Single-cell RNA sequencing identifies CCR4 as a therapeutic target for sepsis with DB07845 as a potential inhibitor

Sepsis results in lethal organ malfunction due to dysregulated host response to infection, which is a condition with increasing prevalence worldwide. Transglutaminase 2 (TG2) is a crosslinking enzyme that forms a covalent bond between lysine and glutamine. TG2 plays important roles in diverse cellular processes, including extracellular matrix stabilization, cytoskeletal function, cell motility, adhesion, signal transduction, apoptosis, and cell survival. We have shown that the co-culture of Candida albicans and hepatocytes activates and induces the translocation of TG2 into the nucleus. In addition, the expression and activation of TG2 in liver macrophages was dramatically induced in the lipopolysaccharide-injected and cecal ligation puncture-operated mouse models of sepsis. Based on these findings and recently published research, we have reviewed the current understanding of the relationship between TG2 and sepsis. Following the genetic and pharmacological inhibition of TG2, we also assessed the evidence regarding the use of TG2 as a potential marker and therapeutic target in inflammation and sepsis.

Recently, Fiusa and colleagues [1] described that a high angiopoietin (Ang)-2/Ang-1 ratio is associated with a high risk of septic shock in patients with febrile neutropenia. Although this study failed to detect a statistically significant difference in the levels of vascular endothelial growth factor (VEGF) between both outcome groups, the observed increased Ang-2 concentrations and Ang-2/Ang-1 ratio in the febrile neutropenia subgroup of patients with septic shock address the concept that angiogenic factors could be a promising therapeutic target in sepsis. The imbalance of angiogenic factors and their receptors, including the Ang/Tie2 and VEGF/VEGF receptor pathways, has been shown to predict poor prognosis in sepsis and has been implicated as a contributing factor in multiple organ dysfunction. For these reasons, several strategies targeting Ang/Tie2 and VEGF/VEGF receptor are being explored in preclinical sepsis models; however, most of these agents are unavailable or not applicable in clinical practice. Efforts to develop anticancer agents that target angiogenesis have led to the approval of inhibitors of VEGF and its receptor, specifically bevacizumab. As recently reported in Critical Care, bevacizumab has proven effective in the treatment of sepsis in an animal model [2]. The current study reinforces and extends previous findings that Ang-2 levels are increased in the septic state and have been established as a prognostic biomarker for identification of the patient group with the highest mortality rates. Interestingly, most recently by using Ang-2 antibody treatment, Ziegler and colleagues [3] showed that inhibition of Ang-2 has a significant effect on survival of mice with sepsis. AMG 386 is an investigational peptide-Fc fusion protein that inhibits angiogenesis by preventing the interaction of Ang-1/2 with their receptor, Tie2. The human study demonstrated that intravenous AMG 386 appeared well tolerated, and its safety profile seemed to be distinct from that of VEGF axis inhibitors [4]; it has reached late stages of clinical development. This provides a basis for Ang-2 inhibition as a potential clinical-translational therapeutic target in sepsis. The distinct safety profiles of AMG 386 and the VEGF-axis inhibitors suggest that they could be combined to achieve cooperative action, which may thereby improve clinical efficacy without significant overlapping toxicities. Thus, recent findings indicate that angiogenic factors are potential therapeutic targets in sepsis. However, the safety profiles of these anti-angiogenic agents in septic patients are often complicated by organ dysfunction and may be different from those in cancer patients. Future clinical trials may be needed to evaluate the safety and efficacy of these anti-angiogenic agents in sepsis patients, either alone or in combination.

