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Immunotherapies have recently gained traction as highly effective therapies in a subset of late-stage cancers. Unfortunately, only a minority of patients experience the remarkable benefits of immunotherapies, whilst others fail to respond or even come to harm through immune-related adverse events. For immunotherapies within the PD-1/PD-L1 inhibitor class, patient stratification is currently performed using tumor (tissue-based) PD-L1 expression. However, PD-L1 is an accurate predictor of response in only ~30% of cases. There is pressing need for more accurate biomarkers for immunotherapy response prediction. We sought to identify peripheral blood biomarkers, predictive of response to immunotherapies against lung cancer, based on whole blood microRNA profiling. Using three well-characterized cohorts consisting of a total of 334 stage IV NSCLC patients, we have defined a 5 microRNA risk score (miRisk) that is predictive of overall survival following immunotherapy in training and independent validation (HR 2.40, 95% CI 1.37–4.19; P  < 0.01) cohorts. We have traced the signature to a myeloid origin and performed miRNA target prediction to make a direct mechanistic link to the PD-L1 signaling pathway and PD-L1 itself. The miRisk score offers a potential blood-based companion diagnostic for immunotherapy that outperforms tissue-based PD-L1 staining.

We investigate the feasibility of molecular-level sample classification of sepsis using microarray gene expression data merged by in silico meta-analysis. Publicly available data series were extracted from NCBI Gene Expression Omnibus and EMBL-EBI ArrayExpress to create a comprehensive meta-analysis microarray expression set (meta-expression set). Measurements had to be obtained via microarray-technique from whole blood samples of adult or pediatric patients with sepsis diagnosed based on international consensus definition immediately after admission to the intensive care unit. We aggregate trauma patients, systemic inflammatory response syndrome (SIRS) patients, and healthy controls in a non-septic entity. Differential expression (DE) analysis is compared with machine-learning-based solutions like decision tree (DT), random forest (RF), support vector machine (SVM), and deep-learning neural networks (DNNs). We evaluated classifier training and discrimination performance in 100 independent iterations. To test diagnostic resilience, we gradually degraded expression data in multiple levels. Clustering of expression values based on DE genes results in partial identification of sepsis samples. In contrast, RF, SVM, and DNN provide excellent diagnostic performance measured in terms of accuracy and area under the curve (>0.96 and >0.99, respectively). We prove DNNs as the most resilient methodology, virtually unaffected by targeted removal of DE genes. By surpassing most other published solutions, the presented approach substantially augments current diagnostic capability in intensive care medicine.

Innate immune memory describes the functional reprogramming of innate immune cells after pathogen contact, leading to either a boosted (trained immunity) or a diminished (immune tolerance) response to a secondary stimulus. Immune tolerance or \"sepsis-induced immunosuppression\" is a typical hallmark of patients after sepsis survival, characterized by hypo-responsiveness of the host's immune system. This condition renders the host vulnerable for a persisting infection or the occurrence of secondary, often opportunistic infections, along with an increased mortality rate. The mechanisms involved in the maintenance of this long-lasting condition are not examined yet. Polymicrobial abdominal sepsis was induced in 12 week old male C57BL/6 mice by cecal ligation and puncture. Mice were euthanized 3 months after insult. Immune cell composition of the spleen and whole blood, as well as stem and progenitor cells of the bone marrow, were assessed by flow cytometry. Whole blood and bone marrow monocytes were stimulated with LPS and supernatant levels of TNF and IL-6 detected by ELISA. Furthermore, naïve bone marrow monocytes were analyzed for metabolic (Seahorse technology) and transcriptomic (RNA sequencing) changes. Flow cytometric analysis revealed an increase of inflammatory monocytes and regulatory T cells in the spleen, whereby immune composition of whole blood kept unchanged. Granulocyte-monocyte progenitor cells are increased in sepsis survivors. Systemic cytokine response was unchanged after LPS challenge. In contrast, cytokine response of post-septic naïve bone marrow monocytes was increased. Metabolic analysis revealed enhanced glycolytic activity, whereas mitochondrial indices were not affected. In addition, RNA sequencing analysis of global gene expression in monocytes revealed a sustained signature of 367 differentially expressed genes. We here demonstrate that sepsis via functional reprogramming of naïve bone marrow monocytes induces a cellular state of trained immunity, which might be counteracted depending on the compartmental localization of the cell. These findings shed new light on the complex aftermath of sepsis and open up a new pathophysiological framework in need for further research.

