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BackgroundCritical illness such as sepsis is a life-threatening syndrome defined as a dysregulated host response to infection and is characterized by patients exhibiting various impaired immune profiles. In the field of diagnosis, a gap still remains in identifying the immune profile of critically-ill patients in the ICU. The availability of an immune profiling tool holds a great potential in providing patients at high risk with more accurate and precise management. In this study, a multiplex immune profiling panel prototype was assessed for its ability to semi-quantify immune markers directly from blood, using the FilmArray® System.ResultsThe Immune Profiling Panel (IPP) prototype consists of 16 biomarkers that target both the innate and adaptive immune responses, pro- and anti-inflammatory mediators as well as genes involved in diverse regulatory pathways. The analytical studies carried out on healthy volunteers showed minimal inter- and intra-variability in testing the samples across the tested lots. The majority of the assays were linear with an R2 higher than 0.8. Results from the IPP pouch were comparable to qPCR and were within the limits of agreement. Finally, quantification cycle values of the target genes were normalized against reference genes to account for the different composition of cells among specimens. The use of the selected panel of markers in IPP demonstrated various gene modulations that could distinctly differentiate three profiles: healthy, borderline mHLA-DR septic shock patients and low mHLA-DR septic shock patients.ConclusionThe Immune Profiling Panel allowed host transcriptomic analysis of immune response biomarkers directly from whole blood in less than an hour. The use of IPP showed great potential for the development of a fully automated, rapid and easy-to-use immune profiling tool, enabling the stratification of critically-ill patients at high risk in the ICU.

Sepsis, defined as life-threatening organ dysfunction caused by dysregulated host response to infection, has recently been acknowledged as a worldwide health priority. Sepsis remains the leading cause of mortality in intensive care units and accounts for 6 million deaths every year. Few therapeutic options targeting host immune response in sepsis have demonstrated their efficacy so far. Increasing evidence suggests that a profound immune suppression develops following sepsis, affecting innate and adaptive immune response, of which intensity and duration is associated with increased risk of death and nosocomial infection. Immunostimulant treatments are thus now evaluated in sepsis, and recombinant human IL-7 (rhIL-7) represents a promising candidate. rhIL-7 has been evaluated in several clinical trials in patients with altered lymphocytic responses (HIV infection, hematopoietic stem cell transplantation, and cancer). Recent studies in animal models and in patients' samples ex vivo demonstrated its efficacy in improving sepsis-induced T cell alterations. Finally, the first clinical trial evaluating rhIL-7 in septic shock patients has just been published. This review will discuss the use of rhIL-7 to treat sepsis-induced T cell dysfunction by introducing the pathophysiology of sepsis and sepsis-related lymphocyte alterations before focusing on rhIL-7 and its potential use of as a therapeutic intervention in patients.

Item does not contain fulltext Increasing evidence supports a central role of the immune system in sepsis, but the current view of how sepsis affects immunity, and vice versa, is still rudimentary. The European Group on Immunology of Sepsis has identified major gaps that should be addressed with high priority, such as understanding how immunological alterations predispose to sepsis, key aspects of the immunopathological events during sepsis, and the long-term consequences of sepsis on patient's immunity. We discuss major unmet topics in those three categories, including the role of key immune cells, the cause of lymphopenia, organ-specific immunology, the dynamics of sepsis-associated immunological alterations, the role of the microbiome, the standardisation of immunological tests, the development of better animal models, and the opportunities offered by immunotherapy. Addressing these gaps should help us to better understand sepsis physiopathology, offering translational opportunities to improve its prevention, diagnosis, and care.

Purpose Several remarks have been raised regarding the variables and cut-points used in the Sequential Organ Failure Assessment (SOFA) score. This study revisited the SOFA score and created a new simplified and accurate sa-SOFA score. Methods The study grouped four prospective cohorts (2005–2016) of patients with Systemic Inflammatory Response Syndrome. It collected 28-day mortality, sociodemographic characteristics, and the SOFA score with all variable values at Day 1. A logistic regression analysis was used to select the most relevant variables and a minimum p value approach with a 10-fold cross-validation were used to find the optimal partition of selected variables. The minimum number of cut-points (2, 3, or 4) was also tested by comparing the distributions of areas under receiver operating characteristic (AUROC) curves. Results Among the 1436 participants, 416 died within 28 days (28.9%). The sa-SOFA kept one variable per dimension and two cut-points per variable. The AUROC curves that investigated the abilities of the sa-SOFA and SOFA scores to predict 28-day mortality were 0.739 [0.712–0.768] and 0.687 [0.656–0.717], respectively (p-value of DeLong test Conclusion Keeping the conventional SOFA dimension variables, the new sa-SOFA proved to be simpler and more accurate in predicting 28-day mortality.

A dynamical pathophysiological model is presented in the case of septic shock. The proposed approach takes into account the environment and the different subsystems. The considered immune system is the blood, the subsystems are leukocyte cells and cytokines. The interactions between blood cells, plasma and the environment are described via mass balances. The transport phenomena between cells and blood and thermodynamic equilibrium are considered. The quantities of pro and anti-cytokines present in the body are combined to propose global information on the inflammatory state of the patient. The proposed model is strongly nonlinear. Simulations showed how the global information evolves after septic shock. In particular, the model has the ability to evolve to alternative homeostatic equilibrium in case of septic shock.

Background Septic shock patients exhibit an increased incidence of viral reactivation. Precise timing of such reactivation—as an early marker of immune suppression, or as a consequence of the later—is not known precisely. Here, using a fully designed nucleic acid extraction automated procedure together with tailored commercial PCR kits, we focused on the description of early reactivation within the first week of ICU admission of several herpes viruses and Torque Teno virus (TTV) in 98 septic shock patients. Results Most of septic shock patients had at least one viremia event during the first week (88%). TTV and herpesviruses were detected in 56% and 53% of septic shock patient, respectively. The two most frequent herpesviruses detected within the first week were EBV (35%) and HSV1 (26%). Different kinetic were observed among herpesviruses, faster for EBV and HSV1 than for CMV and HHV6. Although no association was found between herpes viremia and secondary infections, patients with herpesviridae-related viremia were more severe, e.g., higher SOFA scores and plasma lactate levels. While reactivating only 1 virus was not associated with mortality, patients with multiple viremia events had higher ICU mortality. Surprisingly, EBV + TTV early reactivation seemed associated with a lower D28 mortality. No clear association was observed between viremia and immune biomarkers. Conclusion Applying a semi-automated process of viral DNAemia determination to this cohort of 98 patients with septic shock, we observed that the number of patients with positive viremia increased during the first week in the ICU. Of note, there was no improvement in predicting the outcome when using viremia status. Nevertheless, this pilot study, introducing standardized procedures from extraction to detection, provides the basis for future standardized diagnostic criteria. A prospective longitudinal clinical study using these procedures will enable determination of whether such viremia is due to a lack of a latent virus control by the immune system or a true clinical viral infection. Electronic supplementary material The online version of this article (10.1186/s40635-019-0256-z) contains supplementary material, which is available to authorized users.

