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1 Cincinnati Children's Hospital Medical Center and Cincinnati Children's Hospital Research Foundation, Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio
2 C. S. Mott Children's Hospital at the University of Michigan, Ann Arbor, Michigan
3 Children's Hospital and Research Center Oakland, Oakland, California
4 Children's Hospital of Philadelphia, Philadelphia, Pennsylvania
5 Children's Mercy Hospital, Kansas City, Missouri
6 Penn State Children's Hospital, Hershey, Pennsylvania
7 University of Virginia Medical Center, Charlottesville, Virginia
8 Newark Beth Israel Medical Center, Newark, New Jersey
9 University of Alabama at Birmingham, Birmingham, Alabama
10 DuPont Hospital for Children, Wilmington, Delaware
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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inflammation; pediatrics; innate immunity
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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42,000 cases per year of pediatric septic shock in the United States, with a mortality rate of 10%, and higher mortality rates in children with comorbidities such as cancer and prematurity (39). A great deal of basic research efforts have focused on the biological processes that occur in septic shock. While highly informative, a relative paucity of this information has been readily translated to the bedside in the form of meaningful therapeutic advances for children (12, 34). For example, multiple trials focused on immune modulation strategies have been conducted in adults with septic shock (1). Despite strong preclinical data, as well as strong phase I and II data, the majority of these strategies have failed when subjected to large-scale, randomized placebo-controlled trials. Consequently, the majority of these strategies have not been effectively tested in the pediatric population. One notable exception is activated protein C, which recently received Food and Drug Administration approval for use in adults with septic shock (2, 4). Unfortunately, a phase III trial of activated protein C in children with septic shock was recently terminated early due to lack of efficacy (14). Current care for pediatric septic shock remains fundamentally based on antibiotics and supportive care (7, 34). Zinc homeostasis appears to be required for normal function of both the innate and acquired immune systems. For example, King and colleagues (16, 23) have demonstrated that zinc deficiency causes a cumulative loss of T and B cell maturation, which subsequently leads to lymphopenia. Natural killer cell function and phagocytic cell function are impaired by zinc deficiency (20), as is expression of specific cytokines that modulate the immune system (15, 33). In clinical states associated with immune suppression (e.g., sickle cell disease, human immunodeficiency virus infection, Down syndrome, and senescence), zinc supplementation has been shown to restore natural killer cell activity, lymphocyte production, mitogen responses, wound healing, and resistance to infection (33). Finally, in clinical trials involving children in developing countries or in rural communities, zinc supplementation has been demonstrated to reduce the incidence and severity of gastroenteritis and upper respiratory tract infections (5, 32). Thus, the established literature would suggest that zinc homeostasis may be an important research paradigm in the context of pediatric septic shock.
The clinical problem presented by pediatric septic shock, coupled with the relative lack of specific therapies, warrants a more comprehensive understanding of this condition at the translational level. This is a complex task given the inherent complexity of clinical septic shock and patient heterogeneity. We are addressing this complexity through genome-level expression profiling based on microarray technology. This approach has led to advances in specific forms of cancer (11, 31,36 ) and is beginning to show promise in the more heterogeneous condition of septic shock (6, 8, 9, 26, 29). Our goal for these studies is to define the genome-level expression profiles that occur in pediatric septic shock as a means of substantially advancing our understanding of this condition.
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METHODS |
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Sample and data collection.
After obtaining informed consent, we obtained blood samples (for RNA and serum isolation) within 24 h of admission to the PICU, heretofore referred to as day 1 of septic shock. Severity of illness was calculated from the PRISM III score (27), and organ failure was defined by pediatric-specific criteria (18, 28, 40). Annotated clinical and laboratory data were collected daily while in the PICU. All study patients were followed for 28 days to determine survival. We entered and stored clinical, laboratory, and biological data using a web-based database developed locally.
RNA extraction, microarray hybridization, and microarray analysis.
