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1.  Neutrophil Derived Microvesicles: Emerging Role of a Key Mediator to the Immune Response 
In response to infection and trauma, exquisite control of the innate inflammatory response is necessary to promote an anti-microbial response and minimize tissue injury. Over the course of the host response, activated leukocytes are essential for the initial response and can later become unresponsive or undergo apoptosis. Leukocytes, along the continuum of activation to apoptosis, have been shown to generate microvesicles. These vesicles can range in size from 0.1 to 1.0 µm and can retain proteins, RNA and DNA of their parent cells. Importantly, neutrophil-derived microvesicles (NDMV) are robustly increased under inflammatory conditions. The aim of this review is to summarize the research to date upon NDMVs. This will include describing under which disease states NDMVs are increased, mechanisms underlying formation, and the impact of these vesicles upon cellular targets. Altogether, increased awareness of NDMVs during the host innate response may allow for diagnostic tools as well as potential novel therapies during infection and trauma.
PMCID: PMC4267953  PMID: 25051983
Apoptosis; inflammatory; immune response; microvesicles; neutrophil derived microvesicles; sepsis
2.  Substance P Mediates Reduced Pneumonia Rates After Traumatic Brain Injury 
Critical care medicine  2014;42(9):2092-2100.
Objectives
Traumatic brain injury results in significant morbidity and mortality and is associated with infectious complications, particularly pneumonia. However, whether traumatic brain injury directly impacts the host response to pneumonia is unknown. The objective of this study was to determine the nature of the relationship between traumatic brain injury and the prevalence of pneumonia in trauma patients and investigate the mechanism of this relationship using a murine model of traumatic brain injury with pneumonia.
Design
Data from the National Trauma Data Bank and a murine model of traumatic brain injury with postinjury pneumonia.
Setting
Academic medical centers in Cincinnati, OH, and Boston, MA.
Patients/Subjects
Trauma patients in the National Trauma Data Bank with a hospital length of stay greater than 2 days, age of at least 18 years at admission, and a blunt mechanism of injury. Subjects were female ICR mice 8–10 weeks old.
Interventions
Administration of a substance P receptor antagonist in mice.
Measurements and Main Results
Pneumonia rates were measured in trauma patients before and after risk adjustment using propensity scoring. In addition, survival and pulmonary inflammation were measured in mice undergoing traumatic brain injury with or without pneumonia. After risk adjustment, we found that traumatic brain injury patients had significantly lower rates of pneumonia compared to blunt trauma patients without traumatic brain injury. A murine model of traumatic brain injury reproduced these clinical findings with mice subjected to traumatic brain injury demonstrating increased bacterial clearance and survival after induction of pneumonia. To determine the mechanisms responsible for this improvement, the substance P receptor was blocked in mice after traumatic brain injury. This treatment abrogated the traumatic brain injury–associated increases in bacterial clearance and survival.
Conclusions
The data demonstrate that patients with traumatic brain injury have lower rates of pneumonia compared to non–head-injured trauma patients and suggest that the mechanism of this effect occurs through traumatic brain injury–induced release of substance P, which improves innate immunity to decrease pneumonia.
doi:10.1097/CCM.0000000000000486
PMCID: PMC4215933  PMID: 25014065
infection; innate immunity; leukocytes; lung inflammation; neuropeptide; vagus nerve
3.  Mechanisms Underlying Mouse TNF-α Stimulated Neutrophil Derived Microparticle Generation 
Despite advances in understanding and treatment of sepsis, it remains a disease with high mortality. Neutrophil derived microparticles (NDMPs) are present during sepsis and can modulate the immune system. As TNF-α is a cytokine that predominates in the initial stages of sepsis, we evaluated whether and how TNF-α can induce NDMPs in a mouse model. We observed that TNF-α treatment results in increased NDMP numbers. We also determined that the activation of either TNF receptor 1 (TNFr1) or TNF receptor 2 (TNFr2) resulted in increased NDMP numbers and that activation of both resulted in an additive increase. Inhibition of Caspase 8 diminishes NDMPs generated through TNFr1 activation and inhibition of NF-κB abrogates NDMPs generated through activation of both TNFr1 and TNFr2. We conclude that the early production of TNF-during sepsis can increase NDMP numbers through activation of the Caspase 8 pathway or NF-κB.
doi:10.1016/j.bbrc.2013.06.118
PMCID: PMC3745991  PMID: 23850678
Sepsis; TNF-α; Caspase 8; NF-κB; innate immunity
4.  RON RECEPTOR TYROSINE KINASE DEPENDENT HEPATIC NEUTROPHIL RECRUITMENT AND SURVIVAL BENEFIT IN A MURINE MODEL OF BACTERIAL PERITONITIS 
Critical care medicine  2008;36(5):1585-1593.
