Deleterious effect of sustained neuroinflammation in pediatric traumatic brain injury.

INTRODUCTION: Despite improved management of traumatic brain injury (TBI), it still leads to lifelong sequelae and disability, particularly in children. Chronic neuroinflammation (the so-called tertiary phase), in particular, microglia/macrophage and astrocyte reactivity, is among the main mechanisms suspected of playing a role in the generation of lesions associated with TBI. The role of acute neuroinflammation is now well understood, but its persistent effect and impact on the brain, particularly during development, are not. Here, we investigated the long-term effects of pediatric TBI on the brain in a mouse model. METHODS: Pediatric TBI was induced in mice on postnatal day (P) 7 by weight-drop trauma. The time course of neuroinflammation and myelination was examined in the TBI mice. They were also assessed by magnetic resonance, functional ultrasound, and behavioral tests at P45. RESULTS: TBI induced robust neuroinflammation, characterized by acute microglia/macrophage and astrocyte reactivity. The long-term consequences of pediatric TBI studied on P45 involved localized scarring astrogliosis, persistent microgliosis associated with a specific transcriptomic signature, and a long-lasting myelination defect consisting of the loss of myelinated axons, a decreased level of myelin binding protein, and severe thinning of the corpus callosum. These results were confirmed by reduced fractional anisotropy, measured by diffusion tensor imaging, and altered inter- and intra-hemispheric connectivity, measured by functional ultrasound imaging. In addition, adolescent mice with pediatric TBI showed persistent social interaction deficits and signs of anxiety and depressive behaviors. CONCLUSIONS: We show that pediatric TBI induces tertiary neuroinflammatory processes associated with white matter lesions and altered behavior. These results support our model as a model for preclinical studies for tertiary lesions following TBI.

Whole blood no-lyse no-wash micromethod for the quantitative measurement of monocyte HLA-DR.

BACKGROUND: Monocyte (m)HLA-DR expression appears to be a potent marker of immunosuppression in critically ill patients. The persistence of low mHLA-DR expression is associated with an increased risk of nosocomial infections and mortality. To adapt this measurement to pediatric requirements and provide extensive 24/7 access, we have developed a whole blood no-lyse no-wash micromethod (MM) and compared it with the standardized method (SM). METHODS: mHLA-DR was quantified by flow cytometry using Quantibrite™ Anti-HLA-DR PE/Monocyte PerCP-Cy™5.5 with either the SM performed in a diagnostic hematology laboratory using manufacturer protocol, or a whole blood no-lyse no-wash MM using an Attune flow cytometer located in the pediatric ICU. Median fluorescence intensity was measured in both techniques and converted to antibodies per cell (AB/C) calibrated with BD Quantibrite™ PE beads. Blood and Quantibrite™ reagent volume used with the MM was reduced by 5-fold compared to SM. In addition to Quantibrite™ Anti-Human HLA-DR PE/Monocyte PerCP-Cy™5.5, MM required anti-CD45 and anti-CD19 labeling. RESULTS: We determined the expression of mHLA-DR in 34 patients, 20 adults, and 14 children admitted to ICU. Correlation between MM and SM was excellent (Pearson's correlation: y = 0.8192x + 678.7, r = 0.9270, p < 0.0001). The estimated bias was 2467 ± 1.96 × 3307 AB/C; CI 95% [-4016; +8949]. CONCLUSIONS: The no-lyse no-wash whole blood microvolume method for measuring mHLA-DR expression allows for simplified sample preparation without compromising accuracy of the data. This method may simplify immune monitoring of critically ill patient by the deployment of a point of care method.

G protein-coupled receptor 17 is regulated by WNT pathway during oligodendrocyte precursor cell differentiation.

