Plasticity of the systemic inflammatory response to acute infection during critical illness: development of the riboleukogram.

BACKGROUND: Diagnosis of acute infection in the critically ill remains a challenge. We hypothesized that circulating leukocyte transcriptional profiles can be used to monitor the host response to and recovery from infection complicating critical illness. METHODOLOGY/PRINCIPAL FINDINGS: A translational research approach was employed. Fifteen mice underwent intratracheal injections of live P. aeruginosa, P. aeruginosa endotoxin, live S. pneumoniae, or normal saline. At 24 hours after injury, GeneChip microarray analysis of circulating buffy coat RNA identified 219 genes that distinguished between the pulmonary insults and differences in 7-day mortality. Similarly, buffy coat microarray expression profiles were generated from 27 mechanically ventilated patients every two days for up to three weeks. Significant heterogeneity of VAP microarray profiles was observed secondary to patient ethnicity, age, and gender, yet 85 genes were identified with consistent changes in abundance during the seven days bracketing the diagnosis of VAP. Principal components analysis of these 85 genes appeared to differentiate between the responses of subjects who did versus those who did not develop VAP, as defined by a general trajectory (riboleukogram) for the onset and resolution of VAP. As patients recovered from critical illness complicated by acute infection, the riboleukograms converged, consistent with an immune attractor. CONCLUSIONS/SIGNIFICANCE: Here we present the culmination of a mouse pneumonia study, demonstrating for the first time that disease trajectories derived from microarray expression profiles can be used to quantitatively track the clinical course of acute disease and identify a state of immune recovery. These data suggest that the onset of an infection-specific transcriptional program may precede the clinical diagnosis of pneumonia in patients. Moreover, riboleukograms may help explain variance in the host response due to differences in ethnic background, gender, and pathogen. Prospective clinical trials are indicated to validate our results and test the clinical utility of riboleukograms.

Mechanisms of decreased intestinal epithelial proliferation and increased apoptosis in murine acute lung injury.

OBJECTIVES: The aim of this study was to determine the effects of acute lung injury on the gut epithelium and examine mechanisms underlying changes in crypt proliferation and apoptosis. The relationship between severity and timing of lung injury to intestinal pathology was also examined. DESIGN: Randomized, controlled study. SETTING: University research laboratory. SUBJECTS: Genetically inbred mice. INTERVENTIONS: Following induction of acute lung injury, gut epithelial proliferation and apoptosis were assessed in a) C3H/HeN wild-type and C3H/HeJ mice, which lack functional Toll-like receptor 4 (n = 17); b) C57Bl/6 mice that received monoclonal anti-tumor necrosis factor-alpha or control antibody (n = 22); and c) C57Bl/6 wild-type and transgenic mice that overexpress Bcl-2 in their gut epithelium (n = 21). Intestinal epithelial proliferation and death were also examined in animals with differing degrees of lung inflammation (n = 24) as well as in a time course analysis following a fixed injury (n = 18). MEASUREMENTS AND MAIN RESULTS: Acute lung injury caused decreased proliferation and increased apoptosis in crypt epithelial cells in all animals studied. C3H/HeJ mice had higher levels of proliferation than C3H/HeN animals without additional changes in apoptosis. Anti-tumor necrosis factor-alpha antibody had no effect on gut epithelial proliferation or death. Overexpression of Bcl-2 did not change proliferation despite decreasing gut apoptosis. Proliferation and apoptosis were not correlated to severity of lung injury, as gut alterations were lost in mice with more severe acute lung injury. Changes in both gut epithelial proliferation and death were apparent within 12 hrs, but proliferation was decreased 36 hrs following acute lung injury while apoptosis returned to normal. CONCLUSIONS: Acute lung injury causes disparate effects on crypt proliferation and apoptosis, which occur, at least in part, through differing mechanisms involving Toll-like receptor 4 and Bcl-2. Severity of lung injury does not correlate with perturbations in proliferation or death in the gut epithelium, and acute lung injury-induced changes in intestinal epithelial proliferation persist longer than those in apoptosis.

High-dose exogenous iron following cecal ligation and puncture increases mortality rate in mice and is associated with an increase in gut epithelial and splenic apoptosis.

OBJECTIVES: Despite having dysregulated iron metabolism, critically ill patients may receive exogenous iron for the treatment of anemia. Iron is associated with increased tissue apoptosis and may facilitate bacterial growth. We hypothesized that exogenous iron administration given after the onset of sepsis would lead to increased mortality rate. To discriminate between elevated cell death and bacterial overgrowth as potential mediators of mortality, we examined gut epithelial and lymphocyte apoptosis and systemic bacterial counts in animals given iron supplementation after the onset of sepsis. DESIGN: Prospective, randomized, controlled study. SETTING: Animal laboratory in a university medical center. SUBJECTS: Male C57BL/6 mice, 6-10 wks old. INTERVENTIONS: C57BL/6 mice were subjected to cecal ligation and puncture (CLP), a well-accepted model of intra-abdominal sepsis, followed by daily subcutaneous injections of either 1 mL of iron dextran (5 mg/mL) or 0.9% NaCl for a total of five doses. Animals (n = 78) were followed for survival for 8 days. Separate cohorts (n = 76) were killed 24 or 48 hrs after cecal ligation and puncture or sham laparotomy and were assayed for gut epithelial and splenic apoptosis as well as for quantitative blood cultures. MEASUREMENTS AND MAIN RESULTS: Eight-day survival was 7% in animals that received iron and 26% in mice that received 0.9% NaCl (p < .005). Iron supplementation after cecal ligation and puncture increased apoptosis by both active caspase 3 and hematoxylin and eosin staining in both the intestinal epithelium and spleen at 24 hrs (p < .05). Iron supplementation after sham laparotomy did not cause mortality or elevated apoptosis. Quantitative blood cultures revealed no detectable differences between septic animals that received iron and those that received 0.9% NaCl. CONCLUSIONS: High-dose iron supplementation with iron dextran after the onset of sepsis significantly increases mortality rate in this animal model. Iron-induced mortality may be mediated by an increase in gut epithelial and splenic apoptosis, whereas severity of bacteremia does not appear to play a causative role.

