b, Sirt1, and FOXO3a in intestinal tissues and the transcription and translation of the apoptosis-related genes Bim and puma, which are downstream of FOXO3a were analysed. Secondly, the effect of Cx43 levels on miR-181b and Sirt1/FOXO3a signalling pathway activity was explored by using the Cx43 inhibitor heptanol. Finally, luciferase assays were used to determine miR-181b binding to the predicted target sequence. Results: The results show that during sepsis, intestinal injury becomes increasingly worse with time, and the expression of Cx43 and miR-181b increase. In addition, we found that heptanol could significantly reduce intestinal injury. This finding indicates that inhibiting Cx43 regulates the transfer of miR-181b between adjacent cells, thereby reducing the activity of the Sirt1/FOXO3a signalling pathway and reducing the degree of intestinal injury during sepsis. Conclusions: In sepsis, the enhancement of Cx43 gap junctions leads to an increase in miR-181b intercellular transfer, affects the downstream SIRT1/FOXO3a signalling pathway and causes cell and tissue damage.

Nigericin attenuates lipopolysaccharides induced sepsis and alleviates acute lung injury in in vivo model

Nigericin is a potent antibiotic derived from Streptomyces hygroscopicus which acts as a polyether ionophore. The anticancer property of nigericin was extensively studied, and it has been proven to inhibit cancer cell proliferation, progression, and invasion in prostate, colorectal, nasopharyngeal, and lung cancer. It also stimulates bacterial clearance in macrophages. The protective role of nigericin in sepsis, which is the foremost risk factor for acute lung injury and acute respiratory distress syndrome. Since bacteremia is frequently associated with lung injury, in this study we evaluated the potency of nigericin against an lipopolysaccharide (LPS)-induced sepsis mice model. BALB/c mice were challenged with intratracheal administration of LPS and simultaneously treated with 10 mg/kg and 20 mg/kg nigericin for 3 consecutive days. The mice were euthanized, and the lung tissue was excised to determine the lung wet/dry ratio. Bronchoalveolar lavage fluid was collected and assessed for protein content, LDH, and immune cell count. The antioxidant activity of nigericin was evaluated by measuring the MPO, MDA, SOD, and GSH activities in the lung tissues. The proinflammatory cytokines TNF-α and IL-6 were quantified to assess the anti-inflammatory potency against LPS-induced inflammation. iNOS, COX-2 and PGE-2 levels in lung tissue of nigericin-treated LPS-challenged mice were estimated to assess the immunomodulatory activity of nigericin. Finally, the antiapoptotic potency and cytoprotective effect of nigericin were analyzed by estimating apoptotic protein levels in lung tissue and assessing lung tissue histopathology with hematoxylin & eosin staining. Nigericin treatment significantly reduced immune cell infiltration, synthesis of proinflammatory cytokines, immunomodulatory proteins, and proapoptotic proteins in LPS-challenged mice. It also increased antioxidants and antiapoptotic protein Bcl2, thereby ameliorating lung tissue from LPS-induced inflammation. Our results indicate nigericin suppressed the LPS induced sepsis in an in vivo model and has the therapeutic potency to cure sepsis induced acute lung injury.

CD1d-dependent natural killer T-cells inactivation aggravates sepsis-induced myocardial injury via T lymphocytes infiltration and IL-6 production in mice

Myocardial edema mediated by endothelial dysfunction plays an important role in sepsis-induced cardiomyopathy (SIC); however, its mechanism is unclear. The current study aimed to provide evidence on the cardioprotection of CD1d-dependent natural killer T (NKT) cells and clarify the possible mechanism in a mouse model of sepsis. Wild-type (WT) and CD1d-dependent NKT-cells inactivation (CD1dko) mice were subjected to sepsis induced by intraperitoneal injection of lipopolysaccharide (LPS). The NKT-cells number and CD1d expression were both increased in the hearts and blood of WT mice after LPS treatment. Compared with WT mice, CD1dko mice exhibited remarkably accelerated LPS-induced mortality, cardiac dysfunction, myocardial injury, endothelial apoptosis, microvascular damage, microvascular permeability and cardiac edema. Mechanistically, CD1d deficiency further increased LPS-induced accumulation of T lymphocytes in the myocardium and upregulation of IL-6 protein levels. Administration of an IL-6 neutralizing antibody to CD1dko mice improved cardiac dysfunction, myocardial injury and edema induced by LPS. Our study identified that CD1d-dependent NKT-cells inactivation exacerbated SIC via T lymphocytes infiltration and IL-6 production. Hence, activation of CD1d-dependent NKT cells may be a potential candidate strategy for SIC treatment.