G protein-coupled receptor 17 (GPR17) and the WNT pathway are critical players of oligodendrocyte (OL) differentiation acting as essential timers in developing brain to achieve fully-myelinating cells. However, whether and how these two systems are related to each other is still unknown. Of interest, both factors are dysregulated in developing and adult brain diseases, including white matter injury and cancer, making the understanding of their reciprocal interactions of potential importance for identifying new targets and strategies for myelin repair. Here, by a combined pharmacological and biotechnological approach, we examined regulatory mechanisms linking WNT signaling to GPR17 expression in OLs. We first analyzed the relative expression of mRNAs encoding for GPR17 and the T cell factor/Lymphoid enhancer-binding factor-1 (TCF/LEF) transcription factors of the canonical WNT/ß-CATENIN pathway, in PDGFRα+ and O4+ OLs during mouse post-natal development. In O4+ cells, Gpr17 mRNA level peaked at post-natal day 14 and then decreased concomitantly to the physiological uprise of WNT tone, as shown by increased Lef1 mRNA level. The link between WNT signaling and GPR17 expression was further reinforced in vitro in primary PDGFRα+ cells and in Oli-neu cells. High WNT tone impaired OL differentiation and drastically reduced GPR17 mRNA and protein levels. In Oli-neu cells, WNT/ß-CATENIN activation repressed Gpr17 promoter activity through both putative WNT response elements (WRE) and upregulation of the inhibitor of DNA-binding protein 2 (Id2). We conclude that the WNT pathway influences OL maturation by repressing GPR17, which could have implications in pathologies characterized by dysregulations of the OL lineage including multiple sclerosis and oligodendroglioma.

Targeting the brain 5-HT7 receptor to prevent hypomyelination in a rodent model of perinatal white matter injuries.

Approximately 15 million babies are born prematurely every year and many will face lifetime motor and/or cognitive deficits. Children born prematurely are at higher risk of developing perinatal brain lesions, especially white matter injuries (WMI). Evidence in humans and rodents demonstrates that systemic inflammation-induced neuroinflammation, including microglial and astrocyte reactivity, is the prominent processes of WMI associated with preterm birth. Thus, a new challenge in the field of perinatal brain injuries is to develop new neuroprotective strategies to target neuroinflammation to prevent WMI. Serotonin (5-HT) and its receptors play an important role in inflammation, and emerging evidence indicates that 5-HT may regulate brain inflammation by the modulation of microglial reactivity and astrocyte functions. The present study is based on a mouse model of WMI induced by intraperitoneal (i.p.) injections of IL-1ß during the first 5 days of life. In this model, certain key lesions of preterm brain injuries can be summarized by (i) systemic inflammation, (ii) pro-inflammatory microglial and astrocyte activation, and (iii) inhibition of oligodendrocyte maturation, leading to hypomyelination. We demonstrate that Htr7 mRNA (coding for the HTR7/5-HT7 receptor) is significantly overexpressed in the anterior cortex of IL-1ß-exposed animals, suggesting it as a potential therapeutic target. LP-211 is a specific high-affinity HTR7 agonist that crosses the blood-brain barrier (BBB). When co-injected with IL-1ß, LP-211 treatment prevented glial reactivity, the down-regulation of myelin-associated proteins, and the apparition of anxiety-like phenotypes. Thus, HTR7 may represent an innovative therapeutic target to protect the developing brain from preterm brain injuries.

CIITA promoter polymorphism impairs monocytes HLA-DR expression in patients with septic shock.

Low monocyte (m)HLA-DR expression is associated with mortality in sepsis. G-286A∗rs3087456 polymorphism in promoter III of HLA class II transactivator (CIITA), the master regulator of HLA, has been associated with autoimmune diseases but its role in sepsis has never been demonstrated. In 203 patients in septic shock, GG genotype was associated with 28-day mortality and mHLA-DR remained low whereas it increased in patients with AA or AG genotype. In ex vivo cells, mHLA-DR failed to augment in GG in comparison with AG or AA genotype on exposure to IFN-γ. Promoter III transcript levels were similar in control monocytes regardless of genotype and exposure to IFN-γ. Promoter III activity was decreased in GG genotype in monocyte cell line but restored after stimulation with IFN-γ. Hereby, we demonstrated that G-286A∗rs3087456 significantly impact mHLA-DR expression in patients with septic shock in part through CIITA promoter III activity, that can be rescued using IFN-γ.

