Kevin W. McConnell

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.

A comparison of ventilator-associated pneumonia rates as identified according to the National Healthcare Safety Network and American College of Chest Physicians criteria.

Objective:The objective of this study was to compare the observed rates of ventilator-associated pneumonia when using the National Healthcare Safety Network vs. the American College of Chest Physicians criteria. Design:Prospective, observational cohort study. Setting:A 1250-bed academic tertiary care medical center. Patients:Adult medical and surgical intensive care unit patients requiring mechanical ventilation for >48 hrs. Interventions:None. Measurements and Main Results:Patients were prospectively and independently screened for ventilator-associated pneumonia from January 2009 to January 2010 using the National Healthcare Safety Network and American College of Chest Physicians criteria. All American College of Chest Physicians classifications, including the corresponding radiographs and laboratory data, were prospectively reviewed by one of the investigators (JD) and confirmed by a second investigator (MHK). All National Healthcare Safety Network classifications were administratively determined using the hospitals infection prevention surveillance system. Over 1 yr, 2060 patients met the inclusion criteria. Of these, 83 patients (4%) had ventilator-associated pneumonia according to the American College of Chest Physicians criteria as compared with 12 patients (0.6%) using the National Healthcare Safety Network criteria. The corresponding rates of ventilator-associated pneumonia were 8.5 vs. 1.2 cases per 1,000 ventilator days, respectively. Agreement of the two sets of criteria was marginal (&kgr; statistic, 0.26). Cultures were positive in 88% of ventilator-associated pneumonias in the American College of Chest Physicians group as compared to 92% in the National Healthcare Safety Network group. Conclusions:There is poor agreement between clinical and administrative surveillance methods for the diagnosis of ventilator-associated pneumonia. Although there may be some benefit to using more stringent criteria for surveillance of ventilator-associated pneumonia, use of the administratively applied National Healthcare Safety Network criteria may significantly underestimate the scope of the clinical problem.

Neutrophil depletion causes a fatal defect in murine pulmonary Staphylococcus aureus clearance

BACKGROUND Staphylococcus aureus is the most common cause of healthcare-associated pneumonia. Despite the significant morbidity and mortality associated with the disease, animal models of S. aureus pneumonia are rare. MATERIALS AND METHODS We examined the pathogenicity of four different strains of S. aureus (both methicillin-sensitive and -resistant as well as Panton-Valentine leukocidin-positive and -negative) in four strains of immunocompetent inbred and outbred mice (FVB/N, C57Bl/6, BALB/c, ND4; n = 148). The immunological basis for the development of murine S. aureus pneumonia was then determined by selectively depleting neutrophils, lymphocytes, or pulmonary macrophages prior to the onset of infection. An additional cohort of animals was rendered immunosuppressed by induction of abdominal sepsis via cecal ligation and puncture 2, 4, or 7 d prior to the onset of pneumonia. RESULTS Nearly all immunocompetent mice survived, regardless of which strain of S. aureus was used or which strain of mouse was infected. Among animals with immune depletion or prior immunosuppression, survival was decreased only following neutrophil depletion (26% versus 90% alive at 7 d, P < 0.0001). Compared to immunocompetent animals, neutrophil-depleted mice with S. aureus pneumonia had delayed pulmonary bacterial clearance at 16 and 40 h but had no difference in levels of bacteremia. Neutrophil-depleted mice also had elevated levels of pulmonary monocyte chemotactic protein-1 (822 pg/mL versus 150 pg/mL, P < 0.05). In contrast, pulmonary histological appearance was similar in both groups as was dry/wet lung weight. CONCLUSIONS These results suggest that neutrophils play a critical role in the host response to S. aureus pneumonia, and the survival differences observed in neutrophil-depleted mice are associated with alterations in bacterial clearance and pulmonary cytokine response.

