Importance of phenol-rich virgin olive oil in the host modified polar fatty acids against L.monocytogenes infection in mice

Jasminka Giacomettia, Maja Abramb, Ivana Bogeticd, Hrvoje Krizana and Damir Muhvicc

University of Rijeka, School of Medicine, aDept. of Chemistry and Biochemistry, bDept. Microbiology and Parasitology, cDept. of Physiology and Pathophysilogy; dStudent of the School of Medicine, Brace Branchetta 20, HR-51000 Rijeka, CroatiaE-mail: jasminka@medri.hr

Animal studies generally support the idea that olive oil is capable of modulating functions of cells of the immune system. The major components of olive oil that influence some biological activities are high oleic acid and polyphenols. The Gram-positive facultative intracellular bacterium Listeria monocytogenes is a model pathogen for elucidating important mechanisms of the immune response. Olive oil diet produces an immunosuppressive state responsible for the impairment of host immune resistance against L.monocytogenes that hinders the efficient elimination of microorganisms. Here, we examine the influence of olive oil diet and isolated olive oil polyphenols on liver host polar fatty acids modification against L.monocytogenes infection in mice, helps in the current state of knowledge concerning the effects and interactions of olive oil on immune system functions, as well as the their action in the preservation of host immune defense.

Female Balb/c mice, 8–10 weeks old were divided into 3 groups as follows: Group 1 – fed standard diet; Group 2 – fed olive oil (5%,w/w); Group 3 - fed standard diet and then i.p. simultaneously injected saline phenolics extract (10 mg/kg) and subletal dose L. monocytogenes suspension (200 l, 2 x 107 CFU/ml). Each group had custom control (uninfected) animals. The evaluation of L.monocytogenes infection was carried out at the days 1, 3 and 6 after induced infection by the collection and processing of the serum, liver, brain and spleen. A third of the liver was used for histological analyses (H&E strain), and the second third was used for the bacterial growth determination by the colony forming unit (CFU per g of tissue). The last third was used for the determination of the polar fatty acid composition (PL FAs). Liver PL FAs were isolated and purified by solid-phase-extraction (SPE) method using an aminopropylsilica column and subsequently determined by gas chromatography (GC).

Calculations and Statistical Analysis

SFAs=% (14:0+16:0+18:0+20:0+22:0+24:0); MUFAs=% (14:1+16:1+18:1+20:1); PUFAs=% (PUFAn-3 + PUFAn-6); PUFAs n–

3 =% (20:5n-3+22:5n-3+22:6n-3);

PUFAs n–6 =% (18:2n-6+18:3n-6+20:2n-6+20:3n-6+20:4n-6+22:4n-6); D9 C18 index =18:1n-9/18:03; D6D index=[(18:3n-

6+20:3n-6)/18:2n-6]1; D5D index=[(20:4n-6/(20:3n-6)]1. Data are reported as mean ±S.D. Statistical significance was assumed with p<0.05. All statistical analyses were conducted using the Statistica software package for Windows, StatSoft, Inc. (2005), Version 7.1. The significance of the differences was analyzed by the Descriptive Statistics by Groups (Breakdown) - analysis of variance and Post-hoc Comparisons of Means using Scheffe test.

1. de Pablo, M.A., Puertollano, M.A., Galvez, A., Ortega, E., Gaforio, J.J., de Cienfuegos G.A. 2000. Determination of natural resistence of mice fed dietary lipids to experimental infection induced by Listeria monocytogenes. FEMS Immunol Med Microbiol. 27:127-133.2. Rodrigez Vaquero, M.J., Alberto, M.R., Manca de Nadra, M.C.. 2007. Influence of phenolic compounds from wines on the growth of Listeria monocytogenes. Food Control.18:587-593.3. Puertollano, M.A., de Pablo, M.A. i Álvarez de Cienfuegos, G. 2002. Relevance of Dietary Lipids as Modulators of Immune Functions in Cells Infected with Listeria monocytogenes. Clin Diagnostic Lab Immunol. 2002, 352–357.4. de Pablo, M.A., Puertollano, M. A. i de Cienfuegos, G.A. 2004. Olive oil and immune system functions: potential involvement in immunonutrition. Grasas y Aceites, 55(1):42-51.5. Puertollano, M.A., Puertollano, E., de Cienfuegos, G.A., de Pablo, M.A. 2007. Significance of olive oil in the host immune resistance to infection. Br J Nutr. 98(1):S54-S58.

INTRODUCTION

EXPERIMENTAL

References

CONCLUSIONS

L.monocytogenes significantly altered the fatty acid composition of the host liver membrane phospholipids during 6-day infection monitoring.The n-3/n-6 ratio was significantly increased in the group fed olive oil and olive oil polyphenols compared to the control.MUFA was higher in the group fed olive oil and olive oil polyphenols compared to the control.Bacterial growth depended on the examined organs; major bacteria were found in the spleen, and minor in the brain.The protective role of olive oil diet reflected on found minor bacterium number and the absence on pathohistological and metabolic liver damage.

Figure 2. a. Bacterial growth by the determination of colony forming unit (CFU per g of tissue) in the liver, brain and spleen;b. Histological analysis of the liver from female Balb/c mice: H & E-stained liver sections at the 1st, 3th and 6th days after infection treated mice in the group 1, 2 and 3; magnification = 100X.

DYNAMI CS OF EXPERIMENTS

Group 1 – fed standard diet and then 2x107 CFU/ml L. monocytogenes suspension i.p. injected into each mouse;

Group 2 – fed 3-wks before olive oil diet and then 2x107 CFU/ml L. monocytogenes suspension i.p. injected into each mouse;

Group 3 – fed standard diet and then phenolics extract and 2x107 CFU/ml L. monocytogenessuspension i.p. simultaneously injected into each mouse

OOP=10 mg/kg olive oil phenolics extracts (saline solution, i.p.)

Group 1Group 2Group 3

Serum, liver, spleen and brain collectionSTART of experiments

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Figure 1. a. Differences in the liver PL FAs profile.* significantly differences among groups were found by the Descriptive Statistics by Groups (Breakdown) - analysis of variance (p<0.05);b. Differences in the liver n-6, n-3, SFA, MUFA i PUFA FAs profile.* significantly different from the control were determined by the Descriptive Statistics by Groups (Breakdown) - Post-hoc Comparisons of Means using Scheffe test (p<0.05).

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Acknowledgements

This work was supported by the Croatian Ministry of Science, Project No. 062-0000000-0221 and 062-0621273-1235, DIOKI,d.d. Zagreb and The university of Rijeka foundation.