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The Journals of Gerontology Series A: Biological Sciences and Medical Sciences 60:688-694 (2005)
© 2005 The Gerontological Society of America

Caloric Restriction Decreases Survival of Aged Mice in Response to Primary Influenza Infection

Elizabeth M. Gardner

Department of Bioscience and Biotechnology, Drexel University, Philadelphia, Pennsylvania.

Address correspondence to Elizabeth M. Gardner, Drexel University, Department of Bioscience and Biotechnology, 3141 Chestnut Street, Philadelphia, PA 19104. E-mail: eg25{at}drexel.edu


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
Caloric restriction (CR) extends life span of healthy rodents compared to those fed ad libitum. Previous studies have shown positive effects of CR on the immune response of aged mice after influenza immunization. To extend these studies, a mouse model of CR was used to determine if CR could modulate primary responses of aged mice to influenza. Although CR delayed the age-related decrease in mitogen-induced lymphoproliferation of aged mice, in stark contrast, CR decreased survival, increased virus titers, and reduced natural killer cell activity in lungs of aged mice after primary influenza infection. Thus, CR has differential effects on immunity of aged mice, as general indices of immune response are maintained, but primary responses to influenza infection are impaired. This suggests that, although CR may positively affect many long-term parameters of aging, increased susceptibility after primary exposure of aged mice to virus, such as influenza, may not be correctable by CR.

NUMEROUS studies in both humans and in animal models have clearly shown that immune function declines with advancing age, as evidenced by thymic atrophy, decreased T-cell proliferative responses to both mitogenic and antigenic stimulation, as well as reduced antibody production and altered cytokine production in response to specific antigens [for review, see (1–4)]. It is believed that this overall dysregulation in immune function is related to the increased incidence in cancers, autoimmunity, and infectious diseases prevalent in elderly persons.

Influenza is a major cause of morbidity and mortality in the general population, but is a serious concern for the elderly population, as influenza and its secondary complications represent the fourth leading cause of death in persons over the age of 65 in the United States (5,6). Thus, a major focus of research in our laboratory has been to use a mouse model to characterize the age-related decline in the immune response to influenza infection. Studies in mice have shown reduced antibody titers, impaired cytotoxic T cell (CTL) responses, and increased virus burden after infection with influenza virus (7–11). We have extended these findings and have shown that aged C57BL/6 (B6) mice demonstrated reduced and delayed expansion of influenza-specific CD8+ T cells in lung during primary infection that was paralleled by a decrease and delay in maximal CTL activity, a delay in lung virus clearance, and impaired interferon-{gamma} (IFN-{gamma}) production (12). These data indicate that a decrease in both the number and function of influenza-specific CD8+ T cells during primary influenza infection contribute to the age-related decline in the immune response to influenza.

A second focus of our research is to develop methods that may postpone, or even abrogate, these age-related changes in the immune response to influenza, with the ultimate goal of reduction of influenza-related morbidity and mortality. It is well established that dietary caloric restriction (CR; 40% reduction in kilocalories), without malnutrition, extends both median and maximal life span in healthy rodents, compared to those fed ad libitum (AL), presumably by reducing age-related accumulation of oxidative damage (13–16). A further benefit of CR has been to induce positive effects on several specific physiologic and metabolic systems, including the immune system (13–16). Aged rodents fed CR diets exhibit extended life span, decreased incidence of cancers, increased antibody production to antigens, and enhanced lymphoproliferation in response to mitogenic stimulation (17–20). Importantly, it has also been reported previously that CR improved the immune response of aged mice to influenza vaccination, as evidenced by improved antigen-specific proliferation, antigen presentation, antibody production, and T-cell function (20). Thus, the extension of life span in rodents by CR, coupled with its positive effects on numerous immune parameters in aged rodents, suggests that CR may be a useful experimental tool to better understand age-related changes in the primary response to influenza.

In the current studies, we have used a mouse model of CR to determine whether CR could modulate the response of aged mice to primary influenza infection. Our data indicate that, although CR delayed the onset of the age-related decline in T-cell proliferative responses after mitogenic stimulation, in stark contrast and surprisingly, increased virus burden, reduced natural killer (NK) cell activity in lungs, and decreased survival were observed early on during primary influenza infection of aged CR mice. Importantly, CR appears to have differential effects on immunity of aged mice, as general indices of immune response were maintained, but immune responses to primary influenza infection in the lungs were impaired. Importantly, these data suggest that the age-related increased susceptibility and impaired recovery to a primary infection with influenza, and possibly other viruses, may not be correctable by long-term CR.


