Quorum sensing molecule N-(3-oxododecanoyl)-l -homoserine lactone: An all-rounder in mammalian cell modification

Journal Pre-proof Quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone: An allrounder in mammalian cell modification Guo Jiajie, Kaya Yoshida, Mika Ikegame, Hirohiko Okamura PII:

S1349-0079(20)30001-3

DOI:

https://doi.org/10.1016/j.job.2020.01.001

Reference:

JOB 261

To appear in:

Journal of Oral Biosciences

Received Date: 1 December 2019 Revised Date:

9 January 2020

Accepted Date: 14 January 2020

Please cite this article as: Jiajie G, Yoshida K, Ikegame M, Okamura H, Quorum sensing molecule N-(3oxododecanoyl)-L-homoserine lactone: An all-rounder in mammalian cell modification, Journal of Oral Biosciences, https://doi.org/10.1016/j.job.2020.01.001. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V. on behalf of Japanese Association for Oral Biology.

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Quorum sensing molecule N-(3-oxododecanoyl)-L-homoserine lactone: An all-rounder

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in mammalian cell modification

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Guo Jiajiea, b*, Kaya Yoshidac, Mika Ikegamea, Hirohiko Okamuraa* a

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Department of Oral Morphology, Graduate School of Medicine, Dentistry and

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Pharmaceutical Sciences, Okayama University, 2-5-1 Shikata, Kitaku, Okayama 770-8525,

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Japan. b

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Department of Endodontics, School of Stomatology, China Medical University, Nanjing

north street 117, Shenyang 110002, China c

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Department of Oral Healthcare Education, Institute of Biomedical Sciences, Tokushima

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University Graduate School, Tokushima, 770-8504, Japan.

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*Address correspondence to:

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Guo Jiajie, DDS, PhD

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Tel: 81-86-235-6630; Fax: 81-86-235-6634

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E-mail: [email protected]

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Hirohiko Okamura, DDS, PhD,

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Tel: 81-86-235-6630; Fax: 81-86-235-6634;

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E-mail: [email protected]

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Abstract:

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Background: Bacteria exhibit multi-cellular social behavior, such as biofilm formation,

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virulence generation, bioluminescence, or sporulation, through cell-to-cell communication

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involving a quorum sensing (QS) system capable of sensing species density. Pseudomonas

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aeruginosa (P. aeruginosa) is a ubiquitous gram-negative opportunistic pathogen that is

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frequently isolated from immunocompromised patients. It is particularly detected in patients

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with severe periodontitis and persistent endodontic infections, forcing a rethink of the role of

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this opportunistic pathogen in oral lesions.

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Highlight: N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) is a pivotal QS molecule,

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which regulates numerous virulence genes in P. aeruginosa and exhibits broad biological

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modulation effects in mammalian cells. In this review, we highlight the diverse

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OdDHL-mediated apoptosis and immunomodulatory effects on host cells. The structural

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properties, signaling pathways, targeted genes and proteins, and intracellular metabolism of

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OdDHL are also discussed to clarify the interactions between P. aeruginosa and the host.

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Conclusion: The purpose of this review is to identify a valid target for quenching OdDHL,

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which could potentially eliminate the pathogenic effect of P. aeruginosa.

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Key words: Quorum sensing, N-(3-oxododecanoyl)-L-homoserine lactone, apoptosis,

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immunomodulatory, mammalian cells

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1. Introduction

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Humans have always cooperated to perform complex social activities that any one individual

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cannot accomplish, thereby promoting progress and development. Research over the past 50

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years has revealed that bacteria, which preceded human beings and have evolved for hundreds

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of millions of years, have similar cooperative relationships based on intercellular

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communication, known as quorum sensing (QS) [1]. In 1976, Nealson found that the

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bioluminescence of Vibrio fischeri (V. fischeri) was positively correlated with bacterial

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density [2]. In 1994, Fuqua proposed the idea of QS to describe the luxR-luxI gene regulation

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process controlled by bacterial population [3]. In general, QS is defined as microbial

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cell-to-cell communication that operated by sensing population density through signaling

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molecules termed autoinducers (AIs). AIs are synthesized by LuxI and then diffuse into the

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surroundings. If the local bacterial density reaches a certain threshold, AIs will be transported

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into bacteria and bind to the cytoplasmic receptor LuxR, which enables individual bacterial

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cells to simultaneously initiate an array of gene expression and cellular responses, such as

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biofilm formation, virulence generation, bioluminescence, sporulation, antibiotic production,

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and competence [3-6]. Four QS systems that utilize diverse AIs have been confirmed. These

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are the N-acyl homoserine lactone (AHL)-based QS in gram-negative bacteria, the

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oligopeptide-based QS in gram-positive bacteria, the 4, 5-dihydroxy-2, 3-pentanedione (DPD,

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AI-2)-based QS between gram-negative bacteria and gram-positive bacteria, and the

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quinolone-based QS in some special bacteria, including species of the genera Alteromonas,

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Burkholderia and Pseudomonas [4, 7, 8]. Information exchange between these QS systems is

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akin to that in multi-cellular organisms that demonstrate social behavior, thereby enabling

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completion of biological processes that are impossible for individual cells, and permitting

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increasingly complex bacterial biological activities from a community perspective [6, 9].

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Pseudomonas aeruginosa (P. aeruginosa) is a ubiquitous gram-negative opportunistic

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pathogen that has multiple QS systems. It is an important nosocomial pathogen-associated

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with hospital acquired infections, including wounds, burns, and pulmonary infections,

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especially in debilitated or immunocompromised patients [10, 11]. Bacteremia and sepsis

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associated with these infections through blood transport can cause death. The presence P.

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aeruginosa in oral flora can result in severe periodontitis as well as persistent endodontic

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infections, which are not merely a transitory event [12-17]. The ability to produce biofilms

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and the resulting resistance to broad spectrum antibiotics may help P. aeruginosa to cause

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these prolonged infections [18, 19]. However, most importantly, P. aeruginosa may use QS to

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deploy its own arsenal in defending its niche from host immune counterattack [20]. The first

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confirmed QS system in P. aeruginosa was lasR-lasI, which is homologous to the V. fischeri

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luxR-luxI, which employs N-(3-oxododecanoyl)-L-homoserine lactone (OdDHL) as an AI [21,

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22]. The second QS system discovered was rhlR-rhlI, which is responsible for the

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biosynthesis of rhamnolipids and utilizes N-butyryl-L-homoserine lactone (BHL) for

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cell-to-cell

communication

[23,

24].

