Assessment of Interleukin-12, Gamma Interferon, and Tumor Necrosis Factor Alpha Secretion in Sera from Mice Fed with Dietary Lipids during Different Stages of Listeria monocytogenes Infection

Recent experimental observations have determined that long-chainn-3 polyunsaturated fatty acids suppress immune functions andare involved in the reduction of infectious disease resistance.BALB/c mice were fed for 4 weeks with one of four diets containingeither olive oil (OO), fish oil (FO), hydrogenated coconut oil,or a low fat level. Interleukin-12p70 (IL-12p70), gamma interferon(IFN- ${gamma}$ ), and tumor necrosis factor alpha (TNF- ${alpha}$ ) production inthe sera of mice fed these diets and challenged with Listeriamonocytogenes were determined by enzyme-linked immunosorbentassay. In addition, bacterial counts from spleens of mice werecarried out at 24, 72, or 96 h of infection. Here, we quantifiedan initial diminution of production of both IL-12p70 and IFN- ${gamma}$ ,which appear to play an important role in the reduction of hostresistance to L. monocytogenes infection. In addition, an efficientelimination of L. monocytogenes was observed in spleens of micefed a diet containing OO at 96 h of infection, despite reductionsin IL-12p70 and TNF- ${alpha}$ production, suggesting an improvement ofimmune resistance. Overall, our results indicate that the initialreduction of both IL-12 and IFN- ${gamma}$ production before L. monocytogenesinfection represents the most relevant event that corroboratesthe impairment of immune resistance by n-3 polyunsaturated fattyacids during the different stages of infection. However, wespeculate that the modulation of other cytokines must be alsoinvolved in this response, because the alteration of cytokineproduction in mice fed an FO diet in a late phase of L. monocytogenesinfection was similar to that in mice fed OO, whereas the abilityto eliminate this bacterium from the spleen was improved inthe latter group.

INTRODUCTION

Top

Introduction

Evidence from numerous investigations has revealed that nutritionalstatus is a crucial factor that contributes to the modulationof the host immune response (6). Hence, the interactions betweencertain nutrients and immunity play a relevant role that shouldbe evaluated from biological and clinical points of view (11).In the last few years, a large number of epidemiological, clinical,and experimental studies have indicated that certain dietarylipids are capable of altering different immune functions inboth animals and humans (5, 9). Accordingly, diets containingmoderate levels of oily fish (mainly constituted by long-chainn-3 polyunsaturated fatty acids) exert numerous health benefits,such as reduction of autoimmune disorders (17, 18) or diminutionof cancer incidence (25). As a direct consequence, differentinvestigations have applied several fatty acids in clinicalnutrition, because long-chain n-3 polyunsaturated fatty acidsderived from fish oil have been demonstrated to be more immunosuppressivethan n-6 polyunsaturated fatty acids or n-9 monounsaturatedfatty acids. Despite this, several studies have reported thatan excess intake of n-3 polyunsaturated fatty acids may causeundesirable effects in both humans and animals, because theyare able to reduce host immune resistance against infectiousmicroorganisms (1, 11). Thus, numerous investigations have indicatedthat diets containing n-3 polyunsaturated fatty acids enhancesusceptibility to infection by Listeria monocytogenes (8, 13,23), Salmonella enterica serovar Typhimurium (7), Mycobacteriumtuberculosis (21), or Pseudomonas aeruginosa (22), whereas otherrecent observations have reported that these diets increaseimmune resistance against Klebsiella pneumoniae (2, 3, 27),although these effects could not be demonstrated in infectionwith Streptococcus pneumoniae (27). Therefore, it is possiblethat the consequences derived from the reduction of immune resistanceby n-3 polyunsaturated fatty acids may also depend on the typeof microorganisms and their pathogenic mechanisms. Based onthe exposed arguments, it is important to consider the factorsinvolved in the reduction of immune resistance caused by manydietary lipids. In fact, cytokines are important immune mediatorsthat participate in the development of immune responses. Thus,production of interleukin-12p70 (IL-12p70), an inflammatorycytokine that plays a critical role in the Th1-type response,is reduced after fish oil diet administration (13, 24), whereasproduction of IL-4 (an anti-inflammatory cytokine) is increased,although the alteration in the levels of this cytokine doesnot appear to constitute a critical factor responsible for thereduction of immune defense after dietary lipid administration(24). Nevertheless, other studies have indicated that fish oildiets reduce the secretion of proinflammatory cytokines suchas tumor necrosis factor alpha (TNF- ${alpha}$ ), IL-1, or IL-6 (26), andtherefore this leads to an increase in percent survival afterlipopolysaccharide injection (20). We and others have suggestedthat the reduction of cytokine synthesis caused by n-3 polyunsaturatedfatty acids indicates that these fatty acids could affect thehost response to bacterial infections (10, 11, 24). As a directconsequence of these effects, a reduction in percent survivalof mice as well as an inefficient ability to eliminate the microorganismsfrom liver and spleen has been reported (8, 13).