Magnetic Isolation of Microglial Cells from Neonate Mouse for Primary Cell Cultures.

Microglia, as brain resident macrophages, are fundamental to several functions, including response to environmental stress and brain homeostasis. Microglia can adopt a large spectrum of activation phenotypes. Moreover, microglia that endorse pro-inflammatory phenotype is associated with both neurodevelopmental and neurodegenerative disorders. In vitro studies are widely used in research to evaluate potential therapeutic strategies in specific cell types. In this context studying microglial activation and neuroinflammation in vitro using primary microglial cultures is more relevant than microglial cell lines or stem-cell-derived microglia. However, the use of some primary cultures might suffer from a lack of reproducibility. This protocol proposes a reproducible and relevant method of magnetically isolating microglia from neonate pups. Microglial activation using several stimuli after 4 h and 24 h by mRNA expression quantification and a Cy3-bead phagocytic assay is demonstrated here. The current work is expected to provide an easily reproducible technique for isolating physiologically relevant microglia from juvenile developmental stages.

The Impact of Mouse Preterm Birth Induction by RU-486 on Microglial Activation and Subsequent Hypomyelination.

Preterm birth (PTB) represents 15 million births every year worldwide and is frequently associated with maternal/fetal infections and inflammation, inducing neuroinflammation. This neuroinflammation is mediated by microglial cells, which are brain-resident macrophages that release cytotoxic molecules that block oligodendrocyte differentiation, leading to hypomyelination. Some preterm survivors can face lifetime motor and/or cognitive disabilities linked to periventricular white matter injuries (PWMIs). There is currently no recommendation concerning the mode of delivery in the case of PTB and its impact on brain development. Many animal models of induced-PTB based on LPS injections exist, but with a low survival rate. There is a lack of information regarding clinically used pharmacological substances to induce PTB and their consequences on brain development. Mifepristone (RU-486) is a drug used clinically to induce preterm labor. This study aims to elaborate and characterize a new model of induced-PTB and PWMIs by the gestational injection of RU-486 and the perinatal injection of pups with IL-1beta. A RU-486 single subcutaneous (s.c.) injection at embryonic day (E)18.5 induced PTB at E19.5 in pregnant OF1 mice. All pups were born alive and were adopted directly after birth. IL-1beta was injected intraperitoneally from postnatal day (P)1 to P5. Animals exposed to both RU-486 and IL-1beta demonstrated microglial reactivity and subsequent PWMIs. In conclusion, the s.c. administration of RU-486 induced labor within 24 h with a high survival rate for pups. In the context of perinatal inflammation, RU-486 labor induction significantly decreases microglial reactivity in vivo but did not prevent subsequent PWMIs.

A unique cerebellar pattern of microglia activation in a mouse model of encephalopathy of prematurity.

Preterm infants often show pathologies of the cerebellum, which are associated with impaired motor performance, lower IQ and poor language skills at school ages. Using a mouse model of inflammation-induced encephalopathy of prematurity driven by systemic administration of pro-inflammatory IL-1ß, we sought to uncover causes of cerebellar damage. In this model, IL-1ß is administered between postnatal day (P) 1 to day 5, a timing equivalent to the last trimester for brain development in humans. Structural MRI analysis revealed that systemic IL-1ß treatment induced specific reductions in gray and white matter volumes of the mouse cerebellar lobules I and II (5% false discovery rate [FDR]) from P15 onwards. Preceding these MRI-detectable cerebellar volume changes, we observed damage to oligodendroglia, with reduced proliferation of OLIG2+ cells at P10 and reduced levels of the myelin proteins myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) at P10 and P15. Increased density of IBA1+ cerebellar microglia were observed both at P5 and P45, with evidence for increased microglial proliferation at P5 and P10. Comparison of the transcriptome of microglia isolated from P5 cerebellums and cerebrums revealed significant enrichment of pro-inflammatory markers in microglia from both regions, but cerebellar microglia displayed a unique type I interferon signaling dysregulation. Collectively, these data suggest that perinatal inflammation driven by systemic IL-1ß leads to specific cerebellar volume deficits, which likely reflect oligodendrocyte pathology downstream of microglial activation. Further studies are now required to confirm the potential of protective strategies aimed at preventing sustained type I interferon signaling driven by cerebellar microglia as an important therapeutic target.