The

pseudomonas

quinolone

signal

(PQS)

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2-heptyl3-hydroxy-4-quinolone, which is regulated by the pqsR-pqsABCDH gene, was

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identified later. It is structurally distinct from the homoserine lactone (HSL) signals of the las

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and rhl QS systems [25]. In 2013, the fourth intercellular communication signal QS (IQS)

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was identified. It is based on the 2-(2-hydroxyphenyl)-thiazole-4-carbaldehyde AIs [26].

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These QS systems govern more than 10% of the P. aeruginosa genes. These genes are mainly

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involved in virulence factors including elastase, alkaline protease, exotoxin A, pyocyanin,

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phospholipase C, rhamnolipids, and others [27, 28] (Figure 1). However, numerous studies

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have suggested that the AIs, especially OdDHL, regulate a range of complex biological

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processes with respect to immunity and disease in host cells, in addition to communication

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across species. This review provides an overview of OdDHL, focusing on the regulatory

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crosstalk between itself and mammalian cells.

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2. Characteristics of OdDHL

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The conserved residues at the N-terminus of LasI form an enclosed active site for

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S-adenosylmethionine (SAM), which is the substrate of the homoserine lactone ring. The

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acyl-acyl carrier protein (acyl-ACP) then binds to SAM to generate acyl-SAM through the

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removal of ACP. OdDHL is produced in the next step through the release of

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5’-methylthioadenosine (MTA) from acyl-SAM by the lactonization of methionine [29-31]

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(Figure 2). OdDHL is composed of the homoserine lactone ring carrying acyl chains that are

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12 carbons in length. The L-homoserine lactone unit, the oxo group, as well as the length of

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the carbon chains, is crucial for the structural stability and biological activity of OdDHL [32,

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33]. Esterase hydrolyzes the lactone ring to yield an open-loop structure, which inactivates

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OdDHL. The homoserine lactone structures with long acyl carbon chains are less prone to

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hydrolysis than the shorter chains with changes in temperature and pH [34]. BHL, which has

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only four-carbon chains, is another class of AIs in P. aeruginosa. BHL displays weaker

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biological activity in mammalian cells in many cases, compared to OdDHL. Therefore, full

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functionality of OdDHL is dependent on its structural characteristics and stability. OdDHL

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also exhibits structural and functional similarities to mammalian lipid-based hormones, which

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are excellent candidates for mediating biological functions of mammalian cells [35]. OdDHL

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can be rapidly transported into mammalian cells and displays intracellular activity through

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passive mechanisms because of its lipid solubility and membrane permeability. The

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intracellular concentration of OdDHL trapped in the cells is proportional to the extracellular

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concentration [36]. We have demonstrated that green fluorescence tagged OdDHL enters

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osteoblasts within 15 minutes and quickly assembles in many organelles, such as

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mitochondria and endoplasmic reticulum (ER) (Figure 3). In human epithelial and fibroblast

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cell lines, the maximum concentration of cytosolic OdDHL is achieved within 20 to 30

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minutes without the requirement for energy. The OdDHL is then released from the cells

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through an ABC transporter in an energy-dependent manner. During this process, many

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transport-related and metabolic genes are expressed in response to OdDHL, not as a general

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response to bacterial cell products or AHL, since BHL and AI-2 have only minimal effects

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[37].

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3. OdDHL-induced cell death is associated with apoptosis

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Apoptosis is a mechanism of programmed cell death that occurs through a series of

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endogenous gene regulation processes. It can be stimulated by three different pathways: The

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first is the intrinsic pathway (mitochondria pathway), which releases cytochrome c from

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mitochondria and activates the Caspase pathway as a downstream signal. The second is the

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extrinsic pathway, also known as the death receptor pathway, which activates the intracellular

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Caspase-8 and Caspase-10 by the binding of death receptor ligands (such as tumor necrosis

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[TNF] or Fas) to the membrane-bound death receptors. The third is the ER pathway induced

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by ER stresses, such as protein misfolding or unfolding, which lead to intracellular calcium

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overload and activation of Caspase-12 and Caspase-9-dependent apoptosis. Each of these

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pathways lead to a final execution phase with the activation of Caspases-3 and/or -7, and

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generally lead to the cleavage of different proteins [38, 39]. Ultimately, apoptosis involves

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cell shrinkage, pyknosis, chromatin condensation, nuclear DNA cleavage, extensive plasma

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membrane blebbing, as well as apoptosome formation [40]. Many studies have shown that

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OdDHL triggers apoptosis in eukaryotic cells through these three apoptotic pathways (Figure

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4).

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3.1 OdDHL and intrinsic apoptosis

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Induction of apoptosis by OdDHL in several mammalian cell types have been well

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documented. These include epithelial cells [41, 42], endothelial cells [43], fibroblasts [43, 44],

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mesenchymal stem cells [45], sperm cells [46], lymphocytes [47], dendritic cells [48], mast

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cells [49], macrophages, and neutrophils [50]. Among the three aforementioned apoptotic

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mechanisms, OdDHL-mediated cell death predominantly employs the intrinsic pathway.

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Apoptosis induced by OdDHL is concentration-dependent and generally occurs within 1 hour

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at the micromolar level [42]. Apoptosis primarily begins with OdDHL-induced mitochondrial

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damage, which is accompanied by changes in membrane depolarization and permeabilization.

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Cytochrome c is then released from the mitochondria to the cytosol and in turn activates a

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cascade of signaling events involving Caspases-9, -3, and -7. Apoptosis follows with

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cytoskeletal lysis and DNA degradation [42, 47, 51].

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Multiple genes are involved in the regulation of OdDHL-mediated apoptosis in host cells.

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Paraoxonase 2 (PON2) is an enzyme that acts as a cellular antioxidant to protect cells from

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oxidative stress. In the epithelial cells, OdDHL is rapidly hydrolyzed intracellularly by PON2

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to OdDHL acid, which becomes trapped in the mitochondria and rapidly induces intrinsic

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apoptosis [52]. In the Caco-2 human intestinal epithelial cell line, suppression of Akt

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phosphorylation or activation of extracellular signal-regulated kinase (ERK)1/2 enhances

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OdDHL-induced apoptosis. During this process, a vital component of the gut barrier protein

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Mucin 3 (MUC3), which is regulated by ERK1/2, alleviates the cell death effect of OdDHL,

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since OdDHL does not induce cell death in differentiated Caco-2 cells that express higher

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levels of MUC3, compared to undifferentiated cells [53, 54]. X-box binding protein 1

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transcription factor (XBP1s) is a transcription factor belonging to the CREB/ATF basic

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region-leucine zipper family. Its leucine zipper and transcriptional activation domains

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mediates the apoptotic response in mouse embryonic fibroblasts (MEFs) treated with OdDHL

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[55].