Objective
To determine if Ron receptor tyrosine kinase signaling impacts the in vivo response to bacterial peritonitis.
Design, Setting and Subjects
A genetic approach comparing wild type mice to mice with a targeted deletion of the Ron tyrosine kinase signaling domain (TK−/−) was undertaken to determine the influence of Ron receptor in the in vivo response to a well-characterized model of bacterial peritonitis and sepsis induced by cecal ligation and puncture (CLP).
Measurements
Several clinical (ie. survival curves, blood and tissue bacterial burdens, and neutrophil oxidative burst), morphologic (ie. liver histology and leukocyte trafficking) and biochemical parameters (ie. serum aminotransferases and select serum cytokine and chemokine levels) important for assessing inflammatory responses to bacterial infection were assessed in mice following CLP.
Main results
Ron TK−/− mice had a significant decrease in survival time compared to controls, and this was associated with a significant increase in bacterial colony forming units found in the blood and several end-organs. Moreover, this increased bacterial load was associated with increased liver necrosis and serum alanine aminotransferase levels. Neutrophils isolated from TK−/− mice exhibited decreased spontaneous oxidative burst capacity ex vivo, and by intravital microscopy, a reduced level of neutrophil migration to and translocation within the liver was observed. Loss of Ron signaling resulted in significantly reduced production of serum MCP-1 and IL-6 levels following CLP, and peritoneal macrophage isolated from TK−/− mice exhibited blunted production of MCP-1, IL-6 and MIP-2 following stimulation with endotoxin ex vivo.
Conclusion
Ron signaling negatively regulates the response to polymicrobial infection by regulating the activation and recruitment of inflammatory cells necessary for clearing a systemic bacterial burden. This effect may be regulated in part through the Ron-dependent, macrophage-mediated, production of cytokines and chemokines, namely MCP-1, IL-6 and MIP-2, important for neutrophil mobilization.
doi:10.1097/CCM.0b013e318170a8c2
PMCID: PMC4105018  PMID: 18434891
5.  Characterization of Microparticles after Hepatic Ischemia-Reperfusion Injury 
PLoS ONE  2014;9(5):e97945.
Background
Hepatic ischemia-reperfusion (I/R) is a well-studied model of liver injury and has demonstrated a biphasic injury followed by recovery and regeneration. Microparticles (MPs) are a developing field of study and these small membrane bound vesicles have been shown to have effector function in other physiologic and pathologic states. This study was designed to quantify the levels of MPs from various cell origins–platelets, neutrophils, and endolethial cells–following hepatic ischemia-reperfusion injury.
Methods
A murine model was used with mice undergoing 90 minutes of partial hepatic ischemia followed by various times of reperfusion. Following reperfusion, plasma samples were taken and MPs of various cell origins were labeled and levels were measured using flow cytometry. Additionally, cell specific MPs were further assessed by Annexin V, which stains for the presence of phosphatidylserine, a cell surface marker linked to apoptosis. Statistical analysis was performed using one-way analysis of variance with subsequent Student-Newman-Keuls test with data presented as the mean and standard error of the mean.
Results
MPs from varying sources show an increase in circulating levels following hepatic I/R injury. However, the timing of the appearance of different MP subtypes differs for each cell type. Platelet and neutrophil-derived MP levels demonstrated an acute elevation following injury whereas endothelial-derived MP levels demonstrated a delayed elevation.
Conclusion
This is the first study to characterize circulating levels of cell-specific MPs after hepatic I/R injury and suggests that MPs derived from platelets and neutrophils serve as markers of inflammatory injury and may be active participants in this process. In contrast, MPs derived from endothelial cells increase after the injury response during the reparative phase and may be important in angiogenesis that occurs in the regenerating liver.
doi:10.1371/journal.pone.0097945
PMCID: PMC4039439  PMID: 24879335
6.  Microparticles from Stored Red Blood Cells Activate Neutrophils and Cause Lung Injury after Hemorrhage and Resuscitation 
BACKGROUND
Transfusion of stored blood is associated with increased complications. Microparticles (MPs) are small vesicles released from RBCs that can induce cellular dysfunction, but the role of RBC-derived MPs in resuscitation from hemorrhagic shock is unknown. In the current study, we examined the effects of RBC-derived MPs on the host response to hemorrhage and resuscitation.
STUDY DESIGN
MPs were isolated from murine packed RBC units, quantified using flow cytometry, and injected into healthy mice. Separate groups of mice underwent hemorrhage and resuscitation with and without packed RBC–derived MPs. Lungs were harvested for histology and neutrophil accumulation and assessed by myeloperoxidase content. Human neutrophils were treated with human RBC-derived MPs and CD11b expression, superoxide production, and phagocytic activity were determined.
