Claudio Lagoa

THE ACUTE INFLAMMATORY RESPONSE IN DIVERSE SHOCK STATES

A poorly controlled acute inflammatory response can lead to organ dysfunction and death. Severe systemic inflammation can be induced and perpetuated by diverse insults such as the administration of toxic bacterial products (e.g., endotoxin), traumatic injury, and hemorrhage. Here, we probe whether these varied shock states can be explained by a universal inflammatory system that is initiated through different means and, once initiated, follows a course specified by the cellular and molecular mechanisms of the immune and endocrine systems. To examine this question, we developed a mathematical model incorporating major elements of the acute inflammatory response in C57Bl/6 mice, using input from experimental data. We found that a single model with different initiators including the autonomic system could describe the response to various insults. This model was able to predict a dose range of endotoxin at which mice would die despite having been calibrated only in nonlethal inflammatory paradigms. These results show that the complex biology of inflammation can be modeled and supports the hypothesis that shock states induced by a range of physiologic challenges could arise from a universal response that is differently initiated and modulated.

IN SILICO MODELS OF ACUTE INFLAMMATION IN ANIMALS

ABSTRACT Trauma and hemorrhagic shock elicit an acute inflammatory response, predisposing patients to sepsis, organ dysfunction, and death. Few approved therapies exist for these acute inflammatory states, mainly due to the complex interplay of interacting inflammatory and physiological elements working at multiple levels. Various animal models have been used to simulate these phenomena, but these models often do not replicate the clinical setting of multiple overlapping insults. Mathematical modeling of complex systems is an approach for understanding the interplay among biological interactions. We constructed a mathematical model using ordinary differential equations that encompass the dynamics of cells and cytokines of the acute inflammatory response, as well as global tissue dysfunction. The model was calibrated in C57Bl/6 mice subjected to (1) various doses of lipopolysaccharide (LPS) alone, (2) surgical trauma, and (3) surgery + hemorrhagic shock. We tested the models predictive ability in scenarios on which it had not been trained, namely, (1) surgery ± hemorrhagic shock + LPS given at times after the beginning of surgical instrumentation, and (2) surgery + hemorrhagic shock + bilateral femoral fracture. Software was created that facilitated fitting of the mathematical model to experimental data, as well as for simulation of experiments with various inflammatory challenges and associated variations (gene knockouts, inhibition of specific cytokines, etc.). Using this software, the C57Bl/6-specific model was recalibrated for inflammatory analyte data in CD14−/− mice and was used to elucidate altered features of inflammation in these animals. In other experiments, rats were subjected to surgical trauma ± LPS or to bacterial infection via fibrin clots impregnated with various inocula of Escherichia coli. Mathematical modeling may provide insights into the complex dynamics of acute inflammation in a manner that can be tested in vivo using many fewer animals than has been possible previously.

The role of initial trauma in the host's response to injury and hemorrhage: insights from a correlation of mathematical simulations and hepatic transcriptomic analysis.

ABSTRACT Trauma and hemorrhagic shock (HS) elicit severe physiological disturbances that predispose the victims to subsequent organ dysfunction and death. The general lack of effective therapeutic options for these patients is mainly due to the complex interplay of interacting inflammatory and physiological elements working at multiple levels. Systems biology has emerged as a new paradigm that allows the study of large portions of physiological networks simultaneously. Seeking a better understanding of the interplay among known inflammatory pathways, we constructed a mathematical model encompassing the dynamics of the acute inflammatory response that incorporates the intertwined effects of inflammation and global tissue damage. The model was calibrated using data from C57Bl/6 mice subjected to endotoxemia, sham operation (i.e., surgical trauma induced by cannulation [ST]) or ST + HS+ resuscitation (ST-HS-R). An in silico simulation, made at whole-organism level, suggested that similar pathways of different magnitudes were operant as the degree of total body damage increased. We sought to validate this hypothesis by subjecting mice to HS and comparing the models predictions to circulating markers of inflammation and tissue injury as well as the global transcriptomic response of the liver. C57Bl/6 mice were subjected to ST or ST-HS (without resuscitation). Liver gene expression was assessed using an Affymetrix DNA microarray (GeneChip Mouse Expression Set 430A, Affymetrix, Santa Clara, CA), which contains 22,621 probe sets and effectively interrogates 12,341 mouse genes. The microarray data sets were subjected to hierarchical clustering and pathway analysis. In agreement with model predictions, circulating levels of inflammation/tissue injury markers and the microarray analysis both demonstrated that ST alone accounts for a substantial proportion of the observed phenotypic and genetic/molecular changes versus untreated animals. The addition of HS further increased the magnitude of gene expression, but relatively few additional genes were recruited. Mathematical simulations and DNA microarrays, both systems biology tools, may provide valuable insight into the complex global physiological interactions that occur in response to trauma and hemorrhagic shock.ABBREVIATIONS-ST-surgical trauma, HS-hemorrhagic shock, ST-HS-surgical trauma + hemorrhagic shock, R-resuscitation, MAP-mean arterial blood pressure, IL-interleukin, TNF-&agr;-tumor necrosis factor alpha, AST-aspartate aminotransferase, iNOS-inducible nitric oxide synthase, eNOS-endothelial nitric oxide synthase, NO-nitric oxide, NO2−-nitrate, NO3−-nitrite, LPS-bacterial lipopolysaccharide (endotoxin), IP-ingenuity pathway, IPKB-ingenuity pathway knowledge base

