More than 50,000 patients die of pneumonia in the United States each year, despite the vast majority receiving antibiotics targeted against the initiating microorganism (1). The most common organism causing pneumonia in the intensive care unit is Staphylococcus aureus, accounting for a quarter of all new cases (2). Regardless of the site of infection, methicillin-resistant S. aureus (MRSA) infection is becoming increasingly common in both the hospital setting and the community setting, and nearly 100,000 patients are diagnosed with invasive MRSA annually (3). Unfortunately, compared with methicillin-sensitive S. aureus, MRSA is associated with increased length of stay, hospital costs, and mortality.
Essentially, all patients with pneumonia requiring intensive care unit admission meet the Society of Critical Care Medicine/American College of Chest Physicians diagnostic criteria for sepsis (4), with the systemic inflammatory response syndrome criteria in the setting of infection. Of these, a significant proportion of patients with pneumonia-induced sepsis develop organ dysfunction remote from the lungs.
The intestine has long been characterized as the “motor” of systemic inflammatory response syndrome (5) and has been hypothesized to play a central role in the pathophysiology of sepsis. Intestinal integrity is abnormal in sepsis induced by pneumonia (caused by bacteria other than MRSA), with increased intestinal epithelial apoptosis, decreased crypt proliferation, and intestinal hyperpermeability (6–9). Preventing sepsis-induced apoptosis leads to improvement in survival following Pseudomonas aeruginosa pneumonia, although the pathways responsible for this are unknown. It is unclear whether pneumonia changes intestinal integrity via a generic host response (10, 11) or via an organism-specific response (12, 13).
Despite the increasing prevalence of MRSA pneumonia in intensive care unit patients, there are few studies examining the extrapulmonary effects of this virulent organism. We sought to determine whether MRSA pneumonia induces a secondary injury in the intestinal epithelium and, if so, to identify whether the mechanisms responsible for these alterations are distinct from P. aeruginosa pneumonia.
MATERIALS AND METHODS
Six- to 15-week-old mice were used for experiments. All mice were on an FVB/N genetic background unless otherwise indicated. Transgenic mice containing nucleotides −596 to +21 of a rat fatty acid–binding protein (Fabpl) linked to human Bcl-2 were generated as previously described (14). Fabpl–Bcl-2 mice have intestine-specific overexpression of Bcl-2 and appear phenotypically identical to wild-type (WT) littermates. Bid−/− mice on a C57Bl/6 background were a generous gift from Dr. Richard Hotchkiss (Washington University, St Louis, Mo). Fas ligand−/− mice (Fas-Lgld) and control C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, Me). All mice received water and mouse chow ad libitum and were kept on a 12 h light-dark schedule. All studies were approved by the Animal Studies Committees of both Emory University and Washington University in accordance with the National Institutes of Health laboratory animal use guidelines.
MRSA and P. aeruginosa preparation
Methicillin-resistant Staphylococcus aureus strain 313 was isolated from a patient in the BJC HealthCare System (St Louis, Mo) (15). This strain is Panton-Valentine leukocidin negative by polymerase chain reaction analysis, multilocus sequence type 5, and staphylococcal cassette chromosome (SCCmec) type II. Experiments using P. aeruginosa used the ATCC 27853 strain (16). Bacteria were grown overnight in trypticase soy broth (Becton Dickinson, Sparks, Md) at 37°C, centrifuged at 6,000g, washed in sterile 0.9% NaCl twice, and resuspended in sterile 0.9% NaCl. Methicillin-resistant S. aureus was brought to a final density of 0.5 at 600 nm, which corresponds to approximately 5 × 108 colony-forming units (CFUs)/mL, whereas P. aeruginosa final density ranged between 5 × 108 and 1 × 109 CFUs/mL.
Animals underwent isoflurane anesthesia followed by midline cervical incision and blunt dissection down to the trachea. Septic animal received either 40 μL (2 × 107 CFUs) of MRSA inocula or 20 μL (2–4 × 106 CFUs) P. aeruginosa inocula via intratracheal injection. Of note, we have previously demonstrated that all animals subjected to P. aeruginosa pneumonia are bacteremic 24 h after the induction of sepsis (9). Sham-operated mice were treated identically except they received an injection of the same volume of 0.9% NaCl. Mice received a single 1-mL subcutaneous injection of 0.9% NaCl for fluid resuscitation following incision closure and were held vertically for 10 s to enhance the delivery of bacteria into the lungs. Mice were killed 24 h later (6, 7, 12).