Sepsis is characterized by a systemic host response to infection. Monocytes, as major mediators of acute infection, are implicated in complications among critically ill patients. Identifying key monocyte subsets and their activation states is essential for diagnosis and delineating new therapeutic targets for sepsis. Here, single cell transcriptome sequencing and mass cytometry are used to assess alterations in the composition and function of peripheral monocytes of patients with sepsis, and CD38high monocytes in circulation are specifically accumulated within the first 24 h of sepsis. CD38high monocytes are detectable by conventional flow cytometry to discriminate sepsis and sterile inflammation, and are associated with 28‐day mortality in bacterial sepsis. Targeting CD38 therapy markedly reduces inflammatory response in primary monocytes and in sepsis mice model. Mechanistically, CD38high monocytes in sepsis exhibit hyperactivated glycolysis with activation of hypoxia‐inducible factor‐1α (HIF‐1α) due to NAD+ consumption. Glycolytic metabolite methylglyoxal (MGO) is found to regulate expression of CD38, establishing a CD38‐HIF‐1α/glycolysis/MGO loop that exacerbates sepsis‐induced immune dysregulation. These findings demonstrate that CD38high monocytes might serve as a candidate diagnostic biomarker and therapeutic target for sepsis.

Background: The exact molecular mechanisms underlying sepsis remain unclear. Accumulating evidence has shown that noncoding RNAs (ncRNAs) are involved in sepsis and sepsis-associated organ dysfunction (SAOD). Methods: We performed this updated systematic review focusing mainly on research conducted in the last 5 years regarding ncRNAs associated with sepsis and SAOD. The following medical subject headings were used in the PubMed database from October 1, 2016, to March 31, 2022: "microRNA," "long noncoding RNA," "circular RNA," "sepsis," and/or "septic shock." Studies investigating the role of ncRNAs in the pathogenesis of sepsis and as biomarkers or therapeutic targets in the disease were included. Data were extracted in terms of the role of ncRNAs in the pathogenesis of sepsis and their applicability for use as biomarkers or therapeutic targets in sepsis. The quality of the studies was assessed using a modified guideline from the Systematic Review Center for Laboratory Animal Experimentation. Results: A total of 537 original studies investigated the potential roles of ncRNAs in sepsis and SAOD. Experimental studies in the last 5 years confirmed that long ncRNAs have important regulatory roles in sepsis and SAOD. However, studies on circular RNAs and sepsis remain limited, and more studies should be conducted to elucidate this relationship. Among the included studies, the Systematic Review Center for Laboratory Animal Experimentation scores ranged from 3 to 7 (an average score of 3.78). Notably, 94 ncRNAs were evaluated as potential biomarkers for sepsis, and selective reporting of the sensitivity, specificity, and receiver operating characteristic curve was common. A total of 117 studies demonstrated the use of ncRNAs as potential therapeutic targets in sepsis and SAOD. At a molecular level, inflammation-related pathways, mitochondrial dysfunction, cell apoptosis, and/or oxidative stress were the most extensively studied. Conclusion: This review suggests that ncRNAs could be good biomarkers and therapeutic candidates for sepsis and SAOD. Prospective, large-scale, and multicenter cohort studies should be performed to evaluate specific ncRNAs as biomarkers and test the organ-specific delivery of these regulatory molecules when used as therapeutic targets.

Sepsis is a life-threatening condition characterized by a dysregulated host response to infection, resulting in high mortality rates and complex clinical management. This study leverages transcriptomics and machine learning (ML) to identify critical biomarkers and therapeutic targets in sepsis. Analyzing microarray data from the Gene Expression Omnibus (GEO) datasets GSE28750, GSE26440, GSE13205, and GSE9960, we discovered three pivotal biomarkers that BMX (bone marrow tyrosine kinase gene on chromosome X), GRB10 (growth factor receptor bound protein 10), and GADD45A (growth arrest and DNA damage inducible alpha), exhibiting exceptional diagnostic accuracy (AUC >0.9). Functional enrichment analyses revealed that these genes play key roles in reactive oxygen species metabolism and immune response regulation. Specifically, GADD45A was positively correlated with eosinophils and inversely associated with activated NK cells, CD8 T cells, and activated memory CD4 T cells. BMX showed positive correlations with eosinophils, mast cells, and neutrophils, while GRB10 was linked to eosinophils and M2 macrophages. Additionally, we constructed a comprehensive mRNA-miRNA-lncRNA regulatory network, identifying key interactions that may drive sepsis pathogenesis. Molecular docking and dynamics simulations validated Bendroflumethiazide, Cianidanol, and Hexamidine as promising therapeutic agents targeting these biomarkers. In conclusion, this integrated approach provides profound insights into the molecular mechanisms underlying sepsis, pinpointing BMX, GRB10, and GADD45A as pivotal biomarkers and therapeutic targets. These findings significantly enhance our understanding of sepsis pathophysiology and lay the groundwork for developing personalized diagnostic and therapeutic strategies aimed at improving patient outcomes.