Hemoadsorption reprograms inflammation in experimental gram-negative septic peritonitis: insights from in vivo and in silico studies.

Improper compartmentalization of the inflammatory response leads to systemic inflammation in sepsis. Hemoadsorption (HA) is an emerging approach to modulate sepsis-induced inflammation. We sought to define the effects of HA on inflammatory compartmentalization in Escherichia coli-induced fibrin peritonitis in rats. Hypothesis: HA both reprograms and recompartmentalizes inflammation in sepsis. Sprague Dawley male rats were subjected to E. coli peritonitis and, after 24 h, were randomized to HA or sham treatment (sepsis alone). Venous blood samples collected at 0, 1, 3 and 6 h (that is, 24–30 h of total experimental sepsis), and peritoneal samples collected at 0 and 6 h, were assayed for 14 cytokines along with NO2−/NO3−. Bacterial counts were assessed in the peritoneal fluid at 0 and 6 h. Plasma tumor necrosis factor (TNF)-α, interleukin (IL)-6, CXCL-1, and CCL2 were significantly reduced in HA versus sham. Principal component analysis (PCA) suggested that inflammation in sham was driven by IL-6 and TNF-α, whereas HA-associated inflammation was driven primarily by TNF-α, CXCL-1, IL-10 and CCL2. Whereas peritoneal bacterial counts, plasma aspartate transaminase levels and peritoneal IL-5, IL-6, IL-18, interferon (IFN)-γ and NO2−/NO3− were significantly lower, both CXCL-1 and CCL2 as well as the peritoneal-to-plasma ratios of TNF-α, CXCL-1 and CCL2 were significantly higher in HA versus sham, suggesting that HA-induced inflammatory recompartmentalization leads to the different inflammatory drivers discerned in part by PCA. In conclusion, this study demonstrates the utility of combined in vivo/in silico methods and suggests that HA exerts differential effects on mediator gradients between local and systemic compartments that ultimately benefit the host.

The role of hepatic type 1 plasminogen activator inhibitor (PAI‐1) during murine hemorrhagic shock

Hemorrhagic shock (HS) followed by resuscitation (HS‐R) is characterized by profound physiological changes. Even if the patient survives the initial blood loss, these poorly understood changes can lead to morbidity. One of the tissues most often affected is liver. We sought to recognize specific hepatic changes induced by this stressor to identify targets for therapeutic intervention. Gene array analyses using mouse liver mRNAs were used to identify candidate genes that contribute to hepatic damage. To verify the role of one of the genes identified using the arrays, mice were subjected to HS‐R, and multiple parameters were analyzed. A profound increase in plasminogen activator inhibitor type 1 (PAI‐1) mRNA was observed using hepatic mRNAs from C57Bl/6 mice after HS, both with and without resuscitation. Constitutive loss of PAI‐1 resulted in notable tissue preservation and lower (P < .05) alanine aminotransferase (ALT) levels. Fibrin degradation products (FDPs) and interleukins 6 and 10 (IL‐6 and IL‐10) were unaffected by loss of PAI‐1; however, enhanced urokinase activity, an elevation of active hepatocyte growth factor (HGF), an increase in unprocessed transforming growth factor‐β1 (TGF‐β1), and retention of ERK phosphorylation after HS‐R were associated with improved hepatic function. In conclusion, PAI‐1 protein is a negative effector of hepatic damage after HS‐R through its influence on classic regulators of hepatic growth, as opposed to its role in fibrinolysis. (HEPATOLOGY 2005;42:390–399.)

