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(The FASEB Journal. 2004;18:1185-1191.)
© 2004 FASEB

Improved survival in experimental sepsis with an orally administered inhibitor of apoptosis

JOEL G. R. WEAVER*,{dagger}, MARK S. ROUSE{ddagger}, JAMES M. STECKELBERG{ddagger} and ANDREW D. BADLEY{dagger},{ddagger},1

* Division of General Surgery, University of Ottawa, Ottawa, Ontario, Canada; and
{dagger} Translational Immunovirology and Biodefense Research Program,
{ddagger} Division of Infectious Diseases, Mayo Clinic and Foundation, Rochester, Minnesota, USA

1 Correspondence: Division of Infectious Diseases, Translational Program in Immunvirology and Biodefense, Mayo Clinic, 200 First St. NW, Rochester, MN 55905, USA. E-mail: badley.andrew{at}mayo.edu


   ABSTRACT
TOP
 ABSTRACT
BACKGROUND
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The pathophysiology of sepsis involves excessive lymphocyte apoptosis, which correlates with adverse outcomes, and disordered cytokine production, which may promote host injury. As the protease inhibitor (PI) class of antiretroviral agents is known to prevent apoptosis in vitro, we evaluated their effect on survival, lymphocyte apoptosis, and consequent cytokine production in mice with sepsis induced by cecal ligation and perforation. Mice pretreated with PIs have improved survival (67%; P<0.0005) compared with controls (17%) and a significant (P<0.05) reduction in lymphocyte apoptosis. Even mice receiving therapy beginning 4 h after perforation demonstrated improved survival (50%; P<0.05) compared with controls. PI therapy is also associated with an increase in the Th1 cytokine TNF-{alpha} (P<0.05) early in sepsis and a reduction in the Th2 cytokines IL-6 and IL-10 (P<0.05) late in sepsis; despite no intrinsic antibacterial effects, PI also reduced quantitative bacterial blood cultures. The beneficial effects of PI appear to be specific to lymphocyte apoptosis, as lymphocyte-deficient Rag1–/– mice did not experience benefit from treatment with PI. Thus, inhibition of lymphocyte apoptosis by PI is a candidate approach for the treatment of sepsis.—Weaver, J. G. R., Rouse, M. S., Steckelberg, J. M., Badley, A. D. Improved survival in experimental sepsis with an orally administered inhibitor of apoptosis.


Key Words: SIRS • cytokine • lymphocyte • CLP • protease inhibitor


   BACKGROUND
TOP
 ABSTRACT
 BACKGROUND
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
SEPSIS IS A LEADING CAUSE of death in critically ill patients. It develops in 750,000 Americans annually, and more than 210,000 of them die (1) . Despite improved understanding of the underlying pathophysiology of sepsis as well as technical advances in supportive care, mortality from sepsis remains high. In fact, between 1979 and 2000 the number of sepsis-related deaths increased (2) .

Previous theories regarding the pathogenesis of sepsis have implicated proinflammatory cytokines (e.g., TNF-{alpha}) as harmful mediators, and numerous strategies aimed at attenuating these mediators have been explored. However, these interventions have proved largely ineffective, and recent work suggests this may be because exaggerated hyperinflammatory responses occur less frequently than originally thought (3 4 5 6) . Indeed, several theories now implicate the later ensuing hypoinflammatory phase of sepsis as being more important in determining the outcome of sepsis (7 8 9) .

Apoptosis is the programmed death of cells essential for homeostatic cell turnover. However, disordered apoptosis has been linked to the pathogenesis of numerous disease states. Recently, the role of apoptosis in the pathogenesis of sepsis has been explored. Numerous animal studies have shown that sepsis induces extensive lymphocyte (10 11 12 13 14 15 16 17 18) and intestinal epithelial cell (11 , 19 20 21 22) apoptosis. Autopsy studies of patients who have died of sepsis and multiple organ dysfunction syndrome (MODS) also demonstrate enhanced lymphocyte apoptosis in the spleen, intestinal lamina propria, and in lymphoid aggregates throughout the body (11 , 23 , 24) , as well as apoptosis of intestinal epithelial cells (19 , 23) . Moreover, the severity and outcome of sepsis are correlated with plasma concentrations of nucleosomes, products specific to apoptotic cells (25) .