Here, we have focused on the evaluation of IL-12p70, gamma interferon- ${gamma}$ (IFN- ${gamma}$ ), and TNF- ${alpha}$ production after the administration of dietarylipids in mice infected with L. monocytogenes, which has establishedan important model system for understanding host resistanceto intracellular bacteria. The measurement of the cytokinesin sera from mice fed with dietary lipids was carried out duringdifferent stages of murine listeriosis.

MATERIALS AND METHODS

Top

Materials and Methods

Animals and experimental diets. BALB/c mice 8 to 10 weeks old (from a breeding colony of theresearch technical services from the University of Jaén)were allocated to receive one of four diets for 4 weeks, andeach group had unlimited access to food and water during theexperiment. Mice were housed in cages in an environmentallycontrolled room at a temperature of 24°C with a 12-h light-darkcycle. Diets contained either olive oil (OO) (20%), fish oil(FO) (20%), or hydrogenated coconut oil (HCO) (20%). In addition,another group was fed a diet containing a low fat (LF) (2.5%of fats) level and was used as a control. The diet containingHCO was supplemented with 1% corn oil in order to prevent essentialfatty acid deficiency. The compositions of the experimentaldiets are illustrated in Table 1.

View this table:
[in this window]
[in a new window]
TABLE 1. Compositions of the experimental diets^a

Bacterial growth, experimental infection, and serum collection. L. monocytogenes was grown in blood tryptic soy agar medium(Scharlau Chemie, Barcelona, Spain) for 24 h at 37°C. Theconcentration of the cell suspension was adjusted spectrophotometricallyat 550 nm. At the end of dietary lipid administration, eachmouse was infected with a virulent strain of L. monocytogenesinjected through the tail vein (10⁵ CFU/ml), and peripheralblood was isolated at 0, 24, 72, and 96 h of experimental infection.Mice were anesthetized with diethyl ether, and the blood wasremoved from the retro-orbital plexus into tubes containingheparin (20 U/ml blood). Mice were sacrificed by cervical dislocation,and the spleens were removed into sterile tubes. Serum was separatedfrom freshly heparinized blood by centrifugation of the tubesat 1,500 x g for 30 min at 4°C. Finally, serum samples werestored in aliquots at –80°C for later use.

Determination of numbers of viable L. monocytogenes in spleens. Mice were infected with 100 µl of a L. monocytogenes cellsuspension (10⁵ CFU/ml) and sacrificed at 24, 72, and 96 h postinfection.Spleens were removed under sterile conditions in phosphate-bufferedsaline (pH 7.4). Spleens were then prepared by being homogenizedbetween frosted-glass slides in distilled water in order torelease intracellular bacteria at 24, 72, and 96 h after infectionwith the bacteria. Serial 10-fold dilutions of each sample wereincubated on blood tryptic soy agar medium to determine thenumber of viable L. monocytogenes organisms. Finally, plateswere incubated at 37°C for 24 h, and the number of bacterialcolonies was counted. Results were expressed as the log₁₀ viablebacteria in spleens.