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The B cell lymphoma-2 (Bcl-2) family of proteins including anti-apoptotic (Bcl-2, Bcl-xL,

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and others) and pro-apoptotic members (Bax, Bak, Bid, and others) are important for the

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integrity of the mitochondrial membrane in the intrinsic apoptosis pathway [56]. OdDHL

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rapidly induces apoptosis in human Jurkat T lymphocytes by reducing the mitochondrial

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transmembrane potential. Overexpression of Bcl-2 completely abrogates the apoptotic effects

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such as the processing of Caspase-8 and poly ADP-ribose polymerase (PARP) [47]. However,

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the pro-apoptotic effect of OdDHL likely occurs without the involvement of Bak or Bax,

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since wild-type and Bak−/−Bax−/− (DKO) MEFs exhibit similar mitochondrial depolarization

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and cytochrome c release. Bak or Bax expression in double knock-out (DKO) MEFs also did

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not promote Caspase-3/7 expression during the treatment with OdDHL [44]. This presumes

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that the apoptotic response triggered by OdDHL is not limited to mitochondria-dependent

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intrinsic pathways.

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3.2 OdDHL and extrinsic apoptosis

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The death receptor family includes eight species. Two prominent species are the TNF receptor

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1 (TNFR1) and the Fas receptor. TNFR1 undergoes trimerization after binding with TNF

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followed by binding of the activated TNF/TNFR1 complex with the TNFR-associated death

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domain (TRADD), leading to the formation of death-inducing signaling complex, which

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results in pro-Caspase-8 activation. Active Caspase-8 either induces Bid or Caspase-3/7. Both

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are involved in the final intrinsic apoptosis pathway [57]. Although extensive research has

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confirmed that OdDHL can trigger pro-Caspase-8 activation, indicating that the extrinsic

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pathway probably participates in OdDHL-mediated cell death [42, 44, 50, 55], there is no

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evidence confirming the existence of death receptors targeting OdDHL in host cells. However,

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a novel mechanism supporting this theory was recently reported. Song et. al proposed that

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OdDHL disrupted the well-organized lipid domains in the mammalian cell membrane. The

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TNFR1that originally inserted in the lipid membrane is expelled into the disordered phase of

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the membrane, activating TNFR1 through spontaneous trimerization without an external

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ligand. The downstream Caspase-8-Caspase-3 axis is successively activated, leading to

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apoptosis [58]. These findings imply that the unique structure of OdDHL with its long acyl

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chains as well an oxo group at position 3 allows it to be uniquely recognized by mammalian

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cells, compared with other pathogen-associated molecular patterns (PAMPs).

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3.3 OdDHL and ER- Ca2+-related apoptosis

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ER is an important organelle responsible for post-translation protein modification, folding,

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and oligomerization. It is also involved in calcium (Ca2+) storage, signal transduction, and

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oxidative stress. Emerging evidence suggests that the ER also mediates apoptosis by

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sensitizing the mitochondria to various intrinsic and extrinsic death stimuli, or by initiating

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apoptotic signals of its own [59]. Ca2+ is a ubiquitous intracellular signal messenger

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responsible for a variety of physiological and pathological processes. The disturbance and

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overload in intracellular Ca2+ released by ER has a strong relationship with ER stress and

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mitochondria-induced apoptosis [60]. OdDHL triggers a dramatic Ca2+-dependent apoptotic

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process mainly through the disruption of mitochondrial activity in human vascular endothelial

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cells [43], epithelial cells [42, 61, 62], sperm cells [46], and murine fibroblasts [43]. Typically,

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the release of Ca2+ from the ER is primarily achieved by the inositol 1,4,5-triphosphate

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receptor (IP3R) or the Ryanodine receptor (RyR) channels. But, OdDHL induces intracellular

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Ca2+ release mainly through IP3R and not RyR [61, 63]. In fact, OdDHL targets

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phospholipase C (PLC), which can cleave phosphatidylinositol into 1,2-diacylglycerol and

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IP3. IP3 rapidly diffuses through the cytosol and binds to IP3 receptors, leading to the

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opening of the Ca2+ channel valve. Hence, Ca2+ release triggered by OdDHL can be blocked

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by a PLC inhibitor. Inhibition of Ca2+ signaling rescues apoptosis, but not the

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immunomodulatory effects, in MEFs treated with OdDHL, which indicates the existence of at

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least two signal transduction pathways in mammalian cells in response to OdDHL [43].

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Apart from apoptosis, OdDHL can also induce the release of reactive oxygen species (ROS)

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in human platelets [63] and intestinal goblet cells [64, 65], causing premature loss of the

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acrosome in sperm cells [46]. This leads to pro-inflammatory cytokine production [66] or

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decrease in cell gap junctional intercellular communication (GJIC) [41] in airway epithelial

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cells, which are Ca2+-dependent processes. The transient intracellular Ca2+ changes activated

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by OdDHL are critical for diverse signal transduction mechanisms in mammalian cells [67].

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3.4 Application of the pro-apoptotic effect of OdDHL in tumor therapy

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Although OdDHL has a strong pro-apoptotic effect, not all cell types are sensitive to it.

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Several types of cells, such as human polarized epithelial cells [41], CD8 (+) dendritic cells

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[48], and human primary macrophages [68], do not undergo apoptosis in response to OdDHL.

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The anti-tumor effect of OdDHL has been extensively studied in different kinds of cells. It

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has been shown that OdDHL induces apoptosis and alters viability in both prostate

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adenocarcinoma and prostate small cell neuroendocrine carcinoma cells (PC3) in a

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concentration-dependent manner, but not in RWPE1 noncancerous prostate epithelial cells

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that are resistant to modification by OdDHL [69]. OdDHL also has potent effect on apoptosis

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promotion and colony inhibition in pancreatic carcinoma cell lines (Panc-1 and Aspc-1)

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compared with the minimal effect on human pancreatic duct epithelial cells (HPDE) [70].

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Similarly, OdDHL can markedly decrease the viability of the more malignant human breast

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adenocarcinoma cells (MDA-MB-231) and an intermediate ductal carcinoma cell line

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(MCF-DCIS). In contrast, non-malignant breast epithelial cells (MCF-10A) do not display

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any change in viability in any culture condition under OdDHL treatment [71]. The

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diametrically opposite response of tumor cells and normal cells to OdDHL may be attributed

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to PON2 overexpression in tumors, which induces mitochondrial membrane permeabilization

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that is independent of both pro- and anti-apoptotic Bcl-2 proteins in tumor cells [72].