Innovative single cell technologies such as mass cytometry (CyTOF) widen possibilities to deeply improve characterisation of immune alterations mechanisms in human diseases. So far, CyTOF has not been used in sepsis – a condition characterized by complex immune disorders. Here, we evaluated feasibility of CyTOF analysis in patients with septic shock. We designed a mass cytometry panel of 25 extracellular markers to study mononuclear cells from 5 septic shock patients and 5 healthy donors. We explored single-cell data with global and specific unsupervised approaches such as heatmaps, SPADE and viSNE. We first validated relevance of our CyTOF results by highlighting established immune hallmarks of sepsis, such as decreased monocyte HLA-DR expression and increased expressions of PD1 and PD-L1 on CD4 T cells and monocytes. We then showed that CyTOF analysis reveals novel aspects of sepsis-induced immune alterations, e.g. B cell shift towards plasma cell differentiation and uniform response of several monocyte markers defining an immune signature in septic patients. This proof of concept study demonstrates CyTOF suitability to analyse immune features of septic patients. Mass cytometry could thus represent a powerful tool to identify novel pathophysiological mechanisms and therapeutic targets for immunotherapy in septic shock patients.

On May 2017, the World Health Organization (WHO) recognized sepsis as a global health priority by adopting a resolution to improve the prevention, diagnosis, and management of this deadly disease. While it has long been known that sepsis deeply perturbs immune homeostasis by inducing a tremendous systemic inflammatory response, pivotal observations based on clinical flow cytometry indicate that sepsis indeed initiates a more complex immune response that varies over time, with the concomitant occurrence of both pro- and anti-inflammatory mechanisms. As a resultant, some septic patients enter a stage of protracted immunosuppression. This paved the way for immunostimulation approaches in sepsis. Clinical flow cytometry permitted this evolution by drawing a new picture of pathophysiology and reshaping immune trajectories in patients. Additional information from cytometry by time of flight mass cytometry and other high-dimensional flow cytometry platform should rapidly enrich our understanding of this complex disease. This review reports on landmarks of clinical flow cytometry in sepsis and how this single-cell analysis technique permitted to breach the wall of decades of unfruitful anti-inflammatory-based clinical trials in sepsis. © 2019 International Society for Advancement of Cytometry.

Model animal; COVID-19; Recerca preclínica Modelo animal; COVID-19; Investigación preclínica Animal model; COVID-19; Pre-clinical research Many milestones in medical history rest on animal modeling of human diseases. The SARS-CoV-2 pandemic has evoked a tremendous investigative effort primarily centered on clinical studies. However, several animal SARS-CoV-2/COVID-19 models have been developed and pre-clinical findings aimed at supporting clinical evidence rapidly emerge. In this review, we characterize the existing animal models exposing their relevance and limitations as well as outline their utility in COVID-19 drug and vaccine development. Concurrently, we summarize the status of clinical trial research and discuss the novel tactics utilized in the largest multi-center trials aiming to accelerate generation of reliable results that may subsequently shape COVID-19 clinical treatment practices. We also highlight areas of improvement for animal studies in order to elevate their translational utility. In pandemics, to optimize the use of strained resources in a short time-frame, optimizing and strengthening the synergy between the preclinical and clinical domains is pivotal.

International audience; Sepsis, which is the leading cause of death in intensive care units (ICU), has been acknowledged as a global health priority by the WHO in 2017. Identification of biomarkers allowing early stratification and recognition of patients at higher risk of death is crucial. One promising biomarker candidate is pentraxin-3 (PTX3); initially elevated and persistently increased plasma concentration in septic patients has been associated with increased mortality. PTX3 is an acute phase protein mainly stored in neutrophil granules. These cells are responsible for rapid and prompt release of PTX3 in inflammatory context, but the cellular origin responsible for successive days' elevation in sepsis remains unknown. Upon inflammatory stimulation, PTX3 can also be produced by other cell types, including endothelial and immune cells. As in septic patients immune alterations have been described, we therefore sought to investigate whether such cells participated in the elevation of PTX3 over the first days after septic shock onset. To address this point, PTX3 was measured in plasma from septic shock patients at day 3 after ICU admission as well as in healthy volunteers (HV), and the capacity of whole blood cells to secrete PTX3 after inflammatory stimulation was evaluated ex vivo. A significantly mean higher (100-fold) concentration of plasma PTX3 was found in patients compared to HV, which was likely due to the inflammation-induced initial release of the pre-existing PTX3 reservoir contained in neutrophils. Strikingly, when whole blood was stimulated ex vivo with LPS no significant difference between patients and HV in PTX3 release was found. This was in contrast with TNFα which decreased production was illustrative of the endotoxin tolerance phenomenon occurring in septic patients. Then, the release of PTX3 protein from a HV neutrophil-free PBMC endotoxin tolerance model was investigated. At the transcriptional level, PTX3 seems to be a weakly tolerizable gene similar to TNFα. Conversely, increased protein levels observed in anergy condition reflects a non-tolerizable phenotype, more likely to an anti-inflammatory marker. Hence, altered immune cells still have the ability to produce PTX3 in response to an inflammatory trigger, and therefore circulating white blood cell subset could be responsible of the sustained PTX3 plasma levels over the first days of sepsis setting.

Septic shock is the primary cause of death in ICUs. A better comprehension of its pathophysiology, in particular, the immune alteration mechanisms, opened new therapeutic perspectives such as the recombinant interleukin-7. The use of biomarkers could improve the identification of eligible patients for this therapy. The soluble form of the interleukin-7 appears as a promising candidate in this regard since an association between its high plasmatic level and mortality in critically ill patients has been demonstrated. Because there are no data available on the transcriptional regulation of the interleukin-7 receptor in such patients, this study aimed to explore the expression level of different interleukin-7 receptor transcripts after septic shock and evaluate their association with mortality.Retrospective discovery cohort (30 patients) and validation cohort (177 patients).Two French ICUs (discovery study) and six French ICUs (validation study).Adult septic shock patients.None.The quantification of several interleukin-7 receptor transcripts using specific reverse transcription quantitative polymerase chain reaction designs allowed for global evaluation of interleukin-7 receptor gene expression in whole blood. In the discovery cohort, all interleukin-7 receptor transcripts studied were expressed at lower levels in septic shock patients than in healthy volunteers. Interleukin-7 receptor gene expression at day 3 after septic shock diagnosis was associated with day 28 mortality. Patients at a lower risk of death showed higher expression levels. These results were confirmed in the independent validation cohort. Interestingly, using a threshold obtained on the discovery cohort, we observed in the validation cohort a high negative predictive value for day 28 mortality for the transcript encoding the membrane form of interleukin-7 receptor (0.86; 95% CI, 0.79-0.93).Interleukin-7 receptor transcripts appear as biomarkers of impaired adaptive immune response in septic shock patients and as a promising tool for patient stratification in clinical trials evaluating immunoadjuvant therapies.

International audience

Background Sepsis-associated hyperlactatemia is a widely used biomarker, associated with initial severity and poor outcomes. This increased circulating lactate concentration has been proposed to result in part from a mismatch between oxygen delivery and demand in organs. However, other mechanisms may participate. In particular, a metabolic reprogramming similar to the Warburg effect initially described in cancer cells could lead to increased lactate production by immune cells such as T lymphocytes after sepsis. The objective of this study was to set up a protocol for lactate measurement in T lymphocytes, and to evaluate whether lactate production by T lymphocytes was increased in septic shock patients. Methods We first optimized protocols for lactate and pyruvate measurements in T lymphocytes purified from healthy volunteers’ blood, either stimulated with phytohaemagglutinine (PHA) or left untreated. We then conducted a pilot study to confirm the feasibility of this protocol in samples from septic shock patients. Results PHA stimulation induced aerobic glycolysis in human lymphocytes ex vivo, with increased lactate and pyruvate productions. To correctly measure this phenomenon, minimal cell number was 250,000 and optimal culture duration was 40 h. We also observed a significant correlation between lactate concentration in T lymphocytes and in their culture supernatants. We were able to measure lactate concentration in T lymphocytes from septic shock patients. Our preliminary results showed that intra-lymphocyte lactate concentration was not different between patients and healthy volunteers. Conclusion This protocol should now be tested in a larger cohort of patients. The association between immune cell metabolic reprogramming as measured by lactate concentration in T cells and functionality represents an exciting field for research.