BmKK2, a thermostable Kv1.3 blocker from Buthus martensii Karsch (BmK) scorpion, inhibits the activation of macrophages via Kv1.3-NF-κB- NLRP3 axis

Ethnopharmacological relevanceInflammation plays pivotal role in the development of chronic diseases. Reducing chronic inflammation is an important strategy for preventing and managing many chronic diseases. In traditional Chinese medicine, the processed Buthus martensii Karsch (BmK) scorpion (also called “Quanxie”) has been used to treat chronic inflammatory arthritis and spondylitis for hundreds of years suggests that “Quanxie” could potentially be utilized as a resource for identifying new anti-inflammatory compounds. However, the molecular basis and the underline mechanism for the anti-inflammatory effect of processed BmK scorpion are still unclear. Aim of the studyThe study aims to determine the potential involvement of macrophage-expressed Kv1.3 in the anti-inflammatory effect of processed BmK scorpion venom, as well as to identify new Kv1.3 blockers derived from processed BmK scorpion. Materials and methodsIn this study, the in vivo and in vitro anti-inflammatory activities were determined using carrageenan-induced paw edema, LPS-induced sepsis mouse models and LPS-induced macrophage activation model respectively. The effect of processed BmK scorpion water extract, processed BmK venom and BmKK2 on different potassium channels were detected by whole-cell voltage-clamp recordings on transfected HEK293 cells or mouse BMDMs. The cytokines were detected using Q-PCR and competitive enzyme-linked immunosorbent assay. High performance liquid chromatography, SDS-PAGE and peptide Mass Spectrometry analysis were used to isolate and identify the BmKK2. SiRNA, western blotting and flow cytometry were used to analysis the anti-inflammatory mechanism of BmKK2. ResultsHere we demonstrate that BmKK2, a thermostable toxin targeting Kv1.3 is the critical anti-inflammatory component in the processed BmK scorpion. BmKK2 inhibits inflammation by targeting and inhibiting the activity of macrophage Kv1.3, thereby inhibiting the activation of NF-κB-NLRP3 pathway and the subsequent release of inflammatory factors. ConclusionsThese findings provide new insights into the molecular basis of the anti-inflammatory effects of “Quanxie” and highlight the importance of targeting Kv1.3 expressed on macrophages as an anti-inflammatory approach.

Clusterin Neutralizes the Inflammatory and Cytotoxic Properties of Extracellular Histones in Sepsis.

Rationale: Extracellular histones, released into the surrounding environment during extensive cell death, promote inflammation and cell death, and these deleterious roles have been well documented in sepsis. Clusterin (CLU) is a ubiquitous extracellular protein that chaperones misfolded proteins and promotes their removal. Objectives: We investigated whether CLU could protect against the deleterious properties of histones. Methods: We assessed CLU and histone expression in patients with sepsis and evaluated the protective role of CLU against histones in invitro assays and invivo models of experimental sepsis. Measurements and Main Results: We show that CLU binds to circulating histones and reduces their inflammatory, thrombotic, and cytotoxic properties. We observed that plasma CLU levels decreased in patients with sepsis and that the decrease was greater and more durable in nonsurvivors than in survivors. Accordingly, CLU deficiency was associated with increased mortality in mouse models of sepsis and endotoxemia. Finally, CLU supplementation improved mouse survival in a sepsis model. Conclusions: This study identifies CLU as a central endogenous histone-neutralizing molecule and suggests that, in pathologies with extensive cell death, CLU supplementation may improve disease tolerance and host survival.