The data and protocols described in this manuscript have been deposited in the National Center for Biotechnology Information Gene Expression Omnibus (GEO,http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE4607.
Total RNA was isolated from whole blood samples using the PaxGene Blood RNA System (PreAnalytiX; Qiagen/Becton Dickson, Valencia, CA) according the manufacturer's specifications. Microarray hybridization was performed by the Affymetrix Gene Chip Core facility at Cincinnati Children's Hospital Research Foundation as previously described using the Human Genome U133 Plus 2.0 GeneChip (Affymetrix, Santa Clara, CA) (42).
Analyses were performed on one patient sample per chip. Image files were captured with an Affymetrix GeneChip Scanner 3000. CEL files were subsequently preprocessed with robust multiple-array average (RMA) normalization (21) using GeneSpring GX 7.3 software (Agilent Technologies, Palo Alto, CA). All signal intensity-based data were used after RMA normalization, which specifically suppresses all but significant variation among lower-intensity probe sets (21). All chips were then normalized to the respective median values of controls. Differences in mRNA abundance between patient samples were determined with GeneSpring GX 7.3. All statistical analyses used corrections for multiple comparisons. The specific statistical and filtering approaches are provided in RESULTS because of their relevance to data interpretation.
Two-dimensional cluster maps were constructed using GeneSpring GX 7.3. Gene trees are represented in the vertical dimension, and condition trees are represented in the horizontal dimension. Both the gene trees and condition trees are based on the Pearson similarity algorithm. The coloring conventions for all maps are as follows: red intensity correlates with increased gene expression, blue intensity correlates with decreased gene expression, and yellow intensity correlates with no change in gene expression relative to the median of controls.
Ontology analyses were performed by uploading specific gene expression lists to the web-based application D.A.V.I.D. (Database for Annotation, Visualization and Integrated Discovery), which allows public access to a relational database of functional gene annotations (13). Canonical pathway analyses were performed by uploading specific gene lists to the Ingenuity Systems Pathways Knowledge Base (Ingenuity Systems, Redwood City, CA), which provides a tool for discovery of canonical pathways within the uploaded gene lists (6). Both applications use specific approaches to estimate significance based on nonredundant representations of the microarray chip and to convert the uploaded gene lists to gene lists containing a single value for each gene.
Ancillary validation studies.
Real-time quantitative PCR was performed for selected genes by a standard approach involving the Superscript First Strand Synthesis kit (Invitrogen, Carlsbad, CA), SYBR green (Bio-Rad, Hercules, CA) and the iCycler Thermal Cycler (Bio-Rad). Serum interleukin (IL)-8 was measured using an ELISA kit, as specified by the manufacturer (Biosource, Camarillo, CA). MT-null mice having mutations for MT-1 and -2 were purchased from The Jackson Laboratory (Bar Harbor, ME; stock no. 002211, strain name: 129S7/SvEvBrd-Mt1tm1Bri Mt2tm1Bri/J). The appropriate control mice were also purchased from The Jackson Laboratory (strain no. 002448, strain name: 129S1/SvImJ mice). Mice were subjected to cecal ligation and puncture (CLP) as previously described (35). All experiments involving animals conformed to the Guide for the Care and Use of Laboratory Animals published by the United States National Institutes of Health (NIH Publication No. 85-23 revised 1996) and received approval of the Cincinnati Children's Hospital Research Foundation Institutional Animal Care and Use Committee.
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RESULTS |
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These 2,482 gene probes were then subjected to two-dimensional cluster analysis as depicted in Fig. 1. All of the patients with septic shock cluster together at the center of the map in a homogenous manner, thus demonstrating a relative commonality of gene regulation on day 1 of pediatric septic shock. This homogenous clustering is dependent on a group of genes in the upper portion of the map having increased expression (1,081 genes) and a group of genes in the lower portion of the map having decreased expression (1,401 genes).
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Differential gene expression between survivors and nonsurvivors of pediatric septic shock.
