PPARα contributes to protection against metabolic and inflammatory derangements associated with acute kidney injury in experimental sepsis

Abstract Sepsis‐associated acute kidney injury (AKI) is a significant problem in critically ill children and adults resulting in increased morbidity and mortality. Fundamental mechanisms contributing to sepsis‐associated AKI are poorly understood. Previous research has demonstrated that peroxisome proliferator‐activated receptor α (PPARα) expression is associated with reduced organ system failure in sepsis. Using an experimental model of polymicrobial sepsis, we demonstrate that mice deficient in PPARα have worse kidney function, which is likely related to reduced fatty acid oxidation and increased inflammation. Ultrastructural evaluation with electron microscopy reveals that the proximal convoluted tubule is specifically injured in septic PPARα deficient mice. In this experimental group, serum metabolomic analysis reveals unanticipated metabolic derangements in tryptophan‐kynurenine‐NAD+ and pantothenate pathways. We also show that a subgroup of children with sepsis whose genome‐wide expression profiles are characterized by repression of the PPARα signaling pathway has increased incidence of severe AKI. These findings point toward interesting associations between sepsis‐associated AKI and PPARα‐driven fatty acid metabolism that merit further investigation

Fas (CD95) induces rapid, TLR4/IRAK4-dependent release of pro-inflammatory HMGB1 from macrophages

Although Fas (CD95) is recognized as a death receptor that induces apoptosis, recent studies indicate that the Fas/FasL system can induce pro-inflammatory cytokine production by macrophages independent of conventional caspase-mediated apoptotic signaling. The precise mechanism(s) by which Fas activates macrophage inflammation is unknown. We hypothesized that Fas stimulates rapid release of high mobility group box 1 (HMGB1) that acts in an autocrine and/or paracrine manner to stimulate pro-inflammatory cytokine production via a Toll-like receptor-4 (TLR4)/Interleukin-1 receptor associated kinase-4 (IRAK4)-dependent mechanism. Following Fas activation, HMGB1 was released within 1 hr from viable RAW267.4 cells and primary murine peritoneal macrophages. HMGB1 release was more rapid following Fas activation compared to LPS stimulation. Neutralization of HMGB1 with an inhibitory anti-HMGB1 monoclonal antibody strongly inhibited Fas-induced production of tumor necrosis factor (TNF) and macrophage inflammatory protein-2 (MIP-2). Both Fas-induced HMGB1 release and associated pro-inflammatory cytokine production were significantly decreased from Tlr4-/- and Irak4-/- macrophages, but not Tlr2-/- macrophages. These findings reveal a novel mechanism underlying Fas-mediated pro-inflammatory physiological responses in macrophages. We conclude that Fas activation induces rapid, TLR4/IRAK4-dependent release of HMGB1 that contributes to Fas-mediated pro-inflammatory cytokine production by viable macrophages

Performance Characteristics of Combinations of Host Biomarkers to Identify Women with Occult Placental Malaria: A Case-Control Study from Malawi

Because of its propensity to sequester in the placental intervillous space, Plasmodium falciparum can evade detection by peripheral smear in women with placental malaria (PM). We evaluated host biomarkers as potential indicators of occult PM infections.Using a case-control design, we evaluated the ability of biomarkers to identify PM in the absence of circulating peripheral parasites (n = 24) compared to placental smear-negative controls (n = 326). We measured levels of biomarkers (C3a, C5a, CRP, angiopoietin-1, angiopoietin-2, sTie-2, sEndoglin, VEGF, sFlt-1, tissue factor, and leptin) in maternal peripheral plasma at delivery. Using ROC curve analysis, we assessed the ability of clinical parameters and biomarkers to accurately detect PM infections identified by placental smear. We show that decreases in sFlt-1 and leptin and increases in CRP were associated with occult PM infections (p<0.01) and correlated with placental parasitaemia (p<0.01). Individually, all markers had moderate ability to diagnose occult PM infections with areas under the ROC between 0.62 and 0.72. In order to improve diagnostic performance, we generated simple scoring systems to identify PM infections using either a clinical score (0-2), a biomarker score (0-3) or a clinical plus biomarker score (0-5). The combinatorial model that incorporated both clinical parameters and biomarkers had an area under curve (AUC) of 0.85 (95% CI, 0.81-0.89), which was significantly better at identifying occult PM infections than the clinical score alone (p = 0.001).These data suggest that host biomarkers in the maternal peripheral blood may improve the detection of PM in the absence of peripheral parasitaemia

