Andrew T. Clark

IL-15 Prevents Apoptosis, Reverses Innate and Adaptive Immune Dysfunction, and Improves Survival in Sepsis

IL-15 is a pluripotent antiapoptotic cytokine that signals to cells of both the innate and adaptive immune system and is regarded as a highly promising immunomodulatory agent in cancer therapy. Sepsis is a lethal condition in which apoptosis-induced depletion of immune cells and subsequent immunosuppression are thought to contribute to morbidity and mortality. This study tested the ability of IL-15 to block apoptosis, prevent immunosuppression, and improve survival in sepsis. Mice were made septic using cecal ligation and puncture or Pseudomonas aeruginosa pneumonia. The experiments comprised a 2 × 2 full factorial design with surgical sepsis versus sham and IL-15 versus vehicle. In addition to survival studies, splenic cellularity, canonical markers of activation and proliferation, intracellular pro- and antiapoptotic Bcl-2 family protein expression, and markers of immune cell apoptosis were evaluated by flow cytometry. Cytokine production was examined both in plasma of treated mice and splenocytes that were stimulated ex vivo. IL-15 blocked sepsis-induced apoptosis of NK cells, dendritic cells, and CD8 T cells. IL-15 also decreased sepsis-induced gut epithelial apoptosis. IL-15 therapy increased the abundance of antiapoptotic Bcl-2 while decreasing proapoptotic Bim and PUMA. IL-15 increased both circulating IFN-γ, as well as the percentage of NK cells that produced IFN-γ. Finally, IL-15 increased survival in both cecal ligation and puncture and P. aeruginosa pneumonia. In conclusion, IL-15 prevents two immunopathologic hallmarks of sepsis, namely, apoptosis and immunosuppression, and improves survival in two different models of sepsis. IL-15 represents a potentially novel therapy of this highly lethal disorder.

Effects of aging on the immunopathologic response to sepsis.

Objective:Aging is associated with increased inflammation following sepsis. The purpose of this study was to determine whether this represents a fundamental age-based difference in the host response or is secondary to the increased mortality seen in aged hosts. Design:Prospective, randomized controlled study. Setting:Animal laboratory in a university medical center. Subjects:Young (6–12 weeks) and aged (20–24 months) FVB/N mice. Interventions:Mice were subjected to 2 × 25 or 1 × 30 cecal ligation and puncture (CLP). Measurements and Main Results:Survival was similar in young mice subjected to 2 × 25 CLP and aged mice subjected to 1 × 30 CLP (p = 0.15). Young mice subjected to 1 × 30 CLP had improved survival compared with the other groups (p < 0.05). When injury was held constant but mortality was greater, both systemic and peritoneal levels of tumor necrosis factor-α, interleukin (IL)-6, IL-10, and monocyte chemotactic protein-1 were elevated 24 hours after CLP in aged animals compared with young animals (p < 0.05). When mortality was similar but injury severity was different, there were no significant differences in systemic cytokines between aged mice and young mice. In contrast, peritoneal levels of tumor necrosis factor-α, IL-6, and IL-10 were higher in aged mice subjected to 1 × 30 CLP than young mice subjected to 2 × 25 CLP despite their similar mortalities (p < 0.05). There were no significant differences in either bacteremia or peritoneal cultures when animals of different ages sustained similar injuries or had different injuries with similar mortalities. Conclusions:Aged mice are more likely to die of sepsis than young mice when subjected to an equivalent insult, and this is associated with increases in both systemic and local inflammation. There is an exaggerated local but not systemic inflammatory response in aged mice compared with young mice when mortality is similar. This suggests that systemic processes that culminate in death may be age independent, but the local inflammatory response may be greater with aging.

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.

Cancer causes increased mortality and is associated with altered apoptosis in murine sepsis

Objective:Whereas most septic patients have an underlying comorbidity, most animal models of sepsis use mice that were healthy before the onset of infection. Malignancy is the most common comorbidity associated with sepsis. The purpose of this study was to determine whether mice with cancer have a different response to sepsis than healthy animals. Design:Prospective, randomized controlled study. Setting:Animal laboratory in a university medical center. Subjects:C57Bl/6 mice. Interventions:Animals received a subcutaneous injection of either 250,000 cells of the transplantable pancreatic adenocarcinoma cell line Pan02 (cancer) or phosphate-buffered saline (healthy). Three weeks later, mice given Pan02 cells had reproducible, nonmetastatic tumors. Both groups of mice then underwent intratracheal injection of either Pseudomonas aeruginosa (septic) or 0.9% NaCl (sham). Animals were killed 24 hrs postoperatively or followed-up 7 days for survival. Measurements and Main Results:Mice with cancer and healthy mice appeared similar when subjected to sham operation, although cancer animals had lower levels of T- and B-lymphocyte apoptosis. Septic mice with cancer had increased mortality compared to previously healthy septic mice subjected to the identical injury (52% vs. 28%; p = .04). This was associated with increased bacteremia but no difference in local pulmonary infection. Septic mice with cancer also had increased intestinal epithelial apoptosis. Although sepsis induced an increase in T- and B-lymphocyte apoptosis in all animals, septic mice with cancer had decreased T- and B-lymphocyte apoptosis compared to previously healthy septic mice. Serum and pulmonary cytokines, lung histology, complete blood counts, and intestinal proliferation were similar between septic mice with cancer and previously healthy septic mice. Conclusions:When subjected to the same septic insult, mice with cancer have increased mortality compared to previously healthy animals. Decreased systemic bacterial clearance and alterations in intestinal epithelial and lymphocyte apoptosis may help explain this differential response.

