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1.  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.
PMCID: PMC3745991  PMID: 23850678
Sepsis; TNF-α; Caspase 8; NF-κB; innate immunity
2.  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
3.  Substance P Mediates Reduced Pneumonia Rates After Traumatic Brain Injury 
Critical care medicine  2014;42(9):2092-2100.
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.
Data from the National Trauma Data Bank and a murine model of traumatic brain injury with postinjury pneumonia.
Academic medical centers in Cincinnati, OH, and Boston, MA.
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.
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.
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.
PMCID: PMC4215933  PMID: 25014065
infection; innate immunity; leukocytes; lung inflammation; neuropeptide; vagus nerve
Critical care medicine  2008;36(5):1585-1593.
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).
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.
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.
PMCID: PMC4105018  PMID: 18434891
5.  Characterization of Microparticles after Hepatic Ischemia-Reperfusion Injury 
PLoS ONE  2014;9(5):e97945.
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.
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.
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.
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.
PMCID: PMC4039439  PMID: 24879335
6.  Microparticles from Stored Red Blood Cells Activate Neutrophils and Cause Lung Injury after Hemorrhage and Resuscitation 
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.
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.
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.
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.
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.
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.
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 
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.
Murine model of sepsis (intraperitoneal fecal slurry injection).
Basic science research laboratory.
C57BL/6 male mice.
Intraperitoneal fecal slurry injection, with or without zinc supplementation (10 mg/kg of intraperitoneal zinc gluconate for 3 days prior to CLP).
Survival over 3 days following CLP, markers of inflammation, bacterial load studies, and immuno-phenotyping studies.
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.
Prophylactic zinc supplementation reduces bacterial load and is beneficial in a murine model of peritoneal sepsis.
PMCID: PMC3438373  PMID: 22760431
9.  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.
PMCID: PMC2830356  PMID: 20097159
sepsis; inflammation; neutrophil; IL-10
10.  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.
PMCID: PMC3491749  PMID: 22693226
11.  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.
PMCID: PMC2868384  PMID: 20427662
12.  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.
PMCID: PMC2914630  PMID: 20200277
13.  The Cannabinoid Receptor 2 Is Critical for the Host Response to Sepsis1 
Leukocyte function can be modulated through the cannabinoid receptor 2 (CB2R). Using a cecal ligation and puncture (CLP) model of sepsis, we examined the role of the CB2R during the immune response to an overwhelming infection. CB2R-knock out (KO) mice showed decreased survival as compared with wild-type mice. CB2R-KO mice also had increased serum IL-6 and bacteremia. Twenty-four hours after CLP, the CB2R-deficient mice had increased lung injury. Additionally, CB2R-deficiency led to increased neutrophil recruitment, decreased neutrophil activation, and decreased p38 activity at the site of infection. Consistent with a novel role for CB2R in sepsis, CB2R-agonist treatment in wild-type mice increased the mean survival time in response to CLP. Treatment with CB2R-agonist also decreased serum IL-6 levels, bacteremia, and damage to the lungs compared with vehicle-treated mice. Finally, the CB2R agonist decreased neutrophil recruitment, while increasing neutrophil activation and p38 activity at the site of infection compared with vehicle-treated mice. These data suggest that CB2R is a critical regulator of the immune response to sepsis and may be a novel therapeutic target.
PMCID: PMC2763235  PMID: 19525393
Shock (Augusta, Ga.)  2009;31(2):157-163.
A dysfunctional immune system is known to be part of the pathophysiology after burn trauma. However, reports that support this have used a variety of methods, with numerous variables, to induce thermal injury. We hypothesized that, all other parameters being equal, an injury infliction by a scald would yield different immunological responses than one inflicted by a flame. Here, we demonstrated that both burn methods produced a full-thickness burn, yet there was more of an increase in subdermal temperature, hematocrit, mortality, and serum IL-6 concentrations associated with the scald burn. On postinjury day 1, the scald-burned mice showed diminished lymphocyte numbers, interferon γ production, and lymphocyte T-bet expression as compared with sham- and flame-burned mice. On postburn day 8, spleens from both sets of thermally injured animals showed an increase in proinflammatory myeloid cells as compared with sham-burned mice. Furthermore, the T-cell numbers, T-bet expression, and phenotype were changed such that interferon γ production was higher in scald-burned mice than in sham- and flame-burned mice. Altogether, the data show that differential immunological phenotypes were observed depending on the thermal injury method used.
PMCID: PMC2674561  PMID: 18650781
Thermal injury; spleen; T cell; macrophage; T-bet; IFN-γ
Shock (Augusta, Ga.)  2008;29(5):591-597.
It is well established that the immune response to sepsis is mediated by leukocytes associated with the innate immune system. However, there is an emerging view that T lymphocytes can also mediate this response. Here, we observed a significant depletion of both CD4 and CD8 T cells in human patients after blunt trauma. To determine what effect the loss of these cells may have during a subsequent infection, we obtained CD4- and CD8-deficient mice and subjected them to cecal ligation and puncture (CLP). We observed that CD4 knockout (KO) mice showed increased CLP-induced mortality compared with CD8-deficient and wild-type (WT) mice especially within the first 30 h of injury. CD4 KO mice also exhibited significantly increased IL-6 concentrations after the CLP. The CD4 KO mice had an increased concentration of bacteremia as compared with WT mice. Antibiotic treatment decreased mortality in the CD4 KO mice as compared with no changes in the wild mice after CLP. Neutrophils isolated from septic CD4 KO mice showed decreased spontaneous oxidative burst compared with neutrophils taken from septic controls. We examined the role of IFN-γ by using mice deficient in this cytokine and found these mice to have significantly higher mortality as compared with WT mice. Finally, we detected a 2-fold increase in CD11b+ cells that exhibited intracellular IFN-γ staining in the peritoneum of WT mice after CLP. The data suggest that CD4+ cells may facilitate the early clearance of bacteria by regulating neutrophils function possibly through an IFN-γ–dependent mechanism.
