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Metagenomic evidence for a polymicrobial signature of sepsis.

ABSTRACT: Our understanding of the host component of sepsis has made significant progress. However, detailed study of the microorganisms causing sepsis, either as single pathogens or microbial assemblages, has received far less attention. Metagenomic data offer opportunities to characterize the microbial communities found in septic and healthy individuals. In this study we apply gradient-boosted tree classifiers and a novel computational decontamination technique built upon SHapley Additive exPlanations (SHAP) to identify microbial hallmarks which discriminate blood metagenomic samples of septic patients from that of healthy individuals. Classifiers had high performance when using the read assignments to microbial genera [area under the receiver operating characteristic (AUROC=0.995)], including after removal of species 'culture-confirmed' as the cause of sepsis through clinical testing (AUROC=0.915). Models trained on single genera were inferior to those employing a polymicrobial model and we identified multiple co-occurring bacterial genera absent from healthy controls. While prevailing diagnostic paradigms seek to identify single pathogens, our results point to the involvement of a polymicrobial community in sepsis. We demonstrate the importance of the microbial component in characterising sepsis, which may offer new biological insights into the aetiology of sepsis, and ultimately support the development of clinical diagnostic or even prognostic tools.

SUBMITTER: Tan CCS

PROVIDER: S-EPMC8715444 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:OBJECTIVES:The intestinal epithelium compartmentalizes the sterile bloodstream and the commensal bacteria in the gut. Accumulating evidence suggests that this barrier is impaired in sepsis, aggravating systemic inflammation. Previous studies reported that cathelicidin is differentially expressed in various tissues in sepsis. However, its role in sepsis-induced intestinal barrier dysfunction has not been investigated. DESIGN:To examine the role of cathelicidin in polymicrobial sepsis, cathelicidin wild-(Cnlp+/+) and knockout (Cnlp-/-) mice underwent cecal-ligation and puncture (CLP) followed by the assessment of septic mortality and morbidity as well as histological, biochemical, immunological, and transcriptomic analyses in the ileal tissues. We also evaluated the prophylactic and therapeutic efficacies of vitamin D3 (an inducer of endogenous cathelicidin) in the CLP-induced murine polymicrobial sepsis model. RESULTS:The ileal expression of cathelicidin was increased by three-fold after CLP, peaking at 4?h. Knockout of Cnlp significantly increased 7-day mortality and was associated with a higher murine sepsis score. Alcian-blue staining revealed a reduced number of mucin-positive goblet cells, accompanied by reduced mucin expression. Increased number of apoptotic cells and cleavage of caspase-3 were observed. Cnlp deletion increased intestinal permeability to 4kD fluorescein-labeled dextran and reduced the expression of tight junction proteins claudin-1 and occludin. Notably, circulating bacterial DNA load increased more than two-fold. Transcriptome analysis revealed upregulation of cytokine/inflammatory pathway. Depletion of Cnlp induced more M1 macrophages and neutrophils compared with the wild-type mice after CLP. Mice pre-treated with cholecalciferol (an inactive form of vitamin D3) or treated with 1alpha, 25-dihydroxyvitamin D3 (an active form of VD3) had decreased 7-day mortality and significantly less severe symptoms. Intriguingly, the administration of cholecalciferol after CLP led to worsened 7-day mortality and the associated symptoms. CONCLUSIONS:Endogenous cathelicidin promotes intestinal barrier integrity accompanied by modulating the infiltration of neutrophils and macrophages in polymicrobial sepsis. Our data suggested that 1alpha, 25-dihydroxyvitamin D3 but not cholecalciferol is a potential therapeutic agent for treating sepsis.