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Interestingly, OdDHL also reduces cell motility in tumor cells (PC3, Panc-1) by modulating

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the

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GTPase-activating protein (IQGAP). These proteins are involved in linking integrin adhesion

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molecules to the actin cytoskeleton and are related to cell metastasis [69, 70].

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4. OdDHL and host immunity

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Immunity is a defense mechanism in organisms that gradually develops during long-term

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development and evolution of germ lines. Immunity is classified as innate and adaptive

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immunity. Innate immunity mainly consists of epithelial and mucosal barriers, phagocytes,

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immune molecules like cytokine, antimicrobial peptides, and complement molecules. The

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innate immune response does not recognize every possible antigen, but focuses on numerous

membrane-cytoskeletal

proteins

vinculin,

RhoC

and

IQ-motif-containing

244

conserved structures presents in diverse pathogens containing PAMPs [73]. Adaptive or

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specific immunity is the specific response of lymphocytes to specific antigens producing

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immune memory effects. Hence, adaptive immunity plays a key role in the complete

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elimination of pathogens and prevention of re-infection. As a bridge molecule for QS signals

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in P. aeruginosa, OdDHL has been widely described as an important component of the host

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immunity, assisting P. aeruginosa to build a comfortable ecological niche or escape the host's

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immune defense.

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4.1 OdDHL disrupts the junctions between epithelial cells

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Epithelial cells are strategically positioned to play a vital role in the first line of host defense

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through physical and immune barriers. The epithelial junctional complex includes gap

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junctions (GJ), tight junctions (TJ), adherent junctions (AJ), and desmosomes. GJs are

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channels between neighboring cells. These channels consist of six donut-like structural

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membrane proteins termed connexons, through which exchange of water, ions, and other

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substances between cells is achieved. This process is defined as GJIC [74]. Src kinases are

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responsible for the remodeling of the cytoskeleton and are linked to Ca2+ homeostasis. In

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nonpolarized airway epithelial cells, OdDHL can induce both Ca2+ influx and Src activation,

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subsequently decreasing GJIC, which indicates the loss of airway epithelial junctional

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integrity [41]. The TJ creates a watertight seal between two adjacent cells. At the site of a TJ,

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cells are held tightly against each other by many individual groups of TJ proteins that include

263

occludin, tricellulin, claudins, zonula occludens (ZO), and junctional adhesion molecule

264

(JAM). AJ is defined as a cell junction whose cytoplasmic face is linked to the actin

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cytoskeleton. The main molecular component of the AJ is the transmembrane protein

266

E-cadherin [75]. In intestinal epithelial cells, OdDHL induces intracellular Ca2+ signaling and

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alteration in the phosphorylation status of E-cadherin, β-catenin, occludin, ZO-1, ZO-3, and

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JAM-A. These events change the association between JAM-A-ZO-3 protein complexes,

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decrease transepithelial electrical resistance, disrupt the function of the epithelial barrier, and

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enhance paracellular permeability [61, 76-78]. Lipid raft- and protease-activated receptor

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(PAR)-dependent matrix metalloprotease (MMP)-2/3 activation is also involved in

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OdDHL-induced alteration of epithelial paracellular barrier function through degradation of

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occludin and tricellulin in intestinal epithelial cells [79]. The collective findings indicate that

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OdDHL is closely related to the destruction of intercellular links, which may provide the

275

means for P. aeruginosa to invade the host (Figure 5).

276

4.2 OdDHL-mediated immune cell chemotaxis

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Polymorphonuclear neutrophils (PMNs) are the main effector cells of the host against

278

extracellular pathogen infection. PMNs usually enter an infected site through phagocytosis

279

and kill pathogens. Most pathogenic infections are terminated at this phase. PMNs also

280

secrete chemokines, recruit macrophages and mast cells to the site of infection, and

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participate in enlarged inflammatory responses and uptake of foreign substances. OdDHL

282

enhances interleukins-8 (IL-8) mRNA and protein expression in the16HBE human bronchial

283

epithelial cell line. The supernatant of 16HBE treated with OdDHL was shown to induce

284

greater chemotaxis of neutrophils compared to that induced by the addition of anti-human

285

IL-8 antibody [80]. However, OdDHL is able to independently induce neutrophil chemotaxis

286

as well, without relying on the well-studied pertussis toxin-sensitive G proteins that

287

complement C5a and IL-8. In contrast, inhibition of tyrosine kinases, phospholipase C (PLC),

288

protein kinase C (PKC), and the mitogen-activated protein kinase (MAPK) reduces

289

chemotaxis towards OdDHL, indicating that OdDHL uses another signaling pathway [81, 82].

290

OdDHL also upregulates the expression of adhesion proteins, such as CD11b/CD18, which is

291

related to chemotaxis and phagocytosis of bacteria by PMNs [83] (Figure 5). However, the

292

migration derived by chemotactic agents prostaglandin E2 (PGE2) and stem cell factor in

293

mouse bone marrow-derived mast cell can be markedly reduced by OdDHL treatment [84].

294

These findings contrast with descriptions that OdDHL can trigger apoptosis in mouse

295

neutrophils [50] and mast cell lines [49]. This may be due to differences in the species used

296

and the concentration of OdDHL, reflecting the biological complexity of OdDHL. However,

297

it is interesting to note that OdDHL also interacts and co-localizes with IQGAP1, modulating

298

epithelial cell migration and promoting wound healing [85].

299

4.3 OdDHL-mediated host immune inflammatory response

300

The inflammatory response is the most important step in the immune system reaction against

301

pathogen infection. Damaged or infected cells release pro-inflammatory factors such as ILs

302

(responsible for the association between leucocytes), chemokines (promote cell chemotaxis),

303

and antiviral interferon (INF), which recruit immune cells to the infection site and eradicate

304

pathogens. There are also anti-inflammatory cytokines that balance this response. If the

305

pathogen releases more anti-inflammatory factors than pro-inflammatory factors, the

306

pathogen may be able to escape damage from the immune system. It has been demonstrated

307

that OdDHL is a versatile immunomodulatory factor which displays opposite effect on the

308

host immune response (Figure 6).