Quantification of cytokine levels in serum by immunoassay. IL-12p70, IFN- ${gamma}$ , and TNF- ${alpha}$ secretion were measured by enzyme-linkedimmunosorbent assay (ELISA) (assay kits were from R&D Systems,Minneapolis, MN) according to the manufacturer's protocols.Cytokine synthesis during different stages of L. monocytogenesinfection was determined at 0, 24, 72, and 96 h. Absorbanceat 405 nm was read using a Microplate reader (Whittaker 2001,Salzburg, Austria). Results were calculated against standardcurves generated using known amounts of recombinant cytokines.The limits of detection of these assays were <2.5 pg/ml (IL-12p70),<5.1 pg/ml (TNF- ${alpha}$ ), and <2 pg/ml (IFN- ${gamma}$ ).

Statistical analysis. Results are presented as means ± standard errors of themeans (SEM). Data were determined by analysis of variance tocompare the effects of experimental and control (LF) diets.When analysis of variance indicated significant differences,the treatment means were compared using Fisher's least-significantdifference test. Differences were considered statistically significantat a P value of <0.05.

RESULTS

Top

Results

Quantification of bacterial loads in spleens of mice. After the administration of dietary lipids, L. monocytogeneswas inoculated in each mouse. We quantified the number of viablebacteria isolated from spleens of mice experimentally infectedwith L. monocytogenes and thus determined the capacity of thehost immune response to eliminate this bacterium from spleensof mice fed dietary lipids. Figure 1 shows a statistically significantincrease of viable bacteria from spleens of mice fed a dietcontaining FO at 24, 72, and 96 h after experimental infection(P < 0.05). In contrast, the group fed a diet containingOO was the most efficient in L. monocytogenes elimination. Ingeneral, it is important to note that the number of viable bacteriafrom spleens of mice at 96 h of experimental infection returnedto the values initially observed at 24 h after infection withL. monocytogenes.

View larger version (17K):
[in this window]
[in a new window]
FIG. 1. Kinetics of the growth of Listeria monocytogenes in the spleens of mice fed dietary lipids and infected with the bacterium. Mice were fed the LF, OO, FO, or HCO diet and were experimentally infected with L. monocytogenes. Spleens were isolated in sterile phosphate-buffered saline and disrupted in sterile water at 24, 72, and 96 h postinfection. Bacterial counts were determined as described in Materials and Methods. The number of bacterial colonies was counted, and the results are expressed as log₁₀ viable bacteria. The bars represent mean values ± SEM for three independent determinations (n = 5 in each dietary group). *, P < 0.05 compared with the values from the control group.

IL-12p70 production. It is well established that cytokines play a crucial role inthe regulation of the immune system, but their production isalso modulated after dietary lipid administration. IL-12 isan important cytokine that participates in the cellular immuneresponse to intracellular microorganisms such as L. monocytogenes.Our results reveal that IL-12p70 production from mice fed dietscontaining OO or FO was significantly reduced at 72 and 96 hafter experimental infection with L. monocytogenes, comparedwith values for the control group (P < 0.05) (Fig. 2B and C).In addition, an initial significant reduction of IL-12p70production was observed in the sera of mice fed an FO diet.In contrast, IL-12p70 secretion from mice fed a diet containingHCO was not altered with respect to values for the control group(Fig. 2D).

View larger version (21K):
[in this window]
[in a new window]
FIG. 2. Determination of IL-12p70 secretion from the sera of peripheral blood of mice fed with different dietary lipids for 4 weeks and subsequently infected with Listeria monocytogenes. BALB/c mice (n = 5 in each dietary group) were fed an LF (A), OO (B), FO (C), or HCO (D) diet and infected with 10⁵ viable L. monocytogenes organisms. The production of IL-12p70 was measured at 0, 24, 72, and 96 h after bacterial infection. Quantification of IL-12p70 secretion in experimental samples was made by extrapolation from ELISA results, using various concentrations of recombinant IL-12 as a standard. Results are means ± SEM of two independent determinations in duplicate. *, P < 0.05 compared with the LF group.