miR-146b Protects the Perinatal Brain against Microglia-Induced Hypomyelination.

OBJECTIVES: In the premature newborn, perinatal inflammation mediated by microglia contributes significantly to neurodevelopmental injuries including white matter injury (WMI). Brain inflammation alters development through neuroinflammatory processes mediated by activation of homeostatic microglia toward a pro-inflammatory and neurotoxic phenotype. Investigating immune regulators of microglial activation is crucial to find effective strategies to prevent and treat WMI. METHODS: Ex vivo microglial cultures and a mouse model of WMI induced by perinatal inflammation (interleukin-1-beta [IL-1ß] and postnatal days 1-5) were used to uncover and elucidate the role of microRNA-146b-5p in microglial activation and WMI. RESULTS: A specific reduction in vivo in microglia of Dicer, a protein required for microRNAs maturation, reduces pro-inflammatory activation of microglia and prevents hypomyelination in our model of WMI. Microglial miRNome analysis in the WMI model identified miRNA-146b-5p as a candidate modulator of microglial activation. Ex vivo microglial cell culture treated with the pro-inflammatory stimulus lipopolysaccharide (LPS) led to overexpression of immunomodulatory miRNA-146b-5p but its drastic reduction in the microglial extracellular vesicles (EVs). To increase miRNA-146b-5p expression, we used a 3DNA nanocarrier to deliver synthetic miRNA-146b-5p specifically to microglia. Enhancing microglial miRNA-146b-5p overexpression significantly decreased LPS-induced activation, downregulated IRAK1, and restored miRNA-146b-5p levels in EVs. In our WMI model, 3DNA miRNA-146b-5p treatment significantly prevented microglial activation, hypomyelination, and cognitive defect induced by perinatal inflammation. INTERPRETATIONS: These findings support that miRNA-146b-5p is a major regulator of microglia phenotype and could be targeted to reduce the incidence and the severity of perinatal brain injuries and their long-term consequences. ANN NEUROL 2022;91:48-65.

Impact of Fetal Growth Restriction on the Neonatal Microglial Proteome in the Rat.

Microglial activation is a key modulator of brain vulnerability in response to intra-uterine growth restriction (IUGR). However, the consequences of IUGR on microglial development and the microglial proteome are still unknown. We used a model of IUGR induced by a gestational low-protein diet (LPD) in rats. Microglia, isolated from control and growth-restricted animals at P1 and P4, showed significant changes in the proteome between the two groups. The expression of protein sets associated with fetal growth, inflammation, and the immune response were significantly enriched in LPD microglia at P1 and P4. Interestingly, upregulation of protein sets associated with the oxidative stress response and reactive oxygen species production was observed at P4 but not P1. During development, inflammation-associated proteins were upregulated between P1 and P4 in both control and LPD microglia. By contrast, proteins associated with DNA repair and senescence pathways were upregulated in only LPD microglia. Similarly, protein sets involved in protein retrograde transport were significantly downregulated in only LPD microglia. Overall, these data demonstrate significant and multiple effects of LPD-induced IUGR on the developmental program of microglial cells, leading to an abnormal proteome within the first postnatal days.

The immune-inflammatory response of oligodendrocytes in a murine model of preterm white matter injury: the role of TLR3 activation.