Background While the process of sepsis-induced immunosuppression is now well described in adults, very little information is available on immune functions in pediatric sepsis. The current study investigated this in children with septic shock by performing immunomonitoring, including both innate (monocyte human leukocyte antigen-DR, mHLA-DR, expression) and adaptive immunity (lymphocyte subsets count), as well as cytokine concentrations (IL-6, IL-8, IL-10, IL-1Ra, TNF-α, IFN-γ). Subsequent objectives were to assess the associations between inflammatory response, potential immunosuppression and secondary acquired infection occurrence. Methods Single-center prospective observational study, including children aged between 1 month and 18 years admitted to pediatric intensive care unit (PICU) for septic shock. Age-matched controls were children hospitalized for elective surgery without any infectious criteria. Blood was sampled at day 1–2, 3–5, and 7–9 after sepsis onset. mHLA-DR and lymphocyte subsets count were measured by flow cytometry and cytokine concentrations by Luminex technology. Results A total of 26 children and 30 controls were included. Patients had lymphopenia, and mHLA-DR levels were significantly lower than controls at each time point (p Conclusions The higher initial inflammatory cytokine production was, the more innate immunity was altered, while evaluated by low mHLA-DR expression. Children with decreased mHLA-DR expression developed more secondary acquired infections. Upon confirmation in multicenter cohorts, these results pave the way for immunostimulation for the most immunosuppressed children in order to prevent nosocomial infections in PICU. Trial registration PedIRIS study NCT02848144. Retrospectively registered 28 July 2016

Profound T-cell alterations are observed in septic patients in association with increased risk of secondary infection and mortality. The pathophysiological mechanisms leading to such dysfunctions are not completely understood and direct and indirect mechanisms have been described. In this study we evaluated whether ex vivo stimulation of lymphocytes with IL-10, an immunosuppressive cytokine released at the systemic level during sepsis, could mimic sepsis-induced intrinsic T-cell alterations. We showed that recombinant human IL-10 priming of T cells altered their proliferative response to anti-CD2/CD3/CD28 antibody-coated beads and PHA stimulations, in a dose-dependent manner independently of accessory cells. This priming also significantly decreased T-cell secretion of IL-2 and IFNγ following stimulation. Furthermore, we demonstrated that IL-10 reduction of T-cell functionality was associated with increased FOXP3 expression in CD4+CD25+CD127- regulatory T cells as observed in sepsis. Finally, we found that blocking the increased IL-10 concentration in plasma from septic shock patients increased the proliferative response of responding T cells from healthy controls. We describe here an ex vivo model recapitulating features of sepsis-induced intrinsic T-cell alterations. This should help, in further studies, to decipher the pathophysiological mechanisms of T-cell alterations induced after septic shock.

It is now well established that profound immunosuppression develops within a few days after sepsis onset in patients. This should be considered additional organ failure because it is associated with increased rate of nosocomial infections, mortality, and long-term complications, thus constituting the rationale for immunomodulation in patients. Nevertheless, the demonstration of the efficacy of such therapeutic strategy in improving deleterious outcomes in sepsis remains to be made. Results from clinical trials based on interleukin 7 and granulocyte macrophage colony-stimulating factor immunoadjuvant therapies in septic shock patients are expected for 2018.

Septic syndromes remain a major although largely under-recognized health care problem and represent the first cause of mortality in intensive care units. While sepsis has, for long, been solely described as inducing a tremendous systemic inflammatory response, novel findings indicate that sepsis indeed initiates a more complex immunologic response that varies over time, with the concomitant occurrence of both pro- and anti-inflammatory mechanisms. As a resultant, after a short proinflammatory phase, septic patients enter a stage of protracted immunosuppression. This is illustrated in those patients by reactivation of dormant viruses (CMV or HSV) or infections due to pathogens, including fungi, which are normally pathogenic solely in immunocompromised hosts. Although mechanisms are not totally understood, these alterations might be directly responsible for worsening outcome in patients who survived initial resuscitation as nearly all immune functions are deeply compromised. Indeed, the magnitude and persistence over time of these dysfunctions have been associated with increased mortality and health-care associated infection rate. Consequently, new promising therapeutic avenues are currently emerging from those recent findings such as adjunctive immunostimulation (IFN-γ, GM-CSF, IL-7, anti-PD1/L1 antibodies) for the most immunosuppressed patients. Nevertheless, as there is no clinical sign of immune dysfunctions, the prerequisite for such therapeutic intervention relies on our capacity in identifying the patients who could benefit from immunostimulation. To date, the most robust biomarkers of sepsis-induced immunosuppression are measured by flow cytometry. Of them, the decreased expression of monocyte HLA-DR appears as a "gold standard." This review reports on the mechanisms sustaining sepsis-induced immunosuppression and its related biomarkers measurable by flow cytometry. The objective is to integrate the most recent facts in an up-to-date account of clinical results, flow cytometry aspects as well as issues in results standardization for multicenter studies. © 2015 International Clinical Cytometry Society.

Severe septic syndromes deeply impair innate and adaptive immunity and are responsible for sepsis-induced immunosuppression. Although neutrophils represent the first line of defense against infection, little is known about their phenotype and functions a few days after sepsis, when the immunosuppressive phase is maximal (i.e., between d 3 and 8). The objective of the present study was to perform, for the first time, a global evaluation of neutrophil alterations in immunosuppressed septic patients (at d 3–4 and d 6–8) using phenotypic and functional studies. In addition, the potential association of these parameters and deleterious outcomes was assessed. Peripheral blood was collected from 43 septic shock patients and compared with that of 23 healthy controls. In the septic patients, our results highlight a markedly altered neutrophil chemotaxis (functional and chemokine receptor expressions), oxidative burst, and lactoferrin content and an increased number of circulating immature granulocytes (i.e., CD10dimCD16dim). These aspects were associated with an increased risk of death after septic shock. In contrast, phagocytosis and activation capacities were conserved. To conclude, circulating neutrophils present with phenotypic, functional, and morphologic alterations a few days after sepsis onset. These dysfunctions might participate in the deleterious role of sepsis-induced immunosuppression. The present results open new perspectives in the mechanisms favoring nosocomial infections after septic shock. They deserve to be further investigated in a larger clinical study and in animal models recapitulating these alterations.