Intestinal epithelial apoptosis was quantified in crypts of 100 well-oriented crypt-villus units using both morphological and functional techniques by an investigator blinded to sample identity (17, 18). Hematoxylin-eosin (H&E)–stained sections were evaluated for morphologic changes characteristic of apoptosis including nuclear fragmentation (karyorrhexis) and cell shrinkage with condensed nuclei (pyknosis). Functional assessment of apoptosis was assessed by active caspase 3 staining. Paraffin-embedded sections were deparaffinized and rehydrated. Endogenous peroxidase activity was blocked by incubation in 30% H2O2 in methanol for 10 min, and slides were then heated in antigen decloaker solution (Biocare Medical, Concord, Calif) for 45 min. Sections were then blocked with 20% goat serum (Vector Laboratories, Burlingame, Calif) for 30 min and incubated overnight at 4°C with rabbit polyclonal anti–active caspase 3 antibody (1:100 diluted in PBS; Cell Signaling Technology, Danvers, Mass). The next day, sections were incubated with goat anti–rabbit biotinylated secondary antibody (1:200 diluted in PBS; Vector Laboratories) for 30 min at room temperature followed by incubation with Vectastain Elite avidin-biotin-peroxidase complex reagent (Vector Laboratories) for 30 min. Sections were developed with diaminobenzidine and counterstained with hematoxylin. Splenic apoptosis was quantified in a similar manner in five random high-power fields.
Western blot analysis
Frozen segments of jejunum were homogenized in 5× volume of ice-cold homogenization buffer and centrifuged at 10,000 revolutions/min at 4°C for 5 min (19). The supernatant was collected, and the Bradford protein assay was used to determine total protein concentration. Protein samples (40 μg) and equal volume of 2× Laemmli buffer were heated at 95°C for 5 min. Samples were run on polyacrylamide gels (Bio-Rad, Hercules, Calif) for 45 min at 180 V and then transferred to Immuno-Blot polyvinylidene difluoride membrane (Bio-Rad) for 2 h at 80 V. Membranes were then blocked in 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (Sigma, St Louis, Mo) at room temperature for 60 min. Membranes were incubated overnight with primary antibody in 4°C. The following primary antibodies were used: rabbit anti–β-actin, rabbit anti-Bax, rabbit anti-Bcl-xL, rabbit anti–Bcl-2, rabbit anti-Bid, rabbit anti-TRADD, rabbit anti-pFADD (Cell Signaling Technology), rabbit anti-Fas ligand, mouse anti–TNF-R1, rabbit anti-Fas, and rabbit anti-FADD (Santa Cruz Biotechnology, Santa Cruz, Calif). The following day, membranes were washed and incubated for 60 min at room temperature with horseradish peroxidase–conjugated goat anti–rabbit or horse anti–mouse immunoglobulin G (Cell Signaling Technology, Santa Cruz Biotechnology). Membranes were developed with chemiluminescent system (Pierce, Rockford, Ill), and proteins were detected following exposure to x-ray film.
Villus length was measured in micrometers on H&E-stained sections of proximal jejunum by measuring the distance from the villus tip to the crypt neck using MetaMorph version 184.108.40.206 (Downingtown, Pa). Twelve well-oriented crypt-villus units were measured in each animal.
Intestinal proliferation was assessed by quantifying S-phase cells in the crypts of 100 well-oriented crypt-villus units in a blinded fashion. Mice were injected with 5-bromo-2′-deoxyuridine (BrdU; Sigma) 90 min before killing using a dose of 5 mg/mL diluted in 0.9% NaCl. After killing, the intestine was removed, fixed, paraffin embedded, and sectioned. BrdU was detected in jejunal sections via immunohistochemistry using a commercially available kit (BD PharMingen, San Diego, Calif).
Mice were gavaged with 0.5 mL of 22 mg/mL fluorescein isothiocyanate dextran (FD4, molecular mass 4.4 kd; Sigma) in sterile phosphate-buffered saline using a 20-gauge feeding needle 19 h after MRSA inoculation (20). Five hours later, animals were killed, and blood was immediately collected. Plasma was isolated by centrifugation at 3,000 revolutions/min for 20 min. The concentration of FD4 in the plasma was measured and compared with a standard using fluorospectrometry (NanoDrop 3300; Thermo Scientific, Wilmington, Del).