Recent research has yielded many interesting and potentially important therapeutic targets in sepsis. Specifically, the effects of antagonistic anti-cytokine therapies (tumor necrosis factor-alpha [TNF-alpha], interleukin-1 [IL-1]) and anti-endotoxin strategies utilizing antibodies against endotoxin or endotoxin receptor/carrier molecules (anti-CD14 or anti-LPS-binding protein) have been studied. Unfortunately, these approaches often failed clinically, and in many cases, the efficacy of these treatments was dependent on the severity of sepsis. Recently, clinical trials using insulin to lock blood glucose levels and activated protein C treatment have showed that while they provided some survival benefit, their efficacy does not appear to be predicated solely upon anti-inflammatory effects. Here, we will review work done in animal models of polymicrobial sepsis and clinical findings that support the hypothesis that apoptosis in the immune system is a pathologic event in sepsis that can be a therapeutic target. In this respect, experimental studies looking at the septic animal suggest that loss of lymphocytes during sepsis may be due to dysregulated apoptosis and that this appears to be brought on by a variety of mediators effecting 'intrinsic' as well as 'extrinsic' cell death pathways. From a therapeutic perspective this has provided a number of novel targets for clinically successful current, as well as future therapies, such as caspases (caspase inhibition/protease inhibition), pro-apoptotic protein-expression (via administration and/or over-expression of Bcl-2) and the death receptor family Fas-FasL (via. FasFP [fas fusion protein] and the application of siRNA against a number pro-apoptotic factors).

According to “Sepsis-3” consensus, sepsis is a life-threatening clinical syndrome caused by a dysregulated inflammatory host response to infection. A rapid identification of sepsis is mandatory, as the extent of the organ damage triggered by both the pathogen itself and the host’s immune response could abruptly evolve to multiple organ failure and ultimately lead to the death of the patient. The most commonly used therapeutic strategy is to provide hemodynamic and global support to the patient and to rapidly initiate broad-spectrum empiric antibiotic therapy. To date, there is no gold standard diagnostic test that can ascertain the diagnosis of sepsis. Therefore, once sepsis is suspected, the presence of organ dysfunction can be assessed using the Sepsis-related Organ Failure Assessment (SOFA) score, although the diagnosis continues to depend primarily on clinical judgment. Clinicians can now rely on several serum biomarkers for the diagnosis of sepsis (e.g., procalcitonin), and promising new biomarkers have been evaluated, e.g., presepsin and adrenomedullin, although their clinical relevance in the hospital setting is still under discussion. Non-codingRNA, including long non-codingRNAs (lncRNAs), circularRNAs (circRNAs) and microRNAs (miRNAs), take part in a complex chain of events playing a pivotal role in several important regulatory processes in humans. In this narrative review we summarize and then analyze the function of circRNAs-miRNA-mRNA networks as putative novel biomarkers and therapeutic targets for sepsis, focusing only on data collected in clinical settings in humans.

The CYP2E1 inhibitor Q11 ameliorates LPS-induced sepsis in mice by suppressing oxidative stress and NLRP3 activation