Elucidating the mechanisms contributing to the dysregulated host response to infection as part of the syndrome is a current challenge in sepsis research. Peripheral blood mononuclear cells are widely used in immunological studies. Density gradient centrifugation, a common method, is of limited use for blood drawn from patients with sepsis. A significant number of low-density granulocytes co-purify contributing to low purity of isolated peripheral blood mononuclear cells. Whole blood anticoagulated with lithium heparin was drawn from patients with sepsis (n=14) and healthy volunteers (n=11). Immediately after drawing, the plasma fraction was removed and PBMC were isolated from the cellular fraction by density gradient centrifugation. Samples derived from patients with sepsis were subsequently incubated with cluster of differentiation 15 MicroBeads and granulocytes were depleted using magnetic-activated cell sorting. Core cellular functions as antigen presentation and cytokine secretion were analyzed in cells isolated from healthy volunteers (n=3) before and after depletion to confirm consistent functionality. We report here that depleting CD15 + cells after density gradient centrifugation is a feasible way to get rid of the low-density granulocyte contamination. Afterwards, the purity of isolated, functionally intact peripheral blood mononuclear cells is comparable to healthy volunteers. Information on the isolation purity and identification of the containing cell types are necessary for good comparability between different studies. Depletion of CD15 + cells after density gradient centrifugation is an easy but highly efficient way to gain a higher quality and more reliability in studies using peripheral blood mononuclear cells from septic patients without affecting the functionality of the cells.

Sepsis is a frequently fatal condition characterized by an uncontrolled and harmful host reaction to microbial infection. Despite the prevalence and severity of sepsis, we lack a fundamental grasp of its pathophysiology. Here we report that the cytokine interleukin-3 (IL-3) potentiates inflammation in sepsis. Using a mouse model of abdominal sepsis, we showed that innate response activator B cells produce IL-3, which induces myelopoiesis of Ly-6Chigh monocytes and neutrophils and fuels a cytokine storm. IL-3 deficiency protects mice against sepsis. In humans with sepsis, high plasma IL-3 levels are associated with high mortality even after adjusting for prognostic indicators. This study deepens our understanding of immune activation, identifies IL-3 as an orchestrator of emergency myelopoiesis, and reveals a new therapeutic target for treating sepsis.

Sepsis is defined as a life-threatening condition, resulting from a dysregulated and harmful response of the hosts' immune system to infection. Apart from this, the (over-)compensating mechanisms counterbalancing the inflammatory response have been proven to render the host susceptible to further infections and increase delayed mortality. Our study aimed to unravel the heterogeneity of immune response in early sepsis and to explain the biology behind it. A systematic search of public repositories yielded 949 microarray samples from patients with sepsis of different infectious origin and early after clinical manifestation. These were merged into a meta-expression set, and after applying sequential conservative bioinformatics filtering, an in-deep analysis of transcriptional heterogeneity, as well as a comparison to samples of healthy controls was performed. We can identify two distinct clusters of patients (cluster 1: 655 subjects, cluster 2: 294 subjects) according to their global blood transcriptome. While both clusters exhibit only moderate differences in direct comparison, a comparison of both clusters individually to healthy controls yielded strong expression changes of genes involved in immune responses. Both comparisons found similar regulated genes, with a stronger dysregulation occurring in the larger patient cluster and implicating a loss of monocyte and T cell function, co-occurring with an activation of neutrophil granulocytes. We propose a consistent-but in its extent varying-presence of immunosuppression, occurring as early in sepsis as its clinical manifestation and irrespective of the infectious origin. While certain cell types possess contradictory activation states, our finding underlines the urgent need for an early host-directed therapy of sepsis side-by-side with antibiotics.