Bronchoalveolar lavage (BAL) and blood samples were taken from mice immediately after killed, diluted serially, and plated on blood agar plates. Twenty-four hours after incubation at 37°C, plates were examined for colony counts.
After determining the groups did not have Gaussian distributions, data were compared using the Mann-Whitney U test. All data were analyzed using the statistical program Prism 4.0 (GraphPad Software, San Diego, Calif) and are presented as mean ± SEM. P < 0.05 was considered to be statistically significant.
MRSA pneumonia increases intestinal epithelial cell apoptosis
FVB/N mice that received intratracheal MRSA and sham mice that received intratracheal 0.9% NaCl were compared for intestinal epithelial apoptosis 24 h postoperatively. Mice with MRSA pneumonia had a marked increase in intestinal apoptosis (Fig. 1) whether assayed by H&E staining (P = 0.01) or active caspase 3 staining (P = 0.0005).
MRSA pneumonia alters expression of apoptotic molecules in both the mitochondrial and receptor-mediated pathways
To determine possible mechanisms through which MRSA pneumonia increased intestinal epithelial apoptosis, Western blots were performed on intestinal isolates from mice receiving sham pneumonia or MRSA pneumonia assaying both the mitochondrial and receptor-mediated pathways of apoptosis.
In the mitochondrial pathway (Fig. 2), proapoptotic proteins Bid and Bax were both increased in mice with MRSA pneumonia (Bid: 6.3 ± 0.9 vs. 1 ± 0.2, P = 0.0005; Bax: 1.5 ± 0.1 vs. 1 ± 0.1, P = 0.02; Fig. 2A, B). Antiapoptotic Bcl-xL was also markedly increased in septic mice (7.1 ± 0.6 vs. 1 ± 0.2, P < 0.0001; Fig. 2C), whereas there was no change in expression of the antiapoptotic protein Bcl-2 (1.1 ± 0.2 vs. 1 ± 0.3, not statistically significant; Fig. 2D).
In the receptor-mediated pathway (Fig. 3), Fas ligand expression was increased in mice with MRSA pneumonia (2.5 ± 0.2 vs. 1 ± 0.3, P = 0.0079; Fig. 3A). In contrast, Fas, FADD, pFADD, TNF-R1, and TRADD were all decreased in mice with MRSA pneumonia compared with sham mice (Fas: 0.2 ± 0.1 vs. 1 ± 0.2, P = 0.01; FADD: 0.2 ± 0.1 vs. 1 ± 0.3, P = 0.03; pFADD: 0.3 ± 0.1 vs. 1 ± 0.3, P = 0.04; TNF-R1: 0.4 ± 0.1 vs. 1 ± 0.1, P = 0.02; TRADD: 0.04 ± 0.01 vs. 1 ± 0.4, P = 0.02; Fig. 3B–F).
Genetic manipulation of the mitochondrial pathway prevents MRSA-induced intestinal epithelial apoptosis
To determine the functional significance of the altered expression of apoptotic mediators in the mitochondrial pathway, MRSA pneumonia was given to animals that had two distinct manipulations of this pathway.
First, mice with a genetic deficiency of the proapoptotic protein Bid were compared with WT C57Bl/6 control animals. Intestinal epithelial apoptosis was decreased in Bid−/− mice subjected to MRSA pneumonia compared with WT mice subjected to the same insult whether assayed by H&E (Fig. 4A, P = 0.04) or active caspase 3 (Fig. 4B, P = 0.007).
Next, mice with intestine-specific overexpression of the antiapoptotic protein Bcl-2 (Fabpl–Bcl-2 mice) were compared with FVB/N littermates. Intestinal epithelial apoptosis was decreased in Fabpl–Bcl-2 mice subjected to MRSA pneumonia compared with WT mice subjected to the same insult whether assayed by H&E (Fig. 4C, P = 0.003) or active caspase 3 (Fig. 4D, P = 0.001). Of note, MRSA induced a lower level of apoptosis in C57Bl/6 WT mice than FVB/N WT mice given the same dose of bacteria (compare Fig. 4A–D).
Wild-type C57Bl/6, Bid−/−, WT FVB/N, and Fabpl–Bcl-2 mice all had MRSA detectable in both their blood and BAL fluid 24 h after injection of MRSA. There were no differences in bacterial levels between WT and transgenic or knockout mice on the same genetic background. Consistent with higher levels of gut apoptosis, BAL levels of MRSA were higher in WT FVB/N and Fabpl–Bcl-2 mice than in WT C57Bl/6 and Bid−/− mice (data not shown).