Background: Sepsis is one of the main causes of death in critically ill patients with high morbidity and mortality. Circular RNAs (CircRNAs) are aberrantly expressed, and play significant regulatory roles in many diseases. However, the expression profiles and functions of circRNAs in sepsis have not yet been fully clarified.Methods: Our present study performed an RNA sequencing (RNA-seq) analysis to assess the expression profiles of circRNAs in vitro. We applied the quantitative real-time polymerase chain reaction (RT-qPCR) to verify the RNA-seq results. The analyses of Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, the competitive endogenous RNA (ceRNA) regulatory networks, were performed to explore the potential mechanism in sepsis. And then, significantly up-regulated differentially expressed (DE) circRNA, hsa_circ_0074158, was selected for further study. Hsa_circ_0074158 was silenced to investigate its regulatory function in sepsis, and the barrier function was also examined in vitro. Endothelial cell junctions were valued using Vascular endothelial cadherin (VE-cadherin), which was detected by immunofluorescence staining. We measured endothelial permeability by transendothelial electrical resistance (TEER) and fluorescein isothiocyanate (FITC)-dextran extravasation.Results: In total, 203 significantly DE circRNAs, including 77 up-regulated and 126 down-regulated, were identified. In vitro, the RT-qPCR assay showed that the expression pattern of hsa_circ_0074158, hsa_circ_RSBN1L_11059, hsa_circ_0004188, and hsa_circ_0005564 were consistent with the results from RNA-seq analysis. The expression of hsa_circ_0074158 detected by RT-qPCR in vivo was also consistent with the RNA-seq results. The ceRNA networks, GO enrichment, and the KEGG pathway analyses revealed that circRNAs may be related to the barrier function in sepsis. The immunofluorescence assay showed that the suppression of hsa_circ_0074158 expression significantly enhanced the expression of VE-cadherin, which was suppressed in lipopolysaccharide (LPS)-induced sepsis. Additionally, hsa_circ_0074158 knockdown could partially reverse the LPS-induced TEER reduction and FITC-dextran extravasation elevation in sepsis.Conclusion: In conclusion, we have found DE circRNAs could serve as potential biomarkers and therapeutic targets for sepsis. Hsa_circ_0074158 plays a vital role in sepsis and is related to the disruption of the endothelial barrier.

BackgroundCuproptosis is a copper-dependent cell death that is connected to the development and immune response of multiple diseases. However, the function of cuproptosis in the immune characteristics of sepsis remains unclear. MethodWe obtained two sepsis datasets (GSE9960 and GSE134347) from the GEO database and classified the raw data with R packages. Cuproptosis-related genes were manually curated, and differentially expressed cuproptosis-related genes (DECuGs) were identified. Afterwards, we applied enrichment analysis and identified key DECuGs by performing machine learning techniques. Then, the immune cell infiltrations and correlation between DECuGs and immunocyte features were created by the CIBERSORT algorithm. Subsequently, unsupervised hierarchical clustering analysis was performed based on key DECuGs. We then constructed a ceRNA network based on key DECuGs by using multi-step computational strategies and predicted potential drugs in the DrugBank database. Finally, the role of these key genes in immune cells was validated at the single-cell RNA level between septic patients and healthy controls. ResultsOverall, 16 DECuGs were obtained, and most of them had lower expression levels in sepsis samples. Afterwards, we obtained six key DECuGs by performing machine learning. Then, the LIPT1-T-cell CD4 memory resting was the most positively correlated DECuG–immunocyte pair. Subsequently, two different subclusters were identified by six DECuGs. Bioinformatics analysis revealed that there were different immune characteristics between the two subclusters. Moreover, we identified the key lncRNA OIP5-AS1 within the ceRNA network and obtained 4 drugs that may represent novel drugs for sepsis. Finally, these key DECuGs were statistically significantly dysregulated in another validation set and showed a major distribution in monocytes, T cells, B cells, NK cells and platelets at the single-cell RNA level. ConclusionThese findings suggest that cuproptosis might promote the progression of sepsis by affecting the immune system and metabolic dysfunction, which provides a new direction for understanding potential pathogenic processes and therapeutic targets in sepsis.

