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. 2001 Sep;8(5):1003-11.
doi: 10.1128/CDLI.8.5.1003-1011.2001.

Effects of Calorie Restriction on Polymicrobial Peritonitis Induced by Cecum Ligation and Puncture in Young C57BL/6 Mice

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Free PMC article

Effects of Calorie Restriction on Polymicrobial Peritonitis Induced by Cecum Ligation and Puncture in Young C57BL/6 Mice

D Sun et al. Clin Diagn Lab Immunol. .
Free PMC article

Abstract

Calorie restriction (CR) is known to prolong the life span and maintain an active immune function in aged mice, but it is still not known if rodents under CR can respond optimally to bacterial infection. We report here on the influence of CR on the response of peritoneal macrophages to lipopolysaccharide, splenic NF-kappaB and NF-interleukin-6 (IL-6) activities, and mortality in polymicrobial sepsis induced by cecal ligation and puncture (CLP). Macrophages from 6-month-old C57BL/6 mice on a calorie-restricted diet were less responsive to lipopolysaccharide, as evidenced by lower levels of IL-12 and IL-6 protein and mRNA expression. Furthermore, in vitro lipopolysaccharide-stimulated macrophages from mice under CR also expressed decreased lipopolysaccharide receptor CD14 levels as well as Toll-like receptor 2 (TLR2) and TLR4 mRNA levels. In addition, the phagocytic capacity and class II (I-A(b)) expression of macrophages were also found to be significantly lower in mice under CR. Mice under CR died earlier (P < 0.005) after sepsis induced by CLP, which appeared to be a result of increased levels in serum of the proinflammatory cytokines tumor necrosis factor alpha and IL-6 and splenic NF-kappaB and NF-IL-6 activation 4 h after CLP. However, mice under CR survived significantly (P < 0.005) longer than mice fed ad libitum when injected with paraquat, a free radical-inducing agent. These data suggest that young mice under CR may be protected against oxidative stress but may have delayed maturation of macrophage function and increased susceptibility to bacterial infection.

Figures

FIG. 1
FIG. 1
Effect of CR on body weight. Six-month-old C57BL/6 mice were fed a semipurified diet containing 5% (wt/wt) corn oil AL or the same diet whose calories were restricted 40% (CR).
FIG. 2
FIG. 2
Effect of CR on macrophage cytokine production after LPS stimulation. Peritoneal macrophages from 6-month-old C57BL/6 mice fed a calorie-restricted diet were stimulated with LPS ex vivo. Twenty-four hours after stimulation, macrophages were incubated with fluorescent antibodies to TNF-α, IL-6, and IL-12. The cells were analyzed by flow cytometry. Each bar represents the mean ± standard error of the mean of six independent measurements. ∗, significantly different from controls fed AL (P < 0.05).
FIG. 3
FIG. 3
Effect of CR on cytokine, TLR2, and TLR4 gene expression in peritoneal macrophages of C57BL/6 mice after LPS stimulation. Peritoneal macrophages were isolated as described in Material and Methods and were stimulated with LPS (1 μg) for 12 h in vitro. Total RNA was isolated and transcribed to cDNA and was then amplified by PCR with primers specific for TLR2, TLR4, IL-12, TNF-α, IL-6, and β-actin, as described in Materials and Methods.
FIG. 4
FIG. 4
Peritoneal macrophage phagocytosis in mice fed a calorie-restricted diet. Six-month-old C57BL/6 mice were fed a semipurified diet containing 5% (wt/wt) corn oil AL or the same diet whose calories were restricted 40% (CR). Peritoneal macrophages were harvested 96 h after injection of thioglycolate intraperitoneally. Phagocytosis of latex beads and zymosan was assessed before and 4 h after LPS stimulation (1 μg/ml) (n = 6). ∗, significant differences between the group fed AL and the group fed a calorie-restricted diet (P < 0.05).
FIG. 5
FIG. 5
Effects of CR on the survival of C57BL/6 mice after CLP. Six-month-old C57BL/6 mice fed AL and a calorie-restricted diet were subjected to polymicrobial sepsis induced by CLP. The survival rate was monitored 72 h after CLP. There is a significant difference between these survival curves (P = 0.004 by the log rank test).
FIG. 6
FIG. 6
Electophoretic mobility shift assay of NF-κB-binding activity. Nuclear extracts were isolated as described in Materials and Methods. Nuclear extracts (2.5 μg) were incubated with radiolabeled probes specific for NF-κB, separated on a 5% polyacrylamide gel, and then exposed to film. The gel is representative of gels from five independent experiments. Band intensity was measured by densitometry and expressed as the mean ± standard error of the mean. ∗, significantly different from the corresponding control group (P < 0.05); †, significantly different from the corresponding group that underwent a sham operation (P < 0.05); §, significantly different from the group that underwent CLP and that was fed AL (P < 0.05).
FIG. 7
FIG. 7
NF–IL-6 activation and binding activity in murine spleen 4 h after CLP. Nuclear extracts were isolated as described in Materials and Methods. Nuclear extract (2.5 μg) was incubated with radiolabeled probes specific for NF–IL-6 and separated on a 5% polyacrylamide gel and then exposed to film. The gel is representative of gels from five independent experiments. Band intensity was measured by densitometry and is expressed as the mean ± standard error of the mean. ∗, Significantly different from corresponding control group (P < 0.05); †, significantly different from the corresponding group that underwent a sham operation (P < 0.05); §, significantly different from the group that underwent CLP and that was fed AL (P < 0.05).
FIG. 8
FIG. 8
Effects of CR on cytokine gene expression after polymicrobial peritonitis induced by CLP puncture. Polymicrobial peritonitis was induced by CLP in C57BL/6 mice. Four hours after the operation total RNA was isolated from the spleens, as described in Material and Methods. The RNA was transcribed to cDNA and was then amplified by PCR with primers specific for TNF-α, IL-6, or β-actin, as described in Materials and Methods. The resulting samples were subjected to electrophoresis on agarose gels and stained with ethidium bromide. Band intensity was quantitated by densitometry. The values are the means ± standard errors of the means for five gels. ∗, significantly different from corresponding control group (P < 0.05); †, significantly different from corresponding group that underwent a sham operation (P < 0.05); §, significantly different from group that underwent CLP and that was fed AL (P < 0.05).
FIG. 9
FIG. 9
Effect of calorie restriction on serum cytokine TNF-α and IL-6 levels of 6-month-old C57BL/6 mice fed a calorie-restricted diet. Mice were treated as described in the legend to Fig. 8. Serum TNF-α (A) and IL-6 (B) levels were quantitated by ELISA. Each bar represents the mean ± standard error of the mean of five independent measurements. The means for bars labeled with different letters are significantly different (P < 0.05).
FIG. 10
FIG. 10
Survival of mice fed a calorie-restricted diet and mice fed AL after challenge with the oxidizing agent paraquat. Six-month-old C57BL/6 mice fed a calorie-restricted diet and mice fed AL were injected intraperitoneally with paraquat at 70 mg/kg. Mice fed AL died significantly earlier than mice fed a calorie-restricted diet (P = 0.0048).

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