Genetic manipulation of the receptor-mediated pathway does not prevent MRSA-induced intestinal epithelial apoptosis
In light of the fact that the only proapoptotic member of the receptor-mediated pathway that MRSA pneumonia increased in the intestine was Fas ligand expression, the functional significance of this was assayed by comparing mice with a genetic deficiency in Fas ligand (Fas-Lgld) to WT C57Bl/6 control animals. No statistically significant difference in intestinal epithelial apoptosis was detected in Fas-Lgld mice subjected to MRSA pneumonia compared with WT mice subjected to the same insult whether assayed by H&E (Fig. 5A) or active caspase 3 (Fig. 5B).
Pathways of gut epithelial apoptosis differ between MRSA pneumonia and P. aeruginosa pneumonias
To determine whether MRSA pneumonia induced gut apoptosis via organism-specific or organism-independent pathways, a different cohort of mice was subjected to P. aeruginosa pneumonia, and Western blots were performed on intestinal isolates as above. Of note, even though this model has previously been shown to cause significant 7-day mortality (9, 12), all mice receiving P. aeruginosa pneumonia survived for 24 h after induction of sepsis, at which time they were killed for gut harvest.
In the mitochondrial pathway (Fig. 6), there was no change in the expression of proapoptotic Bid and Bax or antiapoptotic Bcl-xL with P. aeruginosa pneumonia. Despite the fact that P. aeruginosa pneumonia increases gut epithelial apoptosis (6, 9, 17), the only difference detected in the mitochondrial pathway was a paradoxical increase in the antiapoptotic mediator Bcl-2 (2.4 ± 0.3 vs. 1 ± 0.2, P = 0.016).
In the receptor-mediated pathway (Fig. 7), TNF-R1 was increased in mice with P. aeruginosa pneumonia (5.4 ± 0.4 vs. 1 ± 0.3, P < 0.01, P < 0.01), whereas Fas was decreased in septic mice (0.3 ± 0.05 vs. 1 ± 0.1, P = 0.016). No changes were detected in Fas ligand, FADD, pFADD, or TRADD expression in mice given P. aeruginosa pneumonia compared with sham animals. Pathways of gut epithelial apoptosis were therefore significantly different in both the mitochondrial pathway (compare Fig. 2 with Fig. 6) and the receptor-mediated pathway (compare Fig. 3 with Fig. 7) for animals subjected to either MRSA or P. aeruginosa pneumonia.
MRSA pneumonia decreases intestinal villus length and crypt proliferation but does not affect intestinal permeability
To determine whether the intestinal manifestations of MRSA pneumonia were limited to increased epithelial apoptosis, intestinal proliferation, villus length, and permeability were assayed.
Methicillin-resistant S. aureus pneumonia decreased proliferation in septic mice compared with animals subjected to sham pneumonia as measured by labeling S-phase cells with BrdU (Fig. 8, P = 0.0001). This was associated with decreased villus length in mice with MRSA pneumonia compared with WT animals (Fig. 9, P = 0.0002). Neither crypt proliferation nor villus length was affected by preventing apoptosis as there were no statistically significant differences in either when septic Fabpl–Bcl-2 mice were compared with septic FVB/N littermates (data not shown).
Methicillin-resistant S. aureus pneumonia did not alter intestinal permeability in septic mice compared with shams animals as measured by the appearance of FD-4 in the bloodstream (Fig. 10). Consistent with these findings, there were no differences in intestinal protein expressions of tight junction proteins occludin or claudin 3 (data not shown).
This study demonstrates that MRSA pneumonia–induced sepsis alters intestinal integrity as manifested by increased intestinal epithelial apoptosis, decreased crypt proliferation, and decreased villus length. Furthermore, the mechanisms by which MRSA pneumonia induces intestinal apoptosis do not appear to be part of a common host response as pathways of gut apoptosis differ markedly between MRSA and P. aeruginosa pneumonia.
Although it is known that both the mitochondrial and receptor-mediated pathways contribute to sepsis-induced lymphocyte apoptosis (21, 22), this study is the first to examine pathways of sepsis-induced intestinal epithelial apoptosis in detail. Western blot analysis demonstrated that both the mitochondrial and receptor-mediated pathways in the intestine are altered by MRSA pneumonia. However, the types and significance of these alterations appear to be different between the two pathways.
