Steven J. Schwulst

Multiple triggers of cell death in sepsis: death receptor and mitochondrial-mediated apoptosis

Lymphocyte apoptosis plays a central role in the pathophysiology of sepsis. Lymphocyte apoptosis was examined in mice with defective death receptor pathways due to transgenic expression of a dominant negative mutant of Fas‐associated death domain (FADD‐DN) or Bid−/− and in mice with defective mitochondrial‐mediated pathways due to loss of Bim−/−, Puma−/−, or Noxa−/−. FADD‐DN transgenic and Bid−/− mice had significant albeit incomplete protection, and this protection was associated with increased survival. Surprisingly, splenic B cells were also protected in FADD‐DN mice although transgene expression was confined to T cells, providing evidence for an indirect protective mechanism. Bim−/− provided virtually complete protection against lymphocyte apoptosis whereas Puma−/‐ and Noxa−/‐ mice had modest or no protection, respectively. Bim−/− mice had improved survival, and adoptive transfer of spleno‐cytes from Bim−/− mice into Rag 1−/− mice demonstrated that this was a lymphocyte intrinsic effect. The improved survival was associated with decreased inter‐leukin (IL) ‐10 and IL‐6 cytokines. Collectively, these data indicate that numerous death stimuli are generated during sepsis, and it therefore appears unlikely that blocking a single “trigger” can inhibit apoptosis. If siRNA becomes practical therapeutically, proapoptotic proteins would be potential targets.—Chang, K. C., Unsinger, J., Davis, C. G., Schwulst, S. J., Muenzer, J. T., Strasser, A., Hotchkiss, R. S. Multiple triggers of cell death in sepsis: death receptor and mitochondrialmediated apoptosis. FASEB J. 21, 708–719 (2007)

TAT-BH4 and TAT-Bcl-xL Peptides Protect against Sepsis-Induced Lymphocyte Apoptosis In Vivo

Apoptosis is a key pathogenic mechanism in sepsis that induces extensive death of lymphocytes and dendritic cells, thereby contributing to the immunosuppression that characterizes the septic disorder. Numerous animal studies indicate that prevention of apoptosis in sepsis improves survival and may represent a potential therapy for this highly lethal disorder. Recently, novel cell-penetrating peptide constructs such as HIV-1 TAT basic domain and related peptides have been developed to deliver bioactive cargoes and peptides into cells. In the present study, we investigated the effects of sepsis-induced apoptosis in Bcl-xL transgenic mice and in wild-type mice treated with an antiapoptotic TAT-Bcl-xL fusion protein and TAT-BH4 peptide. Lymphocytes from Bcl-xL transgenic mice were resistant to sepsis-induced apoptosis, and these mice had a ∼3-fold improvement in survival. TAT-Bcl-xL and TAT-BH4 prevented Escherichia coli-induced human lymphocyte apoptosis ex vivo and markedly decreased lymphocyte apoptosis in an in vivo mouse model of sepsis. In conclusion, TAT-conjugated antiapoptotic Bcl-2-like peptides may offer a novel therapy to prevent apoptosis in sepsis and improve survival.

Agonistic Monoclonal Antibody Against CD40 Receptor Decreases Lymphocyte Apoptosis and Improves Survival in Sepsis

Sepsis causes a marked apoptosis-induced depletion of lymphocytes. The degree of lymphocyte apoptosis during sepsis strongly correlates with survival. CD40, a member of the TNFR family, is expressed on APCs and has potent antiapoptotic activity. In this study we determined whether an agonistic Ab against CD40 could protect lymphocytes from sepsis-induced apoptosis. Secondly, we examined potential antiapoptotic mechanisms of the putative protection. Lastly, we aimed to determine whether anti-CD40 treatment could improve survival in sepsis. CD1 mice were made septic by the cecal ligation and puncture method and treated postoperatively with anti-CD40 Ab. Treatment with anti-CD40 completely abrogated sepsis-induced splenic B cell death and, surprisingly, decreased splenic and thymic T cell death as well (p < 0.001). To investigate the mechanism of protection of anti-CD40 therapy on T cells, CD40 receptor expression was examined. As anticipated, the CD40 receptor was constitutively expressed on B cells, but, unexpectedly, splenic and thymic T cells were found to express CD40 receptor during sepsis. Furthermore, CD4+CD8− T cells were the predominant subtype of T cells expressing CD40 receptor during sepsis. Additionally, the antiapoptotic protein Bcl-xL was found to be markedly increased in splenic B and T cells as well as in thymic T cells after treatment with anti-CD40 Ab (p < 0.0025). Lastly, mice that were made septic in a double injury model of sepsis had improved survival after treatment with anti-CD40 as compared with controls (p = 0.05). In conclusion, anti-CD40 treatment increases Bcl-xL, provides nearly complete protection against sepsis-induced lymphocyte apoptosis, and improves survival in sepsis.