RESULTS
Stored murine packed RBC units contained increased numbers of RBC-derived MPs compared with fresh units. Hemorrhaged mice resuscitated with MPs demonstrated substantially increased pulmonary neutrophil accumulation and altered lung histology compared with mice resuscitated without MPs. Intravenous injection of MPs into normal mice resulted in neutrophil priming, evidenced by increased neutrophil CD11b expression. Human neutrophils treated with RBC-derived MPs demonstrated increased CD11b expression, increased superoxide production, and enanced phagocytic ability compared with untreated neutrophils.
CONCLUSIONS
Stored packed RBC units contain increased numbers of RBC-derived MPs. These MPs appear to contribute to neutrophil priming and activation. The presence of MPs in stored units can be associated with adverse effects, including lung injury, after transfusion.
doi:10.1016/j.jamcollsurg.2011.12.032
PMCID: PMC4034387  PMID: 22342784
7.  Roles of Hepatocyte and Myeloid CXC Chemokine Receptor-2 in Liver Recovery and Regeneration after Ischemia/Reperfusion 
Hepatology (Baltimore, Md.)  2013;57(1):331-338.
Previous studies have demonstrated the significance of signaling through the CXCR2 receptor in the process of recovery and regeneration of functional liver mass after hepatic ischemia/reperfusion (I/R). CXCR2 is constitutively expressed on both neutrophils and hepatocytes, however, the cell-specific roles of this receptor is unknown. In the present study, chimeric mice were created through bone marrow transplantation (BMT) using wild-type and CXCR2-knockout mice, yielding selective expression of CXCR2 on hepatocytes (Hep) and/or myeloid cells (My) in the following combinations: Hep+/My+; Hep−/My+; Hep+/My−; Hep−/My−. These tools allowed us to assess the contributions of myeloid and hepatocyte CXCR2 in the recovery of the liver after I/R injury. Flow cytometry confirmed adoption of the donor phenotype in neutrophils. Interestingly, Kupffer cells from all chimeras lacked CXCR2 expression. Recovery/regeneration of hepatic parenchyma was assessed by histologic assessment and measurement of hepatocyte proliferation. CXCR2Hep+/My+ mice showed the least amount of liver recovery and hepatocyte proliferation, while CXCR2Hep−/My− mice had the greatest liver recovery and hepatocyte proliferation. CXCR2Hep+/My− mice had enhanced liver recovery with hepatocyte proliferation similar to CXCR2Hep−/My− mice. Myeloid expression of CXCR2 directly regulated CXC chemokine expression levels following hepatic I/R, such that mice lacking myeloid CXCR2 had markedly increased chemokine expression compared to mice expressing CXCR2 on myeloid cells.
Conclusion
The data suggest that CXCR2 on myeloid cells is the predominant regulator of liver recovery and regeneration after I/R injury, while hepatocyte CXCR2 plays a minor, secondary role. These findings suggest that myeloid cell-directed therapy may significantly impact liver regeneration after liver resection or transplantation.
doi:10.1002/hep.26049
PMCID: PMC3540195  PMID: 22961770
chemokines; neutrophils; hepatocytes; hepatic regeneration; bone marrow transplant
8.  Prophylactic zinc supplementation reduces bacterial load and improves survival in a murine model of sepsis 
Objective
We previously demonstrated that altered zinc homeostasis is an important feature of pediatric sepsis, thus raising the possibility of zinc supplementation as a therapeutic strategy in sepsis. Herein we tested the hypothesis that prophylactic zinc supplementation would be beneficial in a murine model of peritoneal sepsis.
Design
Murine model of sepsis (intraperitoneal fecal slurry injection).
Setting
Basic science research laboratory.
Patients/Subjects
C57BL/6 male mice.
Interventions
Intraperitoneal fecal slurry injection, with or without zinc supplementation (10 mg/kg of intraperitoneal zinc gluconate for 3 days prior to CLP).
Measurements
Survival over 3 days following CLP, markers of inflammation, bacterial load studies, and immuno-phenotyping studies.
Results
Zinc-supplemented mice demonstrated a significant survival advantage compared to control (non-supplemented) mice. Zinc-supplemented mice also demonstrated moderate reductions of inflammation and immune activation. The survival advantage primarily correlated with reduced in vivo bacterial load in zinc-supplemented mice, compared to controls. In addition, peritoneal macrophages harvested from zinc-supplemented mice demonstrated a significantly enhanced phagocytosis capacity for E. coli and S. aureus, compared to peritoneal macrophages harvested from control mice.