Bcl-2 inhibits gut epithelial apoptosis induced by acute lung injury in mice but has no effect on survival.

Gut epithelial apoptosis is increased in human studies and animal models of noninfectious inflammation and sepsis. Elevated intestinal cell death appears to be physiologically significant in sepsis. Previous studies demonstrate that overexpression of the antiapoptotic protein Bcl-2 in the gut epithelium of transgenic mice is associated with improved survival from Pseudomonas aeruginosa pneumonia and cecal ligation and puncture. The functional significance of elevated gut apoptosis in noninfectious inflammation has not been examined. We hypothesized that intestinal apoptosis would be detrimental to survival in noninfectious critical illness. To address this issue, acute lung injury (ALI) was induced with intratracheal injection of lipopolysaccharide (LPS, 800 microg) in wild-type (WT) FVB/N mice and transgenic mice that overexpress Bcl-2 in their intestinal epithelium. Guts were harvested at 12, 24, 48, and 72 h and assessed for apoptosis by both hematoxylin and eosin and active caspase-3 staining in 100 contiguous crypts. ALI increased gut epithelial apoptosis 12 h after LPS instillation compared with shams (P < 0.01), whereas overexpression of Bcl-2 decreased intestinal apoptosis compared with WT animals with ALI when assayed by active caspase-3 (P < 0.05). Plasma levels of tumor necrosis factor alpha, interleukin (IL)-6, and IL-10 were similar between WT and transgenic animals with ALI, both of which had elevated IL-10 levels at 12 h and elevated IL-6 levels at 24 h compared with sham animals. In a separate experiment, transgenic and WT animals with ALI were followed for mortality to determine whether gut overexpression of Bcl-2 conferred a survival advantage. Survival at 10 days was 73% in WT animals (n = 33) and 65% in Bcl-2 animals (n = 23, P = ns). These results indicate that while gut epithelial apoptosis is elevated in multiple models of critical illness, prevention of intestinal cell death by overexpression of Bcl-2 is associated with a disparate survival effect between sepsis and noninfectious inflammation.

Antibiotics improve survival and alter the inflammatory profile in a murine model of sepsis from Pseudomonas aeruginosa pneumonia.

Differing antibiotic regimens can influence both survival and the inflammatory state in sepsis. We investigated whether the addition and/or type of antimicrobial agent could effect mortality in a murine model of Pseudomonas aeruginosa pneumonia-induced sepsis and if antibiotics altered systemic levels of cytokines. FVB/N mice were subjected to intratracheal injection of pathogenic bacteria and were given gentamicin, imipenem, or 0.9% NaCl 2 h after surgery, which continued every 12 h for a total of six doses. Survival at 7 days (n = 24 in each group) was 100% for mice given gentamicin, 88% for mice given imipenem, and 8% for sham mice treated with 0.9% NaCl (P < 0.0001). Systemic interleukin (IL) 6 levels were assayed 6 h postoperatively on all mice to see if they were predictive of outcome. Plasma IL-6 levels above 3,600 pg/mL were associated with a 100% mortality, levels under 1,200 pg/mL were associated with a 100% survival, and levels between 1,200 and 3,600 pg/mL had no utility in predicting mortality. In a separate experiment, mice were sacrificed at 3, 6, 12 or 24 h after instillation of P. aeruginosa and were assayed for levels of TNF-alpha, IL-6, IL-10, and IL-12. Significant alterations in the proinflammatory cytokines TNF-alpha and IL-6 were present at all time points except 3 h between mice treated with antibiotics and sham controls. In contrast, statistically significant differences in the anti-inflammatory cytokine IL-10 were present between the groups only at 6 h, and levels of IL-12 were similar at all time points. These results indicate that both gentamicin and imipenem increase survival at least 10-fold in a model of pneumonia-induced monomicrobial sepsis, and this is predominantly associated with a down-regulation of proinflammatory cytokines.

Role of intestinal epithelial apoptosis in survival.

PURPOSE OF REVIEW: To outline evidence that suggests increased intestinal epithelial apoptosis (programmed cell death) plays an important role in critical illness of infectious and noninfectious origin. RECENT FINDINGS: Both human and animal studies demonstrate that the gut epithelium has increased levels of cellular death in sepsis and noninfectious inflammation. Importantly, gut apoptosis appears to be detrimental to survival in sepsis. Transgenic mice that overexpress the antiapoptotic protein Bcl-2 in their gut epithelia have increased survival compared with wild-type littermates in murine models of ruptured appendicitis and Pseudomonas aeruginosa pneumonia. SUMMARY: These animal studies offer a possible new mechanism underlying the gut's role as the "motor" of the systemic inflammatory response syndrome and suggest that intestinal epithelial apoptosis may be a novel therapeutic target in future critical care research.