Simultaneous host and parasite expression profiling identifies tissue-specific transcriptional programs associated with susceptibility or resistance to experimental cerebral malaria

BACKGROUND: The development and outcome of cerebral malaria (CM) reflects a complex interplay between parasite-expressed virulence factors and host response to infection. The murine CM model, Plasmodium berghei ANKA (PbA), which simulates many of the features of human CM, provides an excellent system to study this host/parasite interface. We designed "combination" microarrays that concurrently detect genome-wide transcripts of both PbA and mouse, and examined parasite and host transcriptional programs during infection of CM-susceptible (C57BL/6) and CM-resistant (BALB/c) mice. RESULTS: Analysis of expression data from brain, lung, liver, and spleen of PbA infected mice showed that both host and parasite gene expression can be examined using a single microarray, and parasite transcripts can be detected within whole organs at a time when peripheral blood parasitemia is low. Parasites display a unique transcriptional signature in each tissue, and lung appears to be a large reservoir for metabolically active parasites. In comparisons of susceptible versus resistant animals, both host and parasite display distinct, organ-specific transcriptional profiles. Differentially expressed mouse genes were related to humoral immune response, complement activation, or cell-cell interactions. PbA displayed differential expression of genes related to biosynthetic activities. CONCLUSION: These data show that host and parasite gene expression profiles can be simultaneously analysed using a single "combination" microarray, and that both the mouse and malaria parasite display distinct tissue- and strain-specific responses during infection. This technology facilitates the dissection of host-pathogen interactions in experimental cerebral malaria and could be extended to other disease models

Whole blood angiopoietin-1 and -2 levels discriminate cerebral and severe (non-cerebral) malaria from uncomplicated malaria

<p>Abstract</p> <p>Background</p> <p>Severe and cerebral malaria are associated with endothelial activation. Angiopoietin-1 (ANG-1) and angiopoietin-2 (ANG-2) are major regulators of endothelial activation and integrity. The aim of this study was to investigate the clinical utility of whole blood angiopoietin (ANG) levels as biomarkers of disease severity in <it>Plasmodium falciparum </it>malaria.</p> <p>Methods</p> <p>The utility of whole blood ANG levels was examined in Thai patients to distinguish cerebral (CM; n = 87) and severe (non-cerebral) malaria (SM; n = 36) from uncomplicated malaria (UM; n = 70). Comparative statistics are reported using a non-parametric univariate analysis (Kruskal-Wallis test or Chi-squared test, as appropriate). Multivariate binary logistic regression was used to examine differences in whole blood protein levels between groups (UM, SM, CM), adjusting for differences due to ethnicity, age, parasitaemia and sex. Receiver operating characteristic curve analysis was used to assess the diagnostic accuracy of the ANGs in their ability to distinguish between UM, SM and CM. Cumulative organ injury scores were obtained for patients with severe disease based on the presence of acute renal failure, jaundice, severe anaemia, circulatory collapse or coma.</p> <p>Results</p> <p>ANG-1 and ANG-2 were readily detectable in whole blood. Compared to UM there were significant decreases in ANG-1 (p < 0.001) and significant increases in ANG-2 (p < 0.001) levels and the ratio of ANG-2: ANG-1 (p < 0.001) observed in patients with SM and CM. This effect was independent of covariates (ethnicity, age, parasitaemia, sex). Further, there was a significant decrease in ANG-1 levels in patients with SM (non-cerebral) versus CM (p < 0.001). In participants with severe disease, ANG-2, but not ANG-1, levels correlated with cumulative organ injury scores; however, ANG-1 correlated with the presence of renal dysfunction and coma. Receiver operating characteristic curve analysis demonstrated that the level of ANG-1, the level of ANG-2 or the ratio of ANG-2: ANG-1 discriminated between individuals with UM and SM (area under the curve, p-value: ANG-2, 0.763, p < 0.001; ANG-1, 0.884, p < 0.001; Ratio, 0.857, p < 0.001) or UM and CM (area under the curve, p-value: ANG-2, 0.772, p < 0.001; ANG-1, 0.778, p < 0.001; Ratio, 0.820, p < 0.001).</p> <p>Conclusions</p> <p>These results suggest that whole blood ANG-1/2 levels are promising clinically informative biomarkers of disease severity in malarial syndromes.</p

Inflammatory and Angiogenic Factors at Mid-Pregnancy Are Associated with Spontaneous Preterm Birth in a Cohort of Tanzanian Women.