   METHODS
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Specific pathogen-free young (3 month old) and aged (23+ month old) male B6 mice were obtained from the mouse colony supported by the National Institute on Aging (NIA). Young mice, designated YAL, were fed an NIH-31 diet AL. Aged mice were divided into two diet groups: OAL, fed the NIH-31 diet AL since weaning, or OCR, fed a 40% CR NIH-31 diet, supplemented with vitamins and minerals, from the age of 3 months onward. Upon arrival, mice were fed their respective diets; i.e., either control diet AL or CR diet (control diet supplemented with vitamins and minerals, but restricted to 60% of the total intake of mice fed AL). Mice were housed individually in microisolator cages in a barrier room of the Association for Assessment and Accreditation of Laboratory and Animal Care (AAALAC)-approved animal facility at Drexel University. Mice acclimated for at least 2 weeks before initiation of experiments. Mice with evidence of disease (e.g., enlarged spleen or gross tumors) were eliminated from the study.

Virus
Influenza A-Puerto Rico/8/34 (PR8; H1N1, a gift from Dr. Walter Gerhardt, University of Pennsylvania) was propagated in specific pathogen-free eggs (B and E Eggs, Lancaster, PA), and cell-free supernatants were stored at –70°C for subsequent use. Mice were anesthetized by intraperitoneal (i.p.) injection with Avertin (2,2,2-tribromoethanol; Sigma, St. Louis, MO) and were infected intranasally (i.n.) with between 0.1 and 100 hemagglutination units (HAU) of PR8.

Weight Loss
All mice were weighed daily to monitor their ability to control infection.

Isolation of Mononuclear Cells from Spleens and Lungs
The procedure for isolation of mononuclear cells from spleens and lungs has been described in detail previously (12). Briefly, mice were killed by CO2 asphyxiation, and spleens and lungs were aseptically removed. Spleens were homogenized (Dounce) and resuspended in RPMI-1640 (BioWhittaker, Walkersville, MD). Lungs were minced with a scalpel and incubated at 37°C for 1.5 hours in a cocktail containing Collagenase A at 2 mg/ml and 80 Kuntz units of DNAase/ml; all from Sigma) with 10% fetal calf serum (Sigma), 1% L-glutamine (Gibco BRL, Gaithersburg, MD), and 50 mM gentamicin (Sigma) in Iscove's Modified Dulbecco's Medium. The digested lung samples were passed through a 40-µm nylon mesh (Fisher Scientific, Pittsburgh, PA) and centrifuged. The pellets were resuspended and washed twice with 10% fetal calf serum in Iscove's Modified Dulbecco's Medium. The cell suspensions from spleens and lungs were layered on Histopaque-1083 (Sigma) and subjected to density gradient centrifugation. Cells from each tissue were resuspended to the appropriate concentration for use in subsequent assays.

Lung Virus Titers
Lungs were disrupted using a tissue homogenizer. The resulting slurry was centrifuged, and supernatants were stored at –70°C until use. Serially diluted supernatants were used to infect Madin-Darby canine kidney (MDCK) cells. After incubation at 37°C for 24 hours, 0.02% trypsin (Sigma) was added, followed by an additional 48-hour incubation. Chick red blood cells (B and E Eggs) were resuspended at 0.05% in phosphate-buffered saline were then added to the cultures. Virus titers were determined based on the presence or absence of hemagglutination, as previously described (12).

NK Cell Activity in Lungs
A standard 4-hour 51Cr-release assay with YAC-1 cells as targets to assess NK cell activity was used as described previously (21,22). Briefly, 1 x 106 YAC-1 cells were incubated with 200 mCi Na 51CrO4 (ICN, Costa Mesa, CA) for 2 hours at 37°C. During this incubation, cells were mixed every 20 minutes by gentle tapping to ensure maximal uptake of Na51 CrO4. The cells were then washed twice with RPMI-1640, resuspended in medium containing 10% fetal bovine serum in RPMI-1640 (complete medium) and then rotated for 1 hour at room temperature. After the final wash, YAC-1 cells were resuspended at 1 x 104 cells/ml in complete medium and plated in round-bottom 96-well microtiter plates (ICN). Effector cell preparations were then added to wells at effector-to-target (E:T) ratios of 100:1, 50:1, or 25:1. All samples were assayed in triplicate. Target cells were incubated with medium alone to assess spontaneous release or with 5% Triton X-100 to quantitate maximum release. After a 4-hour incubation at 37°C, supernatants were harvested using the Skatron harvesting system (Skatron, Sterling, VA), and radioactivity in supernatants was quantitated using a gamma counter (Packard Instruments, Sterling, VA). Percent cytotoxicity was calculated as follows:


{grna-60-05-08-eq1}

where CPM = counts per minute. Spontaneous release was always less than 10% of maximal release.