SESSION TITLE: Tuesday Electronic Posters 2 SESSION TYPE: Original Inv Poster Discussion PRESENTED ON: 10/22/2019 01:00 PM - 02:00 PM PURPOSE: Septic cardiomyopathy (SCM) is common in sepsis and associated with increased morbidity and mortality. Left ventricular global longitudinal strain (LV GLS), measured by speckle tracking echocardiography, is a new and accurate marker of SCM, allowing improved interrogation of the pathophysiology. The peripheral blood transcriptome may be an important window into SCM pathophysiology. We therefore studied the peripheral blood transcriptome in a prospective cohort of patients with sepsis. METHODS: In this single-center observational study, we enrolled adult patients (age >18) with sepsis within 48 hrs of admission to the ICU. SCM was defined as LV GLS > -17% based on echocardiograms performed within 72 hrs of admission. RNA sequencing was performed on blood drawn at the time of enrollment using established methods and differentially expressed genes identified with a 5% false discovery rate. Ingenuity Pathway Analysis (IPA) software was used for analysis of significant genes. RESULTS: We enrolled 27 patients, 24 of whom had high-quality RNA results; 18 had SCM. The cohort was 50% female and had a mean (SD) age of 57 (13) and admission APACHE II score of 24 (10). Forty-six percent of the cohort had septic shock. After filtering for low expression and non-coding genes, 15418 protein coding genes were expressed and 27 were significantly differentially expressed between groups (SCM versus no-SCM: 15 genes upregulated, 12 genes downregulated). Pathway analysis identified enrichment in Interferon signaling (adjusted p<0.00001), with 4 of the pathway genes upregulated in patients with SCM (IFIT1, IFIT3, ISG15, OAS1). CONCLUSIONS: In this hypothesis-generating study, SCM was associated with upregulation of genes in the interferon signaling pathway. Interferons are cytokines that stimulate the innate and adaptive immune response, implicated in the early proinflammatory and delayed immunosuppression phases of sepsis. IFIT1, IFIT3, ISG15 and OAS1 are downstream effectors of Type 1 (IFN alpha/beta) interferon signaling. While type 1 interferons have not been implicated previously in SCM, interferon therapy (for viral hepatitis and Kaposi’s sarcoma) has been associated with reversible cardiomyopathy, perhaps suggesting a causative role of interferon signaling in SCM. CLINICAL IMPLICATIONS: The possible role of interferon signaling in SCM pathophysiology warrants further investigation. Identifying the molecular mechanisms of SCM may ultimately identify therapeutic targets in sepsis. DISCLOSURES: No relevant relationships by Theodore Abraham, source=Web Response No relevant relationships by Sarah Beesley, source=Web Response No relevant relationships by Samuel Brown, source=Web Response No relevant relationships by Meghan Cirulis, source=Web Response No relevant relationships by Colin Grissom, source=Web Response no disclosure on file for Eliotte Hirshberg; No relevant relationships by Michael Lanspa, source=Web Response No relevant relationships by Matthew Rondina, source=Web Response No relevant relationships by Lane Smith, source=Web Response No relevant relationships by Chris Stubben, source=Web Response No relevant relationships by Emily Wilson, source=Web Response

Sepsis is a complex disease due to dysregulated host response to infection. Oxidative stress and mitochondrial dysfunction leading to metabolic dysregulation are among the hallmarks of sepsis. The transcription factor NRF2 (Nuclear Factor E2-related factor2) is a master regulator of the oxidative stress response, and the NRF2 mediated antioxidant response is negatively regulated by BTB and CNC homology 1 (BACH1) protein. This study tested whether Bach1 deletion improves organ function and survival following polymicrobial sepsis induced by cecal ligation and puncture (CLP). We observed enhanced post-CLP survival in Bach1−/− mice with a concomitantly increased liver HO-1 expression, reduced liver injury and oxidative stress, and attenuated systemic and tissue inflammation. After sepsis induction, the liver mitochondrial function was better preserved in Bach1−/− mice. Furthermore, BACH1 deficiency improved liver and lung blood flow in septic mice, as measured by SPECT/CT. RNA-seq analysis identified 44 genes significantly altered in Bach1−/− mice after sepsis, including HMOX1 and several genes in lipid metabolism. Inhibiting HO-1 activity by Zinc Protoporphyrin-9 worsened organ function in Bach1−/− mice following sepsis. We demonstrate that mitochondrial bioenergetics, organ function, and survival following experimental sepsis were improved in Bach1−/− mice through the HO-1-dependent mechanism and conclude that BACH1 is a therapeutic target in sepsis.