309

OdDHL can induce numerous pro-inflammatory cytokines and immunomodulatory factors,

310

such as IL-6 and IL-8, in epithelial cells [66, 86]; IL-1β, IL-8, and cyclooxgenase-2 (Cox-2)

311

in endothelial cells [87]; Cox-2 and PGE2 in fibroblasts [88]; IL-1β and IL-8 in mesenchymal

312

stem cells (MSCs) [45]; IL-6 and histamine in mast cells [49], as well as IL-1, IL-6,

313

macrophage inflammatory protein 2 (MIP-2), MIP-1β, monocyte chemotactic protein 1

314

(MCP-1), inducible protein 10, and helper T cell 1 (Th1) activation in mouse skin [89]. The

315

upstream unfolded protein response (UPR) related to Ca2+, activator protein-2 (AP-2), nuclear

316

factor-kappa B (NF-κB), P38 and the ERK- MAPK signaling pathway regulate the

317

pro-inflammatory effect of OdDHL [49, 66, 80, 87, 90-92]. IL-1 and IL-6 mediate acute

318

phase response and are crucial to the host defense of P. aeruginosa [49]. IL-8, a chemokine

319

produced by macrophages and other cell types, such as epithelial cells and endothelial cells, is

320

required for immunocyte migration and phagocytosis. Thus, the ability of OdDHL-induced

321

inflammation may be important for the development of acute infections caused by P.

322

aeruginosa, such as acute pneumonia [93], since excessive inflammatory reactions can result

323

in extensive destruction of host tissues.

324

On the contrary, OdDHL can also attenuate host activated immune responses stimulated by

325

other PAMPs. Lipopolysaccharide (LPS) acts as the prototypical endotoxin because it binds to

326

toll-like receptors (TLRs) in many cell types, especially monocytes, dendritic cells,

327

macrophages, and B cells, which promote the secretion of pro-inflammatory cytokines, nitric

328

oxide, and eicosanoids. However, OdDHL suppresses LPS-stimulated TNF-alpha (ߙ), MCP-1,

329

and IL-12 in macrophages [90, 94-96], INF-gamma (γ) in spleen cells [97], IL-12 in T-cells

330

[96], and TNF-ߙ in mast cells [84] by inhibiting UPR, NF-κB, or p38 MAPK activity. In

331

addition, OdDHL also promotes the expression of anti-inflammatory factors involving IL-10

332

and human leukocyte antigen-G (HLA-G) in macrophages [90, 94]. IL-10 suppresses the

333

phagocytic activity of neutrophils. Its expression is promoted during bacterial infections as a

334

mechanism of immune suppression [98]. HLA-G is involved in immune tolerance by

335

inhibiting cytolytic functions of natural killer cells, cytotoxic T lymphocytes, and dendritic

336

cells [99]. This implies that OdDHL evades host immune defense by affecting the ratio of

337

pro-inflammatory and anti-inflammatory factors. In addition, the inflammasome is a major

338

inflammatory complex activated by Caspase-1 in the cytoplasm. It triggers pro-inflammatory

339

and antimicrobial responses by inducing IL-1β and IL-18 generation [100]. OdDHL directly

340

suppresses the nucleotide-binding domain and the leucine-rich repeat Caspase recruitment

341

domain 4 (NLRC4), and nucleotide-binding domain and leucine-rich repeat pyrin domain 3

342

(NLRP3)-mediated inflammasome assembly and activation stimulated by LPS. In contrast,

343

mutant P. aeruginosa defective in OdDHL induces robust activation of the NLRC4

344

inflammasome [101].

345

OdDHL can also dampen adaptive immunity by altering the activation of T lymphocytes

346

(T-cells). Dendritic cells (DCs) are the most powerful antigen-presenting cells that mediate

347

the development of T-cells into helper T-cells (Ths) and initiate the adaptive immune response.

348

Normally, Th1 cells secrete IL-2, IFN-γ, and TNF-ߙ, which mainly mediate the

349

inflammation-related immune response and play a key role in the host anti-intracellular

350

pathogen infection. Th2 cells mainly secrete IL-4, IL-5, and IL-10 and can create an

351

immunosuppressive effect by down-regulating the activity of antigen-presenting cells and

352

Th1 cells. Besides polarizing conventional Th1 and Th2 cells, DCs can also generate

353

inducible regulatory T-cells (iTregs), which mainly inhibit the inflammatory autoimmune

354

response. In human monocyte-derived DCs (Mo-DCs) and mixed lymphocyte DCs treated

355

with LPS, OdDHL inhibits the expression of IL-12p70, IFN-γ, IL-6, and TNF-ߙ, which are

356

mainly responsible for the development of Th1 cells. In contrast, OdDHL upregulates IL-10

357

and TGF-β positive iTregs as well as IL-4 and IL-10 positive Th2 cell generation [48, 102]. In

358

bone marrow-derived DCs (BM-DCs), OdDHL suppresses the production of IL-12 (Th1

359

cytokine) by LPS-stimulated BM-DCs without altering their IL-10 release [103]. Similarly,

360

OdDHL can shift immune responses away from host protective Th1 responses to pathogen

361

protective Th2 responses by respectively affecting IFN-γ or IL-4 production in murine spleen

362

cells [97]. Consequently, OdDHL inhibits the adaptive immune response by regulating

363

Th1/Th2 imbalance. The collective data imply that OdDHL might assist P. aeruginosa in host

364

invasion by suppressing the adaptive immune response. Considering that P. aeruginosa is

365

often detected in chronic pulmonary infections and periapical persistent infections, the

366

immunosuppressive effect of OdDHL is most likely to emancipate P. aeruginosa from the

367

immune monitor and allow its survival in the host for decades.

368

The distinct role of OdDHL in host immunity remains unclear. The exact nature of these

369

effects may be dependent on cell types and the concentration and duration of exposure to

370

OdDHL [104]. Environmental conditions underlying the immune status of the host may also

371

lead to contrary results. As described above, the pro-inflammatory effect induced by OdDHL

372

always occurs in resting cells compared to anti-inflammatory effects in activated cells

373

stimulated by LPS. The immunomodulatory effects exhibited by OdDHL may also vary

374

according to the type of P. aeruginosa infection.

375

4.4 Mammalian cell receptor-regulated OdDHL signaling

376

The host organism has a wide-ranging immune response repertoire towards microbial

377

invasion, in order to limit the infection and replication of the pathogens, and eliminate them.

378

This defense mechanism always begins with the recognition of PAMPs by pattern recognition

379

receptors (PRRs), which activate intracellular downstream signaling factors that include the

380

transcription factor NF-κB and MAPK, and then regulate the expression of immune factors.