Concanavalin A-Induced Proliferation of Splenocytes
Proliferation in response to concanavalin A (Con A; Sigma) was assessed using standard procedures in our laboratory (17,23). Briefly, cells (2.5 x 105/well) were plated in triplicate in 96-well round-bottom plates (ICN) containing 2.5 µg of Con A/ml and incubated at 37°C for 48 hours. These conditions produce maximal Con A-induced proliferation in our laboratory (17,23). During the last 4 hours of incubation, 1 µCi of 3H-thymidine was added to the wells, and cells were harvested using a Matrix 9600 Harvester (Packard Instruments). Thymidine incorporation was quantitated on a Top Count plate counter (Packard Instruments), and proliferation was expressed as


{grna-60-05-08-eq2}

Statistics
All statistics were performed using JMP software (version 3.2.6; SAS Institute, Cary, NC). Survival data were analyzed using the Kaplan–Meier test, whereas group comparisons were analyzed using the Mann–Whitney U test or Student's t test, depending on the normality of the data. Statistical significance was accepted at p <.05.


   RESULTS
Top
 Abstract
 Methods
 Results
Discussion
 References
 
CR Decreases Survival of Aged B6 Mice After Primary Infection With Influenza A Virus in a Dose-Dependent Manner
The initial objective of this study was to determine the effects of CR on susceptibility of aged mice to influenza infection. Mice in the three groups, YAL, OAL, and OCR, were infected under avertin anesthesia with 0.1, 1, 10, or 100 HAU of live PR8 influenza A virus. Survival was monitored for 9 days postinfection (p.i.) or until mice were moribund. These doses of virus were chosen based on extensive preliminary studies determining tissue culture infective doses (TCID50) for PR8 as well as lethal dose (LD50) analysis of adult mice in our concurrent studies examining CD8+ T-cell function during primary infection of young (6 month) and aged (22 month) B6 mice (12) (data not shown).

Figure 1 clearly demonstrates, much to our surprise, that infection with all doses of PR8 decreased survival of OCR mice (squares) compared with YAL (triangles) or OAL (diamonds) mice. In addition, doses of PR8 ranging from 1 to 100 HAU significantly reduced survival time of OCR mice (p <.01, Kaplan–Meier test), culminating in 100% mortality 5–8 days after infection, whereas 40% and 60% of YAL and OAL mice, respectively, survived. The rate of mortality was much steeper for OCR mice, with a marked decrease in survival beginning as early as 3 days p.i., whereas the rate of mortality was much more gradual for both YAL and OAL mice at all doses of influenza. Importantly, no further mortality was observed in either YAL or OAL mice monitored up to several weeks p.i. (data not shown), indicating that mortality was not merely delayed. Increased mortality of OCR mice was not attributed to effects of anesthesia given during infection, as 100% survival was observed in control YAL, OAL, or OCR mice that were inoculated i.n. with 0.9% normal saline after avertin anesthesia (data not shown). This effect of CR on mortality after influenza infection of aged mice was highly reproducible, with similar results being observed in two subsequent, independent experiments in which mice were infected with either 1 or 10 HAU of PR8 (data not shown). Collectively, these data clearly indicate that CR increases both the severity and susceptibility of aged mice to influenza in a dose-dependent fashion.



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  		Figure 1. Survival of young and aged C57BL/6 (B6) mice, with or without caloric restriction (CR), after primary infection with various doses of PR8 influenza A virus. Young and old ad libitum fed (YAL and OAL, respectively) and old calorically restricted (OCR) mice were inoculated intranasally with doses of live Influenza A-Puerto Rico/8/34 (PR8) virus ranging from 0.1 to 100 hemagglutinating units (HAU). Survival was monitored for 9 days postinfection. Values represent percent survival of n = 10–12 that were initially in each group. Survival time of OCR mice was significantly reduced compared to that of either YAL or OAL at 1, 10, and 100 HAU (p <.01, Kaplan–Meier test.)

