Lesley Doughty

Interferon alpha/beta-mediated inhibition and promotion of interferon gamma: STAT1 resolves a paradox.

Induction of high systemic levels of type 1 interferons (IFNs) IFN-α and IFN-β is a hallmark of many viral infections. In addition to their potent antiviral effects, these cytokines mediate a number of immunoregulatory functions and can promote IFN-γ expression in T cells. However, during viral infections of mice IFN-γ production is not always observed at the same time as systemic IFN-α/β production and when, elicited at these times, is IFN-α/β–independent. We demonstrate that type 1 interferons not only fail to induce, but also act to inhibit, IFN-γ expression by both NK and T cells. The mechanism of inhibition is dependent upon the IFN-α/β receptor and the signal transducer and activator of transcription 1 (STAT1). In the absence of STAT1, not only are the IFN-α/β–mediated inhibitory effects completely abrogated, but the cytokines themselves can induce IFN-γ expression. These results indicate that endogenous biochemical pathways are in place to negatively regulate NK and T cell IFN-γ expression elicited by IFN-α/β or other stimuli, at times of innate responses to viral infections. They also show that type 1 interferon signaling can occur through STAT1-dependent and independent mechanisms and suggest that efficient induction of IFN-γ expression by IFN-α/β requires STAT1 regulation. Such immunoregulatory pathways may be critical for shaping the endogenous innate and virus-specific adaptive immune responses to viral infections.

Inflammatory cytokine and nitric oxide responses in pediatric sepsis and organ failure

OBJECTIVE To examine the relationship of circulating proinflammatory and anti-inflammatory cytokine concentrations to nitric oxide and organ failure in pediatric sepsis. DESIGN Prospective study. SETTING Pediatric intensive care unit (ICU), childrens Hospital of Pittsburgh, University of Pittsburgh. PATIENTS Nineteen patients with a diagnosis of sepsis admitted to the pediatric ICU. Twelve uninfected critically iII patients served as controls. INTERVENTIONS None. MEASUREMENTS AND MAIN RESULTS Plasma interleukin (IL)-10, IL-6, and nitrite/nitrate concentrations were measured and compared with an index of organ failure daily for 3 days after presentation with the sepsis syndrome. Children with increased plasma IL-6 concentrations (n = 6) had increased plasma nitrite/nitrate concentrations (p < 0.01 on each day), increased organ failure scores (p < .05 on days 1 and 3), and the highest plasma IL-10 concentrations (p < .05 on days 1 and 3, p = .054 on day 2) when compared with children with sepsis and undetectable IL-6 concentrations. Children with sepsis and detectable IL-6 concentrations, and children with undetectable IL-6 concentrations, both had increased nitrite/nitrate concentrations (p < .005 on days 1 through 3) and increased IL-10 concentrations (p < .05 on days 1 and 2) compared with controls. Children with increased IL-6 concentrations had higher organ failure on each day (p < .01), and children with undetectable IL-6 concentrations had higher organ failure on days 1 and 2 only (p < .005) when compared with controls. Organ failure improved over time in the children with undetectable IL-6 concentrations (p < .005). CONCLUSIONS Increased plasma nitrite/nitrates and increased organ failure scores occurred in the children with sepsis who had an exaggerated proinflammatory state, despite a pronounced anti-inflammatory response. When the anti-inflammatory response predominated and the proinflammatory state was dampened, organ failure status improved.

Admission Angiopoietin Levels in Children with Septic Shock

Angiopoietin (angpt) 1 and angpt-2 are circulating proteins first ascribed opposing roles in embryonic angiogenesis. Both bind the tyrosine kinase with immunoglobulin-like loop and epidermal growth factor homology domains (Tie) 2 receptor on endothelial cells, but angpt-1 is a Tie-2 agonist, whereas angpt-2 antagonizes Tie-2 signaling. In the developed vasculature, angpt-1 protects against vascular leak, whereas angpt-2 promotes increased vascular permeability. Because alterations in vascular permeability are common in septic shock, we obtained plasma from critically ill children within 24 h of diagnosis of the systemic inflammatory response syndrome (SIRS, n = 20), sepsis (n = 20), or septic shock (n = 61), as well as 15 healthy controls. Plasma levels of angpt-1 and angpt-2 were measured via a commercially available enzyme-linked immunosorbent assay. Plasma angpt-2 levels were significantly elevated in children with septic shock when compared with healthy children, as well as critically ill children with either SIRS or sepsis, and circulating angpt-2 levels seemed to correlate with disease severity and outcome. In addition, plasma angpt-1 levels were significantly decreased in critically ill children with septic shock compared with critically ill children with either SIRS or sepsis. Given the contrasting effects of angpt-2 and angpt-1 on the vascular endothelium, these two factors may play an important role in the pathophysiology of septic shock in children, and further studies are warranted.