381

OdDHL stimulates various host signaling pathways, especially for the positive and negative

382

regulation of NF-κB and MAPK, implying that OdDHL may also be recognized by PRRs,

383

like other PAMPs. However, it has been shown that the presence of the canonical PRRs, such

384

as TLRs or nucleotide-binding oligomerization domain-like receptors (NLRs), is not required

385

in OdDHL-regulated signaling [105, 106].

386

The hydrophobic nature of OdDHL allows it to attach to the cell membrane and be

387

transported into the cytoplasm. However, OdDHL is unlikely to be directly integrated with

388

NF-κB or MAPK, but rather possibly acts through certain cellular receptor-mediated

389

signaling pathways. Taste receptor type 2 member 38 (T2R38), first identified on taste bud

390

cells for sensing bitter taste, has been confirmed to bind to OdDHL [107, 108]. Meanwhile,

391

T2R38 is not limited to the tongue, but is also expressed on the apical surface of the human

392

upper respiratory epithelium as well as peripheral blood neutrophils, monocytes, lymphocytes,

393

and intestinal cells. T2R38 may serve a sentinel role in detecting microbial QS

394

communications and regulating localized innate biocidal defenses [107, 109-111]. For

395

example, in sinonasal epithelial cells, T2R38 can be stimulated by OdDHL and trigger nitric

396

oxide (NO) production in a Ca2+-dependent manner, increasing ciliary beat frequency and

397

mucus clearance [109, 111, 112]. However, antibodies of T2R38 only partially inhibit the

398

binding of OdDHL and do not inhibit OdDHL-mediated activation of neutrophils [107],

399

implying that OdDHL relies on other recognition mechanisms besides the T2R38 pathway.

400

Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor

401

family. They act as ligands for transcriptional regulators stimulated by liposoluble signaling

402

compounds. Ligand activated PPARγ represses NF-κB transcriptional activity and alleviates

403

the inflammatory response. Rosiglitazone and pioglitazone are agonist ligands of PPARγ.

404

They are widely used in the treatment of inflammation-related nephropathy. But, it has been

405

demonstrated that even 1 nM OdDHL can interfere with rosiglitazone for binding with PPARγ,

406

which possibly relieves the NF-κB transcriptional activity by acting as an antagonist and

407

exacerbating infection-associated inflammation [113, 114]. This has been partially confirmed

408

by the low level of PPARγ mRNA in cystic fibrosis patients who also display a

409

hyperinflammatory response caused by P. aeruginosa [115]. Other studies have indicated that

410

activation of PPARγ can enhance macrophage clearance of P. aeruginosa and attenuate P.

411

aeruginosa biofilm formation [116, 117]. These collective findings indicate that OdDHL is

412

likely to interfere with important inflammatory signaling mechanisms in eukaryotic cells by

413

inhibiting PPARγ activation.

414

However, the aforementioned receptors are only involved in the conduction of

415

pro-inflammatory signals induced by OdDHL. The observations cannot explain all the

416

biological effects of OdDHL, such as immunosuppression. Therefore, in view of the complex

417

biological functions of OdDHL, it is necessary to continue to find possible binding targets in

418

eukaryotes.

419

5. Crosstalk between PON2 and OdDHL

420

Crosstalk between OdDHL and mammalian cells is mutually inhibitory. For instance, OdDHL

421

can induce a cellular immune response and cells can secrete a broad range of enzymes to

422

destroy the structural integrity of OdDHL. The paraoxonase (PONs) family (PON1, PON2,

423

and PON3) is a class of Ca2+-dependent esterases that have organophosphate detoxification

424

and oxidative stress mediation activities [118]. Due to lactonase activity, PONs can

425

effectively hydrolyze OdDHL to their ring-opened biologically inactive carboxylic acid

426

counterparts [119-121]. Unlike PON1 and PON3, which are mainly present in serum, PON2

427

is ubiquitously expressed in many tissues and cell types. PON2 has the strongest OdDHL

428

inactivation activity compared with PON1 and PON3 [122]. PON2 protects the integrity of

429

the epithelium in human or murine airway epithelial cells as a key defense mechanism against

430

the OdDHL. PON2-deficient mice or nonpolarized epithelial cells that express a low level of

431

PON2 are impaired in their ability to hydrolyze OdDHL, and P. aeruginosa QS is enhanced in

432

cultures of these epithelial cells [41, 123, 124]. In PON2-defcient mice, clearance of P.

433

aeruginosa is also dramatically slower. The impaired ability of macrophages to inactivate

434

OdDHL results in reduced phagocytosis by inhibiting phosphoinositide 3 kinase/AKT

435

activation, but promotes ER and mitochondrial oxidative stress [125]. These findings

436

demonstrate that PON2 plays a critical role in the innate defense against OdDHL. In

437

comparison, OdDHL also suppress PON2 hydrolytic activity and can subvert the protection

438

afforded by PON2. PON2 has been detected in mitochondria, ER, and the perinuclear region

439

with its lactonase and critical Ca2+ binding site [126]. An intracellular Ca2+ burst leads to the

440

degradation of PON2 mRNA by its 5'-untranslated region and protein degradation by a

441

calpain-mediated mechanism [127]. Therefore, it is reasonable to assume that OdDHL can

442

also mediate PON2 activity because of its ability to promote cytosolic Ca2+, as mentioned

443

before. In fact, OdDHL can down-regulate hydrolytic activity of PON2, rather than PON2

444

protein, by a rapid post-translational mechanism through the induction of cytosolic Ca2+ in

445

epithelial and endothelial cells. This process is rapid, reversible, and can be blocked by the

446

Ca2+ chelator BAPTA/AM, implying that PON2 protein can be potentially modified by

447

uncertain methylation, acetylation, ubiquitination, or glycosylation in a Ca2+-dependent

448

mechanism [128]. PON3, the PON2 paralogue, can also be inactivated through a similar Ca2+

449

signal triggered by OdDHL [129]. Furthermore, OdDHL damaged cellular PON2 confers

450

protection against redox-active, pyocyanin-mediated oxidative stress, which makes cells more

451

vulnerable to the invasion of P. aeruginosa [128, 129]. A class of cell-active enzymes

452

represented by PON2 is involved in the invasion of OdDHL, with the product of the lactonase

453

reaction spontaneously undergoing ring formation in acidic environments, reverting back to

454

an active form [9]. The result of this interaction ultimately affects the fate of the cells. This

455

may explain why OdDHL triggers distinct effects in different cell species.