IFN- ${gamma}$ production. Similarly, IFN- ${gamma}$ production is crucial to an efficient immuneresponse to this intracellular pathogen. Here, IFN- ${gamma}$ secretionwas significantly increased in the groups fed diets containingOO, FO, or HCO at 96 h of experimental infection compared withdata from the LF group (P < 0.05) (Fig. 3B, C, and D). However,a statistically significant reduction of IFN- ${gamma}$ production wasobserved in the group fed a diet containing FO before experimentalinfection with L. monocytogenes (P < 0.05) (Fig. 3C).

View larger version (24K):
[in this window]
[in a new window]
FIG. 3. Determination of IFN- ${gamma}$ secretion from the sera of peripheral blood of mice fed with different dietary lipids for 4 weeks and subsequently infected with Listeria monocytogenes. BALB/c mice (n = 5 in each dietary group) were fed an LF (A), OO (B), FO (C), or HCO (D) diet and infected with 10⁵ viable L. monocytogenes organisms. The production of IFN- ${gamma}$ was measured at 0, 24, 72, and 96 h after bacterial infection. Quantification of IFN- ${gamma}$ secretion in experimental samples was made by extrapolation from ELISA results, using various concentrations of recombinant IFN- ${gamma}$ as a standard. Results are means ± SEM of two independent determinations in duplicate. *, P < 0.05 compared with the LF group.

TNF- ${alpha}$ production. To evaluate the action of one of the most important proinflammatorycytokines, we analyzed the production of TNF- ${alpha}$ from sera of micefed dietary lipids. These results demonstrate a significantenhancement of TNF- ${alpha}$ secretion from mice fed diets containingOO or FO before and at 24 h of experimental infection with L.monocytogenes (P < 0.05) (Fig. 4B and C). However, TNF- ${alpha}$ secretionwas significantly reduced at 72 or 96 h of experimental infectionwith L. monocytogenes in the groups fed diets containing OOor FO (P < 0.05), although the production of this proinflammatorycytokine was very similar at each time point. In contrast, anenhancement of TNF- ${alpha}$ production was observed at 96 h of infectionin the groups fed diets containing LF or HCO with respect tovalues before experimental infection with L. monocytogenes (Fig.4A and D). In fact, the kinetics of TNF- ${alpha}$ production were similarin these two groups, with the exception of a significant increasein the HCO group at 96 h of infection with L. monocytogenes.

View larger version (21K):
[in this window]
[in a new window]
FIG. 4. Determination of TNF- ${alpha}$ secretion from the sera of peripheral blood of mice fed with different dietary lipids for four weeks and subsequently infected with Listeria monocytogenes. BALB/c mice (n = 5 in each dietary group) were fed an LF (A), OO (B), FO (C), or HCO (D) diet and infected with 10⁵ viable L. monocytogenes organisms. The production of TNF- ${alpha}$ was measured at 0, 24, 72, and 96 h after bacterial infection. Quantification of TNF- ${alpha}$ secretion in experimental samples was made by extrapolation from ELISA results, using various concentrations of recombinant TNF- ${alpha}$ as a standard. Results are means ± SEM of two independent determinations in duplicate. *, P < 0.05 compared with the LF group.

DISCUSSION

Top

Discussion

The investigation of the events that occur during the infectiousprocesses contributes to establishment of the action of dietarylipids on the immune system in the course of an infection promotedby L. monocytogenes. Numerous studies have indicated that thealteration in secretion of different cytokines by the actionof certain dietary lipids constitutes a critical factor responsiblefor the modulation of immune and inflammatory responses (5,9, 32). Therefore, the relationship between dietary lipids andtheir role in the reduction of the host immune defense againstinfectious agents acquires substantial importance.