Despite these advances, the significance of apoptosis to the pathogenesis of sepsis remained correlative until it was shown that administration of caspase inhibitors, which prevent apoptosis, significantly improves survival in murine cecal ligation and perforation (CLP) -induced sepsis (26 , 27) . Repeating these experiments in animals lacking functional lymphocytes showed no benefit with caspase inhibition, implicating lymphocyte apoptosis as a critical event in the pathogenesis of sepsis (26 , 27) . However, due to unacceptable toxicities, caspase inhibitors currently are not in clinical use (28) . Other strategies designed to prevent apoptosis, including overexpression of the antiapoptotic protein Bcl-2 (29) and inhibition of Fas/FasL signaling with Fas receptor fusion proteins (30) , have shown similar survival benefits in animal models of sepsis. On the basis of these findings, antiapoptotic strategies have been suggested as a novel approach to treat sepsis (28 , 31 , 32) , although none so far are developed sufficiently to enter widespread clinical use.

It has been well documented that the HIV protease inhibitor (PI) class of antiretrovirals are associated with a reduction in HIV-associated CD4 T cell apoptosis in vivo (33 34 35) and are directly antiapoptotic in vitro against a variety of stimuli (36) . However, the ability of PIs to modulate apoptosis in nonviral apoptotic disease states has not been examined. Here, we investigate the effect of PI therapy on survival, apoptosis, microbiology, and plasma cytokine profile in a murine CLP model of sepsis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 BACKGROUND
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cecal ligation and perforation
Female ND4 (Harlan, Indianapolis, IN, USA) and Rag1–/– along with their appropriate C57BL/6 controls (Jackson Laboratory, Bar Harbor, ME, USA), 6–8 wk, 15–20 g, were housed in a germ-free environment and given access to standard laboratory chow and water. The murine CLP model that reproduces many of the clinical features of sepsis in patients was used to induce intra-abdominal sepsis (37) . This is a clinically relevant model of sepsis that has been validated in many laboratories (38 , 39) . Briefly, after externalization of the cecum, two perforations were made on the antimesenteric border of the cecum with a 19 gauge needle. Postoperatively, mice were resuscitated with 1 cc of normal saline subcutaneously and given access to standard laboratory chow and acetaminophen-supplemented water. Animals were killed if they displayed any of the following characteristics: moribund, lateral recumbency, and/or hypothermia (rectal temperature<32°C). All surviving animals were killed at 48 h.

All animal treatments were reviewed and approved by the Mayo Foundation Institutional Animal Care and Use Committee.

PI treatments
Beginning at the times indicated, animals were treated every 8 h by oral gavage with a PI mixture consisting of 125 mg/kg nelfinavir (Agouron Pharmaceuticals, La Jolla, CA, USA) and 13 mg/kg ritonavir (Abbott Pharmaceuticals, Abbott Park, IL, USA) in 2% ethanol in distilled water or vehicle control (2% ethanol). Pharmacokinetic studies in mice demonstrated that with this dosing regimen, nelfinavir plasma levels were similar to those achieved in humans receiving a standard dose of nelfinavir (data not shown). To achieve these levels, it was necessary to co-dose nelfinavir with a small dose of ritonavir (another PI), which inhibits CYP3A-mediated nelfinavir metabolism, thus increasing nelfinavir plasma levels.

Immunohistochemistry
At the time of euthanasia, the heart, lungs, thymus, liver, spleen, kidneys, and ileum were harvested and placed in 10% neutral buffered formalin (Sigma, St. Louis, MO, USA). A random selection of tissues from five animals per treatment group (pretreated and vehicle control) was made. These tissues were embedded in paraffin and terminal deoxynucleotide UTP transferase (TUNEL) staining was performed (Molecular Histology, Little Rock, AK, USA). The number of TUNEL-positive nuclei were enumerated for two random high power fields (hpf) per tissue section by three reviewers blinded as to sample origin. Averaging the number of TUNEL-positive nuclei from each field for all three reviewers resulted in an overall TUNEL positivity score for the tissue.

Microbiology
To evaluate for any antibacterial effects, we performed standard minimal inhibitory concentration (MIC) analyses of nelfinavir against Pseudomonas aeruginosa, Enterococcus, Escherichia coli, and Streptococcus bovis. Five different clinical isolates of each organism were tested according to the National Committee for Clinical Laboratory Standards (NCCLS) guidelines (40) . Organisms were subcultured twice from freezer stocks, then incubated in Tryptic Soy Broth to logarithmic growth. These cultures were diluted to 105 colony forming units (cfu)/mL and tested. Nelfinavir was dissolved in DMSO to 1000 µM and further diluted in Mueller Hinton Broth.