Plasma nitrite and nitrate concentrations and multiple organ failure in pediatric sepsis

OBJECTIVE To determine whether plasma nitrite and nitrate concentrations are associated with the development of sepsis-induced multiple organ failure. DESIGN Prospective study. SETTING University childrens hospital. PATIENTS Fifty-three consecutive children meeting criteria for sepsis and not receiving exogenous sources of nitric oxide. INTERVENTIONS Plasma nitrite and nitrate concentrations were measured, and the number of organs failing was scored using an organ failure index on the first 3 days of sepsis. MEASUREMENTS AND MAIN RESULTS Children with three or more organs failing on day 3 of sepsis had higher plasma nitrite and nitrate concentrations than children who had resolution of failure of three or more organs by day 3 of sepsis (days 2 and 3) and children who never had three organs failing in the first 3 days of sepsis (days 1, 2, and 3). Children who developed sequential pulmonary/hepatic/renal organ failure had significantly higher plasma nitrite and nitrate concentrations (days 1, 2, and 3). Nonsurvivors had significantly higher plasma nitrite and nitrate concentrations (days 2 and 3) than survivors. Plasma nitrite and nitrate concentrations on day 1 predicted the development of persistent failure of three of more organs and sequential multiple organ failure but not mortality. CONCLUSION Increased plasma nitrite and nitrate concentrations are associated with the development of multiple organ failure in pediatric sepsis.

Intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 are increased in the plasma of children with sepsis-induced multiple organ failure.

Objectives To determine concentrations of circulating adhesion molecules endothelial (E)-selectin, intercellular adhesion molecule (ICAM)-1, and vascular cell adhesion molecule (VCAM)-1 in children with sepsis-induced multiple organ failure (MOF), and to determine associations among increased concentrations of these circulating adhesion molecules and important outcome measures. Design Prospective study. Setting University pediatric intensive care unit. Patients A total of 77 consecutive children with sepsis and 14 acutely ill children without sepsis. Interventions Plasma E-selectin, ICAM-1, and VCAM-1 concentrations and organ failure index (indicating number of failed organ systems) were determined in 77 children on days 1 and 3 of sepsis, and in 14 control children on pediatric intensive care unit day 1. Multivariate logistic regression analysis was used to determine associations between adhesion molecule concentrations and clinically relevant outcome measures. Measurements and Results Plasma concentrations of E-selectin, ICAM-1, and VCAM-1 were increased in children with sepsis vs. control on day 1 (p < .05). Plasma VCAM-1 (but not ICAM-1 or E-selectin) was increased in children with more than three organ failures vs. children with less than three organ failures (p < .05). Plasma ICAM-1 and VCAM-1 (but not E-selectin) concentrations independently predicted number of organs failed and development of more than three organ failures. Plasma ICAM-1 and VCAM-1 also predicted mortality and development of sequential (pulmonary/hepatic/renal) MOF (p < .05). Conclusions The pronounced and persistent increase in plasma VCAM-1 and ICAM-1 that occurs in children with sepsis and persistent MOF may indicate a phenotypic change in endothelium toward a more proinflammatory state. Alternatively, the source for these adhesion molecules may be activated leukocytes and other cell types. Future studies are required to determine the role of ICAM-1 and VCAM-1 in the pathogenesis of sepsis-induced MOF.