In the mitochondrial pathway, the Bcl-2 family contains both proapoptotic and antiapoptotic proteins, and the balance between these plays a critical role in cell survival (23). This study indicates that both proapoptotic (Bid and Bax) and antiapoptotic (Bcl-xL) proteins are increased by MRSA pneumonia–induced sepsis. This is consistent with findings that these three mediators are elevated in the intestinal epithelium following cecal ligation and puncture (CLP) (19, 24). Bcl-xL inhibits apoptosis by binding to proapoptotic proteins (25), and the ratio of Bax to Bcl-xL or Bax to Bcl-2 is often used as a rheostat for cellular survival (26). These ratios are also correlated with mortality in septic patients (27). However, these ratios do not explain the increased apoptosis in septic mice compared with sham mice following MRSA pneumonia because the relative increase in Bcl-xL was much greater than the relative increase in Bax (and Bcl-2 was unaffected). The truncated form of Bid translocates to the mitochondria to activate Bax and Bak (28). This suggests that MRSA may induce intestinal epithelial apoptosis by inducing Bid, which secondarily induces Bak as its primary downstream mediator.
The functional significance of the mitochondrial pathway is reinforced by data demonstrating that Bid−/− mice and Fabpl–Bcl-2 mice are both able to inhibit sepsis-induced intestinal apoptosis. Although intestinal Bcl-2 levels are unaffected by MRSA pneumonia, either knocking out Bid or overexpressing Bcl-2 should alter the Bax/Bcl-xL or Bax/Bcl-2 ratio directly or indirectly, leading to the phenotype seen. The ability to genetically manipulate the mitochondrial pathway could theoretically have therapeutic benefit in more lethal models of sepsis where elevated gut apoptosis is associated with increased mortality.
The results from the receptor-mediated pathway were more complicated. Wesche-Soldato and colleagues (29) and Perl and colleagues (30) have previously shown that preventing signaling through Fas/Fas ligand prevents apoptosis in a wide variety of cell types following either CLP or a two-hit model of critical illness. Based on these results and previous work from our laboratory showing that both FADD and pFADD are elevated following CLP (19), it was reasonable to assume that multiple mediators within the receptor-mediated pathway would be increased following MRSA pneumonia, consistent with the increase in intestinal epithelial apoptosis. However, whereas MRSA increased Fas ligand levels, the other five mediators examined (Fas, FADD, pFADD, TNF-R1, and TRADD) were all decreased.
Although the data suggest that the mitochondrial pathway plays a critical role in pneumonia-induced apoptosis, the importance of the receptor-mediated pathway is less clear. First, whereas a single mediator (Fas ligand) was increased by MRSA pneumonia, the other five mediators were decreased, and preventing signaling through the only proapoptotic mediator upregulated by MRSA failed to prevent pneumonia-induced intestinal epithelial apoptosis. There are a number of possible explanations for this. First, the changes in apoptotic mediators in the receptor pathway may cancel themselves out, rendering the pathway unimportant in the final live/die decision a cell faces. Next, this may be explained by the fact that Western blot analysis was performed on whole-bowel homogenates and may represent an artifact of immune cells. Although Fas is expressed in intestinal epithelial cells, the expression of Fas ligand in these cells is more controversial (31). It is therefore possible that the Western blot analysis was measuring Fas ligand in the lamina propria, although even if that were the case, Fas ligand in this location has been demonstrated to activate Fas on intestinal epithelial cells resulting in apoptosis (31). It is also possible that elevated Fas ligand is a significant cause of MRSA-induced apoptosis and the fact that no difference was seen in Fas-Lgld mice relates to either they were studied at a single timepoint or there is a large variance seen in WT C57Bl/6 subjected to MRSA at 24 h (note error bars in Fig. 5), and our findings represent a type II error.