Conclusion
Prophylactic zinc supplementation reduces bacterial load and is beneficial in a murine model of peritoneal sepsis.
doi:10.1097/PCC.0b013e31824fbd90
PMCID: PMC3438373  PMID: 22760431
9.  Interleukin-7 Ameliorates Immune Dysfunction and Improves Survival in a 2-Hit Model of Fungal Sepsis 
The Journal of Infectious Diseases  2012;206(4):606-616.
Background. Secondary hospital-acquired fungal infections are common in critically-ill patients and mortality remains high despite antimicrobial therapy. Interleukin-7 (IL-7) is a potent immunotherapeutic agent that improves host immunity and has shown efficacy in bacterial and viral models of infection. This study examined the ability of IL-7, which is currently in multiple clinical trials (including hepatitis and human immunodeficiency virus), to improve survival in a clinically relevant 2-hit model of fungal sepsis.
Methods. Mice underwent cecal ligation and puncture to induce peritonitis. Four days later, surviving mice had intravenous injection with Candida albicans. Following Candida infection, mice were treated with IL-7 or saline control. The effect of IL-7 on host immunity and survival was recorded.
Results. IL-7 ameliorated the loss of immune effector cells and increased lymphocyte functions, including activation, proliferation, expression of adhesion molecules, and interferon-γ production. These beneficial effects of IL-7 were associated with an increase in global immunity as reflected by an enhanced delayed type hypersensitivity response and a 1.7-fold improvement in survival.
Conclusions. The present findings showing that IL-7 improves survival in fungal sepsis, together with its previously reported efficacy in bacterial and viral infectious models, further supports its use as a novel immunotherapeutic in sepsis.
doi:10.1093/infdis/jis383
PMCID: PMC3491749  PMID: 22693226
10.  Genome-wide DNA methylation analysis reveals novel epigenetic changes in chronic lymphocytic leukemia 
Epigenetics  2012;7(6):567-578.
We conducted a genome-wide DNA methylation analysis in CD19+ B-cells from chronic lymphocytic leukemia (CLL) patients and normal control samples using reduced representation bisulfite sequencing (RRBS). The methylation status of 1.8–2.3 million CpGs in the CLL genome was determined; about 45% of these CpGs were located in more than 23,000 CpG islands (CGIs). While global CpG methylation was similar between CLL and normal B-cells, 1764 gene promoters were identified as being differentially methylated in at least one CLL sample when compared with normal B-cell samples. Nineteen percent of the differentially methylated genes were involved in transcriptional regulation. Aberrant hypermethylation was found in all HOX gene clusters and a significant number of WNT signaling pathway genes. Hypomethylation occurred more frequently in the gene body including introns, exons, and 3′-UTRs in CLL. The NFATc1 P2 promoter and first intron was found to be hypomethylated and correlated with upregulation of both NFATc1 RNA and protein expression levels in CLL suggesting that an epigenetic mechanism is involved in the constitutive activation of NFAT activity in CLL cells. This comprehensive DNA methylation analysis will further our understanding of the epigenetic contribution to cellular dysfunction in CLL.
doi:10.4161/epi.20237
PMCID: PMC3398986  PMID: 22534504
DNA Methylation; NFATc1; chronic lymphocytic leukemia; next-generation sequencing; reduced representation bisulfite sequencing
11.  The Properties of Genome Conformation and Spatial Gene Interaction and Regulation Networks of Normal and Malignant Human Cell Types 
PLoS ONE  2013;8(3):e58793.
The spatial conformation of a genome plays an important role in the long-range regulation of genome-wide gene expression and methylation, but has not been extensively studied due to lack of genome conformation data. The recently developed chromosome conformation capturing techniques such as the Hi-C method empowered by next generation sequencing can generate unbiased, large-scale, high-resolution chromosomal interaction (contact) data, providing an unprecedented opportunity to investigate the spatial structure of a genome and its applications in gene regulation, genomics, epigenetics, and cell biology. In this work, we conducted a comprehensive, large-scale computational analysis of this new stream of genome conformation data generated for three different human leukemia cells or cell lines by the Hi-C technique. We developed and applied a set of bioinformatics methods to reliably generate spatial chromosomal contacts from high-throughput sequencing data and to effectively use them to study the properties of the genome structures in one-dimension (1D) and two-dimension (2D). Our analysis demonstrates that Hi-C data can be effectively applied to study tissue-specific genome conformation, chromosome-chromosome interaction, chromosomal translocations, and spatial gene-gene interaction and regulation in a three-dimensional genome of primary tumor cells. Particularly, for the first time, we constructed genome-scale spatial gene-gene interaction network, transcription factor binding site (TFBS) – TFBS interaction network, and TFBS-gene interaction network from chromosomal contact information. Remarkably, all these networks possess the properties of scale-free modular networks.
doi:10.1371/journal.pone.0058793
PMCID: PMC3594155  PMID: 23536826
12.  Reduced PPARα Expression is Associated with Decreased Survival and Increased Tissue Bacterial Load in Sepsis 
Shock (Augusta, Ga.)  2012;37(2):164-169.
