Neutrophil derived microparticles increase mortality and the counter-inflammatory response in a murine model of sepsis.

Although advances in medical care have significantly improved sepsis survival, sepsis remains the leading cause of death in the ICU. This is likely due to a lack of complete understanding of the pathophysiologic mechanisms that lead to dysfunctional immunity. Neutrophil derived microparticles (NDMPs) have been shown to be the predominant microparticle present at infectious and inflamed foci in human models, however their effect on the immune response to inflammation and infection is sepsis has not been fully elucidated. As NDMPs may be a potential diagnostic and therapeutic target, we sought to determine the impact NDMPs on the immune response to a murine polymicrobial sepsis. We found that peritoneal neutrophil numbers, bacterial loads, and NDMPs were increased in our abdominal sepsis model. When NDMPs were injected into septic mice, we observed increased bacterial load, decreased neutrophil recruitment, increased expression of IL-10 and worsened mortality. Furthermore, the NDMPs express phosphatidylserine and are ingested by F4/80 macrophages via a Tim-4 and MFG-E8 dependent mechanism. Finally, upon treatment, NDMPs decrease macrophage activation, increase IL-10 release and decrease macrophage numbers. Altogether, these data suggest that NDMPs enhance immune dysfunction in sepsis by blunting the function of neutrophils and macrophages, two key cell populations involved in the early immune response to infection. This article is part of a Special Issue entitled: Immune and Metabolic Alterations in Trauma and Sepsis edited by Dr. Raghavan Raju.

Impact of caspase-8 and PKA in regulating neutrophil-derived microparticle generation.

The morbidity and mortality from sepsis continues to remain high despite extensive research into understanding this complex immunologic process. Further, while source control and antibiotic therapy have improved patient outcomes, many immunologically based therapies have fallen short. Microparticles (MPs) are intact vesicles that serve as mediators of intercellular communication as well as markers of inflammation in various disease processes. We have previously demonstrated that MPs can be produced at the infected foci during sepsis, are predominantly of neutrophil derivation (NDMPs) and can modulate immune cells. In this study, we sought to elucidate the molecular mechanisms underlying NDMP generation. Using thioglycolate (TGA) to recruit and activate neutrophils, we first determined that intra-peritoneal TGA increase NDMP accumulation. We next utilized TGA-elicited neutrophils in vitro to investigate signaling intermediates involved in NDMP production, including the intrinsic and extrinsic caspase pathways, cAMP dependent PKA and Epac activation as well as the role myosin light chain kinase (MLCK) as a final mediator of NDMP release. We observed that NDMP generation was dependent on the extrinsic caspase apoptotic pathway (caspase 3 and caspase 8), cAMP activation of PKA but not of Epac, and on activation of MLCK. Altogether, these data contribute to an overall framework depicting the molecular mechanisms that regulate NDMP generation.

Thymic stromal lymphopoietin mediates the host response and increases mortality during sepsis.

BACKGROUND: Sepsis and subsequent multiorgan system failure is associated with high rates of mortality and morbidity. Thymic stromal lymphopoietin (TSLP) is a cytokine that can be produced by keratinocytes and epithelial cells. Primarily, TSLP has been shown to promote counter-inflammatory processes. However, its potential expression or role in the pathogenesis of sepsis is largely unexplored. We hypothesized that TSLP is expressed during sepsis and TSLP blockade would alter the immune response and mortality. MATERIALS AND METHODS: Mice underwent cecal ligation and puncture (CLP) to produce a physiologically relevant murine model for sepsis. Cohorts were either treated with neutralizing TSLP antibodies or isotype controls before the CLP to determine changes in survival, bacterial loads, cytokine levels, and neutrophil function. RESULTS: It was observed that TSLP levels peaked at 6 h and remained detectable up to 48 h after CLP. Mice pretreated with neutralizing TSLP showed decreased mortality and bacterial load after CLP. Additionally, we determined that septic mice pretreated with the anti-TSLP antibody had increased tumor necrosis factor alpha and oxidative burst as well as increased interleukin 17 and neutrophil numbers compared with mice pretreated with isotype controls. CONCLUSIONS: TSLP levels peak early but are sustained during the first 48 h of sepsis. We speculate that TSLP blunts the neutrophil response resulting in increased bacterial load and mortality.

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.

Interleukin-7 ameliorates immune dysfunction and improves survival in a 2-hit model of fungal sepsis.

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.

Interleukin-7 (IL-7) treatment accelerates neutrophil recruitment through gamma delta T-cell IL-17 production in a murine model of sepsis.

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.

Predictors of retained hemothorax in trauma: Results of an Eastern Association for the Surgery of Trauma multi-institutional trial.