The comparison between mice given MRSA pneumonia and P. aeruginosa pneumonia demonstrates that the host response in the intestinal epithelium to these septic insults is not uniform. Depending on which component of the host response studied, the literature contains evidence of both a common host response independent of the initiating microorganism (10, 11) and an organism-specific response (12, 13, 32). The differences between apoptotic pathways between MRSA and CLP outlined above can potentially be attributed to different anatomic locations of initiating infection (intrathoracic vs. intra-abdominal) and to complexity of infection (polymicrobial vs. monomicrobial). To control for this, we examined two models of monomicrobial pneumonia and examined pathways of gut apoptosis. The results demonstrated a striking difference in both the mitochondrial and receptor-mediated pathways. Of the four mitochondrial mediators examined, three were altered in MRSA pneumonia (Bid, Bax, Bcl-XL), whereas one was altered in P. aeruginosa pneumonia, without any overlap detected. The differences were nearly as pronounced for the receptor-mediated pathway where overlap was noted in a single mediator (decreased expression of Fas in both), with differing results noted in all other mediators examined (FasL, TNF-R1, FADD, pFADD, TRADD).
An additional surprising result of this study was that intestinal permeability was not altered by MRSA pneumonia. Although intestinal apoptosis, proliferation, length, and permeability have autonomous control, they are often linked, and alterations in one aspect of intestinal integrity frequently affect another. In addition, multiple models of critical illness including P. aeruginosa pneumonia–induced sepsis cause intestinal hyperpermeability (8). It is unclear if the fact that intestinal permeability was unaffected when all other components of gut integrity were altered is secondary to the initiating organism involved (MRSA vs. P. aeruginosa) or differences in study design between our data and published literature.
This study has a number of limitations. The MRSA model used in this study was nonlethal. Although MRSA is a virulent pathogen in patients, it does not have the same degree of virulence in mice, which are highly resistant to infections with S. aureus compared with humans secondary to the microorganism’s preferential ability to take iron needed to proliferate from host hemoglobin in humans (33). This is consistent with recent studies from our group demonstrating that multiple isolates of S. aureus taken from patient samples (including MRSA, methicillin-sensitive S. aureus, Panton-Valentine leukocidin (PVL) positive, PVL negative) caused essentially no mortality in multiple strains of inbred and outbred mice (15, 34). Thus, even though preventing intestinal epithelial apoptosis has been associated with improved survival following multiple models of sepsis, it is difficult to know if the findings presented herein translate to more lethal models of sepsis. It is possible that the differences in apoptosis pathways between MRSA and P. aeruginosa were related to differences in mortality as opposed to differences in initiating microorganism. The study also did not examine the truncated form of Bid, which mediates crosstalk between the mitochondrial and receptor-mediated pathways of apoptosis, and also did not examine levels of Bak in the intestinal epithelium. Furthermore, because Bid−/− mice have a germline deletion, there is no way of knowing whether the decreases in MRSA-induced gut epithelial apoptosis in Bid−/− mice are due to direct effects on the intestinal epithelium or are secondary to other factors initiated outside the intestine, although it should also be noted that these mice have alterations in lymphocyte apoptosis following CLP. Along these lines, this study focused specifically on the intestine, while not examining other organ systems in any degree of detail. In addition, based on prior studies from our laboratory using either CLP or P. aeruginosa pneumonia, which showed maximal apoptosis at 24 h (7, 17, 35), all end points were measured at this timepoint in this study. However, it is important to note that gram-positive bacteremia may have a much more indolent time course than polymicrobial or gram-negative bacteremia, so important changes that may have occurred earlier or later would not have been determined in our study design.
Despite these limitations, this is the first description of how the most common type of pneumonia in the intensive care unit affects the intestinal epithelium. Because extrapulmonary effects of pneumonia may account for some of its morbidity, understanding the mechanisms through which MRSA pneumonia affects the host response may yield targets for future therapeutic intervention.
1. Heron M, Hoyert DL, Murphy SL, Xu J, Kochanek KD, Tejada-Vera B: Deaths: final data for 2006. Natl Vital Stat Rep 57 (14): 1–134, 2009.
2. Hidron AI, Edwards JR, Patel J, Horan TC, Sievert DM, Pollock DA, Fridkin SK: NHSN annual update: antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the National Healthcare Safety Network at the Centers for Disease Control and Prevention, 2006–2007. Infect Control Hosp Epidemiol 29 (11): 996–1011, 2008.
3. Klevens RM, Morrison MA, Nadle J, Petit S, Gershman K, Ray S, Harrison LH, Lynfield R, Dumyati G, Townes JM, et al.: Invasive methicillin-resistant Staphylococcus aureus
infections in the United States. JAMA 298 (15): 1763–1771, 2007.
4. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: definitions for sepsis
and organ failure and guidelines for the use of innovative therapies in sepsis
. Crit Care Med 20 (6): 864–874, 1992.