The peroxisome-proliferator activated receptor alpha (PPARα) is a member of the nuclear receptor family with many important physiologic roles related to metabolism and inflammation. Previous research in pediatric patients with septic shock revealed that genes corresponding to the PPARα signaling pathway are significantly downregulated in a subgroup of children with more severe disease. In this study, PPARα expression analysis using whole blood derived RNA revealed that PPARα expression was decreased in patients with septic shock and that the magnitude of that decrement correlated with the severity of disease. In a mouse model of sepsis, induced by cecal ligation and puncture (CLP), knockout mice lacking PPARα had decreased survival compared to wild type animals. Plasma cytokine analysis demonstrated decreased levels of IL-1β, IL-6, IL-17, KC, MCP-1, MIP-2, and TNFα at 24 hours in PPARα knockout animals. Cell surface markers of activation on splenic dendritic cells, macrophages, and CD8 T-cells were reduced in PPARα null animals and the bacterial load in lung and splenic tissues was increased. These data indicate that reduced or absent PPARα expression confers a survival disadvantage in sepsis and that PPARα plays a role in maintaining appropriate immune functions during the sepsis response.
doi:10.1097/SHK.0b013e31823f1a00
PMCID: PMC3261332  PMID: 22089192
pediatric sepsis; septic shock; PPARα; nuclear hormone receptors; bacterial load
13.  Pathway Correlation Profile of Gene-Gene Co-Expression for Identifying Pathway Perturbation 
PLoS ONE  2012;7(12):e52127.
Identifying perturbed or dysregulated pathways is critical to understanding the biological processes that change within an experiment. Previous methods identified important pathways that are significantly enriched among differentially expressed genes; however, these methods cannot account for small, coordinated changes in gene expression that amass across a whole pathway. In order to overcome this limitation, we use microarray gene expression data to identify pathway perturbation based on pathway correlation profiles. By identifying the distribution of gene-gene pair correlations within a pathway, we can rank the pathways based on the level of perturbation and dysregulation. We have shown this successfully for differences between two experimental conditions in Escherichia coli and changes within time series data in Saccharomyces cerevisiae, as well as two estrogen receptor response classes of breast cancer. Overall, our method made significant predictions as to the pathway perturbations that are involved in the experimental conditions.
doi:10.1371/journal.pone.0052127
PMCID: PMC3527387  PMID: 23284898
14.  The first step in utilizing immune-modulating therapies: immune status determination 
Critical Care  2011;15(1):108.
Recently, a single center study conducted by Oiva and coworkers and published in Critical Care demonstrated that phospho-specific whole blood flow cytometry could be used to assess activated signaling pathways in leukocytes isolated from pancreatitis patients. The authors demonstrated that this methodology had the potential to determine the current status of a patient's immune state. Although the experimental cohort was clinically homogeneous, the observed data were heterogeneous. Altogether, these results suggest that prior to administering immune-modulatory therapies in inflammatory diseases, it will be beneficial to first determine immune status. Rapid results from whole blood phospho-specific flow cytometry may allow for determination of immune status, improve early diagnosis, and provide a rational basis for immunomodulatory therapies.
doi:10.1186/cc9397
PMCID: PMC3222027  PMID: 21349138
15.  CXC Chemokine Receptor-1 is Expressed by Hepatocytes and Regulates Liver Recovery after Hepatic Ischemia/Reperfusion Injury 
Hepatology (Baltimore, Md.)  2010;53(1):261-271.
CXC chemokines mediate hepatic inflammation and injury following ischemia/reperfusion (I/R). More recently, signaling through CXC chemokine receptor-2 (CXCR2) was shown to delay liver recovery and repair after I/R injury. The chemokine receptor, CXCR1 shares ligands with CXCR2, yet nothing is known about its potential role in liver pathology. In the present study, we examined the role of CXCR1 in the injury and recovery responses to I/R using a murine model. CXCR1 expression was undetectable in livers of sham-operated mice. However, after ischemia CXCR1 expression increased 24 hours of reperfusion and was maximal after 96 hours of reperfusion. CXCR1 expression was localized largely to hepatocytes. In order to assess the function of CXCR1, CXCR2-/- mice were treated with the CXCR1/CXCR2 antagonist, repertaxin. Prophylactic treatment with repertaxin had no effect on acute inflammation or liver injury. However, when repertaxin was administered 24 hours post-reperfusion there was a significant increase in hepatocellular injury and a delay in recovery compared to control-treated mice. CXCR1-/- mice also demonstrated delayed recovery and regeneration after I/R when compared to wild-type mice. In vitro, hepatocytes from CXCR2-/- mice that were stimulated to express CXCR1 showed increased proliferation in response to ligand. Hepatocyte proliferation was decreased in CXCR1-/- mice in vivo.