Traumatic brain injury-induced alterations in peripheral immunity

BACKGROUND The complex alterations that occur in peripheral immunity after traumatic brain injury (TBI) have been poorly characterized to date. The purpose of this study was to determine the temporal changes in the peripheral immune response after TBI in a murine model of closed head injury. METHODS C57Bl/6 mice underwent closed head injury via a weight drop technique (n = 5) versus sham injury (n = 3) per time point. Blood, spleen, and thymus were collected, and immune phenotype, cytokine expression, and antibody production were determined via flow cytometry and multiplex immunoassays at 1, 3, 7, 14, 30, and 60 days after injury. RESULTS TBI results in acute and chronic changes in both the innate and adaptive immune response. TBI resulted in a striking loss of thymocytes as early as 3 days after injury (2.1 × 107 TBI vs. 5.6 × 107 sham, p = 0.001). Similarly, blood monocyte counts were markedly diminished as early as 24 hours after TBI (372 per deciliter TBI vs. 1359 per deciliter sham, p = 0.002) and remained suppressed throughout the first month after injury. At 60 days after injury, monocytes were polarized toward an anti-inflammatory (M2) phenotype. TBI also resulted in diminished interleukin 12 expression from Day 14 after injury throughout the remainder of the observation period. CONCLUSION TBI results in temporal changes in both the peripheral and the central immune systems culminating in an overall immune suppressed phenotype and anti-inflammatory milieu.

Lymphocyte Phenotyping to Distinguish Septic from Nonseptic Critical Illness

BACKGROUND Clinical signs and symptoms of sepsis are nonspecific and often indistinguishable from those of nonseptic critical illness. This ambiguity frequently delays the diagnosis of sepsis until culture results can confirm the presence or absence of an infectious organism. Lymphocyte phenotyping can be conducted rapidly and may provide information on the presence of infection before culture results are available. In this study, we hypothesized that lymphocyte phenotype can distinguish between septic and nonseptic critical illness. STUDY DESIGN C57Bl/6 mice were subjected to either P aeruginosa pneumonia or lipopolysaccharide-induced acute lung injury (ALI). Animals were sacrificed 24 hours postinjury and splenic lymphocytes were harvested. Additionally, 13 patients in a surgical ICU were enrolled in the study. Whole blood was obtained and lymphocytes were isolated by density gradient centrifugation. Lymphocyte phenotype was identified through flow cytometry after labeling lymphocytes for CD3, CD4, CD8, CD20, CD40, CD69, and CD86 with fluorochrome-conjugated antibodies. RESULTS CD69 expression on B cells and CD8+ splenocytes from septic mice was significantly increased compared with acute lung injury mice (p < 0.001 and p < 0.05, respectively). Similarly, CD4+ and CD8+ lymphocytes from septic patients had a two- to threefold increase in the expression of CD69 compared with nonseptic critically ill patients (p < 0.05). CONCLUSIONS These data indicated that CD69 expression on lymphocytes may be useful in distinguishing between septic and nonseptic critical illness. Continued investigation into the expression of CD69 during sepsis is warranted.

Surgical prophylaxis and other complication avoidance care bundles.

Individual health care quality measures that have been shown to improve outcome can be combined together into what are called care bundles, with the expectation that this set of practices produces further improvements in outcome. Prevention of surgical site infection is the focus of several quality measures put forward by the Surgical Care Improvement Project; these can collectively be considered a bundle as well. Whether these process measures, which include several components related to the administration of antibiotic prophylaxis, are effective in decreasing rates of surgical site infection has come under considerable debate recently.