Serum bactericidal titers (SBT) were determined as per NCCLS guidelines (40) . Briefly, mice were treated with PI or vehicle control every 8 h. Two hours after the fourth dose, which corresponds to peak plasma drug levels (data not shown), animals were anesthetized with pentobarbital (60 mg/kg; Abbott Labs, Chicago, IL, USA) and exsanguinated. Whole blood was collected in glass tubes and allowed to clot at room temperature for 2 h before being placed at 4°C overnight. The clot was then removed and the sample spun (4000 rpm, 10 min, 4°C). The serum was removed and stored at –80°C. The same bacterial isolates used for MIC analyses were used to determine SBT. Briefly, 5 x 105 of each isolate was added to serial dilutions of serum from PI and vehicle control-treated animals, incubated overnight, and read for inhibition the next day.

For quantitative blood cultures, blood was obtained via cardiac puncture from pretreated and vehicle control animals 12 h postperforation; 0.1 mL of blood was diluted in 0.9 mL of sterile saline, plated in 10-fold serial dilutions on blood agar plates (Becton Dickinson, Cockeysville, MD, USA), and aerobically incubated at 37°C. Plates with between 10 and 100 colonies were used to quantify cfu/mL of blood.

Plasma cytokines
Blood was obtained via heparinized ventricular puncture at 6 or 12 h postperforation from animals in the pretreated and vehicle control groups. After centrifugation (14,000 rpm, 5 min, 4°C), the plasma was removed and stored at –80°C. Using standard ELISA procedures as outlined by the manufacturer (Bio-Rad, Hercules, CA, USA), determination of the plasma levels of TNF-{alpha}, IL-6, and IL-10 was performed. The lower limit of quantification for each cytokine was 6.9 pg/mL.

Statistical analyses
Survival analyses were performed by Kaplan-Meier analysis. Blood counts, cytokines, and TUNEL-positive cells were analyzed with Student’s t test.


   RESULTS
TOP
 ABSTRACT
 BACKGROUND
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PI therapy is associated with improved survival in sepsis
To evaluate the effect of PI therapy on survival in sepsis, animals were gavaged with PI cocktail (nelfinavir and ritonavir) beginning either 24 h before (n=33) or 4 h after (n=28) cecal perforation. Control animals (n=30) received vehicle control alone beginning 24 h prior to perforation. Sham-operated animals (n=10) underwent laparotomy and cecal manipulation, but no ligation or perforation was performed. Survival at 48 h was 17% in the control group, 67% (P<0.0005) in the pretreated group, and 50% (P<0.05, compared with control) when treatment began 4 h after perforation (Fig. 1 ). Sham-operated animals experienced no deaths (data not shown).



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  		Figure 1. Effect of PI treatment on survival in CLP-induced sepsis. Mice undergoing CLP were treated with PI or control for 24 h before cecal perforation or PI therapy beginning 4 h after CLP. Survival was monitored for 48 h.

PI therapy attenuates lymphocyte apoptosis in the spleen and thymus
To investigate whether the observed survival advantage conferred by PI was associated with a reduction in apoptosis, TUNEL staining of all major organs was performed. No appreciable apoptosis was detected in the heart, lungs, liver, ileum, or kidneys (data not shown). Analysis of control animals (n=5) demonstrated an average of 48 ± 6 and 61 ± 6 TUNEL-positive nuclei per hpf in the spleen and thymus, respectively. In contrast, those animals pretreated with PI (n=5) displayed a reduced average splenic and thymic TUNEL positivity of 8±2 (P<0.05) and 15±4 (P<0.05) TUNEL-positive nuclei per hpf, respectively (Fig. 2 ).



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  		Figure 2. Effect of PI treatment on lymphocyte apoptosis. Representative photomicrographs of TUNEL stained splenic and thymic sections from vehicle control and PI pretreated animals. TUNEL-positive (apoptotic) nuclei appear in red (arrows).

PIs do not directly affect bacterial growth
One possible explanation for the improved survival observed with PI treatment is that PI may possess unanticipated antibacterial properties. We therefore used NCCLS Guidelines to formally test whether nelfinavir had intrinsic antibacterial effects against P. aeruginosa, Enterococcus, E. coli, and S. bovis. In all cases, the MIC for nelfinavir was greater than 10 µM. In our pharmacokinetic studies of mice (data not shown) and in humans receiving nelfinavir, Cmax is ~8 µM (41) . Thus, even at concentrations greater than those observed clinically, nelfinavir does not appear to inhibit growth of any of the isolates analyzed.