Conclusion
This is the first report to show that CXCR1 expression is induced in hepatocytes after injury. Furthermore, the data suggest that CXCR1 has divergent effects from CXCR2 and appears to facilitate repair and regenerative responses after I/R injury.
doi:10.1002/hep.24028
PMCID: PMC3058860  PMID: 21254176
chemokines; chemokine receptors; liver repair; hepatocyte proliferation; regeneration
16.  A pathologist-in-the-loop IHC antibody test selection using the entropy-based probabilistic method 
Background:
Immunohistochemistry (IHC) is an important tool to identify and quantify expression of certain proteins (antigens) to gain insights into the molecular processes in a diseased tissue. However, it is a challenge for pathologists to remember the discriminative characteristics of the growing number of such antigens across multiple diseases. The complexity of their expression patterns, fueled by continuous discoveries in molecular pathology, gives rise to a combinatorial explosion that places an unprecedented burden on a practicing pathologist and therefore increases cost and variability of IHC studies.
Materials and Methods:
To tackle these issues, we have developed antibody test optimized selection method, a novel informatics tool to help pathologists in improving the IHC antibody selection process. The method uses extensions of Shannon's information entropies and Bayesian probabilities to dynamically build an efficient diagnostic tree.
Results:
A comparative analysis of our method with the expert and World Health Organization classification guidelines showed that the proposed method brings threefold reduction in number of antibody tests required to reach a diagnostic conclusion.
Conclusion:
The developed method can significantly streamline the antibody test selection process, decrease associated costs and reduce inter- and intrapathologist variability in IHC decision-making.
doi:10.4103/2153-3539.93393
PMCID: PMC3307231  PMID: 22439121
Antibody test selection; clinical decision support; computer--aided pathology diagnosis; entropy maximization; knowledge representation
17.  Interleukin-7 (IL-7) Treatment Accelerates Neutrophil Recruitment through γδ T-Cell IL-17 Production in a Murine Model of Sepsis ▿  
Infection and Immunity  2010;78(11):4714-4722.
The sepsis syndrome represents an improper immune response to infection and is associated with unacceptably high rates of mortality and morbidity. The interactions between T cells and the innate immune system while combating sepsis are poorly understood. In this report, we observed that treatment with the potent, antiapoptotic cytokine interleukin-7 (IL-7) accelerated neutrophil recruitment and improved bacterial clearance. We first determined that T cells were necessary for the previously observed IL-7-mediated enhanced survival. Next, IL-7 increased Bcl-2 expression in T cells isolated from septic mice as early as 3 h following treatment. This treatment resulted in increased gamma interferon (IFN-γ) and IP-10 production within the septic peritoneum together with local and systemic increases of IL-17 in IL-7-treated mice. We further demonstrate that the increase in IL-17 was largely due to increased recruitment and production by γδ T cells, which express CXCR3. Consistent with increased IL-17 production, IL-7 treatment increased CXCL1/KC production, neutrophil recruitment, and bacterial clearance. Significantly, end-organ tissue injury was not significantly different between vehicle- and IL-7-treated mice. Collectively, these data illustrate that IL-7 can mediate the cross talk between Th1 and Th17 lymphocytes during sepsis such that neutrophil recruitment and bacterial clearance is improved while early tissue injury is not increased. All together, these observations may underlay novel potential therapeutic targets to improve the host immune response to sepsis.
doi:10.1128/IAI.00456-10
PMCID: PMC2976361  PMID: 20823197
18.  Neutrophils are significant producers of IL-10 during sepsis 
Sepsis is a syndrome involving systemic inflammation as well as an infectious focus. Accordingly, the host immune response to sepsis involves complex leukocyte interplay that is incompletely understood. It is known that the immunoregulatory cytokine, IL-10, is rapidly expressed during the early stages of sepsis. In a murine model of sepsis, we sought to elucidate which leukocytes are early IL-10 producers. Using a novel IL-10 transcriptional reporter mouse, we observed that splenic leukocytes produced little IL-10. At the site of infection, peritoneal neutrophils produced the highest levels of IL-10 among leukocytes. Using cytokine antibody labeling, we further show that peritoneal neutrophils had high amounts of intracellular IL-10. We next depleted neutrophils and found a 40% decrease in peritoneal IL-10 levels. Altogether, this report demonstrates that among leukocytes, neutrophils are significant contributors of IL-10 at the site of infection during sepsis.
doi:10.1016/j.bbrc.2010.01.066
PMCID: PMC2830356  PMID: 20097159
sepsis; inflammation; neutrophil; IL-10
20.  Epigenetic regulation of WNT signaling in chronic lymphocytic leukemia 
Epigenomics  2010;2(1):53-70.