Background Pneumatic tube system (PTS) for transportation of blood specimens may present with advantages for hospital organization as it provides faster tube transfer from medical wards to routine labs. These characteristics are expected to result in faster sample processing and decreased turnaround time, therefore benefiting the patient particularly in emergency units. However, PTS could affect sample quality and therefore laboratory results. Within the context of routine lab certification, effects of PTS on routine cellular immunology analyses, especially on determination of T lymphocyte subpopulations, need to be evaluated. Methods Paired EDTA blood samples were collected from 30 healthy donors. For each pair, one sample was hand-delivered by a courier while the other was transported through a PTS of 2.4 km long (1.6 miles) with two 90-degree turns and one-U turn generating a speed of 5 m/s with a maximal acceleration of 2 g-force. The percentages of CD3+ cells, CD4+ and CD8+ T-cells and their absolute counts were assessed by flow cytometry. Results For every parameter, results measured by flow cytometry were not significantly different after transport by PTS or hand-delivery. Results comparison revealed an excellent linear correlation between both delivery methods (R ranged from 0.969 to 0.982; P

Background In various ICU conditions, measurement of diminished expression of human leukocyte antigen-DR on circulating monocytes (mHLA-DR) by flow cytometry appears to be a reliable marker of acquired immunosuppression. Low mHLA-DR is associated with an increased risk of nosocomial infections and mortality. Nevertheless, its use remains somewhat limited and has not been adopted in common medical practice. The main drawback of mHLA-DR measurement is likely related to the use of flow cytometry that is not accessible everywhere on a 24/7 basis. Recently, the Accellix system, a fully automated table top cytometer, was developed for use at bedside or emergency labs. Methods The objective was to assess the performance of the Accellix (beta site evaluation including repeatability and method comparison with reference protocol) for the measurement of mHLA-DR expression. Results Accellix repeatability at low and high expression levels of mHLA-DR was

Purpose Intensive care unit (ICU)-acquired infections (IAI) result in increased hospital and ICU stay, costs and mortality. To date, no biomarker has shown sufficient evidence and ease of application in clinical routine for the identification of patients at risk of IAI. We evaluated the association of the systemic mRNA expression of two host response biomarkers, CD74 and IL10, with IAI occurrence in a large cohort of ICU patients. Methods ICU patients were prospectively enrolled in a multicenter cohort study. Whole blood was collected on the day of admission (D1) and on day 3 (D3) and day 6 (D6) after admission. Patients were screened daily for IAI occurrence and data were censored after IAI diagnosis. mRNA expression levels of biomarkers were measured using RT-qPCR. Fine and Gray competing risk models were used to assess the association between gene expression and IAI occurrence. Results A total of 725 patients were analyzed. At least one IAI episode occurred in 137 patients (19%). After adjustment for shock and sepsis status at admission, CD74 and IL10 levels were found to be significantly associated with IAI occurrence [subdistribution hazard ratio (95% confidence interval) 0.67 (0.46–0.97) for CD74 D3/D1 expression ratio and 2.21 (1.63–3.00) for IL10 at D3]. IAI cumulative incidence was significantly different between groups stratified according to CD74 or IL10 expression (Gray tests p

Functional testing protocols are thought to be the gold standard for the exploration of the immune system. However, in terms of routine analysis, they present numerous drawbacks and consequently their use is mainly limited to research applications. In the clinical context of septic shock, characterized by marked lymphocyte alterations, a new approach for lymphocyte intracellular cytokine measurement in whole blood upon was evaluated in a proof-of-concept study. Following lymphocyte activation, simultaneous intracellular labeling of Interferon-γ (IFN-γ), Tumor Necrosis Factor-α (TNF-α), and Interleukin-2 (IL-2) was performed in CD4+ and CD8+ T cells (identified by surface marking). The analysis was carried out by flow cytometry (6 colors). Results obtained in septic patients (n = 22) were compared to those of healthy volunteers (n = 8). Independently of lymphopenia, there were significant differences between groups. In particular there was significant decrease in the production of IL-2 and TNF-α in septic patients, while the production of IFN-γ was not significantly altered. Polyfunctional results showed that patients presented with increased percentages of triple negative lymphocytes. In contrast, volunteers had higher proportions of triple positive cells. The approach could be performed in a robust and consistent way, taking 4.5 hours to complete. Moreover, clear differences could be observed between clinical groups with this modified method. These characteristics illustrate the potential of this novel whole blood protocol for clinical applications. However, further research is required to determine the applicability compared to alternative test and to evaluate clinical performances in larger cohorts of patients.

Septic shock is accompanied by the development of immune dysfunctions whose intensity and duration are associated with increased risk of secondary infections and mortality. Although B lymphocytes play a pivotal role in the immune response to infections, no comprehensive exploration of circulating B cell status has been performed during the immunosuppressive phase of septic shock. Thus, our aim was to extensively characterize the phenotype and function of B cells in septic shock, including IL-10 production. Circulating B lymphocyte phenotype and function were evaluated by flow cytometry on fresh whole blood and after ex vivo stimulation in adult septic shock patients sampled at day 1, 3, and 6 after the onset of shock. The circulating B cell number was reduced in septic shock patients, whereas the B cell proportion among total lymphocytes was increased. The remaining circulating B lymphocytes presented with decreased MHC class II expression and increased CD21low CD95high exhausted-like phenotype but showed no change in maturation status. Circulating B cell functions were markedly altered after sepsis with reduced ex vivo activation and proliferation capacities. Finally, B cell response after septic shock was characterized by a clear plasmacytosis and an increased IL-10 production in remaining B cells from patients after ex vivo stimulation. During the sepsis-induced immunosuppression phase, B cell response is altered and is oriented toward an exhausted-like/immunoregulatory profile. Further studies are now needed to confirm the immunoregulatory properties of B lymphocytes and evaluate their role in sepsis-induced immunosuppression.

Introduction The host response to septic shock is dynamic and complex. A sepsis-induced immunosuppression phase has recently been acknowledged and linked to bad outcomes and increased healthcare costs. Moreover, a marked suppression of the immune response has also been partially described in patients hospitalized in intensive care unit (ICU) for severe trauma or burns. It has been hypothesized that immune monitoring could enable identification of patients who might most benefit from novel, adjunctive immune-stimulating therapies. However, there is currently neither a clear definition for such injury-induced immunosuppression nor a stratification biomarker compatible with clinical constraints. Methods and analysis We set up a prospective, longitudinal single-centre clinical study to determine the incidence, severity and persistency of innate and adaptive immune alterations in ICU patients. We optimized a workflow to describe and follow the immunoinflammatory status of 550 patients (septic shock, severe trauma/burn and major surgery) during the first 2 months after their initial injury. On each time point, two immune functional tests will be performed to determine whole-blood TNF-α production in response to ex vivo lipopolysaccharide stimulation and the T lymphocyte proliferation in response to phytohaemagglutinin. In addition, a complete immunophenotyping using flow cytometry including monocyte HLA-DR expression and lymphocyte subsets will be obtained. New markers (ie, levels of expression of host mRNA and viral reactivation) will be also evaluated. Reference intervals will be determined from a cohort of 150 age-matched healthy volunteers. This clinical study will provide, for the first time, data describing the immune status of severe ICU patients over time. Ethics and dissemination Ethical approval has been obtained from the institutional review board (no 69HCL15_0379) and the French National Security agency for drugs and health-related products. Results will be disseminated through presentations at scientific meetings and publications in peer-reviewed journals. Trial registration number Clinicaltrials.gov Registration number: NCT02638779. Pre-results.

Background Increase in mortality and in recurrent infections in the year following ICU discharge continues in survivors of septic shock, even after total clinical recovery from the initial septic event and its complications. This supports the hypothesis that sepsis could induce persistent long-term immune dysfunctions. To date, there is almost no data on ICU discharge and long-term evolution of sepsis-induced immunosuppression in septic shock survivors. The aim of this study was to assess the persistence of sepsis-induced immunosuppression by measuring expression of human leukocyte antigen DR on monocytes (mHLA-DR), CD4+ T cells, and regulatory T cells (Treg) at ICU discharge and 6 months after ICU discharge in patients admitted to the ICU for septic shock. Methods In this prospective observational study, septic shock survivors with no preexisting immune suppression or treatment interfering with the immune system were included. mHLA-DR, CD4+ T cells, and Treg expression were assessed on day 1–2, 3–4, and 6–8 after ICU admission, at ICU discharge, and 6 months after ICU discharge. Results A total of 40 patients were enrolled during their ICU stay: 21 males (52.5%) and 19 females, median age 68 years (IQR 58–77), median SOFA score on day 1–2 was 8 (IQR 7–9), and median ICU length of stay was 11 days (IQR 7–24). Among these 40 patients, 33 were studied at ICU discharge and 15 were disposed for blood sampling 6 months after ICU discharge. On day 1–2, mHLA-DR expression was abnormally low for all patients [median 4212 (IQR 2640–6047) AB/C] and remained abnormally low at ICU discharge for 75% of them [median 10,281 (IQR 7719–13,035) AB/C]. On day 3–4, 46% of patients presented CD4+ lymphopenia [median 515 (IQR 343–724) mm−3] versus 34% at ICU discharge [median 642 (IQR 459–846) mm−3]. Among patients with a 6-month blood sample, normal values of mHLA-DR were found for all patients [median 32,616 (IQR 24,918–38,738) AB/C] except for one and only another one presented CD4+ lymphopenia. Conclusions While immune alterations persist at ICU discharge, there is, at cellular level, no persistent immune alterations among septic shock survivors analyzed 6 months after ICU discharge.

