Project description:Background: Sepsis involves aberrant immune responses to infection, but the exact nature of this immune dysfunction remains poorly defined. Bacterial endotoxins like lipopolysaccharide (LPS) are potent inducers of inflammation, which has been associated with the pathophysiology of sepsis, but repeated exposure can also induce a suppressive effect known as endotoxin tolerance or cellular reprogramming. It has been proposed that endotoxin tolerance might be associated with the immunosuppressive state that was primarily observed during late-stage sepsis. However, this relationship remains poorly characterised. Here we clarify the underlying mechanisms and timing of immune dysfunction in sepsis. Methods: We defined a gene expression signature characteristic of endotoxin tolerance. Gene-set test approaches were used to correlate this signature with early sepsis, both newly and retrospectively analysing microarrays from 593 patients in 11 cohorts. Then we recruited a unique cohort of possible sepsis patients at first clinical presentation in an independent blinded controlled observational study to determine whether this signature was associated with the development of confirmed sepsis and organ dysfunction. Findings: All sepsis patients presented an expression profile strongly associated with the endotoxin tolerance signature (p < 0.01; AUC 96.1%). Importantly, this signature further differentiated between suspected sepsis patients who did, or did not, go on to develop confirmed sepsis, and predicted the development of organ dysfunction. Interpretation: Our data support an updated model of sepsis pathogenesis in which endotoxin tolerance-mediated immune dysfunction (cellular reprogramming) is present throughout the clinical course of disease and related to disease severity. Thus endotoxin tolerance might offer new insights guiding the development of new therapies and diagnostics for early sepsis. For the RNA-Seq study reported here, 73 patients were recruited with deferred consent at the time of first examination in an emergency ward based on the opinion of physicians that there was a potential for the patient's condition to develop into sepsis. These were retrospectively divided into groups based on clinical features and compared to 11 non-urgent surgical controls.
Project description:Background: Sepsis involves aberrant immune responses to infection, but the exact nature of this immune dysfunction remains poorly defined. Bacterial endotoxins like lipopolysaccharide (LPS) are potent inducers of inflammation, which has been associated with the pathophysiology of sepsis, but repeated exposure can also induce a suppressive effect known as endotoxin tolerance or cellular reprogramming. It has been proposed that endotoxin tolerance might be associated with the immunosuppressive state that was primarily observed during late-stage sepsis. However, this relationship remains poorly characterised. Here we clarify the underlying mechanisms and timing of immune dysfunction in sepsis. Methods: We defined a gene expression signature characteristic of endotoxin tolerance. Gene-set test approaches were used to correlate this signature with early sepsis, both newly and retrospectively analysing microarrays from 593 patients in 11 cohorts. Then we recruited a unique cohort of possible sepsis patients at first clinical presentation in an independent blinded controlled observational study to determine whether this signature was associated with the development of confirmed sepsis and organ dysfunction. Findings: All sepsis patients presented an expression profile strongly associated with the endotoxin tolerance signature (p < 0.01; AUC 96.1%). Importantly, this signature further differentiated between suspected sepsis patients who did, or did not, go on to develop confirmed sepsis, and predicted the development of organ dysfunction. Interpretation: Our data support an updated model of sepsis pathogenesis in which endotoxin tolerance-mediated immune dysfunction (cellular reprogramming) is present throughout the clinical course of disease and related to disease severity. Thus endotoxin tolerance might offer new insights guiding the development of new therapies and diagnostics for early sepsis.
Project description:Identifying at first clinical presentation gene expression signatures that predict subsequent severity will allow clinicians to identify the most at risk groups of patients, and also enable appropriate antibiotic use. Accordingly, we characterized the blood immune profiles of patients with early/pre-sepsis to identify signatures reflecting disease severity, organ dysfunction, mortality, and specific endotypes/mechanisms.