 
Aged CR Mice Demonstrate Marked Weight Loss During Primary Influenza Infection
To assess the severity of illness during infection using another parameter, weight loss was recorded daily to assess the ability of mice to control infection. It was also important to identify possible reasons for increased mortality of aged CR mice. Therefore, YAL, OAL, and OCR mice were infected with either 1 or 10 HAU of PR8 because these doses yielded the most reproducible survival data for mice in all groups. In confirmation of previous observations, by 4 days p.i., OCR mice demonstrated a sharp drop in survival, compared with that of YAL or OAL mice (data not shown). Figure 2 shows representative weight-loss curves for animals after infection with 10 HAU of PR8; similar data were observed after infection with 1 HAU (data not shown). Mice in all groups lost weight through day 4 after infection with 10 HAU of PR8. However, weight began to stabilize, or increase, for YAL and OAL 5 days p.i., which was consistent with their complete recovery from infection by 10 days p.i. In marked contrast, weight continued to steadily decline throughout the duration of infection of OCR mice, such that 100% mortality was seen by 7 days p.i. (Figure 2). These weight loss data parallel survival curves generated in Figure 1 and are consistent with our observation that weight begins to stabilize or increase in mice that survive infection.



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  		Figure 2. Weight loss in young and old mice, with or without calorie restriction (CR), after infection with 10 hemagglutinating units (HAU) of Influenza A-Puerto Rico/8/34 (PR8) virus. Body weights were recorded daily for all mice in each group for 9 days postinfection or until moribund. Each line represents mean weights ± standard error of the mean [n = 5 old mice fed ad libitum (OAL); n = 5 young mice fed ad libitum (YAL), and n = 5 old mice fed a calorie-restricted diet (OCR)] during the course of infection. OAL mice weighed significantly more than did either YAL or OCR mice through 5 days postinfection (p <.05, Student's t test). Weight was not significantly different among groups at any other time points

 
Aged CR Mice Demonstrate Increased Virus Titers and Reduced NK Activity in Lung During Influenza Infection
Our previous report indicated that there was an age-related delay in the expansion of CD8+ T cells, which was reflected in a delay in lung virus clearance during primary influenza infection (12). Therefore, an important question for the current studies was to determine whether increased susceptibility of OCR mice to influenza was related to higher lung virus burden during infection. Due to the limited number of animals available from the NIA, we could only examine virus titers 4 days p.i. in YAL, OAL, and OCR mice, the point at which AL mice began to recover from infection, but the point at which OCR mice showed increased mortality. As shown in Table 1, OCR mice demonstrated 10-fold higher (p <.05) lung virus titers, relative to those seen in YAL and OAL mice 4 days p.i. These data indicate that lung virus clearance is delayed or impaired in OAL mice.


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  		Table 1. Lung Virus Titers Four Days after Infection of AL and CR Mice with 10 HAU PR8.

 
Our initial intention was to measure the effects of CR on CD8+ T-cell function during the course of primary influenza infection in aged mice. Our rationale was that CD8+ T cells are necessary for virus clearance after challenge with influenza (8–10), and data generated in our laboratory demonstrated both an age-associated decrease and delay in influenza-specific CD8+ T-cell number and cytotoxic activity in lung during primary influenza infection (12). However, data in Figures 1 and 2 clearly indicate that the most dramatic decrease in survival and weight loss occurred within the first 4–6 days p.i. in all groups, before a measurable CD8 + T-cell response is detectable. Given the importance of NK cells in controlling virus infection, NK activity was assessed in lungs from YAL, OAL, and OCR mice at 0 (basal), 24, and 48 hours after infection with 10 HAU of PR8. Figure 3 shows that NK cytotoxicity in lungs of both YAL and OAL mice increased by 48 hours p.i., relative to basal activity of YAL mice assessed on the day of infection. However, the activity of OCR mice did not change between 24 and 48 hours after infection with influenza, was not greater than basal levels of YAL mice at any time point, and was lower than inducible levels seen in OAL mice. Importantly, influenza-induced NK activity of OAL mice was about 50% of that of YAL mice, in accord with our previous studies demonstrating an age-related decrease in inducible splenic NK activity (21). Due to limited availability of animals from the NIA, these data were generated from two mice per group; nonetheless, highly reproducible values were obtained (<0.5% difference for animals within each group, data not shown). These data suggest that altered NK activity may contribute to increased susceptibility of aged CR mice to influenza infection.