Sepsis, an infection-induced systemic inflammatory disorder, is often accompanied by multiple organ dysfunction syndromes with high incidence and mortality rates, and those who survive are often left with long-term sequelae, bringing great burden to social economy. Therefore, novel approaches to solve this puzzle are urgently needed. Previous studies revealed that long non-coding RNAs (lncRNAs) have exerted significant influences on the process of sepsis. The aim of this review is to summarize our understanding of lncRNAs as potential sepsis-related diagnostic markers and therapeutic targets, and provide new insights into the diagnosis and treatment for sepsis. In this study, we also introduced the current diagnostic markers of sepsis and discussed their limitations, while review the research advances in lncRNAs as promising biomarkers for diagnosis and prognosis of sepsis. Furthermore, the roles of lncRNAs in sepsis-induced organ dysfunction were illustrated in terms of different organ systems. Nevertheless, further studies should be carried out to elucidate underlying molecular mechanisms and pathological process of sepsis.

Morbidity and mortality from severe sepsis remain high, despite decades of research and improvements in intensive care unit (ICU) care. There have been over 100 failed clinical trials of biological response modifiers aimed at single therapeutic targets, mostly to suppress the early pro-inflammatory responses. In the last decade, extracellular HMGB1 has emerged as a late mediator of sepsis in murine sepsis models, whose blockade improves mortality and has a wider therapeutic window than previous efforts. Although this review promulgates the use of HMGB1 inhibitor as a therapeutic target, it should be recognized that it may not be an optimal approach to the early systemic inflammatory response syndrome (SIRS) response and cytokine storm, but rather for those patients who survive their cytokine storm and present with a persistent inflammatory, immunosuppressive and catabolism response (PICS). With earlier implementation of evidence-based best care principles for treating sepsis, fewer patients are dying from early septic shock, and there is an endemic increase in sepsis survivors with dismal long-term outcomes. These patients have ongoing inflammatory processes that may well be driven by the late and continued release of HMGB1 and other damage-associated molecular patterns receptors (DAMPRs). HMGB1 therapeutics, whether antibodies or natural herbal approaches, may be one novel approach for targeting not the early, but the late persistent inflammation of sepsis survivors.

Introduction: Sepsis is a life-threatening complication of a bacterial infection. It is hard to predict which patients with a bacterial infection will develop sepsis, and accurate and timely diagnosis as well as assessment of prognosis is difficult. Aside from antibiotics-based treatment of the causative infection and supportive measures, treatment options have remained limited. Better understanding of the immuno-pathophysiology of sepsis is expected to lead to improved diagnostic and therapeutic solutions.Functional activity of the innate (inflammatory) and adaptive immune response is controlled by a dedicated set of cellular signal transduction pathways, that are active in the various immune cell types. To develop an immune response-based diagnostic assay for sepsis and provide novel therapeutic targets, signal transduction pathway activities have been analyzed in whole blood samples from patients with sepsis.Methods: A validated and previously published set of signal transduction pathway (STP) assays, enabling determination of immune cell function, was used to analyze public Affymetrix expression microarray data from clinical studies containing data from pediatric and adult patients with sepsis. STP assays enable quantitative measurement of STP activity on individual patient sample data, and were used to calculate activity of androgen receptor (AR), estrogen receptor (ER), JAK-STAT1/2, JAK-STAT3, Notch, Hedgehog, TGFβ, FOXO-PI3K, MAPK-AP1, and NFκB signal transduction pathways.Results: Activity of AR and TGFβ pathways was increased in children and adults with sepsis. Using the mean plus two standard deviations of normal pathway activity (in healthy individuals) as threshold for abnormal STP activity, diagnostic assay parameters were determined. For diagnosis of pediatric sepsis, the AR pathway assay showed high sensitivity (77%) and specificity (97%), with a positive prediction value (PPV) of 99% and negative prediction value (NPV) of 50%. For prediction of favorable prognosis (survival), PPV was 95%, NPV was 21%. The TGFβ pathway activity assay performed slightly less for diagnosing sepsis, with a sensitivity of 64% and specificity of 98% (PPV 99%, NPV 39%).Conclusion: The AR and TGFβ pathways have an immunosuppressive role, suggesting a causal relation between increased pathway activity and sepsis immunopathology. STP assays have been converted to qPCR assays for further evaluation of clinical utility for sepsis diagnosis and prediction of prognosis, as well as for prediction of risk at developing sepsis in patients with a bacterial infection. STPs may present novel therapeutic targets in sepsis.