BACKGROUND: The natural history of traumatic hemothorax (HTX) remains unclear. We aimed to describe outcomes of HTX following tube thoracostomy drainage and to delineate factors that predict progression to a retained hemothorax (RH). We hypothesized that initial large-volume HTX predicts the development of an RH. METHODS: We conducted a prospective, observational, multi-institutional study of adult trauma patients diagnosed with an HTX identified on computed tomography (CT) scan with volumes calculated at time of diagnosis. All patients were managed with tube thoracostomy drainage within 24 hours of presentation. Retained hemothorax was defined as blood-density fluid identified on follow-up CT scan or need for additional intervention after initial tube thoracostomy placement for HTX. RESULTS: A total of 369 patients who presented with an HTX initially managed with tube thoracostomy drainage were enrolled from 17 trauma centers. Retained hemothorax was identified in 106 patients (28.7%). Patients with RH had a larger median (interquartile range) HTX volume on initial CT compared with no RH (191 [48-431] mL vs. 88 [35-245] mL, p = 0.013) and were more likely to be older with a higher burden of thoracic injury. After controlling for significant differences between groups, RH was independently associated with a larger HTX on presentation, with a 15% increase in risk of RH for each additional 100 mL of HTX on initial CT imaging (odds ratio, 1.15; 95% confidence interval, 1.08-1.21; p < 0.001). Patients with an RH also had higher rates of pneumonia and longer hospital length of stay than those with successful initial management. Retained hemothorax was also associated with worse functional outcomes at discharge and first outpatient follow-up. CONCLUSION: Larger initial HTX volumes are independently associated with RH, and unsuccessful initial management with tube thoracostomy is associated with worse patient outcomes. Future studies should use this experience to assess a range of options for reducing the risk of unsuccessful initial management. LEVEL OF EVIDENCE: Therapeutic/care management study, level III.

Staying connected: Service-specific orientation can be successfully achieved using a mobile application for onboarding care providers.

Communicating service-specific practice patterns, guidelines, and provider information to a new team of learners that rotate frequently can be challenging. Leveraging individual and healthcare electronic resources, a mobile device platform was implemented into a newly revised resident onboarding process. We hypothesized that offering an easy-to-use mobile application would improve communication across multiple disciplines as well as improve provider experiences when transitioning to a new rotation. A mobile platform was created and deployed to assist with enhancing communication within a trauma service and its resident onboarding process. The platform had resource materials such as: divisional policies, Clinical Practice Guidelines (CMGs), and onboarding manuals along with allowing for the posting of divisional events, a divisional directory that linked to direct dialing, text or email messaging, as well as on-call schedules. A mixed-methods study, including an anonymous survey, aimed at providing information on team member's impressions and usage of the mobile application was performed. Usage statistics over a 3-month period were analyzed on those providers who completed the survey. After rotation on the trauma service, trainees were asked to complete an anonymous, online survey addressing both the experience with, as well as the utility of, the mobile app. Thirty of the 37 (81%) residents and medical students completed the survey. Twenty-five (83%) trainees stated that this was their first experience rotating on the trauma service and 6 (20%) were from outside of the health system. According to those surveyed, the most useful function of the app were access to the directory (15, 50%), the divisional calendar (4, 13.3%), and the on-call schedules (3, 10%). Overall, the app was felt to be easy to use (27, 90%) and was accessed an average of 7 times per day (1-50, SD 9.67). Over half the survey respondents felt that the mobile app was helpful in completing their everyday tasks (16, 53.3%). Fifteen (50%) of the respondents stated that the app made the transition to the trauma service easier. Twenty-five (83.3%) stated it was valuable knowing about departmental events and announcements, and 17 (56.7%) felt more connected to the division. The evolution of mobile technology is rapidly becoming fundamental in medical education and training. We found that integrating a service-specific mobile application improved the learner's experience when transitioning to a new service and was a valuable onboarding instrument. Level of evidence IV.

Human microparticles generated during sepsis in patients with critical illness are neutrophil-derived and modulate the immune response.

BACKGROUND: Microparticles (MPs) are 0.3 µm to 1.0 µm vesicles generated after cell activation or apoptosis that may play a role in the pathophysiology of sepsis. We sought to elucidate the role of MPs in patients with critical illness and hypothesized that MPs are generated at the site of inflammation and can modulate the immune response. METHODS: Surgical patients with critical illness with ongoing sepsis were enrolled from the intensive care unit of an urban, Level I trauma center from March to June 2011. Abdominal washings and bronchoalveolar lavage fluid were collected from sites of inflammation. MPs were isolated using differential centrifugation, then characterized by flow cytometry. Immunologic assays were conducted by incubating neutrophil-derived MPs (NDMPs) with a human monocytic cell line (THP-1). A p value ≤0.05 was considered significant. RESULTS: MPs were absent in noninflamed foci in patients, whereas NDMPs were present in locations of inflammation. NDMPs were added to cultured THP-1 cells to quantify immunomodulatory effects. THP-1 cells were able to phagocytose NDMPs. Cells that ingested NDMPs demonstrated increased activation. In contrast, bystander THP-1 cells without ingested NDMPs demonstrated decreased activation. CONCLUSION: NDMPs are generated at the site of inflammation in patients with critical illness during sepsis. They have a divergent effect on the immune response by activating phagocytic cells and deactivating bystander cells. NDMPs may play an important role in regulating the inflammatory response to sepsis in patients with critical illness.

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 enhanced 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.

The leptin system: a potential target for sepsis induced immune suppression.

Sepsis, which is defined as a systemic inflammatory response syndrome that occurs during infection, is associated with several clinical conditions and high mortality rates. As sepsis progresses immune paralysis can become severe, leaving an already vulnerable patient ill equipped to eradicate primary or secondary infections. At present the predominant treatments for sepsis have not demonstrated convincing efficacy of decreased mortality. During sepsis, it has been observed that leptin levels initially increase but subsequently decline. A body of evidence has demonstrated that central or systemic leptin can beneficially regulate immune function. In this report expression of leptin and its receptor, signaling, and function on leukocytes will be reviewed. Furthermore, the effects mediated by central and systemic leptin during sepsis will be reviewed. Altogether, the ability of leptin to beneficially enhance inflammation and the host response during sepsis supports its use as a therapeutic agent, particularly during the latter phases of the syndrome.