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Department of Microbiology, Biochemistry and Immunology, Morehouse School of Medicine, Atlanta, Georgia 30310-1495
Submitted 21 October 2002 ; accepted in final form 4 March 2003
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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antiorthostatic; stress; spaceflight
Ground-based models, which simulate some of the spaceflight conditions, have provided critical data for the design of spaceflight experiments (23). Among those models, the hindlimb unloading rodent model has been widely accepted and used by the scientific community (14, 21–23, 25, 32). In the model, rodents are suspended by the tail with no load bearing on the hindlimbs and with a head-down tilt of 15–20° (14, 21–23, 25, 32). These conditions induce muscle and bone loss and a fluid shift to the head, which are similar to changes observed during spaceflight (14, 21–23, 25).
Pseudomonas aeruginosa bacteria are gram-negative aerobic rods that are widespread in soil and water (37). Infection by P. aeruginosa is not common in healthy people; however, under certain circumstances, particularly in weakened hosts, this organism can infect the urinary tract, burns, and wounds, and it also can cause septicemia and meningitis (37, 38). It can produce opportunistic respiratory infections in people compromised by immune deficiencies or by chronic pulmonary disease, including cystic fibrosis (37). Most importantly, this organism has shown to cause problems during spaceflight (33–36). In fact, P. aeruginosa was isolated from one astronaut who developed a urinary tract infection during the Apollo 13 mission (33–36). As plans for longer term missions in space develop, the possibility of problems related to infectious agents takes on increasing importance.
The present study was carried out to determine the effects of exposure of mice to hindlimb unloading on resistance to infection with P. aeruginosa and possible mechanisms that could be involved in spaceflight conditions resulting in compromised resistance to infection.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Animals. Specific pathogen-free female Swiss Webster mice, 9–11 wk old and each weighing 21–25 g, were purchased from Harlan Sprague Dawley Laboratories (Indianapolis, IN). Animals were housed in a quiet, isolated room with controlled temperature and light cycle, and they had access to food and water ad libitum. Experimental procedures commenced after a 1-wk acclimation. All experimental manipulations were approved by the Atlanta University Center Institutional Animal Care and Use Committee and were carried out under the supervision of a veterinarian.
Experimental groups. Animals were randomly assigned to three groups (n = 8–10/group): 1) hindlimb-unloaded mice were suspended by the tail at 15–20° head-down tilt with no load-bearing on hindlimbs (4); 2) restraint control mice were restrained by the tail parallel to the ground, and all limbs were allowed to touch the floor of the cage; and 3) normally housed control mice were housed individually in standard cages. Animals in all groups were infected 2 days after the initiation of treatment. Additional uninfected control mice were maintained in individual cages. Experiments were repeated at least twice under the same experimental conditions.
Inoculum preparation. A standard loop containing bacteria from a previously frozen stock culture of P. aeruginosa was inoculated into 5 ml of TSB for 5 h at 37°C. One loopful of the suspension was inoculated on tryptic soy agar (TSA) plates for colony isolation. After 24 h of incubation at 37°C, one single colony was transferred to fresh TSB medium and incubated at 37°C, and standard growth curve fits were prepared by plotting absorbance readings at 595 nm vs. the corresponding bacterial counts at different time points. Counts were expressed as colony-forming units (CFU) per milliliter.
For the LD50 determination, bacteria were grown to midlog phase in TSB for 4 h at 37°C with gentle shaking. Cells were harvested and washed twice with PBS by spinning at 3,000 g for 10 min. Cell pellets were subsequently resuspended in 10 ml of PBS and serially diluted to the desired concentrations. Mice were subcutaneously inoculated with 500 µl of PBS containing doses ranging from 1 x 109 to 1 x 104 CFU/ml, and the LD50 was determined to be 1.5 ± 0.18 x 108 CFU/mouse by using the Reed-Muench estimation (27). Concentrations were confirmed by plating three consecutive 10-fold dilutions of the suspension on TSA solid medium.
Experimental infection with bacteria. Infections were begun 48 h after hindlimb-unloading treatment. For each infection, a total inoculum of 500 µl · mouse-1 · injection-1 containing the previously determined LD50 dose was administered via the subcutaneous route. To ensure that the actual LD50 dose of bacteria was injected into each mouse, a sample of the P. aeruginosa suspension was serially diluted and plated on TSA solid medium and incubated at 37°C. The CFU/ml counts were obtained after 24 h. Animal survival was assessed four times a day for 15 days.