Reorganization of resident macrophages and recruited monocytes in the heart during sepsis

Sepsis is defined as life-threatening organ dysfunction, caused by a dysregulated host response to infection. Survivors of sepsis have long-lasting consequences and notably an increased risk of developing cardiovascular disease. Changes in intra-organ immune homeostasis may be responsible for the observed chronic failures and lower resilience to new infections. To characterize reorganization in number and phenotype of resident macrophages and recruited monocytes in the heart during sepsis using a mouse model of sepsis. Using a sublethal cecal ligation and puncture (CLP) model of sepsis, macrophages subtypes were analyzed by flow cytometry for 21 days, and cardiac function was assessed by echocardiography and histology. Cx3cr1CreER/+R26tdTomato/+ mice were used for fate-mapping studies. In a model of sublethal sepsis induced by CLP (25% mortality, 15% weight loss), a fleeting and rapidly reversible alteration of cardiac function (24 h to 48 h) characterized by a decrease in the shortening fraction was observed. This alteration was not associated with cardiac fibrosis after histological analysis using Sirius red (0% at day 28). Surviving mice recovered their weight and normal activity on day 14. Leukocyte count showed a significant increase in intracardiac macrophages (CD45+, CD11b+, CD64+), with a maximum increase on day 14. Phenotypic analysis revealed a decrease in the pool of Timd4+ macrophages (resident macrophages), and a significant increase in the pool of CCR2+ macrophages (recruited macrophages). This increase persisted for 14 days. The MHC2+ macrophages (resident macrophages) population was globally stable over time. Fate mapping analysis showed a high turnover of the intracardiac macrophages pool (CD45+, CD11b+, CX3CR1+) with 40% turnover in 21 days in control mice. After CLP, we observed a significant decrease in the pool of resident macrophages, which is consistent with the disappearance reaction hypothesis. In this study, we described the evolution of tissue macrophages and variations in all three subsets of macrophages in the heart after sepsis. Using our fate mapping approach, we confirmed that the increased number of tissue macrophages after sepsis was mainly due to the recruitment of monocytes.

Homeodomain-interacting protein kinase 2 regulates NLRP3 inflammasome activation through endoplasmic reticulum stress in septic liver injury.

Septic liver injury is a major burden for the clinical management of sepsis. Hepatocyte cell death plays a crucial pathophysiological role in sepsis. A recent study proposed that NLRP3 inflammasome-mediated pyroptosis participates in septic liver injury. Therefore, investigating the mechanism controlling this process may help manage sepsis. We investigated the role of homeodomain-interacting protein kinase 2 (HIPK2) in regulating the NLRP3 inflammasome in vivo using mouse models and in vitro in primary hepatocytes. HIPK2 could improve liver injury and survival in a mouse model of sepsis. Overexpression of HIPK2 could suppress NLRP3 and caspase-1-p20 expression, while HIPK2 knockdown led to higher levels of these two molecules. Importantly, HIPK2 could suppress endoplasmic reticulum (ER) stress. Pharmacologically inhibiting ER stress could abolish activation of the NLRP3 inflammasome in hepatocytes with HIPK2 knockdown. HIPK2 can regulate ER stress and NLRP3 inflammasome activation in the liver during sepsis, and HIPK2-mediated suppression of ER stress participates in regulating NLRP3 inflammasome activation. The present study highlights the role of HIPK2 in regulating the inflammasome in septic liver injury, which may serve as a target for managing sepsis.

Poly(lactic acid)-based immunomodulatory nanoparticles promote survival in a polymicrobial mouse model of sepsis

Abstract Sepsis is a life-threatening multifactorial organ dysfunction caused by a dysregulated host response to infection. Despite being recognized as a global health priority, current therapeutic approaches for sepsis have yet to demonstrate significant improvements in patient outcomes. Our group previously demonstrated that cargo-less immunomodulatory nanoparticles (iNPs) could suppress innate immune cell proinflammatory responses and increase survival in an LPS-induced endotoxemia mouse model. In the present work, we examine the impact of poly(lactic acid)-based iNP delivery on survival and comprehensively characterize the host immune response using a clinically relevant and lethal mouse model of polymicrobial sepsis, cecal ligation and puncture (CLP). iNPs, when administered intraperitoneally in combination with broad spectrum antibiotics following CLP surgery, resulted in a significant improvement in disease symptoms and survival compared to CLP + antibiotics only. We also observed that iNP treatment reduced the levels of proinflammatory cytokines in the peritoneal fluid. Analysis of immune cell populations suggested that iNP treatment modulated macrophage, myeloid-derived suppressor cell, and inflammatory monocyte populations both locally and systemically. Our study demonstrates that local administration of iNPs promotes survival through a multimodal immunomodulatory mechanism to improve sepsis outcomes. This research was supported by startup funds from the University of Maryland School of Pharmacy, National Institute of General Medical Sciences of the National Institutes of Health under Award Number R35GM142752, and the Shock Society Faculty Research Award awarded to Ryan Pearson. Nhu Truong is supported by a PhRMA Foundation Pre-Doctoral Fellowship from Washington, DC. This publication was supported by funds through the National Cancer Institute - Cancer Center Support Grant (CCSG) - P30CA134274, and Maryland Department of Health's Cigarette Restitution Fund Program