Despite MIC data indicating that at supraphysiologic concentrations nelfinavir does not directly inhibit bacterial growth, it is possible that an active metabolite of nelfinavir or ritonavir may affect bacterial growth in vivo. To further investigate this, SBT were determined. Serum from PI (n=12) and vehicle control (n=12) -treated animals displayed no ability to inhibit growth of those bacterial isolates tested, suggesting that serum from PI-treated animals does not directly affect bacterial growth.

With the knowledge that serum from animals treated with PI possess no antibacterial properties, we next looked at the effect of PI therapy on bacterial blood counts. Twelve hours after perforation, control animals (n=10) had a mean bacterial blood count of log10 5.8/mL whereas pretreated animals (n=10) had an average of log10 1.6/mL (P<0.05) (Fig. 3 ).



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  		Figure 3. Effect of PI therapy on aerobic blood cultures. Blood was harvested 12 h after CLP from vehicle control and PI pretreated animals and cultured under aerobic conditions.

Protease inhibitor therapy affects the biphasic plasma cytokine profile in sepsis
Cytokine profiles in sepsis are biphasic, with a proinflammatory/Th1 predominance early in sepsis and an anti-inflammatory/Th2 profile late in sepsis. To examine the effect of PI treatment on the plasma cytokine profile, the prototypic Th1 cytokine TNF-{alpha} and Th2 cytokines IL-6 and IL-10 were measured in groups of 10 animals at each time point in both treatment groups. At 6 h, PI treatment was associated with an elevation of TNF-{alpha} from 54 pg/mL in control treated animals to 212 pg/mL (P<0.05) (Fig. 4 A). There was no significant effect on IL-6 or Il-10 levels at 6 h (Fig. 4B ). However, by 12 h after perforation, both IL-6 and IL-10 plasma levels in PI pretreated animals were significantly lower (1139 pg/mL and 1496 pg/mL, respectively; P<0.05) when compared with IL-6 and IL-10 levels in vehicle control-treated animals (4809 pg/mL and 4815 pg/mL, respectively) (Fig. 4D ). TNF-{alpha} plasma levels at 12 h were not significantly altered (Fig. 4C ). Based on this, PI appear to augment TNF-{alpha} levels 6 h after CLP yet attenuate IL-6 and IL-10 levels at 12 h.



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  		Figure 4. Effect of PI therapy on plasma TNF-{alpha} (A, C) and IL-6 and Il-10 (B, D) levels 6 and 12 h after CLP in vehicle control and PI pretreated animals.

Protease inhibitor therapy does not alter survival in Rag1–/– mice
To examine the significance of PI-mediated inhibition of lymphocyte apoptosis, we repeated these studies using mice lacking functional lymphocytes. Rag1–/– mice on a C57BL/6 background were pretreated with either PI (n=12) or vehicle control (n=12) for 24 h prior to perforation. Forty-eight hours after CLP there was no difference in survival between the PI and vehicle control-treated Rag1–/– mice (25% vs. 17%, respectively, P=0.25) (Fig. 5 ). Repeating these experiments with wild-type C57BL/6, survival results similar to those with ND4 mice were obtained (data not shown).



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  		Figure 5. Effect of PI therapy on 48 h survival in Rag1–/– mice receiving either vehicle control or PI treatment before CLP.


   DISCUSSION
TOP
 ABSTRACT
 BACKGROUND
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Theories regarding the pathogenesis of sepsis have long held that the harmful event is the body’s uncontrolled hyperinflammatory response directed against the invading microbes. These theories are based on animal models of sepsis using large doses of LPS and/or endotoxin, which, in retrospect, poorly mimic the actual conditions of sepsis (42 43 44) . Indeed, human studies have revealed that the frequency of an exaggerated systemic inflammatory response is lower than originally thought (3 4 5 6) . In contrast, more recent work has suggested that the inflammatory cytokines, once thought to be harmful, may in fact be beneficial. This is suggested by animal models of peritonitis (45 , 46) and in trials of humans with sepsis (47) , which found that anti-TNF-{alpha} antibodies either had no effect or actually worsened sepsis-related survival.