456

Recently, it was hypothesized that OdDHL exhibits PON2-dependent apoptosis. This

457

hypothesis is contrary to previous theories. In Bak-/- Bax-/- DKO MEFs, one clone expressed

458

very low levels of PON2 and did not undergo OdDHL-induced apoptosis (termed DKONR

459

MEFs), but another clone expressed a high level of PON2 and underwent OdDHL-induced

460

apoptosis (termed DKOR MEFs). However, overexpression of human PON2 in DKONR MEFs

461

rendered them responsive to OdDHL, which indicated that PON2 associated lactonase

462

activity uniquely triggered apoptosis in response to OdDHL without the involvement of Bax

463

and Bak [130]. This phenomenon was further elaborated in primary human bronchial

464

epithelial cells. OdDHL was hydrolyzed by PON2 to carboxylic acid, which becomes trapped

465

within the mitochondria, causing an intracellular acidification triggering pH-dependent

466

apoptosis [52]. Treatment with the PON2 inhibitor Triazolo [4,3-a] quinoline (TQ416) could

467

recover viability and block the oxidative stress of intestinal cells induced by OdDHL [64,

468

131]. These counterintuitive results may partly be explained by the hydrolysis of OdDHL by

469

PON2 in an enzymatic reaction, with the concentration of OdDHL as a substrate being critical

470

for the overall reaction rate [52]. If OdDHL reaches the threshold concentration, it will be

471

rapidly hydrolyzed to it acidic counterparts by PON2, inducing apoptosis. Conversely, this

472

process will be much slower if the concentration of OdDHL is below the threshold, with the

473

receptors like T2R38 and PPARγ related to immunoreaction being predominant. However,

474

depending on the diverse level of lactonase in different cell lines, the same concentration of

475

OdDHL tends to trigger different cellular responses. In summary, although the relationship

476

between PON2 and OdDHL is still unclear, it is undeniable that PON2 plays a very important

477

regulatory role in OdDHL-mediated biological effects.

478

6. Conclusion

479

P. aeruginosa is a leading pathogen that can cause critical illnesses particularly in

480

immunocompromised patients. P. aeruginosa often causes persistent infections due to the

481

acquisition of antibiotic resistance that is related to rapid development of drug resistance

482

mutations and the biofilm mode of growth. A better understanding of P. aeruginosa requires a

483

better understanding of the QS molecule OdDHL, which is an important regulator of the

484

expression of virulence factors. Although OdDHL controls P. aeruginosa functions only when

485

the density of the flora reaches a certain threshold, OdDHL may also contribute in

486

constructing a niche suitable for P. aeruginosa invasion and reproduction by interacting with

487

host cells. As is illustrated in this review, OdDHL has a huge impact on eukaryotes, such as

488

multiple pathway-mediated apoptosis, destruction of cell connections, chemotaxis of immune

489

cells, NF-κB dependent pro-inflammatory role or immunosuppression, unusual cell

490

receptor-mediated immune responses, and crosstalk with lactonase that is present in

491

mammalian cells. OdDHL may also facilitate virus dissemination [132] and increased

492

accumulation of autophagosomes in cells [133]. Thus, OdDHL is an active bioregulator and

493

cannot be easily eliminated by regulating certain signaling pathways in cells. However,

494

knowledge of the OdDHL target binding protein remains limited since T2R38 and PPARγ are

495

only involved in a minor way in OdDHL-mediated signaling pathways. Further studies on the

496

OdDHL receptor are necessary for effective blocking of OdDHL communication with

497

eukaryotes.

498

As research on OdDHL has broadened, contradictory results have been obtained, especially

499

concerning its immunological enhancement or immunosuppression. This may reflect the

500

different effects of varying concentrations of OdDHL on different cell types. Examination of

501

cells stimulated with OdDHL after various times would yield valuable information. We

502

observed a potent effect of OdDHL on cells in the early stage, with a gradual weakening with

503

time. Cell density is also related to OdDHL function because the different levels of lactonase

504

produced by cells can hydrolyze OdDHL. It is notable that diverse enzymes in distinct cells

505

may degrade OdDHL into different secondary products, which may elicit certain biological

506

regulatory activities. The degraded OdDHL can also revert to an active structure by ring

507

closure in acidic environments [65]. Therefore, the state of OdDHL in the cell is likely to be a

508

dynamic process that is difficult to monitor. Further investigations will be required to provide

509

clarity. The metabolic pathways of OdDHL in cells also facilitate targeted development of

510

OdDHL quenchers.

511

It needs to be recognized that OdDHL also has a uniquely positive role in anti-tumor activity

512

and skin wound healing. OdDHL is not a simple PAMP of P. aeruginosa. Further explorations

513

of the interactions between OdDHL and its mammalian host are required.

514

Ethical approval

515

Ethical approval is not required for this review.

516

Conflicts of Interest

517

All authors declare no conflict of interest.

518

Acknowledgements

519

This study was supported by a grant-in-Aid for Scientific Research from the Ministry of

520

Education, Science, Sports, and Culture of Japan (19H0405111, HO;16K11506, KY), the

521

SHISEIKAI Scientific Award, SHISEIKAI ASSOCIATION (KY), Suzuken Memorial

522

Foundation, Bayer Academic support, Astellas Academic support, Daiichi Sankyo Academic

523

support, Shionogi Academic support, and the Novartis Foundation (HO).

524

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891 892

893

Figure legends

894

Figure 1

895

AHL-based quorum sensing (QS) in P. aeruginosa

896

This diagram shows two AHL-based QS in P. aeruginosa. The lasI and rhlI genes are

897

responsible for the synthesis of OdDHL and BHL respectively, collectively known as AHL.

898

The lasR and rhlR genes synthesize the corresponding cytoplasmic receptors. AHL binds to

899

the cytoplasmic receptor at a certain threshold local density of P. aeruginosa and forms the

900

transcriptional regulator complex, which regulates numerous virulence factors of P.

901

aeruginosa.

AHL-based QS in Pseudomonas aeruginosa

lasI / rhlI

lasR / rhlR

Complex

AHL

R Protein Activation

Proteases Exotoxin A

902

903 904

Pyocyanin Elastase

Rhamnolipids

905

Figure 2

906

Synthesis of OdDHL

907

The transfer of the acyl group from acyl-ACP to the methionine on SAM through an amide

908

bond is catalyzed by LasI. OdDHL is then produced by the release of MTA from acyl-SAM

909

through lactonization of the methionine.