Project description:IntroductionVarious immune cell types play critical roles in sepsis with numerous distinct subsets exhibiting unique phenotypes even within the same cell population. Single-cell RNA sequencing (scRNA-seq) enables comprehensive transcriptome profiling and unbiased cell classification. In this study, we have unveiled the transcriptomic landscape of immune cells in sepsis through scRNA-seq analysis.MethodsWe induced sepsis in mice by cecal ligation and puncture. 20 h after the surgery, the spleen and peritoneal lavage were collected. Single-cell suspensions were processed using a 10× Genomics pipeline and sequenced on an Illumina platform. Count matrices were generated using the Cell Ranger pipeline, which maps reads to the mouse reference transcriptome, GRCm38/mm10. Subsequent scRNA-seq analysis was performed using the R package Seurat.ResultsAfter quality control, we subjected the entire data set to unsupervised classification. Four major clusters were identified as neutrophils, macrophages, B cells, and T cells according to their putative markers. Based on the differentially expressed genes, we identified activated pathways in sepsis for each cell type. In neutrophils, pathways related to inflammatory signaling, such as NF-κB and responses to pathogen-associated molecular patterns (PAMPs), cytokines, and hypoxia were activated. In macrophages, activated pathways were the ones related to cell aging, inflammatory signaling, and responses to PAMPs. In B cells, pathways related to endoplasmic reticulum stress were activated. In T cells, activated pathways were the ones related to inflammatory signaling, responses to PAMPs, and acute lung injury. Next, we further classified each cell type into subsets. Neutrophils consisted of four clusters. Some subsets were activated in inflammatory signaling or cell metabolism, whereas others possessed immunoregulatory or aging properties. Macrophages consisted of four clusters, namely, the ones with enhanced aging, lymphocyte activation, extracellular matrix organization, or cytokine activity. B cells consisted of four clusters, including the ones possessing the phenotype of cell maturation or aging. T cells consisted of six clusters, whose phenotypes include molecular translocation or cell activation.ConclusionsTranscriptomic analysis by scRNA-seq has unveiled a comprehensive spectrum of immune cell responses and distinct subsets in the context of sepsis. These findings are poised to enhance our understanding of sepsis pathophysiology, offering avenues for targeting novel molecules, cells, and pathways to combat infectious diseases.

Project description:OBJECTIVES:Cell death in lymphatic organs, such as the spleen, is in part responsible for immunosuppression and contributes to mortality during sepsis. An early and noninvasive detection of lymphoid cell death could thus have significant clinical implications. Here, we tested in vivo imaging of lymphoid cell death using a near-infrared annexin V (AV-750). DESIGN:Animal study. SETTING:Laboratory investigation. SUBJECTS:C57BL/6J wild-type and toll-like receptor 3 knockout mice. INTERVENTIONS:Mild and severe polymicrobial sepsis was induced with cecum ligation and puncture. Serum cytokines and acute kidney injury markers were tested by immunoassay and quantitative reverse transcription-polymerase chain reaction, respectively. Sepsis-induced lymphoid cell death was detected by fluorescent AV-750 accumulation in the thorax and abdomen (in vivo), in isolated organs (ex vivo), and in isolated cells (flow cytometry). Caspase-3 cleavage/activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling staining were tested for apoptosis. MEASUREMENTS AND MAIN RESULTS:Severe sepsis induced marked apoptosis in the thymus, spleen, and liver as demonstrated by cleaved caspase-3 and an increase in caspase-3 activity and terminal deoxynucleotidyl transferase dUTP nick-end labeling-positive cells. A significant increase in fluorescent AV-750 signal was seen in the thoracic and upper abdominal fields and associated with the severity of sepsis. The in vivo thoracic and abdominal AV-750 fluorescent signal was attributed to the thymus, liver, and spleen as determined by ex vivo imaging and highly correlated with the levels of cell death in thymocytes and splenocytes, respectively, as measured by flow cytometry. Compared with wild-type septic mice, toll-like receptor 3 septic mice had attenuated abdominal AV-750 fluorescent signal, reduced ex vivo fluorescence in the spleen, and decreased splenocyte cell death. CONCLUSIONS:In vivo AV-750 fluorescent imaging provides spatially resolved and organ-specific detection of lymphoid cell death during polymicrobial sepsis. The AV-750 fluorescent intensity in the thoracic and abdominal fields is associated with sepsis severity and well correlated with sepsis-induced cell death in the thymus and spleen, respectively.

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Metagenomic evidence for a polymicrobial signature of sepsis.

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