Since our initial attempt at elucidating the genome-level response of children with septic shock yielded biologically plausible data, we next tested the hypothesis that there is a differential pattern of gene expression between survivors and nonsurvivors of pediatric septic shock. To this end, we conducted a three-group ANOVA (Benjamini-Hochberg false discovery rate of 5%) using controls, septic shock survivors, and septic shock nonsurvivors as the comparison groups and all gene probes within the microarray (54,681 gene probes). This statistical filter yielded a working list of 13,054 gene probes that were differentially regulated between the three groups. A post hoc Tukey test indicated that 589 of these 13,054 gene probes were differentially regulated between the survivors and the nonsurvivors. To further refine this 589-gene list, we applied an expression filter that selected only the genes, within the above 589-gene list, having at least twofold expression difference in at least 50% of the survivors, compared with the median of the nonsurvivors. This expression filter yielded a final working list of 63 gene probes that were differentially regulated between survivors and nonsurvivors.
These 63 gene probes were then subjected to two-dimensional cluster analysis as depicted in Fig. 2. All of the nonsurvivors cluster to the left side of the map, thus demonstrating a relative commonality of gene regulation in nonsurvivors and survivors, respectively. As shown in Table 7, D.A.V.I.D.-based analysis of the 63 gene probes depicted in Fig. 2 yielded several biologically relevant functional annotations, including the "metal-binding" functional annotation previously noted in Table 4. Among the 63 gene probes depicted in Fig. 2, 36 gene probes (corresponding to 34 individual genes, Table 8) were upregulated and 27 gene probes (corresponding to 26 individual genes, Table 9) were downregulated in the nonsurvivors relative to survivors, respectively. The two gene lists provided in Tables 8 and 9 represent potential biomarkers for poor outcome and/or potential novel therapeutic targets in the context of pediatric septic shock.
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MTs are cysteine-rich, low-molecular-weight, intracellular metal-binding proteins (10). In particular, MTs are capable of avidly binding zinc in the intracellular compartment. Given this biochemical property of MT, the recurrence of zinc-related ontologies in our microarray data, and the demonstration that nonsurvivors had increased expression of MT, we hypothesized that nonsurvivors would have decreased serum (extracellular) zinc levels compared with survivors. Accordingly, we measured zinc levels in the parallel serum samples from the same patient cohort by way of atomic absorption.
The 50th percentile range of normal serum zinc concentrations in children <10 yr of age is between 75 and 80 µg/dl, and the 2.5th percentile range is between 50 and 55 µg/dl (19). As shown in Fig. 4, survivors had normal serum zinc concentrations. In contrast, nonsurvivors had serum zinc concentrations that were significantly lower than the survivors, and at the 2.5th percentile range. These data indirectly indicate that increased MT expression in nonsurvivors of pediatric shock may have a functional consequence on zinc homeostasis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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The data demonstrate that day 1 of pediatric septic shock is characterized by broad alterations of gene expression and that these broad alterations can be identified through genome-level expression profiles generated from accessible, clinically relevant biological samples. Importantly, the coordinately regulated genes fit well within biologically relevant gene ontologies and canonical pathways. The gene ontologies (Table 3) and canonical pathways (Table 5) detected within the 1,081 upregulated genes in this patient cohort do not necessarily represent novel concepts. The existing experimental and clinical literature has well established that inflammation-, immunity-, and stress response-related genes are highly regulated in the context of septic shock (3, 34). Nevertheless, the current demonstration of these ontologies and pathways provides confidence that the overall data are biologically relevant, rather than being artifacts of this high-throughput approach.