An Exploratory Pathways Analysis of Temporal Changes Induced by Spinal Cord Injury in the Rat Bladder Wall: Insights on Remodeling and Inflammation

Background Spinal cord injuries (SCI) can lead to severe bladder pathologies associated with inflammation, fibrosis, and increased susceptibility to urinary tract infections. We sought to characterize the complex pathways of remodeling, inflammation, and infection in the urinary bladder at the level of the transcriptome in a rat model of SCI, using pathways analysis bioinformatics. Methodology/Principal Findings Experimental data were obtained from the study of Nagatomi et al. (Biochem Biophys Res Commun 334: 1159). In this study, bladders from rats subjected to surgical SCI were obtained at 3, 7 or 25 days post-surgery, and Affymetrix GeneChip® Rat Genome U34A arrays were used for cRNA hybridizations. In the present study, Ingenuity Pathways Analysis (Ingenuity® Systems, www.ingenuity.com) of differentially expressed genes was performed. Analysis of focus genes in networks, functional analysis, and canonical pathway analysis reinforced our previous findings related to the presence of up-regulated genes involved in tissue remodeling, such as lysyl oxidase, tropoelastin, TGF-β1, and IGF-1. This analysis also highlighted a central role for inflammation and infection, evidenced by networks containing genes such as CD74, S100A9, and THY1. Conclusions/Significance Our findings suggest that tissue remodeling, infection, inflammation, and tissue damage/dysfunction all play a role in the urinary bladder, in the complex response to SCI.

Transcriptomic response of murine liver to severe injury and hemorrhagic shock: a dual-platform microarray analysis

Trauma-hemorrhagic shock (HS/T) is a complex process that elicits numerous molecular pathways. We hypothesized that a dual-platform microarray analysis of the liver, an organ that integrates immunology and metabolism, would reveal key pathways engaged following HS/T. C57BL/6 mice were divided into five groups (n = 4/group), anesthetized, and surgically treated to simulate a time course and trauma severity model: 1) nonmanipulated animals, 2) minor trauma, 3) 1.5 h of hemorrhagic shock and severe trauma (HS/T), 4) 1.5 h HS/T followed by 1 h resuscitation (HS/T+1.0R), 5) 1.5 h HS/T followed by 4.5 h resuscitation (HS/T+4.5R). Liver RNA was hybridized to CodeLink and Affymetrix mouse whole genome microarray chips. Common genes with a cross-platform correlation >0.6 (2,353 genes in total) were clustered using k-means clustering, and clusters were analyzed using Ingenuity Pathways Analysis. Genes involved in the stress response and immunoregulation were upregulated early and remained upregulated throughout the course of the experiment. Genes involved in cell death and inflammatory pathways were upregulated in a linear fashion with elapsed time and in severe injury compared with minor trauma. Three of the six clusters contained genes involved in metabolic function; these were downregulated with elapsed time. Transcripts involved in amino acid metabolism as well as signaling pathways associated with glucocorticoid receptors, IL-6, IL-10, and the acute phase response were elevated in a severity-dependent manner. This is the first study to examine the postinjury response using dual-platform microarray analysis, revealing responses that may enable novel therapies or diagnostics.

Augmenter of liver regeneration (ALR) is a novel biomarker of hepatocellular stress/inflammation: in vitro, in vivo and in silico studies.

The liver is a central organ involved in inflammatory processes, including the elaboration of acute-phase proteins. Augmenter of liver regeneration (ALR) protein, expressed and secreted by hepatocytes, promotes liver regeneration and maintains viability of hepatocytes. ALR also stimulates secretion of inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-6) and nitric oxide from Kupffer cells. We hypothesized that ALR may be involved in modulating inflammation induced by various stimuli. We found that hepatic ALR levels are elevated at 24 h, before or about the same time as an increase in the mRNA expression of TNF-α and IL-6, after portacaval shunt surgery in rats. Serum ALR also increased, but significantly only on d 4 when pathological changes in the liver become apparent. In rats, serum ALR was elevated after intraperitoneal administration of lipopolysaccharide alone and in a model of gram-negative sepsis. Serum ALR increased before alanine aminotransferase (ALT) in endotoxemia and in the same general time frame as TNF-α and IL-6 in the bacterial sepsis model. Furthermore, mathematical prediction of tissue damage correlated strongly with alterations in serum ALR in a mouse model of hemorrhagic shock. In vitro, monomethyl sulfonate, TNF-α, actinomycin D and lipopolysaccharide all caused increased release of ALR from rat hepatocytes, which preceded the loss of cell viability and/or inhibition of DNA synthesis. ALR may thus serve as a potential diagnostic marker of hepatocellular stress and/or acute inflammatory conditions.