The Role of Heat Shock Protein 70 in Mediating Age-Dependent Mortality in Sepsis

Sepsis is primarily a disease of the aged, with increased incidence and mortality occurring in aged hosts. Heat shock protein (HSP) 70 plays an important role in both healthy aging and the stress response to injury. The purpose of this study was to determine the role of HSP70 in mediating mortality and the host inflammatory response in aged septic hosts. Sepsis was induced in both young (6- to 12-wk-old) and aged (16- to 17-mo-old) HSP70−/− and wild-type (WT) mice to determine whether HSP70 modulated outcome in an age-dependent fashion. Young HSP70−/− and WT mice subjected to cecal ligation and puncture, Pseudomonas aeruginosa pneumonia, or Streptococcus pneumoniae pneumonia had no differences in mortality, suggesting HSP70 does not mediate survival in young septic hosts. In contrast, mortality was higher in aged HSP70−/− mice than aged WT mice subjected to cecal ligation and puncture (p = 0.01), suggesting HSP70 mediates mortality in sepsis in an age-dependent fashion. Compared with WT mice, aged septic HSP70−/− mice had increased gut epithelial apoptosis and pulmonary inflammation. In addition, HSP70−/− mice had increased systemic levels of TNF-α, IL-6, IL-10, and IL-1β compared with WT mice. These data demonstrate that HSP70 is a key determinant of mortality in aged, but not young hosts in sepsis. HSP70 may play a protective role in an age-dependent response to sepsis by preventing excessive gut apoptosis and both pulmonary and systemic inflammation.

Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia induce distinct host responses

Objective:Pathogens that cause pneumonia may be treated in a targeted fashion by antibiotics, but if this therapy fails, then treatment involves only nonspecific supportive measures, independent of the inciting infection. The purpose of this study was to determine whether host response is similar after disparate infections with similar mortalities. Design:Prospective, randomized controlled study. Setting:Animal laboratory in a university medical center. Interventions:Pneumonia was induced in FVB/N mice by either Streptococcus pneumoniae or two different concentrations of Pseudomonas aeruginosa. Plasma and bronchoalveolar lavage fluid from septic animals was assayed by a microarray immunoassay measuring 18 inflammatory mediators at multiple time points. Measurements and Main Results:The host response was dependent on the causative organism as well as kinetics of mortality, but the pro-inflammatory and anti-inflammatory responses were independent of inoculum concentration or degree of bacteremia. Pneumonia caused by different concentrations of the same bacteria, Pseudomonas aeruginosa, also yielded distinct inflammatory responses; however, inflammatory mediator expression did not directly track the severity of infection. For all infections, the host response was compartmentalized, with markedly different concentrations of inflammatory mediators in the systemic circulation and the lungs. Hierarchical clustering analysis resulted in the identification of five distinct clusters of the host response to bacterial infection. Principal components analysis correlated pulmonary macrophage inflammatory peptide-2 and interleukin-10 with progression of infection, whereas elevated plasma tumor necrosis factor sr2 and macrophage chemotactic peptide-1 were indicative of fulminant disease with >90% mortality within 48 hrs. Conclusions:Septic mice have distinct local and systemic responses to Streptococcus pneumoniae and Pseudomonas aeruginosa pneumonia. Targeting specific host inflammatory responses induced by distinct bacterial infections could represent a potential therapeutic approach in the treatment of sepsis.

CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis

Lymphocytes help determine whether gut epithelial cells proliferate or differentiate but are not known to affect whether they live or die. Here, we report that lymphocytes play a controlling role in mediating gut epithelial apoptosis in sepsis but not under basal conditions. Gut epithelial apoptosis is similar in unmanipulated Rag‐1–/– and wild‐type (WT) mice. However, Rag‐1–/– animals have a 5‐fold augmentation in gut epithelial apoptosis following cecal ligation and puncture (CLP) compared to septic WT mice. Reconstitution of lymphocytes inRag‐1–/– mice via adoptive transfer decreases intestinal apoptosis to levels seen in WT animals. Subset analysis indicates that CD4+ but not CD8+, γδ, or B cells are responsible for the antiapoptotic effect of lymphocytes on the gut epithelium. Gut‐specific overexpression ofBcl‐2 in transgenic mice decreases mortality following CLP. This survival benefit is lymphocyte dependent since gut‐specific overexpression ofBcl‐2 fails to alter survival when the transgene is overexpressed inRag‐1mice. Further, adoptively transferring lymphocytes toRag‐1mice that simultaneously overexpress gut‐specificBcl‐2 results in improved mortality following sepsis. Thus, sepsis unmasks CD4+ lymphocyte control of gut apoptosis that is not present under homeostatic conditions, which acts as a key determinant of both cellular survival and host mortality.—Stromberg, P.E., Wool‐sey, C.A., Clark, A.T., Clark, J.A., Turnbull, I.R., McConnell, K.W., Chang, K.C., Chung, C.‐S., Ayala, A., Buchman, T.G., Hotchkiss, R.S., Coopersmith, C.M. CD4+ lymphocytes control gut epithelial apoptosis and mediate survival in sepsis. FASEB J. 23, 1817–1825 (2009)

The endogenous bacteria alter gut epithelial apoptosis and decrease mortality following Pseudomonas aeruginosa pneumonia.

