Role of PON2 in innate immune response in an acute infection model

doi:10.1016/j.ymgme.2013.07.003

Molecular Genetics and Metabolism

Volume 110, Issue 3, November 2013, Pages 362-370

https://doi.org/10.1016/j.ymgme.2013.07.003 Get rights and content

Highlights

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3OC₁₂-HSL is a quorum sensor produced by Pseudomonas aeruginosa (PAO1)
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PAO1 clearance is significantly reduced in PON2-def mice
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The phagocytosis function of PON2-def macrophages is inhibited by 3OC₁₂-HSL
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Increased nitration of PI3 kinase correlates with reduced phagocytosis function in PON2-def macrophages
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PON2 plays an important role in innate immune defense against PAO1 infection

Abstract

N-(3-oxododecanoyl)-l-homoserine lactone (3OC₁₂-HSL) is a quorum-sensing molecule produced by gram-negative microbial pathogens such as Pseudomonas aeruginosa (PAO1). 3OC₁₂-HSL is involved in the regulation of bacterial virulence factors and also alters the function of the host immune cells. Others and we have previously shown that paraoxonase 2 (PON2), a member of the paraoxonase gene family expressed in immune cells, hydrolyzes 3OC₁₂-HSL. In this study, we examined i) whether macrophage PON2 participates in 3OC₁₂-HSL hydrolysis, ii) the effect of PON2 deficiency in acute PAO1 infection in mice and iii) the effect of 3OC₁₂-HSL on PON2 deficient (PON2-def) macrophages. When compared to wild type macrophages, both intact cells and membrane-enriched protein lysates obtained from PON2-def macrophages show a marked impairment in their ability to hydrolyze 3OC₁₂-HSL. PON2 expression (message and protein) is not altered in response to 3OC₁₂-HSL in macrophages. 3OC₁₂-HSL treated PON2-def macrophages showed i) an increase in ER stress and oxidative stress, ii) defective phosphatidylinositol 3-kinase (PI3 kinase)/AKT activation, and iii) reduced phagocytosis function. Moreover, the nitration to phosphorylation ratio of Tyr458 in p85 protein, the regulatory subunit of PI3-kinase that has been correlated with the phagocytosis function of macrophages, was increased in PON2-def macrophages. Antioxidant treatment reversed the effects of PON2 deficiency in macrophage phagocytosis function. Furthermore, following administration of 1.6 × 10⁷ CFU of PAO1, bacterial clearance was significantly reduced in the lungs (5.7 fold), liver (2.5 fold), and spleen (14.8 fold) of PON2-def mice when compared to wild type mice. Our results suggest that PON2 plays an important role in innate immune defense against PAO1 infection.

Introduction

Pseudomonas aeruginosa (PAO1) is an opportunistic pathogen that causes a wide variety of acute infections in immune-compromised patients, as well as chronic respiratory infections in patients suffering from cystic fibrosis, cancer, and other chronic respiratory diseases [1]. To facilitate the establishment of infection, PAO1 produces cell-associated and extracellular virulence factors, which are regulated by quorum sensing (QS) [2]. QS is a mechanism wherein small diffusible molecules, specifically acyl homo serine lactones (AHL), are produced and detected by the organisms' surrounding molecules. As the number of bacteria increase, intracellular levels of AHLs achieve a threshold concentration, including the activation of targeted transcriptional regulators [3]. This process is well described for the formation of biofilm in chronic infection by PAO1 and 3OC₁₂-HSL; a member of the AHL family is shown to be the primary QS molecule [4]. Biofilms typically display a marked resistance to antibiotic killing and therefore infections associated with biofilm-forming bacteria are difficult to eradicate [5]. 3OC₁₂-HSL is shown to induce stress signaling in immune cells, reduce immune cell function, and improve bacterial survival. Hence, inactivation of this molecule or its effect on host cells is a therapeutic target for PAO1 associated disease.

Paraoxonases, a family of Ca^2 +dependent esterases consisting of PON1, PON2 and PON3 [6], hydrolyze 3OC₁₂-HSL [7]. The PON gene family is located on chromosome 6 in mice and on chromosome 7 in humans [8]. In humans, PON1 mRNA expression is limited to the lung and liver [8]. In contrast, hPON2 is ubiquitously expressed including kidney, liver, lung, placenta, small intestine, spleen, stomach, testis, and vascular cells. In humans, PON3 is restricted to the lung, liver, and kidney [8]. In addition, hPON1 and hPON3 are associated with HDL in the circulatory system whereas hPON2 protein is undetectable in HDL, LDL, or cell supernatants; it is, however, associated with intracellular membrane fractions [9]. A property shared by all PON proteins is the capacity to hydrolyze lactones [7]. In particular, AHLs have been identified as substrates common for all PON proteins, with PON2 exhibiting the highest specific activity [7].

Macrophages play a central role in response to extracellular and intracellular pathogens [10]. Our previous studies have shown that PON2 is expressed in this immune effector cell and plays an important role in macrophage function under chronic inflammatory conditions [11]. Based on this evidence, we hypothesized and tested whether PON2-def influences the innate immune response in an acute PAO1 infection model.