Certain WNT and WNT network target genes are expressed at higher or lower levels in chronic lymphocytic leukemia compared with normal B-cells. This includes upregulation of nuclear complex genes, as well as genes for cytoplasmic proteins and WNT ligands and their cognate receptors. In addition, epigenetic silencing of several negative regulators of the WNT pathway have been identified. The balance between epigenetic downregulation of negative effector genes and increased expression of positive effector genes demonstrate that the epigenetic downregulation of WNT antagonists is one mechanism, perhaps the main mechanism, that is permissive to active WNT signaling in chronic lymphocytic leukemia. Moreover, constitutive activation of the WNT network and target genes is likely to impact on additional interacting signaling pathways. Based on published studies, we propose a model of WNT signaling that involves mainly permissive expression, and sometimes overexpression, of positive effectors and downregulation of negative regulators in the network. In this model, DNA methylation, histone modifications and altered expression of microRNA molecules interact to allow continuous WNT signaling.
doi:10.2217/epi.09.43
PMCID: PMC2869094  PMID: 20473358
chronic lymphocytic leukemia; DNA methylation; epigenetics; histone modification; microRNAs; WNT pathway
21.  CNS leptin action modulates immune response and survival in sepsis 
Sepsis describes a complex clinical syndrome that results from an infection, setting off a cascade of systemic inflammatory responses that can lead to multiple organ failure and death. Leptin is a 16 kDa adipokine that, among its multiple known effects, is involved in regulating immune function. Here we demonstrate that leptin deficiency in ob/ob mice leads to higher mortality and more severe organ damage in a standard model of sepsis in mice (cecal ligation and puncture, CLP). Moreover, systemic leptin replacement improved the immune response to CLP. Based on the molecular mechanisms of leptin regulation of energy metabolism and reproductive function, we hypothesized that leptin acts in the central nervous system (CNS) to efficiently coordinate peripheral immune defense in sepsis. We now report that leptin signaling in the brain increases survival during sepsis in leptin-deficient as well as in wild-type mice and that endogenous CNS leptin action is required for an adequate systemic immune response. These findings reveal the existence of a relevant neuroendocrine control of systemic immune defense, and suggest a possible therapeutic potential for leptin analogues in infectious disease.
doi:10.1523/JNEUROSCI.4875-09.2010
PMCID: PMC2868384  PMID: 20427662
22.  IL-7 Promotes T Cell Viability, Trafficking, and Functionality and Improves Survival in Sepsis 
Sepsis is a highly lethal disorder characterized by widespread apoptosis-induced depletion of immune cells and the development of a profound immunosuppressive state. IL-7 is a potent antiapoptotic cytokine that enhances immune effector cell function and is essential for lymphocyte survival. In this study, recombinant human IL-7 (rhIL-7) efficacy and potential mechanisms of action were tested in a murine peritonitis model. Studies at two independent laboratories showed that rhIL-7 markedly improved host survival, blocked apoptosis of CD4 and CD8 T cells, restored IFN-γ production, and improved immune effector cell recruitment to the infected site. Importantly, rhIL-7 also prevented a hallmark of sepsis (i.e., the loss of delayed-type hypersensitivity), which is an IFN-γ– and T cell-dependent response. Mechanistically, rhIL-7 significantly increased the expression of the leukocyte adhesion markers LFA-1 and VLA-4, consistent with its ability to improve leukocyte function and trafficking to the infectious focus. rhIL-7 also increased the expression of CD8. The potent antiapoptotic effect of rhIL-7 was due to increased Bcl-2, as well as to a dramatic decrease in sepsis-induced PUMA, a heretofore unreported effect of IL-7. If additional animal studies support its efficacy in sepsis and if current clinical trials continue to confirm its safety in diverse settings, rhIL-7 should be strongly considered for clinical trials in sepsis.
doi:10.4049/jimmunol.0903151
PMCID: PMC2914630  PMID: 20200277
23.  Genome-Wide DNA Methylation Maps in Follicular Lymphoma Cells Determined by Methylation-Enriched Bisulfite Sequencing 
PLoS ONE  2010;5(9):e13020.