Given that some elements of our microarray data are well substantiated in the established literature, we were intrigued to find that a large number of genes that either are dependent on zinc homeostasis or play a direct role in zinc homeostasis were downregulated in this cohort of patients. The biological plausibility of these observations is supported by the literature discussed above regarding zinc homeostasis, zinc supplementation, and immunity (5, 15, 16, 20, 22, 32, 33). In addition, a recent review focused on the link between zinc homeostasis and immunity (30), and number of previous reports involving experimental endotoxemia/sepsis indicate that altered zinc homeostasis may indeed play a role in pediatric septic shock. For example, Kitamura et al. (24) recently established a link between zinc homeostasis and lipopolysaccharide (LPS)- mediated signaling through Toll-like receptor-4 in dendritic cells. Liuzzi et al. (25) demonstrated that systemic administration of LPS or turpentine in mice induced liver expression of the zinc transporter protein, Zip14, in an IL-6-dependent manner. The induction of Zip14 in the liver was postulated to represent a mechanism by which the acute-phase response/sepsis induces hypozincenemia. In another study involving systemic LPS injection in mice, Zhou et al. (41) demonstrated that zinc administration protected mice from LPS-mediated liver injury in an NF-
B-dependent manner. Finally, von Bulow et al. (37) demonstrated that zinc inhibits in vitro, LPS-mediated release of tumor necrosis factor (TNF)-
and IL-1ß in primary human monocytes. Collectively, these data involving LPS-mediated signaling and injury support a role for altered zinc homeostasis in sepsis and septic shock. Our current data significantly add to this body of literature by demonstrating an analogous principle at the level of the entire genome and, in human children with clinical septic shock, a far more complex process than experimental endotoxemia.
We are ultimately interested in determining whether or not there exist biologically significant gene expression profiles that distinguish survivors and nonsurvivors of pediatric septic shock. The rationale for addressing this question is to discover novel biomarkers of poor outcome and novel therapeutic targets as means for developing more effective therapeutic strategies. The current data represent an initial approach to this question given the relatively small number of nonsurvivors in the current patient cohort. The data demonstrate, however, that our microarray-based approach is a feasible means of addressing this question. Furthermore, the validity of these data is suggested by the demonstration that increased IL-8 mRNA expression in nonsurvivors (microarray) correlated with increased IL-8 protein levels (ELISA).
Within the current patient cohort, we have elucidated a relatively small group of genes that are differentially regulated between survivors and nonsurvivors of pediatric septic shock. The veracity of this particular gene expression profile to effectively predict survival vs. nonsurvival is the subject of ongoing studies in which the current data set will serve as a learning data set and a separate, future group of patients will serve as a validation data set. Nevertheless, the current data provide a foundation to begin formulating testable, novel hypotheses regarding the pathophysiology of poor outcome in pediatric septic shock.
The current cohort of nonsurvivors was characterized by increased expression of MT. One interpretation of this finding is that increased MT expression in nonsurvivors is simply an epiphenomenon of illness severity, rather than being directly involved in the pathobiology of septic shock. As such, MT expression could still potentially serve as a robust biomarker for poor outcome in pediatric septic shock. The development of this type of biomarker would be a powerful clinical tool for the selective implementation of high risk, experimental forms of therapy for pediatric patients with septic shock (e.g., plasmapheresis, extracorporeal membrane oxygenation, etc.), or for patient stratification in future therapeutic trials.
More intriguing is the alternative interpretation that increased MT expression plays a direct role in the pathobiology of septic shock. We have begun to address this possibility by conducting initial experiments directed toward understanding the functional role of MT in septic shock. Using a well-established murine model of polymicrobial sepsis, we have demonstrated that MT ablation confers a survival advantage in mice subjected to CLP, thus supporting the concept that MT expression plays a direct role in the pathobiology of septic shock. This assertion is further supported by two notable reports in the literature. Using a rat model of polymicrobial sepsis, Chinnaiyan et al. (8) assessed multiorgan gene expression profiles (microarray) and found that there was differential gene expression of a variety of genes, including MT. In support of our clinical data, MT was part of the gene cluster defining a "systemic sepsis signature." Waelput and colleagues (38) evaluated the role of MT in the context of TNF lethality, a model of severe systemic inflammation having many common biological and physiological features with septic shock. Based on the assumption that MT would be protective against TNF-mediated oxidant stress, these investigators sought to demonstrate that MT-null mice would be more sensitive to TNF-mediated lethality, whereas MT-overexpressing mice would be more resistant to MT-mediated lethality. Their results were completely opposite of what they expected: MT-null animals were the most resistant to TNF lethality, MT-overexpressing mice were the most sensitive to TNF-mediated lethality, and wild-type mice had an intermediate phenotype between these two extremes. Interestingly, however, Waelput et al. (38) demonstrated that zinc supplementation was protective against this model of TNF-lethality, but the protective effect of zinc seemed to be independent of MT expression.