5. Clark JA, Coopersmith CM: Intestinal crosstalk: a new paradigm for understanding the gut
as the “motor” of critical illness. Shock 28 (4): 384–393, 2007.
6. Coopersmith CM, Stromberg PE, Dunne WM, Davis CG, Amiot DM, Buchman TG, Karl IE, Hotchkiss RS: Inhibition of intestinal epithelial apoptosis and survival in a murine model of pneumonia-induced sepsis
. JAMA 287: 1716–1721, 2002.
7. Coopersmith CM, Stromberg PE, Davis CG, Dunne WM, Amiot DM, Karl IE, Hotchkiss RS, Buchman TG: Sepsis
from Pseudomonas aeruginosa
pneumonia decreases intestinal proliferation and induces gut
epithelial cell cycle arrest. Crit Care Med 1 (6): 1630–1637, 2003.
8. Yu P, Martin CM: Increased gut
permeability and bacterial translocation in Pseudomonas
. Crit Care Med 28 (7): 2573–2577, 2000.
9. Dominguez JA, Vithayathil PJ, Khailova L, Lawrance CP, Samocha AJ, Jung E, Leathersich AM, Dunne WM, Coopersmith CM: Epidermal growth factor improves survival and prevents intestinal injury in a murine model of Pseudomonas aeruginosa
pneumonia. Shock 36 (4): 381–389, 2011.
10. Fry DE: The generic septic response. Crit Care Med 36 (4): 1369–1370, 2008.
11. Tang BM, McLean AS, Dawes IW, Huang SJ, Lin RC: Gene-expression profiling of peripheral blood mononuclear cells in sepsis
. Crit Care Med 37 (3): 882–888, 2009.
12. McConnell KW, McDunn JE, Clark AT, Dunne WM, Dixon DJ, Turnbull IR, Dipasco PJ, Osberghaus WF, Sherman B, Martin JR, et al.: Streptococcus pneumoniae
and Pseudomonas aeruginosa
pneumonia induce distinct host responses. Crit Care Med 38 (1): 223–241, 2010.
13. Sousse LE, Jonkam CC, Traber DL, Hawkins HK, Rehberg SW, Traber LD, Herndon DN, Enkhbaatar P: Pseudomonas aeruginosa
is associated with increased lung cytokines and asymmetric dimethylarginine compared with methicillin-resistant Staphylococcus aureus
. Shock 36 (5): 466–470, 2011.
14. Coopersmith CM, O’Donnell D, Gordon JI: Bcl-2
inhibits ischemia-reperfusion–induced apoptosis in the intestinal epithelium of transgenic mice. Am J Physiol 276 (3 Pt 1): G677–G686, 1999.
15. Robertson CM, Perrone EE, McConnell KW, Dunne WM, Boody B, Brahmbhatt T, Julia DM, Van RN, Hogue LA, Cannon CL, et al.: Neutrophil depletion causes a fatal defect in murine pulmonary Staphylococcus aureus
clearance. J Surg Res 150 (2): 278–285, 2008.
16. Fox AC, Breed ER, Liang Z, Clark AT, Zee-Cheng BR, Chang KC, Dominguez JA, Jung E, Dunne WM, Burd EM, et al.: Prevention of lymphocyte apoptosis in septic mice with cancer increases mortality. J Immunol 187 (4): 1950–1956, 2011.
17. Vyas D, Robertson CM, Stromberg PE, Martin JR, Dunne WM, Houchen CW, Barrett TA, Ayala A, Perl M, Buchman TG, et al.: Epithelial apoptosis in mechanistically distinct methods of injury in the murine small intestine. Histol Histopathol 22 (6): 623–630, 2007.
18. Fox AC, Robertson CM, Belt B, Clark AT, Chang KC, Leathersich AM, Dominguez JA, Perrone EE, Dunne WM, Hotchkiss RS, et al.: Cancer causes increased mortality and is associated with altered apoptosis in murine sepsis
. Crit Care Med 38 (3): 886–893, 2010.
19. Clark JA, Clark AT, Hotchkiss RS, Buchman TG, Coopersmith CM: Epidermal growth factor treatment decreases mortality and is associated with improved gut
integrity in sepsis
. Shock 30 (1): 36–42, 2008.