Background
Follicular lymphoma (FL) is a form of non-Hodgkin's lymphoma (NHL) that arises from germinal center (GC) B-cells. Despite the significant advances in immunotherapy, FL is still not curable. Beyond transcriptional profiling and genomics datasets, there currently is no epigenome-scale dataset or integrative biology approach that can adequately model this disease and therefore identify novel mechanisms and targets for successful prevention and treatment of FL.
Methodology/Principal Findings
We performed methylation-enriched genome-wide bisulfite sequencing of FL cells and normal CD19+ B-cells using 454 sequencing technology. The methylated DNA fragments were enriched with methyl-binding proteins, treated with bisulfite, and sequenced using the Roche-454 GS FLX sequencer. The total number of bases covered in the human genome was 18.2 and 49.3 million including 726,003 and 1.3 million CpGs in FL and CD19+ B-cells, respectively. 11,971 and 7,882 methylated regions of interest (MRIs) were identified respectively. The genome-wide distribution of these MRIs displayed significant differences between FL and normal B-cells. A reverse trend in the distribution of MRIs between the promoter and the gene body was observed in FL and CD19+ B-cells. The MRIs identified in FL cells also correlated well with transcriptomic data and ChIP-on-Chip analyses of genome-wide histone modifications such as tri-methyl-H3K27, and tri-methyl-H3K4, indicating a concerted epigenetic alteration in FL cells.
Conclusions/Significance
This study is the first to provide a large scale and comprehensive analysis of the DNA methylation sequence composition and distribution in the FL epigenome. These integrated approaches have led to the discovery of novel and frequent targets of aberrant epigenetic alterations. The genome-wide bisulfite sequencing approach developed here can be a useful tool for profiling DNA methylation in clinical samples.
doi:10.1371/journal.pone.0013020
PMCID: PMC2947499  PMID: 20927367
24.  DNA Hypermethylation Accompanied by Transcriptional Repression in Follicular Lymphoma 
Genes, chromosomes & cancer  2009;48(9):828-841.
High-throughput microarray technologies were used to study DNA methylation accompanied by transcriptional changes in follicular lymphoma (FL). Using Methylated CpG Island Amplification with Microarrays (MCAM) to study CpG Island (CGI) DNA methylation in follicular lymphoma (FL) we discovered widespread hypermethylation of homeobox genes and previously identified targets of polycomb repressive complex 2 (PRC2) in cell lines and primary tumors, but not in benign follicular hyperplasia (BFH). DNA methylation for HOXA11, HOXD10, HOXB7, HOXC12, PAX6, LHX9, SFMBT2, EN2 and PAX7 was independently validated in the RL cell line and HOXA11, HOXD10, PAX6 and EN2 in primary tumors. Combined Bisulfite Restriction Analysis (COBRA) also established DNA methylation for the previously identified PRC2 targets DCC, DES, GAD2, AQP5, GPR61, GRIA4, GJD2 and AMPH in FL but not in BFH. Gene expression analyses revealed 411 genes that were hypermethylated and transcriptionally repressed in RL, 74% of which were re-activated by the demethylating agent 5-aza-2′-deoxycytidine (5-azaD) plus or minus the histone deacetylase inhibitor trichostatin A (TSA). Forty genes were also down-regulated in primary FL. Our results suggest that extensive hypermethylation in promoters of polycomb target genes is a characteristic of FL and that loss of expression of certain SUZ12 target genes could be functionally relevant for lymphomagenesis.
doi:10.1002/gcc.20687
PMCID: PMC2738630  PMID: 19530241
25.  Next Generation Sequencing: Advances in Characterizing the Methylome  
Genes  2010;1(2):143-165.
Epigenetic modifications play an important role in lymphoid malignancies. This has been evidenced by the large body of work published using microarray technologies to generate methylation profiles for numerous types and subtypes of lymphoma and leukemia. These studies have shown the importance of defining the epigenome so that we can better understand the biology of lymphoma. Recent advances in DNA sequencing technology have transformed the landscape of epigenomic analysis as we now have the ability to characterize the genome-wide distribution of chromatin modifications and DNA methylation using next-generation sequencing. To take full advantage of the throughput of next-generation sequencing, there are many methodologies that have been developed and many more that are currently being developed. Choosing the appropriate methodology is fundamental to the outcome of next-generation sequencing studies. In this review, published technologies and methodologies applicable to studying the methylome are presented. In addition, progress towards defining the methylome in lymphoma is discussed and prospective directions that have been made possible as a result of next-generation sequencing technology. Finally, methodologies are introduced that have not yet been published but that are being explored in the pursuit of defining the lymphoma methylome.
doi:10.3390/genes1010143
PMCID: PMC3954092  PMID: 24710039
lymphoma; leukemia; next-generation sequencing; methylation; epigenome

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