ABSTRACT The endogenous bacteria have been hypothesized to play a significant role in the pathophysiology of critical illness, although their role in sepsis is poorly understood. The purpose of this study was to determine how commensal bacteria alter the host response to sepsis. Conventional and germ-free (GF) C57Bl/6 mice were subjected to Pseudomonas aeruginosa pneumonia. All GF mice died within 2 days, whereas 44% of conventional mice survived for 7 days (P = 0.001). Diluting the dose of bacteria 10-fold in GF mice led to similar survival in GF and conventional mice. When animals with similar mortality were assayed for intestinal integrity, GF mice had lower levels of intestinal epithelial apoptosis but similar levels of proliferation and intestinal permeability. Germ-free mice had significantly lower levels of tumor necrosis factor and interleukin 1&bgr; in bronchoalveolar lavage fluid compared with conventional mice without changes in systemic cytokine production. Under conventional conditions, sepsis unmasks lymphocyte control of intestinal epithelial apoptosis, because sepsis induces a greater increase in gut apoptosis in Rag-1−/− mice than in wild-type mice. However, in a separate set of experiments, gut apoptosis was similar between septic GF Rag-1−/− mice and septic GF wild-type mice. These data demonstrate that the endogenous bacteria play a protective role in mediating mortality from pneumonia-induced sepsis, potentially mediated through altered intestinal apoptosis and the local proinflammatory response. In addition, sepsis-induced lymphocyte-dependent increases in gut epithelial apoptosis appear to be mediated by the endogenous bacteria.

Organ failure avoidance and mitigation strategies in surgery.

Postoperative organ failure is a challenging disease process that is better prevented than treated. Providers should use close observation and clinical judgment, and checklists of best practices to minimize the risk of organ failure in their patients. The treatment of multiorgan dysfunction syndrome (MODS) generally remains supportive, outside of rapid initiation of source control (when appropriate) and targeted antibiotic therapy. More specific treatments may be developed as the complex pathophysiology of MODS is better understood and more homogenous patient populations are selected for study.

Attrition of memory CD8 T cells during sepsis requires LFA-1

CD8 T cell loss and dysfunction have been implicated in the increased susceptibility to opportunistic infections during the later immunosuppressive phase of sepsis, but CD8 T cell activation and attrition in early sepsis remain incompletely understood. With the use of a CLP model, we assessed CD8 T cell activation at 5 consecutive time points and found that activation after sepsis results in a distinct phenotype (CD69+CD25intCD62LHI) independent of cognate antigen recognition and TCR engagement and likely through bystander‐mediated cytokine effects. Additionally, we observed that sepsis concurrently results in the preferential depletion of a subset of memory‐phenotype CD8 T cells that remain “unactivated” (i.e., fail to up‐regulate activation markers) by apoptosis. Unactivated CD44HI OT‐I cells were spared from sepsis‐induced attrition, as were memory‐phenotype CD8 T cells of mice treated with anti‐LFA‐1 mAb, 1 h after CLP. Perhaps most importantly, we demonstrate that attrition of memory phenotype cells may have a pathologic significance, as elevated IL‐6 levels were associated with decreased numbers of memory‐phenotype CD8 T cells in septic mice, and preservation of this subset after administration of anti‐LFA‐1 mAb conferred improved survival at 7 d. Taken together, these data identify potentially modifiable responses of memory‐phenotype CD8 T cells in early sepsis and may be particularly important in the application of immunomodulatory therapies in sepsis.

Pathophysiology of septic shock: From bench to bedside.

Our understanding of sepsis and its resultant outcomes remains a paradox. On the one hand, we know more about the pathophysiology of sepsis than ever before. However, this knowledge has not been successfully translated to the bedside, as the vast majority of clinical trials for sepsis have been negative. Yet even in the general absence of positive clinical trials, mortality from sepsis has fallen to its lowest point in history, in large part due to educational campaigns that stress timely antibiotics and hemodynamic support. While additional improvements in outcome will assuredly result from further compliance with evidence based practices, a deeper understanding of the science that underlies the host response in sepsis is critical to the development of novel therapeutics. In this review, we outline immunopathologic abnormalities in sepsis, and then look at potential approaches to therapeutically modulate them. Ultimately, an understanding of the science underlying sepsis should allow the critical care community to utilize precision medicine to combat this devastating disease on an individual basis leading to improved outcomes.