The highly conserved Notch signaling pathway essentially participates in immunity through regulation of developmental processes and immune cell activity. In the adaptive immune system, the impact of the Notch cascade in T and B differentiation is well studied. In contrast, the function, and regulation of Notch signaling in the myeloid lineage during infection is poorly understood. Here we show that TLR signaling, triggered through LPS stimulation or infection with various Gram-negative and -positive bacteria, stimulates Notch receptor ligand Delta-like 1 (DLL1) expression and Notch signaling in human blood-derived monocytes. TLR activation induces DLL1 indirectly, through stimulated cytokine expression and autocrine cytokine receptor-mediated signal transducer and activator of transcription 3 (STAT3). Furthermore, we reveal a positive feedback loop between Notch signaling and Janus kinase (JAK)/STAT3 pathway during infection that involves Notch-boosted IL-6. Inhibition of Notch signaling by γ-secretase inhibitor DAPT impairs TLR4-stimulated accumulation of NF-κB subunits p65 in the nucleus and subsequently reduces LPS- and infection-mediated IL-6 production. The reduced IL-6 release correlates with a diminished STAT3 phosphorylation and reduced expression of STAT3-dependent target gene programmed death-ligand 1 (PD-L1). Corroborating recombinant soluble DLL1 and Notch activator oxaliplatin stimulate STAT3 phosphorylation and expression of immune-suppressive PD-L1. Therefore we propose a bidirectional interaction between Notch signaling and STAT3 that stabilizes activation of the transcription factor and supports STAT3-dependent remodeling of myeloid cells toward an immuno-suppressive phenotype. In summary, the study provides new insights into the complex network of Notch regulation in myeloid cells during infection. Moreover, it points to a participation of Notch in stabilizing TLR-mediated STAT3 activation and STAT3-mediated modulation of myeloid functional phenotype through induction of immune-suppressive PD-L1.

Background Preoperative risk prediction in patients at elevated cardiovascular risk shows limited accuracy. Platelet to lymphocyte ratio (PLR) and neutrophil to lymphocyte ratio (NLR) indicate systemic inflammation. Both have been investigated for outcome prediction in the field of oncology and cardiovascular medicine, as well as risk prediction of adverse cardiovascular events in non-surgical patients at increased cardiovascular risk. Methods For this post-hoc analysis, we included all 38 coronary heart disease patients from the Leukocytes and Cardiovascular Perioperative Events cohort-1 study scheduled for elective non-cardiac surgery. We evaluated preoperative differential blood counts for association with major adverse cardiovascular and cerebrovascular events (MACCE) defined as the composite endpoint of death, myocardial ischemia, myocardial infarction, myocardial injury after non-cardiac surgery, or embolic or thrombotic stroke within 30 days after surgery. We used Youden’s index to calculate cut-off values for PLR and NLR. Additive risk-predictive values were assessed using receiver operating characteristic curve and net reclassification (NRI) improvement analyses. Results Patients with the composite endpoint MACCE had higher PLR and NLR (309 [206; 380] vs. 160 [132; 203], p  = 0.001; 4.9 [3.5; 8.1] vs. 2.6 [2.2; 3.4]), p  = 0.001). Calculated cut-offs for PLR > 204.4 and NLR > 3.1 were associated with increased risk of 30-day MACCE (OR 7, 95% CI [1.2; 44.7], p  = 0.034; OR 36, 95% CI [1.8; 686.6], p  = 0.001). Furthermore, NLR improved risk prediction in coronary heart disease patients undergoing non-cardiac surgery when combined with hs-cTnT or NT-proBNP (NRI total  = 0.23, p  = 0.008, NRI total  = 0.26, p  = 0.005). Conclusions Both PLR and NLR were associated with perioperative cardiovascular adverse events in coronary heart disease patients. NLR proved to be of additional value for preoperative risk stratification. Both PLR and NLR could be used as inexpensive and broadly available tools for perioperative risk assessment. Trial registration NCT02874508 , August 22, 2016.