PMCID: PMC2747655  PMID: 17885647
Interferon-γ; neutrophils; bacteremia; cecal ligation and puncture
16.  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.
PMCID: PMC3261332  PMID: 22089192
pediatric sepsis; septic shock; PPARα; nuclear hormone receptors; bacterial load
17.  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.
PMCID: PMC3222027  PMID: 21349138
18.  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.
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.
PMCID: PMC3058860  PMID: 21254176
chemokines; chemokine receptors; liver repair; hepatocyte proliferation; regeneration
19.  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.
PMCID: PMC2976361  PMID: 20823197
20.  Divergent adaptive and innate immunological responses are observed in humans following blunt trauma 
BMC Immunology  2010;11:4.
The immune response to trauma has traditionally been modeled to consist of the systemic inflammatory response syndrome (SIRS) followed by the compensatory anti-inflammatory response syndrome (CARS). We investigated these responses in a homogenous cohort of male, severe blunt trauma patients admitted to a University Hospital surgical intensive care unit (SICU). After obtaining consent, peripheral blood was drawn up to 96 hours following injury. The enumeration and functionality of both myeloid and lymphocyte cell populations were determined.
Neutrophil numbers were observed to be elevated in trauma patients as compared to healthy controls. Further, neutrophils isolated from trauma patients had increased raft formation and phospho-Akt. Consistent with this, the neutrophils had increased oxidative burst compared to healthy controls. In direct contrast, blood from trauma patients contained decreased naïve T cell numbers. Upon activation with a T cell specific mitogen, trauma patient T cells produced less IFN-gamma as compared to those from healthy controls. Consistent with these results, upon activation, trauma patient T cells were observed to have decreased T cell receptor mediated signaling.
These results suggest that following trauma, there are concurrent and divergent immunological responses. These consist of a hyper-inflammatory response by the innate arm of the immune system concurrent with a hypo-inflammatory response by the adaptive arm.
PMCID: PMC2823662  PMID: 20100328
21.  γδ T cells mitigate the organ injury and mortality of sepsis 
Journal of leukocyte biology  2007;83(3):581-588.
Sepsis is a difficult condition to treat and is associated with a high mortality rate. Sepsis is known to cause a marked depletion of lymphocytes, although the function of different lymphocyte subsets in the response to sepsis is unclear. γδ T cells are found largely in epithelial-rich tissues, and previous studies of γδ T cells in models of sepsis have yielded divergent results. In the present study, we examined the function of γδ T cells during sepsis in mice using cecal ligation and puncture (CLP). Mice deficient in γδ T cells had decreased survival times and increased tissue damage after CLP compared with wild-type mice. Furthermore, bacterial load was increased in γδ T cell-deficient mice, yet antibiotic treatment did not change mortality. Additionally, we found that recruitment of neutrophils and myeloid suppressor cells to the site of infection was diminished in γδ T cell-deficient mice. Finally, we found that circulating levels of IFN-γ were increased, and systemic levels of IL-10 were decreased in γδ T cell-deficient mice after CLP compared with wild-type mice. γδ T cell-deficient mice also had increased intestinal permeability after CLP compared with wild-type mice. Neutralization of IFN-γ abrogated the increase in intestinal permeability in γδ T cell-deficient mice. The intestines taken from γδ T cell-deficient mice had decreased myeloperoxidase yet had increased tissue damage as compared with wild-type mice. Collectively, our data suggest that γδ T cells modulate the response to sepsis and may be a potential therapeutic target.
PMCID: PMC2747639  PMID: 18063696
IFN-γ; intestine; myeloid suppressor cells; neutrophils; bacteremia
22.  Targeted Deletion of HIF-1α Gene in T Cells Prevents their Inhibition in Hypoxic Inflamed Tissues and Improves Septic Mice Survival 
PLoS ONE  2007;2(9):e853.
Sepsis patients may die either from an overwhelming systemic immune response and/or from an immunoparalysis-associated lack of anti-bacterial immune defence. We hypothesized that bacterial superantigen-activated T cells may be prevented from contribution into anti-bacterial response due to the inhibition of their effector functions by the hypoxia inducible transcription factor (HIF-1α) in inflamed and hypoxic areas.
Methodology/Principal Findings
Using the Cre-lox-P-system we generated mice with a T–cell targeted deletion of the HIF-1α gene and analysed them in an in vivo model of bacterial sepsis. We show that deletion of the HIF-1α gene leads to higher levels of pro-inflammatory cytokines, stronger anti-bacterial effects and much better survival of mice. These effects can be at least partially explained by significantly increased NF-κB activation in TCR activated HIF-1 α deficient T cells.
T cells can be recruited to powerfully contribute to anti-bacterial response if they are relieved from inhibition by HIF-1α in inflamed and hypoxic areas. Our experiments uncovered the before unappreciated reserve of anti-bacterial capacity of T cells and suggest novel therapeutic anti-pathogen strategies based on targeted deletion or inhibition of HIF-1 α in T cells.
PMCID: PMC1959117  PMID: 17786224

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