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  		Figure 3. Natural killer (NK) cell activity in lungs from young and old mice fed ad libitum (YAL and OAL, respectively) and old mice fed a calorically restricted diet (OCR) at time 0 (Basal), 24, or 48 hours after infection with 10 hemagglutinating units (HAU) of Influenza A-Puerto Rico/8/34 (PR8) virus. Values represent n = 2 mice per time per age. Due to the limited number of animals available from the National Institute of Aging, basal NK activity was assessed in YAL only. YAL and OAL, but not OCR, mice demonstrate inducible activity after influenza infection

 
CR Maintains Con A-Induced Proliferation in Spleens of Aged B6 Mice
Several published reports have consistently shown that the age-related decline in immunity is abrogated by CR (3,17–20), yet we showed deleterious effects of CR after influenza infection of aged mice. Therefore, the next question was to determine whether our model of CR was effective in ameliorating the age-related decline in general indices of immune function, in accord with previous studies. Mononuclear cells isolated from spleens were incubated for 48 hours with Con A at 2.5 µg/ml, and T-cell proliferation was assessed. These conditions produce maximal T-cell proliferation in our laboratory. As shown in Figure 4, the Con A-induced proliferative response of splenocytes of aged CR mice was not significantly different from that of YAL mice, but was significantly higher (p <.05) than that of OAL mice, confirming previous reports (3,17–20). These data indicate that CR facilitates maintenance of the T-cell proliferative response of aged mice to nonspecific stimulation in vitro, despite our observation of deleterious effects during acute influenza infection.



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  		Figure 4. Concanavalin A (Con A)-induced proliferation of splenocytes 4 days postinfection with 10 hemagglutinating units (HAU) of Influenza A-Puerto Rico/8/34 (PR8) virus. Values represent means ± standard error of the mean of 5 mice per group. Splenocytes were incubated for 48 hours in the presence of Con A at 2.5 µg/ml. Proliferation was measured by 3H-thymidine uptake during the final 4 hours of culture. *Proliferative responses of old mice fed ad libitum (OAL) is significantly lower (p <.05) than those of young mice fed ad libitum (YAL) and old mice fed a calorically restricted diet (OCR); the responses of OCR and YAL mice are not significantly different (Mann–Whitney U test)

 

   DISCUSSION
Top
 Abstract
 Methods
 Results
 Discussion
References
 
The age-related decline in the cell-mediated immune response during primary influenza infection, as evidenced by impaired CTL activity, reduced CD8+ T cells, and prolonged virus shedding has been documented previously (8–10). In our own studies, we showed that aged B6 mice demonstrated reduced and delayed expansion of influenza-specific CD8+ T cells in lung during primary infection that was paralleled by a decrease and delay in maximal CTL activity, lung virus clearance, and impaired IFN-{gamma} production (12). The present study used a mouse model of CR to extend these studies to determine whether CR could modulate the response of aged mice during primary influenza infection.

The data in the present study indicate that CR of aged mice decreases survival after primary infection with influenza, relative to that demonstrated by either young or aged mice fed AL. To our knowledge, this finding is the first to indicate that CR actually increases mortality of aged rodents after a primary exposure to an infectious agent such as influenza. However, positive effects of CR on the immune response of aged mice after influenza immunization have been reported previously (20). In this study, CR-fed aged mice immunized i.p. with influenza virus demonstrated improved influenza-specific T-cell proliferative responses, antigen presentation, and antibody responses, relative to those produced by aged AL-fed mice (20). The difference in the outcome of our study and that of the previous study (20) reflects differences both in study design and in parameters assessed. In the current study, mice were inoculated i.n. with influenza virus and the response to primary infection to influenza was assessed in lung, whereas in the previous study (20), mice were immunized i.p. and the immune response to influenza was assessed in spleen. One could extrapolate from the current findings that the lung, but not other tissues, undergoes age-related changes that are not correctable by CR and that these tissue-specific effects are only detected during a primary pulmonary infection. Thus, further studies are necessary to determine if the ability of aged CR mice to eradicate a pathogen is dependent on the site of virus entry. It is equally possible that these differential effects of CR in aged mice may reflect the inability of CR to maintain, or perhaps initiate, positive changes in early events, such as viral replication or NK cell activity, that may control susceptibility to a new infection. Importantly, long-term CR of aged mice, in the absence of infection, can still induce positive effects on general indices of immune responsiveness and perhaps maintain the function of immune cells that are able to respond to immunization. The current study underscores the need for future studies to determine whether long-term CR of aged mice is detrimental to the primary response to all infectious agents or only to the primary response to respiratory pathogens.