910 911

912

Figure 3

913

OdDHL quickly enters osteoblasts and assembles in mitochondria and endoplasmic reticulum

914

(ER)

915

MC3T3 pre-osteoblast cells were treated with green fluorescence tagged OdDHL (50 µM) for

916

15 minutes together with mitochondria (A) or ER (B) red dye. Confocal microscopy showed

917

that OdDHL was enriched in the cytoplasm and partially colocalized with mitochondria and

918

ER. DMSO denotes dimethylsulfoxide.

919

920

921

Figure 4

922

OdDHL triggers apoptosis in mammalian cells through three different pathways

923

OdDHL induces apoptosis in mammalian cells through three different apoptotic signal

924

pathways. The intrinsic pathway (mitochondria pathway) denoted by the green arrows, causes

925

cytochrome c release from mitochondria followed by activation of Caspase-9. The extrinsic

926

pathway denoted by blue arrows, triggers TNFR1 trimerization by disrupting the lipid domain

927

without an external ligand and then induces Caspase-8 activation. The endoplasmic reticulum

928

(ER) pathway denoted by black arrows, triggers intracellular Ca2+ overload through the

929

IP3R-IP3 calcium channel and promotes cytochrome c redistribution. Each of these pathways

930

leads to a final execution phase of Caspases-3 and/or -7 activation as well as cleaved-PARP

931

expression.

Apoptosis -TNFR1

-TNFR1 trimerization

OdDHL-

-FADD lipid domain disruption

-Pro-Caspase 8 -Caspase 8

to Mi

o ch

r nd

ia -Bcl-2 -Caspase 3 -Caspase 7

-Cytochrome c

-Caspase 9

XBP1 -Ca2+ IP3R

ER-

-PARP

Nucleus IP3

PLC DAG

OdDHLIntrinsic apoptosis Extrinsic apoptosis ER- Ca2+related apoptosis Inhibition 932 933

Apoptosis

934

Figure 5

935

OdDHL mediates cell junction destruction and chemotaxis

936

The epithelial junctional complex includes gap junctions (GJ), tight junctions (TJ), and

937

adherent junctions (AJ). OdDHL disrupts the function of the epithelial barrier by altering the

938

phosphorylation status of TJ- and AJ-related proteins, and blocking the exchange of water and

939

ions through the GJ, permitting P. aeruginosa invasion. In addition, OdDHL enhances IL-8

940

expression in epithelial cells and induces the chemotaxis of neutrophils. OdDHL also

941

upregulates the expression of adhesion proteins, such as CD11b/CD18, through

942

phospholipase C-mitogen activated protein kinase (PLC-MAPK) signaling in neutrophils,

943

which is also related to chemotaxis.

Junctions Destruction

OdDHL-

- P. aeruginosa

TJ Occludin Tricellulin Claudins ZO JAM

AJ

NF-κB

AP-2

E-cadherin IL-8 H2 O ions

GJ

Nucleus

Epithelial cells

CD 1

Endothelial cells 1b-CD 18

MAPK

-OdDHL PLC DAG PKC

Neutrophils 944 945

Chemotaxis

946

Figure 6

947

OdDHL mediates the host immune inflammatory response

948

OdDHL has dual functions on the host immune response. OdDHL can either promote nuclear

949

factor-kappa B (NF-κB) activation from mitogen activated protein kinase (MAPK) and

950

unfolded protein response (UPR) pathway, or can relieve the NF-κB inhibition by interfering

951

with the binding of rosiglitazone with nuclear receptor peroxisome proliferator-activated

952

receptor-gamma (PPARγ). The expression of numerous pro-inflammatory cytokines,

953

including interleukin (IL)-1, IL-8, cyclooxygenase 2 (Cox-2), and prostaglandin E2 (PGE2)

954

regulated by NF-κB are enhanced. OdDHL can physically combine with T2R38 and trigger

955

nitric oxide (NO) and IL-6 production in a Ca2+-dependent manner. On the other hand,

956

OdDHL suppresses the innate and adaptive immune response by activating cells which are

957

stimulated by lipopolysaccharide (LPS). OdDHL either downregulates the LPS-activated

958

pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-ߙ), monocyte chemoattractant

959

protein-1 (MCP-1), interferon-gamma (INF-γ), IL-2, and IL-12 by inhibiting NF-κB, or

960

upregulates the anti-inflammatory factors IL-10. OdDHL also suppresses NLRC4-mediated

961

inflammasome assembly and activation stimulated by LPS. In addition, OdDHL shifts

962

immune responses away from host protective Th1 responses to pathogen protective Th2

963

responses and inducible regulatory T-cells (iTregs) response.

964

Pro-Inflammatory

Nucleus

Innate Immunity

IL-1-� MIP NO

MCP

IL-1-�

LPS-

IL-8

R UP

NOS

Cox2

AP-2

OdDHL-TLR 4

R� PPA

IL-6

PGE2 NF-κB

C/EBP-� I P3 R

Ca2+

R UP

MAPK

CHOP

MAPK

AP-2 NF-κB

-T2R38 INF-�

IL-10

IL-12

OdDHL NLRC4

TNF-�

LPS signal OdDHL signal Promotion Inhibition

MCP

Anti-Inflammatory

INF-� 12 I L-

LPS

INF-�

Th1

IL-12 TNF-�

Immunity

IL-10

IL-4

CD4+T

Th2

IL-4 IL-5

Mo-DCs

Allergy, Th1 inhibition

IL2

TGF�

Adaptive Immunity 965 966

967

IL-2

iTreg Tolerance

TGF-� IL-10

OdDHL

Japanese Association for Oral Biology

Journal of Oral Biosciences Author Contribution Statement

Manuscript Title: Quorum-sensing molecule N-(3-Oxododecanoyl)-L-Homoserine lactone: An all-rounder in mammalian cell modification

Authors’ names: Guo Jiajie, Kaya Yoshida, Mika Ikegame, Hirohiko Okamura

Article type:

Review

Please list contribution made by each author to this manuscript – eg, literature search, figures, study design, data collection, data analysis, data interpretation, writing etc. If all authors contributed equally, please state this. If one page is not enough, please use any pages as possible. Author Name Guo Jiajie

Contribution made to this manuscript Literature search, Figures, Writing - original draft, Writing - review & editing

Kaya Yoshida

Writing - review & editing

Mika Ikegame

Writing - review & editing

Hirohiko Okamura

Funding acquisition, Methodology, Project administration, Writing - review & editing

This statement will be kept for 2 years after the publication of the manuscript.

Date of Completion:

9/10/2019

Corresponding author's Name: Guo Jiajie, Hirohiko Okamura