Differential Effects of Macrophage Inflammatory Chemokine-2 and Keratinocyte-derived Chemokine on Hemorrhage-induced Neutrophil Priming for Lung Inflammation: Assessment by Adoptive Cells Transfer in Mice

Prior studies have shown that hemorrhage (Hem) can serve as a priming stimulus for acute lung injury (ALI) triggered by subsequent septic challenge (cecal ligation and puncture, CLP). Furthermore, we have reported that in vivo antibody neutralization of the chemokines, macrophage inflammatory chemokine-2 (MIP-2) and keratinocyte-derived chemokine (KC), immediately after Hem appears to differentially effect the onset of ALI. However, although we hypothesize that this is due to divergent effects of MIP-2 and KC on Hem-induced neutrophil (PMN) priming, this has not been tested. To examine this hypothesis, PMN donor mice were Sham-Hem or Hem for 90 min at 35 ± 5 mmHg and were then administered anti-MIP-2 (Hem/anti-MIP2), anti-KC (Hem/anti-KC), or nonspecific immunoglobulin (Ig) G (Hem/IgG) during resuscitation (Ringers lactate = four times the amount of drawn blood volume). Twenty-four hours post-Hem, the peripheral blood PMN were purified from these donor animals and were introduced into PMN-depleted recipient mice [depleted by prior anti-Gr1 (mouse PMN-specific marker) antibody treatment]. One hour after PMN transfer, recipient mice were subjected to CLP, euthanized 24 h later, and plasma as well as lung tissue samples were collected. PMN influx was assessed by myeloperoxidase assay (MPO; &mgr;U/mg protein) and histologically (IL-6, MIP-2, KC, and IL-10 levels) by enzyme-linked immunoabsorbant assay (ELISA; ng/mg). The results show that donor PMN from Hem/IgG but not Sham-Hem mice produce increased PMN influx (↑MPO, ↑% esterase+ cells in tissue) into the lung and local tissue inflammation (↑IL-6/MIP-2, ↓IL-10) in PMN-depleted CLP recipient mice, which was attenuated in mice receiving cells from Hem/anti-MIP-2 but not Hem/anti-KC treated donors. Interestingly, although Hem/anti-MIP-2 donor PMN produced comparable effects on blood IL-6/MIP-2 levels, they were ineffective in altering the change in plasma IL-10/KC levels induce by Hem. Taken together, these data demonstrate that Hem-induced priming of PMN not only mediates ALI in the mouse, but also that this process is differentially effected by MIP2 and KC, despite the fact that both signal through CXCR2.

A Role for IFN-αβ in Virus Infection-Induced Sensitization to Endotoxin

Underlying viral infections can heighten sensitivity and worsen cytokine-mediated disease following secondary inflammatory challenges. Mechanisms for this are poorly understood. The impact of the innate response to lymphocytic choriomeningitis virus (LCMV) infection on sensitivity to endotoxin (LPS) was investigated. Compared with uninfected mice, infection with LCMV for 2-days-sensitized mice to LPS by ∼2-fold for lethality and by 2- to 6-fold for serum TNF-α levels. Priming for LPS-induced TNF-α was also seen with splenic and peritoneal leukocytes isolated from infected mice and challenged with LPS ex vivo. The effect on TNF-α production was present in the absence of IFN-γ, its major producers NK and T cells, and the major pathways for its induction through IL-12 and the signal transducer and activator of transcription 4 (STAT4), and therefore was IFN-γ independent. Early LCMV infection induces high concentrations of the type 1 IFNs, IFN-αβ. Administration of recombinant IFN-α alone heightened the TNF-α response to LPS. Innate IFN-αβ and IFN-γ responses to LCMV exist in a delicate balance. To reduce priming for LPS-induced TNF-α during LCMV, deficiencies in both the IFN-αβ and IFN-γ receptors or STAT1, a transcription factor downstream to both IFNs, were required. These data demonstrate that early viral infection can enhance sensitivity to bacterial products, and that this sensitization can occur in part as a result of endogenously expressed IFN-αβ. This work also raises issues about potential complications associated with IFN-αβ therapies.