T lymphocyte alterations are central to sepsis pathophysiology, whereas related mechanisms remain poorly understood. We hypothesized that metabolic alterations could play a role in sepsis-induced T lymphocyte dysfunction. Samples from septic shock patients were obtained at day 3 and compared with those from healthy donors. T cell metabolic status was evaluated in the basal condition and after T cell stimulation. We observed that basal metabolic content measured in lymphocytes by nuclear magnetic resonance spectroscopy was altered in septic patients. Basal ATP concentration, oxidative phosphorylation (OXPHOS), and glycolysis pathways in T cells were decreased as well. After stimulation, T lymphocytes from patients failed to induce glycolysis, OXPHOS, ATP production, GLUT1 expression, glucose entry, and proliferation to similar levels as controls. This was associated with significantly altered mTOR, but not Akt or HIF-1α, activation and only minor AMPKα phosphorylation dysfunction. IL-7 treatment improved mTOR activation, GLUT1 expression, and glucose entry in septic patients’ T lymphocytes, leading to their enhanced proliferation. mTOR activation was central to this process, because rapamycin systematically inhibited the beneficial effect of recombinant human IL-7. We demonstrate the central role of immunometabolism and, in particular, mTOR alterations in the pathophysiology of sepsis-induced T cell alterations. Our results support the rationale for targeting metabolism in sepsis with recombinant human IL-7 as a treatment option.

Resume Les syndromes septiques representent un probleme majeur de sante publique du fait de leur incidence en constante augmentation et du taux eleve de deces associe. La forme la plus grave est le choc septique dont la mortalite approche encore les 40 %. La reponse immunitaire au cours du choc septique associe une importante reponse inflammatoire initiale responsable de l’etat de choc et le developpement secondaire d’alterations du systeme immunitaire. Les polynucleaires neutrophiles (PNN), sentinelles de l’immunite grâce a leur role de phagocytose et leur potentiel antimicrobien, possedent egalement un role central dans la reponse immunitaire du fait d’interactions constantes avec les cellules dendritiques, macrophages et lymphocytes via des cytokines. En consequence, il n’est pas etonnant que les PNN participent a la physiopathologie des etats septiques severes. Au cours de la phase pro-inflammatoire, on observe une augmentation du nombre de PNN, dont une proportion importante de cellules immatures circulantes d’origine medullaire, ainsi qu’une diminution de leur apoptose. Leurs reponses fonctionnelles telles que le metabolisme oxydatif et la phagocytose sont augmentees alors que leurs proprietes de chimiotactisme sont fortement inhibees. Ceci serait a l’origine d’effets deleteres sur certains organes, notamment le poumon. Ainsi, la production sur les sites distants de l’infection par les PNN d’especes reactives de l’oxygene et de cytokines proinflammatoires jouerait un role majeur dans le developpement de defaillances d’organes chez les patients septiques. Enfin, des donnees recentes de la litterature decrivent egalement un nouveau role immunosuppresseur pour les PNN suggerant non seulement un nouvel aspect de l’implication de ces cellules dans la reponse immunitaire mais egalement dans la physiopathologie du choc septique.

Le choc septique induit de profondes alterations immunitaires affectant, notamment, l’ensemble des fonctions des lymphocytes T. L’amplitude et la persistance dans le temps de ces alterations sont associees a une augmentation de la survenue d’infections nosocomiales et du risque de deces. L’interleukine-7 (IL-7) recombinante humaine utilisee en therapeutique a montre des resultats prometteurs dans differentes situations cliniques caracterisees par des lymphopenies marquees. Elle apparait comme une therapeutique immuno-adjuvante innovante chez les patients septiques les plus immunodeprimes.

Flow cytometry has become a basic of biological research and clinical diagnostics, and its application has been crucial to numerous advances in immunology and cell biology. However, several issues remain when considering intracellular stainings, especially in the context of a daily routine use and in multicenter clinical research protocols including large cohorts of patients. The requirements for multiple protocol steps are not only time-consuming but also frequently associated with high cell loss and nonspecific binding or reduced fluorescence. These drawbacks make standardized intracellular flow cytometry use in multicenter studies struggling. As a consequence, intracellular flow cytometry has mostly remained a tool for experimental and clinical research. In the current chapter, we will complete flow cytometry protocols described in the previous edition by presenting novel intracellular protocols usable in clinic. These present with many advantages including shorter time-to-results, one-step whole blood procedures, lyse-no-wash-no-centrifuge protocols, improved staining quality, and lyophilized coated reagents in ready-to-use tubes. This opens novel perspectives for standardization and feasibility in clinical studies, for drug efficacy monitoring and for patients' stratification within a context of personalized medicine. Here, we present illustrative examples taken from septic patients' immunomonitoring. We consider the evaluation of myeloperoxidase and lactoferrin expressions in neutrophils, FOXP3 lymphocyte expression, and STAT5 phosphorylation in lymphocyte subsets.

Flow cytometry has become a basic of biological research and clinical diagnostics, and its application has been crucial to numerous advances in immunology and cell biology. However, several issues remain when considering intracellular stainings, especially in the context of a daily routine use and in multicenter clinical research protocols including large cohorts of patients. The requirements for multiple protocol steps are not only time-consuming but also frequently associated with high cell loss and nonspecific binding or reduced fluorescence. These drawbacks make standardized intracellular flow cytometry use in multicenter studies struggling. As a consequence, intracellular flow cytometry has mostly remained a tool for experimental and clinical research. In the current chapter, we will complete flow cytometry protocols described in the previous edition by presenting novel intracellular protocols usable in clinic. These present with many advantages including shorter time-to-results, one-step whole blood procedures, lyse-no-wash-no-centrifuge protocols, improved staining quality, and lyophilized coated reagents in ready-to-use tubes. This opens novel perspectives for standardization and feasibility in clinical studies, for drug efficacy monitoring and for patients' stratification within a context of personalized medicine. Here, we present illustrative examples taken from septic patients' immunomonitoring. We consider the evaluation of myeloperoxidase and lactoferrin expressions in neutrophils, FOXP3 lymphocyte expression, and STAT5 phosphorylation in lymphocyte subsets.

Preliminary studies suggest that a subgroup of septic patients with severe immune alterations is at high risk of death or nosocomial infection and therefore could benefit from adjunctive immune stimulating therapies. There is thus an urgent need for robust biomarkers usable in routine conditions evaluating rapidly evolving immune status in patients. Although functional testing remains a gold standard, its standardization remains challenging. Therefore, surrogate markers such as monocyte HLA-DR expression, are being developed. Such biomarkers of immune functionality will enable a novel approach in the design of clinical trials evaluating immunostimulating therapies in sepsis at the right time and in the right patient.