Bacterial organ load studies. Mice that survived infection were euthanized by cervical dislocation. Blood and organs (spleen, lungs, liver, brain, and kidneys) were aseptically removed. Twenty microliters of citrated blood were serially diluted and plated in TSA for CFU counting, and the remaining volume was kept at 4°C for 24 h. Plasma was obtained after centrifugation at 3,000 g for 10 min and stored at -20°C until use. Organs were placed in 5 ml of sterile PBS and homogenized with an ultrasonic cell disruptor (Heat Systems, Farmingdale, NY). One hundred microliters of the suspension were plated on TSA (three 1:10 serial dilutions) and incubated for 24 h at 37°C. The number of CFU was determined by averaging the number of colonies counted on the three serially diluted TSA plates.
Kinetics of bacterial growth in organs. Normally housed control and hindlimb-unloaded mice (n = 16/group) were inoculated subcutaneously with a lower dose of bacteria (3.25 x 107 CFU/mouse; a dose equivalent to a LD20) to determine the kinetics of bacterial growth in organs and to decrease the rate of mortality. At least three mice from each group were killed at different time points after infection-.Blood and organs were collected as described previously. Corticosterone and antibody levels were measured in plasma obtained from these groups at different time points. Group of noninfected mice were euthanized at the same time points. For corticosterone and antibodies analyses, the values obtained in this group were subtracted from the values obtained from the infected mice. Restrained control group was excluded because in previous and this experiments we did not see any differences compared with normally housed control mice (3).
P. aeruginosa antigen preparation. An isolated colony of P. aeruginosa was grown in 250 ml of TSB medium for 4 h at 37°C with gently shaking. Bacterial cells were washed two times in PBS at 3,000 g for 10 min and resuspended in 10 ml of distilled water. The bacterial suspension was sonicated with 10 repeated 30-s pulses at high intensity by using an ultrasonic cell disruptor (Heat Systems). Cellular debris and unlysed cells were removed by centrifugation at 3,000 g for 40 min at 4°C. The supernatant containing the antigen was filtered (0.22-µm filter, Sigma Chemical, St. Louis, MO) and aliquoted at -80°C until use. One aliquot was removed for protein determination with the use of a standard bicinchoninic acid assay (Pierce, Rockford, IL).
ELISA for detection of IgG and IgM antibodies to P. aeruginosa. Specific IgG and IgM anti-P. aeruginosa antibodies were detected in plasma collected from mice that survived by using an ELISA as previously described with some modifications (1, 38). Briefly, 96-well Nunc-Immuno MaxiSorp surface microtiter plates (BioWorld Laboratory Essentials, Dublin, OH) were coated with 100 µl containing 5 µg/ml of P. aeruginosa antigen in coating buffer (0.1 M carbonate-bicarbonate, pH 9.6). Plates were kept overnight at room temperature. After the plates were washed three times (PBS, pH 7.2, and 0.05% Tween 20), nonspecific sites were blocked with 300 µl of blocking buffer (1% BSA, 5% sucrose in PBS, pH 7.2, and 0.05% NaN3) for 1 h at 37°C. Plasma samples were diluted at 1:200 vol/vol for IgG and 1:100 vol/vol for IgM detection, and 100 µl of this dilution were plated and incubated at 37°C for 2 h. Secondary antibodies diluted in 1% BSA in PBS (pH 7.2) (reagent diluent) and conjugated to horseradish peroxidase were used; 100 µl of a 1:20,000 vol/vol dilution of goat anti-mouse IgM (Sigma Chemical) and 100 µl of a 1:40,000 vol/vol dilution of rabbit anti-mouse IgG (Sigma Chemical) were plated and incubated at 37°C for 2 h. Reactions were detected by using 100 µl of mixture preparation of the tetramethylbenzidine kit (R&D Systems, Minneapolis, MN). Plates were developed at room temperature for 20 min, and the reaction was stopped with 50 µl of 2 N H2SO4. Optical density was determined by using a Spectramax 250 microplate spectrophotometer system (Molecular Devices, Sunnyvale, CA) set to 450 nm.
Corticosterone measurement. Corticosterone levels were tested by using a competitive enzyme immunoassay and following the manufacturer instructions (Alpco Diagnostics, Windham, NH).
Statistical analysis. At least two separate experiments for each determination were performed in this study. Data were analyzed by using Statview 5.0.1 with 
set a priori at P < 0.05. Results were expressed as percentage of survival at each time point as determined by the Kaplan-Meier method. Differences in survival between the groups were compared by using the Mantel-Cox log-rank test. Student's t-test was used to test statistical significance between any two groups and ANOVA for differences between more than two groups. For organ load studies, the Kruskal-Wallis nonparametric test was used to decrease the effects of outliers.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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6% was observed in the hindlimb-unloaded mice. Survival rates in the range of 32 and 45% were observed in restrained and normally caged controls, respectively. There was no statistical difference between both control groups.