A leading cause of preterm birth is the exposure to systemic inflammation (maternal/fetal infection), which leads to neuroinflammation and white matter injury (WMI). A wide range of cytokines and chemokines are expressed and upregulated in oligodendrocytes (OLs) in response to inflammation and numerous reports show that OLs express several receptors for immune related molecules, which enable them to sense inflammation and to react. However, the role of OL immune response in WMI is unclear. Here, we focus our study on toll-like receptor-3 (TLR3) that is activated by double-strand RNA (dsRNA) and promotes neuroinflammation. Despite its importance, its expression and role in OLs remain unclear. We used an in vivo mouse model, which mimics inflammation-mediated WMI of preterm born infants consisting of intraperitoneal injection of IL-1ß from P1 to P5. In the IL-1ß-treated animals, we observed the upregulation of Tlr3, IL-1ß, IFN-ß, Ccl2, and Cxcl10 in both PDGFRα+ and O4+ sorted cells. This upregulation was higher in O4+ immature OLs (immOLs) as compared to PDGFRα+ OL precursor cells (OPCs), suggesting a different sensitivity to neuroinflammation. These observations were confirmed in OL primary cultures: cells treated with TLR3 agonist Poly(I:C) during differentiation showed a stronger upregulation of Ccl2 and Cxcl10 compared to cells treated during proliferation and led to decreased expression of myelin genes. Finally, OLs were able to modulate microglia phenotype and function depending on their maturation state as assessed by qPCR using validated markers for immunomodulatory, proinflammatory, and anti-inflammatory phenotypes and by phagocytosis and morphological analysis. These results show that during inflammation the response of OLs can play an autonomous role in blocking their own differentiation: in addition, the immune activation of OLs may play an important role in shaping the response of microglia during inflammation.

Therapeutic potential of stem cells for preterm infant brain damage: Can we move from the heterogeneity of preclinical and clinical studies to established therapeutics?

Acquired perinatal brain injuries are a set of conditions that remains a key challenge for neonatologists and that have significant social, emotional and financial implications for our communities. In our perspective article, we will introduce perinatal brain injury focusing specifically on the events leading to brain damage in preterm born infants and outcomes for these infants. Then we will summarize and discuss the preclinical and clinical studies testing the efficacy of stem cells as neuroprotectants in the last ten years in perinatal brain injury. There are no therapies to treat brain damage in preterm born infants and a primary finding from this review is that there is a scarcity of stem cell trials focused on overcoming brain injuries in these infants. Overall, across all forms of perinatal brain injury there is a remarkable heterogeneity in previous and on-going preclinical and clinical studies in terms of the stem cell type, animal models/patient selection, route and time of administration. Despite the quality of many of the studies this variation makes it difficult to reach a valid consensus for future developments. However, it is clear that stem cells (and stem cell derived exosomes) can reduce perinatal brain injury and our field needs to work collectively to refine an effective protocol for each type of injury. The use of standardized stem cell products and testing these products across multiple models of injury will provide a stronger framework for clinical trials development.

Cell Metabolic Alterations due to Mcph1 Mutation in Microcephaly.

A distinctive feature of neocortical development is the highly coordinated production of different progenitor cell subtypes, which are critical for ensuring adequate neurogenic outcome and the development of normal neocortical size. To further understand the mechanisms that underlie neocortical growth, we focused our studies on the microcephaly gene Mcph1, and we report here that Mcph1 (1) exerts its functions in rapidly dividing apical radial glial cells (aRGCs) during mouse neocortical development stages that precede indirect neurogenesis; (2) is expressed at mitochondria; and (3) controls the proper proliferation and survival of RGCs, potentially through crosstalk with cellular metabolic pathways involving the stimulation of mitochondrial activity via VDAC1/GRP75 and AKT/HK2/VDAC1 and glutaminolysis via ATF4/PCK2. We currently report the description of a MCPH-gene implication in the interplay between bioenergetic pathways and neocortical growth, thus pointing to alterations of cellular metabolic pathways, in particular glutaminolysis, as a possible cause of microcephalic pathogenesis.