Section snippets

Animals

Male PON2-def mice on the C57BL6/J background and littermate controls, 8 to 10 weeks old, were used in all experiments. The Animal Research Committee at the University of California, Los Angeles approved all experiments.

Membrane-enriched tissue and cell homogenates

Wild type and PON2-def mice were injected intraperitoneally with 1.5 ml of a solution consisting of 3% thioglycollate broth. Three days later, macrophages were collected from the peritoneal cavity. Cells were homogenized on ice in 1 volume of 25 mM Tris, pH 7.4, containing 1 mM CaCl₂

Quorum quenching is impaired in macrophages from PON2-def mice

Macrophages are vital immune cells that contribute to innate immune defense whose function influences the quality, duration, and magnitude of most inflammatory reactions [17]. PON2 is highly expressed in macrophages and has been shown to alter their activation state [18]. Therefore, we first evaluated the capacity of membrane extracts from PON2-def peritoneal macrophages to inactivate 3OC₁₂-HSL. As shown in Fig. 1, PON2-def macrophage membrane lysates (Fig. 1A) as well as intact macrophages (

Discussion

PAO1 is ubiquitously present in the environment and is known to cause nosocomial infections. 3OC₁₂-HSL, a quorum-sensing molecule secreted by PAO1, participates in bacterial colonization and inhibition of host immune response. Anti-quorum-sensing molecules are therapeutic targets for treating PAO1 associated diseases. PON2 has been shown to inactivate 3OC₁₂-HSL in vitro. PON2 is expressed in immune cells; however, studies to date have only focused on the role of PON2 in atherosclerosis,

Conflict of interest statement

None.

Acknowledgments

We thank Yuen Lin Lee and Ani Shabazian for their expert technical assistance. This work was supported by the National Heart, Lung and Blood Institute grant 1RO1HL71776 (S.T.R).

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Previously, we have demonstrated that β-estradiol-3-benzoate (EB) has a protective effect on the neurodegenerative experimental model of Parkinson's disease. The protective effect is through the induction of the expression of paraoxonase-2 (PON2) in the striatum. PON2 has proven to have antioxidant and anti-inflammatory activity, this protein has a beneficial effect in MPP⁺ model in rats decreasing the lipid peroxidation and the oxidative stress. Furthermore, the molecular effect and the pathway by which EB induces protection were not further pursued. This study shows the regulation by EB of the anti-inflammatory effect through the modulation of cytokines, antioxidant enzymes and PON2 in the rat striatum. Rats were gonadectomized and 30 days after were randomly assigned into four experimental groups; only vehicles (Control group); EB treatment (EB group); MPP⁺ injury (M group); EB plus MPP⁺ injured (EB/M group). EB treatment consisted of 100 μg of the drug administered every 48 h for 11 days. Results showed that EB (group EB/M) treatment decrease significantly (40%) the number of ipsilateral turns respect to the M group and prevents significantly the dopamine (DA) decreased induced by MPP⁺ (~75%). This results are correlate with a significant decrease in the level of lipid peroxidation (60%) of the EB/M group respect to the M group. The EB treatment showed protection against neurotoxicity induced with MPP⁺, this could be due to EB capacity to prevent the increase in the expression level of proinflammatory cytokines TNF-α, IL-1 and IL-6 induced by MPP⁺. While, TGF-β1 and TGF-β3 expression was reduced in the rats treated only with MPP⁺, in the rats of EB/M group the expression of both cytokines was increased. EB protective effect against MPP⁺ neurotoxicity is related to antioxidant effect of PON2, pro-inflammatory cytokines and GSHR but not to SOD2, catalase, GPX1 or GPX4.
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¹: Present address: Eulerstrasse 64, 4051 Basel, Switzerland.

²: Present address: Department of Pathology and Lab Medicine, University of California at Los Angeles, Los Angeles, CA 90095, USA.

³: Present address: Shanti Clinical Trials (1880 East Washington St. Suite-A, Colton, CA 92324, USA.

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Role of PON2 in innate immune response in an acute infection model

Highlights

Abstract

Introduction

Section snippets

Animals

Membrane-enriched tissue and cell homogenates

Quorum quenching is impaired in macrophages from PON2-def mice

Discussion

Conflict of interest statement

Acknowledgments

Int. J. Med. Microbiol.

Free Radic. Biol. Med.

J. Lipid Res.

Free Radic. Biol. Med.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

FEBS Lett.

Mol. Genet. Metab.

Free Radic. Biol. Med.

FEMS Microbiol. Lett.

J. Investig. Dermatol.

Regulation of gene expression by cell-to-cell communication: acyl-homoserine lactone quorum sensing

Annu. Rev. Genet.

Quorum sensing in bacteria

Annu. Rev. Microbiol.

Listening in on bacteria: acyl-homoserine lactone signalling

Nat. Rev. Mol. Cell Biol.

Pseudomonas aeruginosa tssC1 links type vi secretion and biofilm-specific antibiotic resistance

J. Bacteriol.

Is it just paraoxonase 1 or are other members of the paraoxonase gene family implicated in atherosclerosis?

Curr. Opin. Lipidol.