Abstract 163: Thrombocytopenia Independently Leads To Monocyte Immune Dysfunction

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and leukocytes, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. We have now found that thrombocytopenia (a low platelet count) leads to monocyte dysfunction, independent of the cause of thrombocytopenia, in a manner that is dependent on direct platelet-monocyte CD47 interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) responses, including increased IL-6 production. Furthermore, ex vivo co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming and durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed persistently open chromatin at LPS response genes. Using mouse models of thrombocytopenia and sepsis we found that normal platelet numbers are needed to limit immune dysregulation in sepsis. IL6 and TNF expression in monocytes from human patients with sepsis also inversely correlated with patient platelet counts. Our studies demonstrate that in healthy conditions resting platelets maintain monocyte immune tolerance by regulating monocyte immunometabolic processes that lead to epigenetic changes in TLR-related genes. Platelets therefore function as immune rheostats in both health and disease.

Identification of nascent cardiovascular proteomes by cell-type-specific metabolic labeling in experimental sepsis mouse model

BACKGROUND – Sepsis is characterized by an overwhelming systemic inflammatory response to infection that may lead to multiple organ failure and death in over 20% of the estimated 49 million annual cases worldwide. Because sepsis-related inflammatory processes disturbing cardiac and endothelial cells homeostasis are still poorly understood, only few therapeutic strategies reached added-value efficacy in terms of mortality reduction in the critical care units. Using an innovative transgenic mouse line to detect the acute changes of newly synthetized proteins, we propose to assess the protein expression profile of cardiomyocytes and endothelial cells in a murine model of polymicrobial sepsis. METHODS – The Cre-recombinase mediated expression of mutant L274G-methionyl-tRNA synthetase (MetRS*) in transgenic mouse crosses enables the selective in vivo incorporation of azidonorleucine (ANL), a non-canonical methionine analog, in newly synthetized proteins. MetRS* mice expressing CDH5-creERT2 (CDH5, endothelial cells) or αMHC-MerCreMer (MCM, cardiomyocytes) underwent CLP (cecal ligation and puncture). The sepsis model was validated by echocardiography, inflammatory cytokine profiling and histology. Labeling efficiency of nascent proteins from MCM and CDH5 cells (heart and lung) were visualized by FUNCAT (Fluorescence non-canonical amino acid tagging) or purified by BONCAT (bioorthogonal non-canonical amino acid tagging) for mass spectrometry analysis. RESULTS – Significant elevation of circulating inflammatory cytokines proves the systemic inflammatory state of mice submitted to CLP compared to SHAM mice. Our experimental model also displays a significant impairment of the LV function at both systolic (reduced cardiac index) and diastolic levels (increased E/A and isovolumic relaxation time) confirming hemodynamics alteration related to sepsis. Metascape analysis showed expression changes for several proteins involved in the acute response to stress in both MCM and CDH5 samples. Overall, nascent protein synthesis is mainly reduced in MCM samples in sepsis (14 up/98 down), something not observed in CDH5 samples both in the heart (69 up/97 down) and in the lungs (137 up/43 down). Alterations in metabolic pathways such as pyruvate metabolism and glycolysis/gluconeogenesis were only observed in MCM samples while protein associated with focal adhesion, for example, was observed in heart and lungs CDH5 samples. CONCLUSIONS – The results of this study provide a better understanding of which proteins are altered in specific cell types in a sepsis model. We confirmed previously identified markers of inflammatory response in tissues, while identifying new protein candidates and signaling pathways that may contribute to key steps of sepsis detrimental effects and thus, offering potential new therapeutic avenues to curb the fatal outcomes of this disease. Supported by Canadian Institute of Health Research (CIHR) - Project Grants (399567) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Premorbid use of selective beta-blockers improves sepsis incidence and course: Human cohort and animal model studies.