Current thinking is that sepsis is characterized first by a hyperimmune, hyperinflammatory state with a Th1 predominant cytokine profile (e.g., IFN{gamma}, IL-2, TNF-{alpha}), followed by a hypoimmune, hypoinflammatory state characterized by a Th2 cytokine profile (e.g., IL-4, 6, 10). Indeed, this model is supported by observations in septic patients including loss of delayed type hypersensitivity (7) , an inability to clear infection (8) , and a predisposition to nosocomial infections (9) . Potential mechanisms of immune suppression in septic patients include a shift from a Th1 to a Th2 response (48) , anergy (49 , 50) , loss of macrophage expression of major histocompatability complex class II and costimulatory molecules (51) , immunosuppressive effects of apoptosis (52 53 54) , and apoptosis of CD4 T cells, B cells, and dendritic cells leading to decreased antibody production, macrophage activation, and antigen presentation, respectively (23 , 55 , 56) . In our current study, we demonstrate that reduced T cell apoptosis is associated with improved outcomes, improved control of bacterial replication, and enhanced proinflammatory cytokine production early in the septic response. However, due to the pervasive role of CD4 T cells in orchestrating a coordinated adaptive immune response, we predict that inhibition of lymphocyte apoptosis would improve other immune parameters as well.

For example, it has been described how a cell dies can affect the immunologic function of surviving immune cells (52 53 54) . Uptake of apoptotic cellular debris by phagocytic cells stimulates immune tolerance by the release of anti-inflammatory cytokines, including IL-6 and IL-10 (52 , 53 , 57 58 59) . In addition, uptake of apoptotic cells by macrophages and dendritic cells impairs expression of costimulatory molecules (60 , 61) . Thus, T cells that come into contact with antigen-presenting cells that have ingested apoptotic cells are inadequately stimulated and become anergic, hyporesponsive, or undergo apoptosis themselves (52) . Such apoptosis-induced immune suppression is highlighted by the adoptive transfer of apoptotic splenocytes into septic mice, which worsens survival compared with transfer of necrotic splenocytes (62) . In our system, reduced T cell apoptosis associated with PI administration was associated with reduced IL-6 and IL-10 production late in the septic response. A potential mechanism by which this may have occurred is through reduced formation and subsequent phagocytic uptake of apoptotic bodies, resulting in decreased levels of Th2 cytokines, and therefore less immune suppression.

An additional potential benefit of apoptosis inhibition in sepsis may be related to the enhanced bowel epithelial apoptosis seen both in animal models and studies of humans with prolonged sepsis (24 , 63 64 65) . The effect of gut epithelial apoptosis in sepsis is unknown, but it may reduce the barrier function of the gut and promote translocation of intestinal bacteria, which in turn may exacerbate the septic response. Inhibition of sepsis-induced epithelial apoptosis would predictably reduce the bacterial burden. Finally, sepsis-induced tissue hypoperfusion is common in sepsis and results clinically in impaired end organ function, including oligemia and acute tubular necrosis (66) , mental status changes due to neuronal ischemia (67 , 68) , and reduced cardiac output (69) amongst others. Emerging data suggest that, at the cellular level, tissue hypoperfusion results in anoxic apoptosis (12 , 70) —for example, of renal tubular epithelial cells (71) . Therefore, apoptosis inhibition may theoretically improve these hypoperfusion-related complications of sepsis as well, including MODS.

Indeed, much of the cumulative data regarding sepsis may be reconciled in a model whereby the initial sepsis event (e.g., bacteremia) promotes lymphocyte activation and production of Th1 cytokines (72) , including IL-2 and IFN{gamma}, which are beneficial to the host (73 , 74) . Subsequently, enhanced lymphocyte activation and proliferation leads to enhanced lymphocyte apoptosis, immunosuppression, anergy, loss of Th1 cytokine production, and a shift toward a Th2 profile. Indeed, in septic patients, each of these events is seen: lymphocytosis followed by lymphopenia, a shift in plasma cytokines from Th1 to Th2 predominance (48) , loss of delayed hypersensitivity responses late in sepsis (7 , 8) , and high numbers of apoptotic lymphocytes both in animal models (10 11 12 13 14 15 16 17 18) and human autopsy studies (23 , 24) . Here, we show that preventing apoptosis has the functional consequences of reducing blood bacterial counts, increasing TNF-{alpha} early in sepsis, attenuating Th2 cytokine profile in late sepsis, and improving survival in murine CLP-induced sepsis. These effects are not due to direct antibacterial properties of PI, and the survival benefit is not seen in animals lacking lymphocytes. Thus, lymphocyte apoptosis in sepsis may both directly and indirectly affect host outcome and therefore be an appropriate target for therapeutic intervention.


   ACKNOWLEDGMENTS
 
A.D.B. is funded by the National Institutes of Health (R01 AI40384-06 and R21 AI54187-01A1). The authors would like to Teresa Hoff for her administrative expertise.

Received for publication November 24, 2003. Accepted for publication April 21, 2004.


   REFERENCES
TOP
 ABSTRACT
 BACKGROUND
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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