A new pathway of three protein tyrosine kinase receptors, namely, the TAM receptor family [Tyro-3, Axl and Mer tyrosine kinase (MerTK)], has recently been described to negatively control immune responses. The objective of this prospective, observational, clinical study was to investigate the expression patterns of TAM receptors in circulating white blood cells collected from patients with septic shock.The expression of TAM receptors was measured by flow cytometry in circulating leukocytes from patients with septic shock sampled on days (D) 1-2 (n = 47) and D3-4 (n = 37) after the onset of shock, severe trauma patients at D1-2 after trauma (n = 51) and healthy individuals (n = 23).On D1-2 after injury, MerTK was overexpressed in monocytes and neutrophils collected from patients with septic shock in comparison with those collected from healthy volunteers and trauma patients. This phenomenon was also observed for mRNA. Conversely, the expression of Tyro-3 and Axl was higher in monocytes from trauma patients versus healthy volunteers or those in septic shock. MerTK expression between D1-2 and D3-4 remained elevated in patients suffering from septic shock who died or developed an intensive care unit-acquired infection, whereas it decreased in patients who recovered uneventfully. This in vivo observed expression pattern was reproduced ex vivo after the incubation of healthy volunteer cells with plasma from septic shock or trauma patients.MerTK expression in circulating innate immune cells is increased in patients with septic shock in comparison with healthy volunteers and trauma patients. Persistent MerTK overexpression after septic shock is associated with adverse outcome. The role of this family of receptors in the pathophysiology of injury-induced immune dysfunctions deserves to be specifically investigated.

Background: By measuring multiple parameters on a single-cell basis, flow cytometry is a potent tool to dissect the phenotypes and functions of cell subsets. However, because this technique may be time-consuming, particularly for intracellular staining, it could be problematic for its use in daily routine or in large cohorts. Recently, a novel reagent has been developed to perform intracellular staining in one step. The objective of our study was thus to assess this new method in comparison with the reference technique by focusing on FOXP3 staining in clinical samples. Methods: Peripheral blood was collected from 15 HIV-1-infected patients, 5 critically ill patients, and 5 healthy volunteers and stained using the two different methods. Different subsets of FOXP3 positive cells were investigated by flow cytometry. Results: When comparing results obtained with the two techniques, no statistical differences between the percentages of CD4+FOXP3+, CD4+CD25+FOXP3+, and CD4+CD25+CD127−FOXP3+ cells were observed. Besides, a strong correlation between percentages of CD4+FOXP3+CD25+CD127− lymphocytes measured with both techniques was found in patients (r: 0.843, P < 0.001, intra-class correlation coefficient: 0.820, P < 0.001). Importantly, flow cytometry stainings obtained with the one-step method were very robust with an excellent intra-assay precision, a better discriminative power and correct stability and reproducibility of the staining even after blood storage. Conclusions: With a strong correlation between the percentages of FOXP3+ Tregs when compared with the reference method, a better staining quality, a shorter realization time and no need of isotype control, this one step procedure may represent an important improvement for a daily routine use of intracellular staining. © 2012 International Clinical Cytometry Society

International audience; Objective: Septic syndromes are the leading causes of mortality in intensive care units. In patients, the occurrence of sepsis-induced immune suppression is associated with delayed mortality, although the exact role of lymphocyte dysfunctions is not well established. The objective of this study was to investigate T-cell receptor diversity, an important feature of T-cell response, in patients with septic shock.Design: Preliminary prospective observational study.Setting: Adult intensive care units in a university hospital.Subjects: Patients with septic shock (n = 41) sampled twice after the onset of shock (early after inclusion [day 1] and at the end of the first week [day 7]).Measurements and Main Results: Using a novel molecular biology technique, the combinatorial diversity of human T-cell receptor β-chain (TRB locus) was measured in peripheral blood. Patients with septic shock presented with a marked decreased T-cell receptor diversity after the onset of shock in comparison with normal values. Importantly, in paired samples, a very steep recovery slope of T-cell receptor diversity, never described in other clinical situations, was observed between day 1 and day 7 (p < 0.0001, Wilcoxon’s paired test). Decreased T-cell receptor diversity was associated with mortality (log-rank test, p = 0.0058; hazard ratio = 4.48; 95% confidence interval 1.96–53.32), and the development of nosocomial infections (p < 0.05, Mann-Whitney U test).Conclusion: Our results show for the first time that septic patients present with a marked decreased T-cell receptor diversity that returned rapidly toward normal values over time. This opens novel cognitive research perspectives that deserve to be investigated in experimental models of sepsis. After confirmation in larger cohorts of these preliminary results, T-cell receptor diversity measurements may become a crucial tool to monitor immune functions in ICU patients.

Septic syndrome is the leading cause of mortality for critically ill patients worldwide. Patients develop lymphocyte dysfunctions associated with increased risk of death and nosocomial infections. In this study, we performed preclinical experiments testing the potential of recombinant human IL-7 (rhIL-7) as a lymphostimulating therapy in sepsis. Circulating IL-7 and soluble IL-7 receptor α-chain (soluble CD127) concentrations were measured in plasma, whereas cellular CD127 expression was evaluated on circulating CD4+ and CD8+ lymphocytes from septic shock patients and healthy volunteers. Lymphocyte proliferation, IFN-γ production, STAT5 phosphorylation, and B cell lymphoma 2 induction were measured ex vivo in response to T cell stimulation in the presence or not of rhIL-7. We show that IL-7 pathway (plasmatic IL-7 concentration and cellular and soluble CD127 expressions) is not overtly altered and remains activable in septic patients. Most importantly ex vivo treatment of patients’ cells with rhIL-7 significantly improves lymphocyte functionality (CD4+ and CD8+ lymphocyte proliferations, IFN-γ production, STAT5 phosphorylation, and B cell lymphoma 2 induction after stimulation). To our knowledge, this constitutes the first report of rhIL-7 ability to restore normal lymphocyte functions in septic patients. These results support the rational for initiating a clinical trial testing rhIL-7 in septic shock.

Septic patients develop immune dysfunctions, the intensities and durations of which are associated with deleterious outcomes. LILRB2 (leukocyte immunoglobulin-like receptors subfamily B, member 2), an inhibitory member of the LILR family of receptors, is known for its immunoregulatory properties. In a microarray study, we identified LILRB2 as an upregulated gene in septic shock patients. On monocytes primed with LPS ex vivo, LILRB2 mRNA and protein expressions were dose-dependently downregulated and subsequently highly upregulated versus non-stimulated cells. This is concordant with clinical data, since both LILRB2 mRNA and protein expressions were significantly increased in septic shock patients at day 3. In a cohort of more than 700 patients, only after septic shock were LILRB2 mRNA levels increased compared with non-infected or less severely infected patients. This was preceded by a phase of downregulated mRNA expression during the first hours after septic shock. Interestingly, the intensity of this decrease was associated with increased risk of death after septic shock. LILRB2 protein and mRNA expressions are deregulated on monocytes after septic shock and this can be reproduced ex vivo after LPS challenge. Considering LILRB2 inhibitory properties, we can hypothesize that LILRB2 may participate in the altered immune response after septic shock.