The early identification of sepsis in surgical intensive care patients is challenging due to the physiological postoperative alterations of vital signs and inflammatory biomarkers. Soluble Delta-like protein 1 (sDLL1) may be a potential discriminatory biomarker for this purpose. For this reason, this study aimed to evaluate sDLL1 for the identification of sepsis in a cohort of surgical intensive care patients. This study comprises a secondary analysis of a prospective observational study including 80 consecutive patients. The study groups included 20 septic shock patients, 20 patients each undergoing major abdominal surgery (MAS) and cardiac artery bypass surgery (CABG), and 20 matched control subjects (CTRL). The surveillance period was 72 h. The plasma concentration of sDLL1 was measured with ELISA. The plasma levels of sDLL1 were significantly elevated in septic patients compared to both surgical cohorts (septic vs. all postoperative time points, data are shown as median and interquartile range [IQR]; septic shock: 17,363 [12,053–27,299] ng/mL, CABG 10,904 [8692–16,250] ng/mL; MAS 6485 [4615–9068] ng/mL; CTRL 5751 [3743–7109] ng/mL; septic shock vs. CABG: p  < 0.001; septic shock vs. MAS: p  < 0.001). ROC analysis showed a sufficient prediction of sepsis with limited specificity (AUCROC 0.82 [0.75–0.82], sensitivity 84%, specificity 68%). The plasma levels of sDLL correlated closely with renal parameters (creatinine: correlation coefficient = 0.60, r 2  = 0.37, p  < 0.0001; urea: correlation coefficient = 0.52, r 2  = 0.26, p  < 0.0001), resulting in a good predictive performance of sDLL1 for the identification of acute kidney injury (AKI; AUCROC 0.9 [0.82–0.9], sensitivity 83%, specificity 91%). By quantifying the plasma concentration of sDLL1, sepsis can be discriminated from the physiological postsurgical inflammatory response in abdominal and cardiac surgical patients. However, sDLL1 has only limited specificity for the detection of sepsis in cardiac surgical patients, which may be explained by impaired renal function. Based on these findings, this study identifies the predictive value of sDLL1 for the detection of AKI, making it a potential biomarker for surgical intensive care patients. Trial registration DRKS00013584, Internet Portal of the German Clinical Trials Register (DRKS), registration date 11.07.2018, https://www.drks.de/drks_web/navigate.do?navigationId=trial.HTML&TRIAL_ID=DRKS00013584 .

Critically ill patients, especially following trauma or extensive surgery, experience a systemic immune response, consisting of a pro-inflammatory as well as a counterbalancing anti-inflammatory response. Pro-inflammation is necessary for the initiation of homeostatic control and wound healing of the organism. However, when the counterbalancing mechanisms dominate, a condition of secondary immunodeficiency occurs, which renders the patient susceptible for opportunistic or secondary infections. However, the incidence of this condition is yet illusive. For a period of 3 months (May to July 2017), 110 consecutive patients admitted to the surgical ICU of the Heidelberg University Hospital, a tertiary university hospital, were enrolled in the study. Monocyte HLA-DR (mHLA-DR), a long-known surrogate of monocyte function, was assessed quantitatively once on admission utilizing a novel point-of-care flow cytometer with single-use cartridges (Accelix system). Patients were followed up for further 28 days and data on ICU stay, antibiotic therapy, microbiological findings, and mechanical ventilation were recorded. Statistical analysis was performed to evaluate the incidence of immunosuppression-defined by different thresholds-as well as its consequence in terms of outcome and clinical course. Depending on the HLA-DR threshold applied for stratification (≤8,000/≤5,000/≤2,000 molecules/cell), a large group of patients (85.5/68.2/40.0%) already presented with a robust decrease of HLA-DR on admission, independent of the cause for critical illness. Analyzed for survival, neither threshold was able to stratify patients with a higher mortality. However, both thresholds of 2,000 and 5,000 were able to discriminate patients with longer ICU stay, ventilation time and duration of antibiotic therapy, as well as higher count of microbiological findings. Moreover, a mHLA-DR value ≤2,000 molecules/cell was associated with higher incidence of overall antibiotic therapy. Single assessment of mHLA-DR using a novel point-of-care flow cytometer is able to stratify patients according to their risk of a complicated course. Therefore, this device overcomes the technical boundaries for measuring cellular biomarkers and paves the way for future studies involving personalized immunotherapy to patients with a high immunological risk profile independent of their background. German Clinical Trials Register; ID: DRKS00012348.