Multicentric experience with interferon gamma therapy in sepsis induced immunosuppression. A case series.

BACKGROUND: The sepsis-induced immunodepression contributes to impaired clinical outcomes of various stress conditions. This syndrome is well documented and characterized by attenuated function of innate and adaptive immune cells. Several pharmacological interventions aimed to restore the immune response are emerging of which interferon-gamma (IFNγ) is one. It is of paramount relevance to obtain clinical information on optimal timing of the IFNγ-treatment, -tolerance, -effectiveness and outcome before performing a RCT. We describe the effects of IFNγ in a cohort of 18 adult and 2 pediatric sepsis patients. METHODS: In this open-label prospective multi-center case-series, IFNγ treatment was initiated in patients selected on clinical and immunological criteria early (< 4 days) or late (> 7 days) following the onset of sepsis. The data collected in 18 adults and 2 liver transplanted pediatric patients were: clinical scores, monocyte expression of HLA-DR (flow cytometry), lymphocyte immune-phenotyping (flow cytometry), IL-6 and IL-10 plasma levels (ELISA), bacterial cultures, disease severity, and mortality. RESULTS: In 15 out of 18 patients IFNγ treatment was associated with an increase of median HLA-DR expression from 2666 [IQ 1547; 4991] to 12,451 [IQ 4166; 19,707], while the absolute number of lymphocyte subpopulations were not affected, except for the decrease number of NK cells 94.5 [23; 136] to 32.5 [13; 90.8] (0.0625)]. Plasma levels of IL-6 464 [201-770] to 108 (89-140) ng/mL (p = 0.04) and IL-10 from IL-10 from 29 [12-59] to 9 [1-15] pg/mL decreased significantly. Three patients who received IFNγ early after ICU admission (<4 days) died. The other patients had a rapid clinical improvement assessed by the SOFA score and bacterial cultures that were repeatedly positive became negative. The 2 pediatric cases improved rapidly, but 1 died for hemorrhagic complication. CONCLUSION: Guided by clinical and immunological monitoring, adjunctive immunotherapy with IFNγ appears well-tolerated in our cases and improves immune host defense in sepsis induced immuno suppression. Randomized clinical studies to assess its potential clinical benefit are warranted.

High plasma level of S100A8/S100A9 and S100A12 at admission indicates a higher risk of death in septic shock patients.

Biomarkers in sepsis for severity, prediction of outcome or reversibility of organ dysfunction are warranted. Measurements of plasma DAMP levels at admission can reflect the severity of cellular damage in septic shock, which might predict the prognosis and reduce the risk of overtreating patients with costly therapies. We measured plasma levels of two DAMPs, S100A8/S100A9 and S100A12 during the first 24 h of admission of septic shock patients. Forty-nine septic shock patients with a similar SOFA scores were selected from our sepsis database to compare a similar proportion of survivors and non-survivors. Plasma levels of S100A8/S100A9 and S100A12 were compared with healthy volunteers using in-house ELISA. Plasma levels of S100A8/S100A9 and S100A12 (5.71 [2.60-13.63] µg/mL and 0.48 [0.22-1.05] µg/mL) were higher in septic shock patients than in healthy volunteers (1.18 [0.74-1.93] µg/mL and 0.09 [0.02-0.39] µg/mL) (P < 0.0001 and P = 0.0030). Levels of S100A8/S100A9 and S100A12 in non-survivors at day 28 (11.70 [2.85-24.36] µg/mL and 0.62 [0.30-1.64] µg/mL) were significantly higher than in survivors (4.59 [2.16-7.47] µg/mL and 0.30 [0.20-0.49] µg/mL) (P = 0.0420 and P = 0.0248) and correlated well (Spearman r = 0.879, P < 0.0001). The high level of plasma calgranulins at admission in septic shock, were higher in non-survivors compared to survivors. These markers could indicate a higher risk of death when SOFA scores are similar and help the stratification of patients for improved care and therapy selection.