The monitoring of septic shock induced immunosuppression has been proposed to identify patients who could benefit from specific immunoadjuvant therapies. Among potential biomarkers to monitor immunological status, functional testing remains the gold standard because it directly measures the capacity of a cell population to respond to an immune challenge. We investigated a new approach in intracellular staining for flow cytometry to measure tumor necrosis factor (iTNF) produced in vitro by monocytes in response to lipopolysaccharide.Observational study in intensive care unit and immunology laboratory of a university medical center. Sixteen septic shock patients and eight control subjects were included.Monocyte iTNF was determined by flow cytometry in whole blood and completed in 2.5 h according to a no-wash, no centrifuge procedure. Lipopolysaccharide challenge induced a tremendous expression of iTNF that was statistically more pronounced in controls than in patients. This was observed when results were expressed as medians of fluorescence intensity (median: 16.1 [IQR: 14.5-19.1] vs. 5 [4.0-8.0], P = 0.0001) or as percentages of positive cells (99.7 [99.6-99.8] vs. 85 [74-97], P = 0.0001). iTNF expression was correlated to monocyte HLA-DR expression in patients and controls.These preliminary results illustrate the feasibility of immune functional testing on a routine manner in septic shock patients. They now deserve to be widely assessed and validated in various intensive care unit conditions. This could be a major step to characterize the rapidly changing immune response overtime and thus permit personalized medicine.

In several clinical contexts, the measurement of ATP concentration in T lymphocytes has been proposed as a biomarker of immune status, predictive of secondary infections. However, the use of such biomarker in lymphopenic patients requires some adaptations in the ATP dosage protocol. We used blood from healthy volunteers to determine the optimal experimental settings. We investigated technical aspects such as the type of anticoagulant for blood sampling, the effect of freeze and thaw cycles, the reagent and sample mixing sequence, and the optimal dilution buffer. We also shortened the incubation time to 8 h, and even showed that a 30 min incubation may be sufficient. To evaluate the ATP rise upon lymphocyte activation, the optimal dose of stimulant was defined to be 4 μg/mL of phytohaemagglutinin. Lastly, we determined that the number of T cells needed for this measurement was as low as 50,000, which is compatible with the existing lymphopenia in clinical settings. This optimized protocol appears ready to be assessed in lymphopenic patients to further investigate the interconnection between T lymphocyte metabolism and impaired phenotype and functions.

International audience; Out-of-hospital cardiac arrest (OHCA) constitutes a major health care problem with the development in immediate survivors of a post-cardiac arrest syndrome including systemic inflammatory response as observed in sepsis. As a decreased monocyte HLA-DR expression (mHLA-DR) has been repeatedly described in septic patients in association with an increased risk of death and nosocomial infections, we tested whether this immune alteration could also be observed after OHCA. Fifty-five non-shockable OHCA patients sampled at Day 0 (D0: within 4 h after OHCA), D1 (the next day), and D3: (after 2 additional days) were included. CD4+ lymphocyte count and mHLA-DR were evaluated by flow cytometry. We observed a marked decrease in mHLA-DR as early as D0 in patients compared with normal values. This decrease persisted till D3 and was associated with a moderate decrease in the number of circulating CD4+ lymphocytes. No correlations were identified between mHLA-DR and usual prognostic markers after OHCA. However, overtime evolution in mHLA-DR values appeared different between survivors and non-survivors with a quasisystematic decrease between D1 and D3 in non-survivors versus an increased expression in survivors. In conclusion, this preliminary pilot study describes the occurrence of OHCA-induced immune alterations as illustrated by a decreased mHLA-DR and CD4+ lymphopenia. Furthermore, we show for the first time the differential overtime evolution in mHLA-DR between survivors and non-survivors without association with usual prognostic markers and multiple organ failure. These initial results should now be confirmed in a larger cohort of OHCA patients.

Severe injuries such as severe sepsis, burn, trauma and major surgery lead to an overlapping development of pro- and anti- inflammatory responses. It is now well established that these injuries are associated with the secondary development of immune suppression, which results in significant morbidity and mortality. Recent data suggest that immunostimulatory drugs might prevent these complications. However, intensive care patients are heterogeneous, making patient stratification essential for a targeted treatment. In the present review, we discuss potential biomarkers of injury-induced immunoparalysis, mainly focusing on these that have been associated with poor outcome in various clinical settings. We namely present clinical data on monocyte human leukocyte antigen DR, lymphopenia, PD-1/PD-L1 and transcriptomic approach.

Background Chemokine (C-X3-C motif) receptor 1 (CX3CR1) was identified as the most differentially expressed gene between survivors and non-survivors in two independent cohorts of septic shock patients and was proposed as a marker of sepsis-induced immunosuppression. Whether such a biomarker is associated with mortality in the heterogeneous group of critically ill patients is unknown. The primary objective of this study was to evaluate the association between CX3CR1 messenger RNA (mRNA) expression and mortality in intensive care unit (ICU) patients. The secondary objective was to evaluate similar endpoints in the subgroup of septic shock patients. Methods We performed a prospective, multicentre, non-interventional study in six ICUs of university hospitals in Lyon, France. Every consecutive adult patient with systemic inflammatory response syndrome and an expected length of stay in the ICU over 2 days was included. Whole-blood CX3CR1 mRNA expression was measured by quantitative real-time polymerase chain reaction at day 1 (D1) and D3 after inclusion. Results In ICU patients (n = 725), decreased CX3CR1 mRNA expression at D1 was associated with high D7 mortality (AUC 0.70, adjusted OR [aOR] 2.03, 95 % CI 1.19–3.46), while decreased expression at D3 was associated with increased D28 mortality (AUC 0.64, aOR 2.34, 95 % CI 1.45–3.77). In septic shock patients (n = 279), similar associations were observed between decreased D1 CX3CR1 mRNA expression and D7 mortality (AUC 0.69, aOR 2.76, 95 % CI 1.32–5.75) as well as decreased D3 expression and D28 mortality (AUC 0.72, aOR 3.98, 95 % CI 1.72–9.23). These associations were independent of lactacidaemia, Simplified Acute Physiology Score II, Sepsis-related Organ Failure Assessment score and Charlson comorbidity index. Conclusions This study represents the largest evaluation of such an mRNA marker in a heterogeneous cohort of severely injured patients. Our results show that decreased CX3CR1 mRNA expression is associated with increased mortality in ICU patients. This suggests a link between injury-induced immunosuppression and mortality in critically ill patients. In this context, the monitoring of such a host response molecular biomarker could prove very helpful for the identification of patients at high risk of death in the ICU. Electronic supplementary material The online version of this article (doi:10.1186/s13054-016-1362-x) contains supplementary material, which is available to authorized users.

Sepsis represents the host's systemic inflammatory response to an infection. In this condition, immune response associates a marked inflammatory response and the delayed development of severe dysfunctions affecting both innate and adaptive responses. As neutrophils are the first line of defense against infection, they are central to the pathophysiology of sepsis in first hours. Nevertheless, their role during immunosuppression phase remains elusive. The main objective of the current work was to perform a transcriptomic study on purified neutrophils from septic shock patients (n=9) so as to identify genes that are differentially expressed during the first week after disease onset both (3-4 and 6-8days) versus healthy donors. Then, 45 septic shock patients were prospectively enrolled to confirm results at the protein level using flow cytometry. Twenty healthy volunteers (HV) were also included for the whole study. By comparing the transcriptome of purified neutrophils, we identified 364 up-regulated and 328 down-regulated genes differentially expressed. Of them, CD177 mRNA, coding for an activation molecule in chemotaxis, had the highest fold change modulation between patients and HV. This increase was then confirmed at the protein level. There was a constant subset of neutrophils that did not express CD177. However, when positive, septic neutrophils presented with significantly increased CD177 expression. Of note, no association between CD177 overexpression and features of immunosuppression has been highlighted. In addition, this up-regulation was negatively correlated with a decreased expression of CD10, a characteristic of immature myeloid cells. In conclusion, in this exploratory work, we shed light on the increased CD177 mRNA and protein expressions in circulating neutrophils after septic shock. Considering the potential dual roles of CD177 neutrophil (i.e., maturation/chemotaxis), negatively correlated in this study, its participation in septic shock pathophysiology deserves further investigation. Furthermore, its potential as a biomarker for sepsis would deserve to be investigated in large cohorts of patients.