20. Clark JA, Gan H, Samocha AJ, Fox AC, Buchman TG, Coopersmith CM: Enterocyte-specific epidermal growth factor prevents barrier dysfunction and improves mortality in murine peritonitis. Am J Physiol Gastrointest Liver Physiol 297 (3): G471–G479, 2009.
21. Chang KC, Unsinger J, Davis CG, Schwulst SJ, Muenzer JT, Strasser A, Hotchkiss RS: Multiple triggers of cell death
: death receptor and mitochondrial-mediated apoptosis. FASEB J 21 (3): 708–719, 2007.
22. Hotchkiss RS, Osmon SB, Chang KC, Wagner TH, Coopersmith CM, Karl IE: Accelerated lymphocyte death in sepsis
occurs by both the death receptor and mitochondrial pathways. J Immunol 174 (8): 5110–5118, 2005.
23. Youle RJ, Strasser A: The BCL-2
protein family: opposing activities that mediate cell death
. Nat Rev Mol Cell Biol 9 (1): 47–59, 2008.
24. Stromberg PE, Woolsey CA, Clark AT, Clark JA, Turnbull IR, McConnell KW, Chang KC, Chung CS, Ayala A, Buchman TG, et al.: CD4+
lymphocytes control gut
epithelial apoptosis and mediate survival in sepsis
. FASEB J 23 (6): 1817–1825, 2009.
25. Billen LP, Kokoski CL, Lovell JF, Leber B, Andrews DW: Bcl-XL inhibits membrane permeabilization by competing with Bax
. PLoS Biol 6 (6): e147, 2008.
26. George NM, Evans JJ, Luo X: A three-helix homo-oligomerization domain containing BH3 and BH1 is responsible for the apoptotic activity of Bax
. Genes Dev 1 (15): 1937–1948, 2007.
27. Bilbault P, Lavaux T, Launoy A, Gaub MP, Meyer N, Oudet P, Pottecher T, Jaeger A, Schneider F: Influence of drotrecogin alpha (activated) infusion on the variation of Bax
/Bcl-xl ratios in circulating mononuclear cells: a cohort study in septic shock patients. Crit Care Med 35 (1): 69–75, 2007.
28. Wei MC, Zong WX, Cheng EH, Lindsten T, Panoutsakopoulou V, Ross AJ, Roth KA, MacGregor GR, Thompson CB, Korsmeyer SJ: Proapoptotic BAX
and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 292 (5517): 727–730, 2001.
29. Wesche-Soldato DE, Chung CS, Gregory SH, Salazar-Mather TP, Ayala CA, Ayala A: CD8+
T cells promote inflammation and apoptosis in the liver after sepsis
: role of Fas-FasL. Am J Pathol 171 (1): 87–96, 2007.
30. Perl M, Chung CS, Lomas-Neira J, Rachel TM, Biffl WL, Cioffi WG, Ayala A: Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis
. Am J Pathol 167 (6): 1545–1559, 2005.
31. Strater J, Moller P: CD95 (Fas/APO-1)/CD95L in the gastrointestinal tract: fictions and facts. Virchows Arch 442: 218–225, 2003.
32. Feezor RJ, Oberholzer C, Baker HV, Novick D, Rubinstein M, Moldawer LL, Pribble J, Souza S, Dinarello CA, Ertel W, et al.: Molecular characterization of the acute inflammatory response to infections with gram-negative versus gram-positive bacteria. Infect Immun 71 (10): 5803–5813, 2003.
33. Pishchany G, McCoy AL, Torres VJ, Krause JC, Crowe JE Jr., Fabry ME, Skaar EP: Specificity for human hemoglobin enhances Staphylococcus aureus
infection. Cell Host Microbe 8 (6): 544–550, 2010.
34. Jung E, Perrone EE, Liang Z, Breed ER, Dominguez JA, Clark AT, Fox AC, Dunne WM, Burd EM, Farris AB, et al.: Cecal ligation and puncture followed by MRSA pneumonia increases mortality in mice and blunts production of local and systemic cytokines. Shock 37 (1): 85–94, 2012.
35. Coopersmith CM, Chang KC, Swanson PE, Tinsley KW, Stromberg PE, Buchman TG, Karl IE, Hotchkiss RS: Overexpression of Bcl-2
in the intestinal epithelium improves survival in septic mice. Crit Care Med 30 (1): 195–201, 2002.
Sepsis; gut; cell death; Pseudomonas aeruginosa; Bid; Bax; Bcl-2; Fas Ligand