Early Sex Differences in the Immune-Inflammatory Responses to Neonatal Ischemic Stroke.

We recently reported that neonatal ischemia induces microglia/macrophage activation three days post-ischemia. We also found that female mice sustained smaller infarcts than males three months post-ischemia. The objective of our current study was to examine whether differential acute neuroinflammatory response and infiltrated immune cells occurs between male and females after three days post-ischemia. Permanent middle cerebral artery occlusion was induced in male and female postnatal 9-day-old (P9) mice, and mice were sacrificed three days after ischemia. Brains were analyzed for mRNA transcription after microglia magnetic cell sorting to evaluate M1 and M2 markers. FACS analysis was performed to assess myeloid infiltration and microglial expression of CX3 chemokine receptor 1 (CX3CR1). Inflammatory cytokine expression and microglia/macrophage activation were analyzed via in situ hybridization combined with immunofluorescence techniques. Lesion volume and cell death were measured. An increase in microglia/macrophages occurred in male versus female mice. The cells exhibited amoeboid morphology, and TNFα and ptgs2 (Cox-2) genes were more expressed in males. More myeloid cell infiltration was found in male versus female brains. However, we did not observe sex-dependent differences in the injured volume or cell death density. Our data show that sex differences in the acute microglial and immune responses to neonatal ischemia are likely both gene- and region-specific.

Downregulation of Blood Monocyte HLA-DR in ICU Patients Is Also Present in Bone Marrow Cells.

BACKGROUND: The downregulation of blood monocyte HLA-DR expression also occurs in tissue infiltrative cells in a context of acute clinical inflammation, especially sepsis. This context favors the development of secondary infections and results from various mechanisms. Little is known about HLA-DR expression on bone marrow (BM) cells of the monocyte lineage, the source of circulating monocytes. This study analyzed the BM HLA-DR expression in ICU patients compared to BM monocytes from non-ICU patients and to blood monocytes of control healthy donors. A potential dysfunction of myeloid differentiation was investigated in a sub-population of these ICU patients to characterize the phenotype of the immature forms of monocytes and granulocytes in BM. METHODS AND FINDINGS: BM and blood were drawn from 33 ICU and 9 non-ICU patients having a BM analysis to precise the etiology of abnormal low count in blood cells. The data were compared with blood cells of 28 control donors. Flow cytometry was used for both HLA-DR expression and phenotyping of immature forms of monocytes and granulocytes. HLA-DR expression was downregulated in both blood and BM monocyte in ICU patients compared to BM of non-ICU patients and blood of control donors. Amplitude of HLA-DR downregulation was comparable in septic and non-septic ICU patients. The phenotype of immature forms of monocytes and granulocytes in BM (n = 11) did not show abnormal myeloid (monocyte + granulocyte) differentiation. CONCLUSION: The downregulation of HLA-DR in BM monocyte lineage is present in ICU patients without major changes in myeloid cells. It may result from a regulation mediated by soluble and/or neuro-endocrine factors present in BM cell microenvironment.

Reactive oxygen species measure for rapid detection of infection in fluids.