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Mean time to death was decreased in hindlimb-unloaded mice. The hindlimb-unloaded group showed a significant decrease (P < 0.05) in the mean time to death after infection (4.68 ± 0.8 days), compared with restrained mice (9.75 ± 1.34 days) and normally housed control mice (7.75 ± 1.47 days). Similarly, the cumulative mean time to death in both control groups combined was significantly increased (8.86 ± 0.99 days) compared with the hindlimb-unloaded mice (4.68 ± 0.8 days) (P < 0.05). No statistical differences were found in the mean time to death between restrained and normally caged control mice.
Survivors of LD50 infection with P. aeruginosa cleared bacteria from the organs. Mice that survived the LD50 infection were subjected to euthanasia by cervical dislocation at the end of the experimental period. Quantitative culture of bacteria from lungs, spleen, liver, kidneys, and blood was performed 15 days after infection. However, no bacteria were detected in any tissue, including blood.
Antibodies against P. aeruginosa were detected in mice that survived 1 LD50 dose. Anti-P. aeruginosa IgG and IgM antibodies were detected in plasma samples collected from mice that survived infection in the different groups. All mice, except one in the control normally housed group, had anti-P. aeruginosa antibodies. Hindlimb-unloaded mice had a trend toward decreased production of IgG and IgM compared with restrained and normally housed control mice. However, no statistical analyses could be attempted because there was only one survivor in the hindlimb-unloaded group in the two experiments performed separately.
Elevated and persistent bacterial organ load was detected in the hindlimb-unloaded group infected with 1 LD20 dose of bacteria. To decrease the rate of mortality and to enable measurement of organ load in infected surviving mice, the dose of infection was reduced from 1.5 x 108 CFU/mouse (LD50) to 3.25 x 107 CFU/mouse (LD20). No differences between restrained and normally housed control were found in this and in all previous studies (2); therefore, restrained group was excluded in the kinetic experiments.
Figure 2 shows the bacterial load present in the organs, and Fig. 3 shows the percentage of infected organs. After subcutaneous infection, only 30% of the organs isolated from control group harbored bacteria at day 1 (Figs. 2, and 3, B–D). However, bacteria were cleared completely after this time point. The exception was liver (Figs. 2 and 3A), in which bacteria persisted until day 3. In contrast, between 60 and 100% of organs were infected in the hindlimb-unloading group at day 3, and the infection persisted until day 6 (Figs. 2 and 3, A–E). On the critical days, when most of mortality occurs (between days 3 and 6), only 6.6% (2 of 30) of organs isolated from control mice showed bacterial load compared with 53% (16 of 30) in the hindlimb-unloaded group (Kruskal-Wallis test, P
< 0.0005; Table 1).
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Earlier production of anti-P. aeruginosa IgG antibodies was detected in control mice infected with 1 LD20. Table 2 shows IgG and IgM antibody levels measured in normally housed control and hindlimb-unloaded mice. IgG antibody levels were detected in 30% (1 of 3) of control mice after 6 h and 1 day and in 100% (3 of 3) after 6 and 10 days of subcutaneous infection. In contrast, IgG antibodies were detected in the hindlimb-unloaded group only after 10 days. Statistical analyses were performed only at days 6 and 10 because of the number of mice with circulating antibodies. Significantly increased levels of IgG were detected in normally housed controls at day 6 compared with the hindlimb-unloaded group at the same time point (P < 0.05). There were no statistical differences in IgM production between both groups at any time point.
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Corticosterone levels were greatly increased in hindlimb-unloaded mice at days 3 and 6. To obtain values above normal levels, corticosterone was measured in groups of noninfected mice at each time point and subtracted from values measured in experimental groups. Relatively low levels of corticosterone were detected in both groups 6 h and 1 day after infection. However, corticosterone levels returned to normal at day 3 in control mice. In contrast, levels of corticosterone increased significantly above normal values in 50% of the hindlimb-unloaded group at the critical time point, days 3 and 6 (Fig. 4).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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The present study demonstrated that hindlimb-unloading suspension increased the susceptibility of mice to subcutaneous P. aeruginosa infection compared with both the restrained and normally housed controls. P. aeruginosa was the bacterium isolated from a urinary tract infection in a crew member during the Apollo 13 mission (33–36). In the present study, we have demonstrated that hindlimb unloading can compromise resistance to a known pathogen that has caused infectious problems during spaceflight (33–36). This finding helps to validate the hindlimb-unloading model for studies of the effects of spaceflight conditions on the immune system and resistance to infection.