Project description:To evaluate whether clarithromycin improves 28-day mortality among patients with sepsis, respiratory and multiple-organ dysfunction syndrome. The INntravenous CLArithromycin in Sepsis and multiple organ dysfunction Syndrome trial was a phase 3, randomized, double blind, placebo-controlled clinical study, conducted in 11 intensive care units and 2 Internal Medicine wards in 2 countries. Patients with sepsis, respiratory failure and total sequential organ failure assessment score of ≥7 were enrolled between December 2017 and September 2019. Follow-up lasted 90 days. Patients were randomized to receive 1 gr of intravenous clarithromycin or placebo once daily for 4 consecutive days.
Project description:Despite the prevalence and recognition of its detrimental impact, clinical complications of sepsis remain a major challenge. Here, we investigated the effects of myeloid ferritin heavy chain (FtH) in regulating the pathogenic sequelae of sepsis. We demonstrate that deletion of myeloid FtH leads to tolerance towards sepsis as evidenced by reduced serum cytokine levels, multi-organ dysfunction and subsequent mortality. We identified that such tolerance is predominantly mediated by the compensatory increase in circulating ferritin (ferritin light chain; FtL) in the absence of myeloid FtH. Our in vitro and in vivo studies indicate that prior exposure to ferritin provides significant tolerance to the septic process by restraining an otherwise dysregulated response to infection. These findings are mediated by an inhibitory action of ferritin on NF-κB activation and its downstream effects. Taken together, our findings suggest an essential immunomodulatory function for circulating ferritin and enhances our understanding of this acute phase reactant.
Project description:There is currently no reliable tool available to measure immune dysfunction in septic patients in the clinical setting. This proof-of-concept study assesses the potential of gene expression profiling of whole blood as a tool to monitor immune dysfunction in critically ill septic patients. Whole blood samples were collected daily for up to 5 days from patients admitted to the intensive care unit with sepsis. RNA isolated from whole blood samples was assayed on Illumina HT-12 gene expression microarrays consisting of 48,804 probes. Microarray analysis identified 3677 genes as differentially expressed across 5 days between septic patients and healthy controls. Of the 3677 genes, biological pathway analysis identified 86 genes significantly down-regulated in the sepsis patients were present in pathways relating to immune response. These 86 genes correspond to known immune pathways implicated in sepsis including lymphocyte depletion, reduced T lymphocyte activation and deficient antigen presentation. Furthermore, expression levels of these genes correlated with clinical severity, with a significantly greater degree of down-regulation found in non-survivors compared to survivors. The results show that whole blood gene-expression analysis can capture systemic immune dysfunctions in septic patients. Our study provides an experimental basis to support further study on the use of a gene expression based assay, to assess immunosuppression and guide immunotherapy in future clinical trials. Daily PAXgene samples for up to 5 days for sepsis survivors (n=26), sepsis nonsurvivors (n=9), and healthy controls (n=18).
Project description:Microbial challenges, such as widespread bacterial infection, induce endotoxin tolerance. This state of hypo-responsiveness to subsequent infections is mainly displayed by monocytes and macrophages. Endotoxin tolerance is generally acquired following a septic episode. In this study, we investigated DNA methylation changes during the acquisition of in vitro tolerance. We identified a set of TET2-mediated demethylation events that are specific to toll like receptor (TLR) 2 and 4 stimulation. Lipopolysaccharide (LPS)-specific demethylation occurs at genomic sites that have low accessibility in quiescent monocytes, concomitantly with the transcriptional activation of many inflammation-related genes, and they are enriched in binding motifs for several signal transducer and activator of transcription (STAT) family members. Indeed, STAT1, STAT3 and STAT5, elements of the JAK2 pathway, are phosphorylated in association with the acquisition of endotoxin tolerance. Inhibition of the JAK2 pathway impairs the activation of tolerized genes at the first encounter with LPS. This supports a crucial role of this pathway in determining the initial response of these genes to bacterial antigens and provides a pharmacological target to prevent exacerbated responses, allowing regulated responses upon subsequent challenges. Finally, we prove the pathological relevance of the JAK2 pathway in monocytes from patients with sepsis.