Validation of a gene expression-based subclassification strategy for pediatric septic shock

Objective:Septic shock heterogeneity has important implications for clinical trial implementation and patient management. We previously addressed this heterogeneity by identifying three putative subclasses of children with septic shock based exclusively on a 100-gene expression signature. Here we attempted to prospectively validate the existence of these gene expression-based subclasses in a validation cohort. Design:Prospective observational study involving microarray-based bioinformatics. Setting:Multiple pediatric intensive care units in the United States. Patients:Separate derivation (n = 98) and validation (n = 82) cohorts of children with septic shock. Interventions:None other than standard care. Measurements and Main Results:Gene expression mosaics of the 100 class-defining genes were generated for 82 individual patients in the validation cohort. Using computer-based image analysis, patients were classified into one of three subclasses (“A,” “B,” or “C”) based on color and pattern similarity relative to reference mosaics generated from the original derivation cohort. After subclassification, the clinical database was mined for phenotyping. Subclass A patients had higher illness severity relative to subclasses B and C as measured by maximal organ failure, fewer intensive care unit-free days, and a higher Pediatric Risk of Mortality score. Patients in subclass A were characterized by repression of genes corresponding to adaptive immunity and glucocorticoid receptor signaling. Separate subclass assignments were conducted by 21 individual clinicians using visual inspection. The consensus classification of the clinicians had modest agreement with the computer algorithm. Conclusions:We have validated the existence of subclasses of children with septic shock based on a biologically relevant, 100-gene expression signature. The subclasses have relevant clinical differences.

Toward a clinically feasible gene expression-based subclassification strategy for septic shock: proof of concept.

Objective:To develop a clinically feasible stratification strategy for pediatric septic shock, using gene expression mosaics and a 100-gene signature representing the first 24 hrs of admission to the pediatric intensive care unit. Design:Prospective, observational study involving microarray-based bioinformatics. Setting:Multiple pediatric intensive care units in the United States. Patients:Ninety-eight children with septic shock. Interventions:None other than standard care. Measurements and Main Results:Patients were classified into three previously published, genome-wide, expression-based subclasses (subclasses A, B, and C) having clinically relevant phenotypic differences. The class-defining 100-gene signature was depicted for each individual patient, using mosaics generated by the Gene Expression Dynamics Inspector (GEDI). Composite mosaics were generated representing the average expression patterns for each of the three subclasses. Nine individual clinicians served as blinded evaluators. Each evaluator was shown the 98 individual patient mosaics and asked to classify each patient into one of the three subclasses, using the composite mosaics as the reference point. The respective sensitivities, specificities, positive predictive values, and negative predictive values of the subclassification strategy were ≥84% across the three subclasses. The classification strategy also generated positive likelihood ratios of ≥16.8 and negative likelihood ratios of ≤0.2 across the three subclasses. The &kgr; coefficient across all possible interevaluator comparisons was 0.81. Conclusions:We have provided initial evidence (proof of concept) for a clinically feasible and robust stratification strategy for pediatric septic shock based on a 100-gene signature and gene expression mosaics.

sFas and sFas ligand and pediatric sepsis-induced multiple organ failure syndrome.

The Fas-Fas ligand system is important for apoptosis of activated immune cells. Perturbation of this system occurs in diseases with dysregulated inflammation. Increased soluble Fas (sFas) occurs in systemic inflammatory response syndrome (SIRS) and can block apoptosis. Increased shedding of FasL (sFasL) occurs in viral infection and hepatitis. Although dysregulated inflammation is associated with sepsis-induced multiple organ failure (MOF) in children, a role for Fas has not been established. We hypothesize that 1) sFas will be increased in children with severe and persistent sepsis-induced MOF and will correlate with inflammatory markers suggesting a role for sFas in inflammatory dysregulation in severe sepsis, and 2) sFasL will be increased when viral sepsis or sepsis-induced liver failure–associated MOF is present in children. Plasma sFas, sFasL, IL-6, IL-10, nitrite + nitrates, and organ failure scores were measured on d 1 and d 3 in 92 children with severe sepsis and 12 critically ill control children. sFas levels were increased in severe sepsis, continued to increase in persistent MOF and nonsurvivors, and were correlated with serum inflammatory markers (IL-6, IL-10, nitrite + nitrate levels). In contrast, sFasL was not increased in severe sepsis and did not correlate with inflammation. sFasL was, however, increased in liver failure–associated MOF and in nonsurvivors, and was associated with viral infection. At autopsy, hepatocyte destruction and lymphocyte infiltration were associated with increased sFas and sFasL levels. sFas may interfere with activated immune cell death and contribute to dysregulation of inflammation, worsening outcome from severe sepsis. sFasL may contribute to hepatic injury and the development of liver failure–associated MOF.