Mechanisms of methicillin-resistant Staphylococcus aureus pneumonia-induced intestinal epithelial apoptosis

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) pneumonia–induced sepsis is a common cause of morbidity in the intensive care unit. Although pneumonia is initiated in the lungs, extrapulmonary manifestations occur commonly. In light of the key role the intestine plays in the pathophysiology of sepsis, we sought to determine whether MRSA pneumonia induces intestinal injury. FVB/N mice were subjected to MRSA or sham pneumonia and killed 24 h later. Septic animals had a marked increase in intestinal epithelial apoptosis by both hematoxylin-eosin and active caspase 3 staining. Methicillin-resistant S. aureus–induced intestinal apoptosis was associated with an increase in the expression of the proapoptotic proteins Bid and Bax and the antiapoptotic protein Bcl-xL in the mitochondrial pathway. In the receptor-mediated pathway, MRSA pneumonia induced an increase in Fas ligand but decreased protein levels of Fas, FADD, pFADD, TNF-R1, and TRADD. To assess the functional significance of these changes, MRSA pneumonia was induced in mice with genetic manipulations in proteins in either the mitochondrial or receptor-mediated pathways. Both Bid−/− mice and animals with intestine-specific overexpression of Bcl-2 had decreased intestinal apoptosis compared with wild-type animals. In contrast, Fas ligand−/− mice had no alterations in apoptosis. To determine if these findings were organism-specific, similar experiments were performed in mice subjected to Pseudomonas aeruginosa pneumonia. Pseudomonas aeruginosa induced gut apoptosis, but unlike MRSA, this was associated with increased Bcl-2 and TNF-R1 and decreased Fas. Methicillin-resistant S. aureus pneumonia thus induces organism-specific changes in intestinal apoptosis via changes in both the mitochondrial and receptor-mediated pathways, although the former may be more functionally significant.

Cecal ligation and puncture followed by methicillin-resistant Staphylococcus aureus pneumonia increases mortality in mice and blunts production of local and systemic cytokines.

ABSTRACT Mortality in the intensive care unit frequently results from the synergistic effect of two temporally distinct infections. This study examined the pathophysiology of a new model of intra-abdominal sepsis followed by methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. Mice underwent cecal ligation and puncture (CLP) or sham laparotomy followed 3 days later by an intratracheal injection of MRSA or saline. Both CLP/saline and sham/MRSA mice had 100% survival, whereas animals with CLP followed by MRSA pneumonia had 67% 7-day survival. Animals subjected to CLP/MRSA had increased bronchoalveolar lavage concentrations of MRSA compared with sham/MRSA animals. Animals subjected to sham/MRSA pneumonia had increased bronchoalveolar lavage levels of interleukin 6 (IL-6), tumor necrosis factor &agr;, and granulocyte colony-stimulating factor compared with those given intratracheal saline, whereas CLP/MRSA mice had a blunted local inflammatory response with markedly decreased cytokine levels. Similarly, animals subjected to CLP/saline had increased peritoneal lavage levels of IL-6 and IL-1&bgr; compared with those subjected to sham laparotomy, whereas this response was blunted in CLP/MRSA mice. Systemic cytokines were upregulated in both CLP/saline and sham/MRSA mice, and this was blunted by the combination of CLP/MRSA. In contrast, no synergistic effect on pneumonia severity, white blood cell count, or lymphocyte apoptosis was identified in CLP/MRSA mice compared with animals with either insult in isolation. These results indicate that a clinically relevant model of CLP followed by MRSA pneumonia causes higher mortality than could have been predicted from studying either infection in isolation, and this was associated with a blunted local (pulmonary and peritoneal) and systemic inflammatory response and decreased ability to clear infection.

Prevention of Lymphocyte Apoptosis in Septic Mice with Cancer Increases Mortality

Lymphocyte apoptosis is thought to have a major role in the pathophysiology of sepsis. However, there is a disconnect between animal models of sepsis and patients with the disease, because the former use subjects that were healthy prior to the onset of infection while most patients have underlying comorbidities. The purpose of this study was to determine whether lymphocyte apoptosis prevention is effective in preventing mortality in septic mice with preexisting cancer. Mice with lymphocyte Bcl-2 overexpression (Bcl-2-Ig) and wild type (WT) mice were injected with a transplantable pancreatic adenocarcinoma cell line. Three weeks later, after development of palpable tumors, all animals received an intratracheal injection of Pseudomonas aeruginosa. Despite having decreased sepsis-induced T and B lymphocyte apoptosis, Bcl-2-Ig mice had markedly increased mortality compared with WT mice following P. aeruginosa pneumonia (85 versus 44% 7-d mortality; p = 0.004). The worsened survival in Bcl-2-Ig mice was associated with increases in Th1 cytokines TNF-α and IFN-γ in bronchoalveolar lavage fluid and decreased production of the Th2 cytokine IL-10 in stimulated splenocytes. There were no differences in tumor size or pulmonary pathology between Bcl-2-Ig and WT mice. To verify that the mortality difference was not specific to Bcl-2 overexpression, similar experiments were performed in Bim−/− mice. Septic Bim−/− mice with cancer also had increased mortality compared with septic WT mice with cancer. These data demonstrate that, despite overwhelming evidence that prevention of lymphocyte apoptosis is beneficial in septic hosts without comorbidities, the same strategy worsens survival in mice with cancer that are given pneumonia.