Holly S. Goetzman

The Cannabinoid Receptor 2 Is Critical for the Host Response to Sepsis

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.

Interleukin-7 (IL-7) Treatment Accelerates Neutrophil Recruitment through γδ T-Cell IL-17 Production in a Murine Model of Sepsis

ABSTRACT 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.

γδ T cells mitigate the organ injury and mortality of sepsis

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.

Thermal injury elevates the inflammatory monocyte subpopulation in multiple compartments.

Recent publications have demonstrated that human resident and inflammatory monocyte (IM) subpopulations have equivalents in rodents. The effect of thermal injury upon these subpopulations has not been studied. Mice were given a scald burn and killed on postburn days (PBDs) 2, 4, and 8. Bone marrow, blood, and spleen white cells were isolated, and the percentage of resident monocytes (CD11b+ LY6C+), IMs (CD11b+ LY6C++), and monocyte progenitors (macrophage-colony-forming unit [M-CFU]) were determined. The ability of each monocyte population to make TNF-α was determined by intracellular cytokine staining. Finally, the ability of sorted fractions from PBD 8 spleen to inhibit lymphocyte proliferation was performed. We noted that there was an increase in M-CFU in the blood and spleen at PBD 8, but the marrow only had a nonsignificant increase in M-CFU. All compartments showed a significant increase in the number of IMs by PBD 8, but no significant changes in resident monocytes were seen. In all compartments, IMs were a major source of TNF-α. The postburn increase in IMs and monocyte progenitors in the spleen was accompanied by an increase in the monocyte chemokine monocyte chemoattractant protein 1 and constitutively high levels of the progenitor chemokine stromal-derived factor 1α. After burn injury, mice deficient in the receptor for soluble TNF-α had equal levels of splenic M-CFU and monocytes, as did wild-type mice, suggesting that this cytokine is not essential for this effect. We conclude that in this model, IMs are a significant source of in vivo TNF-α.

CD4-expressing cells are early mediators of the innate immune system during sepsis.

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.

T-cell activation differentially mediates the host response to sepsis.

Survival during sepsis requires both swift control of infectious organisms and tight regulation of the associated inflammatory response. As the role of T cells in sepsis is somewhat controversial, we examined the impact of increasing antigen-dependent activation of CD4 T cells in a murine model of cecal ligation and puncture using T-cell receptor transgenic II (OT-II) mice that are specific for chicken ovalbumin (OVA) in the context of major histocompatibility complex II. Here, we injected OT-II mice with 0, 1, or 100 &mgr;g of OVA and demonstrate that increased antigen treatment resulted in increased numbers of activated splenic CD4 T cells. Vehicle-treated, septic OT-II mice had decreased survival, increased bacterial load, and increased levels of IL-6. Interestingly, this decrease in survival was abrogated when OT-II mice were injected with 1 &mgr;g OVA, which was correlated with normalized bacterial load and levels of IL-6. However, when OT-II mice were injected with 100 &mgr;g OVA, decreased survival was restored but, in contrast to vehicle-treated OT-II mice, had decreased bacterial load and enhanced IL-6 levels. We also observed that neutrophil oxidative burst and phagocytosis were dependent on CD4 T-cell activation. Further, at extreme levels of T-cell activation, intestinal permeability was significantly increased. Altogether, we conclude that too little CD4 T-cell activation produces dysfunctional neutrophils leading to decreased bacteria clearance and survival, whereas too much CD4 T-cell activation produces a neutrophil phenotype that leads to efficient bacterial clearance but with increased tissue damage and mortality.

Divergent adaptive and innate immunological responses are observed in humans following blunt trauma

BackgroundThe 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.ResultsNeutrophil 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.ConclusionsThese 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.

Early infection during burn-induced inflammatory response results in increased mortality and p38-mediated neutrophil dysfunction

Following burn injury, the host is susceptible to bacterial infections normally cleared by healthy patients. We hypothesized that during the systemic immune response that follows scald injury, the hosts altered immune status increases infection susceptibility. Using a murine model of scald injury under inhaled anesthesia followed by intraperitoneal infection, we observed increased neutrophil numbers and function at postburn day (PBD) 1 compared with sham-burned and PBD4 mice. Further, increased mortality, bacteremia, and serum IL-6 were observed in PBD1 mice after Pseudomonas aeruginosa (PA) infection compared with sham-burned and PBD4 mice infected with PA. To examine these disparate responses, we investigated neutrophils isolated at 5 and 24 h following PA infection from PBD1 and sham-burned mice. Five hours after infection, there was no significant difference in number of recruited neutrophils; however, neutrophils from injured mice had decreased activation, active-p38, and oxidative burst compared with sham-burned mice. In direct contrast, 24 h after infection, we observed increased numbers, active-p38, and oxidative burst of neutrophils from PBD1 mice. Finally, we demonstrated that in neutrophils isolated from PBD1 mice, the observed increase in oxidative burst was p38 dependent. Altogether, neutrophil activation and function from thermally injured mice are initially delayed and later exacerbated by a p38-dependent mechanism. This mechanism is likely key to the observed increase in bacterial load and mortality of PBD1 mice infected with PA.

Roles of hepatocyte and myeloid CXC chemokine receptor‐2 in liver recovery and regeneration after ischemia/reperfusion in mice

Previous studies have demonstrated the significance of signaling through the CXC chemokine receptor‐2 (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 are 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−; and 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 the 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, whereas 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 after hepatic I/R, such that mice lacking myeloid CXCR2 had markedly increased chemokine expression, compared with 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, whereas hepatocyte CXCR2 plays a minor, secondary role. These findings suggest that myeloid cell‐directed therapy may significantly affect liver regeneration after liver resection or transplantation. (HEPATOLOGY 2013)

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 mice. 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-α during sepsis can increase NDMP numbers through activation of the Caspase 8 pathway or NF-κB.