Project description:Microbial challenges, such as widespread bacterial infection, induce endotoxin tolerance. This state of hypo-responsiveness to subsequent infections is mainly displayed by monocytes and macrophages. Endotoxin tolerance is generally acquired following a septic episode. In this study, we investigated DNA methylation changes during the acquisition of in vitro tolerance. We identified a set of TET2-mediated demethylation events that are specific to toll like receptor (TLR) 2 and 4 stimulation. Lipopolysaccharide (LPS)-specific demethylation occurs at genomic sites that have low accessibility in quiescent monocytes, concomitantly with the transcriptional activation of many inflammation-related genes, and they are enriched in binding motifs for several signal transducer and activator of transcription (STAT) family members. Indeed, STAT1, STAT3 and STAT5, elements of the JAK2 pathway, are phosphorylated in association with the acquisition of endotoxin tolerance. Inhibition of the JAK2 pathway impairs the activation of tolerized genes at the first encounter with LPS. This supports a crucial role of this pathway in determining the initial response of these genes to bacterial antigens and provides a pharmacological target to prevent exacerbated responses, allowing regulated responses upon subsequent challenges. Finally, we prove the pathological relevance of the JAK2 pathway in monocytes from patients with sepsis.
Project description:One primary metabolic manifestation of inflammation is the diversion of cis-aconitate within the tricarboxylic acid (TCA) cycle to synthesize the immunometabolite itaconate. Itaconate is well established to possess immunomodulatory and metabolic effects within myeloid cells and lymphocytes, however, its effects in other organ systems during sepsis remain less clear. Utilizing Irg1 knockout mice that are deficient in synthesizing itaconate, we aimed at understanding the metabolic role of itaconate in the liver and systemically during sepsis. We find itaconate aids in lipid metabolism during sepsis. Specifically, Irg1 KO mice develop a heightened level of hepatic steatosis when induced with polymicrobial sepsis. Proteomics analysis reveal enhanced expression of enzymes involved in fatty acid oxidation in following 4-ocytl itaconate (4-OI) treatment in vitro. Downstream analysis reveals itaconate stabilizes the expression of the mitochondrial fatty acid uptake enzyme CPT1a, mediated by its hypoubiquitination. Chemoproteomic analysis revealed itaconate interacts with proteins involved in protein ubiquitination as a potential mechanism underlying its stabilizing effect on CPT1a. From a systemic perspective, we find itaconate deficiency triggers a hypothermic response following endotoxin stimulation, potentially mediated by brown adipose tissue (BAT) dysfunction. Finally, by use of metabolic cage studies, we demonstrate Irg1 KO mice rely more heavily on carbohydrates versus fatty acid sources for systemic fuel utilization in response to endotoxin treatment. Our data reveal a novel metabolic role of itaconate in modulating fatty acid oxidation during polymicrobial sepsis.
Project description:Inflammatory response plays an essential role in the resolution of infections. However, inflammation can be detrimental to the organism and cause irreparable damage, for example during sepsis, when a “cytokine storm” can lead to multiple organ failure and often ends in death. One of the strongest triggers of inflammatory response is bacterial lipopolysaccharide (LPS), acting mostly through Toll-like receptor 4 (TLR4). Prior or prolonged exposure to LPS, however, can induce the state of “endotoxin tolerance” where the macrophages and monocytes do not respond to new endotoxin challenge. The cellular mechanisms regulating this phenomenon remain elusive. Our comprehensive secreted protein analysis comparing LPS-tolerant and -responsive monocyte/macrophage-like cells combined with the extracellular flux analysis reveals the robust switch from the inflammatory to metabolic protein expression as well as points to the involvement of specific proteins that may regulate the change from the responsive to the tolerant state