In parallel with an exaggerated pro-inflammatory response, critically-ill patients develop an immunosuppressive phase, termed immunoparesis/immunoparalysis or immune reprogramming. Innate and adaptive immune responses are affected. In particular, impaired neutrophil recruitment to injury sites and abnormal accumulation in remote sites; monocyte deactivation with preferential anti-inflammatory cytokine production and altered antigen presentation capacity; and a dramatic lymphopenia associated with major induction of apoptosis, functional, and phenotypic alterations have been described. The intensity and duration of this injury-induced immune dysfunction have been associated with an increased risk of death and secondary nosocomial infections. Innovative therapeutic strategies aiming at restoring immunological functions are currently being tested. GM-CSF appears to be an interesting candidate while IFN-γ‎ and IL-7 represent novel future therapeutic approaches. There is thus an urgent need for further clinical trials of such immunoadjuvant therapies that should include large cohorts of critically-ill patients stratified by relevant markers of immune dysfunction.

Objectives Hospital-acquired infections (HAI) are associated with significant mortality and morbidity and prolongation of hospital stay, adding strain on limited hospital resources. Despite stringent infection control practices some children remain at high risk of developing HAI. The development of biomarkers which could identify these patients would be useful. In this study our objective was to evaluate mRNA candidate biomarkers for HAI prediction in a pediatric intensive care unit. Design Serial blood samples were collected from patients admitted to pediatric intensive care unit between March and June 2012. Candidate gene expression (IL1B, TNF, IL10, CD3D, BCL2, BID) was quantified using RT-qPCR. Comparisons of relative gene expression between those that did not develop HAI versus those that did were performed using Mann Whitney U-test. Patients Exclusion criteria were: age

Sepsis is characterized by pro- and anti-inflammatory responses following infection. While inflammation is responsible for widespread organ damage, anti-inflammatory mediators lead to immunoparalysis increasing susceptibility to secondary infections (nosocomial pneumonia). We aimed to investigate the impact of bacterial load on survival and cytokine release in a two-hit murine (C57BL/6J) model of CLP followed by P. aeruginosa pneumonia. Plasmatic TNFα, IL-6, IL-10, sTNFr I and II were quantified until 13 days. At D5, splenocytes were processed for immunological assays or mice were intratracheally instilled with Pseudomonas aeruginosa (5.106, 2.107 and 108 CFU) to evaluate survival and cytokines production. TNFα, sTNFrs, IL-6 and IL-10 increased 2h post CLP. TNFα and sTNFrs declined respectively one and two days later. In CLP mice, IL-6 and IL-10 remained high for the whole experiment, as compared to Sham. At D5, for CLP mice, whereas total T cells population (CD3+) decreased, Treg fraction (CD4+/CD25+) increased. In parallel, T cells proliferation and LPS-stimulated splenocytes ability to release TNFα decreased. At D13, survival was 100% after 5.106 CFU, 50% for CLP mice after 2.107 CFU and 0% for CLP and Sham after 108 CFU. After instillation, IL-10 and IL-6 increased and appeared to be dose and time dependent. Pseudomonas was detected in all CLP and Sham’s lungs; in spleen and liver only in CLP at 2.107 CFU, and in CLP and Sham at 108 CFU. We demonstrated that post-CLP immunosuppression followed by Pseudomonas aeruginosa lung instillation increases mortality reactivates cytokines secretion and is associated with systemic dissemination in septic mice depending on bacterial load.

Background: Although likely pivotal, the role of regulatory T cells (Tregs) in HIV pathogenesis remains elusive. This can be partly explained by analytical issues regarding their phenotypic identification in clinical studies. Instead of intracellular FOXP3 staining, CD4+CD25+CD127− phenotype has been proposed as an alternative to identify Tregs in clinical samples. However, its use remains controversial in viremic patients. Therefore, the objective of the present study was to assess the correlation between frequencies of CD4+CD25+CD127− and CD4+CD25+FOXP3+ lymphocytes in viremic and matched aviremic HIV-infected patients. Methods: Peripheral blood was collected from HIV-1 infected patients. Eleven viremic patients (Viral Load > 40 copies/mL) were matched (age, sex, CD4+ cell number) with 8 aviremic patients under highly active antiretroviral therapy (HAART). Fresh whole blood was immediately stained to analyze by flow cytometry the correlation between CD4+CD25+CD127− and the reference phenotype CD4+CD25+FOXP3+ lymphocytes in the same tube (four color staining CD4/CD25/CD127/FOXP3 for concomitant analysis of cell surface and intracellular markers). Results: In both groups, no significant differences were observed when comparing CD4+CD25+CD127− and CD4+CD25+FOXP3+ cell frequencies. In line, a strong correlation between CD4+CD25+CD127− and CD4+CD25+FOXP3+ lymphocyte percentages was observed in the whole patient population (r: 0.948, P < 0.001) or each group separately: aviremic (r: 0.968, P < 0.001), viremic (r: 0.9, P < 0.001). Finally, we found that most CD4+FOXP3+ cells were indeed CD25+CD127−, both in viremic and aviremic groups (88.5% and 90.9%, respectively). Conclusions: We observed that CD4+CD25+CD127− phenotype is a good and easy-to-perform surrogate identification strategy for FOXP3+ regulatory T cells in both viremic and aviremic HIV-1-infected subjects. Thus, it represents a useful tool for monitoring Tregs in clinical research studies based on large cohorts of patients prospectively monitored, including HIV-infected subjects. © 2012 International Clinical Cytometry Society

Tumor necrosis factor α (TNF-α) has been reported to be a key component of the functional priming, of both myeloid and nonmyeloid cells, that is thought to contribute to the lung's increased susceptibility to injury following shock. Not surprisingly, we found that mice deficient in TNF-α exhibited reduced acute lung injury (ALI) resultant from the combined insults of hemorrhagic shock and sepsis. However, we found that when we adoptively transferred neutrophils from mice expressing TNF-α to neutrophil-depleted mice that lacked TNF-α, they were not able to serve as priming stimulus for the development of ALI. Based on these findings, we proposed that resident lung tissue cells mediate TNF-α priming. To begin to unravel the complex signaling pathway of various resident lung tissue cells in TNF-α-induced priming, we compared the effect of local (intratracheal [i.t.]) versus systemic [intravenous (i.v.)] delivery of TNF-α small interference (siRNA). We hypothesized that alternately suppressing expression of TNF-α in lung endothelial (i.v.) or epithelial (i.t.) cells would produce a differential effect in shock-induced ALI. We found that when in vivo siRNA i.t. or i.v. against TNF-α was administered to C57/BL6 mice at 2 h after hemorrhage, 24 h before septic challenge, that systemic/i.v., but not i.t., delivery of TNF-α siRNA following hemorrhage priming significantly reduces expression of indices of ALI compared with controls. These findings suggest that an absence of local lung tissue TNF-α significantly reduces lung tissue injury following hemorrhage priming for ALI and that pulmonary endothelial and/or other possible vascular resident cells, not epithelial cells, play a greater role in mediating the TNF-α priming response in a mouse model of hemorrhage/sepsis-induced ALI.