To begin assessing the possibility that increased MT expression has a functional effect in clinical septic shock, we measured serum zinc levels in our patient cohort. Based on the ability of MT to avidly bind zinc in the intracellular compartment, we predicted that nonsurvivors would have decreased serum zinc levels and found that this was indeed the case. It should be noted that these data are semiquantitative in that serum samples were not originally collected with the specific intent of measuring heavy metal concentrations (i.e., not collected in metal-free specimen tubes). Thus, while it is possible that the samples are contaminated with exogenous heavy metals, all samples were collected in a similar manner and we have demonstrated a clear trend in that nonsurvivors had substantially lower serum zinc levels compared with survivors.
As previously discussed, zinc is an essential trace element required for normal function of multiple biological processes, and the clinical manifestations of zinc deficiency have been well described in the medical literature and in the context of immunity (22, 30, 33). It is unlikely that the decreased serum zinc levels described in the current cohort of patients represent "classic" zinc deficiency. Rather, they are more likely to represent an acute redistribution of tissue zinc levels, which may nonetheless represent an acute disruption of zinc homeostasis. This assertion is supported by the work of Gaetke et al. (17) involving systemic LPS administration to healthy adult volunteers. These investigators demonstrated acute decreases of serum zinc levels in response to LPS administration. In addition, they found no acute alterations of serum albumin-zinc binding or of urinary excretion of zinc, leading the investigators to postulate that the decreased zinc levels found in their study subjects was a reflection of acute zinc redistribution.
Given the importance of zinc homeostasis to a myriad of biological processes, including immunity, the data presented herein provide a strong rationale for pursuing further research efforts focused on MT expression and zinc homeostasis in clinical septic shock. This assertion is further supported by the demonstration that day 1 of pediatric septic shock is characterized by broad alterations in gene programs that are either dependent on zinc/metal homeostasis or play a direct role in regulating zinc/metal homeostasis.
Several study limitations deserve discussion. The patient population in this study is heterogeneous. Patient heterogeneity is an intrinsic problem to clinical investigations involving septic shock, which is more accurately characterized as a syndrome rather than a disease. Despite this heterogeneity, we have been able to demonstrate coordinately expressed patterns of gene expression that correspond to multiple biologically relevant gene ontologies and canonical pathways, thus indicating that genome-based approaches are effective means of approaching this problem of heterogeneity. We expect to have the opportunity to study more homogenous populations in the future (e.g., patients affected by the same pathogen). Another limitation is a common criticism of microarray-based experiments: exclusively measuring mRNA abundance. We have been able to validate some of our key findings by standard gene expression assays (PCR and ELISA). More importantly, we have validated some of our key findings at the functional level. Finally, another limitation is our reliance on whole blood-derived RNA, which is not necessarily reflective of organ-specific gene expression and contains a mixed population of white blood cells. Whole blood, however, is a readily accessible clinical sample that appears to provide a broad window of systemic gene expression. In addition, we have not found any correlation between differential white blood cell counts and the reported gene expression patterns in our cohort.
These limitations not withstanding, the current data represent an unprecedented first approximation of whole genome expression profiles in children with septic shock. As an initial approximation the data are highly plausible based on the established literature and potentially direct the field of clinical pediatric septic shock into new realms supported by the experimental literature.
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GRANTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).
* Additional members of the Genomics of Pediatric SIRS/Septic Shock Investigators appear in the ACKNOWLEDGMENTS. 
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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