Preterm birth (PTB) is the leading cause of perinatal mortality worldwide, with the greatest burden occurring in resource-constrained settings. Based on the hypothesis that altered placental angiogenesis and inflammation early in pregnancy lead to PTB, we examined whether levels of inflammatory and angiogenic mediators, measured early in pregnancy, were predictive of spontaneous PTB (sPTB).\ud Plasma samples were collected from a prospective cohort of primigravid Tanzanian women between 12-27 weeks gestation. A panel of 18 markers was screened on a training cohort of 426 women. Markers associated with sPTB in the training cohort were repeated in a test cohort of 628 women. All markers were measured by ELISA. In both the training and test cohorts plasma levels of IL-18BP, sICAM-1, sEndoglin and CHI3L1 were elevated and Leptin was lower at enrollment in women who subsequently experienced sPTB. In multivariate analysis women with plasma levels of CHI3L1, C5a, sICAM-1, AngptL3, sEndgolin, sFlt-1 and IL-18BP in the highest quartile had an increased risk of sPTB compared with those in the lowest quartile. Women with Leptin and Ang2 in the highest quartile had a reduced risk of sPTB compared with women in the lowest quartile. Levels of angiogenic and inflammatory mediators measured at mid-pregnancy were associated with subsequent sPTB. These findings provide insight into mechanisms underlying sPTB and suggest biomarkers that may have clinical utility in risk-stratifying pregnancies

Host Biomarkers Are Associated With Response to Therapy and Long-Term Mortality in Pediatric Severe Malaria.

Background. Host responses to infection are critical determinants of disease severity and clinical outcome. The development of tools to risk stratify children with malaria is needed to identify children most likely to benefit from targeted interventions.Methods. This study investigated the kinetics of candidate biomarkers of mortality associated with endothelial activation and dysfunction (angiopoietin-2 [Ang-2], soluble FMS-like tyrosine kinase-1 [sFlt-1], and soluble intercellular adhesion molecule-1 [sICAM-1]) and inflammation (10 kDa interferon γ-induced protein [CXCL10/IP-10] and soluble triggering receptor expressed on myeloid cells-1 [sTREM-1]) in the context of a randomized, double-blind, placebo-controlled, parallel-arm trial evaluating inhaled nitric oxide versus placebo as adjunctive therapy to parenteral artesunate for severe malaria. One hundred eighty children aged 1–10 years were enrolled at Jinja Regional Referral Hospital in Uganda and followed for up to 6 months.Results. There were no differences between the 2 study arms in the rate of biomarker recovery. Median levels of Ang-2, CXCL10, and sFlt-1 were higher at admission in children who died in-hospital (n = 15 of 180; P < .001, P = .027, and P = .004, respectively). Elevated levels of Ang-2, sTREM-1, CXCL10, and sICAM-1 were associated with prolonged clinical recovery times in survivors. The Ang-2 levels were also associated with postdischarge mortality (P < .0001). No biomarkers were associated with neurodisability.Conclusions. Persistent endothelial activation and dysfunction predict survival in children admitted with severe malaria

Mesenchymal stromal (stem) cells suppress pro-inflammatory cytokine production but fail to improve survival in experimental staphylococcal toxic shock syndrome

BACKGROUND: Toxic shock syndrome (TSS) is caused by an overwhelming host-mediated response to bacterial superantigens produced mainly by Staphylococcus aureus and Streptococcus pyogenes. TSS is characterized by aberrant activation of T cells and excessive release of pro-inflammatory cytokines ultimately resulting in capillary leak, septic shock, multiple organ dysfunction and high mortality rates. No therapeutic or vaccine has been approved by the U.S. Food and Drug Administration for TSS, and novel therapeutic strategies to improve clinical outcome are needed. Mesenchymal stromal (stem) cells (MSCs) are stromal cells capable of self-renewal and differentiation. Moreover, MSCs have immunomodulatory properties, including profound effects on activities of T cells and macrophages in specific contexts. Based on the critical role of host-derived immune mediators in TSS, we hypothesized that MSCs could modulate the host-derived proinflammatory response triggered by Staphylococcal enterotoxin B (SEB) and improve survival in experimental TSS. METHODS: Effects of MSCs on proinflammatory cytokines in peripheral blood were measured in wild-type C57BL/6 mice injected with 50 μg of SEB. Effects of MSCs on survival were monitored in fatal experimental TSS induced by consecutive doses of D-galactosamine (10 mg) and SEB (10 μg) in HLA-DR4 transgenic mice. RESULTS: Despite significantly decreasing serum levels of IL-2, IL-6 and TNF induced by SEB in wild-type mice, human MSCs failed to improve survival in experimental TSS in HLA-DR4 transgenic mice. Similarly, a previously described downstream mediator of human MSCs, TNF-stimulated gene 6 (TSG-6), did not significantly improve survival in experimental TSS. Furthermore, murine MSCs, whether unstimulated or pre-treated with IFNγ, failed to improve survival in experimental TSS. CONCLUSIONS: Our results suggest that the immunomodulatory effects of MSCs are insufficient to rescue mice from experimental TSS, and that mediators other than IL-2, IL-6 and TNF are likely to play critical mechanistic roles in the pathogenesis of experimental TSS