BACKGROUND: Early detection of infection is critical to rapidly starting effective treatment. Diagnosis can be difficult, particularly in the intensive care unit (ICU) population. Because the presence of polymorphonuclear neutrophils in tissues is the hallmark of inflammatory processes, the objective of this proof of concept study was to determine whether the measurement of reactive oxygen species (ROS) could be an efficient diagnostic tool to rapidly diagnose infections in peritoneal, pleural and bronchoalveolar lavage (BAL) fluids in ICU patients. METHODS: We prospectively included all patients hospitalized in the 21-bed surgical ICU of a teaching hospital from June 2010 to February 2014 who presented with systemic inflammatory response syndrome with suspicion of a peritoneal or pleural fluid or pulmonary infection needing a BAL. Instantaneous basal ROS production was measured in fluids and after phorbol 12-myristate 13-acetate (PMA) stimulation. We compared patients with infected fluids to those with non-infected fluids. RESULTS: The overall ICU mortality rate was 34 %. A majority of patients were sampled following a delay of 5 days (2-12) after ICU admission, with most receiving antibiotics at the time of fluid sampling (71 %). Fluids were infected in 21/65 samples: 6/17 peritoneal fluids, 8/28 pleural fluids and 7/20 BALs. ROS production was significantly higher in the infected than in the non-infected group at baseline and after PMA stimulation in the peritoneal and pleural fluids but not in BAL. CONCLUSION: Assessing instantaneous ROS production appears as a fast and reliable diagnostic method for detecting peritoneal and pleural fluid infection.

Multicenter testing of the rapid quantification of radical oxygen species in cerebrospinal fluid to diagnose bacterial meningitis.

PURPOSE: Meningitis is a serious concern after traumatic brain injury (TBI) or neurosurgery. This study tested the level of reactive oxygen species (ROS) in cerebrospinal fluid (CSF) to diagnose meningitis in febrile patients several days after trauma or surgery. METHODS: Febrile patients (temperature > 38°C) after TBI or neurosurgery were included prospectively. ROS were measured in CSF within 4 hours after sampling using luminescence in the basal state and after cell stimulation with phorbol 12-myristate 13-acetate (PMA). The study was conducted in a single-center cohort 1 (n = 54, training cohort) and then in a multicenter cohort 2 (n = 136, testing cohort) in the Intensive Care and Neurosurgery departments of two teaching hospitals. The performance of the ROS test was compared with classical CSF criteria, and a diagnostic decision for meningitis was made by two blinded experts. RESULTS: The production of ROS was higher in the CSF of meningitis patients than in non-infected CSF, both in the basal state and after PMA stimulation. In cohort 1, ROS production was associated with a diagnosis of meningitis with an AUC of 0.814 (95% confidence interval (CI) [0.684-0.820]) for steady-state and 0.818 (95% CI [0.655-0.821]) for PMA-activated conditions. The best threshold value obtained in cohort 1 was tested in cohort 2 and showed high negative predictive values and low negative likelihood ratios of 0.94 and 0.36 in the basal state, respectively, and 0.96 and 0.24 after PMA stimulation, respectively. CONCLUSION: The ROS test in CSF appeared suitable for eliminating a diagnosis of bacterial meningitis.

Exocytosis acts as a modulator of the ILT4-mediated inhibition of neutrophil functions.

Neutrophils play a major role in inflammatory responses and immune defense against pathogens. Even though expression of inhibitory receptors has been reported on neutrophils, their role remains poorly defined. Here we show that primary human neutrophils expressed immunoglobulin-like transcript 4 (ILT4) inhibitory receptor and that this expression was induced during differentiation of the myelomonoblast PLB-985 cell line into "neutrophil-like" cells. Functional assays indicated that human leukocyte antigen G, the preferred ligand of ILT4, inhibited the phagocytic function of neutrophils. ILT4 engagement also impaired reactive oxygen species production induced through CD32a and both receptors were found colocalized into neutrophil lipid rafts. Moreover, neutrophil degranulation induced through inflammatory stimuli increased ILT4 expression as a result of the rapid translocation of an intracellular pool to the cell surface. Consequently to this ILT4 up-regulation, the human leukocyte antigen G-mediated inhibition of neutrophil phagocytic function was enhanced. Finally, we found that ILT4 up-regulation induced on healthy donor neutrophils following stimulation was impaired in presence of plasma from patients with sepsis. Similarly, ILT4 up-regulation was inhibited in neutrophils from septic patients. Altogether, our results reveal a unique mechanism of regulation of neutrophil functions through ILT4 and its exocytosis that may have implications in inflammatory disorders.