The present study demonstrated that lung virus titers were significantly higher 4 days after infection of aged CR mice compared with young and aged AL mice, the point at which CR mice began to succumb to infection, but AL mice began to recover. These data are in accord with previous reports from our laboratory (12) and others (8–10) demonstrating impaired virus clearance in lungs from aged mice. These increased lung virus titers suggest that virus clearance is delayed, or perhaps virus replication is increased, during primary influenza infection of aged CR mice. These events may exacerbate influenza infection, resulting in increased mortality early in the primary response of aged CR mice.

The susceptibility of aged CR mice to influenza infection before the generation of detectable levels of functional influenza-specific CD8+ T cells suggested that long-term CR does not positively impact the early stages of the innate immune response after influenza infection. The importance of NK cells in controlling infection prior to the initiation of a virus-specific immune response has been shown (24–26). Depletion of pulmonary NK cells increased mortality of mice infected with influenza and delayed the initiation of a virus-specific CD8+ T-cell response (25). Our data indicated that influenza-induced NK activity in lung was reduced in aged CR mice, relative to both basal and influenza-inducible levels in young, and to inducible levels in aged AL mice. These data are in contrast to one report indicating that aged CR mice demonstrated reduced basal, but not inducible, splenic NK activity after poly I:C stimulation (27). The reasons for the discrepancies between our study and the former report (27) may reflect differences in the agents used to induce NK activity in the two studies (poly I:C in the former vs influenza in the present study), as well as differences in the strain of mouse assessed (C3H.SW/S x C57BL/10/SN (F1)) in the former vs B6 in the present study). Therefore, it is possible that the effect of CR on inducible NK activity of aged mice in the current study was strain-dependent.

Differences in the level of NK cell activity between mouse strains have been reported (21). Although the data generated in the present study were obtained from a limited number of animals, they are in accord with our previous report showing an age-related decline in inducible splenic NK activity after IFN-{alpha}/ß stimulation (21). Our data suggest that increased susceptibility of aged CR mice may be related to alterations in NK cells at the site of infection, the lung. Future studies are necessary to determine whether reduced NK cell activity in lungs of aged mice is strain-dependent or perhaps reflects differences in cell number and/or the kinetics and magnitude of the NK response. These studies are currently underway.

The inability of NK cells to control infection may also reflect decreased production of endogenous cytokines in lung that induce NK activity during virus infections. For example, IFN-{alpha}/ß is a cytokine produced during infection inducing an antiviral state in uninfected cells (28) thus limiting virus replication (24). Both interleukin 12 and tumor necrosis factor-{alpha} are also produced early in the innate immune response and act synergistically to activate NK cells (24,29). Previous kinetic studies of cytokine production in bronchoalveolar fluid after influenza infection in mice have shown early production of interleukin 1, interleukin 6, tumor necrosis factor-{alpha}, and IFN-{alpha}/ß before the initiation of an influenza-specific adaptive immune response in lung (30,31). Therefore, it is possible that reduced NK activity in aged CR mice is due to alterations in endogenous cytokine production at the site of infection, impairing the ability of NK cells to become induced during primary infection. Future studies are required to address this possibility.

Finally, an important finding in this study was that CR of aged mice delayed the age-related decline in the T-cell proliferative response after mitogenic stimulation, confirming numerous reports demonstrating positive effects of CR on the immune response of aged rodents (13,17–20). These data suggest that CR may have a deleterious effect during exposure of aged mice to a "new" infection, which may not be reflective of an impairment in general immune responsiveness. These differential effects of CR should be considered when evaluating the use of CR to modulate the response to infectious agents such as influenza. It will also be necessary to determine whether this model of CR has this same deleterious effect on other viruses and can be extrapolated to other primary infections.

In summary, our study demonstrates that CR exacerbates susceptibility of aged mice to primary influenza infection, while still producing positive effects on general immune responsiveness. This increased susceptibility of aged CR mice to influenza infection may reflect altered immediate responses to the virus, including pulmonary NK activity and the inability to clear virus during the course of infection. Importantly, these data suggest that age-related increased susceptibility to primary influenza infection may not be corrected by long-term CR. This potential deleterious effect should be considered when evaluating CR as a possible method to modulate the age-related decline in the immune response to influenza as well as to other viruses.


   Acknowledgments
 
This work was supported by funds from the Mary Dewitt Pettit Fellowship at MCP Hahnemann University. Dr. Farvardin Anaraki provided excellent technical support during these studies. Dr. Ed J. Gracely provided statistical support. Drs. Donna M. Murasko and Mary K. Howett provided critical review of this manuscript.


   Footnotes
 
Decision Editor: James R. Smith, PhD

Received September 20, 2004

Accepted January 5, 2005


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

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