It is commonly recognized that protective immunity to intracellularbacteria, such as L. monocytogenes, requires a coordinated interactionbetween many cell types and the production of numerous cytokines(12). These responses are efficiently synchronized by the proinflammatorycytokine IL-12, which promotes T-cell differentiation towarda cell-mediated (T-helper 1-type) immune response (28). Indeed,IL-12 is a crucial cytokine for innate and adaptive immunity,contributing to the alteration of the T-cell repertoire afterinfection with intracellularly growing pathogens (4, 16). Inaddition, IL-12 and TNF- ${alpha}$ , two crucial cytokines of the innateimmune system, synergize to cause NK cell secretion of IFN- ${gamma}$ ,leading to macrophage activation. Thus, neutralization of IL-12,TNF- ${alpha}$ , or IFN- ${gamma}$ demonstrated the critical role that these cytokinesplay during the infection with L. monocytogenes (19, 30), suchthat mice lacking any of these cytokines were unable to protectthemselves against an overwhelming infection (31).

As mentioned above, the potential action of several dietarylipids on the modulation of immune system functions (5, 9),and particularly on the alteration of cytokine production (32),has been widely demonstrated. In order to elucidate the actionof diverse dietary lipids on host immune resistance, the presentstudy evaluated the effects of diets containing OO, FO, or HCOon IL-12p70, IFN- ${gamma}$ , and TNF- ${alpha}$ production during different stagesof L. monocytogenes infection.

Previous investigations have determined that diets containingFO show beneficial effects in the reduction of inflammatorydisorders, which leads to a decrease of autoimmune disease incidence(17). Nevertheless, this characteristic may be overshadowedby an impairment of natural host resistance after the administrationof an FO diet, as a consequence of exacerbated immunosuppression(8, 13). Thus, results from our group and others have determinedthat the administration of a diet containing FO is associatedwith a reduction of survival percentage as well as an increasein the number of viable bacteria from spleens and livers ofmice challenged with L. monocytogenes, suggesting an impairmentof the host immune defense (8, 13). Therefore, it is importantto explain the reasons why the host immune response is reducedafter the administration of certain dietary lipids. Previously,some interesting reports from Fritsche and coworkers suggestedthat a reduction of both IL-12 and IFN- ${gamma}$ secretion at 24 h postchallengeplays a relevant role in the impairment of immune resistancein mice fed an FO diet (14, 15). However, irrespective of therelevance of an FO diet in the alteration of immune system functions,it is important to determine the immunomodulation exerted byother fats during different stages of L. monocytogenes infection.Thus, a diet containing OO appears to reduce IL-12p70 production,as well as to significantly decrease the secretion of TNF- ${alpha}$ at72 or 96 h of experimental infection with L. monocytogenes,but it is important to emphasize that this diet is not involvedin an initial reduction of either IL-12p70 or IFN- ${gamma}$ production.Despite the significant reduction of IL-12p70 and TNF- ${alpha}$ productionat 96 h of experimental infection in the group fed an OO diet,an efficient elimination of L. monocytogenes from spleen isexhibited. It is probable that the absence of changes in theproduction of these cytokines before infection with L. monocytogenesis responsible in part for these effects, whereas the initialreduction of IL-12p70 and IFN- ${gamma}$ production in the FO group isassociated with a considerable increase in the number of viablebacteria from spleens. The initial reduction of IFN- ${gamma}$ productionin the FO group constitutes an important result, because noproduction of this cytokine was seen in the LF and HCO groupsat 96 h of experimental infection, whereas the number of L.monocytogenes organisms from spleen varied considerably betweenthe FO and LF groups or the FO and HCO groups. Hence, this resultconfirms again our preliminary argument, which is supportedby the participation of other types of cytokines which shouldbe modulated by dietary lipids.