The mechanisms involved in the modulation of infection after hindlimb unloading procedure remain unclear. Several factors, such as stress-induced alterations of the immune system and the unloading and fluid shift found during the model, could have contributed to the increased mortality seen in the hindlimb-unloaded group.
It is evident that mice in the hindlimb-unloaded group were not able to control P. aeruginosa dissemination. It is also clear that in our model of subcutaneous infection, day 3 through day 6 was a critical time period when events occurred that influenced survival. Using the LD50 dose, the majority of deaths occurred between 3 and 6 days after infection. This was consistent with the results obtained using a lower dose (LD20) of infection where the peaks of both bacterial load and corticosterone levels were detected at these time points. In those experiments, mice in the control group had cleared bacteria from most organs by day 3, which is in contrast with mice in the hindlimb-unloaded group that had massive infiltration of bacteria in most organs at this day and continued dissemination until day 6. In addition, unlike the control group whose levels of corticosterone returned to the normal range on day 3, high levels of corticosterone were detected in the hindlimb-unloaded group at the critical time points. When mice received the LD50 dose of bacteria, the role of antibody was difficult to determine because only one mouse in the hindlimb-unloaded group survived. In an attempt to eliminate this limitation, we decreased the rate of mortality by using a lower dose of bacteria (i.e., a LD20). Results showed delayed production of IgG in hindlimb-unloaded mice. IgG antibody levels were detected in 100% of control mice at day 6 compared with 0% in the hindlimb-unloaded group at this time point. IgG antibodies in the hindlimb-unloaded group were not detected until day 10. The biological significance of the differences seen at day 6 remains unknown. Because of the early bacterial dissemination and peak of mortality (3–6 days), it seems very unlikely that humoral immunity play a critical role for survival in this model. It is possible that other parameters, such as the development of memory cells, could be affected. In previous studies, when an attempt was made to immunize hindlimb-unloaded mice against Listeria monocytogenes, immunologic memory was not able to be induced (20). In this and previous studies, hindlimb-unloaded mice were much less able to clear P. aeruginosa and K. pneumoniae infection than were controls (3). Therefore, the present results support a possible compromise of innate immunity in resistance to bacterial infection as a result of hindlimb unloading. It appears that innate immunity plays a key role in this model in the avoidance of dissemination of infection. Only 6.6% of organs showed bacterial infection in the control group compared with 53% in the hindlimb-unloaded group, suggesting that skin innate immunity is very effective controlling the dissemination of bacteria from the skin to the system. The mechanisms by which bacteria were detected earlier in control group is unclear. However, several studies have shown that acute stress can induce significant enhancement of skin cell-mediated immune response (6, 10). It has been reported that stress hormones enhance skin immunity by increasing leukocyte trafficking and cytokine gene expression at the site of pathogen entry (8). Similar to what happen in response to any stressful situation, stress hormones may prepare the immune system for challenges imposed by a stressor. It is probable that this enhancement of the immune system be transitory. This could explain why we did not find bacteria in the system in the hindlimb-unloaded group until day 3 and why it persisted until day 6, in contrast to control group in which bacteria was detected at day 1 but were promptly cleared from the system. This idea is supported by studies in which immune function was enhanced transiently by acute stress but suppressed after certain period of time or in chronic stage (1, 2, 7, 9).
Neuroendocrine hormones have been shown to have a direct effect on bacterial growth and expression of virulence factors (16, 17, 24). It has been reported that catecholamine levels are increased under spaceflight conditions (5). It is possible that hindlimb unloading could have effects not only on the immune system but also on the neuroendocrine system that resulted in enhanced pathogenesis of bacteria. The spaceflight environment has also been shown to directly influence the virulence of bacteria through yet undiscovered mechanisms (26). These effects could have contributed to the results observed in the present study, but proof of this possibility will require additional experimentation in the future.
As plans for long-term missions and flight opportunities continue to develop, there will be a requirement for further studies focusing on resistance to infection under spaceflight conditions to ensure the safety of potential space travelers. Studies should include identification of the mechanisms involved and development of countermeasures to prevent or ameliorate any compromised resistance to infection that is observed under these conditions.
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ACKNOWLEDGMENTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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, Hindlimb unloaded; 
, restrained; 
control, normally housed.
, Control; 