Nonclassical Monocytes Mediate Secondary Injury, Neurocognitive Outcome, and Neutrophil Infiltration after Traumatic Brain Injury

Traumatic brain injury (TBI) results in rapid recruitment of leukocytes into the injured brain. Monocytes constitute a significant proportion of the initial infiltrate and have the potential to propagate secondary brain injury or generate an environment of repair and regeneration. Monocytes are a diverse population of cells (classical, intermediate, and nonclassical) with distinct functions, however, the recruitment order of these subpopulations to the injured brain largely remains unknown. Thus, we examined which monocyte subpopulations are required for the generation of early inflammatory infiltrate within the injured brain, and whether their depletion attenuates secondary injury or neurocognitive outcome. Global monocyte depletion correlated with significant improvements in brain edema, motor coordination, and working memory, and abrogated neutrophil infiltration into the injured brain. However, targeted depletion of classical monocytes alone had no effect on neutrophil recruitment to the site of injury, implicating the nonclassical monocyte in this process. In contrast, mice that have markedly reduced numbers of nonclassical monocytes (CX3CR1−/−) exhibited a significant reduction in neutrophil infiltration into the brain after TBI as compared with control mice. Our data suggest a critical role for nonclassical monocytes in the pathology of TBI in mice, including important clinical outcomes associated with mortality in this injury process.

Differential activation of infiltrating monocyte-derived cells after mild and severe traumatic brain injury

ABSTRACT Microglia are the resident innate immune cells of the brain. Although embryologically and functionally distinct, they are morphologically similar to peripheral monocyte–derived cells, resulting in a poor ability to discriminate between the two cell types. The purpose of this study was to develop a rapid and reliable method to simultaneously characterize, quantify, and discriminate between whole populations of myeloid cells from the brain in a murine model of traumatic brain injury. Male C57BL/6 mice underwent traumatic brain injury (n = 16) or sham injury (n = 14). Brains were harvested at 24 h after injury. Multiparameter flow cytometry and sequential gating analysis were performed, allowing for discrimination between microglia and infiltrating leukocytes as well as for the characterization and quantification of individual subtypes within the infiltrating population. The proportion of infiltrating leukocytes within the brain increased with the severity of injury, and the predominant cell types within the infiltrating population were monocyte derived (P = 0.01). In addition, the severity of injury altered the overall makeup of the infiltrating monocyte-derived cells. In conclusion, we describe a flow cytometry–based technique for gross discrimination between infiltrating leukocytes and microglia as well as the ability to simultaneously characterize and quantify individual myeloid subtypes and their maturation states within these populations.

The Role of Microglia in the Etiology and Evolution of Chronic Traumatic Encephalopathy.

ABSTRACT Chronic traumatic encephalopathy (CTE) is a progressive neurodegenerative disease that presents as a late sequela from traumatic brain injury (TBI). TBI is a growing and under-recognized public health concern with a high degree of morbidity and large associated global costs. While the immune response to TBI is complex, its contribution to the development of CTE remains largely unknown. In this review, we summarize the current understanding of the link between CTE and the resident innate immune system of the brain–microglia. We discuss the neuropathology underlying CTE including the creation and aggregation of phosphorylated tau protein into neurofibrillary tangles and the formation of amyloid beta deposits. We also present how microglia, the resident innate immune cells of the brain, drive the continuous low-level inflammation associated with the insidious onset of CTE. In this review, we conclude that the latency period between the index brain injury and the long-term development of CTE presents an opportunity for therapeutic intervention. Encouraging advances with microtubule stabilizers, cis p-tau antibodies, and the ability to therapeutically alter the inflammatory state of microglia have shown positive results in both animal and human trials. Looking forward, recent advancements in next-generation sequencing technology for the study of genomic, transcriptomic, and epigenetic information will provide an opportunity for significant advancement in our understanding of prorepair and pro-injury gene signatures allowing for targeted intervention in this highly morbid injury process.

Resuscitative thoracotomy for pediatric trauma in Illinois, 1999 to 2009

BACKGROUND Outcomes in adults who undergo resuscitative thoracotomy are poor. Few studies have examined the procedures use in pediatric trauma. METHODS The Illinois State Trauma Registry was queried for thoracotomy performed in the emergency department from 1999 to 2009, for patients aged 0 to 15. Injury mechanism, vital signs, and mortality were examined while controlling for injury severity. RESULTS Resuscitative thoracotomy was infrequently performed in pediatric trauma (n = 25; 2.3/year). Most patients had suffered penetrating injury. Patients who underwent resuscitative thoracotomy were in extremis, with only 17% demonstrating signs of life upon presentation. Although 6 patients (24%) survived initially, only 2 (8%) survived to hospital discharge. CONCLUSIONS Resuscitative thoracotomy was rarely performed in children in Illinois emergency departments. Survival is low for thoracotomy in the emergency department, but some patients who presented with penetrating injuries did have positive outcomes, supporting a continued role for the procedure in select cases.