A Two-Biomarker Model Predicts Mortality in the Critically Ill with Sepsis.

RATIONALE: Improving the prospective identification of patients with systemic inflammatory response syndrome (SIRS) and sepsis at low risk for organ dysfunction and death is a major clinical challenge. OBJECTIVES: To develop and validate a multibiomarker-based prediction model for 28-day mortality in critically ill patients with SIRS and sepsis. METHODS: A derivation cohort (n = 888) and internal test cohort (n = 278) were taken from a prospective study of critically ill intensive care unit (ICU) patients meeting two of four SIRS criteria at an academic medical center for whom plasma was obtained within 24 hours. The validation cohort (n = 759) was taken from a prospective cohort enrolled at another academic medical center ICU for whom plasma was obtained within 48 hours. We measured concentrations of angiopoietin-1, angiopoietin-2, IL-6, IL-8, soluble tumor necrosis factor receptor-1, soluble vascular cell adhesion molecule-1, granulocyte colony-stimulating factor, and soluble Fas. MEASUREMENTS AND MAIN RESULTS: We identified a two-biomarker model in the derivation cohort that predicted mortality (area under the receiver operator characteristic curve [AUC], 0.79; 95% confidence interval [CI], 0.74-0.83). It performed well in the internal test cohort (AUC, 0.75; 95% CI, 0.65-0.85) and the external validation cohort (AUC, 0.77; 95% CI, 0.72-0.83). We determined a model score threshold demonstrating high negative predictive value (0.95) for death. In addition to a low risk of death, patients below this threshold had shorter ICU length of stay, lower incidence of acute kidney injury, acute respiratory distress syndrome, and need for vasopressors. CONCLUSIONS: We have developed a simple, robust biomarker-based model that identifies patients with SIRS/sepsis at low risk for death and organ dysfunction

Regulation of neutrophil senescence by microRNAs

Neutrophils are rapidly recruited to sites of tissue injury or infection, where they protect against invading pathogens. Neutrophil functions are limited by a process of neutrophil senescence, which renders the cells unable to respond to chemoattractants, carry out respiratory burst, or degranulate. In parallel, aged neutrophils also undergo spontaneous apoptosis, which can be delayed by factors such as GMCSF. This is then followed by their subsequent removal by phagocytic cells such as macrophages, thereby preventing unwanted inflammation and tissue damage. Neutrophils translate mRNA to make new proteins that are important in maintaining functional longevity. We therefore hypothesised that neutrophil functions and lifespan might be regulated by microRNAs expressed within human neutrophils. Total RNA from highly purified neutrophils was prepared and subjected to microarray analysis using the Agilent human miRNA microarray V3. We found human neutrophils expressed a selected repertoire of 148 microRNAs and that 6 of these were significantly upregulated after a period of 4 hours in culture, at a time when the contribution of apoptosis is negligible. A list of predicted targets for these 6 microRNAs was generated from http://mirecords.biolead.org and compared to mRNA species downregulated over time, revealing 83 genes targeted by at least 2 out of the 6 regulated microRNAs. Pathway analysis of genes containing binding sites for these microRNAs identified the following pathways: chemokine and cytokine signalling, Ras pathway, and regulation of the actin cytoskeleton. Our data suggest that microRNAs may play a role in the regulation of neutrophil senescence and further suggest that manipulation of microRNAs might represent an area of future therapeutic interest for the treatment of inflammatory disease