Beta-blockers are widely prescribed to manage hypertension and cardiovascular diseases and have been suggested as an attractive therapy to improve the prognosis of sepsis. Herein, we investigated the potential benefits of premorbid selective beta-blocker use in sepsis with a real-world database and explored the underlying mechanism by in vivo and in vitro experiments. A total of 64,070 sepsis patients and 64,070 matched controls who were prescribed at least one anti-hypertensive drug for more than 300 days within 1 year were selected for the nested case-control study. Female C57BL/6 J mice and THP-1 cells stimulated with lipopolysaccharide (LPS) were used for studying systemic responses during sepsis to validate our clinical findings. The risk of sepsis was lower in current selective beta-blocker users than in non-users (adjusted OR (aOR), 0.842; 95% CI, 0.755-0.939), and in recent users than in non-users (aOR, 0.773; 95% CI, 0.737-0.810). A mean daily dose of ≥0.5 DDD was associated with a lower risk of sepsis (aOR, 0.7; 95% CI, 0.676-0.725). Metoprolol, atenolol, and bisoprolol users had lower risk of sepsis than non-users. In a LPS-induced sepsis mouse model, mice pre-fed with atenolol had significantly reduced mortality. While atenolol had some mild effects on LPS-induced release of inflammatory cytokines in septic mice, it significantly reduced serum soluble PD-L1 levels. Notably, atenolol treatment reversed the negative correlation of sPD-L1 with inflammatory cytokines in septic mice. Moreover, atenolol markedly downregulated the PD-L1 expression on LPS-stimulated THP-1 monocytes/macrophages via targeting ROS-induced NF-κB and STAT3 activation. Atenolol pretreatment can reduce sepsis mortality in mice, and in vivo and in vitro studies of PD-L1 expression suggest a role for atenolol in the modulation of immune homeostasis. These findings may contribute to the reduced incidence of sepsis in hypertensive patients with premorbid treatment with selective beta-blockers, especially atenolol.

Bioactive compound C498-0670 alleviates LPS-induced sepsis via JAK/STAT and NFκB signaling pathways.

The JAK/STAT and NFκB signaling pathways are two major inflammatory signaling pathways that are usually activated simultaneously in the body's inflammatory response to bacterial or viral infections. Hyperactivation of these two prominent signaling pathways is associated with various immune-related diseases and mortality, pointing to an urgent need for drug development targeting JAK/STAT and/or NFκB signaling. In this study, we screened 18,840 compounds using our well-established dual STAT-NFκB driven luciferase reporter based high-throughput screening system and identified a bioactive compound C498-0670, which inhibits both JAK/STAT and NFκB signaling. C498-0670 inhibits the activation of STATs and p-IKKα/β in both the immortalized cell lines and primary peritoneal macrophages, while suppressing the expression of LPS-induced inflammatory mediators in vitro. In addition, the overall anti-inflammatory effects of C498-0670 were investigated using transcriptome sequencing and bioinformatics approaches. C498-0670 was predicted to alleviate sepsis/septic shock by disease/function analysis using IPA software, which was further verified in the LPS-induced mouse sepsis model in vivo. C498 reduced LPS-induced liver and kidney damage, myeloid cell infiltration, and pro-inflammatory cytokine and chemokine production in vivo. Furthermore, the SPR-HPLC-MS-based target fishing approach was used to identify the putative drug targets, and the high affinities of JAK2 (JAK/STAT signaling), NFKBIA (NFκB signaling), and IL-1β, NLRP1b (inflammasome signaling) for C498-0670 were verified by molecular docking approach. These results suggest that C498-0670 can be used as a dual-target inhibitor of JAK/STAT and NFκB signaling pathways for the treatment of various inflammatory diseases, especially septic shock.