Our recent observations have indicated that the type of dietarylipids may affect circulating concentrations of IL-12p70. Thisevent along with inappropriate action of IL-4 contributes toan impairment of host immune susceptibility to infection (24).Here, the reduction of IL-12p70 levels after experimental infectionwith L. monocytogenes gave rise to an important diminution ofIFN- ${gamma}$ secretion in the four groups studied; however, this reductionis more accentuated in the LF and HCO groups at 96 h of infection.Instead, other investigations have underscored that the depletionof IL-12 decreases IFN- ${gamma}$ production and exacerbates infectionin intracellular infection (29). Our results have demonstratedthat despite the reduction of both IL-12p70 and IFN- ${gamma}$ productionat 96 h of experimental infection, a diminution in the numberof viable bacteria from spleens of mice occurs in the four groups.Hence, we can verify that the increase in the number of viablebacteria from spleens of mice fed with an FO diet may be attributedto an initial reduction of both IL-12p70 and IFN- ${gamma}$ secretion,whereas the reduction of viable bacteria from spleens of miceobserved at 96 h after experimental infection with L. monocytogenesin the four groups could be related to an increase in TNF- ${alpha}$ production.In short, although a reduction of viable bacteria has been observedat 96 h of infection, an inefficient resolution of the infectiousprocess has been detected in the group fed a diet containingFO. Thus, the administration of an FO diet does not improvethe host immune resistance during the different stages of L.monocytogenes infection. However, we speculate that other cytokinesmust be also involved in this response, because the alterationof cytokine production in the FO group is similar to that inthe group fed with an OO diet (with the particular exceptionof a significant reduction of both IL-12p70 and IFN- ${gamma}$ initiallyobserved in the group fed a diet containing FO), whereas thenumber of viable bacteria from spleens is considerably reducedin the OO group, indicating an improvement of immune resistanceby the generation of protective immunity.

Concluding remarks. The present results confirm that the reduction of host immuneresistance to infectious microorganisms could be particularlyassociated with an initial decrease of both IL-12p70 and IFN- ${gamma}$ secretion in animals challenged with L. monocytogenes afterthe administration of a diet containing FO. The reduction ofproinflammatory cytokine levels produces a diminution in macrophageactivation that is responsible for an inefficient ability toeliminate L. monocytogenes. Nevertheless, in spite of the factthat an important increase of IFN- ${gamma}$ secretion was observed inthe group fed a diet containing FO in a late stage of L. monocytogenesinfection (72 and 96 h) with respect to the group fed an LFdiet, a relevant impairment of the immune defense has been widelydescribed (8, 13) (Fig. 5). Recent experimental observationshave shown that it is probable that the reduction of IFN- ${gamma}$ receptorexpression participates in part in the inefficient eliminationof L. monocytogenes after FO diet administration (14, 15). Inaddition to this important consideration, we suggest that themodulation of other cytokines by the action of fatty acids containedin FO may constitute a critical factor directly involved inthe capacity of immune cells to eliminate this pathogen.

View larger version (20K):
[in this window]
[in a new window]
FIG. 5. Schematic representation of the effects of the administration of a diet containing fish oil on host immune resistance to infection. Cytokine secretion is altered after FO administration, and cytokine levels are modified depending on the stage of the host response to Listeria monocytogenes. These variations involve a reduction of host natural resistance to infection that leads to different clinical consequences, such as reduction of survival, increase in the number of viable bacteria from spleen, and impairment of bactericidal capacity. The diagram summarizes the data contained in this paper as well as information from reference 24. ${uparrow}$ , increase; ${downarrow}$ , reduction; =, no alteration.

Overall, we hypothesize that the coordinated action of theseand others cytokines represents an important aspect that shouldbe analyzed in further studies to demonstrate the potentialaction of diets containing unsaturated fatty acids on the hostimmune defense against infectious microorganisms.

ACKNOWLEDGMENTS

This study was supported by grants from the Department of Educationand Science (Autonomous Government of Andalusia, Spain, GroupCTS105), the Ministry of Science and Technology (grant BSA2001-3648),and the University of Jaén. M. A. Puertollano receivesa fellowship from the Teaching and Research Staff Training Programme(Department of Education and Science, Autonomous Governmentof Andalusia, Spain). E. Puertollano receives a fellowship fromthe Ministry of Education and Science, Spain.

FOOTNOTES

* Corresponding author. Mailing address: Universidad de Jaén, Facultad de Ciencias Experimentales, Departamento de Ciencias de la Salud, Área de Microbiología, E-23071 Jaén, Spain. Phone: 34 953 212 003. Fax: 34 953 212 943. E-mail: mapablo{at}ujaen.es.

REFERENCES

Top